QSAR and Drug Design: New Developments and Applications

PHARMACOCHEMISTRY LIBRARY- VOLUME 23 QSAR AND DRUG DESIGN" NEW DEVELOPMENTS AND APPLICATIONS

PHARMACOCHEMISTRY LIBRARY, edited by H. Timmerman Other titles in this series Volume 9

Innovative Approaches in Drug Research. Proceedings of the Third Noordwijkerhout Symposium on Medicinal Chemistry, Noordwijkerhout (The Netherlands), September 3-6, 1985 edited by A.F. Harms

Volume 10

QSAR in Drug Design and Toxicology, Proceedings of the Sixth European Symposium on Quantitative Structure-Activity Relationships, Portoro2-Portorose (Yugoslavia), September 22-26, 1986 edited by D. Had2i and B. Jerman-Bla2i~

Volume 11

Recent Advances in Receptor Chemistry. Proceedings of the Sixth CamerinoNoordwijkerhout Symposium, Camerino (Italy), September 6-10, 1987 edited by C. Melchiorre and M. Giannella

Volume 12

Trends in Medicinal Chemistry '88. Proceedings of the Xth International Symposium on Medicinal Chemistry, Budapest, 15-19 August, 1988 edited by H. van der Groot, G. Domany, L. Pallos and H. Timmerman

Volume 13

Trends in Drug Research. Proceedings of the Seventh Noordwijkerhout-Camerino Symposium, Noordwijkerhout (The Netherlands), 5-8 September, 1989 edited by V. Claassen

Volume 14

Design of Anti-Aids Drugs edited by E. De Clerq

Volume 15

Medicinal Chemistry of Steroids

by F.J. Zeelen

Volume 16

QSAR: Rational Approaches to the Design of Bioactive Compounds. Proceedings of the Eighth European Symposium on Quantitative Structure-Activity Relationships, Sorrento (Italy), 9-13 September, 1990 edited by C. Silipo and A. Vittoria

Volume 17

Antilipidemic Drugs - Medicinal, Chemical and Biochemical Aspects edited by D.T. Witiak, H.A.I. Newman and D.R. Feller

Volume 18

Trends in Receptor Research. Proceedings of the Eighth Camerino-Noordwijkerhout Symposium, Camerino (Italy), September 8-12, 1991 edited by P. Angeli, U. Giulini and W. Quaglia

Volume 19

Small Peptides. Chemistry, Biology and Clinical Studies edited by A.S. Dutta

Volume 20

Trends in Drug Research. Proceedings of the 9th Noordwijkerhout-Camerino Symposium, Noordwijkerhout (The Netherlands), 23-27 May, 1993 edited by V. Claassen

Volume 21

Medicinal Chemistry of the Renin-Angiotensin System edited by RB.M.W.M. Timmermans and R.R. Wexler

Volume 22

The Chemistry and Pharmacology of Taxol| and its Derivatives edited by V. Farina

PHARMACOCHEMISTRY

LIBRARY

E d i t o r : H. T i m m e r m a n

Volume

23

QSAR AND DRUG DESIGN: N EW DEVE LO PM E NTS AN D APPLI CATI O N S

Based on Topics presented at the Annual Japanese (Quantitative) StructureActivity Relationship Symposium and the Biennial China-Japan Drug Design and Development Conference

EDITED BY:

TOSHIO FUJITA Department of Agricultural Chemistry, Kyoto University, Kyoto, and EMIL PROJECT, Fujitsu Kansai Systems Laboratory, Osaka, Japan

ELSEVIER Amsterdam

- Lausanne - New York-

Oxford - Shannon

- T o k y o 1995

ELSEVIER SCIENCE B.V. P.O. Box 1527 1000 B M A m s t e r d a m , The N e t h e r l a n d s

IS B N 0-444-88615-X

9 1995 Elsevier Science B.V. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, without the prior written permission of the publisher, Elsevier Science B.V., Copyright & Permissions Department, P.O. Box 521, 1000 AM Amsterdam, The Netherlands. Special regulations for readers in the U.S.A.-This publication has been registered with the Copyright Clearance Center Inc. (CCC), 222 Rosewood Drive, Danvers, MA 01923. Information can be obtained from the CCC about conditions under which photocopies of parts of this publication may be made in the U.S.A. All other copyright questions, including photocopying outside of the U.S.A., should be referred to the publisher. No responsibility is assumed by the publisher for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. This book is printed on acid-free paper. Printed in The Netherlands

Dedicated to

Professor Corwin Hansch Without his heartfelt encouragements, the editing of this volume would never have been completed.

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PHARMACOCHEMISTRY LIBRARY ADVISORY BOARD T. Fujita E. Mutschler N.J. de Souza D.T. Witiak F.J. Zeelen

Department of Agricultural Chemistry, Kyoto University, Kyoto, Japan Department of Pharmacology, University of Frankfurt, F.R.G. Research Centre, Hoechst India Ltd., Bombay, India College of Pharmacy, The Ohio State University, Columbus, OH, U.S.A. Organon Research Centre, Oss, The Netherlands

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PREFACE In this series of Pharmacochemistry Library the preceding volume dealing with the QSAR methodology and related topics is Vol. 16, QSAR: RationalApproaches to the Design of Bioactive Compounds, edited by Carlo Silipo and Antonio Vittoria, both of whom unfortunately passed away recently. Volume 16 was published as the Proceedings of the 8th European Symposium on Quantitative StructureActivity Relationships held in 1990 in Sorrento, Italy. Like the European Symposium, the Japanese Symposium on Structure-Activity Relationships has been organised annually since 1975. A bilateral symposium with Chinese scientists, the "China-Japan Drug Design and Development Conference", has been held biennially since 1989. This volume, instead of taking the form of Proceedings, is an edited volume based on topics selected from those presented at these symposia. Each chapter is thus more complete than the original presentations and includes consecutive series of the same topic originally presented separately. The structure-activity relationship (SAR) studies of bioactive compounds seem to have at least two objectives. One is to obtain insight into the pharmacological modes of action and the other is to deduce possible guiding principles for designing analogues with better bioactive profiles. The quantitative approach to the SAR (QSAR), initiated by Corwin Hansch and his co-workers some 35 years ago, opened up new possibilities in the SAR discipline. Because the Hansch QSAR expanded the Hammett-Taft paradigm in physical organic chemistry toward the biomedicinal (re)activity, the mode of action has been illustrated on the (sub)molecular level in many cases. It also revealed the critical importance of the hydrophobicity of the bioactive molecule. Before the advent of the QSAR, the mode of action had remained mostly on the level of discussions in terms of the "lock-and-key" hypothesis. Because the relationships are represented in the form of mathematical correlation equations with physicochemical (electronic, steric, hydrophobic and others when necessary) parameter terms in the QSAR, the bioactivity of non-measured analogues has sometimes been predicted by extrapolating significant parameters and proved after synthesis and biological tests. This can be regarded as the beginning of the quantitative drug design. Perhaps stimulated by the success of the traditional Hansch QSAR, a number of newer software-based methodologies have been publicized in the SAR and drug design disciplines, supported by the tremendous progress in computer technology in recent years. Among them are those based on theoretical physicochemical and/or molecular orbital calculations, those utilizing molecular modelling and graphics, those managing sophisticated statistical operations and data-base-oriented procedures. Some theoretical calculation softwares do not only deal with the stereo-electronic energy of ligands, but also extend their scope into protein molecules. Thus, the current situation is as if a successful drug design from receptor protein structures could be not entirely impossible.

In this volume topics are covered among almost every procedure and subdiscipline described above. They are categorized into three sections. Section I includes topics illustrating newer methodologies relating to ligand-receptor interactions, molecular graphics and receptor modelling as well as the threedimensional (Q)SAR examples with the active analogue approach and the comparative molecular field analysis. Note that the last two chapters also use the traditional QSAR to cross-validate the results obtained with the newer procedures. In Section II the hydrophobicity parameters, log P (1-octanol/water), for compound series of medicinal-chemical interest are analysed physico-organic chemically. New procedures for the lead generation using databases of aminoacid sequences and structural evolution patterns, as well as a newer statistical QSAR modification utilizable in cases when the bioactivity potency is represented by ratings, are also placed in this Section. Section III contains the examples based on the traditional Hansch QSAR approach. Two contributions are from China illustrating how to identify the lead structures from folk medicine and how to optimize them in clinical applications. Others in this Section are instructive examples of the Hansch approach for various series of bioactive compounds in rationalizing the potency variations, actual designing the clinical candidates and revealing the (sub)molecular mechanism of action. A variety of methodologies and procedures are presented in this single volume. It is recommended that the readers regard each of the methodologies as complementary to others. It must be confessed that editing this volume required a much longer period than I had originally expected. Apologies are due to some of the authors if their chapters have become out of date, because the speed of progress in this field is very fast. If there could be something to mitigate the responsibility, it is the fact that most of the chapters dealing with rapidly growing topics describe their methodological philosophy in some detail. With understanding the background way of thinking, further developments can hopefully be caught up without difficulty. Last but not least, the editor expresses his sincere thanks to Mrs. A. Elzabeth Ichihara for critical correction of the English in most of the original manuscripts. August 1, 1995 Toshio Fujita, at Fujitsu Kansai Systems Laboratory

XI

LIST OF CONTRIBUTORS Dr. G. Appendino Dipartimento di Scienza e Tecnologia del Farmaco via R Giuria 9 10125 Torino ITALY Dr. S.H. Chen Bristol Myers Squibb Pharmaceutical Research Institute RO. Box 5100 Wallingford, CT 06492-7660 U.S.A.

Dr. L. Landino Chemistry Department University of Virginia Charlottesville, VA 22901 U.S.A. Dr. T. MacDonald Chemistry Department University of Virginia Charlottesville, VA 22901 U.S.A.

Dr. T. Cresteil INSERM U75 Universite Rene Descartes 75730 Paris Cedex 15 FRANCE

Dr. B. Monsarrat Laboratoire de Pharmacologie et Toxicologie Fondamentales CNRS 205 Route de Narbonne 31400 Toulouse FRANCE

Dr. R.C. Donehower Division of Pharmacology and Experimental Therapeutics Johns Hopkins Oncology Center Baltimore, MD 21287 U.S.A.

Dr. E.K. Rowinsky Div. of Pharmacology and Experimental Therapeutics Johns Hopkins Oncology Center Baltimore, MD 21287 U.S.A.

Dr. V. Farina Department of Medicinal Chemistry Boehringer Ingelheim Pharmaceuticals 900 Ridgebury Road Ridgefield, CT 06877 U.S.A.

Dr. I. Royer Laboratoire de Pharmacologie et Toxicologie Fondamentales CNRS 205 Route de Narbonne 31400 Toulouse FRANCE

Dr. D. Guenard Institut de Chimie des Substances Naturelles CNRS 91190 Gif-sur-Yvette FRANCE Dr. J. Kant Bristol Myers Squibb Pharmaceutical Research Institute P.O. Box 5100 Wallingford, CT 06492-7660 U.S.A.

Dr. D.M. Was Bristol Myers Squibb Pharmaceutical Research Institute 5, Research Parkway Wallingford, CT 06492-7660 U.S.A. Dr. M. Wright Laboratoire de Pharmacologie et Toxicologie Fondamentales CNRS 205 Route de Narbonne 31400 Toulouse FRANCE

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xIII

CONTENTS T. Fujita: Preface

SECTION I:

.................................

ix

Three-Dimensional Structure-Based Drug Design, Molecular Modelling and Three-Dimensional QSAR.

A. Itai, N. Tomioka, Y. Kato Rational Approaches to Computer Drug Design Based on Drug-Receptor Interactions . . . . . . . . . . . . . . . . . . . . . . . . K. Akahane, H. Umeyama

Drug Design Based on Receptor Modeling Using a System

"BIOCES(E)"

. ...............................

49

T. Matsuzaki, H. Umeyama, R. Kikumoto

Mechanisms of the Selective Inhibition of Thrombin, Factor Xa, Plasmin and Trypsin . . . . . . . . . . . . . . . . . . . . . . . . . . . .

83

H. Koga, M. Ohta Three-Dimensional Structure-Activity Relationships and Receptor Mapping of Quinolone Antibacterials . . . . . . . . . . . . . . . . . . .

M. Yamakawa, K. Ezumi, K. Takeda, T. Suzuki, I. Horibe, G. Kato, T. Fujita Classical and Three-Dimensional Quantitative Structure-Activity Analyses of Steroid Hormones: Structure-Receptor Binding Patterns of Anti-hormonal Drug Candidates . . . . . . . . . . . . . . . . . . . .

97

125

SECTION I1: Quantitative Structure-Parameter Analyses and Database-Oriented and Newer Statistical (Q)SAR Procedures and Drug Design, C. Yamagami, N. Takao, T. Fujita

Analysis and Prediction of 1-Octanol/VVater Partition Coefficients of Substituted Diazines with Substituent and Structural Parameters . . . 153

M. Akamatsu, T. Fujita Hydrophobicities of Di-to Pentapeptides Having Unionizable Side Chains and Correlation with Substituent and Structural Parameters . . 185 T. Nishioka, J. Oda

Analysis of Amino Acid Sequence-Function Relationships in Proteins . 215

xIv

T. Fujita, M. Adachi, M. Akamatsu, M. Asao, H. Fukami, Y. Inoue, I. Iwataki, M. Kido, H. Koga, T. Kobayashi, I. Kumita, K. Makino, K. Oda, A. Ogino, M. Ohta, F. Sakamoto, T. Sekiya, R. Shimizu, C. Takayama, Y. Tada, I. Ueda, Y. Umeda, M. Yamakawa, Y. Yamaura, H. Yoshioka, M. Yoshida, M. Yoshimoto, K. Wakabayashi

Background and Features of EMIL, A System for Database-Aided Bioanalogous Structural Transformation of Bioactive Compounds . . . 235

10

I. Moriguchi, S. Hirono

Fuzzy Adaptive Least Squares and its Use in Quantitative StructureActivity Relationships . . . . . . . . . . . . . . . . . . . . . . . . . . . .

275

SECTION II1: Traditional QSAR and Drug Design. 11

12

13

14

15

16

Z-r. Guo

Structure-Activity Relationships in Medicinal Chemistry: Development of Drug Candidates from Lead Compounds . . . . . . . . . . . . . . .

299

R.-I. Li, S.-y. Wang

Chemical Modification and Structure-Activity Relationship Studies of Piperine and its Analogs: An Example of Drug Development from Folk Medicine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

H. Terada, S. Goto, H. Hori, Z. Taira

Structural Requirements of Leukotriene Antagonists

..........

321

341

K. Mitani

Quantitative Structure-Activity Relationships of a New Class of Ca2+-Antagonistic and 0~-Blocking Phenoxyalkylamine Derivatives . . . 369

H. Ohtaka

Applications of Quantitative Structure-Activity Relationships to Drug Design of Piperazine Derivatives . . . . . . . . . . . . . . . . . . . . .

413

K. Hashimoto, H. Tanii, A. Harada, T. Fujita

Quantitative Structure-Activity Studies of Neurotoxic Acrylamide Analogs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Subject index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

451

481

SECTION I: Three-Dimensional Structure-Based Drug Design, Molecular Modelling and Three-Dimensional QSAR.

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QSAR and Drug Design - New Developments and Applications T. Fujita, editor 9 1995 Elsevier Science B.V. All rights reserved

RATIONAL A P P R O A C H E S TO C O M P U T E R D R U G D E S I G N B A S E D ON D R U G - R E C E P T O R I N T E R A C T I O N S

Akiko Itai*, Nobuo Tomioka* and Yuichi Kato Faculty of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, Japan ABSTRACT

We have developed two novel methods and computer programs for rational drug design on the basis of drug-receptor interaction. The program GREEN is to perform docking studies efficiently and rationally, when the receptor structure is known. The main features of the program are the real-time estimation of intermolecular interaction energy and the informative visualization of the drug binding site. In addition, many functions help to find a p p r o x i m a t e l y the stable positions and conformations of a drug molecule inside the receptor cavity. The other program, RECEPS, is for rational superposition of molecules and for receptor mapping, when the receptor structure is not known. The superposition is performed through the use of spatial grid points and monitored by several goodness-of-fit indices indicating the similarities in physical and chemical properties. Based on the superposed structures, a three-dimensional receptor image can be constructed, which reveals cavity shapes, expected locations and characters of hydrogen-bonding groups, electrostatic potentials of the surface, and other features. 1. I N T R O D U C T I O N

For the development of new drugs, a tremendous number of compounds must be synthesized and assayed for biological activities. As the difficulties in synthesizing compounds have decreased with the technical advances of organic synthesis, the efficient design of bio-active molecules has become more and more important. Usually, drug development starts with the selection of a lead compound, and then the structure is modified to obtain better biological response profiles. But, starting from an appropriate lead compound is the key to success. How to find an appropriate lead compound and how to optimize the lead structure efficiently are the central problems of drug development. As yet, however, no general *Present address: Institute of Medicinal Molecular Design, 4-1-11 Hongo, Bunkyo-ku, Tokyo, Japan

methods for solving these problems are available. Indeed, finding new lead compounds is so difficult as compared with optimizing existing lead compounds that they have never been generated artificially. It has long been desired to design active structures on the basis of logic and calculations, not relying on chance or trial-and-error. Computers have been introduced into drug design for that purpose, and with the remarkable progress of computer technology in the past thirty years, computers have become widely used in drug research for maintaining databases, statistical processing, molecular modeling, theoretical chemical calculation, and so on. Since analyses of the relationships between structures and activities by using computers began more than twenty years ago (1), various approaches have been reported by many researchers. Some of them, however, have fallen by the wayside as our understanding of drug-receptor interactions has deepened.

Drug-Receptor Interactions It is well known now that a drug molecule exerts its biological activities by binding specifically to a target macromolecule, or receptor, in the body. Dozens of receptor molecules for various hormones and neural transmitters have been isolated and characterized, and their amino acid sequences have been determined. None of the three-dimensional structures of such receptors has been elucidated, whereas those of hundreds of proteins have already been elucidated to atomic resolution by X-ray crystallographic analyses. Some solutions have been obtained for complexes of protein and ligand molecules. These results have provided us with details of molecular recognition by the macromolecule as well as the three-dimensional structure of the macromolecule. Such concrete molecular images have validated the key-and-lock model for drugreceptor interaction, which had been vaguely understood for a long time. In most of the complexes, ligand molecules are non-covalently bound to proteins. The complexes are stabilized by intermolecular forces such as hydrogen bonds, electrostatic interactions, van der Waals forces, and hydrophobic interactions. The strength of binding, which is represented experimentally by equilibrium constants of binding or dissociation, can be estimated by empirical energy calculations. The sum of the intramolecular and intermolecular energy values is taken as an index for showing

the binding affinity, although the molecular recognition results from the free energy decrease upon complexation between the molecules. Accordingly, the more energetically favorable the interaction of the ligand molecule with the receptor is, the more efficiently the ligand can bind to the target receptor specifically. There are many examples where agonist and antagonist molecules with quite different chemical structures can bind strongly to the same site of the same receptor as the natural bio-active compounds. This fact is well evidenced by a number of crystallographic studies on protein-ligand or enzyme-inhibitor complexes. It can be seen that it is not the skeletal structure itself but the threedimensional array of submolecular physical and chemical properties of the ligand molecule that is recognized by proteins. As receptors consist mainly of proteins and the main functions of receptors seem to depend on the protein constituents, the molecular recognition between a receptor and drug is supposed to be very similar to that between an enzyme and substrate. The only difference is that reactions proceed in the case of enzymes, whereas signals are transduced between cells in the case of receptors. Many enzyme inhibitors are used as clinical drugs, in order to maintain biological homeostasis by controlling biochemical reactions or to prevent pathogenic microorganisms from proliferating. In this article, we use the term "receptor" in a broad sense, including not only the pharmacological receptors for hormones and neural t r a n s m i t t e r s but also enzymes or other globular proteins or nucleic acids.

Methods for Analysis of Structure-Activity Relationships Various approaches have been proposed for analyzing structure-activity relationships using computers. Among them, there are approaches in which the chemical structural formula is split up into component units. The individual substructural components are regarded as being significant to various extents for the biological activity, and the structureactivity relationships are analyzed a s s u m i n g t h a t the activity is controlled by combinations of the activity-indices assigned to the individual structural units contained in each structural formula. The activities of a series of compounds are expressed as functions of these indices by linear or non-linear combination methods. These approaches seem to be

just for the analyses, but not effective for understanding molecular recognition by biological macromolecules. Some of the substructures may indeed play important roles in interaction with the receptor. But, they can often be replaced by other groups with similar physical and chemical properties. As stated before, it is not just the existence of the particular structural units but the spatial alignments of physical and chemical properties of the units that are important. It seems to be quite difficult to reconstitute the separated pieces of a structural formula to obtain new molecules in the hope that they will have the same biological activity as the original molecule. Among approaches based on the physicochemical properties of molecules, Hansch and Fujita's method (2) is excellent. They have developed a method whereby the relationships between structures and activities can be analyzed quantitatively. In this method, biological activities are correlated with various physicochemical properties of substituent groups at specified positions of molecules in a series of derivatives with the same skeletal structure. By regression analyses, the activities of dozens of compounds can be represented by an equation consisting of a linear combination of several physicochemical variables. Usually, the physicochemical properties of substituent groups, such as inductive, resonance, hydrophobic, and other effects, and those of whole molecules, such as the partition coefficient and molar refractivity, are chosen as variables (3), since they make significant contributions to the activity. From the coefficient for each variable term in the equation, we can determine quantitatively the extent of the contribution of each property to the activity. This method is a powerful tool to indicate quantitatively the direction of subsequent structural modifications in order to improve the biological activity. Although the interpretation of the physical meanings of the variables is not always clear, the equation covers a number of interactions between drugs and biological systems. The method has been shown to be useful for performing lead optimization rationally and used worldwide. But, it is necessary to establish different methods for interpreting the structure-activity relationships for molecules with different skeletal structures, and for designing new molecules with different skeletons. For these purposes, efficient methods using three-dimensional structures, based on new concepts, seem to be essential.

Three-Dimensional Structures of Molecules The three-dimensional structure is the most realistic description of an existing molecule. The chemical structure itself cannot be directly related to biological activities and functions of a molecule, though it is an excellent graphic means to describe chemical bondings. However, all the features of a molecule, such as physical properties, chemical reactivities, dynamical behaviors and molecular interactions, should be interpretable in t e r m s of its three-dimensional structure. With the remarkable advances in techniques of solving crystal structures, it has become more and more easy to obtain three-dimensional structures of molecules. In the last three decades, techniques and equipment for measuring diffraction from crystals, and algorithms for solving the phase problem and for refining structures have made remarkable progress. In the field of small molecules, structure analyses can be routinely performed now. Even in the field of macromolecules, methods for structure analyses have been established (4) and structure elucidations have become progressively easier, although crystallization still remains a difficult problem. The analyses can now be applied to larger, more unstable, and more complicated molecules, and can be done with smaller amounts of samples, with less labor, and in a shorter period than before. The results of these crystallographic analyses have been put into generally available databases. The atomic coordinates of molecules and accompanying crystallographic data of small molecules are available in the Cambridge Crystallographic Database (5). Those of macromolecules are available in the Protein Data Bank (6) (National Laboratory Institute, Brookhaven). These databases have deepened our understanding of the three-dimensional structures of molecules and of molecular interactions. Especially, the crystal structures of protein-ligand complexes or DNA-ligand complexes have clarified the details of molecular recognition by macromolecules in general, as well as in individual cases.

Three-Dimensional Computer Graphics Three-dimensional structures and interactions of protein-ligand and DNA-ligand complexes can be better understood by using threedimensional computer graphics devices (hereafter abbreviated as "3DCG"), which can store images of three-dimensional objects in the

memory and apply three-dimensional transformations to the image, such as rotation, translation and scaling in real time (7). In the past decade, 3D-CG has become an essential tool for computer molecular modeling. Three-dimensional structures in the crystallographic databases or private data files can be displayed directly on 3D-CG and the molecules can be manipulated interactively (rotation, translation, and bond rotation) with input devices such as dials, a joystick, keys, and a mouse connected to the display. After manipulating or modeling the molecule, new atomic coordinates can immediately be stored in files and can be readily used for computation, and the picture can be reproduced at any time. In addition to various representations of molecular structures such as wire-frame, ball-and-stick and space-filling models, physical and chemical properties and virtual characters of molecules, such as electrostatic potentials, molecular orbitals, and expected sites of hydrogen bonding partners, can be displayed on 3D-CG, and compared visually with those of other molecules. Recently, high-performance 3D-CG workstations have become available in place of the combination of 3D-CG terminals with a host computer. Dozens of well-developed softwares for computer-assisted molecular design based on 3D-CG are commercially available and are now widely used (8). The main functions of the softwares are molecular modelling and theoretical calculations. In order to construct threedimensional structures, various procedures are provided with the softwares, and are usually performed interactively on graphic displays. Crystallographic databases or private structure files are referenced, if necessary, and the structures are subjected to further modification, such as addition or deletion of substituent groups, replacement of atomic elements, and conformational changes. Some theoretical calculations are applied for refining the geometries and for obtaining the stable conformation. But, a serious problem is that there are a number of possible three-dimensional structures in non-rigid molecules.

Theoretical Calculations The progress of theoretical calculations in the field of chemistry, such as molecular mechanics (9), molecular orbital (10,11), and molecular dynamics (12) calculations, has been remarkable. The methods are used

for estimating energetic stabilities, electronic properties, and molecular interactions. It is a characteristic of computational methods that they are applicable not only to actually existing molecules but also to imaginary structures. They are useful not only for interpreting various chemical p h e n o m e n a but also for predicting t h e m without experiments. Molecular mechanics and molecular orbital calculations can give us the minimum energy structure with its energy value, although it might not be the global minimum structure but only the local minimum near the starting structure because of the limitations of the energy minimization algorithm. These methods are very useful for refining structures in molecular modeling. Molecular dynamics calculations simulate the motions based on the potential energy calculation by using the force field and Newton's equation of motion, assuming each atom to be a particle. By solving the equation for each short time step in a certain period of time, a trajectory is obtained as a series of positions and velocities of atoms in the system. The dynamic behaviors of molecules can be simulated along the time course by using energy values and other structural features. Unlike the molecular mechanics calculation, the molecular dynamics calculation can override the energy barriers between local minima. But, it still has a limitation in getting over high energy barriers and the global minimum search is not easy even by this technique. Nevertheless, the calculation has come to be used for the purpose of finding the stable structures of super-flexible molecules, including those of solvated states, and estimating free energy difference between two similar states.

Active Conformation of Drugs The calculations described above have become indispensable tools not only in structural organic chemistry but also in analyses of structure-activity relationships in computer-aided drug design. They are of course useful for interpreting the chemical reactivity. For the purpose of drug design or analyses of structure-activity relationships, however, attention has to be paid to the fact that, in general, chemical reactions start from the most stable three-dimensional structures of the molecules involved in the reaction, whereas biological activities arise from the stable interaction of drug molecules with receptor macromolecules. For drug activities, we

10 must consider the stability of the drug-receptor complex, in place of the stability of the drug itself. Therefore, when the three-dimensional structures of receptor macromolecules are not known, we cannot estimate the stability and the stable structure of the drug-receptor complex computationally. Even if the receptor structure is known, it is not easy to find the stable mode of binding of the two molecules, because of the vast number of possibilities arising from the six degrees of freedom of rotation and translation. A "carpet bombing" search for the global energy minimum by changing all degrees of freedom is not realistic in a multidimensional system. A blind calculation of molecular mechanics or molecular dynamics does not yield any stably docked structures owing to the energy barriers. Therefore, we must prepare appropriate starting structures in order to avoid being trapped in unexpected local minima, before starting the calculation. The global energy minimum structure is often assumed to be the most stable structure among them, although this assumption is not necessarily correct. In the case of flexible molecules which have a number of rotatable single bonds, it is especially difficult to find the most stable structure in the complex because of the additional degree of freedom for bond rotation. The conformation which a drug molecule or a natural substrate molecule adopts on its receptor is called the "active conformation". The active conformation for each bio-active molecule is not necessarily the most stable conformation of the molecule itself. The active conformation can be determined most straightforwardly by X-ray crystallography on a crystal of the drug-receptor complex. Those of other drug molecules, which are known to interact with the same receptor, can be estimated based on the structure of the drug binding site. The main problems in docking procedure calculations are as mentioned above. Knowledge of active conformations is quite useful for evaluating structure-activity relationships and designing new structures, especially when the receptor structure is not known. But, it is very difficult to determine the active conformation of a highly flexible molecule without knowledge of the receptor structure. Theoretical calculations are less useful for these purposes.

ll 2. STRATEG1E~S OF OUR APPROACHES Background Because the background is extremely complicated and full of unelucidated factors in spite of recent advances in molecular biology, it seems to be most challenging to establish novel strategies for drug design. First of all, it is important to explore a rational way of drug design in general, r a t h e r t h a n in individual cases. To develop new concepts and new methodologies, effective and efficient utilization of computers seems to be an essential prerequisite, rather than classic procedures utilizing simple mimicry of the process or way of thinking of synthetic chemists, who previously carried out drug development. As it is receptors that hold the keys to biological activities, the most logical approach in drug design is to make use of receptor structures. Even if the receptor structure is unknown, provided that two or more active molecules are known, approaches based on an assumed common receptor are more rational than those based on simple similarities of their structures. We have been developing several program systems based on the receptor, as we will describe later. F u n d a m e n t a l Concepts The key assumptions underlying our concepts are as follows. 1) It is not the chemical structures or atomic positions that are recognized by macromolecules in biological systems. Recognition of a ligand molecule involves the overall intermolecular forces. It is the spatial arrangement of submolecular physical and chemical properties t h a t is important for the proper interaction between two molecules. These properties along with the contact surfaces should be complementary between two molecules. Among various intermolecular forces, the hydrogen bond is very important for discrimination between molecules. Hydrogen bonding works within a limited distance and direction,

whereas the electrostatic interaction works in all directions and over a long distance. In many crystal structures of protein-ligand complexes, ligand molecules have been found to be fixed firmly to the proteins through a number of hydrogen bonds as indicated in Fig. 1 as an example.

12

Fig. 1 Hydrogen bonds ( d o ~ lines) between/~ casei dihydrofolate r e d u c ~ and a potent inhibitor methotrexate (filled bonds) in the crystal structure. (Drawn with the atomic coordinates from the Protein Data Bank entry 3DFR (13)).

2) Molecules with quite different chemical structures can b i n d to the

Many examples are known of competitive inhibition between molecules belonging to different categories of structural types, as found by receptor assay with a radioisotopic ligand. These pairs of molecules, such as those shown in Fig. 2, might have a common three-dimensional shape and common physical and chemical properties such as hydrogen bonding, electrostatic, and hydrophobic interactions. The shape and the properties of these molecules must be complementary with those of the receptor. Furthermore, it is not the existence of the individual properties but their spatial arrangements on the molecule that are important for binding specifically to the receptor site. Flexible molecules must be able to adopt stable conformations that satisfy these requirements.

same site o f a receptor.

13 Natural and Synthetic Estrogens

Natural and Synthetic Retinoids

Substrate and Inhibitor of Cyclooxygenase

OH

~ Estradiol

Retinoic Acid

OH

Hi. ~ ~ N

HO Diethylstilbestrol (14)

0

AM80 (15)

H

Arachidonic Acid

COOH CH30~

N~' CH2COOHcH3 C=O CI

Indomethacin (16)

Fig. 2 Structure-pairs of natural and synthetic ligands (14,15,16) that bind to the same receptor sites. The binding to the same receptor site has been proved by receptor binding assay.

3) The whole structure of the drug molecule is not necessarily required for receptor binding. Inspection of the crystal s t r u c t u r e s of enzymei n h i b i t o r complexes elucidated by X-ray c r y s t a l l o g r a p h y indicates t h a t not all the a t o m s of an inhibitor molecule are necessarily involved in its interaction with a protein, as can be seen, for example, in Fig. 3.

Fig. 3 Three-dimensional structure of/,. case/ dihydrofolate reductase (thin line) and b o u n d inhibitor m e t h o t r e x a t e (thick line) in the crystal. Some atoms in methotrexate at the opening of the binding site may have contacts with molecules outside the protein. (Drawn with the atomic coordinates from the Protein Data Bank entry 3DFR (13))

14 As usual ligand molecules which fill the cavity of the ligand binding site are not totally buried in the protein, an opening cleft exists as an entrance into or an exit from the cavity. Even in the case where most of the atoms in a ligand directly contact protein atoms, the back surface of the ligand might be exposed to the outside. The structure of the exposed portion may be nonspecific, although the functional groups on t h a t portion would contribute to dissolution, partition, transport and permeability through the membrane, together with those in the buried portion. On the other hand, the buried portion of the ligand strongly bound to the receptor should have a specific structure corresponding to the target receptor. Therefore, structural modification for lead optimization should be applied to the exposed portion, if we can distinguish between the two portions. The a p p a r e n t molecular shapes of drugs t h a t are known to bind to the same receptor site often seem to be dissimilar because of the existence of the nonspecific portion. So, conventional shape analysis methods that use the whole three-dimensional structure of drug molecules would have no significance. Comparison of the surface electrostatic potentials between molecules with the same biological activities also seems to have no significance, unless the comparison is limited to the buried surface that is directly involved in receptor binding.

Structure-Activity Relationships and Designing New Structures To establish a correct model of structure-activity relationships is the s t a r t i n g point of designing new structures. For the optimization in a definite skeletal structure, quantitative structure-activity relationships based on two-dimensional structures of molecules (2) are useful to indicate an appropriate course of structural modification in substituents. For molecules with different skeletal structures, however, methods based on the three-dimensional structures of molecules are essential. Several methods have been proposed so far, although they are not sufficiently powerful to guarantee their success in rational drug design at present. When the receptor structure is known, examinations of relationships between three-dimensional structures and activity seem to be r a t h e r easy (8), and the design of new molecules by s t r u c t u r a l modification could be done without difficulty. But, even in these cases, the design of new molecules with different skeletal s t r u c t u r e s cannot be realized

15

easily. When the receptor structure is not known, the examination of structure-activity relationships as well as the design of new molecules becomes much more difficult. The constructed model of structureactivity relationships is necessarily less certain and less reliable because of an insufficiency of information. Each drug molecule may not be wholly complementary to the receptor cavity, only parts of the chemical and physical properties of the drug binding site being reflected. Use of information from multiple molecules with different skeletal structures can give a better image of the receptor cavity. The deduced receptor cavity or the structural requirement for binding to the receptor would give a useful hypothetical basis for structure-activity relationships, and contribute to the design of new structures, although each must be refined or modified repeatedly through synthetic trials. In any case, the design of new structures with different skeletons, so-called "lead generation", is so difficult that it can rarely be attained either by human work or by computer at present. In order to make lead generation possible, it is necessary to develop special methodologies where the h u m a n brain and computer give full play to their particular abilities.

Common Features of the GREEN and RECEPS Programs Based on the principles of drug-receptor interaction described above, we have developed new methods and computer programs for drug design. Among several systems developed for various purposes, we describe here two program systems for evaluating structure-activity relationships using the three-dimensional structures of molecules. One is the program system GREEN for efficient docking studies when the receptor structures are known (17,18), and the other is the program system RECEPS for rational superposition of molecules and receptor mapping when the receptor structures are not known (19). The GREEN program is based on the three-dimensional structures of receptor proteins. It enables the real-time estimation of intermolecular interaction energy between protein and ligand molecules throughout the docking process, describing the physical and chemical environment of the ligand binding site of the protein. It should be helpful in finding the stable relative geometry of protein and ligand molecules in explanations

15

of the m e c h a n i s m s of biochemical reactions and structure-activity relationships of drugs. Without information on receptor structures, the RECEPS program is based on the three-dimensional structures of multiple molecules which are supposed to bind specifically to the same receptor. In the RECEPS program, molecules are superposed in terms of submolecular physical and chemical properties, not in terms of the atomic positions or partial chemical structures as has so far been done conventionally. A threedimensional receptor model can be constructed according to the superposed structures. The model provides the size and shape of the bindingsite cavity, hydrogen bonding sites, the electrostatic character on the surface, and other structural indices. The common features of these two programs are that they (1) are based on the specific interactions between drugs and a target (2) (3) (4) (5)

receptor; make use of a three-dimensional grid to describe the physical and chemical properties spatially; utilize 3D computer graphics interactively, as an interface between the h u m a n brain and computer; yield numerical indices for indicating the validity of docking or superposition in real time; and are useful not only for interpreting structure-activity relationships, but also for designing new structures.

3. APPROACHES BASED ON RECEPTOR STRUCTURE

Docking Studies Techniques for isolation and identification of proteins have made remarkable progress in recent years, and a number of protein structures have been elucidated or are being elucidated at the atomic level. Some of these proteins are bound with small molecules such as inhibitors and cofactors in the crystal. Based on the three-dimensional structure of the protein in such protein-ligand complexes, we can simulate stable interaction modes of ligand molecules with the protein with the aid of computers (20). We can estimate the stability of the ligand molecule with arbitrary conformation at arbitrary relative position, search for the mode

17 of the minimum energy binding and determine its stability. Such approaches have often been called "docking studies" (21). Docking studies are used not only for investigating natural biochemical processes but also for examining the mode and stability of binding of drugs to the target receptor in drug design. Interaction and/or reaction of natural substrates may be difficult to study by crystallographic or other experimental methods, because of the rapid progress of enzymatic reactions. Substrate specificity, site-specific or stereo-specific reactivity, and stability of the possible intermediates can be evaluated by docking simulation. Furthermore, as the binding affinity and the binding mode can be predicted for molecules that have not yet been synthesized, such simulation is useful for designing molecules with enhanced affinity to a target receptor and for selecting candidate molecules for synthesis. A ligand molecule that can bind strongly to the target receptor should have energetically favorable interactions with the receptor with an appropriate relative geometry. In docking simulation, the problem of finding such geometry between ligand and target molecules is too difficult to be accomplished only by computational methods. Besides conformational freedom, six degrees of freedom for rotation and translation of the ligand may give rise to innumerable local minima, from which a global minimum cannot be easily discriminated. Therefore, for the time being, likely stable geometries usually have to be selected by visual judgment using the 3D-CG display before starting computation. To find a likely stable geometry and conformation, the ligand molecule is subjected to a series of interactive three-dimensional manipulations (rotation, translation, and bond rotation) inside the ligand binding site of the protein on the 3D-CG display. During the last ten years, many docking simulation studies for various purposes have been published, based on the known structures of proteins or nucleic acids.

Approaches by Other Research Groups In 1981, Connolly developed an algorithm for rapid calculation of the positions of a group of dots for representing a molecular surface (22) based on the definitions made by Richards (23). Electrostatic properties can be represented by color-coded dots according to electrostatic potentials calculated at the molecular surface from all the atomic charges in

18 the molecule. By using these techniques, Weiner et al. have shown that there is a good complementarity in shape as well as in electrostatic properties between partners in several protein-ligand complexes whose structures had been elucidated by X-ray crystal analyses (24). The representation is not only beautiful but also useful for understanding molecular recognition. Without numerical indices evaluating the goodness of fit, however, this method is not so significant for practical use in finding stable ligand geometry. The protein-ligand interaction energy is a good indicator in selecting or modeling ligand molecules with strong affinity to the target protein. Empirical energy function and force field parameters are usually used for estimating the intermolecular and intramolecular energetic stability of macromolecules. In order to find a stable geometry and conformation of the ligand molecule rapidly and effectively, the estimation should be made on every manipulation of the molecule to provide a guide to the direction and amplitude for the subsequent manipulation. But, because of the large number of atoms in proteins, it takes rather a long time to calculate the energies by using the conventional atom-pair type algorithm even on an efficient workstation at present. In addition to the six degrees of freedom of rotation and translation, the conformational freedom of non-rigid molecules makes the problem very difficult and time-consuming. Therefore, most of the docking processes on 3D-CG are performed without energy estimation, by monitoring only interatomic distances so that the atoms do not come too close to each other. In 1985, Goodford presented a new method to show favored sites for such functional groups as amino, hydroxy, and carboxyl groups, and water inside the ligand binding cavity of a protein (25). The favorable sites for each functional group and water, which are contoured at a certain energy level from the map of total interaction energy consisting of van der Waals, electrostatic and hydrogen bonding interactions, are shown on graphic displays as bird cage models. The method seems to be very useful for designing new structures by adding or modifying functional groups which are expected to enhance the binding. But, it is not suitable for interactive docking studies to find stable relative geometries of the ligand molecule.

19

P a t t a b i r a m a n et al. have presented another approximation method for real-time estimation of interaction energy between a protein and ligand (26). They used the square root of the product of the Lennard-Jones potential parameters of the two interacting atoms to approximate interaction energy between the pair. On each grid point defined in the ligand binding site, they precalculated two sets of data corresponding to the attracting and repulsive terms of the potential function. Although their method enables the real-time estimation of intermolecular van der Waals interaction energy, it is not so useful for practical purposes because other energies such as those of electrostatic and hydrogen-bonding interactions are ignored.

Details of the Program GREEN Intermolecular interaction energy between a protein and a ligand molecule is usually thought to consist mainly of van der Waals, electrostatic and hydrogen-bonding interactions. It can be calculated by the conventional empirical method by Eq. 1, where A and B are the LennardJones parameters, C and D are the hydrogen-bond parameters, rij is the distance between interacting atoms i and j, q is the atomic charge, s is the dielectric constant of the medium, and Nnb and Nhb are the number of atom-pairs included in the calculation of each energy term. E i . r t . . . . . tecutar = Eva,~ ar

W a a l s -3t- E e l e c t r o s t a t i c + E H - b o n d

Nnb Nnb Nhb ___ ~ ( A i j r i j--2 l _ B i j r i j--6 )_jr_ ~ qiqj "~- ~ (CijFij- 2I - - D i j r i j - o1 ) . . erij i,j i,j z,.l

[1]

The calculation takes a rather long computational time because of the large number of atoms in a protein and consequently the l a n e number of atom-pairs between the protein and ligand. We have developed an approximation which greatly speeds up the calculation of the intermolecular interaction energy for real-time use in docking studies. The energy calculations in our approximation method are performed in two phases, the calculation of grid point data by using the protein structure, and the energy calculation by using the grid point data and ligand structures. Once the grid point data have been calculated and stored in a memory or files, the second phase can be performed consecutively for various ligand structures with use of the tabulated data.

20 On each grid point in the ligand binding site, we calculate and store the van der Waals energy term for various probe atoms, electrostatic potential term, expected sites and characters of hydrogen bond partners in the ligand, surface code and other items. Calculation of the Grid Point Data Calculation of the grid point data is as follows. A three-dimensional grid with a regular interval (typically 0.4-1.0 A) is generated inside the binding pocket of the protein molecule (Fig. 4). On each grid point, the van der Waals interaction energy between a probe atom and the whole protein molecule is calculated by using the empirical potential function. Several types of atoms are used as the probe and the energy is calculated and stored separately for each probe atom type. Every atom species that exists in the ligand molecules to be studied is adopted as the probe atom (e.g. carbon, hydrogen, nitrogen, and oxygen). For the van der Waals energy term Gvdw, the Lennard-Jones type potential function as shown in Eq. 2 is used. In Eq. 2, rij is the distance between the probe position on the i-th grid point and thej-th protein atom. As the empirical potential parameters Aij and Bij, those given by Weiner et al. (27,28) are taken currently. Gvdw,i --

protein atoms E ( Z i j r ~ 12 - Bijr[j 6) J

[2]

The electrostatic potential term Gelc is calculated by using the Coulomb potential as in Eq. 3. In Eq. 3, the definition of rij is the same as in Eq. 2. qj is the atomic charge on the j-th protein atom. The value of this term is equivalent to the electrostatic interaction energy in the case that the probe atom bears a positive unit charge. K is a constant to convert the energy unit to kcal/mol. protein atoms

G~l~.i =

~

j

If qj

eriJ

[3]

Determination of the dielectric constant inside the protein molecule is a difficult but an important problem. A constant value, which is often used for simplicity, is not very realistic. We usually use a distance-dependent approximation for the dielectric constant (i.e. ~ = frij where f varies from

21 I to 4). The approximation may still be oversimplified, but it is better than a constant dielectric model when solvent molecules are not explicitly treated in the calculation. The model somehow incorporates shielding of electrostatic interaction by mediating atoms and ions.

Calculation of the Intermolecular Energy When a ligand molecule is placed and manipulated in the gridded region, the interaction energy between the protein and the ligand molecule can be estimated by using the three-dimensionally tabulated energy terms as described above. The tabulated data on the grid point nearest to each ligand atom are used for the calculation. The interaction energy between protein and ligand (Einter) is calculated by using Eq. 4. ligand a t o m s

k

Van der Waals interaction energy is calculated simply by summing up the van der Waals energy term Gvdw(k) on the nearest grid point from the k-th ligand atom. Among the van der Waals energy terms for several probe atom types, the proper term is chosen according to the atom type of each ligand atom. Electrostatic interaction energy is calculated by summing up the product of the electrostatic potential term Gelc(k) on the

ii

LL"k,

J

r

/~

9 9

~\

9.

I/

/

f

X

\

"

.

probe atom (C,H,N,O...) 9

~ f

I

L, ~ . . . j

~'1~ ) ( / \

/

----~

\

,

/•/•

/

~

/,

/

II/

~/f

~

~ %

atom acce~ Lable I /" -"~'~\ region ( ned p ~~ \ \, 9 by Gvdw) "- ~'~\"'~'--( / Il ligand l o l e c u l e ~

9

\

~

\

/

t

protein atoms ~ , . ~ .

Fig. 4 Calculation of the grid point data.

Fig. 5 Calculation of the interaction energy by using the grid point data

22 nearest grid point from the k-th ligand atom and the atomic charge qk on the k-th ligand atom. It would be better to use interpolated values derived from those on the eight neighboring grid points rather than those of the nearest grid point Hydrogen B o n d s

Hydrogen bonds play an important role in the specific recognition of molecules in biological systems. The hydrogen bonding force originates essentially from a combination of van der Waals and electrostatic interactions. But, some empirical force-field calculation methods include the hydrogen-bonding energy term in addition to the van der Waals and the electrostatic energy terms for practical reasons. Several types of potential functions have been proposed to express hydrogen bonding force, where the hydrogen atom as well as the hydrogen donor and acceptor heteroatoms are treated taking into account the atomic distances and angles among them (29,30,31). Hydrogen bonding energy in such functions could easily be calculated, if the coordinates of all atoms involved are known. The positions of hydrogen atoms in protein molecules, however, usually cannot be determined by X-ray crystallography. There are some functional groups such as hydroxy and amino groups whose hydrogen cannot take definite positions because of some degrees of free rotation. Moreover, it seems to be unnecessary to elaborate in calculations of the uncertain energy term in a docking study where the protein structure is assumed to be rigid as a first approximation. Imprecise estimation of hydrogen bonding energy is thought not to be significant, if we consider an allowed flexibility of actual protein atoms. In the GREEN system, we decided not to calculate hydrogen bonding energy using potential functions, but to count the number of hydrogen bonds possibly formed at the current position of the ligand molecule during the docking process. The GREEN system provides a function to calculate the expected region of the hydrogen bonding partner according to each hydrogenbonding functional group, such as hydroxy, primary sp 3 and secondary sp 2 amines, aromatic ring nitrogen, and carbonyl groups, taking into account the directions of lone pairs and hydrogens attached to the heteroatoms as well as the distances. For all the functional groups in a protein molecule, the expected regions are calculated and each grid point is examined to see whether it is inside the region or not. A hydrogen

23 bonding flag, which also expresses the hydrogen bond character, donor or acceptor, is assigned to the grid point inside the region, and stored as one of the grid point data. During the docking study on 3D-CG displays, the hydrogen bonding flag in the grid point data is used to detect possible hydrogen bond formation between the protein and ligand. For each functional group in the ligand molecule, the hydrogen bond flag of the nearest grid point is referenced. In order to refine the ligand geometry to the precise minimum, energy minimization by means of the Simplex algorithm (32) can be performed, where rotation, translation and bond rotation of the ligand molecule are allowed. Optionally, van der Waals and electrostatic energy terms can be calculated by the conventional atom-pair type method in the minimization. More precise energy refinement which takes into account all degrees of freedom of the protein-ligand system should be done by using an external molecular mechanics program such as AMBER (33) or CHARMm (34).

Visualization Tabulated data are used not only for energy calculation but also for visualization of the physical and chemical environment of the drug binding site of the protein on the 3D computer graphic display. This facilitates the initial introduction of a new ligand molecule into the ligand binding site. By using the van der Waals energy term in the tabulated data, an "atom acceptable region" can be displayed. The region is defined as a group of grid points whose van der Waals energy term Gvdw is below a certain level (usually taken as 0.0 kcal/mol). On the 3D-CG display, the region is shown as a "bird cage" r e p r e s e n t a t i o n by threedimensionally contouring the van der Waals energy. As van der Waals energy terms are prepared for several probe atom types, the region can be defined for each atom type. The cage is usually color-coded according to the levels of the electrostatic term of grid point data. Plate 1 shows the structure of horse liver alcohol dehydrogenase, whose structure is solved as a complex with coenzyme NADH, catalytic Zn 2+ ion and inhibitor dimethylsulfoxide. Atomic coordinates were taken from the Protein Data Bank entry 6ADH (35). In Plate 1, the dimethylsulfoxide molecule at the active site was taken away from the crystal

24 structure, and grid point data were calculated on each grid point generated in and around the region which the ligand molecule occupied. The atom acceptable region is represented by a bird cage which is contoured at the energy level of 0.0 kcal/mol for van der Waals term Gvdw of the carbon probe. The color of the cage indicates the electrostatic potential term Gelc from the charges of protein atoms. It is clear that the electrostatically most positive region (red to yellow) extends near the catalytic zinc ion. In Plate 1, substrate ethanol is fitted to the "atom acceptable region" (ball and stick model). With such a cage representation, one can dock molecules much more efficiently and rationally than with the conventional docking procedure as shown in Plate 2. Furthermore, such a representation helps one to model new drug molecules which are highly complementary to the binding site cavity in shape as well as electrostatic character. The "atom acceptable region" may appear similar to the conventional molecular surface representation. But, the molecular surface representation of the ligand binding site is based only on the van der Waals radii of protein atoms, whereas the radii of the ligand atoms are also taken into account to some extent in the "atom acceptable region". The region shows spatial positions which the center of each ligand atom can occupy without severe contacts with protein atoms. The "atom acceptable region" is more useful than the molecular surface, because it clearly shows the energetically favorable region for the binding of drug molecules. The hydrogen bonding flag in the grid point data is used to display the "hydrogen bonding region" representation. The region is either shown as a "bird cage" picture by surrounding the grid points where hydrogen bonding flags are set, or as groups of small symbols at grid points. The cages or symbols are color-coded according to the type of protein functional group affecting the region. The representation shows that the displayed region is affected by the hydrogen-bonding functional group on the protein molecule. If a hydrogen bonding partner exists in this region, then a strong interaction would be expected between the partner and the protein.

25 Plate 3 shows the "hydrogen bonding region" in a part of the substrate binding site of E. coli dihydrofolate reductase (13). The colors of the cages indicate the hydrogen-bonding characters expected from the protein functional groups affecting the region. The characters are divided into three types: hydrogen donor, hydrogen acceptor and ambivalent. Red: hydrogen donor region which is affected by hydrogen-donating functional groups of protein, such as arginine and lysine side chains and main-chain amide N-H. Blue: hydrogen acceptor region which is affected by hydrogen-accepting functional groups, such as main-chain carbonyl oxygen and aspartate and glutamate side chains. Yellow: ambivalent region from functional groups which work either as hydrogen donor or as hydrogen acceptor (free-rotating hydroxy and water molecule). The protein structure is shown by a pale-colored skeleton, and the inhibitor methotrexate, which is bound in the crystal, is shown by a yellow skeleton. It can easily be seen that the functional groups of methotrexate are located at complementary positions to the hydrogen bonding regions of the protein. Representation of the "hydrogen bonding region" is useful for locating the positions of hydrogen bonding functional groups of drug molecules during the docking operation. Furthermore, the representation helps one to design positions of complementary hydrogen-bonding functional groups, when one wants to create drug molecules with more specific hydrogen-bonding capability. Plate 4 simulates the position of an inhibitor, trimethoprim, in the atom acceptable region of dihydrofolate reductase. The position of inhibitor methotrexate in the crystal structure is also shown for comparison.

Designing New Structures Using the Program GREEN. The program GREEN is useful not only for docking studies, but also for designing new structures directly based on the receptor structures. The program provides functions for model building, such as connecting fragment structures, addition or deletion of atoms or groups and replacing atomic elements. With the stable structures of the complex obtained by docking studies or the crystal structures of the drug-receptor complexes, it is possible to modify the drug structures by adding or replacing substructural fragments so as to obtain more favorable structures for interaction with the receptor. The various energy calculations and

25 visualizations provided in this program serve this purpose. In addition to lead optimization, the program is also useful for lead generation. One can construct new molecular structures interactively on 3D-CG, so as to fit well the cavity shape and properties. Structures should be constructed so that functional groups can interact with those of the receptor as much as possible, and so that the atoms can fit well inside the cavity. At the same time, the structures should be stable, or at least not unstable, intramolecularly, and not be too close to receptor atoms. The validity of the constructed structure is monitored by real-time energy estimation at eve,--] step of the procedure. In addition to this interactive approach, we are developing methods for automatic generation of new drug structures t h a t satisfy the shape and various properties of the receptor cavity. By these methods, it should be possible to obtain structures with new skeletons and new functional groups, among which a new lead compound might be found.

Summary of the Program GREEN The program GREEN has been developed for rational docking simulation and also for the construction of new structures based on the receptor structures. As regards docking simulation, the program covers almost all the necessary functions. In addition to the functions that are commonly implemented in the conventional programs for computer-aided drug design, the program GREEN provides the following features: (1) Real-time estimation of the intermolecular interaction energy by the approximation method, together with precise calculation of the energy in the conventional atom-pair-type calculation. (2) Representation of the "atom acceptable region" and physical and chemical properties, such as electrostatic potentials and expected hydrogen bonding sites in ligands. These features facilitate the initial introduction of new ligands to appropriate positions inside the receptor cavity on 3D-CG. (3) Real-time calculation of the intramolecular energy of the drug molecule, for every operation of bond rotation, by using the AMBER force field.

27

(4) Memorization of trajectories of 3D manipulation. Stable geometries can easily be retrieved after a series of interactive docking studies by use of the memorized geometries and energies. (5) Partial energy estimation, which enables a head-to-tail fitting for flexible drug molecules. (6) Interactive optimization of geometry and conformation of the drug molecule by the Simplex method. (7) Display of the contribution of each atom in the drug molecule to the total intermolecular interaction energy. (8) Display of the electron density map from crystallographic analyses of protein-ligand complexes. For determination of the position and structure of the ligand, energetically stable ones can be referenced by superposing them on the ligand electron density. (9) Interactive molecular-modeling functions which enable us to design molecules fitting well to the shape and various properties of the cavity. These are expected to be useful not only for lead optimization but also for lead generation as indicated before. In order to select the most probable structure of the protein-ligand complex, it would be desirable to compare several possible structures of the complex. If necessary, they should be fully optimized by energy minimization, taking into account the flexibility of the protein molecule. In our method, structures are refined by calculations which are done outside the GREEN program by using the AMBER or other molecular mechanics/dynamics packages developed for macromolecules. The GREEN program should provide an efficient tool not only for interpretation of the structure-activity relationships of various drug molecules, but also for the design of new structures based on the known receptor structure. 4. A P P R O A C H E S BASED ON MOI~ECULAR S U P E R P O S I T I O N

When the receptor structure is known, rational approaches seem to be feasible to some extent. However, it seems to be very difficult to find rational approaches, when the receptor structure is unknown. Nevertheless, most drug development studies have to be made without any knowledge of receptor structure, at least initially. So, drug design is done on the basis of comparison of the structures of a number of known active

28

and inactive compounds. In this situation, the elucidation of the structure-activity relationships is very important and is the starting point for designing new structures. The QSAR method has been developed mainly for this purpose. However, the method has a limitation that the design of new molecules as well as the interpretation of the structureactivity relationships must usually remain within the framework of derivatives with the same skeletal structure. It is necessary to establish approaches with three-dimensional structures of molecules, in order to compare the structures and properties of known drugs with different skeletons. The comparison of three-dimensional structures has been done for a long time by inspecting molecular models made from bamboo, metal or plastic from appropriate directions. Superposition of molecules is one of the most efficient ways to compare the structures and properties of multiple molecules. But, this is impossible with the above types of material molecular models. On the other hand, it is possible to superpose molecules on 3D-CG displays interactively or to superpose them computationally followed by visualization of the results. Such computer-aided methods enable us to store structures of the superposed molecules and to compare not only molecular structures but also physical properties with quantitative measures.

Methods for Superposing Molecules Comparison of the structures and properties of drug molecules would be meaningless, unless their biological activities are based on binding to the same receptor site in spite of their superficial similarity. This is because drugs i n t e r a c t i n g with different receptors should have different requirements for structures and properties. Molecules with apparently different chemical structures often exhibit the same kind of biological activities and pharmacological behaviors. Among them, there are many examples where bindings to the same receptor have been confirmed by receptor binding assay with radioisotopic ligands. There are many crystal structures in which a protein molecule stably binds ligand molecules whose structures are quite different from that of the natural substrate or the natural bio-active molecule. Such ligand molecules are tightly trapped inside the cavity or surface

29 cleft through hydrogen bonding, electrostatic, and van der Waals interactions, which work through space between the two molecules. This fact strongly suggests t h a t the physical and chemical properties are much more important than the chemical structure itself in these intermolecular interactions to be recognized by receptor. Therefore, the abilities of various molecules to bind to the same receptor are determined not only by similarities in molecular shape (not necessarily overall, but in part, as described before) but also more importantly by the relative arrangements of their submolecular physical and chemical properties in the threedimensional structures of the molecules. Accordingly, for the purpose of structure-activity relationships, molecules should be superposed in terms of their physicochemical properties but not in terms of their atomic positions or chemical structures. Methods for superposition conventionally used so far are: (1) l e a s t - s q u a r e s calculation specifying the a t o m - p a i r s between molecules (2) 3D manipulation of individual molecules on 3D-CG with visual judgment of the goodness of fit. The least-squares method cannot be applied easily to molecules in which the atom-pair specifications are difficult when large discrepancies exist between their chemical structures. If it can be applied, this method gives the least-squares residual as a measure of"goodness of fit". Specification of at least three atom-pairs is required for this calculation. This superposing method is routinely performed for the common skeletal part of two structures to reveal the similarities and differences in other parts. The biological activities of a series of compounds are often discussed on the basis of the similarities and differences of the volumes occupied by the two molecules. In cases where the two structures look alike, the differences in structure and properties are so clear t h a t superposing the molecules is not necessary. Superposition by the positions of heteroatoms is also often performed to examine biological equivalence, when the two structures are different from each other. But, it is not always easy to assign the corresponding atoms in the two molecules. Moreover, most of the superposition methods are done without taking into account the properties of the heteroatoms and the direction of interaction with possible partners in the

30 receptor. Although an approximate superposition might give information for substructural correspondence in a set of structurally different molecules, a significant superposition of such molecules seems to be very difficult. Another problem with the superposing method is the conformations of flexible molecules. Usually, superposition has been performed assuming the conformation of each molecule to be the same as in the crystal s t r u c t u r e , or the energetically most stable s t r u c t u r e obtained from molecular mechanics or molecular orbital calculations. But, it is doubtful whether the active conformation is the same as t h a t found in the crystal or in solution, or that of the stable state of the isolated single molecule; the active conformation may not coincide with any of these local energym i n i m u m structures. It seems to be pointless to superpose molecules with conformations other than the active conformation. In the superposition of flexible molecules, the conformations of two molecules can be varied by 3D manipulation interactively so as to fit as well as possible with each other by visual judgement. As the specification of pairs of corresponding atoms in the two molecules is not necessary, the method can be applied to very different structures. The disadvantage of such a superposition method is, however, t h a t it does not give us any numerical index of the goodness of fit. To obtain quantitative and reproducible results of superposition, appropriate indices to show the goodness of fit are necessary.

Receptor Models Three-dimensional models of the receptor cavity can be made based on the superposed structures. More accurate or more probable models would be produced based on multiple molecules which bind to the same receptor, t h a n based on a single molecule. The structure-activity relationships cannot be interpreted at all by a single active molecule. The greater the difference in structures used for the superposition, the more useful is the information obtained. In the "Active Analog Approach", Marshall et al. proposed useful definitions for the volume occupied by the receptor, based on the superposition of active or inactive molecules (36,37). They are the receptor-excluded volume defined as union of the volume of the active molecules, and the receptor-essential volume

31

defined as union of the volume of the inactive molecules minus the receptor-excluded volume. It seems to be useful for drug designers to consider the common volume, the differences in volumes of molecules, and the volume occupied by at least one molecule. The validity of the receptor model completely depends on the validity of the superposition. Therefore, superposition of molecules should be done as rationally and logically as possible. We have developed a rational method for superposing molecules based on the prerequisite of specific binding to a common receptor, and for threedimensional receptor mapping to describe the environment of the receptor cavity.

,..Program RECEPS~

Conventional Methods.)

Drug Structures

Drug Structures

in terms of spatial arrangement of physical & chemical

in terms of atomic positions

,I,

properties

9no structural correspondence required 9numerical indices to show "goodness of fit"

,I, /

\

least-squares method manual superposition specifying the atom-pairs with visual judgement 9structural correspondence required 1

Atomic Coordinates of Superposed Molecules

j

9no numerical index

Fig. 6 Superposition of molecules.

Details of the Program System RECEPS In our method, molecules are superposed in terms of physical and chemical properties by using a three-dimensional grid, whereas in the conventional methods, they are superposed in terms of the atomic positions. The specification of atom-pairs is not necessary, although a template molecule to which other molecules are superposed is required, as in other superposition methods. First, the template molecule must be chosen whose structure should be rigid or conformationally well-defined (although this limitation has been removed to some extent by the devel-

32 opment of functions for automatic superposition). On the 3D-CG, a rectangular box is set up in order to extract the essential region for specific binding to the receptor, and to determine the range of grid point calculation (Plate 5). The lengths of three edges and the position of the box are determined interactively so as not only to cover the region required by the template molecule, but also to have a sufficient reserve space for the subsequent superposition of other molecules. Then, a threedimensional grid with a regular interval of 0.4-1.0 .~ is generated inside the box. For each grid point, the following physical and chemical properties are calculated and stored: electrostatic potential, charge distribution, expected hydrogen-bonding character, flag on occupancy by each molecule, and flag for molecular surface. New molecules (hereafter called trial molecules) are superposed on the graphic expression of these three-dimensionally tabulated data. The goodness-of-fit values are calculated on the basis of spatial similarity of the physical and chemical properties of molecules by using the tabulated data. The values are displayed on the 3D-CG and updated during interactive manipulation (rotation, translation and bond rotation) of the trial molecule during the superposing process. The molecule is manipulated until satisfactory goodness-of-fit values are obtained. Trial molecules are superposed one after another, and the resultant atomic coordinates are stored in a file successively. From the atomic coordinates of every superposed molecule, the grid point data are calculated, from which united grid point data are obtained by applying weights for biological activities. These united grid point data describe the threedimensional environment of the receptor pocket. A receptor cavity model, which provides information on cavity size and shape, surface electrostatic potentials, locations of hydrogen-bonding heteroatoms and other features, can be obtained from the united grid point data. The receptor cavity model can be presented on the 3D-CG in various ways and can be further modified (including its enlargement) by superposing additional molecules. The correct superposition enables us not only to extract the structural and physicochemical requirements for the biological activity, but also to determine their required spatial arrangement. One of the major characteristics of our method is that the goodness-of-fit values can be estimated in real time t h r o u g h o u t the interactive

33

superposing process on the 3D-CG. Such values provide a quantitative measure of the extent of superposition. Goodness of Fit The current version of the grid point data file tabulates the address of each grid point, flag of occupancy by molecules, charge distribution, electrostatic potential and hydrogen bonding character. They are used to r e p r e s e n t the spatial a r r a n g e m e n t of properties of s u b s t r u c t u r e s in molecules and to calculate the goodness of fit of each molecule in real time. Goodness-of-fit values are calculated by using the tabulated data for the template molecule and the atomic data for the trial molecule, which are varied by the interactive manipulation. The goodness-of-fit terms t h a t we currently use are summarized as follows: Fshap e - - _

Number of common occupied grid points Number of occupied grid points of template tool.

Fchar9 e = __ E i

cj -

qil 2

Ei ~jl ~ j" grid point nearest to atom i

cj" charge distribution of grid point j qi" charge of atom i E i ( Vtemp,i Vtrial,i ) Felpo -- - V~/~-~i Vtemp,i 2 / ~ / E i

]Vt,-i~,,i 2

v

Vt~mp,i" electrostatic potential at the grid point i of the template molecule Vt,~ial,i" electrostatic potential at the grid point i of the trial molecule FH_bond z --

Number of common H-bonding grid points Number of H-bonding grid points of template tool.

Equations for the calculation of"goodness~f-fit" indices

The charge distributions, which we have tentatively defined from the atomic charges so as to be distributed on the grid points around the atoms in a Gaussian distribution, are calculated inside the van der Waals volume of each molecule, whereas the electrostatic potentials are calculated outside it. To improve these indices for goodness of fit, further modification of the equations, and replacement of terms or addition of new terms

34 may be required. For this purpose, the program has been designed to allow alterations to be made easily by users. Suitable terms and equations should be selected on the basis of their effectiveness by applying them to distinguish effectively the correct superposition from incorrect ones.

Hydrogen Bonds and Electrostatic Potential Atomic charges should be calculated in advance by molecular orbital calculations. In the case of a flexible molecule, the calculations are made based on the crystal structure or the energetically most stable conformation of the molecule, as the active conformation cannot easily be identified. Hydrogen-bond category numbers are assigned in advance to all hydrogen-bonding heteroatoms in the molecule. The geometries of the attached hydrogen atoms and ambiguity of their position by free rotation, as well as the hydrogen-bonding character (donor, acceptor or both) are judged according to the category number. The category number corresponds to each hydrogen-bonding functional group, such as a hydroxy O, carbonyl O, ether O, carboxyl O, amino N, amide N, aromatic N and sulfhydryl S. For the formation of hydrogen bonds, matching between the expected locations and the character of the hydrogen bonding partners of two molecules is judged during the superposition process. Allowable locations are assumed to be 2.5 to 3.1 .~ in distance and allowable deviation from the orientation vector of X-H or Y-lone-pair electrons (X, Y = N or O) is taken as 30 ~. For all hydrogen-bonding functional groups, the program provides functions for generating the positions of lone-pair electrons automatically and for predicting the possible locations of hydrogen bonding partners, taking into account the freedom of bond rotation of the C-X bond in C-X-H, and the C-Y bond in C-Y-lone-pair electrons. The correlation of electrostatic potentials between the template and the trial molecules is always calculated at the surface grid points of superposed plural molecules as discussed afterwards. The surface grid points vary at every stage of manipulation of the trial molecule.

Application to Dihydrofolate-Methotrexate System Methotrexate (MTX) is a potent inhibitor of the enzyme dihydrofolate reductase, which reduces dihydrofolic acid (DHF) to tetrahydrofolic acid

35 with the aid of the coenzyme NADPH. The structures of MTX and DHF resemble each other well, both having a pteridine ring.

H2N

N

H

(CH2)2COOH

dihydrofolate(DHF)

NH2 N H2N

N

N

I

N

II C -- N ~ CHCOOH

CH3

H

I

(CH2)2COOH

methotrexate (MTX)

Fig. 7 Chemical structures of dihydrofolate (D/IF) and methotrexate (MTX).

The enzyme has been well studied for a long time as an attractive target of rational drug design (38,39,40,41). The crystal structures of a number of isozymes from various sources and in various complexed states have been elucidated (13,42,43,44). The structure of dihydrofolate reductase

101

Fig. 8 Schematic picture of the ternary complex of dihydrofolate reductase from L. casei, the inhibitor methotrexate (MTX), and the cofactor NADPH. (Reproduced from (13) by permission of Prof. Joseph Kraut.)

35

from L. casei elucidated as a ternary complex with the inhibitor MTX and NADPH by X-ray crystallography by Bolin et al. (13) is shown in Fig. 8. The atomic coordinates are taken from the Protein Data Bank. The active conformation of MTX is assumed to be the same as in the crystal. In order to verify the validity of the program RECEPS, we have attempted the superposition of the DHF molecule on the active conformation of the MTX molecule (45). Although we can simulate the active conformation of the natural substrate DHF by means of a docking study using the known structure of the enzyme, here we discuss it by the superposition method with the MTX molecule whose active conformation is known and without using the enzyme structure. For the conformation of the DHF molecule trapped in the enzyme active site, two representative models have been proposed so far (13,40), as shown in Fig. 9 and Plate 6.

~N

TRP 21

R

~_.~TRP

N

-b - H

H

N

H--N8

~

,, H/b-H .......o\~j.~/'N~o ~ ~<~H

N

O

O

O.......H--N '~0

H

H

mode] A

"b ........H-N/' O.......H--N

I ...........H .6.H.. ......0 = : ~

THR 116

21

LEU 114

~'~N/'H

H

" ~ 0 I ...........H.6.. H........ THR 116

mode] B

:O LE

0"~ LEU 114

Fig. 9 Two representative models of the binding of the substrate dihydrofolate (DHF) to dihydrofolate reductase.

In model A, the DHF molecule takes the same orientation as the MTX molecule in the crystal of the ternary complex, whereas in model B, the pteridine ring of the DHF molecule is reversely placed with rotation of the C6-C9 bond by 180 ~ In model A, the carbonyl group at C4 seems to destabilize the complexed structure with an electrostatic repulsion. In the crystal, the amino group at the corresponding position of MTX is hydrogen-bonded to the backbone carbonyl oxygens of Leu-4 and Ala-97

37

in the enzyme. In model B, on the other hand, the complexed structure seems to be stabilized by additional hydrogen bonds to the enzyme. Moreover, it has been suggested t h a t the DHF molecule binds to the enzyme in the model B orientation from the stereochemistry of the enzyme reaction product, tetrahydrofolic acid. The hydrogen atom which is introduced at C6 by the enzymatic reduction should come from the NADPH molecule in the ternary complex. If the DHF molecule binds to the enzyme in the model A orientation, an epimer at C6 would be obtained, but this has not been observed. 0

0 N

H2N

N H

0

R

H

.

H2N

8 N H

.R

H

H

dihydrofolate ( D H F )

N ,.

H2N

N H

hH

H

tetrahydrofolate (THF)

Fig. 10 Stereochemistry of the reduction of dihydrofolic acid (DHF) to tetrahydrofolic acid (THF) by dihydrofolate reductase.

Each of these orientation models of the DHF molecule was independently superposed on the template-molecule MTX and the similarities of their submolecular physical and chemical properties were monitored in terms of the goodness-of-fit values displayed on the upper part of the screen. Plate 7 illustrates the wire-skeletal pictures for the two models of the superposition. Table 1 shows a comparison between models A and B employing the various goodness-of-fit criteria (final values after manipulations). The reference values, which are expected for perfect superposition of the same molecules, are shown in the first line of the table. The values for shape and charge distribution favor model A, whereas those for electrostatic potential and hydrogen bonding favor model B.

Table I Comparison of Model A and Model B by "goodness~f-fif' value&

reference (MTX) Model A Model B

Fshape 1.00 0.85

0.67

Fcharge 0.00 0.94 1.67

Felpo

FH-bond

1.00

1.00

0.07 0.29

0.29

0.71

38

Accordingly, the goodness-of-fit for electrostatic potential and hydrogen bonding was shown to be effective for determining the correct superposition in this case. As two molecules with a similar shape were superposed with the same ring-orientation in model A, it seems quite natural that the goodness-of-fit value for the molecular shape, and consequently the value for the charge distribution in the molecules are favorable for model A. This example strongly suggests that the coincidence of molecular shapes is not always essential for binding to the same receptor and the superposition of molecules in terms of atomic positions is ineffective in some cases. It is widely known that among various intermolecular forces between drugs and receptors, hydrogen bonding, electrostatic and hydrophobic interactions are very important for the binding specificity to the receptor. The correlation of electrostatic potentials can directly indicate the similarity of electrostatic requirements for receptor binding between molecules better than the charge distribution in the molecules. For the correlation of the electrostatic potential values between two molecules, the goodness-of-fit value must be calculated with the grid point data at the same positions. The molecular surfaces of two different molecules never coincide with each other even in the best superposed state. Moreover, each surface varies owing to bond rotations. Therefore, we have decided to use the values on the grid points that are close to and outside the surface of superposed molecules, as shown in Fig. 11. [ ] T " ' : ' " ' [ " ]

.... F " : F T F ' . . ' " ' ~ ' " T ' " F ' : ' ]

"''] .........

F"]

..... i

.... " - - i - - - i .... i-------i .... i---!-------i----i---i .... i---i----i---~---i---i ....... - - - ! .... i .... . . . . . :~---i----~ . . . . ~----~----i . . . . i - - - ~ - - - - ~ - - - - - ~ - - - - i - - - ~

~..!.._}....!....

.... i---~---~-/~-d

.... ~---i---i .... i---~---i .... i---i---~---i----i---i .... i - - : .............. ~.... .... ---+--~ .... ~---§ using using grid

surface points

molecular surface

of

the

enzyme

Model A Model B

0.07 0.29

.... "--+-----

'- -i----i-- '

i ....

0.10 0.30

..... , ~ . . . i . . . ~ , _ . _ ~ . ! b _ .... :...L..-..'

.'...i

.... "...'...'..

....

i

:

i

i

i

i

i

i

if.:'.

!~:'-,~,

....

correlation.

of the method for of the electrostatic potential

Fig.

....

! ....

i ....

,

i

'

~

,

O-i ....i.... ~--~--J

....

i ....

:,:-i ....i....

:.----,---t ..... O

---~---t

....

1 ....

- t i ....i.... . . . . ~.._.! . . . . ~ . . . .

,---i .... i-

:.... i---i ....

.... : . : . : .: .: : : : : : : : : : : : : : : : : : : : : : : : : : : : :

2 Evaluation

-J

"':.,~( ....

.....

.... f . : b .

....

O--i....i....

.... t - - - i - - + - ~ ~ - - i : ~ .... .... 4---i---::---' ~---i----~ ,----i---i---o---4---i---i ....

.... ~---~----: . . . .

calculation

" ? " !

i .... ~...L..:

. . . . .

Table

q ~ ....]....

: : i : i i i: : ii---i : : : :.... i iii,i ! i~i i .i i.i.i.~. i l i4--i - ~- i--i---

.

.

_' ...:.._.

.

11

potential points

.

....

.

.

:...•

.

.

.

....

.

.

.

.

,...,...,...,....,...,

.

.

Calculation

correlation

.

.

....

.

,...,....,...,...,...,

~---i---i .... ....

- - - ~ -,- - t , . . . . 1, . . . . . . ,

"YI

.... ~....

...

....

:...:

9. . . .

of the electrostatic at surface grid

39 To calculate the goodness-of-fit value for electrostatic potential, the potential values of hundreds of grid points should be used. As the surface grid points vary depending on the relative positions, orientations, and conformations of the two molecules, recalculation of the surface grid point is necessary after every 3D manipulation of molecules. The validity of the method was confirmed in the following way. The goodness-of-fit values for electrostatic potential with the surface grid-point procedure were compared with the ideal values, which were calculated by using electrostatic potential values at equally distributed points on the van der Waals surface of the actual enzyme structure. The goodness-of-fit values by the surface grid-point method were in good agreement with the ideal ones as shown in Table 2. The constructed receptor model comprising the two molecules based on model B is shown in Plate 8, in which the image has been clipped off at the front and back planes for clarity. The size and shape of the cavity are represented by cage-expression which is color-coded according to the surface electrostatic potential. The colored dots express the expected locations of hydrogen-bonding functional heteroatoms in the receptor. Plate 9 shows a comparison of the cavity size and shape of the constructed receptor models and the actual enzyme, dihydrofolate reductase. The constructed receptor cavity is expressed as the white cage and the actual enzyme surface is expressed by red dots. For model B (Plate 9 right), the boundaries of the two agree well. The tentative receptor model constructed from the MTX molecule only (Plate 9 left), which is regarded as equivalent to the receptor model based on model A, shows a large vacant space between the cage and the actual enzyme surface. This indicates that the receptor model based on multiple molecules can depict the receptor cavity more accurately than that based on a single molecule, if the superposition is made rationally. Moreover, the correct receptor cavity model can be derived from the correct superposition.

Automated Superposition of Molecules Initially, the program RECEPS was developed for interactive use on 3DCG. But, the interactive superposition procedures required a long time and much labor. Elaborate pre-examination was also required for considering possible superposition models, by using molecular models or

40 graphic displays. Still, the results were apt to be affected by various subjective factors. Moreover, the problems of how to determine the conformation to be used for the superposition, and how to find the correct correspondences of functional groups between molecules are very difficult, when all the molecules to be superposed are flexible and have many hydrogen-bonding functional groups. These situations apply to any superposition method, although it is not the correspondences in the atomic positions of molecules, but those in the interacting groups in the receptor that are important in the RECEPS program. So, we have developed a function to superpose molecules automatically (46). By the automation, it has become possible to examine superpositions based on combinations of all possible correspondences of the functional groups between two molecules and all possible conformations of each molecule. The best superposed structure can be chosen on the basis of the goodness-of-fit indices from all the combinations without prejudice. This function would give q u a n t i t a t i v e character, reproducibility, and reliability to the results of superposition. Removal of subjective choice and exploration of all possibilities seem to be the greatest advantage of this automated technique. Furthermore, the structures obtained from the automatic superposition can be used as starting structures for more precise manual superposition. In such systems as r e c e p t o r - d r u g and e n z y m e - s u b s t r a t e (or inhibitor) complexes, there are many examples where two or more hydrogen bonds play important roles in the molecular recognition (47). Therefore, we have developed a new method using least-squares calculation minimizing differences in the direction vectors and the positions of the possible hydrogen-bonding heteroatoms between superposed molecules in the common receptor site. The procedure is as follows. First, we generate all combinations of hydrogen-bonding groups between two molecules. For each combination, possible conformers are prepared by the systematic rotation of bonds or the input from a conformer file prepared in advance by an appropriate method such as molecular dynamics calculation. Then, the least-squares calculations are performed for all the combinations successively. The program sorts the results on the basis of the indices (residual of least-squares calculation) for selecting the promising superposed models. In the sorting, we can

41

take into account other physical properties using the grid point data as in the interactive procedure. We have also developed a function for refining superposed structures by the Simplex method, considering the similarity of electrostatic potentials, molecular shape and other factors, in addition to the hydrogen bonds. The details of this function will be published elsewhere. We have applied the above least-squares method to the dihydrofolatemethotrexate system (48). The trials were performed for six hydrogen bonding groups on the pteridine ring of both MTX and DHF molecules. As there was no conformational ambiguity by neglecting the other part of the molecules, the number of combinations was 720 ( = 6!). By the conventional least-squares calculation which minimizes only the sum of the deviations of corresponding atom positions between the two molecules, model A was the best and model B was the second best among the 720 combinations. This merely indicates that model A is the best superposition with regard to similarity in molecular shape. By our new leastsquares method, however, model B was the best and model A was the 156th. Thus, model B was shown to be the best superposition, taking into consideration the hydrogen bonds available for interaction with the receptor.

Applications to Other Systems The program RECEPS has also been applied to several other systems and has given successful results. In the superposition of four potent TPA-type tumor promoters (TPA, teleocidin, aplysiatoxin and ingenol ester), the common structural and physical features were extracted, in spite of the apparent structural dissimilarity of the four compounds (49). It was concluded that three hydrogen-bonding groups (two hydrogen-donor groups and one hydrogen-acceptor group) and a large lipophilic group are essential for the potent tumor-promoting activity. Although the relative positions of the groups are very important, the positions of the heteroatoms in the molecules do not coincide, but the expected positions of the interacting hydrogen-bonding groups in the receptor do coincide. In the superposition of phosphodiesterase inhibitors (50), a tentative receptor model was constructed based on the superposed structures of three compounds including the substrate, namely cyclic AMP. Fifteen other

42 j /

a~Lc.

P l a t e 1 "Atom acceptable region" of h o r s e liver alcohol d e h y d r o g e n a s e . NADH a n d Zn 2§ ion are d i s p l a y e d in purple. S u b s t r a t e e t h a n o l is s h o w n as a ball a n d stick model.

J,

9

P l a t e 3 " H y d r o g e n b o n d i n g region" of E. coli d i h y d r o f o l a t e reductase. Cage colors i n d i c a t e the type of protein f u n c t i o n a l g r o u p t h a t affects the region. (See text.)

l

1

Plate 2 Conventional d o c k i n g m e t h o d w i t h the h o r s e liver alcohol dehydrogenase only s h o w n by the skeleton model of molecules.

!

Plate 5 A r e c t a n g u l a r box for determ i n i n g the r a n g e for grid p o i n t calculation. The MTX molecule is displayed as a wire-frame model. (Plates 5-9 are r e p r o d u c e d from Ref. 45 by permission of P e r g a m o n Press.)

Plate 4 "Atom acceptable region" of E. coli d i h y d r o f o l a t e reductase. (left) S i m u l a t e d position o f t h e i n h i b i t o r t r i m e t h o p r i m . (right) I n h i b i t o r m e t h o t r e x a t e in the crystal structure.

43

l}

i

1

f:

t

P l a t e 6 S u p e r p o s i t i o n of DHF molecule (sky blue) on MTX molecule (yellow). (left) Model A. (right) Model B.

a] ,2'i

o.

9 If

l

P l a t e 7 S u p e r p o s i t i o n of DHF molecule on the grid p o i n t data. T h e e x p e c t e d locations of h y d r o g e n - b o n d i n g h e t e r o a t o m s in the r e c e p t o r a r e i n d i c a t e d by dots (pink: donor, light blue: acceptor). C u r r e n t values of"goodness of fit" are s h o w n in the u p p e r p a r t . (left) Model A. (right) Model B.

P l a t e 8 R e c e p t o r cavity model b a s e d on m o d e l B. MTX a n d D H F m o l e c u l e s a r e also shown.

P l a t e 9 C o m p a r i s o n of t h e r e c e p t o r cavity model by R E C E P S (white cage) a n d m o l e c u l a r surface of the a c t u a l e n z y m e (red dots). (left) R e c p t o r model A (right) R e c e p t o r model B

44 compounds were superposed successively on this receptor model, just as in the docking study when the receptor structure is known. The fit to the receptor model was analyzed in the light of the potency of the inhibiting activity. The common volume between each compound and the receptor model was taken as an index for the fit, and the relationships between the potencies and the volumes were analyzed by regression analysis. The relative potencies of the fifteen compounds were well explained by an equation with a fairly high correlation coefficient. The potencies predicted by the equation were in good agreement with the observed values. In this work, all the superpositions were made by using the function for automatic superposition. Further, through the superposition of artificial estrogenic compounds on natural estrogen, estradiol, the active conformation of the flexible synthetic estrogens was estimated.

Summary of the Program RECEPS We have developed a new rational method for superposing molecules interactively on 3D-CG or automatically. This method offers the following advantages" (1) It can be used to superpose multiple molecules whose chemical structures are quite different. (2) It can take into account interactions from different directions to the same receptor site. For example, heteroatoms in different molecules participating in the interaction with a common receptor site are not necessarily superposed at the same atom position. (3) It enables the real-time estimation of "goodness-of-fit" values throughout the interactive superposing process. The values indicate the similarities of submolecular physical and chemical properties of the superposed molecules. (4) It can be used to construct a receptor cavity model from the structural information of superposed molecules, which provides the cavity size and shape, surface electrostatic potentials, expected hydrogen-bonding sites and so on. Our new method for the superposition of molecules was shown to be useful for various purposes. It should greatly assist us not only to explain the relationships between three-dimensional structures and biological

45 activities, but also to predict the potencies of unknown or not yet synthesized compounds. 5. C O N C L U S I O N

We have developed new methods using computers for rational design of drugs based on drug-receptor interactions. Drug molecules must reach the target receptor site in the body and bind to the receptor specifically. From studies on structure-activity relationships based on threedimensional structures, the structural and physicochemical requirements for specific binding to the target receptor are extracted. At the same time, the structural part that participates directly in the receptor binding can be distinguished from the part that does not. A certain specific structure is required for the former part, whereas diverse and nonspecific structures may be allowed for the latter part, although the macroscopic physicochemical properties of the whole molecule must be within a certain range. We should deal with the structural changes of these two parts independently, although they have been dealt with together in QSAR and other methods so far. Further development of the rational computer-aided procedures are still required for designing and constructing new skeletal structures based on the concept of drug-receptor interaction. These further procedures in combination with the methods described here might enable artificial lead generation and thus might change the entire approach to drug development. REFERENCES

1 2 3 4 5

C. Hansch, T. Fujita, J. Am. Chem. Soc. 86 (1964) 1616-1626. C. Hansch, J. Med. Chem. 19 (1976) 1-6. C. Hansch, A. Leo, "Substituent Constants for Correlation Analysis in Chemistry and Biology", John Wiley & Sons, New York, 1979. T.L. Blundell, L. N. Johnson, "Protein Crystallography", Academic Press, London, 1976. F.H. Allen, S. Bellard, M. D. Brice, B. A. Cartwright, A. Doubleday, H. Higgs, T. Hummelink, B. G. Hummelink-Peters, 0. Kennard, W. D. S. Motherwell, J. R. Rodgers, D. G. Watson, Acta Cryst. B35 (1979) 2331-2339.

45 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

21 22 23 24 25 26

F.C. Bernstein, T. F. Koetzle, G. J. B. Williams, E. F. Meyer, M. D. Brice, J. R. Rodgers, O. Kennard, T. Shimanouchi, M. Tasumi, J. Mol. Biol. 112 (1977) 535-542. S. Harrington, "Computer Graphics, A Programming Approach", McGraw-Hill, Tokyo, 1983. T.J. Perum, C. L. Propst (Eds.) "Computer-Aided Drug Design", Marcel Dekker, New York, 1989. N.L. Allinger, Adv. Phys. Org. Chem. 13 (1976) 1-76. W.J. Hehre, L. Radom, P. v. R. Schleyer, J. A. Pople, "ab initio Molecular Orbital Theory", John Wiley & Sons, New York, 1986. J . J . P . Stewart, J. Comp.-Aid. Mol. Des. 4 (1990) 1-105. J.A. McCammon, S. C. Harvey (Eds.), ~Dynamics of Proteins and Nucleic Acids", Cambridge University Press, Cambridge, 1987. J.T. Bolin, D. J. Filman, D. A. Matthews, R. C. Hamlin, J. Kraut, J. Biol. Chem. 257 (1982) 13650-13662. E.C. Dodds, L. Goldberg, W. Lawson, R. Robinson, Nature 141 (1938) 247-248. H. Kagechika, E. Kawachi, Y. Hashimoto, T. Himi, K. Shudo, J. Med. Chem. 31 (1988)2182-2192. H.J. Robinson, J. R. Vane (Eds.), "Prostaglandin Synthetase Inhibitors ~ Their Effects on Physiological Functions and Pathological States", Raven Press, New York, 1974. N. Tomioka, A. Itai, Y. Iitaka in: Y. Iitaka, A. Itai (Eds.), "Proceedings of Symposium on Three-Dimensional Structures and Drug Action", Univ. Tokyo Press, Tokyo, 1986, pp.186-196. N. Tomioka, A. Itai, Y. Iitaka, J. Comp.-Aid. Mol. Des. 1 (1987) 197-210. A. Itai, Y. Kato, Y. Iitaka in: Y. Iitaka, A. Itai (Eds.), "Proceedings of Symposium on Three-Dimensional Structures and Drug Action", Univ. Tokyo Press, Tokyo, 1986, pp.195-205. G.M. Cole, E. F. Meyer Jr., S. M. Swanson, W. G. White, in: E. C. Olson, R. E. Christoffersen (Eds.), ~Computer-Assisted Drug Design", ACS Symposium series 112, American Chemical Society, Washington, 1979, pp. 189-204. B. Busetta, I. J. Tickle, T. L. Blundell, J. Appl. Cryst. 16 (1983) 432-437. M.L. Connolly, J. Appl. Cryst. 16 (1983) 548-558. F.M. Richards, Ann. Rev. Biophys. Bioeng. 6 (1977) 151-176. P.K. Weiner, R. Langridge, J. M. Blaney, R. Schaefer, P. A. Kollman, Proc. Natl. Acad. Sci. USA 79 (1982) 3754-3758. P.J. Goodford, J. Med. Chem. 28 (1985) 849-857. N. Pattabiraman, M. Levitt, T. E. Ferrin, R. Langridge, J. Comp. Chem. 6 (1985) 432-436.

47 27 S.J. Weiner, P. A. Kollman, D. & Case, U. C. Singh, C. Ghio, G. A]agona, S. Profeta Jr., P. Weiner, J. Am. Chem. Soc. 106 (1984) 765-784. 28 S.J. Weiner, P. A. Kollman, D. T. Nguyen, D. A. Case, J. Comp. Chem. 7 (1986) 230-252. 29 E.R. Lippincott, R. Schroeder, J. Phys. Chem. 23 (1955) 1099-1106. 30 R. Chidambaram, R. Balasubramanian, G. N. Ramachandran, Biochim. Biophys. Acta 221 (1970) 182-195. 31 D.N.A. Boobbyer, P. J. Goodford, P. M. McWhinnie, R. C. Wade, J. Med. Chem. 32 (1989) 1083-1094. 32 J.A. Nedler, R. Mead, Computer J. 7 (1965) 308. 33 P.K. Weiner, P. A. Kollman, J. Comp. Chem. 2 (1981) 287-303. 34 B.R. Brooks, R. E. Bruccoleri, B. D. Olafson, D. J. States, S. Swaminathan, M. Karplus, J. Comp. Chem. 4 (1983) 187-217. 35 H. Eklund, J.-P. Samama, L. Wallen, C.-I. Branden, A. ,~keson, T. A. Jones, J. Mol. Biol. 146 (1981) 561-587. 36 G.R. Marshall, C. D. Barry, H. E. Bosshard, R. A. Dammkoehler, D. A. Dunn in: E. C. Olson, R. E. Christoffersen (Eds.), "ComputerAssisted Drug Design", ACS Symposium series 112, American Chemical Society, Washington, 1979, pp. 205-226. 37 G.R. Marshall in: G. Jolles, K. R. H. Wooldridge (Eds.), "Drug Design: Fact or Fantasy ?", Academic Press, New York, 1984, pp. 35-46. 38 C. Hansch, R. Li, J. M. Blaney, R. Langridge, J. Med. Chem. 25 (1982) 777-784. 39 L.F. Kuyper, B. Roth, D. P. Baccanari, R. Ferone, C. R. Beddell, J. N. Champness, D. tL Stammes, J. G. Dann, F. E. A. Norrington, D. J. Baker, P. J. Goodford, J. Med. Chem. 25 (1982) 1120-1122. 40 C.R. Beddell in: A. S. Horn, C. J. DeRanter (Eds.), "X-ray Crystallography and Drug Action", Clarendon Press, Oxford, 1984, pp.169-193 41 A.K. Ghose, G. M. Crippen, J. Med. Chem. 27 (1984) 901-914. 42 D.A. Matthews, J. T. Bolin, J. M. Burridge, D. J. Filman, K. W. Volz, B. T. Kaufman, C. R. Beddell, J. N. Champness, D. tC Stammers, J. Kraut, J. Biol. Chem. 260 (1985) 381-391. 43 C. Bystroff, S. J. Oatley, J. Kraut, Biochemisty 29 (1990) 3263-3277. 44 J.F. Davies, II, T. J. Delcamp, N. J. Prendergast, V. A. Ashford, J. H. Freisheim, J. Kraut, Biochemistry 29 (1990) 9467-9479. 45 Y. Kato, A. Itai, Y. Iitaka, Tetrahedron 43 (1987) 5229-5236. 46 Y. Kato, A. Inoue, A. Itai, in preparation. 47 H. Luecke, F. A. Quiocho, Nature 347 (1990) 402-406. 48 A. Inoue, Y. Kato, A. Itai in: ~Abstract of the 17th National Symposium on Structure-Activity Relationships", Osaka, 1989, pp.292-295.

48

49 A. Itai, Y. Kato, N. Tomioka, Y. Iitaka, Y. Endo, M. Hasegawa, I~ Shudo, H. Fujiki, S. Sakai, Proc. Natl. Acad. Sci. USA 85 (1988) 3688-3692. 50 S. Sekiya, H. Sugawara, H. Okushima, Y. Kato, A. Itai in: "Abstract of the 17th National Symposium on Structure-Activity Relationships", Osaka, 1989, pp. 296-299.

QSAR and Drug Design - New Developments and Applications T. Fujita, editor 0 1995 Elsevier Science 6.V. All rights reserved

49

DRUG DESIGN BASED O N RECEPTOR MODELING USING A SYSTEM

" B I O C E S [El "

KENJI AKAHANEl and HIDEAKI UMEYAMA2 Central Research Laboratories, Kissei Pharmaceutical Co.,Ltd.,Matsumoto-city, Nagano 3 9 9 , J a p a n 1 * of Pharmaceutical Sciences, Kitasato and School University, Shirokane, Minato-ku, Tokyo 108, Japan2

A B S T R A C T : A computer system BIOCES [ E l enables medicinal chemists to study the interaction between biologically active molecules and their receptors. It is capable of building receptor protein models, docking drug molecules into the receptor model, and analyzing their interacting modes. By using BIOCES[E], the three-dimensional structures of human and rat cathepsin H were predicted based on the crystal structure of papain. T h e binding sites of both enzymes are highly conserved and the only amino acid replacement found was residue 6l(papain numbering scheme). A w e l l - k n o w n inhibitor of thiol proteases, benzyloxyc a r b o n y l - L - p h e n y l a l a n y l - L - a l a n y l e t h y l e n ewas , fitted i n to the binding site by a Monte Carlo method. Steric, electrostatic and hydrophobic aspects of the interactions of the inhibitor and the enzymes were analyzed.

1.

INTRODUCTION Bioactive molecules including synthetic compounds are

thought to exert their activities through interaction with receptors. The interactions of drug molecules with receptors are highly s t e r e o - s p e c i f i c , and this situation has long been compared to a "lock and k e y " . According to this idea, the shape of the key

( i.e., the drug molecule)

should be

complementary to the lock

(i.e., the receptor

molecule).

In

evidence

the

past

decade, much

supporting

has been accumulated (1). Many crystal of complexes formed between non-membrane enzymes, which are regarded as the simplest forms of receptors, and their inhibitors have been determined by X ray diffraction studies. The results support the concept of complementarity not only in shape but also in physical properties. this

situation

structures

50 Just as complexes have

structural studies on enzyme-inhibitor proved efficient in molecular design of

effective

and

selective

structures

should

drugs,

contribute

to

studies the

proteins such as enzymes and vaccines. However, determination of protein

design

on of

protein useful

structure by X - r a y

crystallography is difficult: The protein must be purified and h i g h - q u ality crystals must be grown. Gene-cloning techniques are often useful for obtaining sufficient amounts of pure protein. However, there is no easy way to obtain suitable crystals for collecting diffraction data. Thu s , this method for determining the three-dimensional structure of a protein is very time consuming. The primary structure of a protein its

tertiary

can be obtained more

structure by using

easily

than

gene-cloning techniques.

Thu s , many attempts have been made to predict the tertiary structure of a protein from its amino acid sequence. Chou Fasman ( 2 - 3 ) , predicted the secondary structures of proteins using empirical rules based on the structures of spherical proteins. Since then, many methods to predict &

( 4 - 8 ) .Because the tertiary structure of a protein is defined by the spatial arrangement of secondary structures, it is necessary to determine the relative spatial positions of individual secondary structures to predict protein folds. However, no critical method for this purpose has been reported. On the other h a n d , for estimation of the stable conformations of relatively small peptides, energy calculations, including molecular mechanics and molecular dynamics, have been applied. If the initial geometry is not too distant from secondary structures have been reported

the global minimum, i . e . , in the same "concavity" of the energy

hypersurface,

usual

effective for determining

minimization

techniques

are

the global minimum. Otherwise,

the conformation is trapped in the nearest local minimum. Many methods to avoid this trouble have been proposed

(9-

13).

Besides the procedures mentioned above, a "distance geometry" method to determine the three-dimensional structure of a protein in solution has been proposed

17).

This

method

involves

use

of

a

combination

(14of

51

experimental data, e.g., NOE data obtained by nuclear magnetic resonance spectroscopy, and a mathematical calculation. This method is very useful because it does not require crystalline protein, and provides significant information on the structure of a protein. Homologous

proteins

are

reported

to

show

striking

(18.20). of proteins have been conserved to maintain their functions. Therefore, homologous proteins are expected to have similar tertiary structures. Browne et a l . (21), predicted the three-dimensional structure of a-lactalbumin based on its structural similarity to homologous lysozyme. A renin inhibitor is a suitable target of antihypertensive drugs. For the purpose of developing antihypertensive drugs, three-dimensional models of human renal renin have been constructed ( 2 2 - 2 4 ) based on structural similarities to aspartic proteinases. The three-dimensional structures of proteins have been predicted by comparative studies ( 2 5 31). W e have been developing a computer system BIOCES[E], BIOChemical Expert System[Extended Version], for protein modeling, protein engineering and medicinal chemistry. In this article, w e describe the general aspects of the system and its applications to drug design based on the method of receptor fit. similarities

in

three-dimensional

Presumably, during

2.

evolution

the

structure

tertiary

structures

G E N E R A L A S P E C T S OF BIOCES [ E ] A general procedure of protein modeling

in Fig.1. Before model building

is outlined

of a target protein, the

most suitable reference protein is selected from a protein data base. It is necessary that the reference protein should

have

preferably

high the

homology

same

with

sequence

the

target

length.

Then

protein

and

hydrophobic

“core s c o r es” (32) are calculated for each residue of the reference protein acid

alignment

(step 2). These values are used in amino

(step 3).

Details of

the

alignment

using

the core values are given in the following section. Modelbuilding, known as “comparative m o d e l i n g ” , is carried out by

using

this

amino

acid

alignment

(step

4).

The

52 Step 1

Search for reference protein

Step 2

Calculate hydrophobic core scores of reference protein

Step 3

Align sequence of reference protein and target protein using hydrophobic core scores

Step 4

Construct main-chain and side-chains for conserved region

Step 5

Calculate hydrophobic core-distance of tarqet protein

step 6

Determine "window" for searching for structurally homologous regions of other proteins I

4

1

1

Step 7

l ; _ ;

Is there a suitable
Step 9

,

automa tica11y

Build loop using selected fragment

+

Step 10

Determine energetically stable conformation of side-chains

Step 11

Relax hole structure by molecular mechanics or molecular dynamics

Fig.

1.

Step 8

Procedure for protein modeling using B I O C E S [ E l

53 coordinates of the conserved amino acid residues in the target protein are taken from the corresponding residues

-

-

in the reference protein. For conservatively substituted amino acid residues such as Ser Thr and Phe T y r , the conformations of the side-chains are generated to be as similar to that of the reference protein a s possible. However, for more varied side-ch a i n substituents such as Gly +Trp

and Ala - A r g ,

the coordinates of the s i d e - c h a i n s

are taken from crystal data

for

the

corresponding

amino

acid derivatives. The coordinates of the m a i n - c h a i n s for the amino acids mentioned above are taken from those of the corresponding main-chains in the reference protein. Two methods are used to generate the coordinates of the inserted amino acid residues. The first is a mathematical method

using

method

allows generation

second

a generalized

method

conformations

is

in

a

based data

on

base

subsequently

for

search

(step 9).

these methods will be given later. is

(step 8 ) .

inverse matrix

This

of the coordinates in s i t u . The

The

suitable details

of

The structure obtained

"energy-optimized" using

an

empirical

force-field (step 11). Another important feature of BIOCES [ E l is simulation of binding of a drug to its receptor and analysis of its mode of interaction. An outline of the steps is shown in Fig. 2. First, the drug molecule is "docked" into the active site of the receptor protein (step 1 ) . If a crystal structure of the complex formed between ligand and homologous enzyme is provided, one c a n use a least-squares fitting method or a flexible molecular-fitting method to superimpose the trial molecule onto the crystal structure of the ligand. If a n appropriate reference protein is not provided, the initial docking is carried out manually. It should

be

noted

that, in

this

process,

a

problem

may

arise about the orientation of the t r i a l molecule. It has been

pointed

out

that

the

orientations

of

analogous

ligands in the same enzyme are not always the same, even if structural differences of the ligands a r e small. For example, methotrexate (MTX) is a n inhibitor of dihydro-

54 F i g . 2 . G e n e r a l a p p r o a c h to r a t i o n a l d r u g t h e method of receptor fit.

I Dock a

Step 1

drug into the active site

design by

I

Step 2

Optimize binding conformation of the drug

Step 3

Analyses of: Steric interaction Hydrophobic interaction Electrostatic interaction Hydrogen bonding

Step 4

Design new molecules based on the method of receptor fit

folate reductase (DHFR). This enzyme plays an important role in the folate metabolic pathway and reduces 7 , 8 dihydrofolate(FH2) to 5 , 6 , 7 , 8 - t e tr a h y d r o f o l a t e (FH4). The crystal structure of the complex formed between DHFR and MTX has been determined ( 3 3 - 3 4 ) . Although MTX has high structural similarity to the substrate, FH2, the orientations of MTX and FH2 in the enzyme are reported to differ (35). E - 6 4 (l-[N-[L-3-trans-carboxyoxirane-2-carbonyl) -Lleucyl] amino] - 4 - g u a n i d i n o butane) is an inhibitor of thiol proteases

(36). It has a reactive trans-epoxysuccinic acid

a t its N - t e r m i n u s . E - 6 4 has been proposed leaving group side

( S ' )

a recent study showed rather than S '

of the binding

to bind

in the

site (37). However,

( 3 8 ) , that E - 6 4 binds to S subsites

subsites, and that the mode of binding

is

very similar to that of another thiol protease inhibitor benzyloxycarbonyl-L-phenylalanyl-L-alanylmethyl-ene (ZFA) chloride (3 9 1 , which has a reactive chloro-methyl ketone group, at the C-terminus. After rough

model

obtained

is

the

initial docking, the

energetically

empirical force-field (step 2).

optimized

by

an

55 If

the

trial

molecule

is

not

rigid,

another

difficulty arises: the multiple-minima problem. To avoid this problem, the molecular dynamics are usually calculated.

A

specially

devised

Monte

Carlo

simulation

method (40) would also be effective. Finally, the binding modes at the energetically favorable orientation are analyzed considering various intermolecular (step 3 ) . Free-energy calculations using a method have been successfully applied to

interactions perturbation predict the

relative binding energies of many ligand-enzyme complexes ( 4 1 - 4 4 ) . These calculations provide valuable information about energetic and geometrical features of protein-ligand interaction. However, they take much computation time. In usual studies on development of n e w drugs, the structures of

dozens

more

of

candidates

practical

methods

must are

be

considered.

required.

The

that govern the energy change in the binding to

a

receptor

hydrophobic

protein

and

are

steric,

hydrogen-bonding.

factor is separately calculated information for drug design. 3 .

ALIGNMENT

PROTEINS

-

OF

AMINO

ACID

to

In

Therefore,

major

factors

of a ligand

electrostatic, BIOCES[E],

obtain more

RESIDUES

IN

TWO

each

definite

RELATED

Generally, in the process of protein evolution, a protein undergoes occasional changes in its amino acid sequence, including replacements, deletions and insertions, by erroneous replications of D N A bases. The functions of a protein are not impaired by a conservative replacement, but are affected significantly by drastic changes i n amino acid residues. Consequently, only slight mutations that do not cause serious damage to the protein functions are the

fact

cores,

that

which

allowed. the take

IThis concept

catalytic part

in

site the

is

substantiated

and

protein

the

by

hydrophobic

functions

and

folding, are preserved better than surface regions.

on the gives erroneous results in some cases. This is probably due to neglect of the fact that serious mutations, including The traditional

method

of

exact

sequence alignment is based

match.

This

method,

however,

56 insertions and deletions of amino acids, rarely occur the active site and hydrophobic et

al.

reported

(45),

formation

of

gaps

alignment procedure

an

in

algorithm

such

used

in

cores. Recently, Kanaoka that

regions.

in B I O C E S [ E ]

depressed

The

amino

is based

on

the acid their

method. The fundamental algorithm of the method is based on the method proposed by Needleman & Wunsch(46). First, a P matrix is defined, in which the element P ( i , j ) is

where Ri and Tj are the i - t h and j - t h amino acid residues in

proteins

R

and

T,

respectively.

is the (47), for Ri and Tj. The number of amino acid residues is m for R and n for PAM250 score defined by Dayhoff

et

PAM(Ri,Tj)

al.

T . T h e n , a D matrix is formulated from the P matrix in the decrement manner. That i s ,

r21

In this equation, H ( i ) is the hydrophobic core score of amino acid R , and k is a coefficient to adjust H ( i ) . G is a g a p - p e n a l ty to depress the formation of gaps in the hydrophobic cores appropriately. The values of k and G are determined empirically to obtain a good result using values of zero to 10 (k) and zero to 20 ( G ) . The best alignment is determined from the D matrix according to the algorithm of Needlman In

this

score, the more into

the

Wunsch

&

method,

the

depressed

hydrophobic

(46).

larger are the

cores.

Fig.3

the

hydrophobic

introductions shows

a

core

of

gaps

"path"

that

expresses an alignment of rat cathepsin H and papain. The amino

acid

replacement

that

influences

the

specific

bindings of these enzymes will be discussed later. 4 .

PROTEIN MODELING

T h e most difficult protein modeling

and

time-consuming work

in usual

is the introduction of gaps. Originally,

57

\

\

I

F

.o..I...

.I I".**,.. 1*1.,1..

n.. i:

I O I C 1 * m a n ? "' O- -.

4

Fig. 3. ( a ) F i g u r e of p a t h f o r r e p r e s e n t i n g a u n i q u e ( b )E n l a r g e m e n t alignment of two protein sequences. of the boxed part in (a). One can interactively alter the path.

58 comparative modeling was programs such as FRODO

conducted by computer graphics (49), which were designed to

superimpose amino acid residues onto a three-dimensional electron-density map obtained from an X - r a y diffraction experiment. In these programs, the amino acids to be inserted are'manually

put

in a suitable position with a

desirable conformation. BIOCES[E] provides two convenient methods to construct these regions easily.

4.1

Geometrical method Fig.4 schematically shows the geometrical method

(50)

for insertion or deletion of amino acid residues. In this figure, the amino acids to be linked are A 2 and A3. N' is a dummy atom corresponding to an amide nitrogen atom of residue A 3 , and the relative position of N' is decided by + * considering the geometry of the amide nitrogen. ex-e, and * e',-e', are orthogonal unit vectors. elx and el, are

4

4

-

6

CA

'p3

H- N

A2

A3

I

\

Fig. 4 Schematic representation m e t h o d f o r i n t r o d u c t i o n o f a gap.

'p6

c=o

geometrical

of

-.

placed on the plane defined by C A , C and N' of A 2 , and ex and

gz are on that determined by N , C A and C of A3. The

angle

defined

by

4

e,,

N

and

CA

of

A3

is

decided

by

considering the geometry of the amide bond. The bond + angles 'PI - ~ 8are varied to superimpose e x - e z onto elx4

4

-

0

elZ. Generally, these conditions are satisfied when both

[3] and [4] hold for n rotatable bonds.

59

In these equations, Yf i s the 3-dimensional vector defined by the differences in the coordinates of N and N', and Of is the between [31 and n

3-dimensional vector defined by Euler's angle + + e x - e z and e ' x - e ' z . Under linear approximations,

-

[4] are expressed as [ 5 ] and [6], respectively. [51

n

[ 5 1 and

AP =

[6] are combined into [71

[71

F

where,

C81

p=[j A'P 1

I:::[

1101

=

is a 6 x n matrix, P is an n-dimensional vector (unknown) and f is a 6-dimensional vector. Although [7] has many solutions, [ll] is a solution with the minimum minimum rotations of ' ~ 1 . q ~ .

[PI,

i.e.,

60

Fig.

5.

Modification of m a i n - c h a i n conformation.

One planarity

can of

introduce a

proline

constraint ring

or

to

to

maintain

satisfy

the

the

special

geometries required for some amino acids. This method

can

also b e applied to "draw" a peptide backbone in a desired direction (Fig.5). In Fig.5, the peptide backbone indicated between the two small arrows is moved in the direction indicated by the long arrow. These operations are treated geometrically, and no energetic consideration is taken into account.

4.2 Utilization of known geometries In this method, the main-c h a i n conformation to be generated is taken from data bases such as the Brookhaven Protein Data Bank. The most suitable peptide b a c k - b o n e structure is extracted from the data base considering the following requirements: ( 1 ) The spatial orientation of amino acids located on both edges of peptide fragments should be conserved between the peptide chain under consideration and that to be extracted from the data base. The boxed regions shown in Fig.6 indicate these amino acids. In this figure, the target protein (T.P) is rat cathepsin H and the reference protein (R.P) is papain. The distance of each amino acid

residue

61

from

the

hydrophobic

core

of

the

reference

protein

is

plotted.

Fig. 6 . Hydrophobic indicate gaps. (2)

core

distances.

Dotted

lines

T h e numbers of amino acids in the two peptides

should

be equal and their sequence similarity should be high. The similarity index is taken from the literature (51). 5.

DRUG-RECEPTOR INTERACTION AND DRUG DESIGN The discoveries of effective drugs having

structures or novel largely based on:

biological

activities

have

novel been

(1) Separation and identification of active substances from herbs and plants; (2) Massive screening of naturally occurring or synthesized compounds; and (3)

Studies

on

side-effects

on

unexpected

biological

activities. These having

methods

all

aim

at

discovery

some of the desired characteristics

of

compounds

of a drug. In

cases where compounds are not suitable for clinical use because of undesirable side-eff e c t s , low potencies and problems in ADME (absorption, distribution, metabolism and

excretion), the test compounds have to be modified to overcome these drawbacks. In designing better drugs, many different approaches have been used ( 5 2 - 5 4 ) . Drug design by the method of receptor fit is explicitly based on the three-dimensional structure of a drug-receptor complex; ideas for designing n e w drugs arise through in-depth investigations of drugreceptor interactions at the (sub)molecular level. 5.1 T r e a t m e n t of m o l e c u l a r

surface Previously ( 5 5 1 , w e defined the molecular surface as a set of area-preserving spherical triangles with the intention of calculating the surface area accurately and obtaining a uniform surface. Briefly, this method is as follows. The surface of a molecule is divided into an appropriate number of uniform spherical triangles. We used a regular icosahedron or a regular hexahedron as the starting polyhedron. In the case of triangulation from an

(a)

(b)

F i g . 7. S c h e m a t i c r e p r e s e n t a t i o n o f t r i a n g u l a t i o n b a s e d on a n i n s c r i b e d r e g u l a r i c o s a h e d r o n . icosahedron, each of the 2 0 spherical triangles defined by an inscribed regular icosahedron is divided into 9 spherical triangles (Fig .7). The triangulation of a spherical triangle A B C is carried out using points Q1-Q6 and P ’ in Fig.8. The points Q1-Q6 are determined so as to give area-preserving spherical triangles S 1 - S 3 by using a parameter t as shown in [ 1 2 1 ,

63 where S 1 = @l(t) , S 2

=

@2(t)

and S3

=

@3(t)

as shown in

Fig. 8 . A

F i g . 8 . S c h e m a t i c r e p r e s e n t a t i o n of d i v i s i o n of t h e s p h e r i c a l triangle(AE3C) i n t o n i n e s p h e r i c a l t r i a n gles. S 1 - S 3 are the surface areas of the spherical and Q J P ' Q ~ , respectively. t triangles B Q l Q 3 , Q l Q - , P ' i s a p a r a m e t e r ( s e e [12]). These spherical triangles are further divided by the same operations. Likewise, a spherical triangle defined by an inscribed regular hexahedron is divided into two areapreserving spherical triangles(Fig.9 and Fig.10). The surface area S of a spherical triangle calculated by the well-known formula,[l3].

is

S = R2(a+O+y-7T)

and y are the interior angles, and

In this equation, a ,

R is the radius of the sphere(Fig.11). The angles a , 0 and y are determined by [141 - [ 1 6 ] . +

-

+

-

+

-

cosa=

c o s ( ~ - O a )= -eb.ec

cosb

=

cos(7r-Ob) = -ec.ea

cosy

=

cos (n-Oc)

=

-ea.eb -0

4

8a is the angle determined by unit vectors eb and ec. Ob and Oc are similarly determined by

gc

and ;a,

4

and ea and

64

--

+

-

D

+

e b , respectively. e a , eb and z c are the unit vectors

of

a , b and ?, respectively, defined as [171 - [19].

+

-

D

-

a = B x C

-

-

+

b = C x A

-

D

+

-

c = A x B

Schematic representation of triangulation Fig. 9. based o n a n inscribed regular hexahedron.

E

F

0 Fig. 1 0 . S c h e m a t i c r e p r e s e n t a t i o n o f d i v i s i o n of t h e spherical triangle(EFG) into two spherical triang l e s . S 4 a n d S 5 a r e t h e s u r f a c e a r e a s of s p h e r i c a l t r i a n g l e s E F Q a n d EQG. t i s a p a r a m e t e r d e f i n i n g Q . The summing

overall up

the

molecular areas

of

surface the

area

spherical

is

obtained

triangles.

by The

overlapping spherical triangles placed on the boundary between the neighboring atoms are further divided into an appropriate number of spherical triangles.

65

0 Fig. 11. spherical and y ) .

Calculation of the triangle(ABC) using

s u r f a c e a r e a of t h e interior angles(a, 0

5.2 Hydrophobic effect on the drug-receptor interact ion For the association of a drug(D) and a receptor(R) in the aqueous phase, the hydrophobic interaction energy (AGHI) is defined ( 5 6 ) by [ 2 0 ] using free-energy changes W for association in the aqueous phase (AGasso) and in the gaseous phase ( A G : ~ ~ ~ ) . W

AGHI = AGasso -

Pol

4 5 . 0

W

Since AGasso is thought to be thermodynamically equal to the free-energy changes as represented by [21] for the process I-+II-+III (Fig.12), AGHI is expressed by [ 2 2 ] ,

where AGI and AGII1 are shown as [ 2 3 1 and

[241.

66 A G I = A G ~ ++ ~AG:-+~

D

Then, [ 2 5 ] is formulated AGHI

A GD ~ - ++ ~A GR~ - ' ~ -D A- G R~+~

In these equations, L\GW*' M transfer of a molecule M

[251

represents the free-energy of from the aqueous phase to the

is assumed to be divided gaseous phase. In [ 2 5 ] , AGW-' D-R into the contributions from the drug ( A G ; ~ ~ ) and from the receptor (AGW:g) R

as indicated in [ 2 6 1 .

Fig. 12. S c h e m a t i c r e p r e s e n t a t i o n o f a drug ( D ) and a receptor ( R ) .

association

of

where D' and R' stand for the drug and the receptor in the complex. Thus, [ 2 5 ] can be rewritten as [ 2 7 1 .

small molecules

such

as drugs can be estimated from solubility data, but

The free-energy of

transfer of

that

of large molecules such as enzymes or D - R complexes is difficult to estimate experimentally. Here, the following assumptions are made in estimation of the value.

( 1 ) The overall free-energy of transfer can be obtained as

the sum of the contributions of all the individual groups that constitute the molecule (57-58). (2) The contributions of the groups are proportional their solvent accessible surface areas ( A S A ) (59-60). These assumptions are formulated as [281 and

[30] is derived from [27], [281 and

[29],

[291,

A A S A ~is the change in solvent

where

to

accessible

surface

TABLE 1 f - V a l u e s f o r g r o u p s and s u b s t r u c t u r e s in calculated (55) based on hydrop r o t e i n s and Z F A , p h o b i c f r a g m e n t - c o n s t a n t s estimated b y R e k k e r and d e Kort. ( 5 7 ) -

Guanidinium -

SH

19.30 -24.10

-S-

0.17

Imidazolium

1.27

Indolyl - NH3

I

-C6H5 - CONH2

- 12.56

45.28 - 12.88

11.30

- coo-

18.63

-OH(aliphatic)

11.26

-OH(aromatic)

15.78

Hydrocarbon Back-bone amide

-20.87 29.34

-

40.46

OCONH -

-co-

31.36

Hydrocarbon (aliphatic)

-

24.42

Hydrocarbon (aromatic)

-

22.81

68 area ( 4 8 1 , of the i - t h group in the process of association, and fi is the free-energy change per unit ASA for transferring the i - t h group from the aqueous phase to the fj gaseous phase for the drug molecule. AASA, and correspond

with

those

for

the

groups

existing

in

proteins (Table 1 ) . In order to determine the contribution of each parts, [301 is further converted to [311 ( 5 6 ) ,

x1

complex molecules groups AGHI =

x

x

m

[ ((p'fm'ASAm'S1) /SAmI

where

In this equation,

S 1

is

the

area

of

the

1 - t h spherical

triangle defined on the van der Waals surface of the m - t h group, and SAm is the van der Waals surface area of the m - t h group interacting with a solvent in the free state (see Fig.13). When the upper limit of 8 in Fig.13 was arbitrarily

set

as 55'.

in

[321 was

-0.1312 to reproduce the hydrophobic methane interaction correctly.

determined to be

energy

of

methane-

Fig. 13. C a l c u l a t i o n of hydrophobic interaction e n e r g y ( d e s o l v a t i o n e n e r g y ) o f a p a t c h b y u s e of [311 and [ 3 2 1 . R k i s the van der Waals radius of the atom k. S1 is the area of the 1 - t h spherical triangle defined o n the van der Waals surface of the 1 - t h atom. SAm and ASAm are the van der Waals surface area and solvent accessible surface area, r e s p e c t i v e l y , o f t h e m - t h atom. ylk i s t h e distance between the centers of spherical triangle 1 and atom k i n a hydrophobic-bonding partner.

69 5.3

Hydrophobic Indices

Hydrophobic effects contribute to important biological events such as formation o f micells, holding of proteins and binding of small molecules to biopolymers. These events involve structural change of the surrounded water. Previously, w e reported two empirical indices ( 5 5 ) accounting for the hydrophobic nature of the binding site of a receptor (the Hf index), and the hydrophobic correspondency between a drug and receptor

(the Hc index).

The Hf index is defined for the 1 - t h spherical triangle of an atom m in a guest molecule as [ 3 3 ] .

of

In this equation, fk is the unit f r e e - e n e r g y change transfer and cp is a dumping factor dependent on the

interacting distance(Fig.14). These factors are defined in [ 2 8 1 and

[321.

host molecule

guest molecule Fig. 14. T h e projection o f t h e h y d r o p h o b i c effect from the k - t h atom onto the 1 - t h spherical triangle i s d e f i n e d a s fk'q. T h e t o t a l e f f e c t o f t h e h o s t molecule o n the 1 - t h spherical triangle is calcul a t e d b y eq.33. The Hc index is then formulated a s Hf index and fm (f -value o f atom m).

[34]

by using

the

70

According to these equations, spherical triangles that interact with hydrophobic binding sites show negative Hf values. Positive Hc values indicate interaction between the hydrophobic part of a drug and the hydrophobic binding pocket

of

a

receptor,

or

interaction

between

the

hydrophilic part of a drug and hydrophilic pocket. These indices are strictly empirical, but they are simple to use, and also suitable for graphical representation. 5.4

Electrostatic effects The electrostatic potential

represented by

'pQ

at

a

point

Q

is

[351,

where qi is the Mulliken net-charge of the i - t h atom i-n a molecule or a drug-receptor complex, and ri is the distance

between

point

Q

and

the

i - t h atom.

&

is

the

dielectric constant. Nakamura e t a l . (61-62) defined two kinds of electrostatic potential. One is an ordinal "Guest-on-Guest''electrostatic potential. The other is a "Host-on-Guest"potential that indicates the electrostatic nature of a binding site of a "host" molecule such as a receptor. Nakamura e t a l . also defined the electrostatic correlation p~tential(C;~) (63) for the point Q on the drug surface by using a "Guest-on-Guest"potential "Host-on-Guest"potential

6.

'pQG

H

'pQ

and

as shown in [36].

MODEL BUILDING OF CATHEPSIN H

Cathepsins B , H and L are thiol proteases containing

(64). These enzymes are intracellular thiol proteases and are thought to participate in the processing of proteins (65-66), and in diseases such as cancer (67), and muscular dystrophy (68). Accordingly, development of inhibitors of the cathepsins should be very useful therapeutically. We have

a

catalytic

cysteine

in

the

active

site

71

reported comparative model-building of rat liver cathepsin B (69). Here, we describe model-building and the binding specificities of rat and human cathepsin H by using BIOCES [El . 20

10

*

50

*

*

AFSAWTIEGIIKIRTGNLNQYSEQEL TFSTTGALESAVAIATGKMLSLAEQQL TFSTTGALESAVAIASGKMMTLAEQQL

70

60 ab*

40

30

*

* a PA IPEWDWRQKGAV-TPV HH RH

*

90

80

a

100

*

*

*

PA LDCDRR--SYGCNGGYPWSALQLVAQ-YGIHYRNTYPYEGVQRYCRSREKGPYA HH VDCAQDFNNYGCQGGLPSQAFEYILYNKGIMGEDTYPYQGKDGYCKFQPG~IG RH VDCAQNFNNHGCQGGLPSQAFEYILYNKGIMGEDSYPYIGKNGQCKFNPE~VA 110

120

140

130

*

*

*

150

*

*

abc PA AKTDGVRQVQPYNQGALLYSIANQPVSWLQAAGKDFQLYRGGIFVGPCGN- HH FVKDV~-ITIYDEEAMVEAVALYNPVSFAFEVTQD~YRTGIYSSTSCHKTPD RH FVKNVQITLNDEAAMVEAVALYNPVSFAFEVTEDFMMYKSGVYSSNSCHKTPD 1 70

160

*

PA HH RH

abed *

180

*

190

*

200

*

VLAVGYGEKNGIPYWIVKNSWGPQWGMNGYFLIERGK---NMCGLAAC 210

*

PA SFYPVKN HH ASY PIPLV RH ASY PIPQV Fig. 1 5 . A l i g n m e n t o f a m i n o acid s e q u e n c e s of p a p a i n and cathepsins: PA = papain, H H = human cathepsin H , R H = r a t c a t h e p s i n H. T h e p o t e n t i a l g l y c o s y l a t i o n s i t e i s underlined. T h e a r r o w i n d i c a t e s a c l e a v a g e site. B o x e d a m i n o a c i d s a r e c a t a l y t i c a l l y i m p o r t a n t residues. T h e p a p a i n numbering s c h e m e i s a p p l i e d i n t h i s f i g u r e , so t h e l e t t e r s a - d a r e u s e d to s p e c i f y t h e a m i n o a c i d s of c a t h e p s i n s a l i g n e d to g a p r e g i o n s o f papain. We used papain as a reference protein for comparative model-building. Papain is classified and

its

crystal

resolution

(39)

.

structure

has

been

as a thiol protease, determined

at

2.8A

In the following description, the papain

numbering scheme is used.

72 The complete amino acid

sequences

of

rat

and

human

cathepsin H's have been reported by Takio et a l . (64) and Fuchs et a l . (70), respectively. Fig.15 shows an alignment of the amino acid sequences of the rat and human and papain. Rat cathepsin cathepsin

H

cathepsin H

and

H

35%

shows

85%

homology

shows 37% homology

homology

with

with

human

and

human

papain,

with papain.

enzymes

As

shown

in

Fig.15, six gaps (five insertions and one deletion) are found in the sequences of the cathepsins and papain. Fig.6 (section 4 )

shows

the hydrophobic

core distances.

Amino

acids situated in the surface region of proteins have high values. Figure 6 clearly shows that the gaps mainly occur in the surface regions. The catalytically essential residue i s Cys25, which is conserved in all three enzymes. Other

important

residues

Asn175 are also conserved

including in all

Gln19,

His159

three enzymes.

and

Gln19

is

thought to play an important role in stabilizing the tetrahedral intermediate by forming the oxyanion hole ( 3 9 ) . The role of Asn175 is to orient the His159 imidazole ring by forming a hydrogen bond. The regions including these residues are also highly conserved in the cathepsins and papain.

F t q - 16. P e p t i d e b a c k b o n e o f r a t line) and papain (dotted line) (b) Thr155a-Asp155c (see text)

cathepsin H (solid (a) Phe59a-Asn59b.

73 The three-dimensional structures of rat cathepsin H and papain are shown in Fig.16. Cathepsin H has a single glycosylated polypeptide chain (71). A potential glycosylation site is Asnlll (indicated by an underline in Fig.15), and a putative cleavage site for proteolytic processing Fig.15).

is Asn168b-Gly168c

I n our model,

these

(indicated by regions

are

an

arrow

located

on

in the

protein surface. As shown in Fig.16, there are two insertions near the active site in cathepsin H which would affect the substrate specificity of this enzyme; Phe59a and Asn59b, (b) Thr155a, Pro155b and Asp155c. 7.

BINDING

The

SPECIFICITY

skeleton

of

a

OF CATHEPSIN

well-kn o w n

(a)

H

inhibitor

of

thiol

proteases, ZFA chloride, the benzyloxycarbonyl-l-phenylalanyl-L-alanylmethylene moiety, was placed in the binding site of rat cathepsin H with the same conformation as the crystal structure in the complex with papain. T h e n , an energetically stable conformation was determined by a Monte Carlo simulation using the Metropolis method (72) at 3OoC. I n this treatment, the protein was fixed at a starting structure, and only rotational degrees of freedom of ZFA were taken into account. Torsional energies for ZFA was calculated by a simple energy function. Non-bonded energies for ZFA and interaction between ZFA and cathepsin H were considered. The force-field parameters were taken from the literature (73). The most stable structure through the simulation is shown in Fig.17. There are 4 hydrogen bonds between ZFA and rat cathepsin H , i.e., a P1 carbonyl oxygen t* N E 2 of Gln19, P1 carbonyl oxygen ++ backbone NH of Cys25, P2 carbonyl oxygen Gly66, and

P2 NH

*-t

backbone-CO of

t*

Gly66.

backbone NH of These

hydrogen

bonds are also found in the complex formed between ZFA and papain (39). The electrostatic effects from the rat enzyme are

shown

potentials. +25kcal/mol

in

Fig.18

as

Host-on-Guest

electrostatic

this figure, + indicates the regions or more and - indicates the regions

In

of

of

+5kcal/mol or less. The hydrogen bonding donor (P2 N H ) has negative values and the hydrogen bonding acceptors(P1 CO

14 and P2 C O ) have positive values. The major component included in the hydrogen bond has been proposed to be the electrostatic energy by using an energy decomposition technique (74). The negative values on the benzene ring of P2 Phe would be due to Asp155C.

Fig. 17. A c t i v e s i t e region of r a t c a t h e p s i n H w i t h t h e i n h i b i t o r ZFA.

Y

Y

Fig. 18. H o s t - o n - G u e s t p o t e n t i a l s c a l c u l a t e d o n the v a n d e r W a a l s s u r f a c e of Z F A i n r a t c a t h e p s i n H . + i n d i c a t e s the r e g i o n o f +25kcal/mol o r m o r e , and i n d i c a t e s the r e g i o n of +5kcal/mol o r less. Fig.19 shows the distance between the molecular surface of ZFA and rat cathepsin H, in which + indicates the regions of 3 . O A or more. Figs. 17 and 19 show that half the benzene ring of the P3 benzyloxy-carbonyl group interacts with solvents. In the association with the enzyme, this part does not suffer effective desolvation.

Fig. 19. Graphical representation of interacting d i s t a n c e b e t w e e n t h e v a n d e r W a a l s s u r f a c e of ZFA and r a t c a t h e p s i n H. + i n d i c a t e s t h e r e g i o n of 3 A o r more.

Hydrophobic energy on the van der Waals Fig. 2 0 . s u r f a c e o f ZFA. F o r e a c h s p h e r i c a l t r i a n g l e of a n a t o m m , t h e v a l u e i s c a l c u l a t e d b y ~Y~,-ASA,/SA,. T h e r e g i o n s of 1 0 c a l / m o l . A 2 o r m o r e and t h e r e g i o n of - 1 O c a l / m o l - A 2 o r l e s s a r e r e p r e s e n t e d b y + and - , respectively, at the center of the spherical triangle.

ZFA

The hydrophobic interaction energies obtained for the are listed in Table 2. The calculation was based on

the assumption that no conformational change occurred during the complexation. Furthermore, the methylene group (group 11) of

the C-terminus is assumed

not

to be

in a

chloromethyl form, but in the form of a methylene radical in a state free from the enzyme. -l.Skcal/mol for this group This

calculation

shows

that

Therefore, the value of

is a somewhat over-estimate. groups

1 , 5 , 6 , 9 and

11

gain

hydrophobic stabilization energies by forming the complex,

TABLE 2 Hydrophobic energy association with calculated by eq.31.

(kcal/mol) obtained the enzymes. These

by ZFA on values are

9 p

3 0

---

CH2-OCNH-CH-CONH-CH-E-CH2-

1

4

3

2

7

81011

on association with

cathepsin H

Papain (a)

Group

rat

human

1

-1.5

-1.6

-1.6

2

-0.1

-0.3

-0.7

3

1.1

1.3

1.3

4

-0.1

-0.1

-0.0

5

-0.5

-0.5

-0.5

6

-1.4

-1.4

-1.8

7

0.8

0.8

0.9

8

-0.3

-0.3

-0.3

9

-0.8

-0.8

-

10

1.0

1.0

1.2

11

-1.5

-1.5

-1.4

(a) Taken from ref.5 5

0.7

77 while

groups

3,7

and

10

lose

hydrophobic

energies.

AS

mentioned above, the latter three gain hydrogen bonding energies. T h e major regions in ZFA that contribute to hydrophobic energies are depicted in Fig.20 by (stabilization) or + (destabilization).

, 157,160

F i g . 21. H y d r o p h o b i c f i e l d - e f f e c t i n d e x ( H f ) o f t h e c o m p l e x f o r m e d b e t w e e n Z F A a n d r a t c a t h e p s i n H. T h e 100 or more and -70 or less are regions of r e p r e s e n t e d b y + a n d - , r e s p e c t i v e l y , o n t h e van d e r Waals surface of ZFA. Allows indicate residues included i n the hydrophobic pocket. The

P2

s i d e - c h a i n interacts

with

the

hydrophobic

pocket

consisting of Leu67, Pro68, Ala133, Val157 and Ala160. It is clearly shown in Fig.21. Fig.22 shows the active site of human cathepsin H with

ZFA.

The

rat

and

human

enzymes

have

high

sequence

homology without gap (Fig.15). Only 33 of 220 amino acids are replaced. The binding sites of both enzymes are highly conserved, and no replacement of amino acids i s found. The only replacement in the active site is the 61st residue, which

corresponds

with

Tyr61

in

papain.

In

the

enzyme this residue is Tyr61, while in the rat is His61

(Fig.15). This amino acid

residue

is

human

enzyme

it

located

on

the 53 subsite in the enzyme and is in close contact with the

P3

benzene

specificity replacement.

of

ring. the

It P3

is

expected

s i d e - c h a i n is

that

the

affected

substrate by

this

78

F i g . 2 2 . S t e r e o d r a w i n g of t h e a c t i v e s i t e r e g i o n of h u m a n c a t h e p s i n H w i t h t h e i n h i b i t o r Z F A . Try61 i s i n d i c a t e d by a n arrow.

a. and

CONCLUSION

BIOCES[E] is a powerful tool for protein engeneering drug design. This system is capable of building a

three-dimensional model of a protein based on the wellknown evidence that the tertiary structures of homologous proteins are very similar. By this method, a hydrophobic core score was successfully used to align the amino acid sequence of the target protein with the sequence of the BIOCES [ E l provides convenient reference protein. procedures to facilitate the most difficult and timeconsuming work of introducing gaps in protein modeling. BIOCES [El can also be used to deduce the mode of interaction of a drug and its receptor. If the crystal structure of a drug-receptor complex is unknown, the drug molecule is docked into the active site of the receptor protein in the energetically favorable conformation. Then,

modes are analysed considering steric, electrostatic and hydrophobic factors, hydrogen bonds, etc. This method can provide medicinal chemists with useful information for designing new and effective drugs. the

interacting

79 REFERENCES 1

2

3 4 5 6 7 8 9

10

11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29

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80 30 31 32 33 34 35 36 37 38 39 40

41 42 43 44 45 46

47 48 49 50

51 52

J.C. Fontecilla-Camps, J . Mol. Evol., 2 9 (1989) 6 3 67. K. Toma, S. Yamamoto, Y. Deyashiki and K. Suzuki, Protein Engineering, 6 (1987) 4 7 1 - 4 7 5 . Y. Umezawa and H. Umeyama, Chem. Pharm. Bull., 3 6 (1988) 4 6 5 2 - 4 6 5 8 . D.J. Filman, J.T. Bolin, D.A. Matthews and J. Kraut, J . Biol. Chem., 2 5 7 (1982) 1 3 6 6 3 - 1 3 6 7 2 . K.W. Volz, D.A. Matthews, R.A. Alden, S.T. Freer, C. Hansch, B.T. Kaufman and J. Kraut, J . Biol. Chem., 2 5 7 (1982) 2 5 2 8 - 2 5 3 6 . C. Oefner, A. D’arcy and F.K. Winkler, Eur. J . Biochem., 174 (1988) 3 7 7 - 3 8 5 . K. Hanada, M. Tamai, M. Yamagishi, S. Ohmura, J. Sawada and I . Tanaka, Agric. Biol. Chem., 4 2 (1978) 523 - 528. A.J. Barrett, A.A. Kembhavi, M.A. Brown, H. Kirschke, C.G. Knight, M. Tamai and K. Hanada, Biochem. J., 2 0 1 (1982) 189.198. K.I. Varughese, F.R. Ahmed, P.R. Carey, S. Hasnain, C.P. Huber and A.C. Storer, Biochemistry, 28 (1989) 1330-133 2 . J. Drenth, K.H. Kalk and H.M. Swen, Biochemistry, 1 5 (1976 ) 373 1 - 3738. K. Akahane and H. Umeyama, in:I. Moriguchi (Ed.), Proceedings of the 15th Symposium on StructureActivity Relationships, Tokyo, Japan, 5 - 7 December 1987, P3 5 0 - 3 5 3 . B.L. Tembe and J.A McCammon, Computer & Chemistry , 8 (1984) 2 8 1 - 2 8 3 . S.N. R a o, V.C. Singh, P.A. Bash and P . A . Kollman, Nature, 3 2 8 (1987) 5 5 1 - 5 5 4 . P.A.Bash, V.C.Singh, F.K.Brown, R.Langridge and P.A.Kollman, Science, 2 3 5 (1987) 5 7 4 - 5 7 6 . V.C. Singh, Proc. Natl. Acad. Sci. USA, 8 5 (1988) 4280-428 4 . M. Kanaoka, F. Kishimoto, Y. Ueki and H . Umeyama, Protein Eng., 2 (1989) 3 4 7 - 3 5 1 . S.B. Needlman and C.D. Wunsch, J. Mol. Biol.., 48 (1970) 4 4 3 - 4 5 3 . M.O. Dayhoff, W.C. Baker and L.T. Hunt, Methods in Enzymol., 9 1 (1983) 5 2 4 - 5 4 5 . B. Lee and F.M. Richards, J. Mol. Biol., 5 5 (1971) 379-400. T.A. Jones, J. Appl. Crystallogr., 1 1 (1978) 2 6 8 272. T . Takinaka and H. Umeyama, in:I. Moriguchi (Ed.), Proceedings of the 15th Symposium on StructureActivity Relationships, Tokyo, Japan, 5 - 7 December 1987, P3 5 4 - 3 5 7 . Y. Kubota, K. Nishikawa, S. Takahashi and T. O o i , Biochim. Biophys. Acta, 7 0 1 (1982) 2 4 2 - 2 5 2 . R. Franke (Ed.), Theoretical Drug Design Method, Elsevier, Amsterdam, 1984.

81

53

54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74

J.C. Emmett ( E d . ) , Second S C I - R S D Medical Chemistry Symposium, The Proceedings of a Symposium Organized by the Fine Chemicals and Medicinals Group of the Industrial Division of The Royal Society of Chemistry and the Fine Chemicals Group of the Society of Chemical Industry, Cambridge, England, 1 2 - 1 4 September 1 9 8 3 , The Royal Society of Chemistry Burlington House, London WIVOBN. E.C. Olson and R.E. Christofferson (Eds.), ComputerAssisted Drug Design, ACS Symposium Series 1 1 2 , American Chemical Society, Washington, D . C . , 1979. K. Akahane, Y. Nagano and H. Umeyama, Chem. Pharm. Bull., 3 7 (1989) 8 6 - 9 2 . K. Akahane and H. Umeyama, Chem. Pharm. Bull., 3 4 (1986) 3 4 9 2 - 3 4 9 5 . R.F. Rekker and H.M. de Kort, Eur. J . Med. Chem., 1 4 (1979) 4 7 9 - 4 8 8 . C. Hansch and T. Fujita, J. Am. Chem. S O C . , 8 6 (1964) 1 6 1 6 - 1 6 2 6 . R.B. Hermann, J. Phys. Chem., 7 6 (1972) 2 7 5 4 - 2 7 5 9 . J.A. Reynolds, D.B. Gilbert and C. Tanford, Proc. Natl. Acad. Sci. U S A , 7 1 (1974) 2 9 2 5 - 2 9 2 7 . K. Komatsu, H. Nakamura, S. Nakagawa and H. Umeyama, Chem. Pharm. Bull., 3 2 (1984) 3 3 1 3 - 3 3 1 6 . H. Nakamura, K. Komatsu, S. Nakagawa and H. Umeyama, J . Mol. Graph., 3 (1985) 2 - 1 1 . H. Nakamura, K. Komatsu and H . Umeyama, J. Phys. SOC. J a p a n , 5 4 (1985) 3 2 5 7 - 3 2 6 0 . K.Takio, T. Towatari, N. Katunuma, D.C. Teller and K. Titani, Proc. Natl. Acad. Sci. USA, 80 ( 1 9 8 3 ) 3 6 6 6 3670. K. Docherty, R.J. Carroll and D.F. Steiner, Proc. Natl. Acad. Sci. U S A , 7 9 (1982) 4 6 1 3 - 4 6 1 7 . A. S u h a r and N. Marks, Eur. J. Biochem., 1 0 1 (1979) 23-30. A.R. Roole, K.J. Tiltman, A.D. Recklies and T.A.M. Stoker, Nature, 2 7 3 (1978) 5 4 5 - 5 4 7 . N . C . Kar and C.M. Pearson, Biochem. Med., 1 8 (1977) 126 - 129. K. Akahane and H. Umeyama, Enzyme., 3 6 (1986) 1 4 1 149. R. Fuchs, W. Machleidt and H.G. Gassen, Biol. Chem. H o p p e - S e y l e r . , 3 6 9 (1988) 4 6 9 - 4 7 5 . W.N. Schwartz and A.J. Barrett, Biochem. J . , 1 9 1 (1980) 4 8 7 - 4 9 7 . N. Metropolis, A.W. Rosenbluth, N.M. Rosenbluth, A.H. Teller and E. Teller, J.Chem.Phys., 2 1 (1953) 1 0 8 7 1092. S.J. Weiner, P.A. Kollman, D.A. Case, V.C. Singh, C . G h i o , G. Alagona, S. Profeta, Jr. and P. Weiner, J. Am. Chem. S O C . , 1 0 6 (1984) 7 6 5 - 7 8 4 . H. Umeyama and K. Morokuma, J. Am. Chem. S O C . , 9 9 (1977) 1 3 1 6 - 1 3 3 2 .

Figures 7 , 8 , 9 , 1 0 , 1 1and 14 are reproduced from reference 55 by permission of the Pharmaceutical Society of Japan. Figure 12 is reproduced from reference 56 by permission of the Pharmaceutical Society of Japan.

This Page Intentionally Left Blank

OSAR and Drug Design - New Developments and Applications T: Fujita, editor 0 1995 Elsevier Science B. V. All rights reserved

MECHANISMS

OF

THE

SELECTIVE

INHIBITION

83

THROMBIN,

OF

FACTOR

Xa.

P L A S M I N AND T R Y P S I N

T a k a o M A T S U Z A K I + , Hideaki U M E Y A M A * + and Ryoji KIKUMOTO.

*

Research Center, Mitsubishi Kasei Corporation, Midoriku, Yokohama 227. Japan * * School of Pharmaceutical Sciences, K i t a s a t o University, Minatoku. T o k y o 108, Japan ABSTRACT:

T h e three-dimensional structures of bovine trypsininhibitor c o m p l e x e s w e r e determined by X - r a y analysis. The selective inhibition of thrombin, factor Xa, plasmin and trypsin exhibited by a r g i n i n e and lysine derivatives w a s c l e a r l y explained based o n the structures and the homology in the a m i n o acid T h e d i f f e r e n c e s in the s e q u e n c e s at sequences o f these enzymes. the positions corresponding to Ile-63, L e u - 9 9 and S e r - 1 9 0 of trypsin w e r e shown to g i v e e a c h e n z y m e different binding affinity toward the inhibitors and result in the s e l e c t i v e inhibition. This study provides design strategies for enzyme specific inhibitors and suggestions for site-directed mutagenesis experiments.

1.

INTRODUCTION

Thrombin, blood

factor X a and plasmin are serine proteases

c o a g u l a t i o n and

regulation

of

their

Intrinsic system factor X

I

fibrinolytic c a s c a d e s activity

is

1).

(Fig.

clinically

in t h e

Since

important

in

Extrinsic system I

> factor X a .I

prothrombin

+

fibrinogen

thrombin I

fibrin I

Polymerization (Coagulation) I

fibrin polymer plasmin

+& Degradation (Fibr ino lys i s )

Fig.

1. B l o o d c o a g u l a t i o n a n d f i b r i n o l y s i s c a s c a d e s .

the

the

84 treatment

of

thrombosis

(1,2).

have been carried out Kikumoto et al. inhibitory

bleeding,

or

In a search for thrombin inhibitors,

found that arginine derivatives exhibit selective

activities

toward

different

though the active site structures o f similar amino

to that o f

acid

extensive inhibitor studies

trypsin

sequences.

judging

The

purpose

serine

these enzymes

seems

from the homology our

of

(3).

proteases

study

to be

in

their

(4,5) was

to

elucidate the mechanisms of the selective inhibition based on the three-dimensional thereby

to

structures

find

useful

of

trypsin-inhibitor

suggestions

for

the

complexes

design

of

and

enzyme-

specific inhibitors. 2.

X-RAY

ANALYSES OF T R Y P S I N - I N H I B I TO R

COMPLEXES

T a b l e 1 summarizes crystal data on the five complexes whose

In this paper, the

structures were determined by X-ray analysis.

trypsin-(2R,4R)MQPA complex will be described in detail. Table 1 Crystal

d a t a o f bovine tryps'tn

-

i n h i b i t o r complexes. .-

Inhibitor

MQPA (2R.4R)

PNPA

p31 21 6 55.34

PSI21 6 55.35 109.38 2. 5 0. 215

Space Group

Z a

b

(A)

C

Fig.

109.51 2. 5 0. 172

(A)

Resolution R-f ac t or

2 shows the

structural

MQP P21 21 21 4 55.37 56. 73 66.91 3. 0 0. 401

formula of

MQPA (2R,4s)

MQPA (2s.4R)

P21 21 21 4 55.49 56. 79 67. 07 2. 4 0.265

P21 21 21 4 63. 51 69. 07 63. 81 2. 4 0. 244

the thrombin

inhibitor,

(2R.4R)MQPA ( ( 2 R . 4 R ) - 4 - m e t h y l - 1 - I N 2 - [ ( R , S ) - 3 - m e t h y l - l , 2 , 3 , 4 - t e t r a h y d r o - 8 - q u i n o l i n e s u l f o n y l l - L - a r g i n y l l - 2 - p i p e r i d i n e c a r b o x y l i c acid)

(3).

MQPA

quinoline

is composed o f portion

and

three

portions:

piperidine

an

portion.

arginine There

portion,

are

three

asymmetric carbons in addition to the a carbon of the L-arginine. The configuration at

the 3 position of

the quinoline ring

is a

mixture of R and S .

Depending on the configurations at the 2 and

4

piperidine.

positions

of

the

(2R.4R)MQPA

is

to

be

used

there for

are

four

clinical

stereo-isomers.

purposes

as

an

antithrombotic agent. Hereafter, M Q P A is used to denote ( 2 R , 4 R ) M Q P A and other stereo-isomers are always referred to with stereo notations.

85

I b Il -I

quinol ine port ion

Fig.

2.

I

I

I.

I

Structural formula of ( 2 R . 4 R ) M Q P A .

Fig. 3 shows the formulae of PNPA( (2R,4R) - 4 - p h e n y l - l - [ N 2 - ( 7 - m e t h o x y - 2 - n a p h t h a l e n e s u l f o n y l ) - L - a r g i n y l 1 - 2 - p i p e r i d i n e c a r b o x y l i c acid) (6)

and MQP(4-methyl-l-[Nz

-[

(R, S) -3-methyl-1, 2, 3, 4-tetrahydro-8-quino-

8:"

l i n e s u l f o n y l l - L - a r g i n y l l p i p e r i d i n e ) (3).

0 - A r g -N

3

COOH

PNPA Fig.

3.

w

MQPA

has

strong

thrombin

toward

bovine

and

procedure

Crystallization I I I),

selective

(Ki=O. 0 9pM),

for

X-ray

was

inhibitory

but

still

(Ki=5.OwM).

trypsin

crystallize the complex o

0 . 01 ml

MQP

Structural formulae o f PNPA and MQP.

bovine

the

- A I- g - N >

activity

significant

We

therefore

carr ed

ys

of

is

out

by

the

the

t ryps i n-MQPA

hanging

length g r e w

in 2 weeks.

Crystals of

T h e crystals are isomorphous w i t h

the native trypsin crystal registered in the P r o t e i n D a t a Bank with

an

identification code

collected

o n an Enraf-Nonius

reflections had

measured

within

3PTN

(8).

were

calculated

with

The

2.5

A

the

resolution, in

In all

5.400

of

5.967

reflections

the calculation.

coordinates

(7)

intensity data were

C A D 4 diffractometer.

I F o ( > l . O a ( l F o ~ ) and were used

phases

A

(Sigma T y p e

1. 0 mg/ml CaCle, and 0.26 M ammonium sulfate

was kept in a reservoir w i t h 0.97 M ammonium sulfate.

1 mm

to

complex.

d r o p method.

d r o p of solution containing 60 mg/ml trypsin

2. 5 mg/ml MQPA,

tried

T a b l e 2 shows

bovine trypsin and MQPA. ana I

toward activity

trypsin

Starting in

the

86 Table 2 X-Ray analysis of the trypsin-MQPA complex at 2.5 A resolution. (1) Crystallization by the hanging drop method w i t h a-55. 3420. 0 3 , c-109. 5 1 2 0 . 2 0 P3121. Z = 6

(NH4)2SO4

A,

isomorphous w i t h the native trypsin crystal, PDB 3PTN

(2) Electron density m a p calculated with the 3 P T N parameters R=O.31 for 5400 reflections w i t h lFol 2 I.Oa(lFol) (R-0.24 for 3 P T N 1Fols) (3) Hendrickson-Konnert refinement

trypsin. MQPA’s quinoline and arginine R=O. 328 3 R=O. 258 trypsin, whole MQPA and 70 waters R=O.172. RMS deviation of bond distances RMS deviation of angle distances

native trypsin crystal.

A

reliability factor

= =

0.014 0.033

A

i\

(R-factor) of 0.31

was obtained, while the corresponding value for the 3PTN data w a s

0.24.

The

small difference

3 P T N coordinates were complex crystal.

Fig.

a good

in the R-factors approximation

indicates that

for

the

the

trypsin-MQPA

4 shows the electron density map o f

Fig. 4. Electron density map of the trypsin-MQPA complex. The quinolinesulfonyl group i s shown.

the

87 trypsin active site of the complex calculated at this stage. map quality w a s s o

good

that

the directions of

The

the two sulfonyl

oxygens and the 3-methyl group attached to the quinoline ring were easily

identified.

piperidine quinoline refined

portion and

the

But, was

(9).

and

isotropic

of

three-dimensional

group,

and

temperature

factors

of

the Hendrickson-Konnert the piperidine

After all non-hydrogen atoms of MQPA identified and

included

Fig.

in

5 shows the

structure of the trypsin-MQPA complex.

refinement

not

the carbonyl

T h e present R-factor i s 0. 1 7 2 .

the refinement.

the the

showed clear electron density for

ring and the carboxyl group.

could

first

of

fitted

MQPA with

were refined, 70 water molecules were

We

we

so

R-factor was decreased from 0.328 to 0.258 and

The

the second map

Similar

interpretat ion

possible,

arginine portions without coordinates

trypsin and the fitted part program

definite

not

see

the

is

in progress

electron

density

for of

the other

complexes.

(2s. 4S)MQPA, probably

because of its weak activity (Ki>500pM).

Gly193

Ser-2 1 7

Trp215

5. Three-dimensional structure o f the trypsin-MQPA complex. Bold line, MQPA; thin line, trypsin. Fig.

3.

THREE-DIMENSIONAL STRUCTURE OF THE TRYPSIN-MQPA COMPLEX In Fig. 6.

of

and

trypsin-BPTI trypsin-APP

the trypsin-MQPA structure is compared w i t h those (bovine basic

pancreatic trypsin

(p-amidinophenyl

pyruvate)

inhibitor)

(10) complexes.

(10)

BPTI

and A P P are bound to trypsin in a similar way through the specific hydrogen bonds at two locations ; ( 1 ) at the bottom o f active site hole and ( 2 ) other hand. MQPA

at the so-called 'oxyanion

the trypsin

hole'.

shows unique hydrogen bonding ; (1)

On the

the hydrogen

88 bonds

at

the bottom

of

the

hydrogen

bonds

at

the

carboxyl

group

and

Gly-193

active s i t e hole

oxyanion

through a water molecule,

hole

and

and

are

Ser-195

(3)

are preserved, formed

main

(2)

between

chain

the

nitrogens,

the carbonyl o x y g e n of the M Q P A

arginine is hydrogen bonded to the G l y - 2 1 6 main c h a i n nitrogen and the

nitrogen

carbonyl those

of

the

MQPA

arginine

oxygen of Gly216.

found

which

the case, does

not

8-pleated

have

the

bonded

carboxyl

the to

T h e carboxyl But this

the trypsin-MQP c o m p l e x

group

was

shown

to that of the trypsin-MQPA

scheme seems to be the result of

to

bonds a r e similar sheet.

lead to the unique scheme.

since the structure of

analysis to be similar present

hydrogen

T h e s e hydrogen

in an anti-parallel

g r o u p at the piperidine might is not

is

by

X-ray

complex.

The

the stable conformation

of the M Q P A molecule itself because the conformation is similar to that found in M Q P A single crystals.

trypsin-APP

trypsin-BPTI

trypsin-MQPA

F i g . 6. Comparison o f three-dimensional structures. Bold lines indicate hydrogen bonds.

4.

MECHANISMS OF SELECTIVE INHIBITION T h e inhibitory activities of

(3), MNP (4-methyI-l-[N2-

MQPA

( 7 - m e t h o x y - 2 - n a p h t h a l e n e s u l f o n y l ) - L - a r g i n y l ~ p i p e r i d i n e ~(6),

PNP

(4-phenyl-l-CN2- (7-methoxy-2-naphthalenesulfonyl) - L - a r g i n y l l p i p e r idine)

(6) and BAP (4-benzyl-l-IN2 - (2-anthraquinonesul fonyl) - L - l y -

syllpiperidine) trypsin,

all

(11)

toward

from bovine

a-thrombin,

sources,

are

factor shown

Xa.

plasmin

in T a b l e

3.

and

These

data c a n be interpreted o n the bases of the X - r a y s t r u c t u r e of the trypsin-MQPA c o m p l e x s h o w n in Fig. 7 and the molecular models of thrombin.

factor X a and plasmin

built

according to the homology

of the amino acid s e q u e n c e s shown in T a b l e 4. Table

3 describes

the

selective inhibition.

three

binding

sites

T h e lower half of

responsible

for

the

T h e s e are indicated by arrows in Fig. 7 and

89 Table 3 I n h i b i t o r y a c t l v i t i e s and d e s c r i p t i o n s o f b i n d i n g s i t e s . a ~

(Ki

Activity lnhibi tor

Thrombin

CH30@@SOZ-Arg-N3 MNP

or

Factor X a

0. 0 7 2

pM) toward

158'.

Plasmin

Trypsin

NA

NA

1. 4

NA

NA

0.

NA

33

23

NA

D e s c r i p t i o n o f binding site

Binding site

A1 a

Arginine binding s i t e

Q u i n o l i n e binding s i t e

Wide

A1 a Wide

Leu

T Yr

Med i u m

Deletion of 6 residues Wide

Narrow

Insertion o f 10 residues Narrow

P i p e r i d i n e binding s i t e

S er Nar r o w

Insertion

Leu99 Medium

Insertion of 5 residues Med i u m

2

of

Serl9O Na r r o w

residues Med i urn

I le63 Wide

a NA: D a t a not available.

Table

4.

T h e arginine binding

trypsin.

The

quinoline

piperidine binding s i t e data

raise several

site

binding

is near

questions

is a region near S e r - 1 9 0 o f

site

Ile-63

concerning

is (*

near

Leu-99

His-57).

and

the

T h e activity

the selectivities, w h i c h

c a n be answered as follows 4 . 1 Why a r e a r g i n i n e d e r i v a t i v e s s u c h a s MQPA a n d MNP i n g e n e r a l

more a c t i v e o n t h r o m b i n t h a n o n t r y p s i n ?

T h e reason is that the arginine binding s i t e in thrombin h a s Ala

at

the

position

arginine binding

corresponding

site o f

thrombin

of trypsin. T h e arginine portion o f

to

SerlSO

of

trypsin.

The

is consequently w i d e r than that the inhibitors c a n thus easily

90

%

Asp I94

x

Gly 193

Fig. 7. Three binding sites of the trypsin-MQPA complex r e s p o n s i b l e f o r s e l e c t i v e i n h i b i t i o n ( i n d i c a t e d by arrows). Table 4 C o m p a r i s o n o f amino a c i d sequences o f b o v i n e enzymes. T r y p s i n - l i k e r e g i o n s o f t h r o m b i n , f a c t o r Xa and p l a s m i n a r e shown. Numbering i s t h a t f o r chymotrypsin. " / " and ' I . ' ' denote a d e l e t i o n and insertion In the chymotrypsin sequence, respectively. A s t e r i s k s w i t h arrows i n d i c a t e s i t e s r e s p o n s i b l e f o r s e l e c t i v e inhibition.

THROMBIN FACT0 XA PLASMIN TRYPSIN

TH FX PL TR

TH

FX PL TR

TH FX PL TR

16

20

I VECQ IVGCR IVCGC IVGGY

80 HSRTRYERKV RNTQ//EGDE HNEKVREQSV DNINVVECNE

140 HAGFKCRVTG /QTKTCIVSG AARTECYITG /AGTQCLISG

40 50 V H L F R K ~ P Q E LL C C A S L I S D R ALLVNE.ENEC FCCCTILNEF VSL/RR.SSRH FCCCTLISPK VSL/N/.SGYH F C C C S L I N S Q

30

DAEVCLSPWQ DCAEGECPWQ VSKPHSWPWQ TCCANTVPYQ

****

.

. . . . . 150

WGNRRETWTTSVAEV F G R T H E K . . . . .C R L W G E T Q / / ..... G T F W C N T K S S . . . . . GTS

200 CECDSGCPFV CQCDSCCPHV CQGDSCGPLV CQCDSCCPVV

90

EKISHLDKIYI EHAHEVEMTVK QEIP.VSRLFR QFIS.ASKSIV

110

RDIALLKLKR FDIAVLRLKT ADIALLKLSR NDIHLIKLKS

160

170

x

********

220 CIVSWCE/CC GIVSWCE/GC GVTSWCL/GC GIVSWCS/GC

PLVERPVCKA PYVDRSTCKL PVIENKVCNR PILSNSSCKS

.....

... ..

70

YPPWNKNFTVDDLLVRIICK Q...AKRFT.....VRV/GD N I L A L S F Y K . . . . .V I L / G A S.....CIQ.....VRL/GE

I(*************

100 HPRYNWKENLO HSRF/VKETYD EP//////.SQ HPSYNSNT.LN

QPSVLQVVNL SS/TLKHLEV GECLLKEAHL YPDVLKCLKA

210 HKSPYNNRWYQN TR..FKDTYFVT CF..EKDKYILQ CS..CK////LQ

60

WVLTAAHCLL YVLTAAHCLH WVLTAAHCLD WVVSAAHCYK

120 P I E L S D YI H P PIRFR/NVAP PAIlTKEVlP AASLNSRVAS

.. 180 S..TRIRITNDM S..SSFTITPNR NEYLDGRVKPTE A..YPCQITSNM

. 230 DRNCKYGFYTH ARKCKFGVYTK ARPNKPCVYVR AQKNKPCVYTK

.. 130 VCLPDKQTAAKLL ACLPEKDWAAETL ACLPP.P..NYHV ISLPT./..SCAS

. .. . . 1 9 0 FCACYKPCEGKRGDA FCACY..DTQ.PEDA LCAGH..LIG.GTDS FCAGY..LEG.CKDS

240 VFRLKKWIQKVIDRLGS VSNFLKWIDK IHKARACAACSR VSPYVPWIEE THRRN VCNYVSWIKQ TIASN

91 enter oy

the active s i t e of thrombin, w h i l e the shorter contact w i t h

of Ser-190

The

result

in

explanation. 0 . 94

trypsin

As

results

the

present

shown

in Fig.

of

in

lower

X-ray 8.

inhibitory

analysis

activity.

supports

this

the O y atom of S e r - 1 9 0 moved

A in the d i r e c t i o n away from the active s i t e hole, leaving Serl90

I

'

F i g . 8 . Movements o f S e r - 1 9 0 Oy a n d L e u - 9 9 C 6 1 upon b i n d i n g w i t h MQPA. Thin l i n e , position i n f r e e trypsin; bold l i n e . position j n the complex; dotted line, steric repulsions leading t o the movements.

more

space

in

of

rotation

the active site.

52'

around

T h i s movement

the C a - C f l

bond.

The

resultant

A,

between S e r - 1 9 0 O y and the arginine NZ is 3.74 been

A without

2.91

explanation

that

the movement.

Ser-190

acts

Oy

This to

w a s achieved by a distance

w h i c h would h a v e

is consistent

repel

arginine

with

rather than to attract them as a hydrogen bond acceptor. case o f

lysine

critical

inhibitors

factor.

determines

the

such a s BAP,

Instead,

the

selectivity.

similar

hydrogen

Accordingly.

bond

in

to

lysine

form

where

hydrogen

inhibitor

in trypsin w h i l e

thrombin

In the

steric repulsion is not a

ability

The

hydrogen bond w i t h S e r - 1 9 0 O y

the

inhibitors

Ala

can

bonds form

cannot

it

replaces

a

form a

Ser-190.

lysine inhibitors h a v e stronger activities on trypsin

than on thrombin.

F r o m this v i e w point,

the f o u r enzymes c a n be

classified into two g r o u p s according to the a m i n o acid at position 190: thrombin and arginine Ser.

factor X a have Ala.

inhibitors

their

selectivity.

pockets The

are

enzyme

narrow

their pockets are w i d e and ;

plasmin

and

lysine

s h o w selectivity

selectivities

c a n be

and

trypsin h a v e

inhibitors explained

in

show this

way, but trypsin w a s found to

h a v e smaller K m v a l u e s for arginine

substrates

substrates

specificity

than

for

could

substrates h a v e

be

lysine

explained

two binding

sites

by (i.e.

(12).

supposing

This that

substrate arginine

two a m i n o groups)

which

92 can

form

trypsin

hydrogen

Asp-189,

bonds

while

directly

lysine

with

the

substrates

carboxyl

have

one

group

amino

of

group

which can form a hydrogen bond with the carboxyl group through a water molecule.

T h e strong hydrogen bonds formed by arginine seem

to predominate over

the steric repulsion between

for

lysine

and

similarly,

will

inhibitors

the amino group

Thus, the K m f o r arginine is smaller than that

and Oy of Ser-190. in

the

general

trypsin

be

inhibition

stronger

than

by

that

arginine by

lysine

inhibitors.

are the activities of arginine derivatives reversed, becoming stronger on trypsin. when a benzene ring is introduced at position 4 of the piperidine as i n PNP? 4.2

Why

The

reason

Thrombin has an trypsin. dine

this

lies

insertion of

in

10

the

piperidine

amino acids near

binding the

and

become

a

barrier

against

the

there is a pocket for the benzene

Ile-63 o f

benzene

the results o f

the X-ray analysis of

shows h o w the benzene ring o f

In

ring.

ring and activity is

increased by the introduction of the benzene ring. on

site.

These inserted amino acids probably surround the piperi-

ring

trypsin.

for

Fig. 9, based

a trypsin-PNPA complex,

P N P A interacts with trypsin in the

pocket near His-57.

Fig. 9. Three-dimensional structure of the trypsin-PNPA complex. 4.3 Why is MQPA not active on factor Xa? The

reason

replaces Leu prevents

in

tight

lies

in

factor X a binding

the

quinoline

position 99 o f

at

with

binding

the

present X-ray analysis supports

quinoline

this

site,

where T y r

trypsin.

This Tyr

ring

explanation,

of

MQPA. as

shown

The in

93 Fig.

The C 6 1

8.

atom of L e u - 9 9 moved

MQPA by a rotation of 2 4 '

around

A

85

0.

the C a - C y

upon binding with

bond.

T h e rotation

increased the distance between Leu-99 C 6 1 and the 3-methyl carbon o f the quinoline from 3.70 A

hindrance.

A

A

to 4 . 5 2

and eliminated the steric

modeling study in which Leu-99 w a s replaced by Tyr

that steric hindrance would occur between T y r C E ~and the

showed

quinoline C 4 with a distance o f 2.66

A. BAP. i n h i b i t p l a s m i n ?

4 . 4 Why d o e s t h e l y s i n e d e r i v a t i v e ,

BAP has

stronger

group of

plasmin

thrombin

and

and

factor

activity on

inhibitory trypsin,

than on

Xa,

explained

as

the narrow pocket

the wide in

pocket

group of

4.1.

section

The

difference in the activities for plasmin and trypsin arises from a

In plasmin,

difference in the quinoline binding site. of 6

amino acids near

able

room a t

position 99 of trypsin provides consider-

the quinoline binding

to

the

sulfonyl

site and changes the surface

T h e bulky anthraquinone can f i l l

structure from that of trypsin. this space and f i t

a deletion

itself to this surface because group

at

the

position

it

and

can

is connected change

orientation of the ring plane by a rotation around the C - S BAP will

not

inhibit

thrombin because

inserted amino acids of

thrombin near

the benzyl

the

bond.

group hits

the

I t will also not

Ile-63.

inhibit factor Xa, because the anthraquinone hits Tyr at position 99.

Thus,

the

mechanisms

of

selective

inhibition were

explained based o n

the X-ray structures of

complexes

difference

and

the

in

amino

clearly

the trypsin-inhibitor

acid

sequences

of

the

enzyme.

SUGGESTIONS FOR DRUG DESIGN A N D PROTEIN ENGINEERING

5.

The related

explanation

For example, by

adapting

hole,

(2)

carbonyl

for

the

selective

inhibition

to the design strategies for enzyme specific

by end

is

directly

inhibitors.

factor X a specific inhibitors will be obtained; (1) the

arginine

backbone

introducing a phenyl to

increase

van der

to or

fit

the w i d e

active

a benzyl piperidine at

Waal's

interaction and

site the

(3) by

introducing a benzene ring o r an alkyl chain at the amino end to avoid the steric repulsion by Tyr. Direct protein

proofs

engineering

for

the

explanation

experiments.

Table

could

5

be

obtained

by

summarizes proposed

site-directed mutageneses and the expected results.

94 Table 5 Proposed site-directed mutageneses and expected results. (I)

(‘goSer+Ala) trypsin mutant Ki for MQPA Ki for BPTI

(2)

J , t ,

(Ala+lgoSer) thrombin mutant Ki for MQPA t ,

(3) (Ala+’goSer) factor X a mutant

Ki for MQPA

(4)

(Tyr+”Leu)

.

t

K m for Arg substrates K m for Lys substrates

J

K m for Arg substrates K m for Lys substrates

t

K m for Arg substrates K m f o r Lys substrates

t

t

J.

J

factor X a mutant Ki for MQPA J. .

T h e first mutation in T a b l e 5 will

increase the activity of MQPA

by eliminating the steric repulsion between MQPA arginine and Ser

BPTI by reducing the A similar effect will be observed for

i t will decrease the activity o f

But,

Oy.

number of hydrogen bonds.

substrates w i t h arginine o r lysine at the S1 site. arginine

will

substrates

become

substrates will become weaker.

stronger

substrates, water

the

activity

a n e w hydrogen

molecule

between

will and

Oy

the

MQPA.

of bond

Ser

lysine

of

T h e r e will be steric repulsion

between MQPA arginine and O y atom by decrease

that

T h e second and third mutations are

suggested from the same purpose. will

and

T h e binding of

introduced

In be

the

the

formed. lysine

Ser

case

and

of

via a

probably

side

it

lysine

chain.

The

fourth mutation will reduce the degree of steric repulsion between MQPA quinoline and T y r by

replacing

the T y r with

less bulky Leu

and increase the activity of MQPA for factor X a mutant. 6.

CONCLUSION

X-Ray analyses of several trypsin-inhibitor complexes provided

three

novel

lines

type of

of

valuable

information.

mechanisms o f selective inhibition. for design reliable

of

First,

trypsin inhibition and lead to enzyme

atomic

specific

coordinates

a

the

Thus, i t indicated strategies

inhibitors.

to

revealed

it

elucidation o f

examine

the

Second,

it

various

provided

simulation

methods which are used to evaluate the free energy change of drugprotein

complex

activities syntheses.

of

formation.

compounds

Third,

by

could

With be

accumulating

a

valid

predicted the

simulation before

method.

their

actual

three-dimensional

struc-

95 tures o f drug-protein complexes, w e may be able to find essential factors

in

drug-protein

interaction

and

utilize

them

in

drug

design. We thank our coworkers, C. Sasaki, C. Okumura, M. and Dr. H. Kubodera.

Miyagawa

REFERENCES

1 2

3 4 5 6

7 8 9 10 11

12

R. Kikumoto. Y. Tamao. K. Ohkubo, T. Tezuka. S. Tonomura, S .

Okamoto and A. Hijikata, J. Med. Chem.. 23 (1980) 1293-1299. J. Sturzebecher. F. Markwardt. 9. Voigt. G . Wagner and P. Walsmann. Thromb. Res., 29 (1983) 635-642. R. Kikumoto, Y. Tamao. T. Tezuka. S. Tonomura. H. Hara. K. Ninomiya. A. Hijikata and S. Okamoto. Biochemistry, 23 (1984) 85-90. T. Matsuzaki. C. Sasaki and H. Umeyama, J. Biochem. 103 (1988) 537-543. T. Matsuzaki. C. Sasaki, C. Okumura and H. Umeyama. J. Biochem. 105 (1989) 949-952. R. Kikumoto and Y. Tamao. Mitsubishi Chem. R&D Rev., 1 (1987) 26-34. Protein D a t a Bank, Brookhaven National Laboratory, Upton. New York. J . Walter. W. Steigemann, T. P. Singh. H. Bartunik. W. Bode and R. Huber. (1981) 3PTN. Protein Data Bank, Brookhaven National Laboratory, Upton, New York. W. A. Hendrickson and J . H . Konnert, Biomolecular Structure, Conformat i o n . Function and Evolution, Pergamon. Oxford, Vol. 1, 1981. pp. 43-57. M. Marquart. J. Walter, J . Deisenhofer. W. Bode and R. Huber. Acta Crystal logr.. 839 (1983) 480-490. T. Naito, personal communication. C. S. Craik, C. Largman. T. Fletcher, S. Roczniak. P. J. B a r r , R. Fletterick and W. J. Rutter. Science 228 (1985) 291 -297.

This Page Intentionally Left Blank

QSAR and Drug Design - New Developments and Applications T. Fujita, editor 9 1995 Elsevier Science B.V. All rights reserved

97

THREE-DIMENSIONAL STRUCTURE-ACTIVITY RELATIONSHIPS AND R E C E P T O R MAPPING OF QUINOLONE ANTIBACTERIALS

HIROSHI KOGA and MASATERU OHTA Fuji-Gotemba Research Laboratories Chugai Pharmaceutical Co., Ltd. 135, 1-Chome Komakado Gotemba-shi, Shizuoka 412 J a p a n . ABSTRACT:

The q u a n t i t a t i v e structure-activity relationship (QSAR) correlation equation previously formulated for a n t i m i c r o b i a l a c t i v i t y of q u i n o l o n e - 3 - c a r b o x y l i c a c i d s i n d i c a t e s that the steric f e a t u r e s of s u b s t i t u e n t s at the I-N-, 6-, and 8p o s i t i o n s are i m p o r t a n t in g o v e r n i n g a n t i m i c r o b i a l a c t i v i t y . We r e e x a m i n e d the steric features of these s u b s t i t u e n t s by a n a l y z i n g t h e i r c o n f o r m a t i o n s by a m o l e c u l a r m o d e l i n g method. The "active" c o n f o r m a t i o n of e a c h s u b s t i t u e n t at e a c h of the p o s i t i o n s was e s t i m a t e d w i t h the c o n f o r m a t i o n a l e n e r g y c a l c u l a t e d by m o l e c u l a r o r b i t a l m e t h o d s and the a n t i m i c r o b i a l a c t i v i t y of the q u i n o l o n e c a r b o x y l i c a c i d m o l e c u l e p o s s e s s i n g that s u b s t i t u e n t . A m o d e l of the receptor supposed to accommodate substituents at the respective positions was constructed by superposing active c o n f o r m a t i o n s of s u b s t i t u e n t s in h i g h l y a c t i v e c o m p o u n d s . With this a c t i v e v o l u m e or r e c e p t o r model, the a c t i v i t i e s of c o m p o u n d s that were not predicted well by the previous QSAR were qualitatively and/or semi-quantitatively rationalized. We b e l i e v e that the p r e s e n t model is useful for p r e d i c t i o n of the a c t i v i t y of compounds to be synthesized in designing new quinolone antibacterials.

1. I N T R O D U C T I O N Nalidixic

acid

antibacterial therapy is

of

urinary

effective

effective Thus,

(NA) is the

drug

family tract

against

against

efforts

and

have

been

made

as

overcome

its m e t a b o l i c

as to

Norfloxacin 1970's

by

Koga

(NFLX, I), (2),

one

to

to

and

expand

1963

which was of

the

it

to

it

synthesized

the

is

it not

bacteria. enhance

and

authors,

for

Although

antibacterial

instability first

use

Pseudomonas

modify

present

(i) .

bacteria,

and its

of a q u i n o l o n e

clinical

gram-negative

activity

well

member

in

since

gram-positive

antibacterial

(1).

known

been

infections

most

most

first has

its

spectrum,

side

effects

in the

opened

up

late new

98

cooH R k?cooH

O

O

CH3 ~ ' c O O H

O

I

C2H5 nalidixic

acid

possibilities clinical

for use

used

However, For

is

I),

further

(Table

are orally

synthesized

developed

I) .

These

quinolone

relatively

is

enoxacin(2)

low

Subsequently,

systemic

toxicity

poorly

and

much b e t t e r

than

(4)

that

a number

are

commonly

called

share

common

a

new

(3) .

absorbed.

AM-833

(see

of NFLX,

of analogs

a few of them have been m a r k e t e d

they

P . and

quinolones

(4) or

6-fluoro-7-amino

structure.

Koga activity

(2b, c)

previously

relationships

structure

3.

From

correlations log

because

including bacteria,

against

NFLX

to

against

acid-resistant

effective has

the

superior

activity

resistant

and

(2,4).

and quite

is

nalidixic

is

absorbed

compounds

fluoroquinolones

potent

stable,

drawback,

and e x p a n d e d

NFLX

bacteria

of

against

orally,

administrated

which

have been

given

this

it has

incidences

metabolically

orally

greatly.

gram-negative

low

when

overcoming

Table

and

in that

cross-resistance

and,

infection,

were

quinolones

causes

incomplete bacteria

antibacterials

of q u i n o l o n e s

gram-positive

aeruginosa,

(norfloxacin,NFLX) (enoxacin)

of quinolone

applications

previously both

I "X=CH 2 X=N 9

(I/MIC)

the

examined

(QSAR's)

of

statistical

the

71

point

was r e p r e s e n t e d by equation =

- 0.362

quantitative

compounds

with

of view,

one

structurethe

generic

of the best

[I] 9

(LI) 2 + 3.036 L1

-

2.499

(Es6) 2 - 3.345 Es 6

-

0.205

(ZK6,7,8) 2 - 0.485 ZK6,7,8

+ 0. 986 I7 - 0. 734 I7N-CO

n=71 In mole/l

-

1.023

-

0.681 ~F6,7, 8 - 4.571

s=0.274

this

equation,

(MIC)

against

(B48)2 + 3. 724 B48 r=0. 964 the

[1]

r2=0. 929

minimum

Escherichia

F=70.22

inhibitory

coli

NIHJ

concentration is

used

as

in the

99 TABLE

i. F l u o r o q u i n o l o n e s . O

F

Ra Compound

R7

AM-833

5

ofloxacin

6

amifloxacin

7

ciprofloxacin

8

CI-934

9

AM-1091

11

difloxacin

activity.

parameters (Es)

R6.

constants terms is

an

carbonyl

the

zero

maximum

and

H

is

width

(R7

inductive

8-substituents.

the

(L) the

to

be

to

V e r l o o p 's

of

the

unity the

B48

of

side) .

when

7-amino

is

it

F6,7, 8

is

the

hydrophobic site(s).

I7 is

not.

is

as

sum (F's)

I7N-CO

unity

when

a

moiety,

and

parameter

for

direction the

quadratic

7-substituent

STERIMOL

parameters almost

The

substituent

another

perpendicular

with

is

expressed

at

parameter

hydrophobic

action the

STERIMOL

substituent steric the

to

their

when

is

electronic

Equations

sum

related to

zero

which

of R8 in the

R 1 substituent

F

and R 8 substituents.

as

within not.

of

compounds

variable

does

for

equal

expressed

exists

one

length

seem of

variable

it

Lupton-Hansch

N-X___/

R 6, R7,

is

group

>

of the

indicator when

F

stands

transport

and

--

the T a f t - K u t t e r - H a n s c h

parameter

the

H

C2H 5 -

7, 8

indicator

another

the

this

on

hydrogen

as

(~'s)

of

effect

~6,

CH3NH--

F

the

Es 6 r e p r e s e n t s

H

_

L1

representing

i.

of

CH3-N

F CH2 CH2 --

--OCH2CH (CH3) - -

/--A N-k__/

~'~N

H

R1

F

/--A N-k__/

~ NH~ N

PDI17558

position

HN

~N

10

biological

CH3-N

R1 R8

/--I CH3-N N -k__Y /---A CH3-N N-k__/

4

/COOH

II

of

opposite the

of the

equivalent,

or

to

Swain6-,

7-,

slightly

100 poorer the

statistical

significance

hydrophobicity

whole

of

Equation

[i] indicates

compounds

having 4.2

fluoroethyl, substituent

piperazinyl,

a

of

to

be

NFLX.

al.

(5)

almost much

this

These since

few

some

[i]

N-l-aryl

activity

analogs,

the

been

good

elaboration effects

quinolone

of

8-positions

of

analyzing

their

the

effects

rationalize quinolones

in

has not been

steric

steric

three-dimensional

of

found

detail

by

are

For

predicted

be

has

predicts

irrelevant However,

and

the

the

cases

of

a

due

general of

applicability

compound

quantitative

substituents

of

(II)

[i]

et

to

attempted.

map

Chu

ii

and

structure-

In this

chapter,

at

i-,

the

systematically and

substituents a possible

6-, in

more

attempted

in

terms

receptor

we and

of

region

to the of

3.

Equation and

not

substituents.

conformations

to

and/or

example,

equation

to

the

antibacterials

structure

been

compounds

ciprofloxacin(7),

2. ANALYSIS OF THE STERIC EFFECT OF 1-SUBSTITUENTS alkyl

have

of

(ii) .

assumed

except

R8

or more

synthesized

activities

compound been

an

methyl,

to

predictions

However

parameters

have

is

relationship

reexamined

have

steric

deviants

equation

and

to p o s i t i o n

bromo,

activities

developed.

NFLX.

for this

deviations of

as

(e.g.,

-1.4

,

of

an R7

(Table i) .

whose

been

of

E s value

comparable

(i0)

that

(L)

l-(p-fluorophenyl)-fluoroquinolone

activity

lower a c t i v i t y

assignments of

that

high

an

opposite

amifloxacin(6), (4)

compounds have

the

the

methylamino,

nitrogen),

chloro,

these

of

PDI17558

industries

some

found as

fluoro,

evaluations

equation

with

oxygen,

activities

ofloxacin(5),

d e v e l o p e d by various Recently,

(e.g.,

and

length

aminopyrrolidinyl)

exhibit

A M - 1 0 9 1 (9) ,

a

approximately

Subsequently, by

such as AM-833(4),

by

i

could

valid

C I - 9 3 4 (8) ,

well

of

for

of the RI, R6,

methoxy,

R 6 substituent

value

P

K7 for

It p r e d i c t s

with

vinyl, chloro,

either log

factors

(B4) in the d i r e c t i o n

1.8

oxygen)

that

ethyl,

aminopiperidinyl,

approximately

shown

substituent an

or

for activity.

fluoro,

z

with a width

methylene, than

R1

(e.g.,

with

substituent

an

(e.g.,

with

[I].

that the steric

important

cyclopropyl),

approximately-0.65

in e q u a t i o n

are A

formulated

R 7 substituent,

instead of ~ 6 , 7 , 8

approximately

of

the

molecule

and R 8 s u b s t i t u e n t s

1

only

were

[i]

was

substituted

derived alkyl

from

groups

compounds as

the

(6)

having

R1

only

simple

substituent,

not

lO1 including

any

prediction of

R1

aryl

of the

should

factor

for

considered

group.

This

activity

be

more

alkyl

could

be

the

of N l - a r y l q u i n o l o n e s .

complex

groups,

than

when

that

reason

expressed

these

for

The

the

steric by

mis-

effect

the

length

Rl-arylquinolones

are

together.

2.1 Compounds and Biological Activity The

listed of

compounds

in T a b l e

biological

negative

2.

E.

relative

to

were

not

five

groups 2.

that

of

overall

activities

to

lowest

ranging

activity.

activities

of t h e s e

a

quinolone

activity

compounds

from

4 to

There

is

an

index

each was

were

calculated was as

tested

The

into

shown

activity,

almost

other

compound

classified

activities

0.5.

gram-

against

of

activity

highest an

as

are

antibacterials

NFLX(1) ,

relative the

chosen

representative

biological

have

activities

activities

drug,

The

their

1 compounds

of

The the

comparable.

is

their

standard which

was

coli

coli

(2,4) .

the

biological

E.

E.

parallel

under

activities

their

against

the

according

Class

the

MIC

bacteria

always

relative shows

and

conditions

and

because

roughly

coli

gram-negative

Table

The

activity

bacterium

against

because

analyzed

and

class

in

their

5 compound

8000-fold

range

in

compounds.

2.2 Conformational Analysis and Molecular Modeling 2.2.1 General Procedure:

quinolone

ring

oxolinic structures the of

was

acid

and

compound force

the

was

The were

in

using as

as

bonds

rotatable of

analyses

were

and

(15). with

5~ .

was

built

initial

of

orbital

standards

for

Nl-substituents

ethyl,

by

from use

Gaussian

the

with

of 82

cyclopropyl,

such

MO

program

the

each

Tripos

minimum-energy for

further

R6=F,

R7=Rs=H)

molecular

rotated

energy methods

(16)

modeling.

conditions

were

and p h e n y l

bond

of

method.

under

minimum

from model

standard

these

(MO)

taken

energy

6-fluoroquinolones(3"

within

The

with

of The

primary

coordinates

preliminarily

continued

were

mechanics

the

(12) .

The

conformational

of

structure

R8=H)

(13).

examined Starting

X-ray

compound

coordinates

primary

all

each

molecular

The

the m o l e c u l a r

were

compounds

by

the

the

structure

-OCH20-,

system

compound

Then,

used

of

SYBYL

7,8-unsubstituted

used

AM1

each

(13) .

Conformations increment

the

minimized were

optimization

of

from

R6-R7 =

substituents

angles.

field

structures

constructed

library

substituents

lengths

three-dimensional

(B : R I = C 2 H 5 ,

of

fragment

The

was

groups

where

with

an

conformations, as

CNDO/2

also

used

as RI.

(14) for

STO-3G

102 TABLE 2 . R e l a t i v e A c t i v i t y of F l u o r o q u i n o l o n e s .

R e lat i v e

1 14

-

1/21

R

R8

1

H

H

5(S)

Me

6 7

11 12 13 14 15

16

17

18 2

[1/4-1/16]

Me H Me H H Me Me H H

Me

H H H H H H H

H H H

30

[1/32-1/128]

31

H

2

H

7

MeNHcyc-Pr4-F-PhFCHzCH2CH2=CH2,4-F2-Ph-

H H H H H

H ~-oH-P~n -CH=CH -S -

-C

*

(CH2)=CH-S-CHJCH7S-

8t9 9 5 2 2 5 5 10

10 11

Me Me Me Me Me Me H Me

Me

-OCHzCH (CH,) (R) P h2-F-Ph2 -Me-Ph4-C1-Ph4 -Me-Ph3 , 4 - ( O C H 2 0 ) -Ph-

32

MC

H H H

33 [1/250-1/1000]

Me

H

4

Et

n-PrCH2=C13CH2HOCH2CH2PhCHz-

Me

3

ref

(S)

20 21 22 23

27 28 29

R2

(CH3)-

-OCH2CH

19

24 25 26

*

R1

H

(Me)>NCH2CH73-F-Ph4-Br-Ph-

H

H

2 5 5

4-MeO-Ph-

H

5

n

basis set w a s used for t h e G a u s s i a n 8 2 c a l c u l a t i o n s .

a n a l y s e s , all r o t a t a b l e bonds i n N 1 - s u b s t i t u e n t s

I n t h e MO w e r e rotated w i t h

103 15 ~ i n c r e m e n t minimum. each

After

of the

group

and

was

determination

introduced

program. of

or

The

using

was

the

the

by

from

Conformational compound

(35) . 35,

quinolones shown

in

energy. three

(Table

bond

Fig.

i.

There

was

methods. where

quinolone

plane.

With

decide

which

the

ring, this of

results of

the

the

information

these

two

the

the

two

energy

moiety

other

to

alone,

is the

more

each

conformation

Activity:

values

of

above

the

it

by

the

to

the

plane

is

is

are

identical

results

corresponds

it

of

conformation, and

where

however,

was

derivative

minima

is

that

active

class,

Nl-substituted

in the

minima

The

classes,

and

calculated

difference

der each

Nl-cyclopropyl

the

of

the

van for

compound

among

showed

with the

7-piperazinyl

energy

cyclopropyl

and

of

from

was

site

system.

Conformation

the

the AM1

optimized

different

in a c t i v i t y .

substantial

One

SYBYL

active

activity

and the the

compounds

volume

The

at

the

the of

rotated,

The no

having

the

(7),

of

it.

of

less

2).

was

using

superposing

started

highest

by

by

Active

was

compounds

Nl-substituents

total

Ciprofloxacin the

that

in

the

between

examined

conformation the

of

local

conformation

compound

volumes

the v a r i a t i o n s

has

NI-RI

MO

the

that

model

to

by

routine

total

analysis

compound

as

Nl-substituents

2.2.2 Relationship

the

energy

the

or N - m e t h y l - p i p e r a z i n y l

each

close

calculated

the

around

optimized

of

occupied

MVOLUME

reflect

the

were

defined

subtracting

class

where

minimum

7 of

value

was

between

to

position

volume

of

difference compound

of the

or an e n e r g y

"total"

estimated

5~ i n c r e m e n t

conformation

conformation

classes

with

structures

receptor

volume

assumed

at

whole

"active"

energy

"active" Waals

of

The

action

scanned

Nl-substituents , a piperazinyl

conformations

minimum

then

below

of the

impossible

responsible

for

to the

activity. Conformation structure more

and

insight

compounds. of

the

(7) .

S

into

isomer

The

S

(17).

[3: R6=F,

two

stable

R7=H,

ofloxacin activity,

the

Ofloxacin

CH2CH2CH(CH3)-] isomer

of

potent

(5)

5(S) isomer is

is of

also

(5) , was

active has

optical than

S-25930 reported

Conformational

analysis

without

in

of

more

of

the

showed

to

the R

5(R)

R8-RI =than

the

R

of

5

compound

the

energy

S

1

activity

isomer

R7=CH3, model

rigid obtain

class

the

active

that

significant

fairly

and

the

R6=F,

be

a

order of

isomers

that

[3 : to

R 8 - R I = - O C H 2 C H ( C H 3)-]

conformations

has

conformation

two

higher

which

analyzed

isomer

has

difference.

104

10

8

6

v

4

35 The d i h e d r a l

2

a n g l e was d e f i n e d as 2-1-1'-3'

0 -60

-120

0

60

120

Rotation 8 (degree)

A : Energy c u r v e c a l c u l a t e d by G a u s s i a n 82 B : Energy c u r v e c a l c u l a t e d by AM1 c : Energy c u r v e c a l c u l a t e d b y CND0/2

(STO-3G)

F i g . 1 . R o t a t i o n a l Energy Map of t h e N1-R1 Bond o f t h e 1C y c l o p r o p y l Compound35 (Reproduced from r e f . 6b by p e r m i s s i o n o f t h e American Chemical S o c i e t y ) .

is

One

that

where

the

branched

methyl

moiety

is

p e r p e n d i c u l a r t o t h e quinolone r i n g p l a n e and t h e o t h e r i s t h a t where it

i s oblique t o t h e plane. From t h e s i m i l a r i t y o f t h e t h e l a t t e r was s e l e c t e d t o match

o v e r a l l shape of N1-substituents,

one o f t h e c o n f o r m e r o f t h e N 1 - c y c l o p r o p y l

compound 35,

i n which

t h e c y c l o p r o p y l g r o u p i s l o c a t e d above t h e p l a n e o f t h e q u i n o l o n e ring.

Consequently,

the

matched

c o n f o r m e r s were

regarded

having t h e "active conformation" f o r t h e N1-substituents

as

of 5 (S)

and 7 ( F i g . 2 ) . F o r t h e N1-ethyl

g r o u p i n compound 3, i n which R1=C2H5, Rg=F,

R7=Rg=H a s t h e model o f n o r f l o x a c i n

(l), t h e r e a r e t h r e e e n e r g y

minima where t h e e t h y l g r o u p i s a b o v e , b e l o w a n d p a r a l l e l t o t h e quinolone r i n g . R1)

The b o n d - r o t a t i o n a l b a r r i e r s a r o u n d t h e bond

b e t w e e n t h e s e t h r e e c o n f o r m e r s were n o t h i g h .

which

the

e t h y l g r o u p was

above t h e

r i n g was

(N1-

The model i n

selected as

the

105 active of

conformation

5(S)

and

For

the

Nl-phenyl

conformational which and

the

showed

those

of the N l - s u b s t i t u e n t s

of

compounds

compounds

I,

5(S),

compounds

of

class

conformers

as

substituents

and

R6=F,

two

the

quinolone

and

benzene

The

latter and

(Fig.

best

highly

RI=C6H5, are

1 were

those of

that

24 7

(3" there

between

respectively.

conformer

it m a t c h e d

derivative

search

angles

i00 ~

because

7 best.

Ii

2) .

selected matching

active

was by

active

as

the

with

from

active

compounds

are

the

the

and

80 ~

active of

of o t h e r

low

energy

conformation

(i,5(S),

in

those

conformers

similarly

a

minima,

rings

selected

comparison

The

R7=R8=H),

energy

7)

of

NI-

(Figs.

2-

4). The

active

superposed

by

total

volume

model

an

the

be

is

activity energy

the

of

2

they

occupies

selected

in

compounds. as

For

the

region

are

of

two

The

form.

conformation the

of

substituents (Figs. We

class

2

former this

L

with

a

well, be

biological

because was

equation the

the

of

a

activity

the

20,

21,

extended as by

22

was

least

and

their

30) ,

a

the

active

the

suggesting in

as

active

and

significant

represented

receptor

this

dimethylaminoethyl

most

[i],

of

low

of the NI-

compound

selected the

of

fairly

occupies

(19, an

end

low is

and

compounds

a number

position

have

6.

arbitrarily

in

of

should

high

are

(22)

conformations"

was

interact

to

hydroxyethyl, 3

to

model

model

model

which

substituent

analysis

length

in

conformation

of N l - s u b s t i t u e n t s

the

there

compound

in Fig.

and

This

prediction

with

The

accommodating

receptor

This

to the m e t a

seemed

active

allyl,

ring.

calculated

5).

and

this

(22),

close 24

shown

(Fig.

were

found.

conformer

Nl-benzyl as

was

receptor

compounds

Nl-benzyl

plausible

in

effect

function

The

fits

compounds

quinolone

activity.

been

a region

1

verification

derivative

The

Nl-propyl ,

substituents there

possible

for

high

have

The

their

antibacterials

compound

later.

unfavorable

the

Nl-substituted

Nl-benzyl

group

described

in

class

Nl-substituents

If a c o m p o u n d show

conformations.

phenyl

the

atoms

standard

to

novel

substituent

for

a

whenever

For

the

quinolone

as

activity.

for

of

superposed

volume"

expected

amended

class

the

active

used

biological it

matching

of

"active

highly

could

conformers

bent

factor

quadratic that

these

extended

forms

6-9). calculated

the

difference

between

the v o l u m e s

occupied

by

106

Fig. 2. S t e r e o v i e w of the s u p e r p o s i t i o n of the p r o p o s e d a c t i v e conformers of I (green), 5(S) (yellow) , 7 (blue), a n d II ( o r a n g e ) ( R e p r o d u c e d f r o m ref. 6b by p e r m i s s i o n of the A m e r i c a n C h e m i c a l Society) .

r

Fig. 3. S t e r e o v i e w of the s u p e r p o s i t i o n of the p r o p o s e d a c t i v e conformers of 6 (yellow) , 13 (green) , 15 (orange) , a n d 16 (cyan) ( R e p r o d u c e d f r o m ref. 6b by p e r m i s s i o n of the A m e r i c a n C h e m i c a l Society) .

Fig. 4. S t e r e o v i e w of the s u p e r p o s i t i o n of the p r o p o s e d a c t i v e conformers of 12 (green), 14 (red), 17 (yellow) , a n d 18 ( v i o l e t ) ( R e p r o d u c e d f r o m ref. 6b by p e r m i s s i o n of the A m e r i c a n C h e m i c a l Society) .

107

Fig. 5. S t e r e o v i e w of the t o t a l v o l u m e (orange) of the NIs u b s t i t u e n t s of the class 1 c o m p o u n d s ( R e p r o d u c e d f r o m ref. 6b by p e r m i s s i o n of the A m e r i c a n C h e m i c a l Society).

Fig. 6. S t e r e o v i e w of the s u p e r p o s i t i o n of the p r o p o s e d a c t i v e c o n f o r m e r s of 19(green), 2 2 ( y e l l o w ) , 5(R) (orange), and 2 4 ( v i o l e t ) and the d i f f e r e n c e (orange) b e t w e e n the t o t a l v o l u m e s of the set of 19, 22, 5 (R) , and 24 and t h o s e of the c l a s s 1 compounds ( R e p r o d u c e d f r o m ref. 6b by p e r m i s s i o n of the A m e r i c a n C h e m i c a l Society) .

108 Nl-substituents of

class

2

occupied

in class

compounds

volumes

are

increases

where

repulsions

steric

8

shows

(28),

and

the

class

(24,

resulting

ends

of

25,

in

the

fact, seen

occupy

for

meta

R8-R 1

for

the

too

compound

quinolone

31.

moiety

ring,

as

receptor.

to

the

fit

the

are

in Fig.

and

compounds in the

of

of

are

19,

the

N l-

wall, The

and

the

meta

to be

21.

In

activity, methyl

the

6, d i s t u r b i n g

(21)

the

assumed

20,

branched

below

region b e l o w the plane

and

p-methyl

receptor

one

reduce

fixed

the

7,

hydroxyethyl

to

regions

Nl-phenyl

and

1 compounds.

and

compounds

6,

those

occupied

p-hydrogen

(20) ,

methylene

(29),

not

of class

These

of

wall

the

regions be

Figs.

Nl-phenyl

corresponding

5 (R)

shown The

the

in

The to

receptor

and

those

group.

The

of

of

(23)

(19) , allyl

activity

8. seem

activity.

regions

small

than

on

and

conformers

increase

m-oxymethylene

occupy

regions

substituents

cyclic

to the

are

activity

of the N l - p h e n y l

unfavorable

(26) ,

Nl-methyl

Nl-propyl

substituents

positions

26)

7,

volume the

the

substituents

The

the

6,

between

reducing

substituent

lower

Figs.

The a c t i v e

and

occupied

occur

(27)

(22)

1 compounds.

phenyl

the

o-methyl

p-chloro

and N l - b e n z y l

in

in

Nl-substituent

that

superposed,

shown

representing end of the

1 and 2 compounds.

were

as

in

plane

the

of

the p r o p e r

the

binding

of the q u i n o l o n e

ring

s h o u l d reduce the activity. The

difference

substituents together the

is

shown

These

at

the

regions

activities

para

In

of the

region

by

occupied compounds.

I0 in

shows the

The

Nl-phenyl

(33)

relevant

binding

is

of

to

Nl-phenyl

cause

are

is too

meta

fluorine

causes

seem

work

31 in class

difference 4

compound

region

occupied

probably the

I, 2, and 3 compounds.

more receptor

between and

the

by the

reduction

the

1 and the

and the of

the in

3. the

total

class

I,

p-methoxy the

in

simultaneously

significantly than

(32) .

(31) ,

small,

by

and the

of class

derivative

factors

the

NI-

occupied

(30)

reductions

to those

position to

by

2 compounds

substituents

further

Nl-(m-fluorophenyl)

of c o m p o u n d

with

1 and

regions

30 and 32 r e l a t i v e

the

class new

occupied

class

Additional

at the para

two

the a c t i v i t y

Fig.

class

thought

the

These

9.

volumes

and the

of the N l - d i m e t h y l a m i n o e t h y l

position

hydrogen

occupied

activity. lowering

in Fig.

groups

were

the

3 compounds

of c o m p o u n d s

compounds.

volume

between

class

two N - m e t h y l

bromine

2

of

volumes 2,

group

and

3

of the

unfavorable

Nl-substituents

for of

109

least

Finally,

the

active

derivative

(34) ,

examined methyl

(Fig.

inhibitory

below

substituents that

of

of

occupied

the

The the

i,

region

total

volumes

of

the

compounds

was

N I- (2, 6 - d i m e t h y l p h e n y l )

2,

3,

and

occupied

quinolone

ring

4

by

one

seemed

provide

to

for

by

of

the

exert

ortho

a marked

the

the

Nl-phenyl

We

of the q u i n o l o n e above

the

group

to the

of the Nl-phenyl.

We

into

one

above

fluorine also

that

NI-

there

corresponds

the

and

propose

the

for

propose

activity"

Nl-cyclopropyl other

insights

relationships

antibacterials.

increasing

and the

the plane

important

structure-activity

quinolone

ring,

position

below

class

analyses

regions

quinolone para

the

the

the

effect upon the activity. present

two

between

compound,

and

three-dimensional are

5

Ii) .

groups

The

difference

class

plane

of

hydroxyl that

to

the

at the

the

regions

ring and a r o u n d the m e t a p o s i t i o n

quinolone

ring

plane

prevent

proper

r e c e p t o r binding. Fig.

12

shows

a modified

volume

occupied

of

Nl-(p-hydroxy)-phenyl.

the

(length) QSAR

is best

equation

phenyl

toward

at

4.2

For

receptor allyl,

activity

as

used

optimum

they w o u l d

and

not

para p o s i t i o n

methyl further

Fig.

12

derive

volume reach

to

has

the

other

of the Nl-phenyl

as

the

the

of

NI-

has

cyclopropyl L

of

L

in

an and

compounds

value

changes

in these

compounds

in terms

receptor is

too

of the

The

group,

the

to

volumes

onto the L to

fit

that

the

but

n-propyl,

the

forbidden

region.

The

favorable

for

Nl-substituents

region

in

dimethylaminoethyl

forbidden

could

to

model

small

extrude

[i]

group.

[i]

group

does,

optimum

optimum

the

situation

groups

above.

why

The

group

equation

of

activity

cyclopropyl

corresponding

the

the

of the

into

total

activity

equation

length

value.

two

explain

the

in

that

group

benzyl

the

hydroxy

for N l - S u s t i t u e n t s

decreases

methyl

on

the

can

predict

variations

described

to

to

shows

projection a

penetrate in

to

based

and

model

parameter

optimum

activity

terminal

model

compounds

[i]

corresponding

The

substituent

L1

the

example,

wall

receptor

the

of

hydroxyethyl

region.

in

the

unable

substituents

side

one-dimensional

axis.

is The

model

group

parameter

corresponding

Nl-phenyl

explain

This

a steric

but

i

receptor

Nl-cyclopropyl

Equation

either

of the

the

as

[i]

groups.

without fact,

the

derivatives.

optimum ethyl

by

be of

of

accommodated cyclopropyl,

corresponding

the only but

to the

110

Fig. I . Stereoview of the superposition of the proposed active conformers of 2 0 (orange), 23 (green), 25 (blue), and 2 7 (yellow) and the difference(0range) between the total volumes of the set of 20, 2 3 , 2 5 , and 2 7 and those of the class 1 compounds. Since the benzene rings of the N1-substituents of 2 5 and 2 7 overlap, this region appears white. The N1-methyl of 23 and N1-ally1 of 20 also overlap and the N1-methyl appears white or yellowish-green (Reproduced from ref. 6b by permission of the American Chemical Society).

Fig. 8 . Stereoview of the superposition of the proposed active conformers of 21 (green), 26 (yellow), 28 (violet), and 2 9 (blue) and the difference (orange) between the total volumes of the set of 21, 2 6 , 28, and 29 and class 1 compounds (Reproduced from ref.6b by permission of the American Chemical Society).

Fig. 9. Stereoview of the superposition of the proposed active conformers of 30 (green), 31 (yellow), and 32 (cyan) and the difference between the total volumes (orange) of 30, 31, and 32 and class 1 and class 2 compounds (Reproduced from ref.6b by permission of the American Chemical Society).

111

~)__...... ~

~

0

.I~N~ ~

0

Fig. I0. S t e r e o v i e w of the p r o p o s e d active c o n f o r m e r of 33 and the d i f f e r e n c e b e t w e e n the v o l u m e s (orange) of c o m p o u n d 33 and class I, 2, and 3 c o m p o u n d s (Reproduced from ref. 6b by p e r m i s s i o n of the A m e r i c a n Chemical Society).

Fig. Ii. S t e r e o v i e w of the p r o p o s e d active c o n f o r m e r of c o m p o u n d 34 and the d i f f e r e n c e b e t w e e n volume (orange) of c o m p o u n d 34 and the total of class i, 2, 3, and 4 c o m p o u n d s (Reproduced from ref. 6b by p e r m i s s i o n of the A m e r i c a n Chemical Society).

Fig. 12. S t e r e o v i e w of the m o d i f i e d r e c e p t o r model for the volume occupied by Nl-substituents of quinolone antibacterials. ( R e p r o d u c e d from ref. 6b by p e r m i s s i o n of the A m e r i c a n C h e m i c a l Society) .

112

3. ANALYSIS OF THE STERIC EFFECT OF 6-SUBSTITUENTS

(18)

According to equation [l], formulated for the entire series of quinolones 3, the effect of substituents at the 6-position on the activity is represented by the Taft-Kutter-Hansch Es Equation [l] reflects equation [2] for the subset of

parameter.

6-monosubstituted compounds 36, the activity of which varies parabolically with the Es of the R g substituent (Fig. 13A) (2). log(l/MIC)

= -3.318(+0.59) ( E S ~ -4.371(?0.85) ) ~ Es6 +3.924 n=8 S = 0 . 1 0 8 r=0.989 F=112.29

[21

In equation [21, the Es value adopted for the nitro group is the one

(-1.01) evaluated from its half-thickness representing the

steric effect in the perpendicular direction and that of methoxy is approximated by the value of the ethyl group

-2

0

-1

1

-2

-1

E s6 Fig. 13

(2).

6 ES

For the

0

Parabolic relationships for the effect of

6-substituents with the Es6 Parameter.

1

113 corresponding

use

of

the

reasonable for

7-piperazinyl

same

E s value

(Fig.

its

13B) .

coplaner

significant significant

correlation

set

of

compounds

for

the

the

greatest

deviation,

observed

calculated

value

changes

of

the

vicinal

is

piperazinyl

relationship

between

0.61) .

log(I/MIC)

the

This

from the

not

For

the

(Es6)2

+1.426(+0.29) s:0.250

6-nitro-7-

for the

Although

being

be

due

value

to

and

conformational

confirmation

of

with

was

the

this,

analyzed

and a c t i v i t y

higher

observed

interaction

36 and 37 was

showed

much

the

a

combined

compound

between

(Es6) 2 -2.682(+0.93) r=0. 984

No

(half-thickness)

steric

conformation

- -2.587 (+0.89)

the

obtained

activity

the

not

half-width

[3].

Es

the

effective.

unless

could

by

R 6 in c o m p o u n d s

s=0.079

n:15

[4] was

however,

apparently

equation

difference

group

:-2.026(+0.68) n=6

give

predicted

(the

the

also

is

6-nitro-7-piperazinyl

group.

of the

to

using

its

6-nitro

conformation

Iog(I/MIC)

37)

value

estimated is

37,

group

formulated

equation the

compounds

6-nitro

E s value

omitted

group,

of

the

was

is

(36 and

6-nitro

subset

effect

correlation compound

the

The

steric

piperazinyl

than

for

the

and

the

examined. [3]

Es 6 +5.561

F-45.50 -3.351 (+1.25)

Es 6

[4]

17 +4. 088 r=0.971

F=60.84

3.1 C o n f o r m a t i o n and Steric Parameters 3.1.1 C o n f o r m a t i o n a l receptor similar

mappings

As R 6 : N O 2)

ring

Thus,

analysis The

should

the

36 for

and

37

were

carried

l-substituents

6-nitro

almost

hand,

group

plane

6-substituents,

14, is

other

6-substituents

substituent. of

Fig.

nitro

used

analyses

and out

in 3.

by

The AM1

for the MO method.

energy

the

the

Conformational

compounds

to those

used in

low

On of

quinolone some

was

shown at

plane. angle

of

procedures

Hamiltonian

Analysis:

in

of

is about could

be

the

steric

low

compound 55 ~ .

of

with (37:

(36: ring

conformation,

the the

the

on

with

conformations by

of the

based

38

quinolone

R6=N02),

influenced

analysis

parameter

compound

the

energy 39

Likewise,

markedly

for q u a n t i t a t i v e a

group

coplaner

the

steric

effect

conformational

be used.

6-methoxy

compound

40

(36:

R6=OCH 3)

has

of

adjacent

two

conformers

114 with

energy

group

is

moiety shown of

minima.

almost

of

the

methoxy

in Fig.

the

14.

methoxy

m e t h o x y group

Fig. 14. right) , 39

The

One

corresponds

coplaner The

group

locates

with

group

locates is that

is

opposite

conformer

was

the

methylthio

conformation

at

group

is

as

the

and

the

the

and

methoxy

the

5-position only

methyl

the

methyl side

as

direction

moiety

of

the

side.

c o n f o r m a t i o n of c o m p o u n d s 38 ( u p p e r (lower right), and 41 (lower left) .

6-methylthio-7-piperazinyl upward,

in w h i c h plane

in w h i c h

at the 7 - p o s i t i o n

the

turning

to that

quinolone

other

Proposed active (upper left), 40

first

the

taken

almost

modeled

as

the

active

compound

41

coplaner

with

in

Fig.

(37"

structure

R6=SMe)

the

14,

since

in

which

quinolone

has

lower

plane energy

than the other.

3.1.2 Quantitative Structure-Activity Relationship using Conformational StericParameters: conformations calculated. sphere the of

Each

with

the

quinolone the

along

New

steric p a r a m e t e r s

for the atom

van

ring

Waals

plane.

projection

substituents

in the

der

6-substituent

the

6-substituents

from of

The the

the

based

on the p r o p o s e d

of q u i n o l o n e s

6-substituent radius. length carbon

bond

The

P.

represented

plane

L is the atom

between

and the C6 onto the plane

was

at

P

is

farthest the

the

active

36 and 37 were a as

extension

6-position

(~ a t o m

as

defined

of

the

A box w h i c h t o u c h e s

(C6) 6-

the

115 van

der

Waals

through 15.

The

sides H2

the

values

of

are

the

the

tangentially

was

widths

defined

of

the

as

from

and

shown

passes in

substituent

respectively.

substituent

compound

H26

The

The the

that

reliable

36. [7]. of

n=8

situations

the

activity

the

6-NO2

plane

for

With

[6]

in

the

are

the

H 1 and

P

and

are

WI,

W2,

HI,

mainly

due

works

well

[5]

the

and

the

the

to

fact

[6] of

new

by

steric

variable,

quality

[4],

than

the the

the

steric

of

Figs.

16A of

the

correlation parameter

about

for

the

is gives

nature

at

give lower more of the

of R 6.

: -5.806(+2.67) s=0.255

r=0.937

(H26) 2 +17.67 (+8.42)

H26

-8.235

[S]

F:18.08

P

P

COOH

H1

H2 H2 --

15.

(half-

[2] to

R6

from

substitution

combined

R6 and

the

Es

equation

for

the

thickness

that

in

I7,

H2

effect

the

were

Es p a r a m e t e r

statistically

selected.

illustrated

with

L,

the

parameter

is

group

and

were

the

that

indicator

Although equation

37

show

accord

equations

information

effect

log(i/MIC)

Fig.

Fig.

to

values

plane

parameters,

36 and

represents

and

on

steric

respectively,

[5]

7-position,

equation

these

compounds

[6],

quinolone

thickness)

with for

[5] and

substituents

steric

are

the

equations,

Equations

than

of

examined

substituents.

the

W2

5-positions,

correlations

equations In

the

and

6-substituent 6-position

as H 2 _> H I.

The

16B.

the

the

W 1 and

7-

H2 w e r e

best

of at

thicknesses

defined and

radii

carbon

Definition

of the

new

steric

parameters.

H1

116 n

R6D3 OoH

7.0-

36

31

-

B

A

6.5-

4

I

GH5

6.0-

6.0

5.5-

5.5

\

a, 4

5.0

m

4.5

0

4

1 .o

1.5

2.0

1 .o

2.5

1.5

2.0

2.5

H2 Fig. 16. Parabolic relationships for t h e effect of 6 - s u b s t i t u e n t s w i t h t h e newly d e f i n e d H

l/MIC)

=

l/MIC)

s=O.211 =

parameters

( H z ~ )+ 6~ . 9 5 9 ( + 6 . 5 2 ) H26 + 0 . 8 8 6

-2.222(+1.99)

n=7

2

r=0.863

-3.427(+1.86)

[61

F=5.81

( H 2 6 ) 2 t 1 0 . 5 7 2 ( + 6 . 0 0 ) H26

+ l .7 0 5 (+O . 3 9 ) I 7 - 3 . 2 8 8

s=O . 3 3 1

n=15

r=O . 9 4 9

[71 F=33.07

V a r i o u s t y p e s of s t e r i c p a r a m e t e r s e t s h a v e b e e n e m p l o y e d f o r

QSAR

analyses.

Although

various

parameter

s u c c e s s f u l l y u s e d d e p e n d i n g upon t h e t y p e o f

sets

have

been

steric i n t e r a c t i o n s

i n v o l v e d , t h e y sometimes d o n o t r e f l e c t t h e s i t u a t i o n based o n t h e biologically

a c t i v e form.

T h e new

s t e r i c parameters

proposed

a b o v e i n a way s i m i l a r t o t h e STERIMOL v a l u e s seem t o be v e r s a t i l e i n o t h e r examples, conformation

from

s i n c e t h e y are b a s e d on t h e p r o p o s e d " a c t i v e " conformational

m a n i p u l a t e d on t h e c o m p u te r g r a p h i c s .

analysis

and

appropriately

1 I7 TABLE 3 . S t r u c t u r e a n d A c t i v i t y o f q u i n o l o n e s a n d fluoroquinolones having 8-substituent.

n

d b - " " O H I RR

log 1/MIC

(mole/l) a g a i n s t E . c o l i

-1

obsd.

'ZH5

calcda)

dif.

43')

H

3.939

4.489

-0.55

44')

F

4.575

4.586

-0.01

4SC)

c1

4.606

4.449

0.16

Me

4.868

4.818

0.05

4 7 ')

OMe

3.694

3.881

-0.19

48')

Et

3.088

3.149

-0.06

2.514

2.386

46

C)

4 gC) OEt

l o g 1/MIC RNJ

R8

R8

1

R

obsd.

ref

0.13

(mole/l) a g a i n s t E . c o l i

R,

R1

b)

calcd?)

dip) calcd?)

difb) ref

C)

H

Et

H

6.629

6.375

0.25

2

50c)

F

Et

H

6.873

6.564

0.31

2

51c)

c1

Et

H

6.892

6.801

0.09

2

H

7.184

7.007d) 0 . 1 8

5.581e)1.60

2

Me

6.859

6.69gf) 0.16

5.798')1.06 h) 6.880 -0.04

2,7

5 2 ')-CH2 5

CH2CH (CH3)

-0CH2 CH (CH3) -

-

53

OMe

Et

H

6.844

5.759

1.08

54

Br

Et

H

6.600

6.746

-0.15

55

CN

Et

H

6.236

6.506

-0.27

56

NO2

Et

H

5.970

6.154

-0.18

20 21 21

i) 6.532 -0.56

C a l c u l a t e d by e q u a t i o n [l] . D i f f e r e n c e between observed and c a l c u l a t e d v a l u e s . I n c l u d e d t o d e r i v e e q u a t i o n [l]. C a l c u l a t e d w i t h B 1 of t h e e t h y l g r o u p i n p l a c e o f B 4 ( 2 b ) C a l c u l a t e d u s i n g B 4 of t h e e t h y l g r o u p f o r B 4 8 . C a l c u l a t e d u s i n g B 1 of t h e 8 - m e t h o x y g r o u p i n p l a c e o f B 4 C a l c u l a t e d u s i n g B 4 o f t h e methoxy g r o u p f o r B 4 8 . C a l c u l a t e d u s i n g B 2 o f t h e methoxy g r o u p f o r B 4 8 . C a l c u l a t e d u s i n g B1 of t h e n i t r o g r o u p i n p l a c e o f B 4 .

21

118

3.2 Proposed Receptor Model The

active

conformers

of

norfloxacin

droxacin,

tioxacin,

and DJ-6783

of

quinolone

rings.

their

substituents 17.

The

positions receptor these

and

total

should

compounds

HN~ . J

helpful

>--S

to the

are very including

volume

for

active

at

of the

against

E.

oxygen,

as

the

estimating

vicinity

fluorine,

occupied

calculated

compounds

oxolinic

acid,

by m a t c h i n g by

shown 5-,

the

6-,

shape

6-position,

coli

with

atoms the and of

a variety

C2H5

C2H5

C2H5

O

i

C2H 5

tioxacin

Fig. 17. Active volume (cyan) of quinolone antibacterials.

acid

the of

and nitrogen. O

oxolinic

7-

because

O

(I)

6-

in Fig.

O

norfloxacin

O

be

was

these

(1),

superposed

total

groups

of

corresponding

6-substituents

The

adjacent volumes

were

droxacin

O

I

C2H5

DJ-6783

of the

6-substituents

and vicinity

119

4. ANALYSIS OF THE STERIC EFFECT OF 8-SUBSTITUENTS

( 19)

The activity (MIC) of 8-substituted quinolones 4 2 ( 4 3 - 4 9 in Table 3 ) has previously been reported as being parabolically related with B48, one of the STERIMOL parameters for the maximum width of the Re as indicated by equation [81 and Fig. 18 (2). The B4 value as the steric parameter of Re substituents also applies to 1, 6, 7, 8-tetra-substituted quinolones 3 ( 5 0 - 5 2 in Table 3) since

the activity

of these

equation

(2).

111

compounds has been

The 8-substituent

well

predicted by

is thought to interact

sterically with the 1-ethyl-substituent in compound 4 2 . Therefore, the maximum width of the Re expressed by B4 has been believed to be that in the direction opposite to the 1-substituent ( R 7 side) and to recognize the receptor wall as such. Depending upon the structure, however, the 8-substituent may be directed above or below the quinolone ring plane with steric repulsions of substituents at positions 1 and 7. log(l/MIC)

=

-1.016(*0.46) (B48)2 +3.726(+2.04) B48 +1.301

n=7

s=O.221

r=0.978

F=44.05

Me

1 .o

2.0

3.0

B48 Fig. 18 Parabolic relationship for the effect of 8-substituents with the STERIMOL B4 parameter.

181

120

Fig. 19. (pink), 48

S t e r e o v i e w of the p r o p o s e d (green), and 49 (blue) .

active

conformers

of

47

proposed

active

conformers

of

53

Stereoview of the a c t i v e of q u i n o l o n e a n t i b a c t e r i a l s .

volume

model

the

8-

Fig. 20. S t e r e o v i e w of (yellow) and 56 (green).

Fig. 21. substituent

the

of

121 Since structure and

equation 3,

50-52

in

quinolones the

[I]

including Table

3,

(5 and 5 3 - 5 6

activities

of

was

formulated

8-substituted some

symmetrical

top

8-substituents

a ring

the

l-substituent

[i],

with

that

group

was

of c o m p o u n d

53

not.

may

conformations

This of

the

were

be

due

by the

We

compounds

with 44-49

some

reported.

Although

spherical

5 with

the

well

by the

or

R 8 forming equation

unsymmetrical

methoxy

differences

between

the

1,8-disubstituted

(47-49)

and

compounds

8-substituents.

quinolones

as

having

predicted

to in

been 55

compound

substituted

8-substituents

8-substituents

and

and

71

such

i, 6, 7, 8 - t e t r a - s u b s t i t u t e d

3) have 54

i, 6, 7, 8 - t e t r a - s u b s t i t u t e d unsymmetrical

new

in Table

compounds

for

ones

such

analyzed

as

the

53

having

conformations

of

to examine this p o s s i b i l i t y .

4.1 Active Conformation and Activity The

compounds

analysis

of

described

above

As ethoxy

the

analyzed

8-substituted

(2.2.1)

shown

in

direction

the

1-ethyl

e),t.',.'.,

19,

opposite

k. " " " . '

<

the

to the

was

shown

compounds 8-methoxy

coplaner 1-ethyl

almost

.~ ~

Fig. 22. Stereoview antibacterials.

.

"

of

< .

the

3.

was

Conformational accomplished

(47) , e t h y l

(48) ,

with the q u i n o l o n e

at the

coplaner

........

V,

in Table

as

only AM1 as the MO method.

are n e a r l y

group

." .

using

Fig.

(49) groups

the

are

and

ring in

e n e r g y minimum,

while

to

the

plane

r , < "~

. " ., . ' . ~ . ~

quinolone

~,

active

volume

model

of

the

quinolone

122 corresponding like

the

with

1-ethylene

suggests

that

required

for

moiety

of

There

are

above

and

methoxy

because

earlier

active

conformer

could

be

instead

better of

expected,

B4 the

the

[i]

of

the

compound

53

is

was

the at

receptor of

20.

compound

predicted

l-

binding

in Fig. is

equation

in p l a c e

the

active the

B 3 parameter in

47.

located

where

of

is shown

or

group

compound

plane

proper

substituent

for the m e t h o x y

that

activity

B2

8-methoxy

group

ring

situation

the

of

also

used

[i] .

very

53

well

As by

of B4 in e q u a t i o n

(Table 3) . In

nitro

plane case,

the

low

quinolone

better

(Fig.

20).

with

substituent 21.

The

the

The

active

nitro

quinolones it

which

was

group.

width

for the

drawn not

(I) with h y d r o g e n

This

of the

in

the

the

as the

be

the

was

53

not

the in

of the

8-

to work 54

in

volume

of

relative

to

maximum

activity

ring

group

seemed

model

8-

predicted

nitro

by B4

compounds

with

reduce

could

receptor

using

corresponds

does

56

represented

volume

group

to the q u i n o l o n e

compound

The m a x i m u m

ring plane

present,

of n o r f l o x a c i n

of

of B4 of the nitro

3).

the

8-substituent

that

activity

(Table

of At

conformation,

(56) is n e a r l y p e r p e n d i c u l a r

56 b e l o w

obstructively. Fig.

energy

B1 i n s t e a d

however

compound

the

when

8-methoxy

activity

u s i n g the B2 value

the

that

the

1

This

is

conformation as

prevent

The

suggests

predicted

for

may

18.

group

of

methoxy

The

below

(2.2.2).

compound

from that

selected

region

norfloxacin

1-ethyl

the

ring.

was

in

conformation

however,

neighborhood

discussed

The

ring

the

the

in w h i c h

quinolone

the

minima

closed-ring of

The

minima

the

its

the

53 differs,

above

and

of

energy

conformation

energy

below

three

activity.

compound

is

position

high

of

moiety

this

two

conformer as

one

and

8-substituent.

5. C O N C L U S I O N The supposed volumes vicinity Since not

"total" to

shown of

steric

active

fit the

in Figs.

the

5,

17,

6-position,

requirements

fully u n d e r s t o o d ,

was p r o v i s i o n a l l y

volume

receptor

of

of

is shown

quinolone in Fig.

and 21 for the and

the

the p i p e r a z i n y l

sum of the

l-N-substituents,

8-substituents,

7-substituents

u s e d as the best

antibacterials

22 as the

for

high

respectively. activity

or N - m e t h y l p i p e r a z i n y l

substituent.

the are

group

123 The

"active

positions,

conformation"

I-N,

information

6,

about

quantitatively

by

receptor

must

the

model

model

compounds believe

was the to

compounds

steric be

model is

for

each

believed

effects [i].

to

in

the the

predicted

well

by

approach

the

method

but

also

for

QSAR

rationalizing

detailed

been

analyzed

active of

volume

newer

in

only

of

several [i] .

this for

biological

or

findings,

equation

used

not

substituent

more

activities

three-dimensional classic

the

have the

light

rationalize

not

of give

which

Although

corrected to

were

that

8,

equation

shown

which

complementary novel

and

We

study

is

developing

activities.

ACKNOWLEDGEMENTS We support our

thank and

thanks

discussion,

Drs.

Shun-ichi

encouragement to

Prof.

advice,

Toshio and

Hata

during Fujita

and

this of

Ikutoshi

work.

Kyoto

We

Matsuura wish

University

for

to

express

for

helpful

comments.

REFERENCES 1 2

3 4 5 6

7 8 9 i0

R. A l b r e c h t , Prog. Drug Res., 21 (1977) 9. (a) H. Koga, A. Itoh, S. M u r a y a m a , S. Suzue a n d T. Irikura, J. Med. Chem., 23 (1980) 1358. (b) H. Koga, in" T. F u j i t a (Ed.), "Structure-Activity Relationships-Quantitative Approaches; Applications to D r u g Design and Mode-of-Action Studies", N a n k o d o , Tokyo, 1982, pp 177-202. (c) T. Fujita, in" G. J o l l e s a n d K. R. H. W o o l d r i d g e , (Eds) , " D r u g D e s i g n - F a c t or F a n t a s y " A c a d e m i c Press, N e w York, 1984, p 19. B. Holmes, R. N. B r o g d e n and D. M. R i c h a r d s , Drugs, 30 (1985) 482. P. B. F e r n a n d e s a n d D. T. W. Chu, Ann. Rep. Med. Chem., 23 (1988) 133, and the r e f e r e n c e s therein. D. T. W. Chu, P. B. F e r n a n d e s , A. K. C l a i b o r n e , E. P i h u l e a c , C. W. N o r d e e n , R. E. M a l e c z k a , Jr. a n d A. G. P e r n e t , J. Med. Chem., 28 (1985) 1558. (a) M. O h t a and H. Koga, in" The 15th S y m p o s i u m on S t r u c t u r e Activity Relationships, Nov. 6-8, 1987, T o k y o . Abstracts of papers, pp. 338-341. (b) M. O h t a and H. Koga, J. Med. Chem., 34 (1991) , 131. S. A t a r a s h i , S. Y o k o h a m a , K. Y a m a z a k i , K. Sakano, M. I m a m u r a and I. H a y a k a w a . Chem. Pharm. Bull., 35 (1987) 1896. M. P. W e n t l a n d , D. M. Bailey, J. B. Cornet, R. A. Dobson, R. G. P o w l e s and R. B. Wagner, J. Med. Chem., 27 (1984) 1103. J. S. W o l f s o n and D. C. Hooper, Antimicrob. A g e n t s Chemother., 28 (1985) 581. H. E n o m o t o , M. Kise, M. O z a k i , M. K i t a n o and I. M o r i t a , J a p a n e s e Patent Kokai 103393, (1983) ; Chem. Abstr., 98 (1983) 53877w.

124 Ii 12 13 14 15 16 17 18 19 20 21

S. Mat sumura, M. Kise, M. Ozaki, S. Toda, K. K a z u n o , H. Watanabe, K. K u n i m o t o a n d M. Tsuda, Japanese Patent Kokai 136588, (1982) ; Chem. Abstr., 98 (1983) 53877w. M. C y g l e r and C. P. Huber, Acta Cryst., C41 (1985) 1052. SYBYL M o l e c u l a r M o d e l i n g System; Tripos A s s o c i a t e s " St. Louis. J. A. Pople and G. A. Segal, J. Chem. Phys., 44 (1966) 3289. M. J. S. Dewar, E. G. Zoebisch, E. F. Healy, and J. P. Stewart, J. Am. Chem. Soc., 107 (1985) 3902. J. S. Brinkley, M. J. Frish, K. R a g h a v a c h a r i , R. A. Whiteside. H. B. Schelgel, E. M. F l u d e r and J. A. Pople, " G a u s s i a n 82", C a r n e g i e M e l l o n University, 1983. J. I. Gerster, S. R. Rolfing, R. M. Pecore, R. M. Winandy, R. M. Stern, J. E. Landmesser, R. A. O l s e n and W. B. Gleason, J. Med. Chem., 30 (1987) 839. H. K o g a a n d M. Ohta, in" The 16th S y m p o s i u m On S t r u c t u r e Activity Relationships, Dec. 5-8, 1988, Kyoto, A b s t r a c t s of Papers, p.260-263. M. Ohta. and H. Koga, in- The 16th S y m p o s i u m On S t r u c t u r e Activity Relationships, Dec. 5-8, 1988, Kyoto, A b s t r a c t s of Papers, p.264-267. K. Iwase, et al. in 9 The 107th Annual Meeting of the Pharmaceutical Society of Japan, Apr. 2-4, 1987, Kyoto, A b s t r a c t s of papers, p.483. K. Iwase, et al. in 9 The 106th Annual Meeting of the Pharmaceutical Society of Japan, Apr. 2-4, 1986, Chiba, A b s t r a c t s of papers, p.490.

QSAR and Drug Design - New Developments and Applications T. Fujita, editor 9 1995 Elsevier Science B.V. All rights reserved

125

CLASSICAL AND THREE-DIMENSIONAL QUANTITATIVE S T R U C T U R E - A C T I V I T Y A N A L Y S E S OF STEROID H O R M O N E S S T R U C T U R E - R E C E P T O R BINDING PATTERNS OF A N T I - H O R M O N A L DRUG CANDIDATES MASUMI YAMAKAWA 1, KIYOSHI EZUMI 1, KEN'ICHI TAKEDA 1, TETSURO SUZUKI 1, ISAO HORIBE1, GORO KATO 1 and TOSHIO FUJITA2 1 Shionogi Research Laboratories, Shionogi & Co., Ltd., Osaka 553, Japan 2 Department of Agricultural Chemistry, Kyoto University, Kyoto 606-01, Japan ABSTRACT: Previous QSAR (quantitative structure-activity relationships) examples of steroid hormones were briefly surveyed. The absorption and distribution processes and pharmacological activities in which transport factors are critical are governed mainly by molecular hydrophobicity. When the expression of the overall biological activity is controlled by the binding-affinity with the receptor sites as the rate-limiting process, the QSAR pattern is more complicated, because stereoelectronic and hydrogen-bonding effects of substituents or substructures of the molecule are usually involved in the structure-affinity relationships. The binding affinities of a number of androstan-1713-ols and estratrien-1713-ols for androgen and estrogen receptor preparations were experimentally measured and their structure-affinity relationships were analyzed using classical and threedimensional (CoMFA) QSAR procedures. The regiospecific stereoelectronic properties of the molecule were found to significantly regulate the affinity in each pair of combinations between ligand and receptor species. The hydrophobicity was of minor importance. The classical and CoMFA procedures were complementary to each other, illustrating the "components" involved in physicochemical and structural requirements for the binding affinity. The structural features of epitiostanol, an antiestrogen, which is an androstanol derivative that has been marketed as an anti-breast cancer agent, agreed very well with the QSAR patterns from the two procedures. 1. I N T R O D U C T I O N Many steroids play extremely important roles as hormones in animal organisms. Estrogens, progestins and androgens are known as sex hormones; the first two maintain female functions and the last, male functions. Glucocorticoids play a major role in the regulation of immune as well as inflammatory responses. A major action of mineralocorticoids is stimulation of active transport of sodium ion across the cell membranes (1).

126

Since cortisone (1), a glucocorticoid, was disclosed as being a very effective drug against rheumatism (2), a number of derivatives and analogs of steroid hormones have been developed and utilized in various chemotherapeutic fields (3). For example, prednisolone (2) and betamethasone (3), widely used as antiinflammatory and antiallergy agents, are analogs of cortisone (1). Oxymetholone (4), a potent anabolic steroid, and dromostanolone propionate (5), an agent for mastopathy, are androstan-3-one derivatives. Also, combinations of estrogen and progestin analogs are sometimes prescribed as contraceptives. The most remarkable aspects of these steroid hormonal agents are that their effects are highly specific and very potent even at low doses, and that, in spite of the diversity of their biological effects, they share the perhydrocyclopentanophenanthrene structure (Fig. 1) as a common skeleton.

1820[,,/21 lC 11~..

16

D ~

3

~7 4

6

Fig. 1. Steroidal Skeleton and Numbering of Atoms. Each category of steroid hormones appears to have a specific target cell species. Although the detailed mechanisms of the interaction with the target sites differ among the categories, there is a similarity in that steroid hormones must form a complex first with the respective receptors (4-6). The receptor complex is then activated and bound with specific target sites, i.e., specific DNA sequences on

?H2OH

?H2OH

R

o

o

OH

C-O

C=O

!

H

O

2: R = H, R'=H 3: R=F, R'-CH 3

OCOC2H5 H3C.~~J~ ''L" H 5 O-"

V , V

H

CH 3 i

C=O

OH

"A"

Ac

0~~. ,O~v~ ,7 H

6 (24a)

CI

127 the chromatin in the target cells. Usually, receptors are located within the nucleus where the chromatin exists (5, 6). For glucocorticoids, however, the receptors are located in the cytoplasm of the target cells, and the receptor complex permeates into the nucleus for the chromatin binding after being activated (5, 6). The specific binding of the receptor complexes with their respective targets results in elevated m-RNA and protein syntheses stimulating target cellular functions and leading to the expression of specific biological activity. There are hormone-antagonists among analogs having similar skeletal structures. For example, epitiostanol (6), an anti-breast cancer agent, and chlormadinone acetate (7), an anti-prostatic cancer drug, are antagonists of estrogen and androgen, respectively. It has been considered that the complex with agonists can bind specifically with chromatin for the time required to promote m-RNA syntheses, but the complex with antagonists can neither regularly bind with the chromatin nor be retained on it for the required time in vivo (4, 6). The threedimensional structure of the antagonist-receptor complex is believed to differ from that of the corresponding agonist-receptor complex (6, 7). The developments of potent steroidal compounds with minimal undesirable side effects have been extensively studied by chemical modifications of natural hormones. Applications of quantitative structure-activity analyses (QSAR) to this field have been making important contributions toward elucidating the physicochemical mechanisms involved in governing the efficacy and potency of steroidhormonal medicines (8). In this article, we first briefly survey past QSAR examples of steroid hormones. We then review our own trials with the use of classical and three-dimensional QSAR procedures to analyze the binding affinities of androgen and estrogen analogs for the androgen and estrogen receptors examined during and after developmental projects of potential antihormonal drugs. 2. PREVIOUS QSAR EXAMPLES FOR STEROID HORMONES As there are comprehensive review articles (8) on previous QSAR examples of steroid hormones, we only descriptively generalize examples from various literature sources.

2.1 Physiological and Pharmacological Processes Permeability (in terms of the log of the permeability constant) of various sets of compounds including a number of steroid hormones through human skin under in vitro (9, 10) and ex vivo (10) conditions has been correlated linearly with the molecular hydrophobicity in terms of log P (P: partition coefficient between organic solvent and water). In these studies, the log P value measured with systems using such organic solvents as diethyl ether (10), n-heptane (9), and 1-octanol (9) are used depending upon the species of the skin samples and the experimental

128

conditions. Absorbability (on the log scale) into rat intestinal lymphatics of testosterone and its ester analogs (8) following oral administration (11) is highly dependent on the log P (n-heptane/water) value. With nandrolone esters (9), the maximum anabolic potency and the time required to exhibit the maximum anabolic effect are related quantitatively with log P (ethyl oleate/water) quadratically and linearly, respectively, in terms of the growth of the levator ani muscles of castrated male albino rats (12). Similar observations are made for the androgenic effect of testosterone esters (8) on the increase in the weight of the capon's comb (13, 14) and the rat seminal vesicle (14). OR

8" R'= CH3, R = H, COR" (R"" C6H13, C10H21, C15H31, CvH14CH=CHC8H17) 9: R'= H, R = COR" (R": C3H 7, C5Hll, C6H13, C7H15, C8H17, C9H19, CloH21)

O

The uterotropic activity of a set of 14-substituted (10) and 14,15-ring condensed 3-methoxyestratrien-1713-ols (11) in terms of the reciprocal of the dose required to double the uterine weight of infantile female mice has been linearly related with the "molecular" hydrophobicity in terms of Zrt (rt: the substituent hydrophobicity parameter in the system of 1-octanol/water) (15). The vasoconstrictory activity in human skin of a set of corticosteroids including prednisolone (2) is also correlated with log P (ether/water) of the molecule and an electronic parameter of the 6~-substituents (10). For the rat liver glycogen deposition

OH CH30~

10

X([3) = NH 2 X(00 = H, OH, NH 2, NHCN

OH

C H 3 0 ~ ~ X X ~ 11 X(13)= CH2,O, NH, NHCH2,NHCO,A~4 X(00= CH2,O,NH, N(CN),N(COOCH3)

CH2OCOCH3

CH3 I

C=O

C=O

O" ~ v

12

X = F, C1, Br, I, OH, H, CH 3

0

•

13

X = CH 3, C1, F, B r, N 3, OCH 3, SCN, CF 3,

CN, OC2H5, H, CHO, OAc, NHAc

129 activity, an indicator of the antiinflammatory activity, of the 9cz-substituted cortisols (12) (16, 17) and for the progestational Clauberg potency of A6-6-substituted progesterone analogs (13) to rabbit uteri (17, 18), the parameters for steric and/or electronic effects of the substituents are required in addition to the hydrophobicity parameter (1-octanol/water) to formulate the QSAR correlation equations. The use of log P values from different solvent systems in the above examples is considered to reflect differences among hydrogen-bonding interactions between compounds and biosystems under the respective experimental conditions (9).

2.2 Receptor Binding Affinity There are a number of QSAR examples for the receptor binding studied using receptor preparations isolated from various animal sources. Wolff and coworkers have analyzed the binding constant (on the log scale unless otherwise noted) of a set of a number of steroids including cortisone (1), prednisolone (2), aldosterone (14), progesterone (15), cortisol (16) and their derivatives for glucocorticoid receptor preparations from rat hepatoma cells (19). Significant determinants of the binding affinity are not only the molecular hydrophobicity, which can be represented by the Bondi molecular surface, but also the stereoelectronic factors, such as the species and position of polar hydrogen-bondable substituents on the molecular skeleton, and the geometric factors, such as the distance between C-3 and C-17 as well as the size of the 9~-substituents.

OH I

CH2OH i

CH3~

CH2OH

HO 0 ~'~',,,~"<; /

14

o

o

c_o

-OH

6

0

,,~ 7

Lee and coworkers (20) have examined the QSAR correlations for the binding constant of a number of 19-norandrost-4-en-3-one (17) derivatives substituted at various positions with progesterone receptor preparations from uteri of humans, sheep, rabbits and guinea pigs originally measured by Kontula and coworkers (21). The hydrophobic effect of the substituents is specific to their position as well as to their configuration on the skeleton, working either to promote or to reduce the affinity depending upon the fit or misfit with the hypothetical hydrophobic pockets on the receptor. The distortions of the molecular conformation from the 4-en-3one structure reduces the affinity according to the number of carbon atoms of which the hybridization state is changed. The lack of the 3-carbonyl group is fatal to the binding affinity. The overall structure-affinity patterns are similar among uterine receptor preparations from various animal species.

130 Bohl and coworkers (22) have carried out QSAR analyses for the binding affinity of the respective sets of a number of progesterone (15) and testosterone (8: R = H, R ' = CH3) derivatives for the progesterone receptor preparation from rabbit uteri. Their results indicate that, the lower the overall three-dimensional dissimilarity of the molecules from a reference compound in each set in terms of the MTD (minimum topological difference) parameter developed by Simon and coworkers (23), and the higher the hydrophobicity of the molecule in terms of the sum of the substituent hydrophobic parameters, the higher is the affinity.

i

; 3

_.-:..:-1

.

2: 18-Deoxyaldosterone(18) 1: Corticosterone (19) 4: Aldosterone (14) 3: 11-Deoxycorticosterone(20) 5: A11_11-Deoxycorticosterone (22) 6:19-Nor-11-deoxycorticosterone (21) Fig.

2.

Side View of Superimposed Aldosterone, Corticosterone and Their Analogs.

We have examined the structure-affinity relationships for the binding of a set of aldosterone (14) and its analog (18) and corticosterone analogs (19-22) for mineralocorticoid receptor preparation from rat kidney cytosol (24). The most significant factor governing the affinity variations is a steric one, the flatness of the molecule, among the compounds tested where no significant variation in electronic structure is assumed to exist. The situation is illustrated in Fig. 2 showing that, with decreasing deviation of the keto-oxygen atom on the A ring from the least squares plane drawn through carbon atoms in the B and C rings, the affinity increases in the order from compound 1 to 6.

CH2OH I C=O

CH20H t C=O

CIH2OH C=O

R O 19: X = OH, R = CH 3 21): X = H, R = CH 3 21: X = H, R=H

0~f~22"

~

131 3. ANALYSES OF THE RECEPTOR BINDING AFFINITY OF ANDROSTANOLS AND ESTRATRIENOLS Duax and coworkers (7) have proposed that the A ring structure of steroids plays a primary role in initiating the receptor binding, whereas the D ring structure participates in controlling the degree of biological response. The male hormone, 1713-testosterone (8: R = H, R'= CH3), and one of the representative female hormones, 17~-estradiol (23), have a common C and D ring structure. Besides affinities for corresponding receptors, they show cross-affinities, although of lower degrees, perhaps indicating antagonistic functions. Thus, in developing potential antagonistic agents of estrogen and androgen, we mainly modified the structure of the A ring of androstanol (24) for the estrogen antagonist and that of estratrienol (25) for the androgen antagonist.

OH HO

OH Xn~2

OH

QCH2CH2-N~

,,.t,,

5

CH30

3.1 QSAR Procedures 3.1.1 Classical Procedure The classical QSAR correlation equation was formulated using conventional software for multiple regression analysis (25). The dependent variable was the logarithm of the affinity index relative to that of a reference compound in the respective series. As the independent variables, various physicochemical molecular and submolecular parameters were used. As the molecular hydrophobicity parameter, we adopted the RM value which was estimated by reversed-phase thin layer chromatography according to Biagi and coworkers (26). The system consisted of a thin-layer plate (Merck 60F-254) impregnated by silicon oil (Toshiba-TSF45-350cs) as the stationary nonpolar phase and various compositions of aqueous dioxane solution as the mobile polar phase. The RM value extrapolated to that for 45% (v/v) aqueous dioxane was taken as the parameter. The experimentally measured 13C chemical shift of the C atom at the 3-position, ~i(3), in CDC13 was adopted for the electronic parameter of androstan-1713-ol (24) analogs because the C-3 atom is the position where most substituent species are bound. The charge density Q(i) on the i-th C atom calculated by the CNDO/2 method (27) was used as the electronic parameter for estratrien-17~-ol (25) analogs.

132 The van der Waals volume in terms of cm3/100, Vw, was used (28) as the steric parameter for substituents in estratrien-1713-01 analogs. Indicator variables were used for the steric features of substituents in androstan-17]3-ol analogs as well as possible hydrogen-bonding effects of the substituents in two series of analogs. These parameters and variables were selected as those of statistical significance which afforded the best results in the analyses. Other parameters were not described because no relevancy was found for them. The quality of the correlation equations was expressed so that n is the number of compounds included, r is the correlation coefficient, s is the standard deviation, F is the ratio of regression and residual variances, and the figures in parentheses after regression coefficients are the 95% confidence intervals. 3 . 1 . 2 Three-Dimensional (CoMFA) P r o c e d u r e Three-dimensional QSAR analysis was performed with the CoMFA (comparative molecular field analysis) option of SYBYL, version 5.41 (29, 30). The initial conformation of compounds such as 23 and 24a, o, p, r, s, t, and u (see Fig. 3) was estimated from the coordinates of the X-ray crystallography (31). Because the structure of steroids is rigid, these X-ray structures were referred to in order to identify the initial conformations for others. The initial conformations were fully optimized by the AM1 method in the MOPAC (version 5) program (32) to obtain the conformer of each molecule. The molecules taking the most stable conformation were placed on a lattice with 2.0 ,~ spaces, being superimposed so that each carbon and other atoms in the C and D rings where the apparent structure is kept unchanged are as close as possible to the corresponding atoms in the reference compound in each series. The steric and electrostatic potential energy fields of each lowest-energy conformer were then calculated at lattice intersections surrounding the entire molecule using an sp3-type carbon with a +1 charge as the probe. For the calculation of the Coulomb electrostatic potential, the atomic charges in each of the molecules were derived from the CNDO/2 calculation (27). The dielectric constant of the medium was taken to be 1.0. The steric interaction potential at the lattice points was calculated using the Lennard-Jones equation (29). The log P value calculated by the CLOGP procedure (33, 34) was introduced as an additional variable whenever relevant. The matrix of the lattice variables was analyzed by the partial least squares method, and these variables were transformed into "latent" ones by linear combinations so as to be orthogonal to each other. The composition of the "latent" variables is so complex that the result of the CoMFA is usually represented by such overall "statistics" as the number of significant latent variables used (component) and the sum of the weights for each of steric, electronic and hydrophobic terms involved in the significant latent variables as well as the correlation qualities in

133 terms of r, s and "press". The "press" is the standard deviation from the leaveone-out cross-validation. More importantly, the substructural or regiospecific physicochemical effects are displayed as contour diagrams of the sum of coefficients in latent variables for each of the field descriptor terms at every lattice intersection to show favorable and unfavorable potential regions. 3.2 5ct-Androstan-17j3-ol Analogs 3 . 2 . 1 Binding Affinity for the Estrogen Receptor Preparation In order to develop an anti-breast cancer agent which functions as an estrogen antagonist, a number of 5ct-androstan-17[3-ol (24) derivatives, in which the A and B ring structures were variously modified, were synthesized at the Shionogi Research Laboratories (35) or were purchased commercially; their chemical structures are shown in Fig. 3. The binding affinity for the estrogen receptor preparation from the JCL-SD rat uteri was measured. In Table 1, the affinity is listed as log RA(E), the logarithm of the affinity index relative to that of nafoxidine (26) as 100. The affinity of nafoxidine, an antiestrogen, for the rat uterine receptor is about 1/10 that of endogenous estrogen, estradiol (23) (36).

X

24a: S 24b: 24c: 24d: 24i: 24j: 24k: 24 1:

R 1 R2 R 3

H

O H NH H CH 2 H S Me S H S H O H

X

24f: S

240: =O

H H H H H H H H MeH H Me Me H

24g: O 24h: NH

24s: 24t: 24v: 24w:

o~-OH ~3-OH ct-OMe [3-OMe

24m

24e

24p

X

H H

24q

24r

24u

Fig. 3. 5ct-Androstan- 1713-ol (24) Derivatives.

134 T a b l e 1. Relative B i n d i n g Affinity Indices of Androstan-1713-ol A n a l o g s for the E s t r o g e n [RA(E)] and the A n d r o g e n R e c e p t o r s [RA(A)] and Q S A R Parameters log RA(E) Compd. 24a 24b 24c 24d 24e 241' 24g 24h 24i 24j 24k 241 24m 24n 240 24p 24q 24r 24s 24t 24u 24v 24w

Obs.

log RA(A)

Calcd. Calcd. Obs. Eq. 1 CoMFA Eq. 2 CoMFA

1.11 0.570 0.407 0.11 0.831 0.002 -1.05 a 0.583 -0.635 0.78 0.447 0.726 0.00 -0.195 0.122 -0.30 -0.443 -0.521 -1.28 -1.762 -1.001 -0.78 -0.301 -1.091 0.05 0.358 0.422 -0.60 -0.551 -0.178 -0.63 -0.630 -0.705 -0.54 -0.327 -0.585 0.68 0.647 0.609 -0.30 -0.416 -0.256 -0.13-0.001-0.342 -1.50 -1.260 ___c -1.04 -1.342 -0.769 0.53 0.400 ___c -0.20 a 0.884 -0.098 1.16 0.986 ___c 1.25 1.014 ___c -1.70 a 0.560 ___c -1.70 -1.157 -1.928

1.56 1.304 1.36 1.378 -0.30 b 1.258 0.48 1.159 1.24 1.761 -0.52 -0.655 0.78 -0.571 -1.22 -0.691 1.23 1.306 1.64 1.352 -1.05 -0.586 2.23 1.418 1.10 1.306 -0.40 -0.664 2.00 2.260 1.97 2.144 2.73 2.151 0.70 b 2.078 -1.40 b 1.455 -0.70-0.453 -0.70 -0.467 -1.30 b 1.502 -0.70 -0.424

RM

1.012 0.74 1.439 0.13 0.584 0.81 0.281 1.25 0.806 1.17 -0.563 0.71 0.437 0.17 -0.865 0.53 1.371 1.11 1.873 0.78 -0.736 0.90 2.007 0.25 0.981 0.60 -0.152 0.68 2.001-0.18 ___c -0.42 2.837 -0.29 ___c -0.52 -1.720 -0.12 ___c -0.38 ___c -0.40 ___c 0.36 -0.832 0.65

5(3) IS(2,3) IS(3) 37.6 51.7 28.7 9.6 125.6 35.2 51.5 28.4 38.0 46.7 48.5 59.4 38.0 33.5 221.7 199.3 200.7 186.5 66.7 74.2 71.5 75.6 79.8

0 0 0 0 0 1 1 1 0 1 1 1 0 1 0 0 0 0 0 0a 0a 0 0'/

Io(3)

0 0 0 0 0 1 1 1 0 0 1 0 0 1 0 0 0 0 0 1 1 0 1

a Not included in the formulation of Eq. 1. b Not included in the formulation of Eq. 2. c Not calculated because they were not included in the CoMFA calculation, d As the oxygen atom of the 313-OH group is almost coplanar with the plane through C-2,-4 and -10, IS(2,3) was taken to be 0. T h e classical Q S A R was p e r f o r m e d with v a r i o u s c o m b i n a t i o n s of p h y s i c o c h e m i c a l p a r a m e t e r s a m o n g w h i c h those s h o w n in Eq. 1 w e r e s e l e c t e d for 20 c o m p o u n d s w i t h o u t including three outliers (24e, s and v). log R A ( E ) = - 0.57(+0.42)RM - 0.59(+0.41)[5(3)/100] - 1.04(+0.42)Is(2,3) - 1.53(+0.50)Io(3) + 1.21(+0.53) n=20,

r=0.924,

s=0.380,

[11 F(4,15)=21.8

In Eq. 1, Is(2,3) is a steric indicator variable taking a value of unity w h e n one of the 2[3 and 313 positions is substituted. Io(3) is another indicator variable for the

135 3-keto and 313-ether (but not alcoholic) oxygen located close by the least squares plane of skeletal carbons. In general, the 13C chemical shift of a certain C atom in a molecule is approximately proportional to the positive charge density on its C atom (37). Thus, the negative 8(3) and RM terms in Eq. 1 suggest that the higher the electron density at the C-3 and its close vicinity in the molecule, the higher is the affinity for the receptor in a rather hydrophilic milieu. The negative Is(2,3) term could mean that the A ring moiety of the molecules has access to and recognizes the receptor from the ]3-face, with the 213- and 313-substituents interfering with the close contact. The negative lo(3) term could indicate that the 3-keto or 313-methoxy oxygen undergoes a hydrogen-bonding interaction by which the proper receptor binding is severely distorted. The possible hydrogen donors may exist on an edge of the binding domain of the receptor. The fact that the 213,313-NH (24h) and 313-OH (24t and u) compounds are not outliers suggests that the proper binding patterns of the amphiprotic 3[3-substituents are not distorted much since possible hydrogen-bond acceptors could counterbalance the unfavorable interaction with the hydrogen donors. The three outliers are the 2o~,3c~-NH (24c), the 3c~-OH (24s) and the 3c~-OCH3 (24v) compounds. In the first two, there is a hydrogen-bondable hydrogen in the (x-configuration, while, in the last, there is a bulky methoxy group on the o~-face. Equation 1 apparently does not properly describe the stereoelectronic effect of the axially suspended 3o~-substituents. The three-dimensional QSAR analysis with the CoMFA procedure for 18 androstan-1713-ol analogs gave an acceptable result after omitting five outliers (24p, r, t, u and v) from the total set. The statistical data are shown in Table 2 and the steric and electronic potential maps are in Figs. 4a and 4b, respectively, with epitiostanol (24a) inserted. The contour maps for the steric field show that 213 and 313 regions are sterically forbidden, whereas 2o~, 3o~ and 4c~ regions are permissible to enhance the activity. The electrostatic field maps indicate that 213, 313 and proximate 3o~ regions are occupied by the electropositive potential field where the more positive electrostatic interaction potentiates the binding affinity, whereas 2o~ and distal 3o~ spaces accommodate the electronegative potential field Table 2. CoMFA Statistical Data for the Receptor Binding of Androstan-171g-ols Receptors

na

Estrogen Androgen

18 18

ra

sa

Fa

0.918 0.332 17.3 0.963 0.391 41.8

Nb 4 4

Contribution (%) Excluded compounds STc ELd Hpe 4 5 . 3 4 0 . 8 13.9 24p, r, t, u, v 3 4 . 5 65.5 --f 24p, r, t, u, v

a See 3.1.1 for the classical procedure, b Number of components, c Contribution of the steric term. d Contribution of the electrostatic term. e Contribution of the hydrophobic term. f Insignificant.

136

,'

i -"

[1 I1

I!1

.:.75_[S~_

~ .........

(a)

:

-..,'

....

(b)

Fig. 4. CoMFA Contour Maps for the Affinity of Androstan-17[3-ols for the Estrogen Receptor with Epitiostanol (24a) Inserted. (a) Steric field: the contours were drawn at the 0.01 level. The yellow and green polyhedra are the forbidden and permissible regions, respectively. (b) Electrostatic field: the contours were drawn at the 0.01 level. The red and blue polyhedra are electropositive and electronegative regions, respectively. The suffices 1, 2 and 3 for subfigures a and b indicate the front, side and top views, respectively.

137 where the more negative electrostatic interaction enhances the receptor binding. In other words, the [3-face at the 2- and 3-positions, being charged positively, interacts with negative charges developed on the corresponding receptor region and the negatively charged substituents at 2o~- and 3cx-positions are attracted by the positively charged domain of the receptor. In addition, the higher hydrophobicity promotes the binding. The sterically forbidden 213 and 313 regions appearing in Fig. 4a correspond well with the negative term of the steric indicator variable, Is(2,3), in Eq. 1. The sterically admissible 2oc, 3c~ and 4a regions shown in Fig. 4a are understandable because many firmly binding compounds with Is(2,3) = 0 such as 24a, b, d and m have substituents at the 2oc and 3o~ positions. The electropositive field appearing in Fig. 4b is in accord with the situation represented by Eq. 1. The more electronegative the C-3 atom, or the more greatly attracted the negative charge by the C-3 corresponding to the negative 8(3) term, the greater the electropositive character of the neighboring atoms and skeletons. Thus, the electropositive space shows up in the 3o~ and 313 regions. The positive space above the 213,313 face is also due to the electron withdrawing inductive effect of the most electronegative atom in the 2o~,3c~ three-membered ring head located below the molecular plane in strongly binding compounds such as 24a, b, d, i and m. The most electronegative atom in these compounds is located in the electronegative 2oc,3c~ region. In the 213,313 three-membered ring compounds such as 24f, g and h, the most electronegative heteroatom is above the molecular plane and located in the electropositive space to lower the affinity. The five outliers (24p, r, t, u and v) were omitted from the analysis. Three compounds (24p, r and u) have no sp3 carbon at the 5-position. Two (24t and u) have the 313-OH group equatorial to the A ring. In compounds 24r and v, the A ring moiety including the 3-carbonyl and the axial 3c~-methoxy groups deviates

CH3

." I

0

t

/

9

/

,// /

CH3

9

/ ,/

l;/

O

S.io,l , ,,,,"

,,,,"

CH3""

9

,/

2 4 o : ~

24a: 24r:

.....

24v:

24p:-

-

-

.......

0/

Fig. 5. Side View of Superimposed Androstan-1713-ol Analogs.

138 largely from the least squares plane for the carbon skeleton of the B, C and D tings as shown in Fig. 5. The differences in the geometry and/or shape from others and specific types of hydrogen-bonding interaction may make these five compounds outliers. The outliers from the CoMFA procedure do not coincide with those from the classical QSAR. A specific amphiprotic hydrogen-bonding effect was considered in compounds 24t and u for their regular behavior in Eq. 1, whereas a "similar" hydrogen-bonding effect was attributed to their outlying behavior in CoMFA. Thus, although the behaviors of outliers could be "rationalized" as far as each of the procedures was considered separately, factors relating to the "hydrogenbonding effects" and geometries of 3~- and 3[3-substituents remain to be solved in the future. The CoMFA result indicating that the higher hydrophobicity is favorable to the binding affinity seems inconsistent with the negative RM term in Eq. 1. This is perhaps due to the deletion of highly active hydrophilic compounds such as 24r, t and u as outliers in the CoMFA.

3.2.2 Binding Affinity for Androgen Receptor Preparation The androstanols (24) as potential anti-breast cancer agents to be mainly administered to females should not exert androgenic effects. Understanding the structure-activity pattern for androgenic effects is important to compare it with that for anti-estrogenic effects and to separate the androgenicity from the antiestrogenicity as much as possible. For the same set of androstan-17[3-ols in Fig. 3, their binding affinity for the androgen receptor preparation from ventral prostates of JCL-SD male rats was measured and listed in Table 1 as log RA(A), the logarithm of the affinity index relative to that of dihydrotestosterone (240) as 100. The affinities of 3-keto compounds, 240, p and q, were high in accord with the suggestions of Cunningham (38), Kirchhoff (39) and their respective coworkers that the 3-keto function has an essential role in the binding process along with the 17[3-hydroxy group in androgens with their receptor. Classical QSAR was carried out using (sub)molecular parameters similar to those used to derive Eq. 1. For 19 compounds, not including four outliers (24c, r, s and v), Eq. 2 was formulated. log RA(A)= 0.52(+0.45)[8(3)/100]- 1.95(_+0.56)Is(3) + 1.11(+0.54) n = 1 9 , r = 0 . 9 1 4 , s=0.538, F(2,16)=40.3

[2]

In Eq. 2, Is(3) is an indicator variable similar to Is(2,3) in Eq. 1, but takes a value of unity when only the 313-position is substituted. A high positive charge density of the C-3 and the lack of the 313 substituent promote the binding affinity. The hydrophobicity term is insignificant in the androgen receptor binding. Similar to the estrogen receptor binding, the A ring moiety of the molecules recognizes the

139 receptor from the l-face. The three outliers 24c, s and v are the same as those for the estrogen receptor binding. The hydrogen-bonding capability and the bulk downward from the molecular plane also seem to be detrimental in this case. Another outlier is the compound 24r. As shown in Fig. 5, the A ring with the 3-carbonyl group of this compound deviates greatly from the molecular plane, as the 3c~-methoxy group in compound 24v. A good result was obtained by CoMFA using steric and electrostatic field parameters as shown in Table 2 with the same five outliers as in the preceding section. The steric and electrostatic field maps are in Figs. 6a and 6b, respectively, with epitiostanol (24a) inserted. Figure 6a clearly indicates that the 213 region is sterically permissible whereas the 313 and 4c~ regions are forbidden. The c~-face of the 2- and 3-positions is permissible only in the vicinity of the ring plane. Figure 6b shows that the 213,313 and 4c~ regions are electropositive, whereas the l c~ and 2a regions are electronegative. In the 3c~ space, the area in the vicinity of the molecule is electropositive but that at a distance is electronegative. The steric potential patterns in Fig. 6a are not inconsistent with the steric parameter Is(3) term in Eq. 2 indicating that the 313 substitution is definitely unfavorable. The high affinity of compounds 24j and 1 having the 213-CH3 substituent is reflected by the sterically admissible 213 region in Fig. 6a. The 3c~OCH3 group in compound 24v and the 3-keto oxygen atom in compound 24r protrude beyond the sterically permissible 3c~ region. Thus, Fig. 6a well rationalizes the outlying behavior of compounds 24r and v in Eq. 2. The electropositive networks found at the 3c~ and 313 regions close to the C-3 atom correspond to the positive 8(3) term in Eq. 2. The electrostatic potential pattern in the 3~ space corresponds to the fact that the 3-carbonyl group is polarized so that the 3-keto carbon is positively charged but the oxo-oxygen is negatively charged in the strongly binding 3-keto compounds such as 240, p and q. The electronegative S and O atoms in the 2c~,3c~-three membered ring in firmly binding compounds such as 24a, b, i, j, l a n d m are accommodated in the electronegative 2c~,3c~-space.

3.3 Estratrien-17[~-ol Derivatives 3.3.1 Binding Affinity for Androgen Receptor Preparation Estradiol (23), an endogenous estrogen, binds to the androgen receptors, although the affinity is low, being about 1/40 that of dihydrotestosterone (24o), an endogenous androgen. The estradiol analogs could be antiandrogens, some of them hopefully working as an anti-prostatic cancer agent. Thus, thirteen estratrien-17[3ol derivatives (25), in which various substituents were introduced into the A ring, were synthesized at Shionogi Research Laboratories. Their affinity indices for the androgen receptor preparation from the JCL-SD rat prostates relative to that of

140

X

/

<"-)--a ~%,")~'~? ',,4..-~'~ ,

QI

II~",

,"ii~ ~: :-"~:~ ." :>

....

i

,

_.

.

.

.-

,.?.--

-

.'

.

i

.

I~

-.-

,'

2 _

(a)

,.-

<

(b)

Fig. 6. CoMFA Contour Maps for the Affinity of Androstan-1713-ols for the Androgen Receptor with Epitiostanol (24a) Inserted. Refer to Fig. 4 for subfigures, the color codes of polyhedra, and the contour levels.

141

dihydrotestosterone (24o) as 100 were measured and listed as log RA(A) in Table 3. Classical QSAR analysis gave Eq. 3 as the best among various combinations of substituent and structural parameters. log RA(A)= -3.84(+1.64)Vw(3)- 6.58(+4.98)Q(2,3) + 1.23(+0.52)Ii~(3) + 0.48(+0.60) n=ll, r=0.949, s = 0 . 2 7 , F(3,7)=21.2

[3]

In Eq. 3, Vw(3) is the value for substituents at the 3-position and Q(2,3) is the sum of the charge density on the 2- and 3-carbon atoms. IH(3) is an indicator variable for the hydrogen-donating substituents at the 3-position. The 3-NHCH3 (25e) and 3-N(CH3)2 (25h) compounds were not used to formulate Eq. 3. The affinity of compound 25e was much lower than that expected from Eq. 3 and that of compound 25h was too low to be measured. Equation 3 indicates that introduction of a

11 .... L7

I~i .... <,

";~ ,

/

< i/ - j

\

i/

(a)

(b)

Fig. 7. CoMFA Contour Maps for the Affinity of Estratrien-1713-ols for the Androgen Receptor with Estradiol (25a) Inserted. Refer to Fig. 4 for subfigures, the color codes of polyhedra, and contour levels.

142 bulky substituent into the 3-position and the increase in the charge density on the C-2 and C-3 atoms are unfavorable to the affinity.

The h y d r o p h o b i c i t y of

substituents is not significant in governing the affinity variations. The hydrogendonor ability of the 3-substituents potentiates the affinity about 20 times. The CoMFA procedure gave Figs. 7a and b with estradiol (25a) inserted along with statistical values in Table 4. Figure 7a indicates that there is a considerably large space, which is sterically forbidden, covering not only the region corresponding to substituents at the 3-position but also a part of the regions into which the end of the substituents at the 2- and 4-positions could be projected. At the 3position, the forbidden region develops outward from the vicinity of the c~-atom of substituents. There is a sterically permissible patch in the region occupied by the 2substituents. Figure 7b shows that the region corresponding to the 3-substituents is definitely electropositive, but regions accommodating 2- and 4-substituents are partly positive and partly negative. Table 3. Relative Binding Affinity Indices of Estratrien-1713-ols (25) for the Androgen IRA(A)] and the Estrogen Receptors [RA(E)] and QSAR Parameters No. 25a 25 b 25c 25d 25e 25f 25g 25h 25i 25j 25k 251 25m

Substituent Xn

Obs.

log RA(A) Calcd.

Obs.

log RA(E) Calcd.

Eq. 3 CoMFA 0.415 0.328 0.034 2.00 0.093 0.256 0.174 1.48 -0.333 0.177-0.197 0.85 0.501 0.477 0.240 0.60 -1.187a-0.118 -0.990 0.48 0.205 0.202 0.352 0.30 -1.736-1.533-1.500 -0.22 .... a -1.967 .... b -0.52 -0.877 -1.079 -0.468 -0.70 3-C1 3-NHCOCH3 -0.831 -0.862-1.203 -1.22 -0.800 -0.798 -1.080 -2.00 3-NO2 3-OH, 2-NO2 0.406 0.151 0.419-3.00 c 3-OH, 2,4-DNe 0.222 -0.053 0.297 .... c

3-OH 3-OH, 4-C1 3-OH, 4-NO2 3-NH2 3-NHCH3 3-H 3-OCH3 3-N(CH3)2

Eq. 4 1.460 1.210 0.761 1.282 0.205 0.476 0.348 -0.872 -1.231 -1.012 -1.577 0.961 -0.487

Q(1,2) Q(2,3) Vw(3) IH(3)

CoMFA 0.580 -0.033 1.146 -0.028 1.256 -0.019 1.004 -0.035 0.404 -0.036 0.722-0.002 -0.015 -0.034 -0.418 -0.037 -0.276 0.006 -1.361 -0.036 -1.993 0.010 .... d -0.023 .... d 0.006

0.129 0.140 0.152 0.083 0.079 0.009 0.128 0.079 0.094 0.090 0.039 0.156 0.187

0.137 0.137 0.137 0.177 0.339 0.056 0.304 0.501 0.244 0.514 0.265 0.137 0.137

1 1 1 1 1 0 0 0 0 1 0 1 1

a Not included in Eq. 3. b Not calculable, c Not included in Eq. 4 and the CoMFA correlation. d Not calculated, e DN: (NO2)2. T a b l e 4. C o M F A Statistical Data for the Receptor Binding of Estratrien-1713-ols a Receptors

n

r

s

F

N

Contribution (%) ST EL

Androgen Estrogen

12 11

0.936 0.889

0.305 0.601

19.0 15.1

3 2

40.2 26.9

a See notes of Table 2.

59.8 73.1

Excluded compounds 25h 25 !, m

143 Among the set of compounds, 1-, 2- and 4-positions were not modified much. The data in Table 3 indicate that the effect of electron-withdrawing 4-C1 and -NO2 groups is to reduce the affinity, whereas that of 2-NO2 is to maintain or promote the affinity. Because the electrostatic field accommodating the 2- and 4substituents could be "neutral" as described above, the difference in the effect between the 2- and 4-substituents is suggested to be mainly due to the difference in the steric field as shown in Fig. 7a. The electropositive network holding 2- and 3substituents in Fig. 7b appears because the periphery of 2- and 3-substituents is electropositive. The electrons are delocalized from the 3-OH (25a) and 3-NH2 (25d) groups into the aromatic A ring and concentrated on the C-2 and C-3 atoms, being consistent with the negative Q(2,3) term in Eq. 3. The log RA(A) value for compounds having electron-donating substituents at the 3-position is either positive or negative depending upon substituent length. While short substituents such as OH and NH2 are favorable to the affinity, "lengthy" substituents such as NHCH3 (in 25e), OCH3 (in 25g) and NHCOCH3 (in 25j) lower the affinity because their end trespasses the sterically forbidden region. Of course, the electron-attracting substituents such as NO2 (in 25k) and C1 (in 25i) at the 3-position lower the affinity. The unmeasurably low affinity of the 3N(CH3)2 compound (25h) is probably due to the bulk of two methyl groups bound to the nitrogen. 3.3.2 Binding Affinity for the Estrogen Receptor Preparation The importance of the A ring of estradiol analogs in the binding with the estrogen receptor has been shown by Brooks and coworkers (40). Estratrien-3-ol has 40% of the affinity of estradiol, whereas estratrien-17~-ol shows only 8%. This does not mean that the D ring structure is not important because the "naked" estratriene retains almost no affinity. As a potential antiandrogenic agent to be administered to males, the estratrienol derivatives should not exert estrogenic activity. The structure-affinity relationship of the same set of estratrienol analogs (25) as in the preceding section for the estrogen receptor was analyzed next. The binding assay of the estratrien-17~ ols (25) was done with the estrogenic receptor preparation from the JCL-SD rat uteri. Classical QSAR was formulated to give Eq. 4 as the best. log RA(E) = -6.95(+2.56)Vw(3)- 49.93(+21.12)Q(1,2) + 0.77(+0.73) n=ll, r=0.929, s=0.49, F(2,8)=25.1

[4]

As in Eq. 1, RA(E) is the relative affinity index, but the reference compound in this case was not nafoxidine (26) but estradiol (23). Equation 4 is similar to Eq. 3 in that the bulk of the 3-substituents and the electron-poor A ring carbon atoms are unfavorable to the affinity. The 3-OH,2-NO2 (251) and 3-OH,2,4-(NO2)2 (25m)

144

compounds were not included in the analysis because their affinities were too low. Figures 8a and 8b display the CoMFA results with accommodation of estradiol (25a); the CoMFA statistical values are given in Table 4. The two outliers omitted were the same as those omitted from Eq. 4. In Fig. 8a, the region covering the 3substituent position is sterically forbidden, but the forbidden region is farther from the (x-atom of the substituent than that observed in the affinity for the androgen receptor in Fig. 7a. The 4-substituent position is permissible, reflecting the affinity of compounds 25b (3-OH,4-C1) and e (3-OH,4-NO2). The effect of the 4-C1 and 4NO2 substituents appears to reduce the affinity of the 3-OH compound (25a). However, it could not be serious, perhaps working promotively under conditions without the 3-OH group. This is supported by Fig. 8b showing that the 4-substituent region is surrounded by the electronegative potential contour being

,.

:

) ~9 :

/; , /

(a)

(b)

Fig. 8. CoMFA Contour Maps for the Affinity of Estratrien-1713-ols for the Estrogen Receptor with Estradiol (25a) Inserted. Refer to Fig. 4 for subfigures, color codes of polyhedra, and contour levels.

145 capable of accommodating electron-withdrawing substituents such as C1 and NO2. The effect of the 2-NO2 group is to greatly lower the affinity. The 2-substituent position is covered by the outskirts of sterically forbidden and electropositive regions. Thus, the very low activity of the two outliers could be justified. The periphery of the 2- and 3-substituents is electron-deficient, being covered by the electropositive network. Electrons in substituents at these positions are withdrawn by and concentrated on the ring carbon atoms similar to the case for the androgen receptor binding. In Eq. 4, however, the sum of the negative charge at the C-1 and C-2 atoms is best to rationalize the affinity variations. There should be some collinearity between Q(1,2) and Q(2,3), even though the Q(1,2) value gave a better correlation as far as the classical QSAR is concerned. 4. DISCUSSION AND C O N C L U D I N G R E M A R K S Steroid hormones and their analogs can express their biological activity only through binding with receptors. Agonistic binding leads to the receptor complex which undergoes normal interaction with target sites, whereas antagonistic binding produces the antagonist-receptor complex which competes with the normal agonistcomplex. Often, antagonists are needed to suppress the excessive target interaction of the normal agonist-receptor complex which may induce pathogenic conditions in animals. Our trials to develop anti-estrogens to treat estrogen-dependent breast cancer led to the marketing of epitiostanol (24a) in 1977, but work on anti-androgens unfortunately had to be terminated. As described in Section 3.2.1 with Figs. 4 and 5, the electronegative S atom at the head of the three-membered ring almost perfectly fits into the sterically permissible and electronegative region of the hypothetical receptor cavity. The binding affinity of this compound for the estrogen receptor is among the highest in the set of androstanols synthesized and tested as shown in Table 1. Although compounds 24t (androstanediol) and 24u (androstenediol) indicate affinities similar to that of epitiostanol, they may exhibit a potent anabolic effect (41) which is undesirable for clinical drugs. In spite of a moderate binding affinity for androgen receptor as also shown in Table 1, that of epitiostanol is not as high as the typical androgens such as compounds 24o (dihydrotestosterone), p (testosterone) and q. Because not many androgenresponsive organs exist in the female body, the androgenicity of epitiostanol could be expected to not exhibit serious undesirable side effects (42). Of course, the pharmacological, pharmacokinetic and toxicological behaviors of epitiostanol have been shown to be either superior or equivalent to those of hither-to-known antibreast cancer agents such as testosterone propionate (8: R'= CH3, R = COCHzCH3) and fluoxymesterone (27) (42, 43). The selection of epitiostanol was justified not only by the QSAR analyses but also by extensive biological examinations.

146 OH

0

Ha

2?

As previous QSAR results indicate, variations in the efficacy and potency of steroid hormonal drugs are mostly governed by the molecular hydrophobicity in the respective families if transport processes such as absorption into and distribution within the body are critical. When the binding process with receptors is ratelimiting, factors other than the hydrophobicity appear in the QSAR formulations. Regiospecific hydrophobic and stereoelectronic effects of substituents and substructures as well as the entire or partial geometry of the molecule are generally significant in determining the affinity variations in individual sets of steroids. The wider the range of structural modifications spatially as well as physicochemically, the more precisely the (sub)molecular requirements for the affinity could be analyzed. In the present analyses, two sets of compounds were those in which the structural modifications were mostly within the A ring structure. Therefore, information about the (stereo)physicochemical requirements for the binding affinity for the receptors was limited. Nevertheless, we believe that the information is invaluable. Classical QSAR results are more readily accepted by ordinary (physical) organic chemists than CoMFA procedures because of the type of information being represented by physicochemical parameter terms. The CoMFA procedure is certainly a sophisticated QSAR variation, but the results are interpreted rather qualitatively on the basis of visualized display of the physicochemical field diagrams. They are supposed to yield a greater amount of information than the classical procedure, but their interpretation is not always so simple. However, information not explicitly foreseeable from the physicochemical parameter terms in the classical QSAR can be often gained with the CoMFA procedure. Thus, structure-affinity relationships were discussed complementarily in the above sections with results from the two procedures. The "contents" involved in the CoMFA field diagrams could be nicely illustrated with the aid of significant parameter terms appearing in classical QSAR formulations. The meaning of the classical QSAR formulations was reinforced by the CoMFA procedure. In fact, the CoMFA procedure was first applied to steroids by Cramer and coworkers who analyzed the affinity of 21 steroids for corticosteroid-binding and testosterone-binding globulins (29). Loughney and Schwender recently published CoMFA results for the binding affinity of a variety of steroids for androgen and progesterone receptors (44). There has been no publication, however, which deals

147 with comparative combinations with the classical procedure in steroid studies. For the receptor-binding of steroid hormones, the hydrogen-bonding interactions of polar substituents on the skeleton have been suggested to be important (7). In Section 3.2.1, we used an indicator variable for the hydrogen-bonding effect of the 3]3-methoxy- and 3-keto groups of androstanols in the classical QSAR (Eq. 1). This is not necessarily in accord with the CoMFA result in which we had to delete quite a few compounds having substituents of the same type as outliers. If the hydrogen-bonding interactions can be properly treated with the CoMFA procedure, this type of discrepancy between the two procedures could be eliminated and the complementarity would become more complete. In the present study, the number of estratrienols included in the analysis (n = 13) and the extent of their structural variety were lower than those for androstanols (n = 23). Thus, the direct comparison as well as generalization among Eqs. 1-4 would not be relevant. It could be said, however, that the affinity variations in these two series of compounds are mostly governed by electrostatic and steric factors of the A ring. Variations in the molecular hydrophobicity are of minor significance, if any. Hydrogen-bonding interaction factors are definitely important especially for the antagonistic binding with the receptors as shown in Eqs. 1 and 3. Among classical and CoMFA QSAR analyses, those for antagonistic binding of androstanols with estrogen receptor (Eq. 1, Table 2 - Estrogen) were most complicated. The correspondence between the two procedures, especially the rationalizations of the outliers, was not as good as others. Because the A ring of the native estrogen, estradiol, is aromatic and "flat", the binding region in the receptor accommodating the A ring could be much narrower than the "thickness" of the alicyclic A ring of androstanols. Conformational change of the binding region in the estrogen receptor would be greater to accommodate the androstanols perhaps leading to more complicated QSAR patterns. In conclusion, comparative as well as complementary examinations of QSAR patterns from classical and three-dimensional procedures should contribute to better understanding of the physicochemical mechanisms of bioactive molecules as well as to optimizing the lead structure in terms of decision-making as to when to interrupt a project and/or how to continue further effort. Because of the conformational rigidity of the steroidal skeleton, the CoMFA procedure in which the three-dimensional structural superimposition of a set of molecules is indispensable could be much more easily applied to the present type of compounds than to structurally flexible compounds. Accumulation of results from comparative studies of this type should be useful for drug development.

148

ACKNOWLEDGEMENTS Dr. Miki Akamatsu of Kyoto University is greatly acknowledged for her assistance in running the CoMFA software. We are grateful to Professor Tanekazu Kubota in Inazawa Women's Junior College and Dr. Takashi Hori in our laboratories for their helpful discussions, and also to Miss Michiko Katayama of our laboratories for the preparation of the manuscript.

REFERENCES 1) N. Applezweig, Steroid Drugs, McGraw-Hill, New York, 1962. 2) P. S. Hench, E. C. Kendall, C. H. Slocumb and H. F. Polley, Arch. Internal Med., 85, 545 (1950).

3) (a) M.J. Green and B. N. Lutsky, Annu. Rep. Med. Chem., 11,149

(1976). (b) M. K. Agarwal (Ed.), Antihormones, Elsevier, Amsterdam, 1979.

4) J. H. Clark and J. W. Hardin, Res. Reproduction, 9, 2 (1977). 5) (a) R. M. Evans, Science, 240,889 (1988). (b) B.W. O'Malley, S. Y. Tsai, M. Bagchi, N. L. Weigel, W. T. Schrader and M.-J. Tsai, Recent Prog. Horm. Res., 47, 1 (1991).

6) F. J. Zeelen, Adv. Drug Res., 22, 149 (1992). 7) W. L. Duax, J. F. Griffin, C. M. Weeks and Z. Wawrzak, J. Steroid Biochem., 31,481 (1988). 8) (a) M. Yamakawa, Kagaku no Ryoiki, Zokan, 136, 95 (1982). (b) M.E. Wolff, in: J. G. Topliss (Ed.), Quantitative Structure- Activity Relationships of Drugs, Chapter 9, Academic Press, New York, 1983, pp. 351-392. (c) F. J. Zeelen, Quant. Struct.-Act. Relat., 5, 131 (1986). (d) M. Bohl, in: M. Bohl and W. L. Duax (Eds.), Molecular Structure and Biological Activity of Steroids, Chapter 3, Boca Raton CRC Press, 1992, pp. 91-155.

9) N. E. Tayar, R.-S. Tsai, B. Testa, P.-A. Carrupt, C. Hansch and A. Leo, J. Pharm. Sci., 80, 744 (1991). 10) E. J. Lien and G. L. Tong, J. Soc. Cosmet. Chem., 24, 371 (1973). 11) T. Noguchi, W. N. A. Charman and V. J. Stella, Int. J. Pharm., 24, 173 (1985). 12) M. A. Q. Chaudry and K. C. James, J. Med. Chem., 17, 157 (1974). 13) G. L. Biagi, A. M. Barbaro and M. C. Guerra, Experientia, 27, 918 (1971). 14) K. C. James, Experientia, 28, 479 (1972). 15) M. Bohl, G. Schubert, M. Koch, G. Reck, J. Strecke, M. Wunderwald, R. Prousa and K. Ponsold, J. Steroid Biochem., 26, 589 (1987).

149 16) (a) M. E. Wolff and C. Hansch, Experientia, 29, 1111 (1973). (b) M.E. Wolff, C. Hansch, P. A. Kollman, D. G. Giannini and W. L. Duax, Experientia, Suppl. 23, 31 (1976). 17) (a) M. E. Wolff, C. Hansch, D. D. Giannini, P. A. Kollman, W. L. Duax and J. Baxter, J. Steroid Biochem., 6, 211 (1975). (b) R. A. Coburn and A. J. Solo, J. Med. Chem., 19, 748 (1976). 18) (a) M. E. Wolff and C. Hansch, J. Med. Chem, 17, 898 (1974). (b) J.G. Topliss and E. L. Shapiro, J. Med. Chem., 18, 621 (1975). 19) (a) M. E. Wolff, J. D. Baxter, P. A. Kollman, D. L. Lee, I. D. Kuntz, E. Bloom, D. T. Matulich and J. Morris, Biochemistry, 17, 3201 (1978). (b) M. E. Wolff, Monogr. Endocrinol., 12, 97 (1979). 20) D. L. Lee, P. A. Kollman, F. J. Marsh and M. E. Wolff, J. Med. Chem., 20, 1139 (1977). 21) K. Kontula, O. J/~nne, R. Vihko, E. de Jager, J. de Visser and F. Zeelen, Acta Endocrinol., 78,574 (1975). 22) (a) M. Bohl, Z. Simon, A. Vlad, G. Kaufmann and K. Ponsold, Z. Naturforsch., 42e, 935 (1987). (b) Z. Simon and M. Bohl, Quant. Struct.Act. Relat., 11, 23 (1992). 23) Z. Simon, A. Chiriac, S. Holban, D. Ciubotam, G. I. Mihalas, Minimum Steric Difference. The MTD Method for QSAR Studies, John Wiley and Sons, New York, 1984. 24) M. Yamakawa, K. Ezumi, M. Shiro, H. Nakai, S. Kamata, T. Matsui and N. Haga, Mol. Pharmacol., 30, 585 (1986). 25) B. F. Ryan, B. L. Joiner and T. A. Ryan, Jr., Minitab Handbook, 2nd Ed., Duxbury Press, Boston, 1985. 26) G. L. B iagi, M. C. Guerra and A. M. Barbaro, J. Med. Chem., 13,944 (1970). 27) J. A. Pople and D. L. Beveridge, Approximate Molecular Orbital Theory, McGraw-Hill, New York, 1970. 28) (a) I. Moriguchi, Y. Kanada and K. Komatsu, Chem. Pharm. Bull., 24, 1799 (1976). (b) I. Moriguchi and Y. Kanada, Chem. Pharm. Bull., 25, 926 (1977). 29) R. D. Cramer, III, D. E. Patterson and J. D. Bunce, J. Am. Chem. Soc., 110,5959 (1988). 30) Tripos Associates, Inc., St. Louis. 31) (a) Cambridge Crystallographic Data Centre, University Chemical Laboratory, Cambridge. (b) W. L. Duax and D. A. Norton (Eds.), Atlas of Steroid Structure, Vol. 1, IFI/Plenum Press, New York, 1975. 32) J. J. P. Stewart, J. Comput.-Aided Mol. Des., 4, 1 (1990). 33) MedChem Software, Daylight, Chemical Information Systems, Inc.,

150 Claremont, 1989. 34) (a) A. J. Leo, Chem. Rev., 93, 1281 (1993). (b) A. Leo, C. Hansch and D. Elkins, Chem. Rev., 71,525 (1971). (c) C. Hansch and A. Leo, Substituent Constants for Correlation Analysis in Chemistry and Biology, John Wiley & Sons, New York, 1979.

35) T. Komeno, S. Hayashi, S. Ishihara, H. Itani, H. Iwakura and K. Takeda,

Annu. Rep. Shionogi Res. Lab., 19, 3 (1969). 36) M. Salman, S. Ray, N. Anand, A. K. Agarwal, M. M. Singh, B. S. Setty and V. P. Kamboj, J. Med. Chem., 29, 1801 (1986).

37) (a) W. J. Hehre, R. W. Taft and R. D. Topsom, Prog. Phys. Org. Chem., 12, 159 (1976). (b) G. L. Nelson and E. A. Williams, Prog. Phys. Org. Chem., 12, 229 (1976).

38) G. R. Cunningham, D. J. Tindall and A. R. Means, Steroids, 33, 261 (1979). 39) J. Kirchhoff, M. Softie and G. G. Rousseau, J. Steroid Biochem., 10, 487 (1979). 40) S. C. Brooks, N. L. Wappler, J. D. Corombos, L. M. Doherty and J. P. Horwitz, in: V. K. Moudgil (Ed.), Recent Advances in Steroid Hormone Action, Walter de Gruyter & Co., Berlin, 1987, pp. 443-466. 41) Lj. Ristovic, M. Cobanovic, Z. Jovanovic and M. Orlic, Kem. Ind., 24, 389 (1975). 42) (a) T. Hori, T. Miyake, K. Takeda and J. Kato, in: W. L. McGuire (Ed.), Hormones, Receptors, and Breast Cancer, Raven Press, New York, 1978, pp. 159-180. (b) Japanese Cooperative Group of Hormonal Treatment for Breast Cancer, Cancer, 31,789 (1973). (c) K. Okazaki, S. Ito, T. Morimoto and M. Hayashi, Jpn. J. Clin. Exp. Med., 50, 1170 (1973). (d) O. Abe, S. Kumaoka and H. Yamamoto, Jpn. J. Clin. Oncol., 12, 99 (1973). (e) Y. Ogawa, Y. Hayashi, M. Matsumura, H. Oikawa, K. Imai and T. Kitakaze, Annu. Rep. Shionogi Res. Lab., 20, 118 (1970). 43) (a) M. Yoshida, Jpn. J. Cancer Chemother., 5, 69 (1978). (b) A. Matsuzawa and T. Yamamoto, Cancer Res., 36, 1598 (1976). (c) T. Miyake and A. Tanaka, Annu. Rep. Shionogi Res. Lab., 19, 20 (1969). 44) D. A. Loughney and C. F. Schwender, J. Comput.-Aided Mol. Des., 6, 569 (1992).

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OSAR and Drug Design - New Developments and Applications T. Fujita, editor 0 1995 Elsevier Science B.V. All rights reserved

153

ANALYSIS AND PREDICTION OF 1-OCTANOL/WATER PARTITION COEFFICIENTS OF SUBSTITUTED DIAZINES WITH SUBSTITUENT AND STRUCTURAL PARAMETERS NARAO TAKAO'

CHISAKO YAMAGAMI',

'Kobe

and TOSHIO FUJITA"

Women's C o l l e g e of Pharmacy,

Motoyamakita-machi, Higashinada, Kobe 658, Japan "Department of A g r i c u l t u r a l Chemistry. Kyoto University, Sakyo, Kyoto 606-01, J a p a n

ABSTRACT: The o c t a n o l / w a t e r p a r t i t i o n c o e f f i c i e n t s (P) of a number of s u b s t i t u t e d d i a z i n e s were measured The composition of t h e H v a l u e of s u b s t i t u e n t s , t h e increment i n t h e l o g P v a l u e accompanied with t h e i n t r o d u c t i o n of s u b s t i t u e n t s , was examined i n t e r m s of physicochemical s u b s t i t u e n t p a r a m e t e r s a n d c o r r e l a t i o n a n a l y s e s . The r e f e r e n c e x v a l u e a l o n g with t h e Hammett-Taft t y p e p a r a m e t e r s r e p r e s e n t i n g s t e r e o e l e c t r o n i c e f f e c t s of s u b s t i t u e n t s o n s o l v a t i o n s of e a c h hydrogen-bonding s u b s t i t u e n t ( r e g a r d i n g t h e ring-N atom as t h e " a z a " s u b s t i t u e n t ) were used i n t h e a n a l y s e s A s t h e reference H value, t h e v a l u e of t h e c o r r e s p o n d i n g s u b s t i t u e n t s in s u b s t i t u t e d p y r i d i n e s w a s used i n t h e a n a l y s e s of monosubstituted d i a z i n e systems For disubstituted pyraz i n e s , t h e sum of s u b s t i t u e n t H v a l u e s from t h e c o r r e s p o n d i n g monosubstitute d p y r a z i n e s was t h e r e f e r e n c e " H " v a l u e The q u a l i t y of t h e c o r r e l a t i o n s was v e r y good i n g e n e r a l and t h e c o r r e l a t i o n e q u a t i o n s were u t i l i z a b l e f o r p r e d i c t i n g t h e l o g P v a l u e of monosubstituted d i a z i n e s and p o l y s u b s t i t u t e d pyrazines Some hydrogen-bonding s u b s t i t u e n t s i n 2 - s u b s t i t u t e d pyrimidine series behaved as o u t l i e r s and s h o u l d be examined f u r t h e r

1.

INTRODUCTION Recently,

the

1-octanol/water

partition

coefficient

predict t h e

l o g P v a l u e of

( l o g P) h a s

been

b i o a c t i v e compounds (1)

widely u s e d as a h y d r o p h o b i c i t y parameter of possible bioactive

To

compounds having c a n d i d a t e

s t r u c t u r e s b e f o r e s y n t h e s i s is of p a r t i c u l a r importance i n q u a n t i t a t i v e modelbuildings f o r s t r u ct u r e- act i v i t y studies tive-constitutive

Since t h e l o g P v a l u e h a s a n addi-

n a t u r e , one may c a l c u l a t e a n approximate l o g P v a l u e of a

given compound from t h a t of t h e r e f e r e n c e p a r e n t molecule and t h e hydrophobicity parameter x values

of

various

of s u b s t i t u e n t s (1,2) A comprehensive compilation of l o g organic

compounds

emphasized. however, t h a t t h e

H

is

now

available

(3)

It

should

P be

v a l u e of c e r t a i n s u b s t i t u e n t s v a r i e s from

o n e s o l u t e system t o a n o t h e r when s i g n i f i c a n t steric a n d / o r e l e c t r o n i c i n t e r a c t i o n s are i n v o l v e d between t h e s u b s t i t u e n t t o be i n t r o d u c e d and t h e f i x e d f u n c t i o n a l g r o u p a l r e a d y e x i s t i n g i n t h e molecule (1.4)

Therefore, t h e l o g

P

v a l u e s for e a c h series of compounds having a common f u n c t i o n a l g r o u p s h o u l d b e a n a l y z e d i n t e r m s of s u b s t i t u e n t e f f e c t s t o d e r i v e empirical c o r r e l a t i o n

154 e q u a t i o n s p r e d i c t i n g t h e l o g P v a l u e of a n y member of t h e compound series A number of

The s i t u a t i o n is even t r u e f o r h e t e r o a r o m a t i c compounds

b i o a c t i v e compounds h a v e v a r i o u s t y p e s of h e t e r o c y c l i c r i n g s as t h e p a r e n t skeleton

Systematic

heterocyclic

log

P v a l u e s have, however, been

meager t o e n a b l e t h e empirical p r e d i c t i o n b i o a c t i v e Compounds

of

l o g P v a l u e s of

too

t h e possible

Not many s t u d i e s have been performed f o r t h e a n a l y s i s

of h e t e r o a r o m a t i c l o g P v a l u e s e x c e p t f o r t h o s e of T a y l o r and h i s coworkers Our earliest h e t e r o c y c l i c s t u d y d e a l t with t h e log P

(5-7) and o u r s (8) value

of

monosubstituted

pyridines

@a).

e f f o r t s t o accumulate and a n a l y z e t h e systems

Since t h e n

w e h a v e been making

l o g P v a l u e of

s u b s t i t u t e d diazine

In t h i s article, o u r r e c e n t s t u d i e s of t h e l o g P v a l u e s for monosub-

s t i t u t e d d i a z i n e s ( p y r a z i n e s , pyrimidines and pyridazines) and m u l t i - s u b s t i t u t e d p y r a z i n e s are reviewed a f t e r a s h o r t i n t r o d u c t i o n d e s c r i b i n g t h e u l t i m a t e p r o c e d u r e for t h e a n a l y s i s and its a p p l i c a t i o n t o t h e p y r i d i n e system.

BIDIRECTIONAL HAMMETT-TAFT-TYPE PROCEDURE FOR THE ANALYSES

2.

2.1 The Original Procedure for Disubstituted Benzenes The

original

procedure

to

analyze t h e

heteroaromatic

d e p e n d s p r i m a r i l y o n t h e " b i d i r e c t i o n a l " Hammett-Taft-type

values

i n which X

In d i s u b s t i t u t u e d benzenes, X-CsH4-Y,

f o r d i s u b s t i t u t e d benzenes.

P

log

treatment proposed

and Y are v a r i a b l e and f i x e d s u b s t i t u e n t s . r e s p e c t i v e l y . t h e v a r i a t i o n s i n t h e and

v a l u e are s u b j e c t t o s t e r e o e l e c t r o n i c i n t e r a c t i o n s between X

n

substituent

Y substituents

F o r meta-

and para-X

(rX/PhY are ) c o r r e l a t e d g e n e r a l l y with

substituents.

R P ~ x ,

i n m o n o s u b s t i t u t e d benzenes. as e x p r e s s e d by Eq

IF^,^^^

=

anphX

I n E q 1, u x"

(

p Y u Z

+

Iut(m)px

+

the n

of t h e s u b s t i t u e n t

x

1 (8)

or u t ( p ) p x l

c

+

111

( p x ) is t h e s u s c e p t i b i l i t y c o n s t a n t of Y(X) t o

t h e solubility-modifying e f f e c t s of s u b s t i t u e n t X(Y), u P and p

For

variables

h a l o g e n s and CFs,

non-hydrogen-bonding

p x is zero

being independ-

X s u b s t i t u e n t s such

as

alkyls,

The p x v a l u e f o r hydrogen-bonding

X sub-

s t i t u e n t s is estimated as t h e c o e f f i c i e n t of t h e analysis

values

u 7 ) is t h e r e g u l a r e l e c t r o n i c s u b s t i t u e n t c o n s t a n t f r e e from t h e

t h r o u g h - r e s o n a n c e e f f e c t , and p

ent

n

their

term i n t h e same t y p e of

for d i s u b s t i t u t e d benzenes i n which X is now fixed and v a r i a b l e

substituents

are

limited

to

non-hydrogen-bondable

one

The

p

u

x"

term

e x p r e s s e s t h e "forward" e l e c t r o n i c e f f e c t of t h e v a r i a b l e X s u b s t i t u e n t s o n t h e hydrogen-bonding s o l v a t i o n of t h e f i x e d Y with 1-octanol r e l a t i v e t o t h a t with

water

The u

tP

x

term is f o r a component i n t h e

increment

of t h e

155 l o g P owing to t h e '"backward" e f f e c t of Y o n t h e hydrogen-bondable (rn)

u

u 7

and

( p 1 depending o n t h e l o c a t i o n of

u

or

X (meta or para) to Y, t h e

term is d i f f e r e n t i a t e d as i n d i c a t e d i n E q

uq p

are determined

as t h e c o e f f i c i e n t s

1

of

u

u

(rn)

(rn)

and

and u

u P (p)

The v a l u e s of "a", p

the

terms, r e s p e c t i v e l y , by t h e r e g r e s s i o n a n a l y s e s plete,

u x"

and

p

If t h e c o r r e l a t i o n is com-

t h u s obtained should be equal t o t h e a u t h e n t i c

( p ) v a l u e s of t h e Y s u b s t i t u e n t

The s l o p e of t h e x

To analyze t h e

PhX

term,

v a l u e of o r t h o - d i s u b s t i t u t e d benzenes, proximity

L X,PhY

s u b s t i t u e n t e f f e c t s should be taken i n t o account o r t h o - s u b s t i t u e n t s comprize

a n d steric components (9) equivalent

JI P h X ,

intercept,"^". s h o u l d b e close to u n i t y and zero, r e s p e c t i v e l y ( 8 )

"a", and t h e

of

X sub-

S i n c e t h e backward e l e c t r o n i c e f f e c t of Y is e x p r e s s i b l e by e i t h e r

stituents

with

substituents.

i

the

e,

In p r i n c i p l e , t h e e f f e c t s

"ordinary" electronic,

"proximity" e l e c t r o n i c

The o r d i n a r y e l e c t r o n i c e f f e c t is defined as being

regular

electronic effect

=

u "(ortho)

u

I

t h e corresponding

para-

For t h e proximity e l e c t r o n i c

(para)

u

e f f e c t , s u c h p a r a m e t e r s as t h e Charton

of

(10) are shown to compensate f o r

t h e u n d e r e s t i m a t i o n of t h e t o t a l e l e c t r o n i c e f f e c t of o r t h o - s u b s t i t u e n t s u "(para)

using t h e corresponding parameters Kutter

and

o r i g i n a l l y defined Hansch

to

by

non-alkyl

which is s h i f t e d so t h a t E,(H)

=

value

Taft

for

by

F o r t h e steric e f f e c t , t h e E, alkyl

groups

and

extended

s u b s t i t u e n t s (11). t h e r e f e r e n c e

point

by of

0. a r e shown t o simulate t h e s i t u a t i o n (9)

Although t h e proximity e l e c t r o n i c and steric e f f e c t s a r e also b i d i r e c t i o n a l , t h e "forward" proximity e f f e c t s on t h e hydrogen-bonding

Y substitu-

e n t s a r e n o t always s i g n i f i c a n t , e s p e c i a l l y when t h e e f f e c t s o n t h e s o l v a t i o n with

1-octanol

and

water are almost e q u i v a l e n t

Likewise, t h e "backward"

proximity e f f e c t s need n o t n e c e s s a r i l y be c o n s i d e r e d when t h e e f f e c t s o n t h e s o l v a t i o n i n two s o l v e n t p h a s e s a r e almost e q u i v a l e n t and/or s u b s t i t u e n t s are i n c a p a b l e of hydrogen-bonding.

the ortho X

Thus, f o r t h e set of

v a l u e s i n c l u d i n g o r t h o , meta, and p a r a d i s u b s t i t u t e d benzenes, E q a p p l i c a b l e u n d e r c o n d i t i o n s i n which =

u 7 ( p a r a ) and t h e

t i o n s involved

u

I

and E,

u x" ( o r t h o )

=

u P (para),

L X,PhY

1 is a l s o u p (ortho)

terms are added depending upon t h e s i t u a -

The c o r r e l a t i o n e q u a t i o n s formulated by E q

1 and its modifi-

c a t i o n s t o i n c o r p o r a t e t h e o r t h o d i s u b s t i t u t e d benzenes have been shown for a number of s o l u t e systems i n o u r e a r l i e r p u b l i c a t i o n s (8a)

2.2 Application t o Monosubstituted P y r i d i n e s The monosubstituted p y r i d i n e s can b e r e g a r d e d as t h e d i s u b s t i t u t e d benz e n e s if w e c o n s i d e r t h e ring-N

atom a s t h e f i x e d s u b s t i t u e n t .

The 2-, 3-,

and 4 - s u b s t i t u t e d p y r i d i n e s (ZPY, 3PY and 4PY) were r e g a r d e d , r e s p e c t i v e l y , as o r t h o , m e t a . and para s u b s t i t u t e d aza-benzenes and t h e s u b s t i t u e n t p a r a m e t e r s

156 We examined

f o r s u b s t i t u t e d benzenes were e x p e c t e d t o b e a p p l i c a b l e bidirectional procedure f o r pyridine substituentl i s t e d i n Table 1

e n t parameters

the

v a l u e s using t h e s u b s t i t u -

R

Among t h e a v a i l a b l e

K

X

-

v a l~u e s

shown i n T a b l e 2, o u r preliminary examination showed t h a t most of t h e v a l u e s f o r t h e hydrogen-bonding

s u b s t i t u e n t s a t t h e 2-position are o u t l i e r s . whereas

o t h e r s are well-behaved c o e f f i c i e n t of t h e

Moreover, a f t e r d e l e t i n g o u t l i e r s . t h e r e g r e s s i o n

term is s i g n i f i c a n t l y lower t h a n u n i t y (ca 0 8 ) a n d

z p h X

Although t h e i n t e r c e p t t e r m i n

t h e i n t e r c e p t is s t a t i s t i c a l l y s i g n i f i c a n t (8a)

1 and its c o u n t e r p a r t s is supposed t o avoid giving t h e u n s u b s t i t u t e d

Eq.

compound a n i n f i n i t e weight, t h e c o r r e l a t i o n may b e s e r i o u s l y o v e r l o a d e d with t h e a d j u s t a b l e i n t e r c e p t t e r m i n t h i s p a r t i c u l a r series Eq

stituted

three

pyridines

including

Thus, by d e l e t i n g

2 w a s formulated f o r 39 mono- and unsub-

t h e i n t e r c e p t t o f o r c e K(H)=O,

compounds

with

2-

hydrogen-bonding

substituents

n

39

=

r

=

0 . 988

s

0 . 107

=

F

335. 2

=

In t h i s a n d following e q u a t i o n s , n is t h e number of d a t a , r is t h e c o r r e l a t i o n

F is t h e v a l u e of t h e F r a t i o

c o e f f i c i e n t , and s is t h e s t a n d a r d d e v i a t i o n between t h e r e g r e s s i o n a n d r e s i d u a l v a r i a n c e s are t h e 95% confidence

difference

practical p

( 0 ,

=

39

=

i n t e r v a l s of t h e r e g r e s s i o n c o e f f i c i e n t s .

exists

between

the

r

0. 941x

(0. 0 5 0 ) =

P

~

0 . 988

0. 2630 Z ( 0 . 098)

X +

s

e l e c t r o n i c parameters f o r

+

0. 104

=

W e preliminarily attempted t o use each

0. 9 4 2 p ( 0 . 113) F

u I

of

and

the

series

For e a c h of t h e t h r e e series, t h e but neither t h e

O .

parameter itself

of t h e u K phX

I

u

R

nor

is independent

u

(0,

of

the

u

2-,

R

3-,

(or and

u

131

G ) v a l u e s (10) as t h e 4-substituted

Because t h e

u I

of t h e s u b s t i t u e n t positions. t h e c o e f f i c i e n t

Moreover, t h e s l o p e of t h e

3 are shown i n Table 2

v a l u e s of some s p e c i f i c 2 - s u b s t i t u e n t s p l a i n e d . Eq

pyridine

u I parameter worked w e l l i n p l a c e

term d e v i a t e d t o a g r e a t e r e x t e n t from u n i t y with by Eq

and

m, p )

R term was s i g n i f i c a n t .

t e r m d i f f e r e d among t h e t h r e e series

rpyrldlne v a l u e s

(m)

3.

u I

t h a n with

A s t h e o v e r a l l c o r r e l a t i o n e q u a t i o n , t h e r e f o r e , w e s e l e c t e d Eq 3 lated

Since n o p

471. 7

=

of

u

coefficients

p ) t e r m s , t h e y were combined t o formulate E q

KDyrldlne

n

The f i g u r e s i n p a r e n t h e s e s

u

The c a l c u -

Although t h e

K

(see below) a r e still l e f t t o b e ex-

3 is b e l i e v e d t o show t h e r e l e v a n c y of t h e b i d i r e c t i o n a l p r o c e -

~

~

TABLE 1 S u b s t i t u e n t P a r a m e t e r s Used as Independent V a r i a b l e s Subst 1tuent

~ a’ X

0. 14 0. 71 0. 8 6 0. 56 1.02 -0. 02 0. 38 I . 05 0. 61 -0.57 -0.01 0. 51 -0.55 -0.28 0 . 18 -1.49 -1.23 -0.97

c1 Br Me Et OMe OEt 0Pr SMe CN C02Me COzEt Ac NO2 NMez CONHz NHz NHAc n-Pr” n-Bu” OCHMe2’

I [ P

0. 00

H F

Phk’

(x)

7t 2 p y b ’

0. 0 0

gpyb’

0. 00 0. 19s’ 0. 12s’ 0. 62 0. 68 0. 73 0. 9 3 0. 46 0. 55 0.95s’ 1.01“’ 0. 69 0. 34s’ 1. 1 6 “ ’ 0.799’ 1.73s’ 1. 0 6 -0.25e’ -0.42e’ -0.29” 0. 16 0. 22 0. 71 0 . 18 -0.22 -0.05 -0. 17 1. 00” 0. 76e’ -0. 50s’ -0.99 -0. 17s’ - 0 . 4 5 -0. 13e’ - 0 . 2 4 -

~

PbY ’

0. 00 0. 6 3 0. 8 6 0. 57 1.00”’ 0 . 35 -

-0.19 0. 22 0. 7 8 - 0 . 11 -

0. 6 9 -0.93 -0.39 -0.15 -

-

’

1. 9 6

-

-

I. 8 0 -

P

XC’

0. 00 0. 0 0 0. 0. 0. 0. 0.

00 00 00 00 27 (0.2 7 ) (0.2 7 ) (0.2 0 ) 0. 0 0 (0. 1 3 ) (0. 1 3 ) (0.1 6 ) -0.14 (0.4 6 ) 0. 45 0.74 0. 91

0. 00

0. 00 0. 00 (0.2 7 ) h ’

a

:*’

0. 00 0. 3 4 0. 37 0. 37 -0. 06 -0. 08 0. 10 0. 10 0. 10 0. 14 0. 62 0. 35 0. 35 0. 36 0.71 -0. 10 0. 2 8 -0.09 0. 14 -

-

a

gd’

0. 00

a

0. 00

0. 15 0. 5 4 0.24 0. 47 0. 47 0. 2 6 -0. 14 -0. 01 -0.13 -0.01 -0. 12 0. 30 -0.14 0. 28 -0. 14h’ 0. 28h’ 0. 06 0. 3 0 0. 71 0. 57 0. 44 0. 32 0. 44 0. 30 0. 3 0 0. 47 0 . 81 0. 6 7 -0.32 0. 17 0. 31 0. 28 -0.30 0. 1 7 0. 00 0. 2 8 -0. 01 -0. 01 0. 05 0. 27

a

Re’

0. 00 - 0 . 48 -0. 25

E,” 0. 00 -0. 46 -0.97

-

-0. 16 -0.14 -0. 58 -0. 57 -0. 52 - 0 . 38 0. 08 0. 11 0 . 11

-1.24 -1.31 -0.55 -0.55 -0.55 -1.07 -0.51 -2.52” -2.52”

-

-

0. 10 -0.88 0. 08 -0.80 - 0 . 35 -0. 16 -0. 16

-1.01 -0. 6 1 -2.52” -0. 60 -0. 6 1 -1. 6 0 -1. 60

-

-0. 52

-0.55

-

a) From Ref 3 b) From Refs 6 and 8a. u n l e s s o t h e r w i s e n o t e d c) From Refs 8a and 13 The v a l u e in pavalue r e n t h e s e s is t h e o r i g i n a l p f o r H-accepting g r o u p s u s e d i n d i s u b s t i t u t e d b e n z e n e systems The p of t h e s e s u b s t i t u e n t s was t a k e n as z e r o in Eqs 20-25 d ) From R e f 15 e) From R e f 10 f ) From Ref 11 g) From Ref 13 h) Estimated from v a l u e s of r e l a t e d s u b s t i t u e n t s i) L ( c o p 1 a n e r ) v a l u e s J) S u b s t i t u e n t s c o n t a i n e d i n E q s 26 and 27, o n l y k) In E q s 2 a n d 3, o n l y

e

cn

I.

TABLE 2 Hydrophobicity Parameter of Substituted Pyridines

Substituent

H

2-F 2-c1 2-Br 2-Me 2-Et 2-OMe 2-OEt 2-OPr 2-SMe 2-CN 2-C02Me 2-COzEt 2-Ac 2-NMez 2-CONHz 2-NHz 2-NHAc 2-NO2 3-F 3-C1 3-Br 3-Me 3-Et 3-0Me

Obsd.

0. 00 0. 19 0. 6 2 0. 73 0. 46 0. 95 0. 69 1. 16 1. 73 1. 06

-0.25 -0.29 0. 22 0. 18 1. 00 -0.50 -0.17 -0. 13 -0.17 0. 12 0. 68 0. 93 0. 55 1. 01 0. 34

Calcd?' 0. 00 0. 17 0.73 0.88 0.49 0. 9 3 0.20"' 0. 58"' 1.21"' 0.78"' -0.35

0. 23"' 0. 72"' -0. 24" 0. 52"' -0. 88"' -0.54"' -0.06 -0.18 0.22 0. 77 0. 91 0. 51 0. 94 0. 26

Dev 0. 00 0. 02 -0.11 -0.15 -0.03 0. 0 2 0.49 0. 58 0.52 0. 28 0. 10 -0.52 -0.50 0.4 2 0.48 0.38 0. 37 -0.07 0. 01 -0.10 -0.09 0. 02 0. 04 0. 07 0. 08

Subs t 1 tuent

Obsd.

Calcd?'

3-OEt 3-CN 3-COzMe 3-COzEt 3-AC 3-NMe2 3-CONHz 3-NHz 3-NHAc 3-NOz 4-C1 4-Br 4-Me 4-Et 4-OMe 4-CN 4-COzMe 4-COzEt 4-AC 4-NMez 4-CONHz 4-NHz 4-NHAc 4-Ph

0. 79 -0. 4 2 0. 16 0. 71 -0.22 0. 76 -0.99 -0.45 -0. 24 -0.05 0. 6 3 0. 86 0. 57 1. 00 0. 35 -0.19 0. 2 2 0. 78 -0. 1 1 0. 69 -0.93 -0.39 -0.15 1. 8 0

0.6 2 -0.37 0. 20 0. 69 -0.27 0. 58 -0.91 -0.48 -0.02 -0.21 0. 73 0.88 0. 49 0. 9 3 0. 20 -0.35 0. 23 0. 72 -0.24 0. 52 -0.88 -0.54 -0.06 1. 86

Dev

0. 17 -0.05 -0.04 0. 02 0. 05 0. 18 -0. 08 0. 03 -0.22 0. 16 -0.10 -0. 02 0. 08 0. 07 0. 15 0. 16 -0. 01 0. 06 0. 13 0. 17 -0. 05 0. 15 -0. 09 -0.06

a) Log P value of pyridine is 0.65 (8a) b) Calculated by Eq. 3. c) Not included in correlation but calculated by Eq. 3.

159

dure

for

the

coefficient

relative

of

the

ring-N

as

varies

approximately

0.9 f o r

the

between the

Such at

along

with

the

meta

in

NH2 , NMe2, a n d

for

C02R,

their

~

from

uents

by

considering

ring

only

in are

substituents

Eq.

the

substituents in

electrons

are

decrease

in in and

substituents

3.

substituents

phase,

log

which

raising

negative

the

log

a

coplanar

value.

In

the

bulky

3-

and

4-

as Eq.

of

OR,

SMe,

3.

Except

the

values

these

substit-

hydration

occur-

of

water-

pair

about

in

0.5

the

electrons

of

(8a).

2-

The

and

coworkers

suggested

groups

containing

lone

be

expected

spite

of

these

behaviors examined

(6).

In

to

such

pair

cases,

a

produce

negative

rationalizations,

factors

of

hydrogen-bonding

in f u t u r e

2-

studies.

HYDROPHOBICITY PARAMETER OF SUBSTITUTED DIAZINES

We t r i e d substituents

(PR), p y r i m i d i n e s

to

Diazlnes

extend

in

each

the of

(PM) a n d

I

procedure t o

bidirectional

the

diazines

series,

pyridazines(PD),

N

~N

as

i.e.,

analyze

the

We o r i g i n a l l y by

fundamental

planned

to

substituents

as

far

the as

log

value

pyrazines

(I-VI)(13).

X -/'N

measure

~

monosubstituted

shown below

N

v,.4

ed

and

equation

from

lone

conformation

systematically

in

hydrogens

would

outlying

NO2,

insignificant

behavior

with

the

signifi-

CN,

various be

deviation

Lewis

and

0.6 t o

not

2-substituents

P value

ring-N

statistically.

included

two as

deviants.

effect

more

well

the

Since

was

as

as

the

from

(12).

bridging-type

the

as

esters

adopt

P

be

in

ring-N

well

not

outlying

of and

of

correlation

could

Such

however,

intermolecular

the

the

as

effects

the

differentiating/ should

in

2-substituents

"l-to-l"

to

3.1 M o n o s u b s t i t u t e d of

amphiprotic

2-substituents.

resonance

the

0.1-0.15,

incorporated

azines,

unable

about

We r a t i o n a l i z e d

were

the

reaction

positive

a-substituted

value meta-

and

The

constant

the

large

with

a

the

well

proximity

simultaneously,

C02R

on

pyridines.

a o

was r e a s o n a b l e

showed

aqueous

the

0.7 f o r

a

a

bonding

that,

defining

3.

to

values,

CONH2 w e r e ,

values

expected

deviations

para the

the

hydrogen-bonding

C02R,

4-substituted

"replacement"

0.5 t o

depending

nonhydrogen-bonding Therefore,

and

# x terms

acceptor

Ac,

molecule

from

of

were

3-

correspond

reported

and

combination

2-position

substituents. general

The

"substituent"

hydrogen

the

of

should

in p a r a l l e l

para-aza

enough,

NHAc

term

a "substituent"

difference cant

partitioning

p x

P value

possible.

of

diazines

Unfortunately,

o substituts ome

de-

160

sirable

compounds

conditions

because

dines

observed

were

ficients.

of

Other

number

of

either

were

their

to

not

isolated

instability.

decompose

nitrodiazines

substituents

For

during have

analyzable

intolerant

or

example,

5-CN

measurement

never

was

been

to

of

the and

their

reported.

inevitably

low

partitioning 5-N02

pyrimi-

partition

coef-

Therefore,

in

some

series

the

of

com-

pounds. The The

log

P

substituent

3 Partition

TABLE

-0 26 0 29 0 7O 0 93 021 0 69 0 73 1 28 1 84 117 -00l -0 23 0 28 0 20 0 93

-0. O. O. O. -0.

labile

the

X

substituent

the

effect

of

to

a

importance obvious on

obtain

of

the The

for

- 0 . 08 - 0 . 27 -

1. 07 - 1 . 20 - 0 . 20

O. 58 - 0 . 68 - 0 . 25

parent

accurate

value,

Inspection

are

generally groups.

the

ring-N

extent

fact

higher

the

system

as

correlation

corresponding

pyridine-

values.

substituted

~

~ of

of

- 0 . 43 -

- 0 . 63 - 0 . 27 O. 23

O. 29 b ) - 0 . 73 -

- 0 . 09 - 0 . 96 - 0 . 53

as

the than

.

- ~) O. 03 O. 52

22

From

-

Her.

1 and

the of

the

regarded

X

in the the as

was

atoms

affect aza same much a

the

pyridine-~ as

the

effect

of

well

as

diazine-~

values.

The

(-N =) functions varies parent better

corresponding having

that

electronic

substituent

values with

reveals

ring-N

and

a given

location

4-9

the

41

6

pyridine-~

substituent of

-0.

corresponding

that

substituent

diazine-~ is

.

corresponding of

values diazine

- 0 . 31 O. 20

.

than

value

well

O. 08 O. 63

suggests

that

interaction the

O. 07 O. 56

Tables

solvation

on

than

that

b)

4PD(VI) log P -0. 73 - 0 . 32

-0.

of

This

relative atoms

electronic

single

O. 0 3

3.

- 0 . 73 O. 10 - 0 . 35

O. 46 - 0 . 92 -

81

Table 4-9.

3PD ( V ) log P

.

.

in

44 03 47 66 O1

.

.

- 0 . 31 - 0 . 71 - 0 . 31

listed in Tables

(13)

-0. -0. O. O. O.

.

alkyl on

the

the

Each

O. 54 O. 97

are given

5PM(IV) log P

P

O. 23 O. 74

Analysis:

greater

from

log

-0. 44 O. 4 7 - 0 . 05

an

are

Diazines

44 02 36 50 05

-0.

diazines (I-VI),

4PM(III)

P

.

values except

series

1.01

Correlation

diazine-~

ing

log

50 O5 O3

to

each

Monosubstituted

2PM(II)

values

values

of

P

-0 -0 -0

3.1.1

monosubstituted

of

2PR(I) log

H F C1 Br Me Et OMe OEt OPr SMe CN C02Me C02Et Ac NMe2 CONH2 NH2 NHAc

for

values

Coefficients

Substituent(X)

a) T o o

values ~

hybrid

is

dependsystem. with

the

benzenefeature

of

161

TABLE 4

Hydrophobicity Parameters o f 2-Substituted Pyrazines(1) x Z P R< l o g P

Substltuent

H

F c1

Br Me Et OMe OEt

OPr SMe CN COzMe COzEt AC

NMez CONHz NHz

NHAc

(Eq.

( p y r a z i n e ) = -0.

6)

26>

( E q . 12)

Obsd.

0. 00 0. 55 0. 96 1. 19 0. 47 0.95 0. 99 1. 54 2 10 1. 43 0 . 25 0. 03 0. 54 0. 46 1. 19 -0.24 0. 21 0. 23

Calcd.

Dev.

0. 01 0. 52 1. 06 1. 19 0. 51 1. 08 0. 92 1. 43 2 12 1. 40 0 . 23 - 0 . 04 0.57 0.53 1. 13 -0.35"' - 0 . 26"' - 0 . 03"'

-0.01 0. 03 -0. 10 0. 00 -0.04 -0. 13 0. 07 0. 1 1 -0.02 0. 03 0. 02 0. 07 -0.03 -0.07 0. 06 0.11 0. 47 0. 26

Calcd.

0.02 0. 49 1.00 1. 12 0.49 1. 03 0. 97 1. 46 2. 1 1 1. 40 0. 20 0. 00 0. 58 0.55 1. 25 -0.17 0. 04 0. 32

Dev.

-0.02 0. 06 -0.04 0. 07 -0.02 -0. 08 0 . 02 0. 08 -0.01 0. 03 0. 05 0. 03 -0.04 -0.09 -0. 06 -0.07 0. 17 -0. 09

a) Not included in the c o r r e l a t i o n b u t c a l c u l a t e d by Eq. 6.

TABLE 5

Hydrophobicity Parameters of 2-Substituted Pyrimidines(I1) ~~

nZPM
Subst 1 tuent

( p y r i m i d i n e ) = -0 ( E q . 7)

Obsd.

Ca 1 c d .

H

F c1

Br

Me OMe OEt SMe CN COzMe COzEt NMez CONHz NHz NHAc

44>

0. 0 0

0. 46 0. 80 0. 94 0 . 39 0. 67 1. 18 1. 45 0. 13 -0.27 0. 13 1. 51 -0. 76 0 . 24 -0.37

0 . 03

0. 32 0 . 82 0. 94 0. 44 0 . 69 1. 17 1. 18"' 0 . 17 -0.03"' 0 . 51"' 0.90"' -0.32"' - 0 . 31"' -0.10"'

Dev.

-0.03 0. 14 -0. 02 0. 00 -0.05 -0.02 0. 01 0. 27 -0.04 -0. 24 -0.38 0. 61 -0.44 0. 55 -0.27

a) Not i n c l u d e d i n the c o r r e l a t l o n b u t c a l c u l a t e d by Eq. I .

162

TABLE 6 Hydrophobicity P a r a m e t e r s of 4 - S u b s t i t u t e d PyrimidinesCIII) z q p M Substltuent

Calcd.

H C1 Me OMe

OEt CN COzMe NMez CONHz NH2 NHAc

(Eq. 1 3 )

(Eq. 8 ) Obsd.

0. 0 0 0. 9 1 0. 39 0. 98 1. 4 1 0. 36 0. 17 1. 0 2 -0.24 0. 1 9 0. 47

Dev.

-0. -0. -0. 0.

0. 06 1. 0 4 0. 5 1 0. 81 1. 3 8 0. 36 0. 08 1. 0 2 -0.25"' - 0 . 42"' - 0 . 11"'

0.

0. 0. 0. 0. 0. 0.

06 13 12 17 03 00 09 00 01 61 58

Calcd.

Dev.

-0. 03 0. 01 -0.05 0 . 11 0. 03 0. 0 8 0. 0 6 -0. 16 -0.25 0. 1 3 0. 04

0. 03 0. 90 0.44 0. 8 7 1 . 38 0. 28 0 . 11 1. 18 0. 0 1 0. 06 0. 43

a) Not i n c l u d e d in t h e c o r r e l a t i o n b u t c a l c u l a t e d b y Eq. 8.

TABLE 7 Hydrophobicity P a r a m e t e r s of 5-Substituted Pyrimidines(IV)

n g p M < l o g P ( p y r i m i d i n e ) = -0. 44> (Eq. 9)

Substituent

Calcd. H

F C1

Br Me OMe

OEt C02Me COzEt NMez CONHz

NHAc

0. 0 0 0. 4 1 0. 9 1 1. 10 0. 4 5 0 . 51 1. 0 0 0.47 0. 96 0. 90 -0.48 0.22

0. 08

Dev.

-0. 08 0. 05 0 . 9 4 -0. 03 1.19 -0.09 0 . 6 1 -0. 16 0. 47 0. 04 0. 8 9 0 . 11 0 . 41 0. 06 0. 9 6 0. 0 0 0. 8 0 0. 10 - 0 . 78"' 0 . 3 0 -0.09"' 0.31 0. 3 6

(Eq. 14)

Calcd.

0. 03 0. 37 0. 9 1 1. 1 4 0.50 0.53 0. 9 1 0. 4 7 0.98 0. 8 6 -0.52 0. 28

(Eq. 17)

Dev.

-0. 0. 0. -0. -0. -0. 0. 0.

-0.

0. 0.

-0.

03 04 00 04 05 02 09 00 02 04 04 06

Calcd.

Dev.

0.10 0.42 0. 97 1.11 0. 5 9 0. 47 0 . 81 0. 45 0. 9 3 0. 78 -0.58 0 . 40

-0.10 -0. 01 -0. 06 -0.01 -0. 14 0. 04 0. 1 9 0. 02 0. 0 3 0. 12 0. 10 - 0 . 18

a) Not included in t h e c o r r e l a t i o n but c a l c u l a t e d b y Eq. 9.

163 TABLE 8 Hydrophobicity Parameters of 3-Substituted Pyridazines(V1 z g p D
Subs t 1 tuent

Obsd. 0. 0 0 0. 8 3 0. 38 0 . 81 1. 3 6 0. 30 1. 0 2 0. 0 0

c1 Me OMe OEt COzMe NMez CONHz

(Eq. 15)

(Eq. 10)

Calcd.

H

P (pyridazine)= -0.7 3 >

0.09 1. 0 1 0.47 0.83 1. 2 3 0. 12 0. 9 5 -0.14"'

Dev. -0.09 - 0 . 18 -0. 09 -0. 02 0 . 13 0 . 18 0. 07 0. 14

Calcd. 0.12 1. 0 1 0.49 0.84 1. 2 1 0. 17 0. 94 -0. 08

Dev. -0.12 - 0 . 18 -0.11 -0. 03 0 . 15 0. 1 3 0. 08 0. 08

~

a) Not included in the correlation but calculated by Eq. 10.

TABLE 9 Hydrophobicity Parameters of 4-Substituted Pyridazines(V1). R ~ P D
Substltuent

Obsd.

( E q . 11)

Calcd.

H

Me OMe CN COzMe COzEt NMez CONHz NHz NHAc

0.00 0. 4 1 0. 42 0. 10 0. 4 6 0. 96 0. 6 4 - 0. 2 3 0. 20 0. 32

P (pyridazine

-0.02 0. 5 1 0. 39 0 . 15 0. 40 0. 9 6 0. 6 1 -0. 78"' - 0 . 46"' - 0 . 09"'

Dev. 0. 02 -0. 10 0. 0 3 -0. 05 0. 06 0. 00 0. 03 0 . 55 0. 66 0. 4 1

=

-0.73>

(Eq. 16)

Calcd. 0. 05 0.42 0.46 0.19 0. 42 0. 81 0. 70 0. 28 0. 08 0.43

Dev. -0. 05

-0. 0 1 -0. 04 -0.09 0. 04 0 . 15 -0. 06 0. 05 0. 12 -0.11

a) Not included in the correlation but calculated by Eq. 11

164 t w o t y p e s of s u b s t i t u t e d p y r i d i n e s s h a r i n g a common s u b s t i t u e n t e x c e p t f o r

F o r such 2 - s u b s t i t u t e d d i a z i n e s as 2PR(I). 4PM

t h e symmetric 2PM and 5PM

(111). and 3PD ( V ) , t h e c o r r e l a t i o n with

the alternative ( n3

or

p

~

A ~ P Y )

KZPY

w a s always b e t t e r t h a n t h a t with

a s shown i n Table 10

In p a r t i c u l a r . f o r

t h e 2PR (I) system, t h e s i n g l e c o r r e l a t i o n with n p P y is much b e t t e r t h a n t h a t with

i n c l u d i n g s u c h o u t l i e r s as OR, SMe, A c . COzR, CONHz. NH2, and N M e z

K 3py,

in Eqs

2 and 3 f o r t h e 2PY system

This i n d i c a t e s t h a t t h e

v a l u e of 2-

A

s u b s t i t u e n t s i n d i a z i n e s u l t i m a t e l y s h a r e s components from complex proximity i n t e r a c t i o n s with

z

K

p

~

The a b o v e o b s e r v a t i o n s l e d u s t o u s e t h e

Then, Eq. 1 c a n b e r e w r i t t e n as

series as t h e r e f e r e n c e independent v a r i a b l e Eq

value f o r t h e pyridine

A

4 c o n s i d e r i n g t h e backward e f f e c t of t h e ring-N by t h e u P ( p ,

Z(ortho)-X-substituents

A s a matter of f a c t , t h e u s e of

0 )

p x

~

o n hydrogen-bondable

term

~

as t h e r /e f e r e n c e means ~ t h a t ~t h e

~

i n t h e r e f e r e n c e p y r i d i n e system is r e g a r d e d as a

s u b s t i t u e n t X p l u s -N=

" s i n g l e " u n i t (N+X), and t h a t t h i s (N+X) u n i t and t h e second -N= function(Y) are bidirectionally interacting partners.

X,

u

x",

but

not

that

of

(N+X)

In Eq. 4, however, t h e forward e f f e c t of

to

is assumed

work

on

the

second

-N=

function(Y1 and t h e backward e f f e c t of Y o n l y o n X is c o n s i d e r e d depending upon t h e r e l a t i v e p o s i t i o n s of X and Y.

TABLE 10 S i n g l e Correlation C o e f f i c i e n t (r) between DiazineA Z P R ( I )

nppy ~ 3 p y

0.954 0. 814

0 . 940

-

-

0. 678 16

0. 680 15

4PY

~ p h x

ne'

nZPM(I1) -

n4PM(III)

x

a n d Reference x

r5PM(IV)

n3PD(V)

-

0. 923 0. 785

0. 881 -

0 . 956 -

0. 783 0 . 589 10

-

0. 840 12

0 . 556 8

Values.

n4pD(vI) -

0. 8 6 5 0. 8 8 5 0 . 710 10

a) Number of s u b s t i t u e n t s whose n v a l u e s are a v a i l a b l e i n common among m o n o s u b s t i t u t e d b e n z e n e and p y r i d i n e systems.

The a n a l y s i s was made n

~

for

each series

For

t h e reference

v a l u e , t h~e c h o i c e ~ was made a~c c o r d i n g t~o which is~ b e t t e r asd t h e

parameter set i n t h e f i n a l c o r r e l a t i o n s

4PM a n d 3PD). 4PD series.

(I-VI)

KZPY

For

a -substituted

w a s , of c o u r s e . t h e parameter of c h o i c e

Z ~ P Yand

A ~ P Y ,

r e s p e c t i v e l y , were s e l e c t e d

d i a z i n e s (2PR. 2PM, F o r t h e 5PM a n d

In f a c t , t h e select-

~

e d s e t of pyridine-Ir

v a l u e s was always b e t t e r c o r r e l a t e d with t h e set of

corresponding diazine

(

v a l u e s even i n t h e s i n g l e c o r r e l a t i o n

K

For non-hydrogen bonding s u b s t i t u e n t s where

p

=

0.

T a b l e 10

)

and some hydro-

g e n a c c e p t i n g s u b s t i t ~ i e n t swhere p x is n o t v e r y l a r g e , t h e "backward" e f f e c t (a

pp

of Y o n X is n o t v e r y s i g n i f i c a n t

x)

e a c h d i a z i n e series. Eq

F o r s u c h a s u b s t i t u e n t set In

4 c a n b e simplified t o E q

5

Thus, w e f i r s t used Eq. 5 f o r e a c h d i a z i n e series e x c l u d i n g t h e amphiprotic

NHz

substituents, NHAc.

examined t h e u s e of a s u b s t i t u e n t series the

u

and CONHz. possessing

large

values

p

4, i n c l u d i n g amphiprotic s u b s t i t u e n t s

p r o c e e d e d t o u s e Eq

I

and a

parameters i n p l a c e of

a

term was

we

in E q

5 f o r each

A s i n t h e case f o r s u b s t i t u e n t s i n t h e p y r i d i n e series.

p a r a m e t e r g a v e almost e q u i v a l e n t statistics with

I

Then,

W e preliminarily

insignificant

The

coefficient

of

the

x

a

but

Dyrldlne

term.

the

a

R

however.

d e v i a t e d from u n i t y s i g n i f i c a n t l y f o r some series In T a b l e 11. t h e c o r r e l a t i o n e q u a t i o n s (Eqs 6-11) formulated with u s e of

E q 5 are shown

values according t o t h e s e

In T a b l e s 4-9, t h e c a l c u l a t e d x

e q u a t i o n s are l i s t e d f o r comparison with t h e o b s e r v e d values. series. N M e z ,

SMe and

From t h e 2PM

COzR had t o b e d e l e t e d , i n a d d i t i o n t o t h e amphiprotic

s u b s t i t u e n t s , t o o b t a i n t h e a c c e p t a b l e q u a l i t y of t h e c o r r e l a t i o n t h e number of

Although

d a t a r e l a t i v e t o t h a t of t h e independent v a r i a b l e terms w a s

n o t s u f f i c i e n t i n some series, E q

5 seems t o h o l d as f a r as

~r

v a l u e s within

e a c h system are c o n c e r n e d A s e x p e c t e d , t h e c o e f f i c i e n t of t h e p y r i d i n e - x

t e r m was close t o u n i t y

b e i n g c o v e r e d almost completely i n t h e r a n g e of t h e 95% confidence i n t e r v a l i n Eqs

6-11

f u n c t i o n (-N=),

The p y v a l u e , t h e s u s c e p t i b i l i t y c o n s t a n t of t h e s e c o n d aza v a r i e d , however, depending on t h e system

j u s t i f i e d o n l y a t t h e 92% l e v e l i n E q

Although it w a s

10 f o r t h e 3PD system, t h e

p y

value

c o u l d b e c a t e g o r i z e d i n t o two g r o u p s : o n e g r o u p l o c a t e d a r o u n d 0 8 7 (Systems I, 111, a n d V), and t h e o t h e r a t a b o u t 0 5 3 (Systems 11, IV. and VI)

I. 111, a n d V. t h e r e were common s t r u c t u r a l f e a t u r e s s u c h t h a t as t h e r e f e r e n c e

In systems

I I ~ P Y

was used

For systems IV and VI where x B P y and n I P y were used,

t h e p y v a l u e was lower t h e reference, t h e p

Although system I1 is one of t h o s e i n which 2PY is

v a l u e w a s lower i n E q 7

t h i s is a v a i l a b l e a t p r e s e n t

much h i g h e r t h a n t h e c o r r e s p o n d i n g s t i t u t e d p y r i d i n e system

No a d e q u a t e e x p l a n a t i o n f o r

I t w a s a l s o noted t h a t t h e s e P Y

values in E q s

p y

v a l u e s are

2 and 3 f o r t h e sub-

The v a r i a t i o n s i n t h e p y v a l u e s with d i f f e r e n t

systems i n d i c a t e t h a t t h e assumptions made t o f o r m u l a t e Eqs

4 and 5 a p p l y

TABLE 11 Correlations of z ( d i a z i n e ) w i t h E q . 5 ” ’

I : 2PR

1I:ZPM

System I I1

III:4PM

IV:5PM

r

Correlation If

ZPR

=

1. 1 9 3 z ~

P

Y+

(0.0 9 3 ) 7C 2 P M

=

1. 0 4 8 n

ZPY

(0. 195)

I11

Z ~ P M =

IV

*SPM

V

~

VI

Z ~ P D=

0 . 8 2 6 u x” ( m )

(0. 241)

*

0 . 555U ; ( p ) (0. 303)

1. 2 4 5 x 2 ~+ ~0 . 8 7 2 u E ( p ) (0.544)

(0. 345) =

1. 0 0 5 n s p y

(0.2 4 1 ) S

P

D=

+

0. 4 7 5 u E ( m ) (0. 398)

0 . 9 5 3 z z ~+ ~0 . 8 8 3 u f ( m ) (0. 385) (1. 055)

0 . 9 9 4 ~ 4 p y+ 0 . 5 7 3 u f ( m ) (0. 331)

(0. 245)

VI : 4PD

V : 3PD

S

F

n”’

E q . No

+

0.014

0.994

0. 073

456. 0

15

6

+

0. 033

0. 988

0. 072

105. 8

8

7

0. 0 5 5 (0. 214)

0.979

0.119

57. 2

8

8

0.081 (0. 158)

0. 9 6 9

0.100

53. 0

10

9

0. 087 (0. 312)

0. 961

0. 1 6 0

23. 8

7

10

0.020 (0. 144)

0.985

0 . 069

63. 6

7

11

(0. 097) (0. 131)

+

+

+

-

a) Reproduced from r e f . 13 with permission o f t h e copyright owner, VCH Publishers, Inc. b) S u b s t i t u e n t s n o t included: I: CONHZ. NHz. NHAc. 11: SMe. COzR. NMez. CONHZ. NHz. NHAc. 111: CONH2, NHz. NHAc. IV: CONHZ. N H A c . NHz(not measured). V: CONHz.NHz(not measured),NHAc(not measured). VI: CONHZ, NHz. NHAc.

167 Eq. I a l s o differed from t h e o t h e r s in t h a t it w a s

only within each series.

unable t o accommodate w e l l non-amphiprotic

s u b s t i t u e n t s such a s NMez, SMe

and COZR. Table

Eq. 4 for

12 lists, t h e c o r r e l a t i o n substituents

including

Tables 4 and 6-9 a l s o show t h e x

equations Eqs.

12-16 formulated

amphiprotic ones having higher

P X

with

values.

values calculated according t o E q s . 12-16.

For t h e 2PM system, no reasonable c o r r e l a t i o n equation was derived.

In

addition t o o u t l i e r s u b s t i t u e n t s from Eq. I , no amphiprotic s u b s t i t u e n t s w e r e accommodated and so t h e c o r r e l a t i o n with t h e bidirectlonal procedure w a s n o t formulated f o r t h i s series.

For t h e 2PR. 4PM. and 5PM systems, however, NHz. w e l l accommodated in Eqs. 12, 13, and 14.

CONHz.

and N H A c were

Again, t h e number of d a t a r e l a t i v e

t o t h a t of t h e independent variable terms w a s n o t enough in some systems. Since t h e c o r r e l a t i o n f o r 3PD was derived from t h e d a t a set including only one amphiprotic s u b s t i t u e n t , t h e significance of t h e coefficient of t h e

p x

t e r m in Eq. 15, which was Justified only a t t h e 90% level, i s somewhat uncerThe coefficient of t h e

tain.

term in Eq. 16 f o r t h e 4PD system was

xqpy

considerably lower t h a n t h a t in t h e corresponding Eq. 11 in Table 11. substituted

pyridazine series, t h e two vicinally

s t r o n g l y i n t e r a c t wlth each other.

N in t h e r e f e r e n c e X-substituted

situated

ring

In t h e

N-atoms may

The e l e c t r o n i c i n t e r a c t i o n s between X and pyridines could be severely d i s t o r t e d be-

tween t h e corresponding X and N

in t h e X-substituted

pyridazines.

Under

such conditions, t h e regression coefficients of t h e x d p y term i n Eq. 16 would b e highly p e r t u r b e d by t h e N-N e n t is amphiprotic.

interactions, especially when t h e X-substitu-

Since t h e numbers of compounds included in t h e s e pyrida-

zine systems are not l a r g e enough, examination of t h i s type of p e r t u r b a t i o n a f f e c t i n g t h e bidirectional

model requires f u r t h e r study, with additionally

synthesized compounds Except f o r t h e s e minor uncertainties, however, t h e general p a t t e r n of v a r i a t i o n s in t h e P Y value is s i m i l a r with t h e sets of equations in Tables 11 and 12. ic terms

The sign of t h e regression coefficient of t h e bidirectional e l e c t r o n in Eqs.

12-16 can be rationalized a s being analogous t o t h a t

in

c o r r e l a t i o n s f o r d i s u b s t i t u t e d benzenes (8). The forward effect of e l e c t r o n withdrawing

X

substituents

tends

to

decrease

the

basicity

of

-N=.

being

unfavorable t o s o l v a t i o n with t h e more acidic water, enhancing t h e p a r t i t i o n ing toward t h e less acidic 1-octanol phase. P

Y

value (coefficient of

u

This process explains why t h e

Z ) i s positive in a l l t h e correlations.

The positive sign of t h e

u?

value (coefficient of

p

f o r t h e back-

ward effect reflects t h e electron-withdrawing n a t u r e of t h e aza (-N=) The c o e f f i c i e n t of t h e P

x

group.

t e r m in Table 12 seems t o be categorized i n t o two

TABLE 12 Correlations of n (dlazlne)

IC

( d i a z i n e ) w i t h Eq. 4

a z (pyridine)

=

-

pya,"

+

O F p x

+

C ~~~~

Systcm

r

Corrclation

F

E q No.

n

0 . 3 8 1 ~+ ~0.016 (0. 175) (0.101)

0. 994

0. 078

355. 8

18

12

0 . 5 9 9 P x - 0.0'27 - (0. 2 1 9 )

0. 973

0. 138

42. 1

11

13

0.9 3 1 ~ +3 0. ~ 6~5 9 a t ( m ) - 0. 1 1 6 +~ 0. ~ 031 (0.0 7 8 ) (0.219) (0. 1 5 8 1 ( 0 . 083)

0.995

0 . 054

278. 0

12

14

0. 026 (0. 239)

0 . 984

0. 1 1 7

40. 5

8

15

0 . 4 6 6 ~- ~0 . 0 4 6 (0. 328) (0. 176)

0.966

0 . 107

27. 8

10

16

I

XZPR

111

X ~ P W=

IV

X 5PM

Y

X ~ P D=

+

vI

z4PD

- 0.447aPIm)

=

1 . 1 3 0 n z ~+ ~0 . 7 5 6 u p ( r n ) (0. 242)

(0.080)

I.115nzpy

(0.2 6 7 )

-

O.7448P(p)

+

+

(0. 4 3 0 )

(0.5 0 1 1

=

0. 8 9 8 x 2 ~ ~0 . 9 4 1 0 Z ( m ) (0.2 4 0 ) (0. 774) 0.706X,,~ (0.191)

=

S

( 0 . 4221

*

+

0. 4 7 4 p x (0.6 3 5 )

+

169 g r o u p s : o n e is 0 6 i n Eq a r o u n d 0 43 i n E q s second

13 f o r t h e 4PM series and t h e o t h e r is c e n t e r i n g

12, and 14-16

13. t h e backward effect of

In E q

atom is d i r e c t e d t o t h e " p a r a " X s u b s t i t u e n t s .

ring-N

systems, it acts i n t h e "meta" d i r e c t i o n group

These

the

in o t h e r

The magnitudes of t h e s e t w o t y p e s ( p ) and

o f c o e f f i c i e n t c o u l d c o r r e s p o n d t o t h o s e of t h e u t h e second -N=

but

( m ) v a l u e s of

u

values, 0 6 and 0 4, are, however, c o n s i d e r a -

u

b l y lower t h a n t h o s e of 0 99 and 0 9 3 , r e s p e c t i v e l y . o b s e r v e d f o r s u b s t i t u t e d pyridines The a b o v e b i d i r e c t i o n a l

p r o c e d u r e with t h e use of

x z P y v a l u e as t h e r e f e r e n c e worked v e r y well f o r t h e

t h e corresponding

~

Z

ries i n c l u d i n g " i r r e g u l a r " s u b s t i t u e n t s i n t h e c o r r e l a t i o n of

x q p M se-

and

P

R

x

~

3,

with E q

P

Y

s u p p o r t i n g t h e a b o v e mentioned a n t i c i p a t i o n t h a t t h e components a t t r i b u t a b l e

t o t h e proximity

effects

between

similar among t h e s e 2 - s u b s t i t u t e d

the

a-substituent

diazine-x

p r o c e d u r e did n o t work. however, f o r t h e c o r r e l a t i o n between x from t h e pyridine- x

2 P M

and x

zpy

are

N-atom

and pyridine-

K ~ P Mseries

s u b s t i t u e n t s were considered

a n d amphiprotic

and

IC

very

values

The

if hydrogen-acceptable

together

was n o t v e r y low ( r

Though t h e simple =

0 94 1, t h e s h i f t s

v a l u e s f o r t h e s e hydrogen-bonding s u b s t i t u e n t s were t o o

i r r e g u l a r t o explain

3.1.2 Physicochemical Meaning of t h e C o r r e l a t i o n s : A s mentioned above, t h e p r e s e n t model r e p r e s e n t e d by E q

4, in which t h e p y r i d i n e - x ,

are u s e d as i n d e p e n d e n t v a r i a b l e s , assumed t h a t t h e (N+X) e n c e p y r i d i n e system a n d t h e second - N = with

each o t h e r

p and

p

unit in the refer-

(Y) i n t e r a c t b i d i r e c t i o n a l l y

The " b i d i r e c t i o n a l " i n t e r a c t i o n s

c o n s i d e r e d f o r t h e second -N=

(N+X) g r o u p

function

u

were,

however,

actually

o n l y with t h e s u b s t i t u e n t X b u t n o t with t h e

The i n t e r a c t i o n between t h e two N-atoms w a s t a k e n as being un-

changed. a t least within e a c h series of s u b s t i t u t e d d i a z i n e s

These assump-

t i o n s a p p a r e n t l y i n c l u d e o v e r s i m p l i f i c a t i o n s as s u g g e s t e d above, i n p a r t i c u l a r , f o r t h e 2PM. 3PD, and 4PD systems We p r e l i m i n a r i l y a t t e m p t e d t o a n a l y z e diazine- x

e a c h system

v a l u e s using x

PhX

for

The 5PM series w a s t h e o n l y one i n which a n a c c e p t a b l e c o r -

r e l a t i o n was found as shown i n E q

17 by r e g a r d i n g t h e d i a z a g r o u p (-N=(C)-N=)

a s t h e i n v a r i a b l e " s u b s t i t u e n t Y"

The c a l c u l a t e d

II

v a l u e s are also shown i n

T a b l e 7.

x n

5 P M

=

=

12

0.9 3 9 x ~ h x+ 0.5 6 2 ;~( m ) ( 0 . 188) (0. 510) r

=

0. 974

s

=

The a c c e p t a b l e q u a l i t y of E q

+

0. 126

1. 2 5 3 +~ 0. ~ 095

(0. 477)

F

=

( 0 . 188)

1171

48. 4

17 a s a c o u n t e r p a r t of Eq. 14 c o u l d b e under-

s t o o d by t h e f a c t t h a t t h e 5PM series

is

unique among o t h e r isomers i n being

170 symmetric, where t h e s u b s t i t u e n t and two N-atoms have "meta" relationships, perhaps without significant d i r e c t resonance e f f e c t s among t h e t h r e e functions. The "forward" e f f e c t of would,

in f a c t , r e p r e s e n t

i n t e r a c t i o n s between t h e two N-atoms a r e neglected. uPp

e f f e c t on s u b s t i t u e n t X in terms of proximately twice t h e e f f e c t of

u

t (m)

values in Eq.

in Eq.

each of

u

x"

in Eq.

17

t h e two N-atoms

if

X in terms of

substituent

twice t h e e f f e c t on each of

p

Likewise, t h e "backward"

17 could account f o r ap-

t h e N-atoms.

When t h e

and

p

17 a r e divided by two, t h e forward and backward ef-

fects between each p a i r of X and -N=

s u b s t i t u e n t s a r e 0.28 and 0.63. respec-

These values are c l o s e r t o t h e corresponding values, 0.26 and 0.94 in

tively.

Eq. 3 f o r

x x

values in t h e monosubstituted pyridine series on t h e basis of

z P h X . This seems t o s u p p o r t t h e relevancy of Eq. 1 in analyzing t h e substituent

II

values of N-heterocycles.

on t h e basis of

Eq. 14.

II S P Y .

p

Y

and

0

When t h e analysis of

S(m)

n,,,,

w a s made

were 0.66 and 0.42, respectively

The "enhanced" forward e f f e c t of X in Eq. 14 in terms of

p yu

in

x" ( m )

compared with 0 . 2 6 ~P in Eq. 3 could be due t o t h e e f f e c t of t h e N-atom

in

t h e (N+X) u n i t on t h e second N-atom in augmenting t h e effect of X so t h a t t h e use of t h e r e g u l a r u P would under-estimate t h e t o t a l effect of (N+X). ing t o a higher

value.

p y

lead-

The reduction of t h e backward e f f e c t could be

due t o a competition f o r e l e c t r o n s of hydrogen-bondable

s u b s t i t u e n t X be-

tween two N atoms a p p a r e n t l y non-additive in n a t u r e so t h a t t h e p

x

value in

Eq. 14 could be over-estimated, leading t o a lower u t ( m ) value. The above t y p e of i n t e r a c t i o n s among X-substituent and two N-atoms a r e c e r t a i n l y r e f l e c t e d in c o r r e l a t i o n equations o t h e r than Eq. 14 f o r t h e o t h e r diazine systems. The forward effect of X on t h e second N atom in terms of p y is higher t h a n t h a t f o r 5PM f o r systems in which t h e reference

(N+X) group

w a s t a k e n as t h e ZPY, such a s 2PR (Eq. 12). 4PM (Eq. 13), and 3PD (Eq. 15). r e v e r s e is t h e case f o r t h e 4PD system (Eq.

The

16) where t h e (N+X) group was

This t r e n d is understandable since t h e e f f e c t of X on

r e g a r d e d a s t h e 4PY.

N within t h e (N+X) group i t s e l f is highest in systems with t h e 2PY r e f e r e n c e and lowest in t h a t with t h e 4PY reference.

The higher t h e e l e c t r o n i c e f f e c t

of t h i s type, t h e g r e a t e r is t h e e x t e n t of t h e under-estimation ward e f f e c t by t h e use of t h e r e g u l a r

d

of t h e for-

P , leading t o t h e higher

The backward e f f e c t of t h e second N atom on t h e (N+X) in terms of coefficient of t h e t h e o t h e r systems. higher t h a n t h e u t p

x

P x

p

value.

u p , the

term. i s higher f o r t h e 4PM system (Eq. 13) t h a n f o r

This is understandable because t h e

u t h e t a ) f o r t h e -N=

uF(para) value i s

" s u b s t i t u e n t " , although t h e " i n t r i n s i c "

term f o r t h e e f f e c t of two N-atoms in diazine systems i s n o t additive

as mentioned above.

171 In E q

7 for t h e ZPM series. OR s u b s t i t u e n t s were w e l l i n c o r p o r a t e d . b u t

s u c h g r o u p s as SMe. N M e z a n d NHz w e r e p o s i t i v e o u t l i e r s and CONHz.

COZR were n e g a t i v e o u t l i e r s between

N H A c and

P o s s i b l e proximity steric and i n d u c t i v e e f f e c t s

X and t h e s e c o n d N atom were examined by i n t r o d u c i n g t h e Taft-

Kutter-Hansch

E,

and t h e C h a r t o n

u

I

terms f o r X. s i n g l y or t o g e t h e r , b u t The OR s u b s t i t u e n t s i n t h e 2PM

t h e y did n o t r a t i o n a l i z e t h e deviations

system would s h a r e proximity i n t e r a c t i o n s with j u s t o n e of common with t h o s e i n t h e 2PY system 2PM series. t h e

the N

atoms i n

For o t h e r outlying su b stitu e n ts in t h e

z Z P M v a l u e seems t o d e v i a t e from t h e c a l c u l a t e d v a l u e i n a

manner similar t o b u t n o t n e c e s s a r i l y t h e same as t h a t o c c u r r i n g with P a r a m e t r i z a t i o n of

t h e proximity e f f e c t s involved

h e t e r o c y c l i c compounds as

2-substituted

pyridines

K z p y

i n such 2 - s u b s t i t u t e d and

pyrimidines

N-

require

f u r t h e r elaboration

3.2 Di- and P o l y - s u b s t i t u t e d P y r a z i n e s Analysis and p r e d i c t i o n of t h e l o g P v a l u e s would b e more d i f f i c u l t f o r m u l t i - s u b s t i t u t e d d i a z i n e s , i n which a d d i t i o n a l e l e c t r o n i c and steric

interac-

2.0-

Q

1.0-

0 3

0 -

0 H

-0.5

I

I

I

I

I

I

Total Carbon Number Fig. 1. P l o t of log P a g a i n s t the c a r b o n number in a l k y l p y r a z i n e s Closed circles. 2-X-substituted p y r a z i n e s : t h e symbols r e p r e s e n t t h e X-substituents Open circles. p o l y s u b s t i t u t e d p y r a z i n e s : t h e numerals indicate t h e substituent positions S l o p e A: , S l o p e B: -------, S l o p e C: - - (Reproduced from r e f 14 with permission of t h e copywrite owner. t h e American Pharmaceutical Association)

172 t i o n s between individual p a r t n e r s of s u b s t i t u e n t s and ring-N atoms a r e generally

involved

A s a f i r s t t r i a l , we investigated possible procedures

t o t h e above mentioned bidirectional Hammett-Taft-type

similar

analyses f o r t h e log P

values of d i s u b s t i t u t e d pyrazines and extended t h e analysis t o p o l y s u b s t i t u t pyrazines (14)

ed

pyrazines

The experimentally measured log P values of

a r e l i s t e d in Tables 13-15, while t h o s e f o r

disubstituted

polysubstituted

pyra-

z i n e s are given in Table 16 In di- and poly-substituted systems. s t e r i c e f f e c t s may o p e r a t e in a

manner t h a t t h e s u b s t i t u e n t k ) n e x t t o t h e ring-N atom would

such

hinder

the

pair

elec-

solvent

molecule

trons

To test t h i s possibility. we first examined various a l k y l p y r a z i n e s

from forming a hydrogen bond with t h e N-lone

in

which t h e e l e c t r o n i c c o n t r i b u t i o n was thought t o be very low Log P values including mono- and poly-alkylsubstituted plotted shown

against in

Fig

pyrazines were

t h e t o t a l carbon number contained in t h e a l k y l 1

F o r t h e s e r i e s of 8-n-alkyl

substituted

chains

pyrazines,

p y r a z i n e t o 2-Bu-pyrazine, t h e increment per C1-unit was very r e g u l a r , mated

a s 048+0 01 (slope A)

When a methyl group was introduced

as from

esti-

in

their

o r t h o position, t h e increment for each alkylpyrazine was 0 3810 03 h = 4 , s l o p e

B).

which

was

lower t h a n t h a t f o r monoalkyl

pyrazines

With

successive

methyl s u b s t i t u t i o n s a t t h e f o u r available pyrazine positions, t h e log P v a l u e increased almost r e g u l a r l y from mono-Me t o Me4 derivatives (omitting with

a s l o p e (C.036i004) c l o s e t o s l o p e B

2,5-Mez)

These r e s u l t s suggest t h a t

di-

s u b s t i t u t i o n s of f a i r l y bulky groups a t t h e 2,3- and Z6-positions. b u t n o t t h e 2,5-position have s t e r i c e f f e c t s

3.2.1 C o r r e l a t i o n Analyses for Disubstituted Pyrazines: The a n a l y s e s were made f o r t h e

A

( d i s u b s t ) ~value ~ defined by E q

For comparison. t h e sum of t h e corresponding s u b s t i t u e n t from

( 2K 15

monosubstituted

It

pyrazines

(2A

zPR ) ,

and 2-substituted pyridines ( 2A

P h X ) ,

monosubstituted

benzenes

a r e presented in Tables 13-

Although t h e s i t u a t i o n is considerably improved with

d e v i a t i o n s from t h e observed values a r e still g r e a t decreased when t h e

XZPR

P

~

A ~ P Y .the

The deviations a r e much

values a r e used, although t h e a d d i t i v i t y t e n d s t o

r e s u l t i n over-estimation of values JC

A

of t h e log P v a l u e f o r most com-

v a l u e s r e s u l t s in g r e a t under-estimation

pyrazine

values derived

c l e a r l y seen t h a t t h e prediction based on t h e a d d i t i v i t y of

IS

pounds

Z P Y )

A

18

Thus, we s e l e c t e d t h e monosubstituted

values a s t h e r e f e r e n c e parameter

For applying E q

1 t o analyze t h e

A

( d i s u b s t ) p R values, some modifica-

X

173 t i o n s were r e q u i r e d

In u s e of

( d l s u b s t ) p R values r e l a t i v e t o t h e log P

II

v a l u e of t h e u n s u b s t i t u t e d p y r a z i n e . no d i s t i n c t i o n was made between X and Y The a n a l y s e s were

s u b s t i t u e n t s as t o which is fixed and which is v a r i a b l e made

for

individual

t y p e s of

disubstituted

pyrazines

t r e a t t h e sum of t h e b i d i r e c t i o n a l e l e c t r o n i c terms, a n i n d e p e n d e n t v a r i a b l e . as shown i n E q

where 2

r e p r e s e n t s t h e sum of

II 2~~

II

we

First, p yu

P and

tried

u pp

to as

19,

f o r X and Y s u b s t i t u e n t s .

ZPR

In t h i s

t r e a t m e n t , b i d i r e c t i o n a l i n t e r a c t i o n s are assumed t o be p r o p o r t i o n a l t o t h o s e i n m-

and p - d i s u b s t i t u t e d b e n z e n e s

The r e g r e s s i o n c o e f f i c i e n t "b" c o u l d b e

a n i n d i c a t i o n of t h e " t r a n s m i t t i n g e f f i c i e n c y ~ 'of t h e b i d i r e c t i o n a l i n t e r a c t i o n s of s u b s t i t u e n t s compared with t h e c a s e of t h e c o r r e s p o n d i n g l y d i s u b s t i t u t e d benzenes

Preliminary

analyses

of

the

values

II

d i s u b s t i t u t e d series l i s t e d in Tables 13 and 14 with

for

v a l u e s i n T a b l e 1. however. demonstrated t h a t Eq

and p

the

u X and

2.6- and 2.5u

constants

19 w a s n o t s u f f i c i e n t

t o s i m u l a t e t h e s i t u a t i o n in "meta" and " p a r a " d i s u b s t i t u t e d p y r a z i n e s W e n e x t t r i e d t o r e p r e s e n t t h e e l e c t r o n i c e f f e c t s by i n d u c t i v e and resou

n a n c e components s e p a r a t e l y using the

P X U I C Y ) ,

P Y U I C X ) ,

I

P Y ~ R ( X ) ,

and

u

and

P X ~ R C Y ,

electronic constants

With

terms, a n a l y s e s were

made by c o n s i d e r i n g whether t h e s u b s t i t u e n t s are amphiprotic (H-donor) or Hacceptor H-donor

W e found t h a t t h e s e t e r m s w e r e s t a t i s t i c a l l y signiflcant only f o r

X and Y s u b s t i t u en t s

In o t h e r words,

v a l u e s f o r H-acceptors

p

c o u l d b e r e g a r d e d as z e r o l i k e t h o s e for non-hydrogen-bonders

w a s t r a n s f o r m e d t o Eq

where p

20,

is t h e o r i g i n a l l y r e p o r t e d P

(AM,

Then, Eq. 19

v a l u e f o r amphiprotic g r o u p s b u t

is t a k e n t o b e z e r o f o r H-acceptors and non-hydrogen b o n d e r s (Table 1). a n d

2

u

p :An,

represents

for amphiprotic X and Y

+

P

:An,

Analysis of a l l t h e 2.6-disubstituted

R

( 2 , 6-PR) v a l u e s i n Table 13 yield-

p

1

Or

R

0 ;

Or

R

0 :

Or

R

substituent pairs

ed Eq. 21

II

( 2 , 6-PR)

=

1. 0 0 8 2

R z p R

+

0. 6 1 5 2 p

(AM) u I

(0.4 8 4 )

(0.0 6 3 ) +

0. 5832 P

(AM,

u

(0.2 5 0 ) n

=

39

r

=

0.987

s

=

0. 125

R

-

0 . 103

(0.1 0 8 ) F B . 35

=

446.2

1211

TABLE 13 Hydrophobicity Parameters of 2.6-Disubstituted Pyrazines (2.6-PR) K

( 2 , 6-PR)

Substituents

x, Y

C1,

F

c1, C1. C1. C1. C1.

c1 Me OMe OEt NH2 NHAc CN C02Me CONHz NMe2

C1. C1. C1. C1, C1, C1. OPr Me, Me Me, OMe Me, N H 2 Me. N H A c Me, C N Me, C02Me Me, COzEt Me. C O N H 2 Me. NMez Me, O P r

log P

Obsd.

Calcd.”’

1 . 15 1. 5 3 1. 03 1. 6 5 2. 2 2 0. 9 5 1. 10 0. 79 0. 4 7 0. 28 1. 9 5 2. 71 0. 5 4 1. 2 9 0 . 35 0 . 38 0. 4 4 0. 1 0 0. 51 -0.13 1. 57 2 . 38

1.41 1.79 1.29 1. 9 1 2. 4 8 1. 2 1 1. 3 6 1. 05 0. 7 3 0. 5 4 2. 21 2. 97 0.80 1. 5 5 0. 6 1 0. 6 4 0. 70 0. 36 0. 7 7 0. 1 3 1. 8 3 2. 64

1.47 1.82 1. 3 4 1 . 88 2. 4 0 1. 2 1 1. 2 5 1. 18 0. 8 2 0. 61 2. 07 2. 9 3 0. 85 1. 4 1 0.5 7 0. 57 0. 70 0 . 30 0.78 -0. 01 1. 5 9 2. 46

dev. -0. -0. -0. 0.

06 03 05 03 0. 0 8 0. 0 0 0 . 11 - 0 . 13 -0. 0 9 -0. 07 0. 1 4 0. 04 -0. 05 0. 1 4 0. 0 4 0. 07 0. 00 0. 06 -0. 01 0. 14 0. 24 0. 18

Z x 2 p R b ’ dev. 1 . 51 1. 9 2 1.43 1. 95 2. 50 1. 1 7 1. 1 9 1.21 0. 9 9 0. 72 2. 15 3. 06 0.94 1 . 46 0.68 0. 7 0 0. 7 2 0. 50 1. 0 1 0. 2 3 1. 6 6 2. 5 7

-0. 10 13 14 04 02 0. 04 0. 1 7 -0. 16 -0. 26 - 0 . 18 0. 06 -0.09 -0.14 0. 0 9 -0.07 -0. 06 -0. 02 -0. 1 4 -0.24 -0. 1 0 0. 17 0 . 07

-0. -0. -0. -0.

Zxphxc’ 0 . 85 1. 4 2 1. 27 0. 6 9 1. 0 9 -0. 5 2 -0. 2 6 0. 1 4 0. 70 - 0 . 78 0. 8 9 1. 76 1. 1 2 0. 5 4 -0. 67 -0.41 -0. 0 1 0.55 1. 07 -0. 9 3 0. 74 1. 6 1

dev.

0. 5 6 0. 3 7 0. 0 2 1. 2 2 1. 3 9 1. 7 3 1. 6 2 0. 9 1 0. 0 3 1. 32 1. 3 2 1. 2 1 -0. 32 1. 0 1 1. 28 1. 0 5 0. 7 1 -0.19 -0. 30 1. 0 6 1. 0 9 1. 0 3

Zzapyd’

0 . 81 1 . 35 1. 0 8 1 . 31 1. 7 8 0. 4 5 0. 4 9 0. 37 0 . 33 0. 1 2 1. 6 2 2. 3 5 0. 9 2 1 . 15 0. 29

0 . 33 0. 21 0. 1 7

0. 6 8 -0. 04 1. 4 6 2. 19

dev. 0. 0. 0. 0. 0.

60 44 21 60 70 0. 7 6 0. 8 7 0. 6 8 0. 4 0 0. 42 0. 59 0. 6 2 -0. 1 2 0. 40 0. 3 2 0. 31 0. 4 9 0. 19 0. 09 0. 1 7 0. 3 7 0. 45

OMe. OMe OMe. O E t OMe. N H 2 OMe. NHAc OMe, C N OMe. C02Me OMe. C O z E t OMe, C O N H 2 OMe. N M e . 2 C N . NMez NMe2. CO2Me NMe2. C 0 2 E t NMe2. C O N H 2 CONHz. O E t F. F OEt, OEt NH2, NH2

1. 5 8 1. 9 8 0.73 0. 82 0.95 0 . 69 1 . 20 0. 13 1. 99 1 . 12 0 . 90 1. 24 0. 38 0. 61 0 . 74 2. 55 -0.45

1.84 2. 24 0.99 1. 08 1.21 0. 95 1. 4 6 0. 39 2. 2 5 1. 38 1. 1 6 1. 5 0 0. 64 0.87 1.00 2.81 -0.19

1. 93 2.45 1.02 1. 0 0 1.23 0. 93 1. 42 0. 58 2. 1 2 1. 41 1. 11 1. 59 0. 6 3 1. 10 1.10 2. 97 -0.22

-0. 09 -0. 21 -0.03 0. 08 -0.02 0 . 02 0 . 04 -0. 19 0 . 13 -0. 03 0. 05 - 0 . 09 0. 01 -0. 23 -0.10 -0. 16 0. 03

1. 9 8 2. 5 3 1.20 1.22 1. 24 1.02 1.53 0. 75 2. 1 8 1. 4 4 1. 2 2 1.73 0.95 1. 3 0 1.10 3. 08 0.42

- 0 . 14 -0. 29 -0.21 -0.14 -0. 0 3 -0.07 -0.07 -0. 36 0. 07 - 0 . 06 - 0 . 06 -0.23 -0.31 -0. 43 -0.10 - 0 . 27 -0.61

- 0 . 04 0 . 36 -1.25 -0.99 -0.59 -0. 03 0. 49 -1. 51 0. 16 -0. 39 0. 17 0. 69 -1.31 -1. 11 0. 28 0. 76 -2. 46

1. 8 8 1. 8 8 2. 24 2.07 1. 8 0 0. 98 0. 97 1. 9 0 2. 09 1. 7 7 0. 99 0 . 81 1. 9 5 1. 98 0. 72 2. 05 2. 27

1 . 38 1. 8 5 0. 52 0. 56 0. 44 0 . 40 0. 91 0. 19 1. 6 9 0. 75 0. 7 1 1. 22 0 . 50 0. 66 0. 38 2. 32 -0.34

0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0.

46 39 47 52 77 55 55 20 56 63 45 28 14 21 62 49 15

a) C a l c u l a t e d b y E q . 23. F o r example, t h e v a l u e for 2-C1-6-NH2-pyrazine 1s c a l c u l a t e d as f o l l o w s : H (2-Cl-6-NHz) = 0 . 9 4 7 { H z p n ( C l ) + a z p n ( N H z ) } + 0. 5 6 8 ( p ( A M ) ( N H ~ ) * u I ( C ~ + ) P ( A M ) ( C ~ ) * ~ I ( N H ~ ) } + 0 . 6 8 0 ( p ( A M ) ( N H 2 ) * o , ( C l ) + P ( A M ) ( C l ) * o , ( N H z ) } - 0 . 080Ee(C1)*E,(NH2) + 0 . 078 = 0. 9 4 7 ( 0 . 9 6 + 0 . 2 1 ) + 0 . 5 6 8 ( 0 . 7 4 * 0 . 4 7 + 0) + 0 . 6 8 0 { 0 . 7 4 ( - 0 . 2 5 ) + 0) - 0 . 0 8 0 ( - 0 . 9 7 ) (-0.6 1 ) + 0 . 0 7 8 = 1 . 2 1 b) For t h e H 2 P R v a l u e of t h e component s u b s t i t u e n t s . see T a b l e 4. c) F o r t h e H~~~ v a l u e of t h e component s u b s t i t u e n t s . see T a b l e 1. d) For t h e H 2 p y v a l u e of t h e component s u b s t i t u e n t s . see T a b l e 1.

176 Although E q

21 seems t o b e s t a t i s t i c a l l y a c c e p t a b l e , examinations of

r e s i d u a l s showed t h a t t h e

v a l u e s of

K

compounds with bulky s u b s t i t u e n t s

s u c h as COzR a n d CONHz g a v e s i g n i f i c a n t n e g a t i v e d e v i a t i o n s

Moreover, t h e

l e v e l of s i g n i f i c a n c e of t h e i n t e r c e p t w a s c l o s e t o 95% in E q

21 These facts

were

effect

thought

Lo

reflect

a

contribution

d i s u b s t i t u t i o n i n lowering t h e derivatives

E

(11)

of

the

steric

of

2.6-

v a l u e as described above f o r 2.6-dialkyl

K

This c o n t r i b u t i o n was examined with u s e of t h e steric 7arameter Eq

ZE, ( 2 , 6 ) v a l u e as an a d d i t i o n a l term

22 o b t a i n e d with t h e

For E, v a l u e s of p l a n a r r-bonded s u b s t i t u -

w a s s l i g h t l y b e t t e r t h a n E q 21

e n t s . t h e v a l u e s f o r t h e steric effect on t h e c o p l a n a r , b u t n o t t h e perpend i c u l a r d i r e c t i o n were used x ( 2 , 6-PR)

=

0. 9502 +

0. 5692

+

K ZPR

(0.0 7 0 )

0. 6 7 1 2 ~ CAM)

+

O R

(0.2 3 5 ) r

n = 39 In E q

0.990

=

u

(AM)

u

I

(0.4 4 0 )

s

0. 0722E, (0.0 5 0 )

0. 1 1 3

=

+

0 . 140 ( 0 . 193)

1221

411. 1

=

22, however, t h e i n t e r c e p t t u r n e d o u t t o be p o s i t i v e and t h e d e v i a t i o n

from z e r o w a s i n c r e a s e d from t h a t i n Eq. 21. effect

of

2.6-substituents.

being

This suggested t h a t t h e steric

overestimated f o r s u b s t i t u e n t s

of

smaller

size, d i d n o t o p e r a t e e x a c t l y additively. b u t p r o p o r t i o n a l l y with t h e bulk of t h e two s u b s t i t u e n t s To e x p r e s s t h i s s i t u a t i o n , t h e cross-product

of

E,(X)

and E,(Y),

Es',

would b e r e l e v a n t , s i n c e it t a k e s v a l u e s c l o s e t o z e r o when one or b o t h of t h e s u b s t i t u e n t s X a n d Y idare) small enough, b u t it i n c r e a s e s Lo a signific a n t l y l a r g e size when b o t h X and produced E q 23.

Y are large

The a d d i t i o n of t h e E,'

term

Although t h e q u a l i t y of t h e c o r r e l a t i o n w a s almost t h e same

as t h a t of E q 22, t h e i n t e r c e p t of E q 23 was much c l o s e r t o z e r o (2. 6-PR)

=

0. 9 4 7 2 II (0.0 7 2 ) -

2PR

0. 080E,'

+

(0.0 5 7 ) n

=

r

39

=

0 . 990

s

=

O. 5682 P (0.443)

+

(AM)

U I

+

0. 6 8 0 2 p

(AM)

0 . 078 ( 0 . 161)

0. 1 1 3

u

R

(0.2 3 8 ) [231

F4. 3 4

=

405. 7

I t is e x p e c t e d t h a t t h e a n a l y s i s f o r 2.5-disubstituted p y r a z i n e s c o u l d b e d o n e in a manner similar t o Eq. 21, s i n c e no s i g n i f i c a n t steric effect would b e induced i n t h e r e l a t i v e s o l v a t i o n of t h e ring-N atoms by i n t r o d u c i n g t h e s e c o n d s u b s t i t u e n t in t h e " p a r a " position. a n d 25 of methoxy

approximately

derivative

was

equivalent omitted

For t h e d a t a in Table 14, Eqs. 24

quality

from t h e

were formulated.

correlation

analyses,

The 2.5-dibecause

it

177 showed l a r g e d e v i a t i o n s i n a l l preliminary t r i a l s

A

( 2 , 5-PR)

0. 9622

=

0. 4752 P

A PPR

(0.0 7 0 ) n

A

n

r

15

=

(2, 5 P R )

15

=

=

0. 994

s

0.9 5 4 2

=

=

K Z P R

+

=

12

0. 5 6 2 2 ~ (AM) O

0. 994

s

0. 0 8 2

R

=

12

493.6

From t h e s t a t i s t i c a l p o i n t of view. it is d i f f i c u l t two e q u a t i o n s 2 p

=

-0 78) among t h e s e f i f t e e n compounds

cal p o i n t of view. however, w e c o n s i d e r t h a t E q

of

the

t o choose between t h e s e

There was a f a i r l y high c o l i n e a r i t y between

(r

u

[251

(0.1 0 9 )

Fz.

0. 077

=

1241

473. 7

=

(0.3 6 2 )

(0.0 6 9 )

r

0. 096 (0.1 0 9 )

(AM) u I

(0.3 2 0 ) 0. 0 7 9 Fz,

2p u

term

is p o s i t i v e ,

2 p

(AM)

u

I

and

From t h e physicochemi-

25, i n which t h e c o e f f i c i e n t

is p r e f e r a b l e

The

electron-withdrawing

effect of s u b s t i t u e n t s t e n d s t o i n c r e a s e t h e a c i d i t y of t h e second s u b s t i t u e n t s . enhancing t h e s o l v a t i o n with 1-octanol, which is and l e a d i n g t o a h i g h e r The b e h a v i o r of t h o s e of from

A

A

( 2 , 5-PR)

m o r e b a s i c t h a n water,

value

A

( 2 , 3-PR)

and

w a s e x p e c t e d t o b e more complicated t h a n

(2. 6-PR),

A

steric i n t e r f e r e n c e as w e l l

because it c o u l d i n v o l v e components possible

as

proximity

electronic

effects

between s u b s t i t u e n t s

Since t h e compounds i n o u r 2,3-PR series d o n o t in-

c l u d e hydrogen-donor

s u b s t i t u e n t s , t h e c o r r e c t i o n for t h e e l e c t r o n i c e f f e c t s

2 p c

i n t e r m s of

2 p

and

AM) u I

(AM) 0 R

was of

no significance

Further

measurements a r e needed t o o b t a i n a g e n e r a l i z e d c o r r e l a t i o n e q u a t i o n t h a t c o u l d a p p l y t o a wider r a n g e of t h e analysis for

A

n

( 2 . 3-PR) =

=

A

1. 0 6 0 2 A z p ~

(0.1 2 6 ) r = 0. 9 8 4

13

2,3-disubstituted pyrazines

( 2 . 3 - P R ) v a l u e s i n Table 15 y i e l d e d Eq 0. 125

C261

(0.2 1 4 ) s

=

0. 131

FI.

1 1

=

343. 3

The c o r r e l a t i o n was improved by t h e a d d i t i o n of t h e E,' a n a l y s i s of

A

( 2 , 3-PR)

=

A

(2. 6-PR),

0. 9 9 2 2

=

The

r

13,

p u

I

=

0. 992.

XZPR

-

0 . 050E,'

+

(0.0 3 4 ) s

=

0 . 096, F P .

term for hydrogen-accepting

test for a p a r t i c i p a t i o n

t e r m as done i n t h e

and gave Eq. 27,

(0.1 0 4 ) n

Nevertheless,

26

=

0 . 082 (0.2 1 3 )

1271

327. 1

s u b s t i t u e n t s was added t o Eq

27 t o

of t h e proximity e l e c t r o n i c (inductive) e f f e c t s be-

tween 2- and 3 - s u b s t i t u e n t s . b u t no improvement was observed.

TABLE 14 Hydrophobicity Parameters of 2,5-Disubstituted Pyrazines (2,5-PR)

K

(2.5-PR)

Substituents

x. Y

c1. C1. C1, C1. C1. C1. C1. C1, Me, Me, Me, Me, Me, OMe, OMe. OMe,

c1 Me OMe

OEt NHz NHAc CN NMez Me CN COzMe COZEt CONHZ OMe NH2 NMe2

log P

Obsd.

1. 58 1. 08 1. 52 1. 99 0. 67 0. 56 0.9 2 1. 70 0. 63”’ 0. 26 0. 17 0. 61 -0.25 1. 14 0. 63 1. 65

1. 84 1 . 34 1. 78 2. 25 0. 93 0.82 1. 18 1. 96 0.89 0.52 0. 43 0. 87 0.01 1. 40f’ 0. 89 1.91

Calcda’

dev.

1. 75 1. 28 1. 78 2.30 0. 93 0. 92 1. 07 1. 97 0.82 0.61 0 . 40 0.88 0.10 1.81 0. 82 2.00

0. 09 0. 06 0. 00 -0. 05 0. 00 -0. 10 0. 1 1 -0.01 0. 07 -0.09 0. 03 -0.01 -0.09 -0.41 0.07 -0.09

Z n

Z p ~ b ’

1. 92 1.43 1.95 2. 50 1. 17 1.19 1.21 2. 15 0. 94 0. 72 0. 50 1.01 0. 23 1. 98 1.20 2. 18

dev

-0. 08 -0.09 -0.17 -0. 25 -0. 24 -0.37 -0.03 -0.19 -0.05 -0.20 -0. 07 -0.14 -0. 22 -0. 58 -0.31 -0. 27

2

K PhXC’

1. 42 1. 27 0. 69 1. 09 -0.52 -0. 26 0. 14 0. 89 1.12 -0.01 0.55 1. 07 -0.93 -0.04 -1.25 0. 16

dev.

0. 42 0. 07 1. 09 1. 16 1. 45 1. 08 1. 04 1. 07 -0.23 0. 53 -0.12 -0.20 0 . 94 1. 44 2. 14 1. 75

2

zpyd’

1. 24 1. 08 1. 31 1. 78 0. 45 0. 49 0. 37 1. 62 0. 92 0. 21 0. 17 0. 68 -0.04 1. 38 0. 52 1. 69

dev.

0. 60 0. 26 0. 47 0. 47 0. 48 0. 33 0. 81 0. 34 -0.03 0. 31 0. 26 0. 19 0. 05 0. 02 0. 37 0. 22

-~ ~~

a) Calculated by Eq. 25. For example, t h e value f o r 2-0Me-5-NHZ-pyrazine 1s c a l c u l a t e d a s follows: n ( 2 - 0 M e - 5 - N H Z ) = 0. 954{n z p R (OMe) + K z p ( N~ H 2 ) } + 0 . 562{p ( A M ) ( N H z ) * U R (OMe) + p ( A M ) (OMe) * - 0 . 082 = 0. 954(0. 99 + 0. 21) + 0. 562{0.74(-0. 57) + 0) 0. 082 = 0.82 b) For t h e n Z P R value of t h e component s u b s t i t u e n t s , see Table 4. c) For t h e K P h X value of t h e component s u b s t i t u e n t s , see Table 1. d) For t h e n z p y value of t h e component s u b s t i t u e n t s . see Table 1. e ) From ref. 6. f ) Omitted from t h e a n a l y s i s b u t calculated by Eq. 25. -

u

R

(NHz) }

TABLE 15 H y d r o p h o b i c i t y P a r a m e t e r s of 2 . 3 - D i s u b s t i t u t e d P y r a z i n e s (2.3-PR)

R

Substituents Y

x.

Me, Me, Me, Me,

Me

Et

Pr n-Bu Et. Et M e , OMe Me, O E t Me, O C H M e z E t . OMe Me, SMe CN. CN COZMe. COzMe COzEt. C O z E t

log P 0. 54 1. 0 7 1. 5 7 2. 1 0 1 . 51 1. 2 4 I. 82 2. 24 1. 8 0 1. 81 0. 38 -0. 02 0. 65

Obsd. 0.80 1. 3 3 1.83 2. 36 1. 77 1. 5 0 2. 08 2.50 2. 06 2. 07 0. 64 0. 24 0. 91

( 2 . 3-PR) Calcda’

dev.

0.94 1.41 1.87 2. 38 1.88 1. 50 2. 04 2.54 1. 9 7 1. 9 0 0. 56 0.26 0. 8 4

-0. 14 -0. 08 -0. 04 -0. 02 - 0 . 11 0. 00 0. 04 -0.04 0. 09 0. 17 0. 08 -0. 02 0. 07

Z

K 2pRb’

0.94 1. 4 2 1.90 2. 4 2 1.90 1. 4 6 2. 0 1 2.51 1. 9 4 1. 9 0 0. 5 0 0. 50 1. 0 8

dev. -0.14 -0. 09 -0.07 -0. 06 -0.13 0. 04 0. 07 -0. 01 0. 12 0. 17 0. 14 -0. 26 -0. 17

Z

A PhXC’

1. 1 2 1. 5 8 2 . 11 2. 6 9 2. 04 0. 54 0. 94 1. 4 1 1. 0 0 1. 1 7 -1. 1 4 -0. 02 1.02

dev.

-0. 32 -0. 25 -0. 28 - 0 . 33 -0. 27 0. 96 1. 14 1. 0 9 1. 06 0. 90 1. 7 8 0. 26 -0.11

Z

dev.

A 2pyd’

0. 92 1.41

-0. 12 -0. 08

-e

-

-e

-e

1.90 1. 1 5 1. 6 2

e

-0.13 0. 3 5 0. 4 6

-e

-e

0. 4 2 0. 55 1. 1 4 0.82 0. 4 7

1. 6 4 1. 5 2 -0.50 -0.58 0. 44

~~

a) C a l c u l a t e d b y Eq. 27. F o r e x a m p l e . t h e value f o r 2-Me-3-OMe-pyrazme IS calculated as f o l l o w s : I[ ( 2 - M e - 3 - O M e ) = 0 . 9 9 2 I Z~P R ( M ~ ) + A ZPR (OMe) } - 0 . 0 5 0 E , (Me) *E, ( O M e ) + 0. 0 8 2 = 0. 9 9 2 ( 0 . 47 - 0 . 0 5 0 ( - 1 . 2 4 ) ( - 0 . 5 5 ) + 0 . 0 8 2 = 1. 50 b) For t h e X ~ P Rv a l u e of t h e c o m p o n e n t s u b s t l t u e n t s . see T a b l e 4. c) F o r t h e z p h X v a l u e of t h e c o m p o n e n t s u b s t i t u e n t s , see T a b l e 1. d) For t h e R Z P Y v a l u e of t h e c o m p o n e n t s u b s t l t u e n t s , see T a b l e 1. e) T h e c o m p o n e n t n 2 p Y v a l u e s are unknown.

+

0. 9 9 )

180 The c a l c u l a t e d v a l u e s according t o E q s

23. 25 and 27 a r e l i s t e d in

Tables 13-15

3.2.2 Physicochernical Meaning of t h e Correlations: The r e s u l t s described above demonstrated t h a t t h e l o g P value of d i - s u b s t i t u t e d p y r a z i n e s having non-amphiprotic s u b s t i t u e n t s could be approximately p r e d i c t e d by t h e additivi t y model of

k 2 P R

v a l u e s provided t h e i r steric effects a r e n o t s i g n i f i c a n t

The b i d i r e c t i o n a l e l e c t r o n i c c o r r e c t i o n terms expressed by p u

p u

and/or

I

a r e needed o n l y f o r amphiprotic substituents(H-donors), possibly because t h e electron-withdrawing

effect of

r i n g N-atoms in t h e pyrazine to

ring tends t o

enhance t h e hydrogen-donating

ability but

reduce t h e proton-accepting

c a p a b i l i t y of t h e s u b s t i t u e n t s

The n e t hydrogen-donating a b i l i t y of c e r t a i n

amphiprotic g r o u p s could be higher t h a n t h a t in s u b s t i t u t e d benzene systems Thus, t h e p

For t h e hydrogen-

v a l u e is s i g n i f i c a n t f o r amphiprotic groups

a c c e p t o r s with lower o r i g i n a l

values. t h e s i t u a t i o n could be d i f f e r e n t

p

Under s u c h conditions, hydrogen a c c e p t o r s would behave as if t h e y were nonis z e r o o r v e r y c l o s e t o zero.

hydrogen b o n d e r s in which t h e "effective" p

T h e r e f o r e , o n l y t h e terms f o r t h e amphiprotic s u b s t i t u e n t s p a r t i c i p a t e i n t h e

20

c o r r e l a t i o n of t h e t y p e of Eq

I t should be noted t h a t t h e bR v a l u e is comparable with br in E q f o r 2.6-PR

Considering t h a t

t h e resonance

importance f o r meta-derivatives.

almost e q u i v a l e n t with t h a t from t h e p u intervention

through

the

ring-N

Eq

P

s u b s t i t u e n t s a r e " p a r a " t o each o t h e r 23 is c l o s e t o t h a t in E q

O R

by

the

two

those

having

two

because

The reason why t h e bR v a l u e i n

25 is n o t c l e a r bulky

minor

substituents

term is significant,

In f a c t . however, examination

of U V s p e c t r a in water showed t h a t t h e absorption of most 2.6-PR

including

of

t e r m t h a t is

p U R

effect might i n d i c a t e a resonance

I

atom sandwiched

For 2.5-PR, it is n o t unexpected t h a t t h e the

is g e n e r a l l y

effect

a c o n t r i b u t i o n from t h e

23

groups.

was

almost

derivatives,

identical

with,

s l i g h t l y more bathochromic t h a n t h a t of t h e corresponding 2.5-PR

but

compounds,

s u g g e s t i n g n o s i g n i f i c a n t d i f f e r e n c e in t h e resonance s t r u c t u r e between t h e two series In any case, considering t h a t

u

=

u

I

+

R

and

u

u

=

+

I

( 1 0 1 , t h e o b s e r v a t i o n t h a t t h e br and bn c o e f f i c i e n t s of t h e ., p u

"

0 4u

t e r m s in

a l l t h e e q u a t i o n s obtained were considerably smaller t h a n u n i t y lead u s t o conclude t h a t t h e p a r t i t i o n i n g behavior in t h e pyrazine system is p e r t u r b e d by t h e X-Y e l e c t r o n i c i n t e r a c t i o n s t o a "lesser" e x t e n t t h a n in t h e benzenoid system

This means t h a t

s u b s t ) PR

ring-N

some of

v a l u e s including t h a t

t h e e l e c t r o n i c component attributable

atoms and s u b s t i t u e n t s has been

reference

t o the

simulated

by

in t h e

x (di-

i n t e r a c t i o n between using

K

z P R as

the

The i n t r o d u c t i o n of b u l k y s u b s t i t u e n t s d e c r e a s e d t h e l o g P v a l u e i n t h e and 2,6-PR series

2,3-

water molecule

Besides t h e fact mentioned above t h a t t h e smaller

is more e a s i l y

a c c e s s i b l e t o t h e crowded ring-N

atom,

the

steric h i n d r a n c e t o t h e a t t a i n m e n t of a p l a n a r conformation might b e considThis p o s s i b i l i t y c o u l d p r o b a b l y b e eliminated. however, a t least i n 2.6-

ered

PR. j u d g i n g from t h e a b o v e mentioned U V spectral f e a t u r e s

2.5-Dimethoxypyrazine was t h e o n l y o u t l i e r among 68 compounds examined here

I n t e r e s t i n g l y , t h e summation of

g a v e a b e t t e r p r e d i c t i o n t h a n t h a t of a n a l y s i s of

(Eq

3)

K

2py,

~ Z P Yv a l u e s

K PPR,

for t h e two OMe g r o u p s

as shown i n Table 14

g r o u p was shown t o b e o n e of

t h e 2-OMe

Our e x p l a n a t i o n is t h a t 2-methoxy-pyridine

In t h e

the outliers

u n d e r g o e s a 1-to-1

che-

s o l v a t i o n i n water b u t n o t i n o c t a n o l t h a t enhances t h e log P

lating-type

value irregularly

Since t h e e f f e c t of s u c h s o l v a t i o n o c c u r r i n g a t b o t h of

two methoxy s u b s t i t u e n t s c o u l d also be simulated on t h e b a s i s of

KZPR.

no

r e a s o n a b l e e x p l a n a t i o n was made o n t h e behavior of t h i s compound

3.2.3 P o l y s u b s t i t u t e d P y r a z i n e s : The empirical c o r r e l a t i o n e q u a t i o n s f o r di-substituted

pyrazine

n

v a l u e s were a p p l i c a b l e t o p r e d i c t

of i n t e r a c t i o n s (2,6-,

t h o s e of t h e

Taking i n t o a c c o u n t a l l t y p e s

p o l y s u b s t i t u t e d p y r a z i n e s given i n Table 16

2.5- and 2 , 3 - i n t e r a c t i o n s ) , t h e g e n e r a l form of t h e p r e -

d i c t i o n e q u a t i o n is w r i t t e n a s E q

28

TABLE 16 Hydrophobicity P a r a m e t e r s of P o l y s u b s t i t u t e d P y r a z i n e s (Poly-PR)

Substituents log P

Obsd.

Calcd?’

H

0 . 95

Me

1. 2 8 1. 9 5 1 . 50

1. 2 1 1 . 54 2. 2 1 1. 7 6 1.21 1.55 2 . 05 2. 56 3. 1 5

1. 1 9 1. 44 2. 1 0 1. 6 9 1.35 1.70 2. 12 2. 51 3. 04

x2

x3

x5

XI3

Me Me

Me Me Et C1 CN CN CN CN CN

Me Me Me

Et Me

CN CN CN CN CN

Me C1 Me Et

Me Bu

H H H C1 C1 Bu C1

0 . 95 1. 2 9 1. 7 9 2 . 30 2. a 9

dev.

I: K

0. 0 2

2pRb)

1.41 1.88 2. 37 1.90 1.46 1.93 2.41 2. 92 3.41

0. 1 0

0. 11 0. 0 7 -0.14 -0.15 - 0 . 07 0 . 05 0 . 11

dev.

-0. 20 - 0 . 34 -0.42 -0.14 -0.25 -0.38 - 0 . 36 - 0 . 36 - 0 . 25

a) C a l c u l a t e d by Eq. 28. For example, t h e v a l u e f o r Z3-di-CN-5-Me-6-CIp y r a z i n e is c a l c u l a t e d as follows: n ( C N , C N , Me. C1) = n ( 2 . 3 - P R ) (CN. C N ) + n (3. 5 P R ) ( C N , Me) JT ( 5 . 6 - P R ) (Me. CI) n ( 2 . G P R ) ( C l , CN) K ( 2 . 5 - p ~ () C N , Me) + (3. 6 P R ) (CN, C l ) - 2(2* ~ z P R ( C N ) n2PR(Me) a Z P R ( C l ) } = Eq. 27(CN. C N ) + Eq. 23(CN, Me) + Eq.27(Me8CI) Eq.23(CI,CN) Eq.25(CNSMe) Eq.25(CN,C1) 2(2nZPR(CN) + nzzpR(Mf?) z z p n ( c 1 ) ) = 0. 56 0.70 1 . 4 5 + 1. 18 + 0 . 6 1 + 1 . 0 7 - 2 ( 2 * 0 . 25 + 0.47 0 . 9 6 ) = 1. 70, i n whlch Eq.N(X,Y) r e p r e s e n t s t h e II v a l u e c a l c u l a t e d by Eq. N f o r X.Y-disubstituted p y r a z i n e s . b) F o r t h e n 2 P R v a l u e o f t h e component s u b s t i t u e n t s , see Table 4. +

+

+

+

+

+

+

+

+

+

+

+

~

182

R

(poly-PR)

r e p r e s e n t s t h e difference in l o g P values between t h e given

compound and t h e u n s u b s t i t u t e d pyrazine, a v a i l a b l e positions, and

K (1,

one of t h e c o r r e l a t i o n E q s t i o n s of XI and X,

J-PR)

X is any s u b s t i t u e n t on t h e f o u r

is t h e value c a l c u l a t e d with t h e use of

23, 25 and 27. depending on t h e r e l a t i v e o r i e n t a is t h e sum of

R 2 P R ( X )

R ZPR

values o v e r a l l s u b s t i t

uents. and n is 1 and 2 f o r tri- and t e t r a - s u b s t i t u t e d derivatives, r e s p e c t i v e ly

By t h e addition of

x (I,

J-PR)

in E q

28, we counted each

R

(X-PR)

one

o r two times e x t r a depending on whether t h e compound is t r i s u b s t i t u t e d or

t e t r a - s u b s t i t u t e d , so t h e negative c o r r e c t i o n was needed

The c a l c u l a t e d

l o g P v a l u e s simulated t h e observed values f o r 9 compounds very w e l l (r

0 99) and a r e l i s t e d in Table 16 of

matter

The good predicting power of E q

course, since t h e s u b s t i t u e n t s included

bonders or "weak" hydrogen a c c e p t o r s

=

28 was a

a r e e i t h e r non-hydrogen

Although t h e r e l i a b i l i t y of

Eq

28

should b e examined f u r t h e r on various pyrazines s u b s t i t u t e d by amphiprotic s u b s t i t u e n t s . it indicates t h a t E q s 23, 25 and 21 a r e p r e d i c t i v e enough as f a r as t h i s d a t a set is concerned 4.

CONCLUSIONS The above analyses are believed t o indicate t h a t bidirectional Hammett-

Taft-type t r e a t m e n t s can be applied t o r a t i o n a l i z e t h e v a r i a t i o n s in

K

values

of s u b s t i t u e n t s o r increment in log P with polysubstitutions in each of t h e

various types

diazine systems, unless

of

solvations

and/or

the

substituents are

intramolecular

involved

interactions

For

monosubstituted diazines, t h e p a i r of one of t h e two ring-N substituent

was

regarded

as

a

"single" s u b s t i t u e n t

between t h e " s u b s t i t u e n t set" and

t h e o t h e r ring-N

and

in

specific

analyses

the

interactions

atom t o r e g u l a t e

r e l a t i v e s o l v a t i o n s with p a r t i t i o n i n g s o l v e n t s were analyzed

of

atoms and t h e

The

the

v a l u e of

K

corresponding s u b s t i t u e n t s in s u b s t i t u t e d pyridines was used a s t h e r e f e r ence

For t h e di- t o p o l y - s u b s t i t u t e d pyrazines. t h e i n t e r a c t i o n s between t h e

s u b s t i t u e n t p a i r s were primarily considered. t h e sum of s u b s t i t u e n t

R

values

in t h e monosubstituted pyrazine system being t h e r e f e r e n c e hydrophobicity Although t h e f a c t o r s governing t h e hydrophobicity of s u b s t i t u t e d diaz i n e s w e r e s e p a r a t e d t o components a s f a r a s well-behaved

compounds a r e

concerned, t h e y were r a t h e r complex and t h e analyses should be done v e r y carefully

These f a c t o r s should be considered in c o n s t r u c t i n g computer-aided

automatic systems t o p r e d i c t t h e log P values of heteroaromatic compounds

183 Because

of

restrictions

relating

to

the

stability

of

compounds, t h e

number of compounds in some monosubstituted diazine systems were low regular

u

v a l u e was t h e b e t t e r

d e s c r i p t o r of

monosubstituted d i a z i n e systems, whereas t h e f o r t h e disubstituted pyrazines

CJ

I

the electronic and

u

The

effect

for

v a l u e s were b e t t e r

This discrepancy may be a t t r i b u t a b l e t o t h e

r e s t r i c t i o n s in accumulating r e l i a b l e d a t a

Since t h e u s e of r e g u l a r

p a r a m e t e r s i n h e t e r o a r o m a t i c systems is sometimes s u b j e c t

a-type

t o skepticism.

a

uniform t r e a t m e n t of t h e e l e c t r o n i c e f f e c t on t h e r e l a t i v e s o l v a t i o n s h o u l d b e examined with

compound sets including g r e a t e r

numbers

of

having v a r i o u s d e g r e e s of e l e c t r o n i c effect and hydrogen-bonding

substituents behaviors

Outlying b e h a v i o r s of s u b s t i t u e n t s in some of t h e 2 - s u b s t i t u t e d series should also be c l a r i f i e d in terms of experimental physical-organic chemistry

REFERENCES 1 2 3 4 5 6 7 8

9 10 11 12 13 14 15

T. F u j i t a , J. Iwasa, and C. Hansch, J. A m e r . Chem. SOC. 86 (1964) 5175. A. Leo, C. Hansch, and D. Elkins, Chem. Rev. 71 (1971) 525. C. Hansch and A. J. Leo, S u b s t i t u e n t Constants f o r C o r r e l a t i o n Analysis i n Chemistry and Biology, John Wiley and Sons, N e w York 1979. J. Iwasa, T. F u j i t a , and C. Hansch. J. Med. Chem., 81 (1965) 150. S. J. L e w i s , M. S. Mirrlees, and P. J. Taylor, Quant. S t r u c . Act. Relat. 2 (1983) 1. S. J. L e w i s . M. S. Mirrlees, and P. J. Taylor. Quant. S t r u c . Act. Relat. 2 (1983) 100. J. Bradshaw and P. J. Taylor, Quant. S t r u c . Act. Relat., 8 (1989) 279. a)T. F u j i t a , Progr. Phys. Org. Chem.. 14 (1983) 75. b)Y. Nakagawa, K. Izumi. N. Oikawa. T. Sotomatsu, M. Shigemura, and T. F u j i t a , Environ. Toxicol. Chem., 11 (1992) 901. T. F u j i t a and T. Nishioka, Progr. Phys. O r g . Chem.. 12 (1976) 49. M. Charton. Prog. Phys. Org. Chem.. 13 (1981) 119. E. K u t t e r and C. Hansch, J. Med. Chem. 12 (1969) 647. M. Charton. in: N. B. Chapman and J. S h o r t e r (Eds.). C o r r e l a t i o n Analysis i n Chemistry. Plenum Press, N e w York, 1978, pp. 175-268. C. Yamagami. N. Takao and T. F u j i t a , Quant. S t r u c . Act. Relat., 9 (1990) 313 C. Yamagami. N. Takao and T. F u j i t a , J. Pharm. Sci., 80 (1991) 772. 0. Exner, in: N. B. Chapman and J. S h o r t e r (Eds.). C o r r e l a t i o n Analysis in Chemistry, Plenum P r e s s , N e w York, 1978, pp.439-540.

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QSAR and Drug Design New Developments and Applications T. Fujita, editor 9 1995 Elsevier Science B. 9'. All rights reserved -

185

H Y D R O P H O B I C I T I E S OF DI- TO PENTAPEPTIDES H A V I N G UNIONIZABLE SIDE CHAINS AND C O R R E L A T I O N WITH SUBSTITUENT AND STRUCTURAL P A R A M E T E R S MIKI AKAMATSU and TOSHIO FUJITA Department of Agricultural Chemistry, Kyoto University, Kyoto 606-01, Japan A B S T R A C T : Under standardized experimental conditions, we measured the partition ratio P' in a 1-octanol/pH 7.0 aqueous phosphate buffer system of a large number of zwitterionized di- to pentapeptides composed of amino acids having unionizable side chains as an approximate "molecular" partition coefficient P. The variations in log P' value of peptides were analyzed with free-energy-related physicochemical parameters for the side chain substituents and substructures. The side chain parameters representing the intrinsic hydrophobicity, the steric effect on the relative solvation of functional groups on the backbone, and the conformational potential index derived from the ChouFasman p-turn propensity parameters were shown to be significant. For polar side chains, specific indicator variables attributable to intramolecular hydrogenbond formations and the "polar proximity effect" for augmentations of hydrophobicity observed when polar groups are crowded together were required in addition. The proline residue was shown to participate in the log P' value depending not only upon its location on the backbone but also upon the total number of residues included in peptides. 1.

INTRODUCTION

The hydrophobicity of component amino acids and peptide segments is believed to govern not only the three-dimensional structure of proteins determining their biological functions (1), but also the affiliation properties of their partial domains into hydrophobic biomembraneous phases (2). In addition, peptides and their analogs have been attracting interest as potential drugs (3). The hydrophobicity of drugs is regarded as a highly important parameter to control transport behaviors from their site of administration to their site of action through a number of biomembranes as well as their binding with hydrophobic receptor sites (4). The log P value, P being the partition coefficient of neutral molecules measured with the 1-octanol/water system, has been widely used to represent molecular hydrophobicity (3, 4). In this article, we show that the log P' values, P' being the partition ratio in the system of 1-octanol/pH 7.0 aqueous buffer at 25~ for a number of di- to

186 pentapeptides having unionizable side chains are analyzable with free-energyrelated physicochemical parameters of the side chain substituents of the component amino acids by the regression technique (5, 6). The log P' value under such conditions is believed to be close to the log P of zwitterionized "neutral" peptides. In tetra- and pentapeptides, such conformational factors as the [3-turn formation (7) are shown to contribute to the net molecular hydrophobicity in addition to factors considered for di- and tripeptides. The correlation equation should be able to predict the log P' values of peptides, at least up to pentapeptides consisting of amino acids with unionizable side chains. 2.

H Y D R O P H O B I C I T Y OF PEPTIDES

2.1 Measurement of Partition Ratio Each peptide dealt with here showed a pH-log P' profile taking a "flat" parabolic form (5). The maximum log P' value, which is expected to be observed at the isoelectric point, should be the "true" hydrophobic parameter, log P, for the zwitterionized molecule in which the electric charges are cancelled. Unfortunately, the isoelectric points of most peptides were difficult to measure because of their limited solubility. Because of the flat form, the pHprofile of the log P' value is almost horizontal within the pH range between 5 and 7. We acertained that the log P' measured at pH 7 indeed parallels that measured at pH 6 for a subset of compounds (5). A theoretically reasonable pH-log P' profile with a "flat" parabolic form was not obtained when NaC1 was used to adjust the ionic strength of the acidic to neutral buffer solutions (5). Therefore, the buffer solution should be prepared from sodium hydrogen phosphate and dihydrogen phosphate only. This was considered to be due to the partitioning of the ion-pairs with counter anions. The phosphate anion, probably existing as a mixture of mono- and divalent species under acidic to neutral pH conditions, is perhaps much less hydrophobic than chloride. 2 . 2 Physicochemical Side-chain and Structural Parameters In the course of preliminary analyses, we found that the variations in the log P'(pH 7) value are governed at least by the hydrophobic and steric effects of side chain substituents of component amino acids. As the hydrophobic parameter of side chain substituents, we used the n value of general utility for aliphatic substituents evaluated under conditions free from components such as intramolecular stereoelectronic and hydrogen-bonding interactions (5, 6). For side chains with a polar group or heteroatom, the "intrinsic" aliphatic n value was evaluated from the log P value of related (but not peptidic) compounds in

187 which the polar group is separated by at least two methylene units from a chromophore (8). Our rc value for alkyl side chains is equivalent to that proposed by Hansch and Leo (9) under consideration of the branching factor, being close to that of Fauchbre and Pligka (10). Our ~ value for polar side chains in serine, threonine, methionine, tryptophan, glutamine, and asparagine is more negative than the corresponding value of Fauchbre and Pliska as will be shown later. For the steric effects of side chain substituents except "that" of proline, we used either the E's or E's c parameter depending upon the situation. The E's is the Dubois steric parameter (11). The E's parameter was defined to improve the Taft Es parameter (12). The E's c is the "corrected" Dubois steric parameter related to the original E's by Eq. 1, where n is the number of o~-hydrogen atoms in aliphatic substituents. E's c = E's - 0.306(3 - n)

[1]

The "correction" term in Eq. 1 takes the same form as that for the Taft Es made by Hancock and coworkers (13) to eliminate possible hyperconjugation effects of alkyl substituents on the reference reaction rate from which Es is defined. As indicated previously (14), however, the E's c (improved Es c) value is the parameter not corrected for the hyperconjugation effect attributable to the (xhydrogen atoms of substituents, but that representing not only the steric bulk but also the effect of a-branching. The coefficient of the correction term is fixed as -0.306 in Eq. 1, but values between -0.25 and -0.35 were found to be equally good. The relevance of the use of E's c for the steric effect of aliphatic substituents is discussed in detail in our previous analyses of the log P value of aliphatic amines and the ion-pair formation-partition constant of aliphatic ammonium ions (14). By definition, the bulkier, as well as the more o~-branched the substituents, the more negative the E's c value becomes. For most side chain substituents dealt with in this article, the E's value has been defined (11). The E's values of the indole-3-methyl group in tryptophan, the aminocarbonyl-methyl group in asparagine, and its higher homolog in glutamine were estimated using a highly linear relationship (5) between the E's value and Charton's ~ steric parameter (15, 16). That of the 4-hydroxybenzyl group in tyrosine was taken to be equivalent to that of the benzyl group in phenylalanine. The reference points of E's and E'sC were shifted so that E's(H) and E'sC(H) of the "side chain" in glycine were zero (5, 6, 17). To deal with the conformational effect arising from the possible 13-turn structure in tetra- and pentapeptides, we adopted the 13-turn "potential" index for

188 component amino acids proposed by Chou and Fasman (7). Their 13-turn index is defined statistically for each amino acid in each of the four consecutive positions from the data for 457 [3-turned backbone substructures found in 29 proteins of known sequence and crystallographic structure. As will be discussed later, the logarithm of the 13-turn index, f, for the i-th amino acid in the four consecutive positions, log fi, was regarded as a free-energy-related 13-turn potential parameter of each amino acid. For the inductive electronic parameter of side chain substituents, the Charton civalue was used (16). The relevant parameter sets are listed in Table 1. Factors governing the value of log P'(pH 7) shown in Table 2 were analyzed by the multiple regression technique in terms of the above-mentioned physicochemical free-energy-related parameters for the side chain substituents and indicator variables for particular substructures. T a b l e 1. Hydrophobicity Scale, Steric and Electronic Parameters, and [3-Turn Potential Indices of Amino Acid Side Chains Amino Acid Gly Ala Val

Leu Ile Phe Tyr Trp Met Ser Thr Asn Gln

Pro

na 0.00 0.32 1.27 1.81

E's b E's c c (YI d 0.00 0.00 0.00 -1.12 -0.20 -0.01 -1.60 -1.29 0.01 -2.05 -1.44 -0.01

log fie log fi+l e log fi+2 e log fi+3 e log ft e 0.09 -0.19 -0.19 -0.18

0.00 -0.02 -0.26 -0.54

0.31 -0.37 -0.46 -0.40

0.25 -0.17 -0.13 -0.09

0.19 -0.19 -0.28 -0.24

1.81 1.95 1.20 1.92

-2.12 -1.51 -1.51 -1.47f

-1.81 -0.90 -0.90 -0.86

-0.01 0.03 0.03 0.00

-0.17 -0.07 0.03 -0.10

-0.39 -0.37 -0.10 -0.89

-0.57 -0.17 0.09 -0.10

-0.14 -0.07 0.18 0.30

-0.27 -0.19 0.05 0.00

0.61 -1.49 - 1.18 -1.95 -1.41 0.86

-1.64 -1.09 - 1.04 -1.60 f -1.43 f -

-1.03 -0.48 -0.73 -0.98 -0.82 -

0.04 0.11 0.04 0.06 0.05 -

-0.07 0.14 0.02 0.25 -0.08 0.13

-0.07 0.17 0.09 -0.02 0.01 0.53

-0.85 g 0.12 -0.09 0.33 -0.35 -0.23

-0.14 0.03 -0.03 0.004 0.09 -0.11

-0.19 0.13 -0.00 0.18 -0.01 0.19

a b c d e

From ref. 5 and 17. From ref. 11, unless noted. The reference point is shifted so that E's(H) = 0. From ref. 5 and 14. The reference point is shifted so that E'sC(H) = 0. From ref. 16. Calculated from ref. 7. f See Text. g Not reliable. The corrected value -0.33 was used in Eq. 17.

2.3 Di- and Tripeptides First, we examined the log P' values of di- and tripeptides composed of the nonpolar amino acids; glycine, alanine, valine, leucine, isoleucine, and phenylalanine, in terms of the summation of the side chain ~ value of component amino acids and derived Eq. 2 with an indicator variable Itri.

189

Table 2. Log P and Physicochemical Parameters of Di- to Pentapeptides log P

~~

No. Compounds 1 2 3 4 5 6 7 8 9 10

FL LF FF LL LV VL A1 I1 LI

vv

11

ww

12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49

WF WA WL WY LY YL VY

FY YY LM ML MV FM SL PF PL PI FP LP IP FFF GFF FVG FVF FVA LVV LII LVL LAL LLL WGG WFA WWL LLY VFY GFY YLV YVF

Zrr

3.76 3.76 3.90 3.62 3.08 3.08 2.13 3.62 3.62 2.54 3.84 3.87 2.24 3.73 3.12 3.01 3.01 2.47 3.15 2.40 3.12 3.12 2.58 3.26 0.32 2.81 2.67 2.67 2.81 2.67 2.67 5.85 3.90 3.22 5.17 3.54 4.35 5.43 4.89 3.94 5.43 1.92 4.19 5.65 4.82 4.42 3.15 4.28 4.42

E,C(RN) ZE,C(RM)

-0.90 -1.44 -0.90 -1.44 -1.44 -1.29 -0.20 -1.81 -1.44 -1.29 -0.86 -0.86 -0.86 -0.86 -0.86 -1.44 -0.90 -1.29 -0.90 -0.90 -1.44 -1.02 -1.02 -0.90 -0.48 -

-

-0.90 -1.44 -1.81 -0.90 0.00 -0.90 -0.90 -0.90 -1.44 -1.44 -1.44 -1.44 -1.44 -0.86 -0.86 -0.86 -1.44 -1.29 0.00 -0.90 -0.90

0.00 0.00 0.00 0.00 0.00

0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

0.00 0.00 0.00 0.00 0.00 0.00 -0.90 -0.90 -1.29 -1.29 -1.29 -1.29 -1.81 -1.29 -0.20 -1.44

0.00 -0.90 -0.86 -1.44 -0.90 -0.90 -1.44 -1.29

E',C(Rc) log Sum log f,+q

-1.44 -0.90 -0.90 -1.44 -1.29 -1.44 -1.81 -1.81 -1.81 -1.29 -0.86 -0.90 -0.20 -1.44 -0.90 -0.90 -1.44 -0.90 -0.90 -0.90 -1.02 -1.44 -1.29 -1.02 -1.44 -0.90 -1.44 -1.81

-

-0.90 -0.90 0.00 -0.90 -0.20 -1.29 -1.81 -1.44 -1.44 -1.44 0.00 -0.20 -1.44 -0.90 -0.90 -0.90 -1.29 -0.90

0.00 0.00 0.00 0.00 0.00

0.00 0.00 0.00 0.00 0.00

0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

0.00 0.00 0.00 0.00

0.00 0.00 0.00 0.00 0.00

0.00 0.00 0.00 0.00 0.00

0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

Obsd.

-1.17 -1.15 -0.85 -1.46 -2.05 -2.07 -2.60 -1.82 - 1.64 -2.82 -0.27 -0.47 -1.98 -0.73 -1.13 -1.94 -1.75 -2.52 -1.68 - 1.87 - 1.87 - 1.84 -2.53 -1.59 -2.49 -2.07 -2.41 -2.56 - 1.36 -1.76 -1.79 -0.02 - 1.33 -2.33 -0.76 -2.19 -2.10 -1.11 -1.57 -2.03 -0.94 -2.72 - 1.oo 0.36 - 1.34 -1.50 - 1.96 - 1.45 -1.37

Calcd. (Q. 18)

(!3+19)

-1.23 -1.36 -0.93 -1.66 -2.12 -2.09 -2.50 -1.98 -1.77 -2.55 -0.21 -0.56 -1.89 -0.86 -1.14 -1.93 -1.80 -2.36 -1.51 -2.08 -2.01 -1.91 -2.37 -1.58 -2.66

-1.24 -1.38 -0.95 -1.67 -2.13 -2.09 -2.50 -1.98 -1.78 -2.56 -0.22 -0.58 -1.91 -0.87 -1.14 -1.94 -1.80 -2.37 -1.51 -2.08 -2.02 -1.91 -2.37 -1.59 -2.67 -1.95 -2.24 -2.35 -1.37 -1.79 - 1.99 0.04 -1.31 -2.29 -0.72 -2.05 -1.90 -1.19 -1.43 -2.01 -0.97 -2.73 -0.92 0.48 -1.24 -1.38 -1.87 -1.57 -1.28

0.05 -1.29 -2.27 -0.71 -2.03 -1.90 -1.20 -1.44 -2.00 -0.97 -2.71 -0.90 0.48 -1.25 -1.38 -1.87 -1.58 -1.28

190 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81

82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101

102 103

YGF YYL AYI IYV MLF LSL ISL IS1 SLI SLL FIT LIT IIT LTI TLI TVL PLL LPL LLP IPI FGGF VAAF LLVF LLLV VGFF AVLL IAGF FFFF LLGF LLAF LLLF IlVV IIGF IAAI FFGF VLVL WLLV WGLL YILG FVYF IYIV VFLT MIL1 VMFI PLLL LPLL LLPL LLLP IPGI VPVL VPGV YPGW YPGI GGFVF

3.15 4.21 3.33 4.28 5.07 2.13 2.13 2.13 2.13 2.13 2.58 2.44 2.44 2.44 2.44 1.90 4.48 4.48 4.48 4.48 3.90 3.86 6.84 6.70 5.17 5.21 4.08 7.80 5.57 5.89 7.38 6.16 5.57 4.26 5.85 6.16 6.81 5.54 4.82 6.37 6.09 3.85 6.04 5.64 6.29 6.29 6.29 6.29 4.48 5.21 3.40 3.98 3.87 5.17

-0.90 -0.90 -0.20 -1.81 -1.02 -1.44 -1.81 -1.81 -0.48 -0.48 -0.90 -1.44 -1.81 -1.44 -0.73 -0.73 -1.44 -1.44 -1.81 -0.90 -1.29 -1.44 -1.44 -1.29 -0.20 -1.81 -0.90 -1.44 -1.44 -1.44 -1.81 -1.81 -1.81 -0.90 -1.29 -0.86 -0.86 -0.90 -0.90 -1.81 -1.29 -1.02 -1.29 -1.44 -1.44 -1.44 -1.81 -1.29 -1.29 -0.90 -0.90 0.00

0.00 -0.90 -0.90 -0.90 -1.44 -0.48 -0.48 -0.48 -1.44 -1.44 -1.81 -1.81 -1.81 -0.73 -1.44 -1.29 -1.44

-1.44

0.00 -0.40 -2.73 -2.88 -0.90 -2.73 -0.20 -1.80 -1.44 -1.64 -2.88 -3.10 -1.81 -0.40 -0.90 -2.73 -2.88 -1.44 -3.25 -2.19 -2.71 -2.34 -3.25 -1.92 -2.88

-2.88

-

-2.19

-0.90 -1.44 -1.81 -1.29 -0.90 -1.44 -1.44 -1.81 -1.81 -1.44 -0.73 -0.73 -0.73 -1.81 -1.81 -1.44 -1.44 - 1.44

-

-1.81 -0.90 -0.90 -0.90 -1.29 -0.90 -1.44 -0.90 -0.90 -0.90 -0.90 -0.90 -1.29 -0.90 -1.81

-0.90 -1.44 -1.29 -1.44 0.00 -0.90 -1.29 -0.73 -1.81 -1.81 -1.44 - 1.44 - 1.44

-

-1.81 -1.44 -1.29 -0.86 -1.81 -0.90

0.00 0.00 0.00 0.00

0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.18 -0.65 - 1.25 -1.24 -0.43 -0.94 0.06 -0.68 -0.48 -1.16 -1.19 -1.14 -0.31 -0.69 -0.20 - 1.28 -1.16 -0.58 -0.51 -0.31 -0.96 -0.99 -0.99 -0.58 -0.89 -0.14 - 1.04 O.0Od 0.54 -0.21 0.53 1.17 0.73 -0.21

0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.31 -0.37 -0.46 -0.40 -0.17 -0.40 0.31 -0.17 0.31 -0.37 -0.40 -0.46 0.31 -0.37 0.31 -0.46 -0.40 -0.40 -0.40 0.09 -0.57 -0.40 -0.40 -0.17 -0.40 -0.40 -0.23 0.00a 0.31 -0.46 0.31 0.31 0.31 -0.17

-1.86 -1.38 -2.04 -1.77 -1.03 -2.35 -2.28 -2.64 -1.99 -2.03 -1.95 -2.14 -2.23 -2.30 -1.66 -1.97 -1.64 -1.56 -1.58 -1.65 -1.51 -1.91 -0.25 -0.51 -0.51 -1.74 -1.78 1.63 -0.42 -1.00 0.24 -1.41 -0.99 -2.82 0.17 -1.23 0.23 0.06 -1.49 -0.32 -1.09 -1.32 -0.49 -0.63 -1.06 -0.92 -1.00 -1.18 -1.69 -1.91 -2.83 -1.25 -1.65 -1.40

-2.08 -1.39 -2.09 -1.92 -0.92 -2.21 -2.41 -2.52 -2.09 -1.97 -1.68 -2.10 -2.31 -2.10 -1.93 -2.28

-1.34 -2.22 -0.27 -0.53 -0.99 -1.25 -1.73 1.43 -0.50 -0.77 0.23 -1.35 -0.82 -2.41 0.23 -1.00 0.27 -0.53 -1.59 0.29 -1.25 -1.21 -0.54 -0.49

-1.26

-2.09 -1.38 -2.08 -1.91 -0.92 -2.21 -2.41 -2.52 -2.08 -1.97 -1.68 -2.10 -2.31 -2.10 -1.93 -2.28 -1.89 -1.44 - 1.44 -1.75 -1.36 -2.25 -0.28 -0.52 -1.01 -1.25 -1.75 1.41 -0.51 -0.78 0.23 -1.34 -0.82 -2.42 0.21 -1.00 0.27 -0.54 -1.58 0.30 -1.24 -1.22 -0.52 -0.48 -1.32 -0.88 -0.75 - 1.09 -1.92 -1.81 -2.50 -1.10 - 1.86 -1.27

191

104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 a

VFVGL VGFVF GAALL AFGVF AGFVF LIIGA GLLGF ALLGF IIIIG IVVVI FGAGI FAAAL WGGFV WLFAA IAYWG GLSVL SLAIV YTGFL LVGTF L-enk b M-enk c

6.30 6.44 4.26 5.49 5.49 5.75 5.57 5.89 7.24 7.43 4.08 4.72 5.14 6.32 5.25 3.40 3.72 3.78 3.85 4.96 3.76

-1.29 - 1.29 0.00 -0.20 -0.20 -1.44 0.00 -0.20 -1.81 -1.81 -0.90 -0.90 -0.86 -0.86 -1.81 0.00 -0.48 -0.90 -1.44 -0.90 -0.90

-2.19 -2.19 -1.84 -2.19 -2.19 -3.62 -2.88 -2.88 -5.43 -3.87 -0.20 -0.60 -0.90 -2.54 -1.96 -3.21 -3.45 -1.63 -2.02 -0.90 -0.90

-1.44 -0.90 -1.44 -0.90 -0.90 -0.20 -0.90 -0.90 0.00 -1.81 -1.81 -1.44 -1.29 -0.20 0.00 -1.44 -1.29 -1.44 -0.90 -1.44 - 1.02

-0.11 -0.49 -0.39 -0.37 -0.48 -0.42 -0.48 -0.48 -0.87 -1.01 0.24 -0.63 0.15 -0.98 0.21 -0.45 -0.89 0.36 -0.16 0.28 O.28

0.31 -0.17 -0.37 0.31 -0.17 0.31 0.31 0.31 -0.57 -0.46 0.31 -0.37 0.31 -0.17 0.09 0.12 -0.37 0.31 0.31 0.31 0.31

-0.97 -0.50 -2.55 -0.59 -1.10 -1.65 -0.18 -0.63 -0.97 -0.89 -1.87 -2.23 -0.44 -0.32 -1.47 -1.64 -1.94 -1.18 -1.18 -0.80 -1.39

-0.70 -0.78 -2.33 -0.71 -1.08 -1.36 -0.73 -0.53 -1.30 -1.11 -2.10 -2.02 -0.75 -0.18 -1.12 -1.59 -1.95 -0.97 -1.30 -1.22 -1.57

-0.70 -0.77 -2.32 -0.70 -1.07 -1.36 -0.73 -0.54 -1.31 -1.13 -2.09 -2.0O -0.75 -0.16 -1.12 -1.60 -1.96 -0.98 -1.30 -1.22 -1.57

These parameter terms were not counted, see text.

b [Leu]enkephalin(YGGFL) c [Met]enkephalin(YGGFM)

log P'= 0.804 En - 0.689 Itri - 4 . 4 2 5 (0.217)

(0.417)

[2]

(0.752)

n = 20

s = 0.333

r = 0.892

F2,17 = 32.9

In Eq. 2 and the following correlation equations, n is the number of compounds, s is the standard deviation, r is the correlation coefficient, F is the ratio of regression to residual variance, and the figures in parentheses are the 95% confidence intervals. Itri is zero for dipeptides and unity for tripeptides. In trying to improve the correlation, we noticed that the log P' values of peptides containing 13-branched amino acids with a-branchings in the side chain, such as valine and isoleucine, were more negative than the value calculated by Eq. 2. Since the steric effect of the "crowded" structure of the branched side chain on the relative solvation of the NHCO moiety and terminal NH3 + and COO- groups with partitioning solvents was anticipated to contribute to these deviations, we introduced steric terms into Eq. 2. Among various steric parameters (18) examined, the E's c parameter worked best, yielding Eq. 3.

192 log P' = 1.031 2;~: - 0.778 Itri + 0.521 E'sC(RN) + 0.337 E'sC(RM) (0.081) (0.225) (0.131) (0.188) + 0.335 E'sC(Rc) - 4.068 (0.128) (0.264) n=20

[31 s=0.113

r=0.990

F5,14 =141

R N and RC represent the side chains of amino acids at the N- and C-termini, respectively. RM is for the side chain of the central amino acid of tripeptides. For dipeptides, the E'sC(RM) term is not counted, i.e., E'sC(RM) = 0. This does not mean that the "phantom" central amino acid in dipeptides is regarded as being glycine. The effect attributed to the "phantom" glycine is compensated by the Itri term. Equation 3 indicates that the steric effects of the side chains on the relative solvation with partitioning solvents depend upon their location in the molecule. When including peptides with polar amino acids, no relevant correlation equation was derived unless indicator variable terms for the presence of respective polar amino acids were introduced to give Eq. 4 (Y, W, M, S, and T are one letter notations for tyrosine, tryptophan, methionine, serine, and threonine, respectively). log P ' = 0.960 z n - 0.635 Itri + 0.561 E'sC(RN) + 0.337 E'sC(RM) (0.075) (0.136) (0.096) (0.123) + 0.255 E'sC(Rc) + 0.165 Iy + 0.352 Iw + 0.637 IM (0.097) (0.079) (0.096) (0.149)

[4]

+ 1.665 (Is + IT) - 3.912 (0.219) (0.210) n=59

s=0.138

r=0.982

F9,49 = 1 4 8

Since the slope values for Is and IT terms were very close in the preliminary calculation, they were combined in Eq. 4. Peptides containing proline were not included in Eq. 4, because the E's c value for the cyclic "side chain" of proline is difficult to estimate. Peptides containing glutamine and asparagine were also not included, because their log P' values were too low to measure accurately. The fact that the slope of the zn term is very close to unity shows that the intrinsic hydrophobic factor of the side chains of constituent amino acids contributes to the total hydrophobicity of peptides almost as such after factors attributed to other effects are separated. The positive sign of the E's c terms

193 indicates that the solvation of backbone functional groups with the bulkier 1octanol is less favorable than that with the smaller water as the side chain substituents are bulkier and more c~-branched, resulting in lower log P' values. Equation 4 also shows that the steric effects of the side chains on the relative solvation depend upon their locations in the molecule. The coefficient of the E'sC(RN) term is the highest and that of the E'sC(Rc) term is the lowest, that of the E'sC(RM) term being intermediate. The NH3 + group as the strongest hydrogen donor in the molecule is solvated more effectively than other sites such as CONH and COO- groups with the more basic octanol than the less basic water. The solvation of the NH3 + group with the bulkier 1-octanol favorable for enhancing the log P' value would suffer the steric hindrance from the N-terminal side chain substituent most sensitively. The coefficient of the Itri term, the Itri value taking unity for tripeptides, indicates that the log P' value decreases by about 0.64 with introduction of one more peptide unit into the dipeptide backbone, other things being unchanged. It also corresponds to the rc value of the CH3CONH group. The value -0.64 is, however, considerably more positive than that [-2.17 (9, 19)] expected under conditions without any intramolecular stereoelectronic effects. This could be rationalized by the "polar proximity factor" (9) for the enhancement of hydrophobicity observed when polar groups are close to each other. Change from a di- to tripeptide backbone increases the proximity interaction between two CONH groups. This factor could be evaluated approximately taking the interaction between two CONH groups separated by CH2 as about 1.73 (9), which is close to the real situation, the increase being about 1.6 (-0.6 + 2.2).

I

I

HC~CH2~O~

I C---'-O I NH

I

HC~CH2~O~ I,

,

R~ ~'

C - - ' - O .... H--O~. NH

I

I

HC~CH2~O~ I

... H

R

ROH

C~O

"~

NH

I

"~ _ H o

..... H----O~"

R

I

Fig. 1. Intramolecular Bridging-Solvation of the Hydroxyl Group of a Serine Residue and Carbonyl Group on the Backbone. R is Either 1-Oct or H. Reproduced from ref. 5 by permission of VCH Verlagsgesellschaft mbH.

Indicator variable terms specific to polar amino acid side chains are always positive. The log P' value of peptides with these side chains is higher than that predicted by the hydrophobicity of the side chains and backbone, as well as

194

factors attributed to the steric effect on the relative solvation. For the side chains of serine and threonine, the size of the coefficient is remarkably high. This is probably due to the fact that the hydroxyl group in the side chain and the carbonyl group in the backbone are well positioned for intramolecular bridgingsolvation as shown in Fig. 1. This type of bridging hydration has been observed in glutamine in the crystal structure of human deoxy-haemoglobin (20). Abraham and Leo (21) discussed the possibility that serine and threonine take this type of bridging-solvation structure in rationalizing the side chain rc value of Fauchbre and Pliska (10), which is significantly higher than the value usually used for aliphatic systems. This type of solvation was estimated to make the log P' value 0.6---0.9 unit higher than that of the structure without such "intramolecular" solvation (22, 23). Subtracting the value attributed to the bridging solvation, the size of the regression coefficient for Ser and Thr residues is about 0.8---1.1. The "corrected" regression coefficient value seems to decrease with the number of bonds separating the polar heteroatom on the side chain from the backbone more regularly than the uncorrected value, as shown in Table 3. When the number of bonds increases, the net inductive electronwithdrawing effect of the side chain polar groups on the backbone functional group is gradually reduced. The electron-withdrawing effect of substituents raises the partition coefficient in series of substituted compounds regardless of whether the functional group is hydrogen-donating or hydrogen-accepting (22). Thus, the greater the number of bonds, the lower should be the increment in the log P' value assigned as the polar proximity factor (9). Table 3. Regression Coefficient of Indicator Variable Terms Amino Acid Ser Thr Met Trp Tyr Asn Gin a b c d

Side Chain RegressionCoefficient a -CH2OH 1.665 (0.8--1.1) c -CH(CH3)OH 1.665 (0.8,--1.1) c -CH2CH2SCH3 0.637 -CH2-(3-Indolyl) 0.352 -CH2-(4-OH-Phenyl) 0.165

nb 2 2 3 4 6

-CH2CONH2 -CH2CH2CONH2

3

1.971 d (1.1 ~- 1.4) c

1.337d (0.5,--0.8) c 4

Unlesss noted, the regression coefficient value of indicator variable terms in Eq. 4. The number of bonds separating the polar heteroatom in the polar group from the a-carbon of the peptides. The value in parentheses is "corrected" by subtracting the intramolecular bridging-solvation factor. Estimated from Eqs. 20 and 21.

195 The intercept of Eq. 4 should correspond with the log P' value of glycylglycine where every independent variable is zero. The very good correlation quality of Eq. 4 could be taken to mean that quite a few component factors contributing to variations of the log P' value are almost completely separated from each other. The contributions of the side chains of asparagine and glutamine residues to the log P' value were analyzed indirectly with use of protected peptides as described later.

2.4 Tetra- and Pentapeptides With the above results for di- and tripeptides in mind, we analyzed the log P' values of tetra- and pentapeptides using parameter terms corresponding to those used in Eq. 4, and formulated Eq. 5. log P ' = 1.025 %rt -0.262 Ipent + 0.575 E'sC(RN)+ 0.491 [ZE's C(RM) (0.157) (0.226) (0.205) (0.137) + E'sC(Rc)] + 0.329 Iw + 0.887 IM + 1.772 (Is + IT) - 4.544 (0.335) (0.432) (0.476) (0.670) n =46

s =0.335

r=0.926

[5]

F7,38 = 32.6

Ipent is an indicator variable taking zero for tetrapeptides and unity for pentapeptides. %E'sC(RM) means the sum of E's c parameters for side chains other than those of the two terminal amino acids. Preliminary examinations indi-cated that the steric effect of RM substituents is almost position-independent, so their E's c values were added together. The coefficients of %E'sC(RM) and E'sC(Rc) terms were also so close that they were combined. Equation 5 might be acceptable, but the quality of the correlation in terms of r and s is consider-ably poorer than that of Eq. 4. The Iy term for the tyrosine side chain is insignificant over the 95% level in Eq. 5. The Iw term for the tryptophan is also only justified over the 94.5% level. Moreover, the coefficient of Ipent corresponding to Alog P with introduction of one more peptide unit is significantly more positive than that of Itri in Eq. 4. The intercept is about 0.6 unit more negative than that in Eq. 4, reflecting the difference between the reference peptide series in Eqs. 4 and 5: dipeptides and tetrapeptides. Since physicochemical factors governing the log P' value of lower peptides could at least be involved as factors for tetra- and pentapeptides on the same standards, these discrepancies should indicate that variables other than those used in Eqs. 3 and 4 are required for log P' of tetra- and pentapeptides. We considered that a specific conformational feature such as the 13-turn formation could be a factor required for tetra- and pentapeptides but not for di- and tripeptides.

196 R3 (i+2)

R4 (i+3)

[3-Turns, classified into at least three types, have been observed as regular conformational patterns in regions of backbone chain reversals of globular proteins (24). ~-Turned substructures consist of four consecutive amino acid residues, mostly with hydrogen-bonding formation between the CO-oxygen of the residue at position i and the NH-hydrogen of the residue at position (i + 3). One of the [3-turn structural types (named type I by Venkatachalam) is shown in Fig. 2 (25).

R2

(i+

i)

Fig. 2. One of the [3-turn Structures (Type I) of Peptides. Reproduced from ref. 25 by permission of the Journal of Biological Chemistry.

We assumed that tetra- and pentapeptides exist as an equilibrium mixture of random and [3-turned structures depending upon the ~-turn potential of component amino acids in partitioning solvents, and so the partition of peptides can be depicted as shown in Fig. 3.

1-Octanol Phase: [C~ Water

Phase:

Koct ~ [Coct]~

[Cw]R ~

[Cw][~

Kw Fig. 3. Partition and Conformational Equilibria of Peptides; [C] represents the concentration, and suffixes, R and [3, express the random and ~-turn structure, respectively. Reproduced from ref. 6 by permission of the American Pharmaceutical Association. The net P' value is expressible by Eq. 6. p

t

.--

[Coct]R+[Coct]13 = [Coct]R [ l + K o c t ] [Cw] R + [Cw][3

[Cw] R

1 + Kw

[6]

In Fig. 3 and Eq. 6, Koct and KW are the conformational equilibrium constants in the 1-octanol and water phases, respectively, being reflected by the [3-turn

197 potential of four consecutive amino acids. Di- and tripeptides are unable to take the [3-turn structure and so Koct=Kw=0 in Eq. 6. Thus, Eq. 7 holds for these lower peptides.

[Coct]R

log P ' = log ~ = log [Cw]R

P'R

[7]

P'R is the P' value for molecules with random structures. It has been shown that, the more hydrophobic the environment, the easier is the intramolecular hydrogen-bond formation (22). For oligopeptides, intramolecular hydrogenbonding could lead to the formation of conformationally fixed structures such as [3-turns and o~-helices. Consistent with CD spectra measured in aqueous buffer (pH 7) and 2,2,2-trifluoroethanol (6), the tetra- and pentapeptides studied here were considered to exist almost entirely as random conformers in the aqueous phase, but to take the [3-turn structure in aliphatic alcohols to various extents according to the [3-turn potential of their component amino acids. Thus, such conditions as I>>Kw and l<
[8]

log P ' = log P'R + log Koct

[91

We examined whether the conformational equilibrium constant in the 1octanol phase, Koct, would be expressible in terms of the Chou-Fasman 13-turn parameter, f, of component amino acids (7). Chou and Fasman have proposed their parameter to reflect the "potential" of certain amino acids at four consecutive positions for the [3-turn formation. For each amino acid involved in the [3-turn substructure, the f parameter is defined in terms of the relative frequency of occurrence at each bend position among the residues i, i+l, i+2, and i+3. It is normalized by dividing the average frequency of occurrence of the amino acid in question in 29 proteins. We first tried to use the product of fi~-fi+3 values, Fturn, as the net potential for [3-tum formation similar to that dealt with by Lewis and coworkers (26). The Koct value was then assumed to be expressible by a linear free-energy relationship with the 13-turn parameters as shown in Eq. 10. log Koct = a log Fturn + c

[101

198 In Eq. 10, log Fturn = Y, log fi(i = i-i+3), "a" (>0) is the slope and c is the intercept. For pentapeptides where the 13-turn formation is possible either with residues 1-4 or residues 2-5, we took the greater of the two Fturn values. The original 13-turn parameter is proposed in terms of the relative probability of each amino acid participating in 13-turn formation. The use of the logarithm of the Chou-Fasman parameter as being free-energy-related could be justified on this basis. For tetra- and pentapeptides, Eq. 11 is the counterpart of Eq. 7. log P ' = log P'R + a log Fturn + c

[11]

With use of the log Fturn as an additional independent variable, Eq. 12 was formulated for tetra- and pentapeptides. log P ' = 1.056I:rt - 0.515 Ipent + 0.580 E'sC(RN) + 0.350 [~:E'sC(RM) (0.142) (0.256) (0.183) (0.150) + E'sC(Rc)] + 0.541 log Fturn + 0.363 Iw+ 0.742 IM (0.335) (0.300) (0.396) + 1.771 (Is + IT) - 4.740 (0.425) (0.610) n=46

[12] s=0.299

r=0.943

F8,37 =37.2

The log Fturn term was indeed significant and the statistical quality was significantly improved from that of Eq. 5. The corresponding terms between Eqs. 4 and 12 were much closer than those between Eqs. 4 and 5. The log Fturn term was positive, showing that the higher the 13-turn propensity of component amino acids, the higher the net hydrophobicity, as expected from Eq. 11 where "a" is positive. The log Fturn term represents a model in which the 13-turn potential of each amino acid at each of the four positions is considered to contribute to 13turn formation with an equivalent significance a priori. We next tested whether this model was best by using individual log fi values as independent variables singly or in various combinations. Interestingly, the use of log fi+2 singly for the third amino acid residue in place of log Fturn was found to be enough, as shown in Eq. 13. For pentapeptides in which there are two choices for the "third" amino acid residue, the higher log f value was used (6). Not only the corresponding terms, except for the pair of log Fturn and log fi+2 terms, but also the correlation quality, are practically equivalent in Eqs. 12 and 13. This was thought to be due to a high collinearity (r = 0.812) between log Fturn and log fi+2 values for 46 tetra- and pentapeptides.

199 log P ' = 0.980 Xrc - 0.459 lpent + 0.539 E'sC(RN) + 0.350 [ZE'sC(RM) (0.136) (0.219) (0.176) (0.137) + E'sC(Rc)] + 0.677 log fi+2 + 0.422 Iw+ 0.769 IM (0.345) (0.291) (0.375) + 1.619 (Is + IT) -4.609 (0.414) (0.573) n=46

s=0.286

r=0.948

[13]

F8,37 =41.1

Although the r and s values are nearly alike in the two equations, Eq. 13 is preferred over Eq. 12, because the conformational parameter in the latter, log Fturn, actually consists of four terms as opposed to the single term, log fi+2, in Eq. 13. Eq. 13 indicates that the ease of 13-turn formation is most significantly governed by the [3-tum potential of the third residue among four consecutively linked amino acids. Besides the fi(i = i--i+3) parameters for each amino acid at each of the four bend positions, Chou and Fasman have estimated the relative frequency of occurrence of each amino acid in the four bend positions, ft, based on 457 13turns in 29 proteins (7). We examined the correlations of log ft derived from their study (Table 1) with each of the log fi(i = i---i+3) values. For the set of ten component amino acids (omitting methionine) in peptides included in Eqs. 12 and 13, Eq. 14 formulated for the log fi+2 value showed the best quality. [14]

log ft = 0.588 log fi+2 + 0.015 (0.137) (0.043) n=10

s=0.051

r=0.962

F1,8 =97.9

Equation 15, formulated for the log fi value for the first residue, followed Eq. 14.

[15]

log ft = 1.319 log fi + 0.006 (0.445) (0.059) n=10

s=0.071

r=0.924

F1,8 =46.6

Neither the log fi+l nor log fi+3 value was able to explain the variance in log ft over 50% (100 x r2). The fi+2 value of methionine is estimated in the original work (7) based on only a single occurrence at the bend position, i+2, so it is not as reliable as that for other residues. Taking the cyclic structure, the

200 conformational effect of the proline residue could differ from those of the other amino acids. For the set of eighteen amino acid residues deleting proline and methionine from the original data of Chou and Fasman, Eq. 16 was obtained. log ft = 0.504 log fi+2 + 0.003

(0.123)

[16]

(0.034) n=18

s=0.066

r=0.909

F1,16=75.7

The fi+2 value was reasonably considered to represent the ease of participation of a certain amino acid residue in 13-turn formation within conformations of natural globular proteins. "Linear free-energy relationships", as shown in Eqs. 14 and 16 for fi+2 with the ft value, that reflect an overall "standard" potential for 13turn formation, were considered to be a background for the formulation of Eq. 13, in which only the log fi+2 term suffices for rationalizing the log P' values of tetra- and pentapeptides. As could be understood from Fig. 2, the side chain R3 of the residue, i+2, would exert a significant effect on the torsion angle of the adjacent CONH plane sterically. In fact, we formulated Eq. 17 for 11 amino acid side chains. The methionine side chain was included after its log fi+2 value was corrected by Eq. 16. [17]

log fi+2 = 0.345 E's + 2.461 ~I + 0.246 (0.213) (3.441) (0.334) n=ll

s=0.166

r=0.842

F2,8 =9.75

The (YI is a parameter for the inductively electron-withdrawing property of aliphatic substituents (16). In Eq. 17, the E's worked much better than E's c. This could mean that the steric effect operating here is similar to that in the reference aliphatic ester system from which Taft Es is defined (12). The physicochemical significance of the log fi+2 term in Eq. 13 is perhaps to represent the steric effect of the side chain of the residue i+2 on the twisting of the adjacent CONH group. The bulkier the side chain substituent, the greater would be the twisting and so the direction of the NH group of the residue i+3 as the hydrogen-donor toward the CO group of the residue i as the acceptor is distorted more severely. Although it is significant only at the 85 % level, the positive (IX term would indicate that the higher the electron-attracting ability of the side chain, the greater the acidity of the NH hydrogen leading to the higher

201 hydrogen-donating property. The most significant driving force for 13-tum formation could be a gain of stabilization energy by intramolecular hydrogen-bond formation. The significant correlation of log ft with log fi as shown in Eq. 15 is also taken to support the above possibility. The carbonyl group of the first residue is the hydrogen-bond acceptor and the relative probability of each residue being found at this position should be related to the relative probability in findings among 13turn substructures. Eqs. 14-17 showing that the stabilization of the 13-turned structure is largely dependent on the steric effect of side chains of amino acids involved are in accord with the result of Charton and Charton (16) analyzed from somewhat different points of view. 2.5 Di- to Pentapeptides To correlate the log P' values for di- to pentapeptides as a set, Eqs. 4 and 13 were combined together with two newly defined indicator variables to give Eq. 18. The one is Iturn which takes zero for di- and tripeptides and unity for tetra- and pentapeptides. The addition of the Iturn term corresponds with the incorportion of the intercept "c" needed only for tetra- and pentapeptides in Eq. 11 after log Fturn is replaced by log fi+2. The other, Ipep, is a combined parameter of Itri and Ipent which takes zero for dipeptides and one, two, and three with ascending numbers of peptide bonds. log P ' = 0.943 En - 0.579 Ipep+ 0.550 E'sC(RN) + 0.307 [ZE'sC(RM) (0.069) (0.105) (0.095) (0.077) + E'sC(Rc)] + 0.521 Iturn + 0.747 log fi+2 + 0.135 Iy (0.206) (0.231) (0.094) + 0.375 Iw+ 0.654 IM + 1.584 (Is + IT) - 3.838 (0.113) (0.170) (0.207) (0.204) n = 105

s = 0.212

r = 0.969

[18]

F10,94 = 144

The correspondence of Eq. 18 for 105 peptides with Eq. 4 for lower peptides as well as with Eq. 13 for higher peptides is very good, supporting the procedure with assumptions made for Eqs. 6, 7, 9, 10 and 11 with use of the Chou and Fasman 13-turn parameter for the conformational effect in higher peptides. The log Koct value was estimated by substituting values for the log fi+2 and Iturn terms in Eq. 18 into the corrected Eq. 10. It ranged between zero and 0.75, however. The value was found not to accord entirely with the conditions of Koct>>l for Eq. 9, but the procedure was admissible at least as a first approximation. In Table 2, the log P' values calculated using Eq. 18 are

202 shown for 105 peptides.

2.6

Peptides Containing Proline

Peptides containing proline were not included in the above correlations, since the E's c value for the "side chain" of proline is not easily estimated. By substituting the values of available parameters for peptides including proline such as En, Ipep, log fi+2, and Iturn into Eq. 18, we calculated the summation of these parameter terms and examined the difference, Alog P', from the observed value. The Alog P' value should correspond with the component of the log P' value attributable to the steric effect together with other effects specific to the Pro residue. As shown in Table 4, the effects seem dependent not only Table 4. Alog P' and Indicator Variables on the location but also on the of Peptides Containing Proline number of residues involved. Compounds ~xlogP' Ip(N) Ip(#pep) When the Pro residue is at the NPI -0.683 1 -1 terminus, the Alog P' value is PL -0.648 1 -1

invariably negative, being -0.5 -0.9. At the C-terminus, however, it shows the reverse effect only in dipeptides. For tripeptides without N-terminal proline, the Alog P' is nearly zero. For tetrapeptides, the Alog P' is always negative. We considered that the effect of a Pro residue at a position other than the Nterminus is to lower the log P' value almost regularly with increase in number of total residues from dipeptides regardless of its location. Although the variation patterns of the Alog P' value looked rather

PF FP IP LP IPI PLL LPL LLP PLLL LPLL LLPL LLLP IPGI VPGV VPVL YPGW YPGI

-0.605 0.325 0.531 0.354 0.090 -0.562 -0.128 -0.148 -0.888 -0.407 -0.607 -0.437 -0.123 -0.688 -0.457 -0.509 -0.140

1 0 0 0 0 1 0 0 1 0 0 0 0 0 0 0 0

-1 -1 -1 -1 0 0 0 0 1 1 1 1 1 1 1 1 1

complex, we assumed that they are represented by two indicator variables. The one is for the effect when the Pro residue is located at the N-terminus, Ip(N), and the other is for the effect of the number of residues, Ip(#pep). The values of these indicator variables were set as zero for tripeptides without N-terminal proline, since their Alog P' value is closest to zero. The values of indicator variables are shown in Table 4. With these two additional indicator variable terms for the Pro residue, Eq. 19 was finally formulated for 124 peptides

203 without any significant decrease in the correlation quality. log P' = 0.942 Zrt - 0.582 Ipep+ 0.546 E'sC(RN) + 0.295 [ZE'sC(RM) (0.064) (0.096) (0.089) (0.071 ) + E'sC(Rc)] + 0.516 Iturn + 0.764 log fi+2 + 0.144 Iy (0.172) (0.211) (0.089) + 0.378 Iw+ 0.659 IM + 1.581 (Is + IT) - 0.807 Ip(N) (0.106) (0.165) (0.197) (0.225) -0.346 Ip(#pep)- 3.866 (0.118) (0.190) n = 124 s =0.209

[191

r=0.967

F12,111 =

134

In Table 2, the log P' values calculated by Eq. 19 are also listed. For Leu-LeuLeu-Pro, where no 13-tum formation with intramolecular hydrogen bonding is possible, the Iturn and log fi+2 terms were ommitted in calculating the log P' values. At the protonated amino group of the N-terminus working as the hydrogen donor, the solvation with the more basic 1-octanol could effectively compete with that with the less basic water. Since the number of polarized N+-H bonds in peptides including proline is lower by unity than that in others without cyclic amino acids at the N-terminus, the solvation with 1-octanol is less significant in peptides including proline than that in other regular peptides, leading to lower log P' values. The slope of the Ip(N) term, -0.81, was in the same order as that previously observed (-0.52) for the effect of the decrease in the number of N+-H bonds on the ion-pair formation-partition equilibrium for various aliphatic ammonium ions and picrate in the 1-octanol/water system (14). At positions other than the N-terminus, one of the amide NH sites working as the hydrogen-donor is reduced by replacing the regular primary amino acid residue with proline. By the same token as that for the N-terminal N+-H sites, the reduction of the NH sites would induce reduction of log P'. On the other hand, the steric inhibition effect of the "side chain" of the Pro residue on the hydrogen-bonding solvation of a neighboring CONH or COO- group could be lowered by the cyclization. This reduced steric effect would be favorable to the solvation of the bulkier 1-octanol leading to the augmentation of log P'. For tripeptides, these two oppositely operating factors may be balanced. The positive effect is predominant for dipeptides, but the negative effect gradually becomes dominant for higher peptides with increase in the number of residues. No theoretical rationalization for variations in the balance between these two

204 opposite factors is available at the moment. Measurements of the log P' values of more peptides containing proline at various positions are needed before drawing definite conclusions.

2.7

Peptides Containing Glutamine and Asparagine

Because the log P' value was very low, it was not always easy to measure the value for zwitterionic peptides including Gln (Q) and Asn (N). To understand the effects of these residues on the hydrophobicity of peptides, we measured the log P values of a number of N-acetylpeptide amides containing these residues under conditions equivalent with those for free peptides (data not shown), and formulated Eq. 20 as the counterpart of Eq. 4 (17). log P = 1.044 1;re- 0.570 Itri + 0.237 XE's c + 0.073 Iy + 0.258 Iw (0.047) (0.054) (0.046) (0.075) (0.080) + 1.476 (Is + IT) + 1.162 IQ + 1.753 IN - 2.375 (0.106) (0.121) (0.154) (0.074) n=53

s=0.072

r=0.997

[20]

F8,44 = 8 4 0

In Eq. 20, the E's c terms for side chain substituents are combined into a single XE's c term. This is due to the fact that, in N-acetylpeptide amides, it is invariably the CONH group toward which the side chain substituents exert the steric effect on the relative solvation. The intercept should correspond with the log P value of Ac-Gly-Gly-NH2. Except for these, the corresponding terms are very similar in Eqs. 4 and 20. Although the indicator variable terms for side chains in Eq. 20 are slightly smaller than the corresponding terms in Eq. 4, the correspondence is very good. In fact, for side chains of Ser, Thr, Trp, and Tyr, Eq. 21 was derived, in which RC is the regression coefficient of the indicator variable terms. RC(Eq. 4) = 1.010 RC(Eq. 20) + 0.092 (0.077) (0.082) n=4

s=0.024

[21]

r=0.9997

F1,2=3146

The slope of the side-chain indicator variable terms for Asn and Gln in Eq. 20 was adjusted to conform to the slope for residues in free peptides with use of Eq. 21 and is indicated in Table 3. Indicator variable terms for Asn (N) and Gln (Q) residues are very large. An intramolecular bridging-type solvation

205 between the side chain amide group and the backbone CONH similar to that shown in Fig. 1 is likely to occur in peptides including these residues (20). In fact, they are even larger than those expected from the simple relationshop with the number of bonds between the side chain heteroatom and the backbone after the correction for the intramolecular solvation is made. This indicates that the size of indicator variable terms is also governed by such factors as the number of hydrogen-bonding sites and electronic effect of the polar groups. In any case, by introducing these indicator variable terms in Eq. 18 or 19, the log P' value of free peptides including Asn and Gln should be estimated with considerable accuracy. 0

A N E W E F F E C T I V E H Y D R O P H O B I C I T Y S C A L E O F SIDE CHAINS

3.1

Definition From the results shown in Eqs. 19 and 20, we propose a new effective hydrophobicity scale, ha, for unionizable amino acid side chains as shown in Eq. 22. The na value is defined as the summation of such factors contributing to the "overall" hydrophobicity of each side chain unit as the "intrinsic" hydrophobicity, steric effects on the relative solvation of backbone functional groups, intra-residue hydrogen-bond formation and the proximity polar effect. In Eq. 22, 8 is 0.55 for N-terminal residues and 0.30 for others. The conformational factors are not included since they are attributable to not only the types of amino acid residues, but also their locations in the sequence. Moreover, they are not applicable to di- and tripeptides or to peptides larger than pentapeptides in which other conformational effects such as a - h e l i x formation should be considered. For proline, the nc~ value varies depending upon its situation. = 0.94 [intrinsic n] + ~5E's c + [coefficient of I for each polar side chain and proline]

[22]

The newly defined na values are listed in Table 5. ha(N) and ncdMC) mean the rta values for N-terminal residue and for others, respectively. The value calculated by Eq. 23 with the na value for the nonconformational components is supposed to be the log P' for an imaginary random form. Comparison of the log P'(random) with the experimentally observed log P' should be useful to obtain information on the component attributable to the effect of the conformation. log P'(random) = Y~na- 0.58

Ipep -

3.87

[23]

206 T a b l e 5. Hydrophobicity Scales for A m i n o Acids or Their Side Chains a,b Amino Acid Gly gla Val Leu Ile Phe Tyr Trp Met Ser Thr Asn Gln Pro d

na n (N) (MC) (FP) 0.00 0.00 0.00 0.19 0.24 0.31 0.48 0.81 1.22 0.91 1.27 1.70 0.71 1.16 1.80 1.34 1.56 1.79 0.78 1.00 0.96 1.71 1.92 2.25 0.67 0.92 1.23 -0.08 0.04 -0.04 0.07 0.25 0.26 -0.51 -0.26 -0.60 -0.51 -0.31 -0.22 e e 0.72

Af (NT) 0.0 0.5 1.5 1.8 2.5 c 2.5 2.3 3.4 1.3 -0.3 -0.4 -0.8 c -0.5 c 0.8

Ef (R) 0.00 0.53 1.46 1.99 1.99 2.24 1.70 2.31 1.08 -0.56 -0.26 _1.05 -1.09 1.01

AG (W) 0.00 -0.45 -0.40 -0.11 -0.24 -3.15 -8.50 -8.27 -3.87 -7.45 -7.27 -12.07 -11.77 -

AHS (C) 0.00 0.05 0.43 0.22 0.58 0.34 -0.68 -0.25 0.10 -0.41 -0.37 -0.84 -1.19 -0.56

AHS AHP (J) (KD) 0.0 0.0 0.0 2.2 0.3 4.6 0.2 4.2 0.4 4.9 0.2 3.2 -0.7 -0.9 0.0 -0.5 0.1 2.3 -0.4 -0.4 -0.5 -0.3 -0.8 -3.1 -1.0 -3.1 -0.6 -1.2

AHS A[-Z] (E) (H) 0.00 0.00 0.09 2.16 0.38 4.92 0.37 6.42 0.57 6.67 0.45 7.15 -0.14 3.62 0.21 6.98 0.10 4.72 -0.42 0.27 -0.34 1.31 -0.80 -0.99 -0.85 0.05 -0.23 3.45

a The reference point is shifted so that each value for Gly is zero. The values for Gly are: G(W) = 2.39, HS(C) = -0.34, HS(J) = 0.3, HP(KD) = -0.4, HS(E) = 0.16, and -Z(H) = -2.23. b For symbols, see text. c Estimated from the value in ref. 31. d Not included in regression analysis. e n~(location, number of residues) of proline; n,x(N, 2): 0.35, n~(MC, 2): 1.16, n~(N, 3): 0.00, na(MC, 3): 0.81, ha(N, 4): -0.34, na(MC, 4): 0.46.

For tetra- and pentapeptides, the conformational effect was represented by Iturn (= 1) and log fi+2 terms. Thus, examination of the difference between experimental log P' and calculated log P'(random) should allow us to predict the 13-turn potential parameter of any amino acids included in tetra- and pentapeptides. Although it does not apply to peptides including proline at the moment, this procedure may be extended to higher peptides in which secondary structural factors differ from those included in tetra- and pentapeptides. To estimate the log P'(random) value for partial domains of proteins, we recommend the use of 8 = 0.30 for the RM and RC side chains to calculate each n~ value by Eq. 22.

3.2

Comparison with Various Hydrophobicity Scales for Amino Acids and Their Side Chains Quite a few sets of parameters supposedly representing the "hydrophobicity" scale of amino acid residues have been proposed. Comprehensive lists of these parameters have been reported by Eisenberg (27), Charton (28) and Nakai and coworkers (29). These parameters are defined and/or estimated on the bases of various standards that are not always consistent.

207 They are broadly categorized into three groups. Parameters in the first group are defined from phase-transfer properties similar to that used in this study but with individual amino acids and their derivatives or related compounds. The scales in the second group are based on the probability of finding a certain amino acid residue in the interior of globular proteins relative to the probability of finding it in the surface. The third group is a composite of parameters of the above two types of scales. The values are listed in Table 5 and the relationships with the rta(MC) are drawn in Fig. 4. Fauchbre and Pligka (10) have measured the log P' value of N-acetylamino acid amides with a system of 1-octanol/aqueous buffer (pH 7), from which they defined the rt value of side chains as the difference from that of N-acetylglycineamide. Because their rt value inherently includes factors such as steric effects on the solvation of backbone CONH functions, the proximity polar effect between the side chain polar group and the backbone CONH functions and the internal hydrogen-bonding in addition to the intrinsic hydrophobicity, our rta(MC) value for 13 unionizable side chains was expected to correspond with theirs. Eq. 24 was formulated for this correspondence.

[24]

rt(FP) = 1.254 rta(MC) - 0.010 (0.175) (0.167) n=13

s=0.198

r=0.979

FI,ll = 2 4 8

The well known classic scale, Af, of Nozaki and Tanford (30) is based on free energy of transfer (kcal/mol) of amino acids from ethanol to water relative to that of glycine, for which Eq. 25 was obtained.

[25]

Af(NT) = 1.819 rta(MC) - 0.080 (0.277) (0.265) n=13

s=0.313

r = 0.975

F I , l l = 208

In the original publication (30), the Af values for lie, Gin, and Asn are not given. In Table 5 and Eq. 25, the values for these residues were estimated from the work of Segrest and Feldman (31). Rekker (32) has proposed a scale, f(R), named the hydrophobic fragment constant for each structural fragment. It is estimated from the 1-octanol/water log P values of a number of organic compounds including substructures appearing in the amino acid side chains statistically based on the additiveconstitutive nature of log P. The summation of the fragment constant values, Y_,f(R), for constituent substructures of amino acid side chains is related to rt~ as shown in Eq. 26.

208 Af (NT) [kcal/mol]

n (FP)

00

% I

I

|

i

i

na(MC)

na(MC) Ef (R)

AG (W) [kcal/mol]

N

0

-10 I

~

I

o

|

]

i

na(MC)

i

2

2

na(MC)

AHS (J) [kcal/mol]

AHP (KD)

-% II

u 9

II

I

,

i

.

1

o

-4

i

I

AHS (E)

i

o

na(MC)

2

na(MC)

zX[-Z (H)] 9

I

I

o0

0 i

I

0

i

I

1

|

71;ot(MC)

i

2

1

I

0

|

i

1

|

71:et(MC)

Fig. 4. Relationships of Various Hydrophobicity Scales with the na(MC) Parameter

209 Zf(R) = 1.538 rca(MC) - 0.673 Ip + 0.088 (0.130) (0.179) (0.161) n=13

s=0.137

r=0.995

[26] F2,10 = 5 0 6

The Ip is an indicator variable taking unity for polar side chains of Ser, Thr, Met, Trp, Asn, and Gln. Because the Rekker fragment parameter is estimated from log P values of compounds without structural characteristics of amino acids or peptides, it seems to underestimate the contribution of such factors in increasing the molecular log P value comprised in the regression coefficient of indicator variable terms for polar side chains listed in Table 3. The Tyr residue did not require the value of Ip = 1 in Eq. 26. This is in accord with the fact that the regression coefficient for the Tyr residue in Eq. 19 is very low. The slope of the rta term is considerably higher than unity. This is due to the fact that the Rekker value neglects the participation of the steric effect of side chains on the relative solvation in lowering the log P' leading to overestimation of the effective hydrophobicity. A set of phase-transfer parameters somewhat special among the category has been proposed by Wolfenden and coworkers (33). Their parameter, G(W), is the free-energy of transfer (kcal/mol) of RH, in which R is the side chain substituent in amino acids, H2NCH(R)COOH, from a gaseous to aqueous phase. Again, this parameter is based on the property of molecules without characteristic features of peptides. Moreover, the phases dealt with in estimation of the parameter are drastically different from the systems in which the above three types of parameters are defined. Therefore, their parameter is not expected to be related with rta. Preliminary examinations showed that G(W) is correlated only with the number of hydrogen-bondable hydrogens, I H D , in the polar groups on the side chain. Eq. 27 shows the situation in which the reference point of G(W) is shifted to that of Gly and so AG(W) = G(W) G(W)gly.

[271

AG(W) = - 5.661 IHD - 1.405 (1.146) (1.101) n = 13 s = 1.385

r = 0.957

FI,ll

=

118

The addition of the rta term to Eq. 27 did not improve the correlation. Eq. 27 indicates that the water-affinity or the hydration potential of the side chains is governed most significantly by the number of hydrogens capable of hydrogenbonding. The higher the number, the less hydrophobic is the residue. Recently, Radzicka and Wolfenden (34) suggested that the vapor phase resembles cyclohexane rather than octanol in its lack of polarity.

210 As parameters of the second category, those proposed by Chothia (35) and Janin (36) are well known. Janin has defined his parameter as the free-energy of transfer (kcal/mol) from the inside to the surface estimated from the ratio of mol fractions in buried and accessible states of each residue in globular proteins. The original Chothia parameter (35), the proportion of each residue 95 % buried in globular proteins, has been modified to place it on the free-energy-related background (37) similar to the Janin parameter. These two parameters were of course very well correlated with the slope of 1.128, s = 0.115, and r = 0.978 taking the Janin parameter as the independent variable. Wolfenden and coworkers showed that their parameter G(W) and the Janin parameter are well correlated with a correlation coefficient of r = 0.90 (33). This is not unexpected because Eq. 28 formulated here for the Janin parameter, HS(J), shows that it is also heavily dependent on IHD. [28]

zxHS(J) = 0.210 rca(MC) - 0.422 IHD - 0.012 (0.117) (0.110) (0.140) n=12

s=0.109

r=0.975

F2,9 =88.0

In Eq. 28, the Tyr residue is not included. Its HS(J) value was significantly lower than that expected. In spite of this, the interior/surface preference of amino acid residues tends to be governed in part by the phase-transfer energy between the two liquid phases. Because the general feature of hydrophobicity scales in terms of the freeenergy of transfer is quite different between scales with the two liquid phases and scales with the gaseous/aqueous system or the interior/surface preference, and also because it seems unrealistic to expect that all aspects of the "hydrophobicity" of residues can be summarized in a single manner, quite a few parameter sets have been proposed by combinations of different categorical parameters for each amino acid residue. One of these third-category parameter sets is the hydropathy scale, HP, proposed by Kyte and Doolittle (2). They defined their scale by somewhat arbitral amalgamation of the Wolfenden G(W) and the Chothia parameters. Because both the G(W) and the Chothia parameters are strongly dependent on the IHD, the HP value is of course related with the IHD as well as the bulkiness in terms of-E's c but not with the rca as shown in Eq. 29. AHP(KD) = -2.271 E's c - 3.039 IHD + 0.882 (0.878) (0.552) (0.965) n-13

s-0.656

r=0.976

[29] F2,10-100

211

Another set of parameters has been put forward by Eisenberg and coworkers (37) as the "consensus" hydrophobicity scale, HS(E). In this scale, not only the gaseous/aqueous, [G(W)], and the interior/surface parameters of Chothia [HS(C)] and Janin [HS(J)], but also a phase-transfer parameter between organic and aqueous liquids theoretically evaluated by von Heijine and Blomberg (38) are amalgamated with normalization and averaging. Because the consensus HS(E) scale involves the component for the phase-transfer between liquids, Eq. 30 shows the significance of our rta as a component factor. In fact, Eq. 30 is very similar to Eq. 28 for the Janin parameter. The amalgamation for the consensus parameter seems to correct the outlying behavior of the Tyr residue from Eq. 28.

[30]

zxHS(E) = 0.303 na(MC) - 0.393 IHD + 0.012 (0.106) (0.099) (0.130) n=13

s=0.103

r=0.979

F2,1o = 113

Hellberg and coworkers (39) have examined a number of descriptors for the amino acid residues characterizing chemical, spectral, phase-transfer, and chromatographic properties statistically by using the principal component analysis and extracted a principal component supposedly related to the hydrophobicity. For their scale, -Z(H), Eq. 31 was formulated.

[3~]

zx[-Z(H)] - 3.638 na(MC)- 1.166 E's c -0.103 (0.767) (1.139) (0.986) n=13

s=0.745

r=0.974

F2,10 = 92.9

Depending upon the selection of the original parameters for the amalgamation, the third-category scales are heavily governed by either the hydration potential represented by IHD and/or the phase-transfer property represented by rta. In Eqs. 29 and 31, a negative E'sC term is significant. The more negative the E ' sC , the more "hydrophobic" is the side chain. This reflects the fact that the steric inhibition effect of side chain substituents on hydration of backbone CONH groups works to make the side chains more burried inside the globular proteins for the hydropathy scale in Eq. 29. In Eq. 31 for the -Z(H) scale, the principal component analysis of the amino acid descripters probably extracted the scale as rta- 8E's c (8 = 0.30) in Eq. 22, because there is no backbone CONH function upon which the steric effect of side chains is exerted in single amino acid residues.

212 4.

CONCLUDING REMARKS

The above examinations are believed to show that the hydrophobicity of peptides, at least up to pentapeptides, that is estimated from the partitioning behavior in an alcohol/aqueous system such as 1-octanol/pH 7.0 buffer, can be analyzed and predicted by combinations of well-defined side chain and substructural parameters. The composition of the hydrophobicity scale was rather complex but each component was rationalized physicochemically very well except for the composition attributable to the Pro residue. The extensions of the present approach toward peptides including ionizable side chains as well as higher peptides should be future projects. The rt (rta) value defined here as the "effective" hydrophobicity index of side chains or residues is unique in that it was estimated from the experimentally measured net "hydrophobicity" of oligopeptides existing in solutions as such. Most of the hydrophobicity indices of amino acid side chains so far published are defined from partition or phase transfer parameters of single amino acids or their analogs or calculated from the solvent-accessible surface area of each residue in globular proteins or composites of these two types of indices, as indicated in the preceding section. We examined the relationship between our rta and each of the existing parameters somewhat in detail because we would like to propose our rta value as the standard hydrophobicity index of amino acid side chains as components of peptides. In this respect, it should be noted that a recent publication of Eisenberg and McLachlan (40) indicates that the solvation energy of globular proteins in water is well rationalized not only by the solvent accessible surface area but also by an "atomic solvation parameter" of each atom included in amino acid side chains accessible to water. The simple ratio of molecular fractions in buried and water-accessible states for amino acid side chains is obviously an oversimplification in estimating the hydrophobicity. The atomic solvation parameter assignable to each atom is very well estimated from the phase-transfer free-energy based on values with the 1-octanol/water system rather than a gaseous/aqueous system. Eisenberg and McLachlan proposed that the interior environment of globular proteins is adequately modeled by nonaqueous but amphiprotic liquids. In a more recent publication of Sharp et al. (41), the changes in the partition free energy of component amino acid residues in a 1-octanol/water system corrected for solute-solvent size differences were shown to agree well with the changes in unfolding free-energy of a variety of mutant proteins. These publications seem to support our proposal that our rta value could be used as the standard hydrophobicity scale.

213 REFERENCES

1. 2. 3.

4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29.

Kauzman, W., Adv. Protein Chem. 14 (1959) 1-63. Kyte, J. and Doolittle, R.F., J. Mol. Biol. 157 (1982) 105-132. a: Hadzi, D. and Jerman-Blazic, B. (Eds.) QSAR in Drug Design and Toxicology, Elsevier Science Publishers, Amsterdam, 1987, pp. 221-297; b: Claassen, V. (Ed.)Trends in Drug Research, Elsevier Science Publishers, Amsterdam, 1990, pp. 73-108. Hansch, C. and Fujita, T., J. Am. Chem. Soc. 86 (1964) 1616-1626. Akamatsu, M., Yoshida, Y., Nakamura, H., Asao, M., Iwamura, H., and Fujita, T., Quant. Struct.-Act. Relat. 8 (1989) 195-203. Akamatsu, M. and Fujita, T., J. Pharm. Sci. 81 (1992) 164-174. Chou, P.Y. and Fasman, G.D., J. Mol. Biol. 115 (1977) 135-175. Iwasa, J., Fujita, T., and Hansch, C., J. Med. Chem. 8 (1965) 150-153. Hansch, C. and Leo, A.J., Substituent Constants for Correlation Analysis in Chemistry and Biology, John Wiley and Sons, Inc., New York, 1979, pp. 17-43. Fauchbre, J.-L. and Pli~ka, V., Eur. J. Med. Chem. -Chim. Ther. 18 (1983) 369-375. MacPhee, J.A., Panaye, A., and Dubois, J.-E., Tetrahedron 34 (1978) 3553-3562. Taft, R.W., Jr., in: Newman, M.S. (Ed.), Steric Effects in Organic Chemistry, John Wiley and Sons, Inc., New York, 1965, pp. 556-675. Hancock, C.K., Meyers, F.A., and Yager, B.J., J. Am. Chem. Soc. 83 (1961) 4211-4213. Takayama, C., Akamatsu, M., and Fujita, T., Quant. Struct.-Act. Relat. 4 (1985) 149-160. Charton, M., Topics Curr. Chem. 114 (1983) 57-91. Charton, M. and Charton, B.I., J. Theor. Biol. 102 (1983) 121-134. Akamatsu, M., Okutani, S., Nakao, K., Hong, N.J., and Fujita, T., Quant. Struct.-Act. Relat. 9 (1990) 189-194. Fujita, T. and Iwamura, H., Topics Curr. Chem. 114 (1983) 119-157. Calculated from the hydrophobic fragmental constants: f(CH3CONH) f(H) = -1.94 - 0.23. The f values were from ref. 9. Fermi, G., Perutz, M.F., Shaanan, B., and Fourme, R., J. Mol. Biol. 175 (1984) 159-174. Abraham, D.J. and Leo, A.J., PROTEINS: Structure, Function, and Genetics, (1987) 130-152. Fujita, T., Prog. Phys. Org. Chem. 14 (1983) 75-113. Leo, A., J. Chem. Soc. PERKIN TRANS. II (1983) 825-838. Venkatachalam, C.M., Biopolymers 6 (1968) 1425-1436. Dickerson, R.E., Takano, T., Eisenberg, D., Kallai, O.B., Samson, L., Cooper, A., and Margoliash, E., J. Biol. Chem. 246 (1971) 1511-1535. Lewis, P.N., Momany, F.A., and Scheraga, H.A., Proc. Nat. Acad. Sci. USA 68 (1971) 2293-2297. Eisenberg, D., Ann. Rev. Biochem. 53 (1984) 595-623. Charton, M., Progr. Phys. Org. Chem. 18 (1990) 163-284. Nakai, K., Kidera, A., and Kanehisa, M., Prot. Eng. 2 (1988) 93-100.

214 Nozaki, Y. and Tanford, C., J. Biol. Chem. 7 (1971) 2211-2217. Segrest, J.P. and Feldman, R.J., J. Mol. Biol. 87 (1974) 853-858. Rekker, R.F., The Hydrophobic Fragmental Constant, Elsevier Science Publishers, Amsterdam, 1977. 33. Wolfenden, R., Andersson, L., Cullis, P.M., and Southgate, C.C.B., Biochemistry 20 (1981) 849-855. 34. Radzicka, A. and Wolfenden, R., Biochemistry 27 (1988) 1664-1670. 35. Chothia, C., J. Mol. Biol. 105 (1976) 1-14. 36. Janin, J., Nature 277 (1979) 491-492. 37. Eisenberg, D., Weiss, R.M., Terwillger, T.C., and Wilcox, W., Faraday Symp. Chem. Soc. 17 (1982) 109-120. 38. von Heijine, G. and Blomberg, C., Eur. J. Biochem. 97 (1979) 175-181. 39. Hellberg, S., Sj/Sstrom, M., Skagerberg, B., and Wold, S., J. Med. Chem. 30 (1987) 1126-1135. 40. Eisenberg, D. and McLachlan, A.D., Nature 319 (1986) 199-203. 41. Sharp, K.A., Nicholls, A., Friedman, R., and Honig, B., Biochemistry 30 (1991) 9686-9697. 30. 31. 32.

QSAR and Drug Design - New Developments and Applications T. Fujita, editor @ 1995 Elsevier Science B.V. All rights reserved

ANALYSIS

OF

PROTEINS

TAKAAKI

ACID

SEQUENCE-FUNCTION

RELATIONSHIPS

IN

N I S H I O K A and JUN' ICHI ODA

Institute

Uji,

AMINO

215

Kyoto

for Chemical

Research,

611, Japan

Kyoto University,

ABSTRACT:

A n e w s t r a t e g y for d r u g d e s i g n is p r o p o s e d , in w h i c h relationships between the function and structure of proteins that are p o s s i b l e t a r g e t s of d r u g s are a n a l y z e d and u t i l i z e d . The f u n c t i o n of p r o t e i n s to r e c o g n i z e the m o l e c u l e s was e x a m i n e d in t e r m s of t h e i r a m i n o a c i d s e q u e n c e r a t h e r t h a n t h e i r t h r e e dimensional structure. Target proteins recognize ligand m o l e c u l e s by f u n c t i o n a l a m i n o a c i d s e q u e n c e s c o r r e s p o n d i n g to chemical substructures of t h e l i g a n d s . The new procedure "Homology Graphing", in c o m b i n a t i o n w i t h the E n z y m e - R e a c t i o n database, could detect sequence segments conserved among a set of sequences of functionally related proteins. Examples of analyses of a m i n o acid sequence-ligand structures showed a great p o t e n t i a l i t y in the lead identification phase in drug design.

1.

INTRODUCTION

Recently,

and-see

drug

complementary

model of

to the

of a t a r g e t

the

binding.

design

approaches

protein For

of

target

protein

and

strategy

finding

the

instance,

site

have been c a l c u l a t e d

advances,

de novo

hundred

be r e a l i z e d

design

in the near

by t r a d i t i o n a l screening.

picoseconds

This

structure-function molecular the

of

(i).

the

supported

Lead-structures

and/or

to

lack

especially

rotations

and

drug

of

by

drug

to

found

large-scale

information

amino

for

technological

is u n l i k e l y

are still

of proteins.

proteins

in

dihydrofolate

these

methods due

static

motions

of 1 f e m t o s e c o n d

structure

relationships of

of

look-

structures

involved

lead

probably

motions

translations

Even w i t h

of a new

relationships,

recognition

simulations like

is

positions

at i n t e r v a l s

future.

beyond

of the m o l e c u l a r

processes

atomic

trial-and-error

chemical

in a t h r e e - d i m e n s i o n a l

dynamic

the

progressed

with

to s i m u l a t i o n

reductase several

has

drugs

acid

on

sequence-

In other words, molecules

of CPK m o d e l s

are

of organic

216 reagents

to find out the

knowledge

of

reaction

relationships, state

"best" rules

no c h e m i s t

structures

of

complementary in

terms

In

this

functions

reactants

proteins,

several are

of o t h e r

relationships structures

chemical 2.

WHY

between

their

of

acid

transition

the

reaction

bases

sequences

must

"molecular of t h e i r

of the

acid

by t h e s e

ANALYZE

proteins:

the the

we are

sequences

that

RELATIONSHIPS,

STRUCTURE-FUNCTION

RELATIONSHIPS

(ligands)

searching

and is,

NOT

amino

acid s e q u e n c e s

their been

in

genes.

in t h e

Protein

Identification

Research

Foundation).

of i n t e r e s t

as targets

crystallographic Laboratory) were

data

rapidly: only

available

number

of

sequence-

THREE-

?

14,372

Protein

Resource

at

DNA

techniques,

M o r e than

90% of the known

from the DNA s e q u e n c e s amino

acid

Sequence the

sequences

Database

National

of

had

(NBRF;

Biomedical

S e q u e n c e data are i n c r e a s i n g

for p r o t e i n s

of drugs and a g r o c h e m i c a l s .

In contrast,

on p r o t e i n s

are

still

in the Protein Data Bank

585

proteins

1989,

NBRF

coordinate

in O c t o b e r

have been registered

biology

have been d e d u c e d

By D e c e m b e r

registered

increasing

molecular

of genes has b e c o m e easy.

for

chemical

2.1 A v a i l a b i l i t y of sequence data progress

as

signals.

to d i f f e r e n t i a t e

sequences;

SEQUENCE-FUNCTION

DIMENSIONAL

by

have

such

of cell

and h o r m o n e s

Therefore,

amino

Proteins

functions

receptors

substrates

chemicals.

between

be defined.

recognition"

and h o r m o n a l

recognized

With

and

sequences

the a p p l i c a t i o n

and transduction

structure relationships.

sequencing

for

three-dimensional

physiological

reactions

in

structures

from t h o s e

amino

We a l s o d i s c u s s

kinds

of e n z y m e s

chemical

biochemical

between

function,

of chemical

interested

ability

the

simulate

in drug design.

the term,

different

catalysis

or

Without

Then, we show how to analyze

relationships.

First,

show

not

and functions.

relationships

We

we

relationships

of

structures

to find

chapter,

the

fitting.

structure-function

can e i t h e r p r e d i c t p o s s i b l e

the

p a t h w a y by energy calculation. examining

of

1989.

entries

known

on

Moreover,

for several proteins with

limited

and

are not

(Brookhaven National crystal

at least

structures

two entries

in the database,

three-dimensional

so the

structures

is

217 actually

less

than

for

drug

available

120. design

interest

for d r u g

localized

in pathogens,

small

design,

quantities,

or

protein

sequences,

"target

(c) h a v e

methods

from

its

the

to

need

for

deduce

sequence, sequence

optimization active

structures,

information

low as

predictions for

relationships

prediction

through

crystal

is

and main-chain

structure

Glutathione

coli enzyme

to

data

on

of

three-

model

of

At present,

the

have

energy

in

the

prediction

We

are

success of

interested

structure

reductase

to

only

-COOH,

catalyzes with

in

not

moiety,

the

from the sequence.

including

two

to

by

the

complex,

to

the

in

the

protein

hydrogen-bonding

misconception

spatial

the r e d u c t i o n

coenzymes,

only at the

where

of

of o x i d i z e d

NADPH

specificity

engineering

and

FAD.

of the E.

(6,7).

The

i/i00 of that to

2'-OH p o s i t i o n

a phosphate

that

orientation

and -NH 2 groups.

enzyme to NADH is only

and NADH d i f f e r

in

sequence

function,

bind

tried to change the c o e n z y m e

adenosine-ribose

of

and e s t i m a t e d

The poor

and p r o t e i n

leads

occurs

glutathione

of and

b e t w e e n the ligand and s i d e - c h a i n

This

such as -OH,

a

a tertiary

molecule,

observed

from NADPH to NADH by protein

NADPH

of

the a v a i l a b i l i t y

interactions

a f f i n i t y of the wild-type NADPH.

as

predictions

drug

(4,5).

assumed

interactions

by p r o t e i n s

P e r h a m et al.

such

W h e n no c r y s t a l l o -

of a p r o t e i n - l i g a n d

generally

groups.

groups

glutathione

in

of a drug to fit into the

between

sequence

a

Even

design.

the

point-to-point

and c h a r g e - c h a r g e

functional

way.

limited

usually roles

structure is recognized by local sequence

molecule

recognition

structure

of the three-dimensional

2.2 Chemical In the

drug

between

has

(2,3).

and

% at most

of

of

of some other protein with

evaluation

protein

the m a t c h

50-60

use

are available,

reliable

the

than

prediction

involve

as a model

in a s a t i s f a c t o r y

is as

structural

ligand

for

less

is increasing.

structure

of the c h e m i c a l

site

secondary values

low

are

weights

a three-dimensional

too

between

far

accurate

however,

so

been

are

for drug design

for related proteins

far

proteins

(a)

larger molecular

graphic data interactions

structures

because

proteins",

data

such as the crystal

a closely related

less

centers and virus coat proteins.

structure

practical template

far

crystallographic

dimensional protein

three-dimensional

are

(b) play important p h y s i o l o g i c a l

those of p h o t o r e a c t i o n Since

The

group

of the

is p r e s e n t

in

218 TABLE

1

Arginines

conserved

in N A D P H - b i n d i n g

NADPH-binding reductases Glutathione reductase E. coli 196 Human 216 Mercuric reductase S__~. aureus 279 S. f l e x n e r i i 298 Trypanothione reductase T__~. c o n q o l e n s e 221

reductases.

*)

F V R K H A P L R S F D M I R H D K V L R S F D m

M Q R S E R L F K T Y D L A R S T L F F R E - D C Y R N N P I L R G F D

NADH-binding reductases Dihydrolipoamide dehydrogenase E. col____!. 202 V E M F D Q V I P S S D Yeast 231 V _E F Q p Q I G A S M D Human 242 V E F L G H V G G V G I *) M o d i f i e d from ref. 6 with p e r m i s i o n of the o r i g i n a l authors. A m i n o acid residues are r e p r e s e n t e d by o n e - l e t t e r symbols. N u m b e r s i n d i c a t e p o s i t i o n s of the first r e s i d u e of each sequence. NADPH, less

but

at

not

the

the

human

ray

analysis

(8,9).

NADH

of

residues recognize residues

NADPH. which with

I) .

might

be

Perham

Arg198

al.

the m u t a n t

positions

was

showed Next,

mutant

less

only to

coenzyme-binding secondary

structures the

site

the

enzyme

of

around

13).

This

requiring

NADH

and

to

in

leucine

mutagenesis. at

the

As two

enzyme,

NADH.

of

NADH

those

site

with around of

reductase

the N A D P H - b i n d i n g type

enzyme

sequence

with

in that

affinity

coli

with

acid

with

arginine

the w i l d - t y p e

glutathione

fold";

the

charges

activity

amino

2'-

to neutral

These

E.

no p o s i t i v e than

the

residues

by m e t h i o n i n e

c a ta l y s i s

by X-

residues

the

site-directed

to N A D P H

human

be

a mutant

the

the

arginine

suppressing

catalytic

"dinucleotide

(12,

dehydrogenases

the

near

replaced

of

charged

dehydrogenases

are

and NADH.

replaced

increased

determined

located

to

compared

In

gy c a l l e d

for

with

catalytic

they

dehydrogenases.

beta-sheet

enzyme

improve

two

NADPH

by u s i n g

slightly

enzyme,

the

charges

strucutre

positively

In o t h e r

constructed were

negative

was

are

concluded

between

side-chains

two

the

residues

unnecessary

et

expected, but

Thus,

and A r g 2 0 4

neutral

NADPH.

were

the d i f f e r e n c e

that

residues

arginine

reductase

are

complex

showed

arginine

these

(Table

glutathione

there

three-dimensional

enzyme-NADPH

of the bound

as coenzyme,

is, The

Results

two

group

that

in NADH.

erythrocyte

side-chains phosphate

in NADH;

2'-OH

the the

other

(i0,ii),

form a topolo-

a beta-sheet-turn-alpha-helixof or

fold

is

NADPH.

found They

commonly showed

in that

219 T A B L E 2 A l i g n m e n t of s e q u e n c e ~ of N A D P H the d i n u c l e o t i d e - b i n d i n g fold.-) NADPH-binding reductases Adrenodoxin reductase Human 151 Octopine synthase Aqrobacterium 8 Malic enzyme Rat 300 Glutamate dehydrogenase Yeast 224 Mercuric reductase S. f l e x n e r i i 276 Glutathione reductase E. coli 174 Human 194 Thioredoxin reductase E. coli 152

and N A D H - e n z y m e s

around

G Q G N V A L D V A R I G A G N V A L T L A G D G A G E A A L G I A H L G S G N V A Q Y A A L K G S S V V A L E L A Q A G A G Y I A V E L A G V G A G Y I A V E M A G I G G G N T A V E E A L Y

NADH-binding reductases Dihydrolipoamide dehydrogenase E. coli 180 G G G I L G Alcohol dehydrogenase Rat 15 G L G G V G_ Lactate dehydrogenase Mouse 25 G V G A V G Glyceraldehyde phosphate dehydrogenase Yeast 7 G F G R I G

L E M G T V L S V V I G M A C A I S R L V M R I

*) M o d i f i e d from ref. 6 with p e r m i s i o n of the authors. Amino acid r e s i d u e s are r e p r e s e n t e d by o n e - l e t t e r symbols. The numbers i n d i c a t e the p o s i t i o n of the first residues of each sequence. dehydrogenases

requiring

GIy-X-GIy-X-X-GIy Gly

is

in the the

replaced E.

by Ala

coli

above

shown

at A l a 1 7 9 ,

mutant

enzyme

have

X is a n y

a highly amino

in d e h y d r o g e n a s e

glutathione

alignments

mutations

NADH

(where

reductase

in T a b l e s Ala183, and

2,

Val197,

finally

they

Lys199,

obtained

the

NADPH

2).

By

further and

a

sequence

while

requiring

)(Table

1 and

conserved

acid),

third

(Ala179

comparing introduced

His200

mutant

in the

enzyme,

Ala179Gly/Ala183Gly/Val197Glu/Arg198Met/Lys199Phe/His200Asp/Arg 204Pro,

enzyme

with

activity

to NADPH.

This

example

phosphate

group,

NADH,

is

phosphate by

group

side-chains

the

structural on

the

interactions

environmental

comparable

illustrates the

recognized

charge-charge

to NADH

enzyme

interactions

and m a i n - c h a i n

important difference not

between

and p o s i t i v e l y of

to that

only

the

fact by

Arg

phosphate

the

2'-

NADPH

and

point-to-point,

negatively

of the

that

between

charged the

of the w i l d - t y p e

charged

side-chains, group

dinucleotide-fold.

2'-

but also with The

the

helix

220 in the

fold

stabilizes

the positive

the n e g a t i v e

helix

by dipoles

tight

turn

that

(14,15). allows

the

The

first

fold

to m a k e

contact with NADH by van der Waals 2.3 Loops are responsible The p e p t i d e

segment

does not always the

a

determined antibody

loop

light

affinity the

of

DNA-binding that,

in

crystallographic

a strict

molecular

Gly

six h y p e r v a r i a b l e

as a s t r u c t u r a l

of

recognition

structure

proteins

(17),

cases,

analysis.

recognition

sites

loops

unit

six

(18,19).

for m o l e c u l a r

For example,

an

the

three-dimensional

structure

forming the loops.

loops

but

to

the

chemical

called the of the heavy

specificity

proteins

synthetase

are r e l a t e d

acid

of

identified

in ras

amino

and

structures

are also

recognition

so

be

of

and tryptophan

of the f l e x i b l e

is

cannot

by the

Loops

such as

but

it

site,

The

are g o v e r n e d

(20), glycogen phosphorylase(21), functions

loops.

a

steric)

(16).

The a n t i g e n - b i n d i n g

consists

form

(less

for m o l e c u l a r

some

by

side of the

residues

a close

region located in the variable domains

chains,

of the b i n d i n g

The

two

interaction

responsible

of its antigen.

hypervariable and

coenzyme

for m o l e c u l a r r e c o g n i t i o n

structure

by X - r a y

makes

structure

of the

take a fixed t h r e e - d i m e n s i o n a l

helix-turn-helix

flexible

charges

charge that is induced at the N-terminal

not

(22).

to the

sequences

2.4 Sequence segments of functional importance are conserved The

related locally

amino

to

each

similar

recognize

and

substitutions importance protein

acid

sequences

other

in

terms

in the regions bind

their

of

fatal

proteins

where

ligand

by r a n d o m m u t a t i o n s

cause

of

their

that

polypeptide

is not inherited.

are

only

fold

Amino

at the p o s i t i o n s

that are conserved among the proteins

closely

chains

molecules.

loss of the p h y s i o l o g i c a l

and this mutation

are

functions,

to

acid

of f u n c t i o n a l

f u n c t i o n of the

Therefore,

are sequences

sequences

of functional

importance. Here,

we

"dinucleotide

briefly fold"

dehydrogenases. evolution

refer

to the b i o l o g i c a l

reasons

is conserved among the sequences

According

of proteins,

to a r e c e n t

the gene

theory

why

the

of different

of the m o l e c u l a r

of a n e w p r o t e i n

evolves

not by

r a n d o m mutations

of the gene of some other protein with different

function,

"exon-shuffling"

but by

(23-26).

In the exon-shuffling

221 theory, acid

exons

coding

residues

rearrangement ferred for

from

of

similarities separate

are

acid

genes,

identical

in

to

residues

function.

same

ancestral

each

other.

sequence, by

find

random

the

not

only

Then

proteins

after begin

mutations. of

the

importance

of m o l e c u l a r

30-50

exons

a new

exons

they

boundaries

of

are

gene

show

trans-

that

codes

whose

genes

local

sequence

divergence

from

amino

of e v o l u t i o n a r y

form

exon would Just

but

of p h y s i o l o g i c a l

a unit

to

the two d u p l i c a t e d

substitutions

difficult are

with

composed

to be a unit

duplications,

and m i x e d

novel

the

segments

supposed

By g e n e

genes

with

inherited

are

genes.

other

a protein

have

sequence

in length

into

the a n c e s t r a l

to

accumulate

With

time,

two exon

nucleic

it b e c o m e s

duplicated

exons.

But,

conserved.

Thus,

exons

are

evolution,

but

also

of

protein

function. 2.5 M o t i f s

are too small

The p o s i t i o n a l different

proteins

are

GIy-X-GIy-X-X-GIy (27,28)

and

motifs.

functionally

to b u i l d only

actions

a peptide

a structural

one

a bound

motif

is u s u a l l y

tif.

Protein

beta-sheets of

five

lowing

supported

secondary

but by the

the m o t i f

composed in t h e called

of

ras the

secondary

27 r e s i d u e s and

including

molecule,

not

of

the

loop"

(35,36).

by w h i c h

is too

Although

than

does the

of

by Chou

and

composed

and

preceding

Fasman

and

fol-

GIy-X-GIy-X-X-GIy

hand,

the

in a l o o p not

motif

a

the mo-

sequence

the m o t i f

and

inter-

folding

"dinucleotide-binding is

short

alpha-helices

local

of

detected

direct

longer

supposed

kinase

Thus,

make

as

On the o t h e r

adenylate

(30),

peptide

far

the

in g r o u p s

a motif

sequences

For e x a m p l e ,

and

recognition.

such

The

zipper

well-known

successfully

the

by a short of the

are

motifs

only

by a s e q u e n c e

(15).

leucine

developed

are

among

patterns).

proteins,

in a motif

as o r i g i n a l l y

"glycine-rich structure

been

residues

find

for m o l e c u l a r

is a p a r t

protein

have

to

residues

"context"

(34).

in d e h y d r o g e n a s e s

DNA-binding

structures

are d e t e r m i n e d

conserved

(or fingers,

methods

ligand

are

serine-proteases

chain

or six r e s i d u e s

(32,33),

of

acid

acid

unit

that

dehydrogenases,

of

unit

or two amino with

(29)

proteins

three-amino

However,

motifs of

several

related

of

(31).

called

sequence

Recently,

patterns

of residues

sequence

zinc-finger

GIy-X-Ser-X-GIy

as a s t r u c t u r a l

patterns

make itself

fold"

same m o t i f structure any

fixed

does

not

222 have

any

fixed

assigned

secondary

depending

is involved.

We have to search

than that of the motifs 3. H O M O L O G Y

GRAPHING:

FUNCTIONAL

If

we

METHOD

could

find

of

molecules

containing

these

nition

common

paring

of a c o m m o n

aligned

There

of

common

segments

similarity addition, of

low

computer

-

may

acid

3.1

amino

be as

(or s u b s t r u c t u r e ) .

as c o n s e r v e d

acid

segments in

to a l i g n m o r e

been

developed

available

by w h i c h among

for

a

set

are

than

usually and

three of

sequences

as

their

In

sequences

although pairs

we d e v e l o p e d regions

of

such

sequences

in p o s i t i o n .

alignment

conserved

acid

length,

(37,38),

Recently,

by com-

in d e t e c t i n g

30 % i d e n t i t y

alignment).

(39,40).

Such

These

sequences

a set of a m i n o

residues

20-

method

are

difficulties

within

low as

has

sequence

of

many

se-

a method

within are

a given

detected

Homology graphing Homology

lative (target

local

graphing

Window

of an a m i n o

and

sequence

from the N H 2 - t e r m i n a l along

eral

The

residues.

step is d e f i n e d 3.1.2 search

acid

as segment-i

with

sequence

segments:

The

stepwise

segment (Figure

against

the cumu-

to be a n a l y z e d

sequences. target

sequence

with

is

a window.

at intervals

in the w i n d o w

of sev-

at the

i-th

i).

of h o m o l o g y value:

is p e r f o r m e d

graphically

to the C O O H - t e r m i n a l

the sequence

sequence

Calculation

is a l i g n e d

and shows

to a set of r e f e r e n c e

The w i n d o w moves

ment-i

calculates

similarity

sequence)

3.1.1 scanned

ity

the

recognize

structure

Graphing"

quantitatively

among

can

chemical

proteins.

programs

OF

present that

longer

(or s u b s t r u c -

related

(pairwise

are

for the recog-

segments

40

that

SEGMENTS

proteins

chemical

it

structure

no p r a c t i c a l

"Homology

SEQUENCE

is

should be r e s p o n s i b l e

certain

similarity

quences amino

20

segments

role

with which

segments

however,

sequence

as

a common

functional

sequences with each other.

functionally

short

its

commonly

related

c o u l d be d e t e c t e d

are,

conserved

TO FIND

segments

functionally

and

of the s e q u e n c e s

sequence

only.

IMPORTANCE

sequences ture),

structure

on the c o n t e x t

For segment-i,

a reference

one of the r e f e r e n c e

sequence sequences,

similar-

set.

Seg-

sequence-

223 Window

NH2-Terminal

COOH-Terminal

i

Target sequence

|

Segment-i

Similarity search Reference sequences m

u

Sequence 1 - - ~

score-i,1

Sequence j - - ~ score-i,j

m

Sequence n - - - ~ score-i,n ~

Homology value of segment-i in Score-ij { if score-ij>Maxd } Score-i1 Fig.

I.

H o m o l o g y graphing.

j, by u s i n g

found,

IDEAS s y s t e m

the d e g r e e

calculated

dent on the amino factors.

limit

acid

If score-ij

is not saved.

is slightly,

composition

is h i g h e r

similarity),

segment-i

than the threshold value.

(42).

length

than a given

the v a l u e

The degree of

of

segment-i.

threshold

(a lower

If not,

it

sequence-(j+l),

and saved if score-i(j+l)

This process

depen-

as to these two

is saved.

reference

is

is

is higher

is repeated until all the

sequences have been compared pairwise with segment-i.

The sum of the score-ij

number

(score-ij)

but significantly,

and the

is a l i g n e d w i t h

then similarity is calculated reference

for the a l i g n m e n t

the v alue is c o r r e c t e d and n o r m a l i z e d

of d e t e c t i n g

Next,

When the best local a l i g n m e n t

from the amino acid mutation data

similarity thus calculated

Therefore,

(41).

of s i m i l a r i t y

of r e f e r e n c e

value of segment-i

(from j=l to n, where n is the total

sequences)

[Equation i].

saved

is d e f i n e d

as the h o m o l o g y

224 H o m o l o g y value of segment-i Score-ij

J

{ if score-ij

The h o m o l o g y v a l u e similarity

and

number

homology

of

of

alignments

3.1.3

segment-i segment.

is

until

showing

calculated

the

for

[i] in the d e g r e e

higher

of

similarity

is r e p e a t e d at each step

COOH-terminal.

each

segment

in

Thus,

the

the

target

Graphing:

To show g r a p h i c a l l y ,

the h o m o l o g y value of

is p l o t t e d

against

at

the

residue

By v a r y i n g three p a r a m e t e r s

movement

}

increase

This p r o c e s s

the w i n d o w

value

sequence.

> threshold

increases with

than the t h r e s h o l d value. of m o v e m e n t

=

of the window,

and t h r e s h o l d

we can detect any sequence

the

center

(window size,

for d e t e c t i n g

segments differing

of

the

step size of

similarity),

in length and simi-

larity. 3.2 H o m o l o g y graphing of glutathione reductase Here, homology human

we

show

graphing.

glutathione

an e x a m p l e The

reductase,

u n d e r the e n t r y name of RDHUU the c r y s t a l composed NADPH-

structure 293),

to 478) domains enzyme

FAD-

central-

(43,44).

of 1.54-2 A

as a t a r g e t Three sequence

sequence

registered

(from r e s i d u e (294 to

analysis acid

364),

using

sequence

of

X-Ray analysis

of

in t h e

(478 residues).

(8-10).

NBRF

database

that this enzyme is 19 to r e s i d u e and

157),

interface-

structures

sequences

(365

of the

includes

those

coenzyme;

19

sequence

are c o m p o s e d

of the F A D - r e l a t e d

reductase

could

detect

are

prepared

from

the

the s e q u e n c e s

NBRF of the

that require NADPH or NADH as a coenzyme; enzymes.

enzymes

of 14 FAD-related

of the s e q u e n c e s

for the c o n t r o l

sets

NAD(P)H-related

27 sequences

graphing

selected

for coenzyme binding.

The first one c o m p r i z e s

enzymes

of

The enzyme was therefore

to test how h o m o l o g y

reference

database.

NAD(P)H-related

enzymes

amino

The three-dimensional

the segments of importance

thione

sequence

the

c o m p l e x e d w i t h FAD and NADPH have also been a n a l y z e d at a

resolution

30

the

is

of the enzyme r e v e a l e d

of four domains:

(158 to

of

target

requiring experiment;

not requiring NADPH,

that

enzymes.

The

second

require

set

FAD as a

These two sets

f u n c t i o n a l l y r e l a t e d to the gluta-

both N A D P H and FAD. sequences NADH,

The third

set is

of n u c l e o t i d e - n o n r e l a t e d

or FAD.

This

set is to detect

225

omain

200

(a)

tO > Cn 0

S

100

f'

100

200

300

400

Residue number

500

NADPH-domain

150

(b) g

100

qJ

ii-,

o 0

E o 50

-r

100

200

,

300

I i

400

Residue number

Fig.

2.

H o m o l o g y graphs of human g l u t a t h i o n e

500

reductase.

A n a l y t i c a l conditions: w i n d o w length = 50 residues, step size = 5 residues, and threshold = 45. R e f e r e n c e sequence sets are ( ) F A D - r e l a t e d and (---) n u c l e o t i d e - n o n r e l a t e d enzymes in graph (a) and ( ) N A D ( P ) H - r e l a t e d and (---) n u c l e o t i d e n o n r e l a t e d enzymes in graph (b). M o d i f i e d from Ref (39) with permission, C o p y r i g h t 1989, A m e r i c a n Chemical Society.

226 the

regions

similar

binding.

A homology

with

a reference

major

peak

130-150,

when

by

graph

170-250,

the

66,

129,

130,

localized

the

other

331,

domains,

peak regions With

homology 245-330. 337,

339,

370

(8-9).

regions

interacting

tively,

as r e f e r e n c e for

and FAD.

tool to detect

cal structures.

4.1

enzyme

cal

combination

unit

of

homology

( i0 ) .

as

These

19 to

The

but

are

51,

two m a j o r peaks

primary 197,

contact

198,

201,

except

all

with

the

the

extracted

chemical

protein

sequence

ligand

at

and nicotinamide

NADPH

molecule

(substructures).

structure

using

respec-

are those

to p r o v i d e

of

a

and chemi-

OF S E Q U E N C E - C H E M I C A L

glutathione

the

290,

structures

sequences

structure recognized

with

and

the bound

224,

enzymes,

is b e l i e v e d

FOR A N A L Y S I S

of h u m a n

the

of

the

370 are e x t r a c t e d

and F A D - r e l a t e d

graphing

on

in the

at 190-245

218,

not

usually

enzymes,

extracted

The regions

57,

are

in the homology graph.

NAD(P)H-related

acid

FAD in

spread

reactions

residues

at

reference

residues

157),

of c a t a l y t i c

between

DATABASE

of m o i e t i e s

chemical

467

recognition

phosphodiester, of

50,

the

with the bound

identified

successfully

RELATIONSHIPS

structure

31,

195,

relationships

interacts

phosphate,

amino

(residues

sequences.

the

complex

The

been

enzymes

Units of chemical In the

the

set.

for

appear

with the bound NADPH and FAD separately

Thus,

ENZYME-REACTION

STRUCTURE

sequence

in the graph.

graphs

sets of N A D ( P ) H - r e l a t e d

responsible

significant

graph

All these residues

regions

homology

as

2a) gave one

are

the

interactions

of

(Figure

These

that make

assigned

reductase

410-460.

2b) showed

residues

been

of g l u t a t h i o n e

peaks

sites

set

to

small

in

segments)

(Figure

The

These

4.

and

coenzyme-

Other

of d o m a i n s .

a reference

graph

have

and

have

because

(conserved

as conserved

NADPH

enzyme

in the F A D - d o m a i n

are on the b o u n d a r i e s

NADPH

and

the

enzymes

80.

peaks

which make primarily complex

related

sequence

50 to

300-340,

with

FAD-enzyme

not

set of F A D - r e l a t e d

nucleotide-nonrelated the

of the

at r e s i d u e s

compared

residues

chance,

recognizable

by proteins

reductase adenine,

moieties.

is

NADPH,

ribose,

3'-

The chemi-

recognized

This by

with

suggests

proteins

as

that is

a a

a

227

I

o

o

', O - P - - O - P - O

/ Fig. into

3. Various possible ways of dividing the structure of NADPH substructures.

substructure twenty).

composed

of

several

atoms

(probably

less

than

The size of substructures recognized by proteins would

be limited by the length of the sequence segments coded by one or two exons. The c o n s e r v e d

graph

of

sequence

glutathione

regions

reductase

detected

are

the

in the h o m o l o g y

sequence

responsible for the recognition of the substructures the NADPH molecule.

segments

contained in

To find the conserved sequence segments for

the r e c o g n i t i o n of the p h o s p h o d i e s t e r moiety, we have to compile a reference

sequence

dehydrogenases,

set

including

but also synthetases,

the

sequences

kinases,

p h o s p h o d i e s t e r m o i e t y is c o m m o n l y p r e s e n t NADPH,

NADH,

substrates

chemical

FAD,

structure

sequence-chemical 4.2

ATP,

and

of these enzymes.

GTP,

relationships

Enzyme-Reaction

only

in the s t r u c t u r e s

are

the

cofactors

the p r o t e i n

we are a n a l y s i n g

"substructure" relationships.

The

of

and

sequence-

are a c t u a l l y

database

There are many possible ways of dividing the chemical struc-

ture of NADPH into substructures tures

which

Therefore,

of not

and ligases.

(Figure 3); from small substruc-

such as -OH and -NH 2 to large ones including the adenosyl-

phosphate problems

moiety,

of

evolutionally

which

and

their

combinations.

substructures

significant,

are recognized by proteins.

are

Here

arise

physiologically

and how many d i f f e r e n t

the

and

substructures

228 /// ENTRY NAME

EC 6.3.1.2 Glut amat e-ammoni a ligase Glu tamine S y n t h e t a s e Lig a s e s bonds For m i n g c a r b o n - n i t r o g e n (or amine) ligases Aci d - a m m o n i a (am i d e s y n t h a s e s ) L-G l u t a m a t e : a m m o n i a ligase (AMP-forming) ATP + L - G l u t a m a t e + NH3 = ADP + O r t h o s p h a t e + L-Glutamine ATP L-Glutamate NH3 ADP Ort h o p h o s p h a t e L-G lutamine L-M e t h i o n i n e s u l f o x i m i n e L-2 - A m i n o - 4 - ( h y d r o x y m e t h y l p h o s p h i n y l ) b u t a n o a t e AJEBQT AJAIQ AJZJQ2 AJAAQ AJE CQ A24714 A05079 A05097 A23970 AJF BO A22 947

CLASS

SYSNAME REACTION SUBSTRATE PRODUCT INHIBITOR NBRF-ENTRY ///

Fig.

4. To

Contents study

database amino This

these

called

acid

types

problems,

we

contains

including

the

their

structure

common

as

classified

by

structures

of s u b s t r a t e s ,

NBRF

inhibitors sequence The

base of

collected

in the

entries

collected

enzymes

by July

1991.

with

each

of

known

2,477

version-up The

the

each

Union

products,

and

is

enzymes.

about

We

IUB

keep

entry

41.5 the

%

codes

the

datanumber

for

and

number

database

in the

The

5,864

a name the

effec-

in the N B R F

was

of

reaction

Databank.

Database.

gave

(46),

45).

the names

of B i o c h e m i s t r y ) ,

registered

1984

(40,

4):

activators,

Protein

database

the

in

of

and E C - n u m b e r s ,

cofactors,

our

a

analysis

1,027

EC-number of

enzymes

biochemically

updated

with

the

of the NBRF database.

total

Enzyme-Reaction with

Since

enzymes

sequences

characterized

in

construct

(Figure

Enzyme-Reaction

NBRF

for

names

the e n z y m e s

to the

relationships

and the B r o o k h a v e n

of all

for

items

(International

reaction

database

entries

are

and

IUB

started

Database

following

chemical tors,

database. have

Enzyme-Reaction

sequence-chemical

database

of e n z y m e s

of E n z y m e - R e a c t i o n

number

updating

compounds

of

Database are

of

the

stored

chemical was

compounds

1,554

database. by

molfile

in

July

The

registered 1991

chemical

format

and

in

the

increases

structures

(Molecular

of

Design

229 Ltd.,

San

MACCS

system

format

Chemical search

Leandro,

are

stored

Chem

A,

of

32

FAD,

Software

coordinates The

substructures.

ring

is

system,

form a n e w

as

compounds

substructures

database.

the

all

the of

into

found

hetero the

another This

now

substructures

to

System.

datafile by

atom

the

hetero

database. atom

are

result

only

connected,

rules

those

in a they

2,764

that

other

of

a

to a set of

project,

out of the

apply

and if

bonds,

(3)atoms

suggests

listed to

(2)

by multiple

these

in

their

substructures in

research

trying

CONCORD

Software

a

substructures

(49). are

using

three-dimensional

substructure,

can be a u t o m a t i c a l l y We

by

to

compounds

indexed

(i)

Pomona

substructure,

When we a p p l i e d in

the

possible

a

a Med-

started

of

registered

form

to the

substructure.

are

follows: it

have

a substructure

list

to

their

database

to

We

in the M e d C h e m

if two or more

were

store

using

Project,

3100. database

and

structures

is i n c l u d e d

different

reduce

have

connected

(4)

substructures 400

the

attached

atom

gives

from m o l f i l e

space

structures

construct

in

substructures

atom

Reaction

to

chemical

atoms

carbon

4,733

We

in the

define

carbon the

is

structures

Chemistry

on V A X s t a t i o n

at Austin)

step

for

substructure-

database

to save disk

Enzyme-Reaction

compounds

on a

Institute

acyl-derivatives

chemical

(Medicinal

CA)

the

next

hydrogen

the

(47,48)

into a THOR d a t a b a s e

the

included We

format

of Texas

the

including

three-dimensional

in

(University

in the

For e x a m p l e ,

against

molfile

and s u g a r - n u c l e o s i d e .

System

the

registered

which

compounds

Claremont,

generate

ester

in

related-enzymes

on F A C O M - 3 8 0

translating

SMILES

structures

of the

University.

NAD(P),

we are

into

College,

Kyoto

chemical

EC-numbers

pyrophosphate

list

Now,

The

the

installed

Research,

Coenzyme format

(MDL)

with

output

CA).

with

about

Enzyme-

rules

to

biological

significance. 5.

APPLICATION STRUCTURE

Previously, tures

of

drugs

OF S E Q U E N C E - S U B S T R U C T U R E

we

showed

supposed

sequence

similarity

segments

detected

substructure

RELATIONSHIPS

IDENTIFICATIONS

and in

to

our

strategy

interact

homology the

relationships

with of

be u s e d

identify target

graphing

analysis could

to

(39,

amino

TO

lead

proteins 50). acid

as f u n c t i o n a l

LEAD

strucusing

Sequence sequencetemplates

230 that

specifically

a sequence a

region matching

protein,

sequence

the

to

with

substructures. listed,

many

a high

be a b l e

chemical

that

combination

of

some

recognizes chemical of

constituting

structure

but

the

modifications

corresponding the

target

suggested

together three

for

on

together.

a

as

For

These

This

combinations

of

structure 5).

phosphate

to

called an

broad

is

more

binding

bind

substrate

be

of

which

"effector"

has

no

a

ligand

new

"modulator".

no

segment

by s c a n n i n g

on

accepts

and

oxidized

from

site

with

various

compound,

and

binding

site

by c y t i d i n e

tri-

to the binding

site

similarity

CTP binds

are

either

binding

A

the

the

structural

nicotineamide,

to

with

by the

structure

site

is i n h i b i t e d

from that of aspartic

or

of

separately

FAD,

of

structure

reductase

a broad

binding

structural

of the enzyme.

domain

to

so

lead

substructures

or

NADPH,

alloxan,

carbamyltransferase

(CTP),

When

All

using

strictly

of the substrates.

a new

by

of the

is d e t e c t e d

site

of

of

A protein

Part

drastic

the

the

than two c o m p o u n d s

us to construct

composed

may

part

glutathione

substrates,

for

interactions

design.

templates,

give

on the w a y

molecule.

by the protein.

substrates

the

chemical

latter

set

substructures

structures

somewhat

example,

substructures

the substrate

on a d i f f e r e n t

its

candidates

lead

structure

is

the

structures.

be r e q u i r e d

substructures the

a

substructures

is r e c o g n i z e d

recognizes

prompts

moieties,

Aspartate

acid,

with

single

binding-affinity

(Figure

lead

usually

compounds

cysteine

the

obtain

lead

of

ligand

The

accept

to c e r t a i n

sites.

glutathione.

whose

may

sequence

ligand

is not.

protein

are as f o l l o w s .

through

the

not to be recognized

An e n z y m e

different

of the

the rest

ligand

find

relationships

its ligand molecule

substructures

as

should

to

The

of

template

containing

of these

the

set

structures

strategies

sequence-substructure

protein,

to i d e n t i f y

of substructures.

Additional

could

combinations

constraint

the

of the t a r g e t p r o t e i n

we

a given

by

compounds

the s e q u e n c e

Among various

different

structure

to

When

in the sequence

by the p r o t e i n .

templates,

combinations

is found

of the leads.

characterized

affinity

By s c a n n i n g

various

we w o u l d

possible

a template

be r e c o g n i z a b l e

show

substructure.

drugs

substructures

substructure

would

expected

characterize

acid

to a s p a r t i c

(51,52).

Since proteins

of

CTP is

interest

231

O I

O

I

NH

HO

j....

-\---/----I

t/),

may

have

scanning

templates,

known The

a binding

the target

L

.

.

.

.

.

.

HN

I

for

sequences

cases,

O

research

Research

the M i n i s t r y

was

from the three substrates

not well

an u n k n o w n

with various

we may find new binding

present

sites

supported

on Priority Areas,

of Education,

oll

HS

, I I

ligands.

Scientific

0

O

in most

site

0

2

Fig. 5. C o m b i n a t i o n of substructures gives n e w lead structures. are,

o

N ~NH

H3C H3C

for drug d e s i g n

o

characterized,

effector

molecule.

conserved

sequences

for compounds

by

a

"Genome

Science and Culture

they

as

other than

Grant-in-Aid

Informatics",

of Japan.

By

for

from

REFERENCES

1 2 3 4 5 6 7 8 9

U.C. Singh, in: The Third Alliant C h e m i s t r y Colloquium in Tokyo, 1989. T.L. Blundell and M.J.E. Sternberg, Trends Biotech., 3 (1985) 228-235. T.L. Blundell, B.L. Sibanda, M.J.E. Sternberg, and J.M. Thornton, Nature, 326 (1987) 347-352. W. Kabsch and C. Sander, FEBS Lett., 155 (1983) 179-182. K. Nishikawa and T. Ooi, Biochem. Biophys. Acta, 871 (1986) 45-54. N.S. Scrutton, A. Berry, and R.N. Perham, Nature, 343 (1990) 38-43. S. Greer and R.N. Perham, Biochemistry, 25 (1986) 2736-2742. E.F. Pai, P.A. Karplus, and G.E. Schulz, Biochemistry, 27 (1988) 4465-4474. P.A. Karplus and G.E. Schulz, J. Mol. Biol., 210 (1989) 163180.

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20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39

40 41

P.A. Karplus and G.E. Schulz, J. Mol. Biol., 195 (1987) 701729. P.A. Karplus, E.F. Pai, and G.E. Schulz, Eur. J. Biochem., 178 (1989) 693-703. M.G. Rossmann, A. Liljas, C.I. Branden, and L.J. Banaszak, Enzymes, ii (1975) 61-102. C.I. Branden, Q. Rev. Biophys., 13 (1980) 317-338. W.G.J. Hol, P.T. Van Duijinen, and H.J.C. Beendsen, Nature, 273 (1978) 443-446. R.K. Wierenga, M.C.H. de Maeyer, and W.G.J. Hol, Biochemistry, 24 (1985) 1346-1357. R.K. Wierenga, P. Terpstra, and W.G.J. Hol, J. Mol. Biol., 187 (1987) 101-107. R. Schkeif, Science, 241 (1988) 1182-1187. P.T. Jones, P.H. Dear, J. Foote, M.S. Neuberger, and G. Winter, Nature, 321 (1986) 522-525. C. Chothia, A.M. Lesk, A. Tramontano, M. Levitt, S.J. SmithGill, G. Air, S. Sheriff, E.A. Padlan, D. Davies, W.R. Tulip, P.M. Colman, S. Spinelli, P.M. Alzari, and R.J. Poljak, Nature, 342 (1989) 877-883. M.V. Milburn, L. Tong, A.M. deVos, A. Brunger, Z. Yamaizumi, S. Nishimura, and S.-H. Kim, Science, 247 (1990) 939-945. E.J. Goldsmith, S.R. Sprang, R. Hamlin, N.-H. Xuong, and R.J. Fletterick, Science, 245 (1989) 528-532. C.C. Hyde, S.A. Ahmed, E.A. Padlan, E.W. Miles, and D.R. Davies, J. Biol. Chem., 263 (1988) 17857-17871. C.C.F. Blake, Nature, 273 (1978) 267. J. Rogers, Nature, 315 (1984) 458-459. M. Cornish-Bowden, Nature, 313 (1985) 434-435. M. Marchionni and W. Gilbert, Cell, 46 (1986) 133-141. W.H. Landschulz, P.F. Johnson, and S.L. McKnight, Science, 240 (1988) 1759-1764. C.R. Vinson, P.B. Sigler, and S.L. McKnight, Science, 246 (1988) 911-916. A. Klug and D. Rhodes, Trends Biochem. Sci., 12 (1987) 464. R.F. Smith and T.F. Smith, Proc. Natl. Acad. Sci. USA, 87 (1990) 118-122. H.O. Smith, T.M. Annau, and S. Chandrasegaran, Proc. Natl. Acad. Sci. USA, 87 (1990) 826-839. P.Y. Chou and G.D. Fasman, Adv. Enzymol., 47 (1978) 45-148. J. Garnier, D.J. Osguthorpe, and B. Robson, J. Mol. Biol., 88 (1978) 873-894. W. Kabsch and C. Sander, Proc. Natl. Acad. Sci. USA, 81 (1984) 1075-1078. E.P. Pai, W. Kabsch, U. Krengel, K.C. Holmes, J. John, and A. Wittinghofer, Nature, 341 (1989) 209-214. E.F. Pai, W. Sachsenheimer, R.H. Schirmer, and G.E. Schulz, J. Mol. Biol., 114 (1977) 37. M. Murata, J.S. Richardson, and J.L. Sussman, Proc. Natl. Acad. Sci. USA, 82 (1985) 7657-7661. D.J. Lipman, S.F. Altschul, and J.D. Kececioglu, Proc. Natl. Acad. Sci. USA, 86 (1989) 4412-4415. T. Nishioka, K. Sumi, and J. Oda, in: P.S. Magee, D.R. Henry, and J.H. Block (Eds), Probing Bioactive Mechanisms, ACS Symposium Series, No. 413, American Chemical Society, 1989, pp.i05-122. K. Sumi, T. Nishioka, and J. Oda, Protein Eng. 4, (1991) 413420. W.B. Goad and M. Kanehisa, Nucleic Acids Res., 10 (1982) 247263.

233 42

43 44 45 46 47 48 49 50 51 52

M.O. Dayhoff, R.M. Schwartz, and B.C. Orcutt, in: Atlas of Protein Sequence and Structure, Vol. 5, Suppl. 3, National Biomedical Research Foundation, Washington, D.C., 1978, pp. 345-352. G.E. Schulz, J. Mol. Biol. 138 (1980) 335-347. R. Thieme, E.F. Pai, R.H. Schirmer, and G.E. Schulz, J. Mol. Biol. 152 (1981) 763-782. M. Suyama, T. Nishioka and J. Oda, unpublished. International Union of Biochemistry, Nomenclature Committee, Enzyme Nomenclature, Academic Press, Orlando, FL., 1984. D. Weininger, J. Chem. Info. Comp. Sci., 28 (1988) 31-36. D. Weininger, A. Weininger, and J.L. Weininger, J. Chem. Info. Comp. Sci., 29 (1989) 97-101. T. Nishioka and J. Oda, unpublished data. H. Kato, M. Chihara, T. Nishioka, K. Murata, A. Kimura, and J. Oda, J. Biochem., i01 (1987) 207-215. K.L. Krause, K.W. Voltz, and W.N. Lipscomb, J. Mol. Biol., 193 (1987) 527-553. K.H. Kim, Z. Pan, R.B. Honzatko, H.-M. Ke, and W.N. Lipscomb, J. Mol. Biol., 196 (1987) 853-875.

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QSAR and Drug Design - New Developments and Applications T. Fujita, editor 9 1995 Elsevier Science B.V. All rights reserved

235

BACKGROUND AND FEATURES OF EMIL, A SYSTEM FOR DATABASEAIDED B I O A N A L O G O U S S T R U C T U R A L T R A N S F O R M A T I O N OF BIOACTIVE COMPOUNDS Toshio Fujita, Michihiro Adachi, Miki Akamatsu, Masaaki Asao, Harukazu Fukami, Yoshihisa Inoue, Isao Iwataki, Masaru Kido, Hiroshi Koga, Takamitsu Kobayashi, Izumi Kumita, Kenji Makino, Kengo Oda, Akio Ogino, Masateru Ohta, Fumio Sakamoto, Tetsuo Sekiya, Ryo Shimizu, Chiyozo Takayama, Yukio Tada, Ikuo Ueda, Yoshihisa Umeda, Masumi Yamakawa, Yasunari Yamaura, Hirosuke Yoshioka, Masanori Yoshida, Masafumi Yoshimoto, and Ko Wakabayashi EMIL Working Group, Department of Agricultural Chemistry, Kyoto University, Kyoto 606-01, Japan* ABSTRACT : Various structural transformation processes observed in a number of past developmental examples of pharmaceuticals and agrochemicals are regarded as being invaluable precedents for the prospective analog design. In certain cases, (sub)structural transformation patterns are interchangeable among various compound series in spite of differences in their pharmacological category. Thus, the patterns extracted with a computer-readable format could be accumulated and integrated as a database for potential "rules" for bioanalogous molecular transformations. EMIL is a system that incorporates the database and a data-processing engine constructed to release "higher-ordered" candidate structures from a "lower-ordered" input structure "automatically". Conceptual background for the database construction and the procedure for the database collection are presented on the basis of some lead evolution examples among pharmaceutical and agrochemical series of compounds. 1. INTRODUCTION There are numerous series of compounds exhibiting specific biological effects. Examples exist among such pharmaceuticals as those acting to nervous, circulatory, respiratory, digestive, and immunoregulatory systems and chemotherapeutics including antimicrobial and anticancer agents as well as among such agrochemicals as insecticides, herbicides, and fungicides. In each series, an ultimate prototype lead compound has been identified or disclosed first. In certain cases, bioactive principles in natural products, including secondary metabolites of animals and plants and endogenous participants such as hormones and signal-transmitters, are the origin of *The corresponding author and the business addresses of authors are listed at the end of this article.

236 the lead compound. In many instances, it is selected from organic compounds synthesized intentionally or unintentionally. The structure of the prototype lead compound is usually modified variously so as to improve the profiles of biological activity and to potentiate the target activity as well as to eliminate undesirable side effects including chronic toxicities and environmentally hazardous behaviors. There seem to exist two aspects in the structural modification processes. The one is the optimization of the lead structure with a systematic replacement of substituents keeping the skeletal structure (almost) unchanged. This is often called the "lead optimization" (1). The other is the structural transformation usually associated with more or less "drastic" variations in the skeletal structure. The structural transformation is usually performed into more elaborated or "higherordered" lead structures one after another consecutively, quite often in different institutions independently and/or competitively. These consecutive structural transformations could be called the "lead evolution" (2). Of course, the lead optimization can be made starting from the "intermediary" lead structure in each step of the consecutive lead evolution processes. How to make the lead evolution, i.e., the lead evolution strategy is also called the analog design (3). Although the disclosure or identification of the ultimate prototype structure is the prerequisite for the structural modifications, the lead evolution is perhaps most important from the synthetic chemical points of view to obtain patentable pharmaceuticals and agrochemicals having newer generation skeletal structures. In the structural transformation or lead evolution series, a majority of individual steps may originally be attempted on trial-and-error bases. However, because structural transformation patterns included in these steps have eventually been "utilized" in improving or at least in retaining the bioactivity profile, they are well regarded as being invaluable precedents for the analog design or "bioanalogous" molecular transformation (4). If these precedents are integrated and organized as a database for the bioanalogous transformation "rules" and the database is incorporated into a system so that any prototype or "lower-ordered" lead structures introduced into the system are processed with the rules to release elaborated or "higher-ordered" candidate structures as the output "automatically", the system could be a great benefit for the synthetic medicinal and agricultural chemists. We have been working on a project to construct a computerized system for the lead evolution or analog design, named EMIL : Example-Mediated-lnnovation-for-Lead-Evolution (5, 6). In this article, after showing some lead evolution examples, we demonstrate that certain (sub)structural transformation pattems are interchangeable among various series of bioactive compounds in spite of differences in the pharmacological category. Then, we illustrate how to collect the database and how to operate the EMIL system for the analog design.

237 2. LEAD EVOLUTION EXAMPLES From among a number of examples, we selected two each for pharmaceuticals and agrochemicals of current interest. In each example, the lead evolution processes were examined according to a "tree" in which structures are arranged not necessarily in the chronological order but from the most primitive (but not always simplest) structure toward the more elaborated (but not always the more complex) one somewhat concisely. If bioactive compounds before and after a certain structural transformation in lead evolution processes elicit analogous biological responses, the transformation could be bioisosteric and the two compounds or two interchangeable substructures be bioisosters in a broader sense. Here, we adopt the terms, "bioanalogous" and "bioanalog", instead of "bioisosteric" and "bioisoster", respectively, as proposed by Floersheim and coworkers (4). The term "bioanalogy" can be used more flexibly than "bioisosterism" without being restricted by the basic definition of the isosterism including isometricity in terms of various physicochemical parameters (7 - 9). 2.1 Cromakalim and Related Potassium Channel Activators. Figure 1 is a simplified lead evolution tree of cromakalim analogs, which are potassium channel activators exhibiting smooth muscle relaxation effects such as antihypertensive and anti-bronchial asthmatic activities (10 - 12). The very prototype was synthesized at Beecham (now SmithKline Beecham) in the early 1980's with an idea that the cyclization of the side chain in such I]-adrenoceptor antagonists (13blockes) as alprenolol (1) to restrict its conformational freedom may give compounds retaining the antihypertensive activity lacking side effects associated with l-blockers (10). The ring-closured compound of the structure 2 was found to indeed show an antihypertensive activity without 13-blocking effects. The geminal dimethyl at the 2position and the nitro group at the 6 position of compound 2 were necessary for the activity but introduced to enhance the cyclization reaction to form the dihydrobenzopyran skeleton originally (10). During the structural modification trials, the pyrrolidine compound 3 was shown to be highly active in vivo but only moderately in vitro. Thus, cromakalim (4) with a lactam ring was designed and synthesized as a possible metabolite of the pyrrolidine compound 3 and proved to be highly active (10). In the course of lead evolution processes starting from cromakalim (4), the lactam structure was successively transformed via the acyclic amide (in 5) and urea (in 6) structures into the cyanoamidine (in 7), cyanoguanidine (in 8), and triazolediamine (in 9) structures. These transformation patterns are shared by quite a few series of compounds of different pharmacological categories as will be shown later in section 3.2.2.

to

2

1: alprenolol

3

4 9cromakalim (lemakalim)

5

6

/ NCN

NCN

~ ~.~ 7 9KP 293

H3C~N--N

~ ~~

~~'~~

8

9

~o

~o

10" NIP 121

~o 12 "bimakalim

11 9emakalim

,

e.~.

P~.o

9~.o N

.~

o

N

~o NC I ~ ~ O . ~ , .

S.-c-N~cN H

O 2 N ~ --- CH2F

13" Ro 31-6930

14" TCV 295

15" YM 099

16" EMD 57283

17" SR 44994

Fig. 1. Simplified Structural Evolution Tree of Cromakalim Analogs.

18" KC 399

239 One of the other pathways is an elaboration of the lactam moiety leading to compounds 10, 11, 12, and 17 and to pyridine N-oxides 13, 14, and 15. A recently reported acyclic thioamide KC 399 (18) from Chugai (12e) is one of members designed and synthesized (13) with a combination of structural features of bimakalim (12), in which the dihydropyranol structure of the preceding compounds is dehydrated into the benzopyran (11), and aprikalim (19) belonging to an independent S~c.NHCH3 series of potassium channel activators (12a), in which a thioamide 6~sk..v o structure is attached at the c~-position to the aromatic system. The compound 18 was reported to be some 1000-fold more potent than 19: aprikalim cromakalim in relaxation of precontracted rat aorta (12e).

2.2 Non-peptide Angiotensin II Receptor Antagonists. The title compound series are recently attracting enormous attention to develop antihypertensive agents which are orally active with a prolonged duration (14). In the course of structural transformations leading to increasingly potent antagonists, it has been shown that there are at least two subtypes of the receptor, AT1 and AT2 (15). Structures arranged in Fig. 2 showing a summarized evolution tree are mostly those of the AT1 antagonists (16 - 25). The ultimate lead compound in this series is CV 2198 (20) which was synthesized by scientists at Takeda in the late 1970's in a series of projects for derivatization and screening of 1-benzylimidazole-5-acetic acid analogs (16). Because this compound 20 and its close analogs were among the first as the nonpeptide angiotensin II receptor antagonists, a number of research groups over the world started projects for transformation of the structure of compound 20 as the lead (14). Among intensive efforts, a great break-through is likely to be the disclosure of DUP 753 (23: losartan) at DuPont (now DuPont Merck) publicized in the late 1980's (17), because numerous analogs developed following losartan either share the 2'tetrazolyl-biphenyl-4-yl-methyl structure in common (in 24 - 26, 30, 31, 36, and 37) or have closely related biarylylmethyl structures carrying an acidic group bioanalogous to the tetrazolyl at the position corresponding to that in the biphenylyl structure (in 28, 29, 32 - 35, and 38) as an indispensable moiety. The imidazole moiety originally included in CV 2198 (20) has been variously transformed into spiro (in 30), oxy-aryl (in 26), and condensed bicyclic (in 31 - 38) systems as well as ring-fissioned structures (in 24 and 25). Candesartan cilexetil (31) is a prodrug. The ester moiety of this compound is metabolized into the free carboxylic acid, candesartan, as the active form in vivo (21a). One of the most recently reported compounds, L 162313 (35), has been revealed to be a partial

( - ~ , N,'r C1 X~/

N

~u-~. ,'r

.N~CH2COOH

C1

N

~u~. ,y

.N~CH2COOH

C1

,

N.~CH2COOMe

N

,'r

C1

N

.o. "v"

~ _ ~.~

~

.N~'~CH20 H ,~,,,,~,,~N,,~COO H V ' ~ N ' ~ ~

N

"~

O ---t~ ~-1~

20:CV 2198 /

~

21 :EXP 6155 O: ~ /2"EXP6803 /

~_~.~,~ ~COOH

~

~

Tet~ j ~ 23ilosartan

~

TetI ~ TetI ' ~ 24"valsartan / 5 " A 8 1 9 8 8

Vet I ~ ~TM 26"ICID8731

~u-~'~~u~ ~u-~'~~~-~o~o~~~. ~ ~. ~o ~.~ ~o-~~z~,

CF3SO2NH

27 9eprosartan

I

H --.t~.~.

28 "saprisartan

3

HO(~" ",,a,"

29 9SC 52458

30 9irbesartan

~-'~o

~ PhC

31 9candesartancilexetil

32 9TAK 536

N~'~Me

BuOC BuOC

33 9telmisartan

34 9MK 996

35 9L 162313

36 9tasosartan

37 9CL 329167

38" L 162393

Fig. 2. Simplified Structural Transformation Tree of Non-peptide Angiotensin II Receptor Antagonists (Tet 9tetrazol-5-yl).

1".9

241 antagonist acting also as the agonist to the AT1 receptor (22). This compound is the first non-peptide agonist of peptide receptors outside the opiate system. Another, L 162393 (38), is one of the balanced angiotensin II antagonists capable of potent binding to both AT1 and AT2 receptor subtypes (23). The AT1 binding potency of this compound in vitro is about 100 times higher than that of losartan at a subnanomolar level. The structure of compound 26 is unique as is that of eprosartan (27). In compound 26, the acidic biarylylmethyl group is attached to the heteroaromatic ring via oxygen. Eprosartan (27) has an acrylic acid side chain and the carboxyphenyl instead of the acidic biarylyl. In leading to these and related structures, threedimensional superimposition pattems of the small-molecule antagonist candidates on a putative pharmacophore model of angiotensin II has been examined iteratively (24, 25). The angiotensin II model has been constructed with structure-activity studies of its peptide analogs containing conformationally constrained replacement of key amino acid residues and conformational analyses of active analogs. The structural modification of this series of compounds is a typical example for the lead evolution associated with the lead optimization from the intermediary lead structures. Substituents at various positions in each structure of compounds shown in Fig. 2 are mostly those optimized with the more or less systematic modifications of the substituent structure in terms of the in vitro binding as well as the oral activity and its duration. The activity potentiation of the order of 10- to 50fold in the optimization phase is not unusual, if the substituent selection has been done appropriately.

2.3 Fungicidal [~-Methoxyacrylates and Analogs. o~-Substituted-aryl-[~-mcthoxyacrylatcs and their analogs such as o~methoxyiminophenyl-acetates and -acetamides are now being developed as agricultural fungicides with a systemic as well as a broad spectrum activity. Figure 3 shows a simplified lead evolution scheme of this series of compounds (26, 27). The original lead compound, strobilurin A (39), is a fungicidal principle included in small agarics belonging to species of Strobilurus and Oudemansiella which grow on decaying woods. There arc a number of analogs differing in substitution patterns on the conjugate polyene moiety and the benzene ring (28). The toxophoric structure of compounds in Fig. 3 is likely to be the "[3-methoxyacryloyl" or "methoxyiminoacetyl" moiety, but the corresponding free acids are known to exhibit only a very low activity. The fungicidal activity is due to the inhibition of the respiratory chain of fungi (29). The target site is believed to be the cytochrome bcl complex located in the inner membrane of fungal mitochondria.

242

OMe

OMe

!

OMe 39 9strobilurin A

40

~ 42

O~oMe I OMe

[~O

OMe !

OMe

41 OMe ~

~

[ ~O

O

i

Ooe OMe~ ~

M

NHMe 43" SSF 126

OMe 44" BAS 490F

,, N,,.Y-'N.o CN

O

OMe I

45" ICIA 5504

OMe

I~NSJ

OMe |

46

OCH3

Fig. 3. Structural Transformation Tree of 13-Methoxyacrylates and Analogs. The structural transformations from strobilurin A (39) to ICIA 5504 (45) have been made to increase the photostability and to decrease the phytotoxicity as well as to increase the systemicity into the plant body suffering from fungal diseases by adjusting the molecular hydrophobicity (26). Although the design principle of SSF 126 (43) is its own being from the ring fission trials of fungicidal carbamoyl isoxazoles (30), it is reasonable to locate this compound following the ICIA compound 41 in the lead evolution tree. Currently (August, 1994), besides ICIA 5504 (45) by Zeneca and SSF 126 (43) by Shionogi, BAS 490F (44) is being under extensive trials for commercialization by BASF (26). 2.4 Arylsulfonylureas and Related Herbicides. The ultimate lead compound of this series, INU 3373 (47), was serendipitously found to show a modest plant-growth retardant activity in the mid-1970's by Levitt and his coworkers at DuPont (31). The discovery of sulfonylureas such as chlorsulfuron (48: a wheat/barley herbicide), metsulfuron methyl (49: a wheat/barleyl/rice herbicide) and thifensulfuron methyl (52: a wheat/barley herbicide) shown in Fig. 4 was the fruits of extensive efforts of DuPont scientists (32). These and a number of analogous DuPont sulfonylureas are characterized by unprecedentedly low dose rates (generally 5 to 50 g a.i./ha with the lowest of 2 g a.i./ha) to eradicate various species of weeds (32). Depending upon structural

~1

.CH3

,COOCH3

SO2NHCONH---(, N - - ~ 47

~

d ON(CH3)2

48 :chlorsulfuron

/

~-

N._ ~,C1 OCH3 ff'-~" g N_--~ ~./~ N~SO2NHCONH-'~q_~ 55 9imazosulfuron

_N~ -N~I~"

Cl

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~

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N_ OCH3

CH3

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r

53 9pyrazosulfuron ethyl

~1~

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I ~

. -
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-CH3

N_--(' N--N-<, ,:>

~5--NHSO~--~.~ N-~,

F

--'~'~C1

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OCH3 61

l~ CH3 CH3

57 9flumetsulam

COOH ~ OCH3

CH3 60

OCH3

l~ll., ~'SO2NHCONH'='('. -

56

~~~

..N~

50

CH3

1

OCH3

OCH3 CH3SO2,~ N_-~ \ ou 3NSO~CO~-~,N~r ZZ v-n3 OCH3 59 9amiclosulfuron

COOC2H5

52 9thifensulfuron methyl

51 9nicosulfuron

OCH3

% 4 -" 'C'~s~176

~ OCH3

OCH3

UOOCH3

49"metsulfuronmethyl OCH3

"CH3 N~

SO2NHCONH'~q~

~~-SO2NHCONH

OCH3

"S"'~COOCH3

N_ OCH3

CH3

CI

58

COOH

N

OCH3

--

H3C4OCH#coocH30CH 3

O ~-

OCH3 62" pyrithiobac

OCH3 63: pyriminobac methyl

Fig. 4. Structural Transformation Tree of Arylsulfonylurea and Related Herbicides.

to 4~

244 features of the aromatic ring, the (sulfonylurea) bridge and the heteroaromatic ring (azine: mostly either pyrimidine or s-triazine) on the opposite side of the bridge as well as properties of substituents on these tings, these compounds exhibit a variety of distinct weed control spectra and crop selectivities (32, 33). Following the discovery of herbicidal sulfonylureas at DuPont, a number of analogs such as compounds 51 and 53 -55 in which aromatic ring structures are modified have been synthesized (32). The structure of the sulfonylurea bridge itself has also been variously manipulated. One of the NH units in the sulfonylurea bridge is omitted in compound 50 (34) and the CONH structure is replaced by nitrogen heterocycles in compounds 56 - 58 (35, 36). Note that the SO2NH bonding in compound 56 is reversed in compounds 57 and 58. The nitrogen biarylylic system in compound 58 seems to have a structure formed by disjoining the condensed bicyclic structure of compounds 56 and 57. In pyrimidinyl(thio)salicylates 61 - 63, the entire sulfonylurea bridge is reduced to just a (thio)ether linkage (37 - 39). Interestingly, in amidosulfuron (59), a sulfamoylsulfonylurea, one of the ring systems is replaced by the N-methyl-methanesulfonamido-substructure (34). In compound 60, the condensed ring system is a promoiety to give the corresponding sulfonylurea in vivo

(40). The mode of herbicidal action of compounds included in Fig. 4 has been shown to be the inhibition of acetolactate synthase in weeds catalyzing the biosynthesis of branched chain amino acids. The selectivity between weed and crop species is mostly due to selective metabolic inactivation with crop plants (33, 41). There is another class of acetolactate synthase-inhibiting herbicides, the representative of which is compound 64, imazapyr, introduced by American Cyanamide (42). Because of the structural evolution process different from sulfonylureas 64: imazapyr / and related compounds, they are not included in Fig. 4.

3. S I M I L A R I T Y IN S T R U C T U R A L F E A T U R E S AND S T R U C T U R A L TRANSFORMATION PATTERNS AMONG VARIOUS BIOACTIVE COMPOUND SERIES Each of the lead evolution examples shown in the preceding section seems to be "unique" as its own. There could exist a number of this type of examples corresponding to a number of bioactive compound series. Depending upon differences in the pharmacology being due to variations in the structural and functional features of "receptor site(s)", (sub)structural requirements for bioactive compounds to fit in with the corresponding receptor site(s) to induce the proper function should not, in principle, be identical among various series. Thus, particular

245 precedents for some bioanalogous structural transformations are not necessarily applicable over a wide range of new bioactive compound series. Nevertheless, there are a number of compounds or compound series which exhibit not a single type but various types of bioactivity. Moreover, there are quite a few examples in which structural transformation patterns are (almost) identical with each other among lead evolution processes of various bioactive compound series irrespective of differences in the pharmacological category.

3.1 Similarity in Structural Features Among Compound Series Exhibiting Various Biological Activities. In general, biologically active compounds exhibit not only a single type of activity. For example, many pharmaceuticals and agrochemicals exert side effects in addition to the principal activity. Sometimes, a certain profitable side effect is separated from others and potentiated with structural manipulations to specifically evolve into other pharmacological compound series. A well known example is that sulfanilamide and its analogs exhibit not only their principal activity as antibacterials, but also antileprosy, antidiabetic, diuretic, and uricosuric activities (43). In each category, series compounds have been developed as briefly shown in Fig. 5. Although the structures located near the end of the structural transformation tree are considerably different, their structural transformations have been initiated from a common origin and structural features are similar to each other on the early stages of evolution.

Antileprosy

H2N-~

HEN--@SO2--~

An~idiabetics

68

69

NH2

N-'-N

SO2NHR

Sulfanilamides

Diuretics

CI CH3CONH-~ SO2NH2 H2NSO2 70 NmN I

Antibacterial

I

c. coN.Zs X so N. 71

N:N

65

C6H5 66

Uricosurics

72

Fig. 5. Development of Various Pharmaceuticals from Sulfanilamides.

246

There are a number of other examples in which structurally closely related compounds exhibit various bioactivities without clean boundaries among pharmacological categories as shown in Table 1. TABLE 1. Structural Series Showing Various Biological Activities. a)

O x~SO2NH~NHY

(32, 43, 4 4 ) d ) x ~ C H c I 2 " ~

X 9p-CH3, Y" n-Bu Antidiabetic (73) N_rCH3 X" o-Cl, y . -.4;,T~N Herbicidal (48) OCH3 X" 3,4-(CH2)3, Y : - ~ _ x C1 Antitumor (74)

b)

x ~

(45)

X" H, Y 92,6-C12 X 92-C1,6-Me, Y ' H

Cytokinin-active (75) Anticonvulsant (76) 2

3

X "p-C1 X' o-C1 e)

Y 93,5-C12, R" X" 2-NO2, Y 93-C1, R"

CF3 Cl ...
Br

Insecticidal

(47)

Insecticidal (79) Antitumor (80) CH3 O-+-COOH Y

(48)

X : C1, Y : H[R] Herbicidal,Auxin-active,

Hypolipidemic, Inhibitoryagainst Platelet Aggregation (81) X :C1, Y :Me

Anti-auxinic, Hypolipidemic (82) X : Et, Y : H[R,S] Antiinflammatory (83) f)

x

x--~

C I ( ~ H

(77)

X-

Antitumor (78)

X"

~,COOCH(CH3)2 X J ~'COOCH(CH3)2

(49)

Rice-Blast Fungicidal (84) I~SS

Hepatotrophic

(85)

3.2 Similarity in Structural Transformation Patterns among Various Bioactive Compound Series. 3.2.1 Arylalkanoic Acid-Type Antiinflammatory Agents and Plant Growth Regulators. The basic lead structures of antiinflammatory agents such as indomethacin (86) and ibuprofen (87) can be traced back to structures of classical plant growth regulators/herbicides such as indole-3-acetic (93) and phenoxyacetic acids (83), respectively (48d). Phenoxyacetic and phenylacetic acids having suitable substitution patterns (96, 97) are bioanalogous as herbicides (50). Similarity in the structure-activity patterns between arylalkanoic acid-type antiinflammatory and herbicidal compounds has long been recognized (48d, 51). Structures of some representative arylalkanoic acid-type antiinflammatory agents and classical plant growth regulators are shown in Fig. 6 (52, 53).

247 Antiinflammatory Agents

CH30~CH2COOH cliO

CH3

86 ,.~

~ ~ ~ i ~CHCOOH c. (s)3 CH30

CH3

CH3

87

O'i 88 89 CH3 0L U CHCOOH CH2=CHcH2Co~CH2COOH

90

91

92

Plant Growth Regulators/Herbicides

H~' NCH2COOH ~CH2COOH 93

94

~COOH CI CI ~~']COOH C1 95

96

OC#2COOH CI Cl

97

Fig. 6. Arylalkanoic Acids as Antiinflammatory Agents and Plant Growth Regulators/Herbicides. Juby and his coworkers at Bristol-Myers (now Bristol-Myers Squibb) synthesized a series of benzocycloalkene-l-carboxylic acids (98 - 1 0 2 ) as the arylalkanoic acids in which the c~-alkyl side chain is cyclized as shown in Fig. 7 (52). Based on structure-activity patterns observed in these and previous compounds, they defined structural requirements for arylalkanoic acids to be antiinflammatory (52a). Most of these requirements are identical with those proposed for the classical plant growth regulators (53a) as compared in Table 2, except for the necessity of large hydrophobic substituents for antiinflammatory activity. TABLE 2. Similarity in Structural Requirements of Arylalkanoic Acid-Type Plant Growth Regulatory and Antiinflammatory Agents. Structural Requirements 1. A flat aromatic ring. 2. A carboxyl group separated by one carbon atom from the ring. 3. The carboxyl group deviating considerably from the ring plane. 4. A free hydrogen atom at the or-position to the carboxyl group. 5. The S configuration at the (x-position if asymmetric. 6. A large hydrophobic substituent on the ring.

Plant Growth Regulators Required

Antiinflammatory Agents Required

Required

Required

Required

Required

Required

Required

Required Not Required

Required Required

248 COOH X~

S

98"X=c-Hex

103 9X = H

COOH X~

S

99"X=c-Hex

104 9X = H

COOH s

COOH

COOH

100 9X = c-Hex

101 9X = c-Hex

102 9X = c-Hex

105 " X = H

106 9X = H

107 9X = H

>>

Fig. 7. Benzocycloalka(di)ene-l-carboxylic Acids as Antiinflamatory Agents (98- 102) and Plant Growth Regulators (103 - 107). >>,---, and > compare the potency between two compounds of both sides in each series in common. We used to study structure-activity relationships of the same type of cyclized arylalkanoic acids (103 - 107) as plant growth regulators (54) the structures of which are also shown in Fig. 7. 1,4-Dihydro-l-naphthoic acid (104) was most potent among them. As the antiinflammatory agent, the indane-l-carboxylic acid derivative (98) was most potent and compound 108 named clidanac was selected as a clinical drug (52a, 55). Of course, the structure-potency patterns need not completely coinside between the two series of compounds. Among partially COOH hydrogenated 1-naphthoic acid series, however, coincidence in C l ~ the potency variations is remarkable suggesting a similarity at ~ J least in the substructural features of the receptor sites between [ 1 the two pharmacologically different series of compounds. ~108: clidanac

3.2.2 Urea, Thiourea, Cyanoguanidine, Nitroethenediamine, and Related Structural Components in Various Bioactive Compound Series. The bioanalogous relationship among the title "polar hydrogen-bonding groups" has been well known since most of them and other related groups were shown as being "interchangeable" with each other in various series of histamine H2antagonists (56). Their general structural feature, as indicated in Table 3, is to consist of the aromatic ring (R), flexible chain (C), and polar hydrogen-bonding grouping (H). Along with thiourea, cyanoguanidine, and nitroethenediamine structures, some other polar hydrogen-bonding groups are arranged in Table 3 as representatives in respective H2-antagonist series in which the aromatic ring (R) and flexible chain (C) are fixed (56, 57). Many of these polar hydrogen-bonding groups are found in various R-C series simultaneously. Although not every combination between the R-C and H moieties is congenial in giving potent compounds, the H structures for the polar hydrogen-bonding group in Table 3 are regarded as being potentially interchangeable. Interestingly, a very similar bioanalogous set of structural components is found in Fig. 1 for the cromakalim series of potassium channel openers. In the consecutive steps from the ring-fissioned acetamino-compound (5) to the methyltriazolediamine

T A B L E 3. Representative H2-Receptor Histamine Antagonists. J R " Aromatic ] Ring j

t C "Flexible Chain k

Ring "R" and Chain "C" H

H 9Polar ] H-Bonding Group

Polar H-Bonding Groups "H" S II

iCH3

)

mNHCNHCH3

109

NCN II

--NHCNHCH 3

CHNO 2

II

---NHCNHCH 3

110: cimetidine

NNO 2

II

--NHCNHCH 3

111

112 o

S

NCN

II

II

mNHCNHCH3

113 NH2

H2N-'J~NANN ~~S~'r

~

O II

--NHCCH2OCCH 3

120 9roxatidine s

~

--CNH 2

N'S'N --NH

~ I! NH 2

115 9ranitidine

116

O ii

II

117 9tiotidine

i

II

--NHCNHCH 3

NSO2NH 2

--NHCNHCH3

O II

---NHCNHCH 3

114

NCN II

S

CHNO2

N"S'N --NH

118" famotidine

~ /1 NH 2

119 o

H3C~N_ N --NH-~NN~.--NH 2

121 9lamtidine

N,,S-N --NH

,, I/' NH 2

122

CHNO 2

II

123

N H

CHNO 2

II mNHCNHCH3 124 9nizatidine

t'~

250 (9), structural components which are replaced one after another are those included in Table 3 as the hydrogen-bonding polar groups. A similar bioanalogous set such as compounds 125 - 127 exhibiting various degrees of smooth muscle relaxant activity have been explored in the synthetic project of compound 18 (12e, 13, 58).

O....C"NHCH3

NCN..~.,NHCH3

O.:.C"~ -

125

126

CN

u CH2F 127

Examples are also found in other series of potassium channel openers, pinacidil (128) and its analogs (129 - 132) (59) and nicorandil (133) and its analogs (134 and 135) (60).

~

N,NcC~_~.Bu

1~ NCN lq@N,, C,,N_~-Bu

128

[~ CHNO2 N J ~ N-C',N_.~t-Bu

129

130

O

N ~ ~ N,i~_~N._~t.Bu H2N,~ NCN ~ N,.C.. N,,.@ 131

~ONO2

132

NCN J~N~ONO2

133

NCN f ~ H2N~I~N~'~'~ -N

135

C1

Further examples exist in imidacloprid and related compounds (136 - 139) which are potent insecticides acting as agonists of the nicotinic receptor of acetylcholine in the insect nervous system (61) and in artificial sweeteners such as cyanosuosan (140 - 142) and superaspartame (143 - 145) series (62).

NNO2 N~NH 136: imidacloprid

A

l -2'Y

137

CHNO2 CI....~N~ C2H5 138: nitenpyram

CHNO2 NXNH NCN

CI 139: acetamiprid

251

N

~ C ~ ~ C O O H

HOOC

140:X=O 141 : X = S 142 : X = NCN

K,~ I

143 : X = O 144 : X = S 145 : X = NCN

It should be noted that, in compounds 5, 7, and 18 in Fig. 1,118 and 120 in Table 3, 125 - 127, 133 - 135, and 139, structural units, which are interchangeable with (thio)urea, N-cyanoguanidine, nitroethenediamine and related structures, have either (thio)amide or N-substituted amidine structures which lack one of the two N atoms in (thio)urea-related structures. The bioanalogous relationship between amide and N-cyanoamidine structures is likely to be disclosed first in penicillins such as 146 and 147 showing an antibacterial activity at comparative levels (63). The possibility for the cyanoamidine compound 147 to be active after hydrolysis giving the amide was excluded. The cyanoamidine is stable enough chemically and tolerable against enzymatic hydrolyses. NCN

O/~-'N ~.,SCOOH 146 :penicillin G

o,~N 147

I,,,COOH

3.2.3 F r o m " A m i d e s " to Cyclic D i c a r b o x i m i d e s a n d R e l a t e d Structural Transformation

Patterns

in A g r o c h e m i c a l s ,

Anticancer

Agents, and

Anticonvulsants.

Compounds having the N-phenyl-amide moiety such as anilides (148),Nphenylcarbamates (149) and N-phenylureas (150) are herbicidally active exhibiting various degrees of the Hill reaction (a component of the photosynthetic system) inhibitory potency (64). The most conventional substitution pattern on the benzene ring in these compound series, 148 - 150, is X = 3,4-C12. Propanil (148: X = 3,4-C12, R = Et), swep (149: X=3,4-C12, R = Me) and diuron (150: X = 3,4-C12, R = R ' = Me) are among representatives. They are regarded as being bioanalogous to each other.

148

149

150

There is a family of agricultural fungicides the structual feature of which is that they are N-phenyl cyclic dicarboximides, such as procymidone (151:R1 - R4 =

252 Me, R2 - R3 = -CH2-), vinclozoline (152: R 1 = Me, R2 = CH=CH2) and iprodione (153:R1 = CONHCHMe2, R2 = R3 = H), sharing the 3,5-dichloro-substitution on the benzene ring in common (65). They are particularly effective on Sclerotinia and Botrytis diseases in vineyards and greenhouses.

R2 3 C

_ CI

151

N

1 O

C1

152

O

2 3

153

Structures of the cyclic imide moiety of above fungicidal compounds, the pyrrolidinedione (in 151), oxazolidinedione (in 152), and imidazolidinedione (in 153), can be regarded as being generated through the cyclization of the side chain structures of the Hill reaction inhibiting anilides (148), carbamates (149) and ureas (150), respectively, with the insertion of another carbonyl component. Structures 151 - 153 are bioanalogous. Regardless of the type of atoms next to the carbonyl function, the open chain "amides" ( 1 4 8 - 150) are the Hill reaction inhibiting herbicides and the ring-closured dicarboximides (151 - 153) are fungicides. N-Phenylcarbamates 154 and 155 having structural features common with the herbicides (149) are also fungicidal against gray mold diseases of vines, vegetables, and beans caused by Botrytis strains resistant against benzimidazole-fungicides (66). Thus, in spite of some differences in the target of the biological activity and the optimum substitution pattern on the benzene ring, the open chain "amides" and cyclic "dicarboximides" can be regarded as being bioanalogous. Examples supporting this respect will be shown below. Cl CH3CH20--~ Cl

154

CH3CH20

NHCOCH(CH3)2 155

Among anilides (148), chloranocryl (X = 3,4-C12, R = -C(Me)=CH2) and pentanochlor (X = 3-C1, 4-Me, R = CH(Me)C3H7) have been used practically to exterminate annual grass and broad-leaved weeds in various crop fields (67). They have the 3,4-disubstitution patterns as X as well as the branched chain alk(en)yl groups as R. Interestingly, a member of compound series 148 similar to the above herbicides, but having X = 3-CF3,4-NO2 and R = CH(Me)2 named flutamide from Schering, is an antiandrogen (68) and has been used as an antiprostatic cancer agent for some 15 years. Flutamide, having the 3,4-disubstitution pattern on the benzene ring and the branched alkyl as R, is reasonably considered to show some Hill reaction inhibitory activity. Although no description about the herbicidal activity has been

253 found, some higher homologs of flutamides in the acyl moiety have been observed to show a potent antibacterial activity (69). Quite interestingly moreover, compound 156 named nilutamide from RousselUCLAF is also a potent and selective antiandrogen being used as an antiprostatic cancer agent (70). The bioanalogous relationship between anilides and N-phenyl cyclic dicarboximides very similar to that described above in agrochemicals is observed in entirely different pharmacological category.

_ ~ O2N F3C

156

O )I.-~H O2N~ N ~ (~-CH3 ~ O CH3 F3C

H _ ~ N OH NC ' ~ (~-CH3 O CH3 F3C

O cH NHC-- CH2SO2- ' ~ F ~H3

157

158

The dicarboximide heterocycle of nilutamide (156) belongs to the imidazolidinediones (in 153). The structural differences of nilutamide (156) from the fungicidal compound series 153 are the substitution patterns on the benzene and imidazolidinedione tings. Flutamide works as its hydroxylated metabolite 157 in vivo (71). The hydroxy group in the metabolite 157 corresponds well with the NH group in nilutamide (156). Thus, nilutamide is regarded also a ring-closured bioanalog of the metabolite 157. By the way, bicalutamide (158) modified further from the "hydroxyflutamide" is now being extensively investigated for clinical use by Zeneca (71).

O~

H

HN _C=O ,C'--~ Et O Ph 159 :phenobarbital

O~

,H

QC--O

f-'<

I

H2N

, .CH

~C-O CH3 CH3NH HC~CH3

/C=O H3C-N~C. C~CH3 HC-Et II Ph O 160: pheneturide 161 : trimethadione

162

Further bioanalogous relationships between amides and cyclic dicarboximides are observed in CNS (central nervous system) agents. Phenobarbital (159), a classic hypnotic/anticonvulsant, is the ring-closured "carbonylog" of pheneturide (160), an acyclic anticonvulsant (72). A similar pattern is found for an oxazolidinedione anticonvulsant, trimethadione (161) with compound 162 (72, 73). A recent example is that between benzanilide (163) and phthalimide (164) (74). Their activity is, respectively, comparable with and higher than that of phenytoin (165), the most important anticonvulsant for various types of epileptic disorders, in the anti-MES (maximum electroshock seizure) test in rats (74).

NH2 CH3 163

CH30 164

HN~~/NH 165

O

254 Examples illustrated above would strongly suggest that, in certain instances, structural characteristics of receptor sites and/or the modes of ligand-receptor interactions are similar among different types of bioactivity at least partially. There could exist other examples showing similarity in features of the structure itself as well as in patterns of structural transformation among compound series of different pharmacologies. Thus, the precedent structural transformation patterns could potentially be extended prospectively and utilizable for the lead evolution into new structural series of compounds regardless of pharmacological differences. 4. D A T A B A S E F O R B I O A N A L O G O U S S T R U C T U R A L TRANSFORMATION "RULES" AND THE OPERATION OF THE EMIL SYSTEM To make the precedent transformation patterns utilizable, the EMIL system uses a database in which patterns from various lead evolution examples are collected in a computer-readable style. Each of the patterns is what to be made up as a potential unit rule for the bioanalogous structural transformation. Because structural transformations accompanied with more or less drastic skeletal variations are inevitably non-isometric, each of the lead evolution processes or bioanalogous structural transformations has been made necessarily with the violation of the basic idea of bioisosterism. Therefore, sometimes, the rules are not easily deduced from and identified in lead evolution examples. Unless bioanalogous structural transformation rules are integrated and systematized, possible mutual relationships as illustrated in the preceding section for those detected between amides and cyclic dicarboximides among agrochemicals, anti-tumor agents, and anticonvulsants may be overlooked easily.

4.1 Identification of Bioanalogous Transformation "Rules". Because the data unit in the EMIL database is primarily for the rule to be utilized for the structural transformation, the core of information is to identify the bioanalogous relationship between the lower-ordered and the higher-ordered structures. Differing from ordinary fact databases in which information is just for a single entry, a specific feature of the EMIL database is that it includes the information about two compounds. Suppose compounds I and II are bioanalogously related, or the substructural modification of the compound I has eventually led to the compound II exhibiting a bioactivity analogous to that of compound I. The identification of substructural modification patterns is done by collating a substructure being modified in the structure I with a substructure having been modified in the structure II, leaving an unchanged substructural part or "evolutionally equivalent" counterparts between structures I and II.

255 4.1.1 Cromakalim and Analogs, Histamine H2-Antagonists and Related Series. The original skeletal structure of cromakalim and analogs such as that in compound 2 is derived from the acyclic alprenolol (1) as indicated in Fig. 8 (10). This structural modification pattern can be schematized as enclosed there. Each of the circled A 1 and A2 is unchanged or evolutionally equivalent in structures I and II.

Structure I

l

StructureII oH

02

1

Qo_

H

(1)

2

(2)

Fig. 8. Substructural Modification Pattern in "Bioanalogous" Transformation of Alprenolol (1).

With this transformation, the pharmacology is changed from the 13adrenoceptor antagonism to the potassium channel activation. Because both are important, the structural transformation of this type had better be included in the database. In this respect, the structures before and after the transformation could be "superbioanalogous", because their bioactivity profiles are not entirely analogous, but the bioactivity is "retained" anyway with the metamorphosis. If compounds exhibiting different pharmacologies are intentionally explored, the superbioanalogous transformation patterns accumulated in the database are to be invaluable precedents. Note that the substituents on the benzene ring are omitted from the patterns in Fig. 8. Modifications of the substituents are to be done in the optimization phase starting from a selected "higher-ordered" compound/structure with information about possible substituent effects on the potency variations for the particular bioactive compound series if any.

HNL 6

(2,

O

~O

( ~ (3)

6

HN~'~CH3 (4)

(~

(5)

HN")~NHCH3

NCN HN"J~CH3 ~

NCN H3CN-N HNJl" NHCH3 ~i~ HN"'~'~N~NH2

(~

(~

(~

Fig. 9.

(6/

(7)

(8)

(~

(9)

Substructural Modification Patterns in Bioanalogous Transformation of Cromakalim Analogs (I).

256 Consecutive patterns from compound 2 to 9 in Fig. 1 including cromakalim (4) are shown in Fig. 9. Each of the patterns between two consecutive structures arranged in Fig. 9 is to be utilized as the unit rule. For the processes from compound 4 to 17 via 11, pattems shown in Fig. 10 are extracted. Note that the process between compounds 4 and 10, two patterns are possible. As described above, each of the circled An's denotes evolutionally "equivalent" moiety between two structures, i.e., the six-membered lactam moiety in compound 10 is regarded as being "equivalent" with the five-membered lactam in compound 4 in Fig. 10a, and the oxadiazole moiety in compound 10 is recognized as a "substituent" on the homocyclic aromatic ring similar to the cyano group in compound 4 in Fig. 10b. b

a

(4)

(10)

OH

(4)

(10)

(11)

(17)

Fig. 10. SubstructuralModification Patterns in Bioanalogous Transformation of Cromakalim Analogs (II). Other notable patterns are shown in Fig. 11. CH3 N,,N~O

a

H3C~N.__N

~ ( ~NOQ 1 2~)/Q(13) )

C ~

HN~ C N

13,

d

9 (11)

(12,13)

(14)

(15)

(18)

CH2F H2F

Fig. 11. SubstructuralModification Patterns in Bioanalogous Transformation of Cromakalim Analogs (III). The structure of cromakalim analogs included in Fig. 1 seems to consist of two substructures. The one corresponds to the dihydrobenzopyran system in cromakalim itself and the other is that accomodates "(cyclic) amides" and related

257 structures. The structural modification patterns arranged in Figs. 9, 10b, and 11 a-c are for the bioanalogous structures of the "amide" moiety, while those listed in Figs. 10a and l ld are for potentially interchangeable structures with the (dihydro)benzopyran system. Interchangeable substructures observed in the processes from structure 5 of the acyclic analog of cromakalim to structure 9 in Fig. 9 are identical with or very similar to those observed as hydrogen-bonding groups (H) in H2-receptor histamine antagonists ( 1 0 9 - 124) which are listed in Table 3 as briefly mentioned before. Figure 9 can be extended by adopting bioanalogous substructures shown in Table 3 for the histamine H2 antagonists. Each of the H structures in Table 3 could be connected with the notation A1 and related to patterns in Fig. 9. Some substructural modification patterns in Fig. 9 extended with those included in Table 3 could also be indicated as shown in Figs. 12 and 13.

NNO2

N,,ON~

|

,,

~

H3C~N - N

@

Fig. 12. Bioanalogous Transformation Patterns of the "Carbonyl" Group. O

O

NSO2NH2

-,-

NCN C-

O -,-

NCN ~

0 II

NCN ~ II M.A~ C-NHCH3 _ ~

Fig. 13. Interchangeability between Amide and Urea Structures and Related Structural Pairs. Figure 12 is for the structures bioanalogous to the carbonyl group, whereas Fig. 13 illustrates the interchangeability between amide and urea and between amidine and guanidine structures including patterns deduced from structural transformations observed in other series of potassium channel activators (compounds 125 - 135) and imidacloprid analogs (136 - 139). For the aromatic ring substructures (R) and flexible chains (C) of histamine H2 antagonists in Table 3, the modification patterns can be drawn as in Fig. 14.

258 a

c

N-H2

Fig. 14. Substructural Modification Patterns in H2-Receptor Histamine Antagonists. From imidacloprid series insecticides (136 - 139), the patterns shown in Fig. 15 can be extracted for N,N'-cyclic guanidines, open-chain ethenediamines and amidines.

(136, 137)

CH2CH3

CH3

(138)

(139)

Fig. 15. Substructural Modification Patterns in Imidacloprid Analogs. 4.1.2 Interchangeability between "Amides" and Cyclic "Dicarboximides". In section 3.2.3, it is demostrated that herbicidal "amide" series of compounds 148, 149, and 150 are bioanalogous as are fungicidal cyclic dicarboximide series of compounds 151, 152, and 153. The situation can simply be schematized as shown in Fig. 16. (~R

-,~=---)- ( ~

(148)

OR -,~---.)~

(149)

R R !

(~

NRR'

(150) R

(151)

(152)

(153)

Fig. 16. Bioanalogy among Alkyl(ene), (Alk)oxy and Alkylamino Moieties.

259 As far as these two series are considered separately, the structural variations seem to follow more or less isometric bioisosteric principles. Among dicarboximide fungicides, an analog with structure 152 in which R1 = CH3 and R2 - H (section 3.2.3) was disclosed first by scientists at Sumitomo (75). The pyrrolidinedione (151) and imidazolidinedione (153) fungicidal structures are likely to be "designed" and synthesized on the basis of structures of anilide (148) and urea (150) herbicides, respectively, following the preceding example showing that the oxazolidinedione fungicides (152) are ring-closured analogs of the carbamate herbicides (149). The structural transformations between "amides" and corresponding dicarboximides common to these three cases are generalized as a single scheme shown in Fig. 17.

O II

(148- 150)

o CH3

-

-

(151- 153)

Fig. 17. Structural Transformation from "Amides" to Cyclic Dicarboximides. The same structural modification pattern can apply to those from flutamide (148: X = 3-CF3, 4-NO2, R - CHMe2) to nilutamide (156), from the benzanilide (163) to the phthalimide (164), and from phenetufide (160) to phenobarbital (159) as well as from compound 162 to trimethadione (161). The bioanalogous relationship between "amides" and dicarboximides is not limited in agrochemicals but extended into series of antiandrogens as well as CNS agents.

4.1.3 Angiotensin II Receptor Antagonists. Most of the structures of potent AT1 receptor antagonists arranged in Fig. 2 seem to be divided into two major substructures : a substituted hetero-aromatic ring or an acyclic counterpart (HT) and a biarylylmethyl moiety with an acidic group (BACH2). Exceptions are eprosartan (27) and compounds 20 - 2 2 in the course toward the disclosure of losartan (23). Therefore, in compounds 23 - 26 and 28 - 38 in Fig. 2, the HT structure is bioanalogous to each other as is the BA moiety. As mentioned before, these compounds are not necessarily arranged chronologically, but according to a similarity in the substructural environment around the connection site of the BACH2 group with the H T moiety in Fig. 2. Structural modification patterns in the HT moiety can be indicated as summarized in Table 4 in which the numeral in parentheses corresponds with the compound number in Fig. 2.

260 TABLE 4. The Mode of Connection with Biarylylmethyl (BACH2) Group and Structural Modification Patterns of "Heteroaromatic" (HT) Moiety in the AT1 Antagonists. Patterns

Features of the HT Moiety and the Connection with the BACH2 Group.

HT(23) ~ ~ HT(26)

Fission of heterocycles; Interposition of heteroatoms for the connection.

HT(23) HT(23) HT(36) HT(25) HT(33)

Conversion of CH2OH to an endocyclic N. Connection as the tertiary amide formation.

~ ~ ~ ~ ~

HT(29) HT(30) -~ HT(37)~ HT(38) HT(31) --, HT(32) HT(34)-~ HT(35)

Benzimidazole and bioanalogous "skeletons" with and without a carboxylic function at the I]-position to the connection site.

Similar to those described in the preceding sections, each pair of consecutive two HT structures is to be patterned as the transformation rule and registered in the database. Some detailed modification patterns in the HT moiety are shown in Fig. 18.

(23)

6

(24)

(25)

(31) c

(32)

COOH

CH 3

(33)

Fig. 18. Substructural Modification Patterns for "Heterocyclic" Moiety of AT1 Antagonists. In Fig. 18a, the CH2OH group in losartan (23) is regarded as being a carboxyl, because the corresponding carboxylic compound is the active form of losartan in vivo (76). The EtO group as A1, in candesartan (31) is taken to be evolutionally equivalent to lower n-alkyl groups in compounds 24 and 25. The EtO group has

261 been shown to be optimal in the candesartan molecule by QSAR (77). In Fig. 18b, the alicyclic spiro structure of compound 30 is divided into two segments, A3 and Y. A3 in compound 30 is regarded as being "equivalent" with such hydrophobic substituents as C1 in compound 23 and Bu in compound 29. Y is a disposable segment which could be selected appropriately, for instance, from lower alkyl groups. In Fig. 18d, detailed substituents are omitted from skeletal structures. Besides the fact that the substituent selection is to be done in the optimization phase, restrictions of the role by defining with specific substituents may reduce the chance of hits with the input structures as described below. For the biarylyl moiety, patterns extracted in processes following losartan is rather simple as arranged in Fig. 19a. Between compound 22 and saprisartan (28), the amide bridge is replaced by a condensed furan ring as shown in Fig. 19b. a

b

N~ O

@- COOH~

--q~(~--~ ~

~(23)

N~N

(32)

R :Ph (34) R : OBu (35, 38)

N

O

(28)

(22)

(28)

Fig. 19. Bioanalogous Transformation of Carboxyl Group and Amide Linkage.

4.1.4 13-Methoxyacrylates and Analogs. In structural transformation processes shown in Fig. 3, the essence is how to elaborate the conjugate diene system leaving the acryloyl double bond. In compound 40, one of the double bonds is replaced with the benzene ring. In compound 41, the second double bond is reduced to an ether bridge. Some specific modification patterns are shown in Fig. 20. b

12

@-o--@ (40)

(41)

(41, 43) N~,. N

(44)

(46)

(42)

(45)

f

(44) OMe

(41)

/OMe

(43, 44)

Fig. 20. Substructural Modification Patterns for 13-Methoxyacrylate Fungicides.

262 The replacement of the double bond moiety with the benzene ring is not unusual. Examples are found in such conjugate polyene compound series as retinoic acids (78) and insect juvenile hormone mimics (79). It should be noted that the modifications shown in Figs. 20d-e are those intentionally made to reduce the molecular hydrophobicity. In the optimization phase of the candidate compounds which are synthesized according to the "rule", the molecular hydrophobicity should be adjusted by introducing substituents having appropriate hydrophilicity or hydrophobicity.

4.1.5 Arylsulfonylureasand Related Herbicides. The structure of this series of compounds shown in Fig. 4 can be divided into three parts, the "ortho" substituted (hetero)aromatic moiety, the six-membered azine system sometimes condensed with another ring and the bridge between the two ring systems. For compounds located closely after chlorsulfuron (48), i.e., for compounds 49 - 53, 56 - 58 and 60, the (hetero)aromatic moiety is "almost" isometric. The 1Narylpyrazole structure in NC 330 (54) is similar to those in sulfaphenazole (66) in Fig. 5 and antipyrine (166). The transformation pattern from NC 330 (54) to imazosulfuron (55), as schematized in Fig. 21a, can be regarded as being that in ~cn3 which two tings connected with a single bond are condensed along O~,,,t4N.cH 3 with minor rearrangements of (hetero)atoms. For structural variations in the non-condensed azine moiety, the rule can be deduced as shown in Fig. 21b, where any type of combinations of two from Me and MeO groups is denoted by the pair of A2 and A3. 166: antipyrine

(54/

(55/

(48,49) ~

(50,51) 0

Fig. 21. Substructural Modification Patterns in Arylsulfonylureas and Related Herbicides (I). The processes from chlorsulfuron (48) to the condensed azine compounds (56) and (57) are regarded as following pattems in Figs. 22a-b. Those from flumetsulam (57) to compound 58, from chlorsulfuron (48) to compound 58 and amidosulfuron (59), and from compound 56 to 60 are shown in Figs. 22c-f.

263

a

(48) = ~

( ~ SO2NH- - ~

(56)

i-iso -Q

(57)

(56)

C

d CH3SO2

N_.

N--@

H3C

(57)

3.,7

(58)

e

(48)

(59)

f o

A~

(48)

N CH3

(58)

(56)

(60)

Fig. 22. Substructural Modification Patterns in Arylsulfonylureas and Related Herbicides (II). It is interesting to note that the methyltriazole structure, which is taken to be equivalent to the amide linkage in Fig. 22e, is isomeric with that included in Fig. 12 which is replaceable with the carbonyl. The transformation pattern from compound 49 to 50 is to delete one of the two NH units in the urea structure. This pattern is also included in Fig. 13 for interchangeability between amide and urea structures. The shortening of the bridge from compound 50 to 61 seems to be very drastic. The SO2NHCO chain could be replaced with just a (thio)ether linkage. In this series of acetolactate synthase inhibitors, an acidic function is required to be located at an appropriate distance from the azine system or its counterparts. The free carboxylic acid form of ester sulfonylureas such as compounds 49, 50, and 52 - 54 is inactive (31). Because the sulfamyl NH works as an acid, the meaningful transformation pattem in this subclass of compounds is perhaps that as shown in Fig. 23.

d

C~~_OOH SOzNHCO- - ~ (50)

~

O--~ (61)

Fig. 23. From N-Acylsulfonamides to O-Arylsalicyclic Acids.

264

Numerous structural evolution patterns in various series of bioactive compounds other than those described above can be explored in past examples and collected as the database. As mentioned above, the structural transformation rules which are to be utilized in the EMIL system are not always identical with patterns with which the past structural modification units were eventually made. The rules to be utilized in the system are somewhat simplified from patterns actually observed in past examples because the detailed substitution types had better not be included in the rules. Certain bioanalogous structural transformation rules are applicable in general regardless of the types of biological activity. The rules found in examples for certain pharmaceuticals could be utilized as the rules for the structural transformation of other bioactive compound series including agrochemicals. The superbioanalogous relationships covering compounds of different pharmacologies could be utilizable to explore "novel" compounds exhibiting bioactivity of any type. Even though we collected rules from existing examples retrospectively, the rules should be utilized prospectively for new trials.

4.2 Operation of the Bioanalogous Transformation System. The operational function of the EMIL system can be simplified as depicted in Fig. 24 (4, 5). IPrimary "Lead"~ Out-ut /'~Higher-ordered-'~ Structure 1 Input._] Data Processing ] P ,--! "Lead" Structure]

[RI-X1] 3

-1

Engine

Jl

]

-L

[R1-Y1] )

atabase of Rules for-'~ Substructural | odification Patterns ]

n-Xn)--~ (An-Yn)] J

Fig. 24. Simplified Operational Function of the EMIL System. First, the structure of the primary lead compound, RI-X1, from which one would like to make structural transformations is introduced into the system. If an example, in which a structure A1-X1 is eventually transformed into A1-Y1, is hit by the database search, then, the system "automatically" constructs a candidate structure, R1-Y1, as that of the higher-ordered lead compound. The substructural modification pattern from X1 to Y1 originally identified in the structural evolution example from the structure I, [A1-X1], to the structure II, [A1-Y1], is utilized here as the rule for the substructural modification of R1-X1 to R1-Y1. Usually, more than a single patterns in the database are hit leading to a number of "brother" structures. The cycles of the operation can be repeated as far as the output structure R1-Y1 which is rewritten as

265

R2-X2, is able to hit another rule with which A2-X2 is transformed to A2-Y2in the database. Depending upon the judgement how many cycles are sufficient to yield a reasonable number of output structures, the operation can be terminated. Of course, the symbol of structures does not mean that the "two" parts are monovalently combined. Instead, they are substructures in a certain structure. 5. CONCLUDING REMARKS Although the output structures are constructed with substructural transformation rules extracted from existing lead evolution examples, the biological activity of compounds having these structures is not always guaranteed. One may also consider that most of the compounds with higher-ordered structures could be synthesized with various combinations of possible bioanalogous substructures accumulated as the personal knowledge of expert practicing chemists without the aid of computerized data processing. Not every possibility could, however, be explored because of the limited memory of the human brain. Some promising candidate structures may be overlooked. The computer-assisted procedure is able to glean such structures. Moreover, the integration as a comprehensive compilation of the information about the bioanalogous structural transformations would be almost impossible without the aid of computer technology. Among a number of output structures as candidates, not every structure need be synthesized. Certain structures, which are attractive for synthetic chemists according to their personal experience and implicit "idea", could actually be synthesized. In addition, it is important to gain insights into or hints as to how to elaborate further promising structures from the output structures instead of following them directly. Such sets of bioanalogous substructures as shown in section 4, if comprehensively deduced and listed, could be used as substructure libraries to support combinatorial syntheses (80). As described earlier in this article, in the process of structural modifications of the primary leads, there are at least two phases according to one's objectives : the one is the lead optimization with systematic modifications of the lead structures and the other is the lead evolution to obtain novel skeletal compounds. For the lead optimization phase, the QSAR procedure has been successfully employed as demonstrated in some chapters of this volume as well as elsewhere (1, 81, 82). For the lead evolution phase, the bioanalogous relationships have been eventually utilized as illustrated above. The EMIL system is trying to integrate the individual information about bioanalogous relationships and to utilize them as the rules for the analog design prescription. In the QSAR procedure, the prescription to optimize the lead structure is deduced from mathematical correlation equations. Therefore, it seems entirely different from the procedure used in the EMIL system. However, both of these procedures use empirical "rules". In the QSAR procedure, the rules are

266 represented by variations in physicochemical numerical parameters, while in the EMIL system, they are expressed by variations in (sub)structural patterns. Thus, within the category of computer-assisted empirical methodologies, the EMIL procedure could be complementary to the QSAR analysis. In the EMIL system, the stereochemistry of candidate structures is not always considered. The 3D structures could be established from the 2D output structural formulas with the aid of crystallographic data of related compounds and theoretical calculations, if necessary. Enantiomeric and diastereomeric conditions for the structural evolution processes are to be included in the database as far as possible within related series of compounds. The candidate structures are, however, presented only two-dimensionally in the present version of the system. The stereochemistry of new compounds is principally unknown before syntheses, dissolution and biological measurements. Especially when the modifications are drastic to make entirely novel compounds, most synthetic pathways have to be prescribed without much information about relationships between stereochemistry and activity. Identification of enantiomeric and diastereomeric effects on the activity could be examined in the optimization phase of compounds selected from candidate 2D structures. The EMIL system can also be combined with such software systems as that to calculate the log P value (83) and/or those to "predict" possible toxicities and environmental behaviors (84). Without using sophisticated theoretical and statistical computations included in various computerized procedures developed recently (85), this system could hopefully be well accepted by practicing synthetic chemists, because the system, in a way, simulates their way of thinking for designing bioactive molecular structures empirically rather than "theoretically". ACKNOWLEDGMENTS The authors are indebted to special coordination funds of the Science and Technology Promotion Bureau, Science and Technology Agency (STA) of the Japanese Government that supported an initial part of the present project, as one of the sections of a comprehensive project research, "Knowledge-Base System for Design of Chemical Substances, 1986-1991", presided by Professor Yukio Yoneda, Tokai University. The authors gratefully extend their appreciation to Messrs. Noriyuki Shiobara, Masahiro Baba, Toshikazu Kubota, Osamu Tezuka and Toshihiko Kuboki of Fujitsu Ltd. for their efforts to construct the EMIL software. The valuable suggestions given by Dr. Takehiko Naka of Takeda Chemical Industries, Ltd. about AT1 antagonists and the skillful assistance of Dr. Yoshiaki Nakagawa of Kyoto University for the artwork are also greatly appreciated.

267 REFERENCES

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272 List of Addresses of Authors

The current address of the corresponding author and business addresses of other EMIL working group members, mostly at the time of the STA project, are shown below. Toshio Fujita (Corresponding Author), EMIL Project, Fujitsu Kansai Systems Laboratory, 2-2-6 Shiromi, Chuoku, Osaka 540, Japan. Michihiro Adachi and Akio Ogino, Research and Development Division, Nippon Shinyaku Co., Ltd., Kyoto 601, Japan. Miki Akamatsu, Department of Agricultural Chemistry, Kyoto University, Kyoto 606, Japan. Masaaki Asao and Ryo Shimizu, Research Laboratory of Applied Biochemistry, Tanabe Seiyaku Co., Ltd., Osaka 532, Japan. Harukazu Fukami, Suntory Institute for Biomedical Research, Shimamotocho, Osaka 618, Japan. Yoshihisa Inoue and Yasunari Yamaura, Central Research Laboratory, The Green Cross Corporation, Hirakata, Osaka 573, Japan. Isao Iwataki and Izumi Kumita, Odawara Research Center, Nippon Soda Co., Ltd., Odawara 250-02, Japan. Masaru Kido, Tokushima Institute of New Drug Research, Ohtsuka Pharmaceutical Co., Ltd., Tokushima 771-01, Japan. Hiroshi Koga, Takamitsu Kobayashi, and Masateru Ohta, Fuji Gotemba Research Laboratories, Chugai Pharmaceutical Co., Ltd., Gotemba, Shizuoka 412, Japan. Kenji Makino, Central Research Institute, Nissan Chemical Industry, Ltd., Funabashi 274, Japan. Kengo Oda, Life Science Laboratory, Mitsui Toatsu Chemicals, Inc., Mobara, Chiba 297, Japan. Fumio Sakamoto, New Drug Research Laboratories, Kanebo Ltd., Osaka 534, Japan. Tetsuo Sekiya, Yokohama Research Center, Mitsubishi Chemical Corporation, Yokohama 227, Japan. Chiyozo Takayama, Takarazuka Research Center, Sumitomo Chemical Co., Ltd., Takarazuka, Hyogo 665, Japan. Yukio Tada, Hanno Research Center, Taiho Pharmaceutical Co., Ltd., Hanno-Shi, Saitama 357, Japan. Ikuo Ueda, Industrial and Scientific Research Institute, Osaka University, Ibaraki, Osaka 567, Japan. Yoshihisa Umeda, Pharmaceutical Research Laboratories, Takara Shuzo Co., Ltd., Otsu, Shiga 520-21, Japan. Masumi Yamakawa, Shionogi Research Laboratories, Shionogi & Co., Ltd., Osaka 553, Japan.

273 Hirosuke Yoshioka, Bioregulator Design and Synthesis Laboratory, Institute of Physical and Chemical Research, Wako, Saitama 351-01, Japan. Masanori Yoshida, Pharmaceutical Research Institute, Nihon Nohyaku Co., Ltd., Kawachi-Nagano, Osaka 586, Japan. Masafumi Yoshimoto, New Lead Research Laboratories, Sankyo Co., Ltd., Tokyo 140, Japan. Ko Wakabayashi, Department of Agricultural Chemistry, Tamagawa University, Machida, Tokyo 194, Japan.

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QSAR and Drug Design - New Developments and Applications T. Fujita, editor 9 1995 Elsevier Science B.V. All rights reserved

275

FUZZY A D A P T I V E LEAST S Q U A R E S AND ITS USE IN QUANTITATIVE STRUCTURE-ACTIVITY RELATIONSHIPS

Ikuo MORIGUCHI and Shuichi HIRONO School of P h a r m a c e u t i c a l Sciences, Kitasato University, Tokyo 108, J a p a n

ABSTRACT

Fuzzy adaptive least s q u a r e s (FALS89) designed to correlate molecular s t r u c t u r e with activity rating h a s been developed. The m o s t novel feature of FALS89 is t h a t the degree of s a m p l e s belonging to activity classes is given using a m e m b e r s h i p function. The a l g o r i t h m involves a n iterative modification of forcing factors to maximize the s u m of the m e m b e r s h i p function values over all samples. This c h a p t e r first describes the m e t h o d a n d calculation procedure of FALS89, and t h e n shows its application to the correlation of s t r u c t u r e with potency rating of three d a t a sets: 33 argininev a s o p r e s s i n inhibitors as an example of small size d a t a and h u m a n acute toxicity (504 samples) and aquatic toxicity (324 samples) of miscellaneous organic chemicals as examples of large size data. The reliability of FALS89 s h o w n in the three examples of the application is considerably high in spite of the diversity of s t r u c t u r e s and vagueness of potencies.

I.

INTRODUCTION

There are two a s p e c t s of p a t t e r n d i s c r i m i n a t i o n for s t r u c t u r e - a c t i v i t y studies as shown in Table 1. One is discrimination of the type of action from molecular structure.

For this p u r p o s e , m e t h o d s for i n d e p e n d e n t - c a t e g o r y

discrimination s u c h as linear discriminant analysis (1), SIMCA (2), and linear l e a r n i n g m a c h i n e (LLM) (3) are used. activity r a t i n g s

The o t h e r is the d i s c r i m i n a t i o n of

(-, +, ++, etc.) w h i c h are o r d e r e d categories.

For this

p u r p o s e , we developed adaptive least s q u a r e s {ALS) in 1977 (4). ALS is a

276 n o n p a r a m e t r i c p a t t e r n classifier, a n d is d e v i s e d to f o r m u l a t e a QSAR in a single m a t h e m a t i c a l

e q u a t i o n i r r e s p e c t i v e of t h e n u m b e r of activity r a t i n g s

b y a n e r r o r c o r r e c t i n g f e e d b a c k a d a p t a t i o n of forcing factors d e s c r i b e d later. B e c a u s e t h e a d a p t a t i o n is d o n e as a s e t c o r r e c t i o n , t h e A I ~ c a l c u l a t i o n is efficient a n d applicable to linearly i n s e p a r a b l e s a m p l e s u n l i k e LLM. TABLE

I

Biological

activity

Type of a c t i o n Independent

Level

and QSAR methods

category

of a c t i o n Interval scale Ordered

(log

category

Linear discriminant analysis (LDA), Statistical isolinear multiple component a n a l y s i s (SIMCA), L i n e a r l e a r n i n g m a c h i n e (LLM), e t c . l / C , C: LD50, ED50 , MIC, e t c . ) Hansch a p p r o a c h , e t c .

(activity r a t i n g s " - , +, ++, e t c . ) A d a p t i v e l e a s t s q u a r e s (ALS), F u z z y ALS, LLbI, e t c .

O r d e r e d c a t e g o r i e s c o m p r i s e n o t only s t a t i s t i c a l v a g u e n e s s s u c h as i n a c c u r a c y of m e a s u r e m e n t , b u t also intrinsic v a g u e n e s s s u c h as subjective criteria

for c l a s s i f i c a t i o n .

Such

c o n c e p t s of fuzzy v a r i a n c e (5). membership

function

(5) w h i c h

indefiniteness

can be grasped

by the

To ALS, t h e r e f o r e , we h a v e i n t r o d u c e d a is a s s u m e d

to b e t h e

fuzzy d e g r e e of

m e m b e r s h i p in a category. T h i s c h a p t e r first d e s c r i b e s t h e m e t h o d of t h e fuzzy v e r s i o n of ALS, FALS89 (6,7), a n d t h e n s h o w s its a p p l i c a t i o n to t h e c o r r e l a t i o n of s t r u c t u r e with p o t e n c y r a t i n g of t h r e e d a t a sets: 33 a r g i n i n e - v a s o p r e s s i n i n h i b i t o r s as a n e x a m p l e of s m a l l size d a t a a n d h u m a n a c u t e toxicity (504 s a m p l e s ) a n d aquatic

toxicity

(324

samples)

of m i s c e l l a n e o u s

organic

chemicals

as

e x a m p l e s of large size data.

2.

FALS89

Like

ALS,

FALS

makes

decisions

for

d i s c r i m i n a t i o n b y a single d i s c r i m i n a n t f u n c t i o n as

ordered

m-class

(m>2)

277 Z = w 0 + WlX I + w2x 2 + .........

where

xk

= kth

descriptor

+ WpXp

[1]

(k=1,2 ..... p)

coefficient; a n d Z = d i s c r i m i n a n t score.

for

structure;

wk

=

weight

For a set of n c o m p o u n d s , [1] can be

r e w r i t t e n as [2].

Z = XW

[2]

Z1 Z2

Z=

1 1

:

x= n

Xll ... X12 - "

:

:

9

o

1

Xln

Xpl Xp2

W0 Wl

: ...

w=

"

Xpn

Wp

In the m a t r i x X, Xik ( k = l , 2 ..... p a n d i = i , 2 ..... n) is the k t h d e s c r i p t o r for the ith c o m p o u n d . S t a r t i n g scores, aj (]= 1,2 ..... m), for the m e m b e r s of class j are a s s u m e d , a n d t h e n b o u n d a r i e s , bj 0=1,2 ..... m-l), between classes are fixed in advance. In fuzzy A I ~ as well as AES, aj is a s s u m e d by [3] or [3'], a n d bj is t a k e n as the m i d p o i n t b e t w e e n aj a n d aj+ I as [4]. aj = 4 (g~_~lng + nj / 2} / n -

2

[3]

w h e r e n g = size of group g a n d nj = size of group j. aj=(4j-2)/m-

2

[3']

bj = { aj + aj+l) / 2

A membership membership

[4]

function,

of c o m p o u n d s

M(Z), is a s s u m e d

to c l a s s e s .

to give t h e

grade

of

The v a l u e of M(Z) ( m e m b e r s h i p

grade) r a n g e s from 0 to 1, a n d is t a k e n to be 0.5 at the class b o u n d a r i e s . Figure 1 s h o w s the function u s e d in FAI~89.

In Fig. 1, fuzzy level, Flj, is the

p a r a m e t e r for fuzziness in the b o u n d a r y b e t w e e n class j a n d class j+ 1. Two levels of slope, steep (Fl=0.1) a n d gentle (Fl=0.5), are generally used. for class j c a n be written as [5].

M(Z)

278 11ll + {(Z-bj_I)IFIj_ 1 - 1}4] M(Z) =

1

Z <_bj_l+Flj_ 1

bj_l+Flj_ 1 < Z _ bj-Flj

I / [ i + {(bj-Z)/FIj - I}4]

[5l

bj-Flj < Z

T h e p r o c e d u r e is d e s i g n e d to m a x i m i z e a p p r o x i m a t e l y t h e total s u m of the membership

g r a d e over all c o m p o u n d s

in t h e set.

The calculation

b e g i n s w i t h t h e s e t t i n g of t h e initial forcing f a c t o r s Si (I) (i=1,2 ..... n), w h i c h are t a k e n to be Si {I} = aj where

[6]

aj = s t a r t i n g

s c o r e for c l a s s j to w h i c h t h e ith c o m p o u n d s

o b s e r v e d to belong. G e n e r a l l y , c l a s s e s are n u m b e r e d biological potency.

was

in a s c e n d i n g o r d e r of

By u s e of Si (I) in place of Z in [1] (or [2]) as

S (I} --- XW

[7]

w h e r e S (I) = {SI(I),$2 (I) ..... Sn(1)) ' (the p r i m e d e n o t e s t h e t r a n s p o s i t i o n ) .

The

l e a s t - s q u a r e s e s t i m a t e of the initial weight, W {I), is w r i t t e n as W (I} - (X'X)-IX'S {I)

[8]

W (1) is computed by ordinary least-squares. Then, Zi(1) for each substance is c a l c u l a t e d from [i] (or [2]) u s i n g W {I} a s Z {I) -- XW (I) The

[9]

membership

grade,

M i, is c a l c u l a t e d

based

on

Zi (I) for all t h e

compounds.

M(Z)

FIj_ 1 <

FIj

~

~

1.0 0.5

.....

0.0

Fig.

.//

"i

bj_ 1 I.

Membership

Class

j

aj function

for

bj class

j

279 At iteration 2 a n d thereafter, the forcing factor Si (t+l) (t_>l) is a d a p t e d u s i n g the correction term C1(t) w h e n c o m p o u n d i a c t u a l l y belongs to class j, as

Si (t+l} = Zi {t) + Ci {t} Ci

{t)

=

[I0] ~{t)< Li _aj

a~/(1-Mi)Flj- 1 -a~/( l - M i ) r l j

[ii]

z(t)>aj i

In [11], r is the c o n s t a n t given below. Then, the l e a s t - s q u a r e s estimate of Wk (t+l) is c o m p u t e d from [12], and Zi (t+l) is c a l c u l a t e d from [1] (or [2]) u s i n g W (t+l) for the

evaluation

of

m e m b e r s h i p grade and classification as illustrated in Fig. 2. w{t+ I) = (X,X)_ ix,s{t+ I}

[ 12]

The adaptive l e a s t - s q u a r e s calculation is iteratively carried out so as to

minimize (Si-Zi)2, or Ci 2. Therefore, we c a n expect to o b t a i n a d i s c r i m i n a n t function giving a l m o s t m a x i m u m M i for the set of c o m p o u n d s . The iterative l e a r n i n g of the d i s c r i m i n a n t f u n c t i o n is a c t u a l l y carried

o u t in two steps.

In step

1 (1
d i s c r i m i n a t i o n r e s u l t is r o u g h l y s e a r c h e d u s i n g all c o m b i n a t i o n s of fuzzy Structure, X

.......

.......

[ ....... .......

class m [Xln

]---,I.tZ

iscriminant function

=W0

W!

1+'''+wp

Xpn] ~

? ]

Xp

Calculation of M (t) and R S

Least-squares calculation of weight vector w w(t)= (X,X) -Ix,s (t)

-_-~

,

,

Adaptation of forcing f a c t o r S

S! l) = startina score a.

when compd i E class j

(t+l) Z (t) ~ (t) Si = i •a (]-M i ) FI

Fig.

2. O u t l i n e

of FALS c a l c u l a t i o n

procedure

MMG.RS

280 levels a n d a greater a value ((x=2.0) to avoid falling into a local o p t i m u m . Since there is some a r b i t r a r i n e s s in the m e m b e r s h i p function, fuzzy levels, a n d (~ values, the p r o d u c t of m e a n m e m b e r s h i p grade (MMG) and S p e a r m a n ' s rank

correlation

discrimination.

coefficient

(R s) is u s e d

as a criterion for the b e s t

Thus, the weight vector giving the g r e a t e s t value of the

p r o d u c t MMG.Rs in step 1 is selected as

the starting vector of step 2.

In

step 2 (t>_11), the iterative calculation with the b e s t c o m b i n a t i o n of fuzzy levels c h o s e n in step 1 and a smaller a value ((~=0.5) is performed until the discrimination is no longer improved within a m a x i m u m of 20 iterations. The results of FALS are validated by leave-one-out prediction (3), The d i s c r i m i n a n t function with a scientifically r e a s o n a b l e s u b s e t of descriptors giving the best leave-one-out prediction is finally adopted.

3.

STRUCTURE

ACTIVITY

CORRELATION

OF

33

VASOPRESSIN

ANTAGONISTS

The first example (6,7) of the application of FALS is for 33 antagonists of

antidiuretic

and

vasopressor

responses

to

arginine-vasopressin.

V a s o p r e s s i n acts on the m e m b r a n e s of the distal convoluted t u b u l e s and collecting d u c t s of the kidney, causing t h e m to become permeable to water. This permits reabsorption of water by osmosis.

Vasopression also acts as a

v a s o c o n s t r i c t o r of v a s c u l a r s m o o t h m u s c l e .

Further,

recent work has

revealed t h a t it acts as a n e u r o h o r m o n e in the central n e r v o u s s y s t e m controlling the c a r d i o v a s c u l a r , renal, a n d t h e r m o r e g u l a t o r y s y s t e m s (8). Vasopressin antagonists are considered as valuable pharmacological tools for investigating physiological and behavioral functions of the posterior pituitary hormone.

A r g i n i n e - v a s o p r e s s i n a n t a g o n i s t s having the following general

s t r u c t u r e were synthesized and their a n t i - a n t i d i u r e t i c a n d a n t i v a s o p r e s s o r activities were m e a s u r e d by Manning et al. (9-12). are listed in Table 2.

The detailed s t r u c t u r e s

TABLE

2

S t r u c t u r e s a n d d e s c r i p t o r s of A r g - v a s o p r e s s l n a n t a g o n i s t s Coipd

no. 1 2 3 4 5 6 7

a

9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

24

25 26 27 28 29 30 31 32 33

Structure

Descriptors used i n e o s C13-181 (Table 3 ) Phe(X) L(X) E(Y) T(Y) HB(Y) H(Y)

x

Y

Z

D(X)

D-Phe D-Phe D-Phe D-Phe D-Phe 0-Phe D-Phe D-Phe D-Phe D-Phe D-Phe L-Tyr I-Tyr L-Tyr L-Tyr L-Tyr L-Tyr L-Tyr L-Tyr D-Tur D-Tyr D-Tyr 0-Tyr 0-Tyr D-Tyr D-Tur D-Tyr D-Tyr D-Tyr D-phe D-phe D-phe D-phe

Va I Ile Thr Ala Gln

L-Arg L-Are L-Arg L-Arg

1 1 1 1 1 1 1 1 1

L-Arg

L-Arg

LYS

Phe Leu

L-Arg

GlU

(Me)a

!;:{ tF'ir) [Et)

i-Pr)

!::{ i!!ir)

(Pr)

'Tyros i n e a l k u I e t h e r . eYorvaiine.

Tyr Ser Va I

Va 1 Va 1 Va I Va I Va I Va I Va I Va I Va I Va I Va I Va I Va I Va 1 Va 1 Va I Va I Abub ChaC Nled Nvae

L-Arg L-Arg L-Arg L-Arp D-Arg D-Arg 0-Ara D-Arg L-Arg L-Arg L-Arg

L-hrg

D-Arg D-Arg D-Arg D-Arg

L-Arg L-Arg L-Arg L-Arg D-Arg

L-Arp

L-Arg

L-Arg

L-Arg L-Arg

1 1 0

0 0

0 0 0

0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1

1 1 1 1

1 1 1 1 1 1 1

0

0

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1

a - A m i n o b u t y r ic a c i d .

2.06 2.06 2.06 2.06 2.06 2.06 2.06 2.06 2.06 2. 0 6 2. 0 6 3. 9 8 4. 9 2 4. 9 2 5.03 3. 9 8 4.92 4.92 5.03 3. 9 8 4.92 4.92 5.03 3.98 4.92 4.92 5.03 2.74 2.74 2.06 2.06 2. 06 2.06

0

0 1 0 1 1 0 0 0 1 1 0

0

0 0 0 0

0 0 0 0 0

0 0 0 0

0 0 0 0 0 0 0

0 0 0 1 1 1 1 1

I

1 0 0

0 0 0 0 0 0 0 0 0 0 0 0

0 0

0 0

0 1 1 1 1

C C v c l o h e x u l a l a n ine.

o. 6 8 0.67 0.13 -0.23 0.12 -0.33 0. 26 0.23 -0.42 0.40 -0. 17

0. 68

-0.62 -0.70 0.06 -0.47 -0.20 0.28 -0.37 -0.62 0.57 0.38 0.21 -0.62 -0.62 -0.62 -0.62 -0.62 -0.62 -0.62 -0.62 -0.62 -0.62 -0.62 -0.62 -0.62 -0.62 -0.62 -0.62 -0.62 -0.62

-

-

0.68

0.63 0.63 0.63 0.68 0.68

0.68 0.68

0. 68 0. 63 0.68 0.68 0.68

0.68 0.68 0.68 o. 68

-

-

-

dNor leucine.

OMHCY) 0.91 1.25 -0.28 -0.40 -0.91 -0.67 1.92 1.22 -0. 67 1.67 -0.55 0.91 0.91 0.91 0.91 0.91 0.91 0.91 0.91 0.91 0.91 0.91 0.91 0.91 0.91 0.91

0.31

0.91 0.91

-

D(Z)

0 0

0 0 0 0 0 0

0 0 0 1 ! 1 1 0 0 0 0 1 1 1

1

0 0 0

0

1 0 0 0

0 0

-

h,

00

282 1

2

3

4

5

6

7

8

9

CH2--(X)--X- Ph e - - Y - A s n - - C y - Pr o - - Z-- Gly --NH 2

CH2

/

C

CH2 --CH 2 S

S

T h e biological activities were observed in vivo as effective dose (ED) in rats, a n d all were not m e a s u r e d at the s a m e time.

Therefore, the potencies

were allotted t h r e e r a t i n g s as listed in Table 4. a n d were s u b j e c t e d to F A I ~ analysis. As the s t r u c t u r a l d e s c r i p t o r s in FALS analysis, those for X, Y, a n d Z in t h e g e n e r a l s t r u c t u r e were c o n s i d e r e d .

As for X, w h i c h i n c l u d e d L-Tyr, D-

Tyr, t h e i r alkyl e t h e r s , a n d D-Phe, two i n d i c a t o r variables, D(X) for X = Da m i n o acid r e s i d u e a n d Phe(X) for X = Phe, a n d L(X) for the l e n g t h of the side c h a i n were i n v e s t i g a t e d . As for Y, w h i c h i n c l u d e d artificial

amino

hydrophobicity

acid

residues,

descriptors

for

11 n a t u r a l a n d 4

hydrogen

bonding,

(13,14), a n d l e n g t h of the side c h a i n a s well as s e v e r a l

c o n f o r m a t i o n p a r a m e t e r s (15-17) were e x a m i n e d .

Among them, descriptors

selected in the QSAR e q u a t i o n s in Table 3 are two i n d i c a t o r variables, HB(Y) for h y d r o g e n b o n d i n g ability of the side c h a i n a n d H(Y) for helix m a k i n g property,

the

optimal

information measures

matching

hydrophobicity

OMH(Y)

(14),

and

the

for e x t e n d e d s h e e t c o n f o r m a t i o n E(Y) (17) a n d for

t u r n s defined as for middle r e s i d u e s T(Y) (17).

H(Y) is t a k e n to be zero for

r e s i d u e s w i t h side c h a i n s b r a n c h i n g at the [3-position as Val a n d lle, a n d t h o s e with a hydroxyl g r o u p a t t a c h e d to the [~-carbon a t o m as T h r a n d Ser; a n d o t h e r w i s e t a k e n to be 1 (16). As for Z, w h i c h included D-Arg a n d L-Arg, a n indicator variable D(Z) for D-Arg w a s u s e d in the analysis. T h e d e s c r i p t o r v a l u e s u s e d in the d i s c r i m i n a n t f u n c t i o n s (Table 3) are listed in Table 2. The v a l u e s of E(Y), T(Y), a n d OMH(Y) are not available for 4 compounds

h a v i n g artificial a m i n o acid r e s i d u e s for Y.

Therefore, FALS

283 calculation was carried o u t with two sets of data, one for 29 c o m p o u n d s excluding these 4 c o m p o u n d s , and the other for all 33 c o m p o u n d s . The resulting d i s c r i m i n a n t functions are expressed as [13-18] in Table 3, where N = n u m b e r of c o m p o u n d s , MMG = m e a n m e m b e r s h i p grade, N = n u m b e r misclassified, and the figure in p a r e n t h e s e s after the value of Nmis is the

number

misclassified

correlation coefficient.

by two r a t i n g s .

Rs is the

Spearman

rank

The figure in b r a c k e t s u n d e r the coefficient of each

t e r m is the c o n t r i b u t i o n index ( = I c o e f i . S D of the descriptor), w h i c h is a m e a s u r e of the c o n t r i b u t i o n of the descriptor to the d i s c r i m i n a n t score (4). The b e s t c o m b i n a t i o n of the fuzzy levels {FI I, Fl2} was {0.5, 0.1} in these e q u a t i o n s except [ 16] {0.1, 0.1}, indicating t h a t the b o u n d a r y between higher p o t e n t classes was clearer. For the anti-antidiuretic activity of 29 antagonists, the five-descriptor equation [13] was the best.

A high correlation (r 2 =0.80) was found between

Phe(X) and L(X). However, there seemed to be no fear of chance correlation, b e c a u s e [14] which was lacking in Phe(X) still gave fairly good results.

For

the d a t a set of all 33 compounds, [15] with the same five descriptors as those in [13] was selected as the best equation. For the a n t i - v a s o p r e s s o r activity of 29 a n t a g o n i s t s , five d e s c r i p t o r equations [16] and [17] were selected.

Although [16] is better in terms of Rs

t h a n [17] both in recognition and in leave-one-out prediction, the predictive MMG is s o m e w h a t inferior. [18] for the set of 33 c o m p o u n d s gave the best predictive results both in MMG and Rs. The resulting recognition and leaveo n e - o u t prediction using these e q u a t i o n s were r e a s o n a b l y good in t e r m s of MMG as well as Rs, all of which indicated a significance level of P < 0.001 (Table 3).

The recognized a n d predicted m e m b e r s h i p g r a d e s (MG) a n d

ratings in both activities for each c o m p o u n d using [15] and [18] was listed in Table 4, along with observed values. Arginine-vasopressin contains L-Cys at position 1, L-Tyr for X, L-Gin for Y, and L-Arg for Z. The equations in Table 3 indicate a clear c o n t r a s t for X

TABLE 3 FALS d i s c r i m i n a n t f u n c t i o n s a n d t h e i r r e l i a b i l i t y

Eq

N

Recognition lbtC Nmis

Rsa

Prediction IMG Nmis

+ 0 . 8 2 4 L(X) C1.091 - 3.458

29

0.925

2(0)

0.955

0.877

3(0)

0.922

+ 0 . 3 9 7 I(X) CO. 521

29

0.877

3(0)

0.935

0.841

4(0)

0.904

33

0.903

3(1)

0.884

0.857

4(1)

0.849

29

0.852

4(0)

0.902

0.785

5(0)

0.860

0.826 T(Y) C0.291 0.240

29

0.857

4(1)

0.818

0.845

4(1)

0.818

0.520 HB(Y) CO. 191 0.885

33

0.870

J(1)

0.862

0.865

4(1)

0.862

no.

[I31

C141

[151

C161

C171

[I81

Anti-antidiuretic Z = 1 . 0 8 7 (XI + CO. 491 -2.167 H(Y) CO. 931

!

Z = 1.329 DCX) CO. 591 -1.637 H(Y) CO. 701 Z = 1 . 0 5 6 D(X) 10. 451 -2.105 H(Y) CO. 991

-

activity 2 . 0 5 2 Phe(X) c1.001 1.569 D(Z) C0.731

2 . 1 2 6 D(Z) C0.981 1 . 9 6 5 Phe(X) 10.981 1 . 5 4 1 O(Z) CO. 691

+

-

Anti-vasopressor activity Z = - 0 . 1 3 6 D(X) - 0 . 3 1 1 P h e ( X ) CO. 151

CO. 061 - 1 . 2 5 8 OMH(Y) CO. 871

-

0 . 1 6 0 D(Z) CO. 071

-

Rs*

1.293

+ 0.802

C1.071 - 3.279

L(X)

+

3.596 E(Y) C1.261 - 0.584

Z = - 0 . 1 8 4 D(X) CO. 081 -0.514 H(Y) co. 2 2 1

-

1 . 1 0 6 Phe(X) 10.541 0 . 1 5 0 D(Z) CO. 071

-

Z = - 0 . 1 3 7 D(X) CO. 061 -0.642 H(Y) CO. 301

- 1 . 0 9 4 Phe(X) CO. 541 - 0.140 D(Z) C0.061

-

+

+

aYighly significant at the level of p<0.001 for all the equations Cl3-181.

b C o n t r i b u t i o n index.

TABLE 4 Observed and calculated activlties o f Arg-vasopressin a n t a g o n i s t s Compd no.

1 2

3 4

1

5

1 1

6

1 1 1 1 3

7

a

9 10

11 12 13

14 15 16 17

18 19

2a

21 22 23 24 25 26 27 28 29 30 31 32 33

I 2 2 2 2 I 2 2 3

3 3 3 3 I 2 3 1 1 1

0.998 0.978 1.000 1.000 1.000 o. a54 1.000 1.000 0.000 1.000 1.000 1.000 1.000 1.000 0.995 0.000 0.982 0.982 0.982

1

2 2 2 2

1

2 2 2 3 3 3 3 1

2 1 1 1

I

o.

gas 0.529 1.000 1.000 o. a97 0.061 1.000 1.000 0.000 1.000 I . 000 I . 000 1.000 1.000 0.956

0.000 0.973 0.973 0.973

1 1 1 1 2 2 2

2

2 2 2 2 3 3 3 3

1

2 1 1 1 1

3 1 1

1 1

1

1

3

3 3

2 3 2 3 3 2 2 7

2 3 3 3 2 2 3 1

1

1

1

0.000 1.000 0.997 0.997 0.997 1.000 0. 9 5 0 0. 936 0. 9 3 6 0.936 0. 1 7 0 0. 946 0.014 1.000 1.000 1.000 1.000 1.000 1.000 0. 946 0. 9 4 6 0. 946 0 . 161 1.000 1,000 0.997 0.997 0. 997 0.997

' 1 , E D > 4 . 0 amo I/k,-; 2 , l . 7 < E D 5 4 , 0 ; 3 , E D 5 I . 7 . b l , E D > 1 . 2 m m o I / k g ;

1 1 1

1 1

1

1 1

3

3

3 3 3

3 3

;2 -2 7

3 3 3 3 2 3 1

1 1 1

0.766 0.996 0.000 1.000 0.996 0.996 0.996 1.000 0.766 0,991 0.991 0.991 0. 0 3 5 1.000 0.000 1.000 1.000 1.000 1.000 I . 000 I . 000 1.000 1.000 I . 000 0.082 1.000 1,000 0.996 0.996 0.996 0.996

2 1 1 1

1 1 1 1 t 1 3 3 3

3

3 3 3

3 2 2 2

2 3 3 3 3

2

3 1 1 1 1

2 , O .45<ED5 1 . 2 ; 3 , ED
N

oc

U I

286 b e t w e e n the two k i n d s of activities; a n t i - v a s o p r e s s o r activity requires L-Tyr, which is the residue at position 2 of vasopressin, w h e r e a s D-Phe, D-Tyr, and Tyr alkyl ether instead of L-Tyr are favorable for antidiuretic activity. As for Y, r e s i d u e s having no t e n d e n c y to form helices, pleated sheets, or t u r n s are favorable for b o t h activities.

The d y n a m i c s a n d conformational energetics of

l y s i n e - v a s o p r e s s i n were s t u d i e d theoretically by Hagler e t al. (18), and the p r e d o m i n a n t role of Phe at position 3 in the d y n a m i c flexibility and multiple c o n f o r m a t i o n a l s t a t e of the cyclic h e x a p e p t i d e ring w a s revealed.

The

i m p o r t a n c e of the conformational property of Y located at position 4 as well as X at position 2 seems to be u n d e r s t a n d a b l e .

As for Z, L-Arg, which is the

residue at position 8 of native vasopressin, is favorable for both activities. Thus, FAI~ analysis successfully generated the significant QSAR models w h i c h c h a r a c t e r i z e d s t r u c t u r a l features favorable for a n t i - a n t i d i u r e t i c a n d a n t i - v a s o p r e s s o r activities.

Interesting r e s e m b l a n c e a n d difference between

t h e i n t e r a c t i o n s with two k i n d s of r e c e p t o r s for A r g - v a s o p r e s s i o n were suggested by the FALS calculation results.

4.

HUMAN ACUTE TOXICITY OF 504 ORGANIC CHEMICALS

The s e c o n d structure-activity

example

(6,19) of the a p p l i c a t i o n

correlation

for p r e d i c t i n g

m i s c e l l a n e o u s organic chemicals. is e x t r e m e l y i m p o r t a n t ,

because

human

of FALS c o n c e r n s acute

toxicity

of

Prediction of h u m a n toxicity by c o m p u t e r human

toxicity c a n n o t

be m e a s u r e d

experimentally. The d a t a were collected m a i n l y from G o s s e l i n ' s c o m p i l a t i o n

(20),

w h i c h c o n t a i n s toxicological information a b o u t a c u t e c h e m i c a l poisonings a r i s i n g t h r o u g h m i s u s e of c o n s u m e r p r o d u c t s .

Some e s t i m a t e d d a t a of

m e d i c i n e s (21) a n d general organic chemicals (22) were also included.

The

d a t a set u s e d for FALS analysis includes 71 h e t e r o a r o m a t i c c o m p o u n d s , 203 c h e m i c a l s b e a r i n g a n a r o m a t i c h y d r o c a r b o n or q u i n o n e ring(s), a n d 230 other m i s c e l l a n e o u s organic c o m p o u n d s .

287 Toxicity involves various combinations of h a z a r d o u s effects on multiple biological r e c e p t o r s .

Therefore, toxicity r a t i n g s are often u s e d for the

expression of toxicity levels (20).

In this FAI~ studies, the following rating

definitions based on a probable lethal dose were used :

Rating i (not or slightly toxic) Rating 2 (toxic)

above 0.5 g / k g - - - 273 compds 0.05 - 0.5 g / k g --- 150 c o m p d s

Rating 3 (severely toxic)

less t h a n 0.05 g / k g - - -

81 compds

Table 5 s h o w s the typical s t r u c t u r e s i n c l u d e d in the t h r e e toxicity classes.

As a m a t t e r of fact, the s t r u c t u r a l and pharmacological features of

each class are not so clear. For instance, sulfisoxazole is assigned to rating 1, b u t sulfamerazine to rating 2; riboflavin (vitamin B2) is assigned to rating 1, b u t m e n a d i o n e (vitamin K3) to rating 2; and m e t h a r b i t a l is assigned to rating 2, b u t amobarbital to rating 3. Since v a r i o u s molecules with diverse s t r u c t u r e s a n d f u n c t i o n s were included in the set of c o m p o u n d s , m o s t of the descriptors investigated were those for molecular fragments and s u b s t r u c t u r e s .

According to their effect

on toxicity, they were divided into numerical and s e m i n u m e r i c a l parameters. As detailed in Table 6, n u m e r i c a l p a r a m e t e r s i n c l u d e p h y s i c o c h e m i c a l properties of c o m p o u n d s a n d n u m b e r s of specified s t r u c t u r a l fragments of molecules.

S e m i n u m e r i c a l p a r a m e t e r s are also for the n u m b e r of specified

s u b s t r u c t u r e s p r e s e n t in the molecules, b u t in this case, they are taken to be 1 and 2 for the presence of a singular and plural n u m b e r , respectively. The r e s u l t s of FALS calculation of s t r u c t u r e - t o x i c i t y rating correlation u s i n g 37 to 47 descriptors are s u m m a r i z e d in Table 7. In the recognition, a 45-descriptor discrimination.

equation

gave

the

best

result

with

88.3%

correct

However, the b e s t prediction w a s o b t a i n e d with the 37-

descriptor equation shown in Table 8.

In

the

table,

descriptors

with

positive d i s c r i m i n a n t coefficients a n d those with negative coefficients are

CD O9

o

O

C~

II

O

~..+.

o._+.

C~ O

CD <=

N

0

0

0

0

0

0

0

0

CZ) O

CZ) O 1 ~

O

CZ) O

OO OO OO

O

O

O

C=) CZ> O

r._o O O

C=) O

C.C> O O

O

CDI~

C=)

CD O

CO0

CZ~ C.O

O

~=) I~C~_IC=) C ~

O

CZ> O

O

CZ~ ~

O

CZ) C D CZ~ CZ~ C D C O O

CZ) O

o ot?oooooooo

O 0

CY~ CYl C.~ C_Y~ CY~ CYl CTI C~m CY~

o

~.~.

~D

0

C~

o

o

,.i.d.

0 ~-~

r,o

~o

CI)

z

9 ~

CD

" I

I

X

I

~o

X

I

A

~

I

:n=

,.-.+o o :=s :=~

0

,-~

~-~

r

c'~ .-

~

~ '-~ s

~

~-~.

"

~

~::~ 0

~-~

0

~

"

"~

,...

~

"0

::~ ~ ~

O)

r./~

.--.

I~

~-~ ;:~

~

C'~

~..~. [/~

~-~

.-.

~

.--.

X

I

~./

0

~-~

~

~

~

,-..b

=~

09

~ CO "

~

O

~-b

==~

'-'~ ~

r

0

0

CO

Cl)

0

o

,-+.

~-~

~-b t::

O)

r..~

9

Q)

0

~

~

O

I

I

I

I

::~

~

~

x

e-~

~.o

=~ 0

,-,,,-

O

~

t'x~O

C"~

r,~

;~

~'~ ~--

~

~-+- ~

I

I

II Z

Z

~

~-~.

e-~

0

CO

~"

O

o

.~

O

~

N ~D :~

~'-~

~--.

~'~

CD

~'~

e'~

~-~.

("D

"~

IE~

~:~

O

~,<

O)

~-~

o

~-~-

Z

[/l

~..+o

~.~o

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290 listed

in

order

of c o n t r i b u t i o n

indices,

c o n t r i b u t i o n to d i s c r i m i n a t i o n .

which

indicate

the

degree

of

D e s c r i p t o r s w i t h p o s i t i v e coefficients a r e

c o n s i d e r e d to c o n t r i b u t e in a positive s e n s e to a n e s t i m a t e of toxicity, while d e s c r i p t o r s w i t h n e g a t i v e coefficients c o n t r i b u t e in a n e g a t i v e way. Unsaturated

lactones,

partially

aromatic

polycyclic

structures,

a,~-

special

c a r b a m a t e s , etc. p r o b a b l y e n h a n c e a c u t e toxicity, w h e r e a s a l i p h a t i c alcohols, s p 2 r i n g c a r b o n s , c a r b o x y l i c a c i d s a n d e s t e r s etc. p r o b a b l y c o n t r i b u t e to l o w e r i n g toxicity.

However, t h o s e c o e f f i c i e n t s c a n n o t be u s e d

to m a k e

i n f e r e n c e s a b o u t t h e c o n t r i b u t i o n of e a c h f r a g m e n t . T h e y are valid only w h e n u s e d in t h e c o n t e x t of this m u l t i d i m e n s i o n a l model. The r e s u l t s of d i s c r i m i n a t i o n of the toxicity r a t i n g s for 504 c o m p o u n d s is fairly s a t i s f a c t o r y a s s h o w n in Table 9.

The a c c u r a c y of classification into

t h r e e r a t i n g s w a s 8 7 . 7 % in the r e c o g n i t i o n a n d 8 2 . 1 % in t h e l e a v e - o n e - o u t p r e d i c t i o n in s p i t e of t h e d i v e r s i t y of t h e m o l e c u l a r s t r u c t u r e of o r g a n i c c h e m i c a l s i n v e s t i g a t e d in this study. It

is

evident

from

these

results

that

a

reasonably

accurate

d i s c r i m i n a t i o n m o d e l could be g e n e r a t e d for t h e e s t i m a t i o n of h u m a n a c u t e toxicity u s i n g FAI~. T ABLE

9

Results

of

Recognition

recognition

Obsd 1 2 3

and p r e d i c t i o n

Calcd 1 249 22 0

2

3

24 124 12

0 4 69

N = 504 MMG = 0.855 C o r r e c t recog = 87.7%

Nmis = 6 2 ( 0 )

Leave-one-out prediction

Calcd

N = 504 Correct

Obsd

M~IG = 0 . 8 1 5 p r e d = 82.1%

using

Rs = O. 866 ( p < O . O0 ] )

1

2

3

237 29 0

35 114 18

7 63

Nmi s = 9 0 ( 1 )

39 d e s c r i p t o r s

1

Rs = 0 . 8 0 5

(p
291 5.

AQUATIC TOXICITY OF 3 2 4 ORGANIC C I I E M I C A I ~ T h e t h i r d e x a m p l e (6,23) of t h e a p p l i c a t i o n of FALS d e a l s w i t h t h e

s t r u c t u r e - a c t i v i t y correlation for p r e d i c t i n g a q u a t i c toxicity of diverse organic compounds.

I n f o r m a t i o n on the a q u a t i c toxicity of c h e m i c a l s u b s t a n c e s is

v e r y i m p o r t a n t for b e t t e r u n d e r s t a n d i n g of p o t e n t i a l o c c u p a t i o n a l h a z a r d s a n d helping to bring a b o u t a more h e a l t h f u l e n v i r o n m e n t . T h e toxicological d a t a (24) u s e d in this s t u d y were m o s t l y on finfish, with i n f o r m a t i o n on s h r i m p s a n d other a q u a t i c o r g a n i s m s to fill in the gaps. T h e original toxicity w a s classified into five g r a d e s on t h e b a s i s of the T L m 9 6 , w h i c h is defined as the c o n c e n t r a t i o n of a s u b s t a n c e t h a t will kill 50% of t h e e x p o s e d t e s t o r g a n i s m s within 96 h o u r s . compounds

in t h e

original

five t o x i c i t y r a t i n g s

Since t h e n u m b e r s of were

unbalanced,

the

c o m p o u n d s were classified into the following three r a t i n g s for F A I ~ analysis.

Rating 1

Practically non-toxic

Rating 2

Slightly toxic

Rating 3

Moderately or highly toxic

100mg/l< TLm96 I 0 m g / l < T L m 9 6 < i 00 m g / l T L m 9 6 < 10 m g / l

T h e n u m b e r of c o m p o u n d s a n d typical s t r u c t u r e t y p e s a p p e a r i n g in e a c h r a t i n g are listed in Table 10. Most aliphatic c o m p o u n d s are included in r a t i n g 1, a n d m o s t a r o m a t i c ones in r a t i n g s 2 a n d 3. T h r e e k i n d s of variables, c o n t i n u o u s variables, discrete variables, a n d i n d i c a t o r v a r i a b l e s were u s e d in the QSAR analysis.

The m o l e c u l a r weight,

h y d r o p h o b i c c o n s t a n t , a n d its s q u a r e d value are u s e d as c o n t i n u o u s variables. A discrete variable is defined as the n u m b e r of specific a t o m s or c o n s t i t u e n t s c o n s i d e r e d to c o n t r i b u t e to the toxicity.

If t h e c o n t r i b u t i o n is p r o b a b l y

different in t h e c a s e s of sp 2 a n d sp 3, in a ring a n d o u t s i d e a ring, in a n a l i p h a t i c m o i e t y a n d in a n a r o m a t i c moiety, the n u m b e r is c o u n t e d in e a c h case.

T h e i n d i c a t o r v a r i a b l e s s h o w n here are defined as 1 for the p r e s e n c e

a n d 0 for t h e a b s e n c e of a n y k i n d of a t o m s

or m o i e t y s t r u c t u r e s t h a t

X

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0

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..--.~ O o

r.O C.O

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C.O

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["0

O0

0

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=~

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X

X

I

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~

,I=~ Oo

=:Z='.

I

~

(--2

(3"J 0"I

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0-1

O0 Oq

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Z

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0

"'0

0

CI)

L~O ~

(3 0 (9 ~b

o

C~

~o

o

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0

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OB

l=.,o

L~

294 c o n t r i b u t e to t h e toxicity r e g a r d l e s s of t h e i r n u m b e r

in a m o l e c u l e .

The

t h r e e k i n d s of v a r i a b l e s are listed in Table 11. FALS c a l c u l a t i o n d e r i v e d a d i s c r i m i n a n t f u n c t i o n w i t h p r e t t y good d i s c r i m i n a n t a n d predictive ability. i n c l u d e d in t h e f u n c t i o n .

As s h o w n in Table 12, 4 0 v a r i a b l e s are

F r o m t h e sign of t h e d i s c r i m i n a n t coefficient for

e a c h variable, it is inferred t h a t n u m b e r s of N, O, S, a n d CI a t o m s , b e n z e n e and naphthalene

rings, h y d r o p h o b i c i t y ,

etc. c o n t r i b u t e to e n h a n c i n g t h e

toxicity, w h e r e a s n u m b e r s of sp 3 c a r b o n a t o m s , carboxylic a c i d s a n d esters, etc. p r o b a b l y c o n t r i b u t e to lowering t h e toxicity.

TABLE

13

Results

of r e c o g n i t i o n

and p r e d i c t i o n

Recognition

Calcd 1

Obsd

1

152

2 3 N

=

324

Correct

MMG

recog

Leave-one-out prediction

12 0

= 0.859 = 87.3%

3

16

0

1

1

142

20 0

MMG = 0 . 8 0 2 pred = 80.2%

Nmi s

1 32

41(0)

Calcd

2 3 N = 324 Correct

2

99 12

Nmi s =

Obsd

u s i n g 40 d e s c r i p t o r s

2

3

26

0

89 13

= 64(2)

Rs

= 0.859

(p
Rs

= 0.754

(p
3 29

The recognition and prediction results by the discriminant function a r e s h o w n in T a b l e 13.

T h e m e a n m e m b e r s h i p g r a d e s w e r e fairly good,

being 0.859 and 0.802, respectively.

T h e a c c u r a c y of c l a s s i f i c a t i o n into

t h r e e r a t i n g s w a s 8 7 . 3 % in the r e c o g n i t i o n a n d 8 0 . 2 % in t h e l e a v e - o n e - o u t prediction.

It is h o p e d t h a t t h e s e r e s u l t s m a y c o n t r i b u t e to k e e p i n g t h e

e n v i r o n m e n t safe a n d clean.

295 6.

CONCLUSION

The reliability of FALS s h o w n in the three examples of the application is considerably high in spite of the diversity of s t r u c t u r e s and v a g u e n e s s of potencies. The new computerized pattern classifier, FALS, should be a useful tool in correlation of semiquantitative biological potencies with a wide variety of s t r u c t u r e s of chemical s u b s t a n c e s which m a y be potentially beneficial or h a z a r d o u s to h u m a n s and the environment. We have p r o p o s e d FALS 91 quite recently (25, 26). In F A I ~ 91, a modification was

made

to the

error-correcting feedback

adaptation

of

d i s c r i m i n a n t functions to avoid falling into a local optimum. The effect of the modification is not always marked, though.

REFERENCES

10 11 12 13 14 15

Y.C. Martin, J.B. Holland, C.H. Jarboe, and N. Protnikoff, J. Med. Chem., 17 (1974) 409-413. W.J. D u n n and S. Wold, J. Med. Chem., 21 (1978) 922-930. A.J. Stuper and P.C. J u r s , J. Pharm. Sci., 6 7 (1978) 745-751. I. Moriguchi and K. Komatsu, Chem. Pharm. Bull., 25 (1977) 28002802; I. Moriguchi, K. Komatsu, and Y. Matsushita, J. Med. Chem., 2 3 (1980) 20-26. D. Dubois and H. Prade, Fuzzy Sets and Systems: Theory and Application. Academic Press, New York, 1980. I. Moriguchi, S. Hirono, and Q. Liu, Abstracts of Papers, 16th S y m p o s i u m on Structure-Activity Relationships, Kyoto, Oct 1988, pp.300-303; Q. Liu, S. Hirono, Y. Matsushita, T. Nakagawa, and I. Moriguchi, Abstracts of Papers, 17th S y m p o s i u m on Structure-Activity Relationships, Osaka, Nov 1989, pp.224-227. I. Moriguchi, S. Hirono, Q. Liu, Y. Matsushita, and T. Nakagawa, Chem, Pharm. Bull., 3 8 (1990) 3 3 7 3 - 3 3 7 9 C.L. Riphagen and Q.J. Pitman, Fed. Proc., 4 5 (1986) 2318-2322. M. Manning, B. Lammek, and A.M. Kolodziejczyk,J. Med. Chem., 2 4 (1981) 701-706. M. Manning, A. Olma, W.A. Klis, and A.M. Kolodziejczyk, J. Med. Chem., 2 5 (1982) 45-50. M. Manning, A. Olma, W.A. Klis, J. Sato, and W.H. Sawyer, J. Med. C h e m . , 26 (1983) 1607-1613. M. Manning, E. Nawrocka, A. Misicka, A. Olma, W.A. Klis, J. Sato, and W.H. Sawyer, J. Med. Chem., 27 (1984) 423-429. J.R. Fauchere and V. Pliska, Eur. J. Med. - Chim. Then, I 8 (1983) 369375. R.M. Sweet and D. Eisenberg, J. Mol. Biol., 171 (1983) 479-488. P.Y. Chou and C.D. Fasman, A n n u . Rev. Biochem., 4 7 (1978) 251-276.

296 16 17 18 19 20

21 22 23 24 25 26

R.L. Kisliuk, in: W.O. Foye (Ed), Principles of Medicinal Chemistry, Lea and Febiger, Philadelphia, 1981, C h a p t e r 24. B. Robson and J. Gamier, Introduction to Proteins and Protein Engineering, Elsevier, A m s t e r d a m , 1986, p p . 3 7 3 - 3 9 3 . A.T. Hagler, D.J. Osguthorpe. P.D. Osguthorpe, and J.C. Hempel, Science, 2 2 7 (1985) 1309-1315. I. Moriguchi, S. Hirono, and Y. Matsushita, Abstracts of Papers, The 1989 International Chemical Congress of Pacific Basin Societies, Honolulu, Dec 1989, 02-513. R.E. Gosselin, R.P. Smith, and H.C. Hodge (Eds), Clinical Toxicology of Commercial Products, 5th Ed., Williams and Wilkins, Baltimore, 1984; available on line as CTCP Data Base, NIH-EPA Chemical Information System. J a p a n e s e Pharmacopoeia, 1 l t h Ed., 1986. D.V. Sweet (Ed), Registry of Toxic Effects of Chemical S u b s t a n c e s , 1985-86 Edition, U.S. Government Printing Office, Washington, 1987. Q. Liu, S. Hirono, Y. Matsushita, T, Nakagawa, and I. Moriguchi, Abstract of Papers, The 1989 International Chemical Congress of Pacific Basin Societies, Honolulu, Dec 1989, 02-406. R.W. Hann, Jr. and P.A. Jensen, Water Quality Characteristics of H a z a r d o u s Materials, National Technical Information Service, Springfield, 1977. I. Moriguchi, S. Hirono, Y. Matsushita, Q. Liu, and I. Nakagome, Chem, Pharm. Bull., 40 (1992) 9 3 0 - 9 3 4 I. Moriguchi, S. Hirono, Q. Liu, and I. Nakagome, Quant. Struct.-Act. Relat., in press.

SECTION 111: Traditional QSAR and Drug Design.

This Page Intentionally Left Blank

QSAR and Drug Design - New Developments and Applications T. Fujita, editor 9 1995 Elsevier Science B.V. All rights reserved

STRUCTURE-ACTIVITY DEVELOPMENT ZONGRU

OF

DRUG

RELATIONSHIPS CANDIDATES

IN FROM

MEDICINAL LEAD

299

CHEMISTRY:

COMPOUNDS

GUO

Department of Synthetic Medicinal Chemistry Institute of Materia Medica Chinese Academy of Medical Sciences Beijing 100050, China

ABSTRACT

Three illustrative examples are presented from our own approaches to development of drug candidates based on structureactivity relationship analyses. Starting from a lead, 3-(S)-nbutyl-phthalide, an anticonvulsive principle, isolated from Chinese celery seeds, a number of congeners were synthesized and tested. Quantitative structure-activity relationship (QSAR) analyses revealed that the potency depends upon the configuration of substituents around the chiral center at the 3-position, in addition to the hydrophobicity and electronic structure of compounds, and the polarity of substituents in the aromatic moiety. R S - 3 - n - B u t y l - 6 - a m i n o - p h t h a l i d e was selected as a candidate for antiepileptic drug. A series of m o d i f i c a t i o n s of an a n t i p e p t i c - u l c e r prototype, furazolidone, led to deduction of a lead skeleton. A series of related (hetero)aromatic aldehyde (thio)semicarbazones were synthesized and tested. The QSAR analyses showed that the activity is significantly dependent on the electron density of the (thio)carbonyl group, which is enhanced by the introduction of e l e c t r o n - d o n a t i n g substituents. Hepatop r o t e c t i v e biphenyl compounds with structures simplified from that of an active principle of a Chinese traditional medicine, were investigated by the analysis of structural parameters obtained from their X-ray crystallography and UV spectrum. The structural requirements for activity were suggested from their c o n f i g u r a t i o n and conformation.

300 i.

INTRODUCTION

The

core

of

medicinal

chemistry

structure-activity

relationships

of drugs.

basis

On

the

is

(SAR)

of the

the

investigation

and mode

elucidation

of actions

of

rational design of drugs

involves careful deductions new

chance

search

findings,

attention

and

for

drugs

is

still

drug

generation closely

on

the

design and

momentum.

processes

may

Drug

lead

design

it

of

new

structural

at

of

candi-

drug

into two phases:

occur

in stages

involves

type(s)

which

important

will

re-

lead

but

are

the discovery with

or

biological

A lead compound per se is not neces-

inselectivity,

is the

modifications

upon

strives

for clinical use owing to pharmacological

with weak potency, But,

They

Lead generation

activity as lead compounds.

ses.

development

divided

optimization.

derivation

sarily suitable

of

be roughly

interrelated.

conceptual

based

although rational design is also receiving great

gathering

Depending

the

and thorough

largely

placing the haphazard practice of trial and error. dates,

(MOA)

SAR and MOA,

praxis.

The

of

lead

and/or

prototype

to,

certain adverse for

further

hopefully,

the

gence of new drugs of high efficacy and safety.

effects

respon-

structural

ultimate

Thus,

emer-

it is seen

that lead generation serves as a starting point to be followed by lead optimization. Although

believed

the

not

to

methodologies

be

available

accurate

methods

and

in

perfect,

summed up in medicinal

is

comparatively principal

our

Institute.

less

amenable

methods

of

lead

to

a

their

origin

Chinese

The

predictions

generation

in

medicine

natural and

country-side

locations,

unexpected

activities

Detailed and

discussion

optimization

relationships.

compounds

being

isolates

from

folklore as

well

of

will as

and

be

well

practice as

delt herbs

serendipitous drugs

devoted

to

aspects

present

examples

with of

here

from have

traditional to

certain

observation

currently

structural of

however,

at

restricted

certain as

and

guidelines.

exercised

introduce a few illustrative lead

are

principles

Lead generation,

still remain at the level of mass screening. This chapter will

optimization

few

chemistry can often be used,

sometimes with moderate effectiveness.

The

lead

in

of

use.

modification

structure-activity

301 2.

PHTHALIDE

DERIVATIVES

WITH

The g r e e n v e g e t a b l e lore

medicine

Prolonged blood

pressure

exhibit and

time

of

of

isolated

from

treat

induced

choline

as a folk-

hypertension

patients

of the plant, effects.

in China

decoctions to

may

some

celery

diseases. reduce

by

was

observed

sodium

and t r e m o r i n e

In

a

found to (I),

to p r o t e c t

glutamate,

as well

the

extent.

seeds w e r e

3-(S)-n-Butylphthalide

from the seeds,

seizures

by acetyl

to

celery

hypertensive

spasmolytic

liquid rats

(1,2) .

long

investigation

some

induced

a

ACTIVITY

c e l e r y has b e e n u s e d

administration

systematic oily

for

ANTICONVULSIVE

and

an

mice

spasms

as e l e c t r i c

shock

benzo-r-lactone

ske-

H

~]~'"' C4H9-n 0 Being leton,

new

tionships,

structural

I was

to p r o b e

is well drug

considerably

to

to

that

upon

the

the

alkyl

aryl

radicals

series,

and hence,

Fourteen luated

in

electric biological

replacement

terms

of

I.

the

Their are

listed

and of

n-butyl

phthalides

protection

(MES).

activities

the c o m p o u n d

action

alter

of

as

to

the

from the

the

the

It was

some

potency.

synthesized

mice

against

physico-chemical in T a b l e

1 along

depend

drug. by

hydrophobicity

were

site

interaction

receptor

group

synrela-

as to de-

of a drug

property

of

so

as well

complementary

the a n t i - c o n v u l s i v e

3-substituted

shock

of

their

would

compound

structure-activity

transport

hydrophobic

that

lead the

of action,

site

and

a

agents.

the

the

molecules

envisaged or

as the

the m e c h a n i s m

known

with

analyze

anti-epileptic

administration

between

type

chosen

compounds,

potential

It of

new

compound

thesize velop

a

(1)

other of

and eva-

the

maximum

parameters with

the

those

and of

302 Table

1

Structure,

activity,

and properties

of

RI

3-substituted

phthalides.

R2

0 No.

R1

1 2 3 4 5 6 I 7 8 9 i0 Ii 12 13 14

R2

H H Me H OMe H OH H i-Pr H n-Bu H (S)-n-Bu H c-Hex H n-Hex H n-Oct H - C H 2 C H 2 C H 2 C H 2Me Me NHCONH 2 H CH2Ph H Ph H

The

quantitative

compounds

were

an e q u a t i o n

log

I/ED50

n = 14

=

Log

P

following dence

represents

of

equations,

compounds,

of F

the

the

residuals

from

R2=R3=H )

2.79 3.08 2.90 2.65 3.29 3.35

P-

of

of

p) 2 +

these

related

2.56(0.19) [i]

In

are

coefficient, coefficient, variances

P being

equation

parentheses

correlation

fit

free-energy

hydrophobicity,

regression ratio

of

follows:

coefficient. in

0.i0 0.i0 0.i0 0.i0 0.i0 0.i0 0.i0 0.i0 0.i0 0.i0 0.i0 0.I0 0.I0 0.i0 0.i0

= 6.21

molecular

the

relationships

0 . i 0 ( 0 . 0 5 ) (log

F2,11

0.80 1.41 1.00 0.57 2.10 2.80 2.80 3.34 3.55 3.94 2.56 1.73 0.49 3.12 2.64

3.30 3.26 3.14 3.34 3.19 2.59 3.33 3.35

linear as

P MR 6

1

the n

is

the

95%

confi-

the

number

s is t h e

between

the

and

standard

observed

and

values.

Obviously, king

is

the

obtained

figures

each

r is t h e

and

calculated

mainly

the

partition

interval

deviation

s = 0.19

log

c a l c d . (eq. 2)

3.25(2.87) 2.94 2.92 3.12(2.73) 3.20 3.42 3.03 3.31 3.22 3.10 3.23 3.28 2.49 3.46 3.37

using

was

0.57(0.21)iog

l-octanol/water

obsd.

H H H H H H H H H H H H H H H

studied

r = 0.73

I / E D 5 0 (mol/kg)

structure-activity

model,

and

log

R3

two

and

of t h e

compounds-

equation data, the

the

1 is

not

deviation

unsubstituted

3-hydroxy-phthalide

satisfactory.

(No.4:

was

found

phthalide

RI=OH ,

Checto

(No.l:

R2=R3=H ) .

stem RI= This

303 was

rationalized

thesized

in

chiral of

this

center

fact,

by

the

sample

No.l

ED50

=

is

presumably

compound,

arguments.

racemic

0.38

This

than

the

atom

of c o n f i g u r a t i o n a l

presence

natural

in

is p r e s e n t ,

and

3-Hydroxy-phthalide form of p h t h a l d e h y d i c

there

isomer the

of

librate

the

are

Using

log

The

2 was

sites.

shows

the

P and

From

the

above

vulsive

activity

related

to

the

no

the

enan-

in fact,

is c a p a b l e

of

a

term, for

it

to

ca-

values

is

and

[2] rere-

receptor

of the hyd-

of the

compounds.

variance Figure

1

the h y d r o p h o b i c i t y .

apparent

that

phthalides

is

coefficient

1 was

chirality

a measure

90 p e r c e n t

upon

= 2.70

equation

molecules as

analysis,

p) 2 + 2.23(0.10)

specific

fourteen

3-substituted

used

rectified

log P(opt)

phthalide

the

in

its r a c e m a t e

and

regression

2 over

that

account

results,

The

interact

i.

of e q u a t i o n

for

4.

= 52.36

of the p o t e n c y

partition

and

and

difference

(I) and

multiple

squared

the

calculated

0.15(0.02)(log

F2,11

between its

data

the d e p e n d e n c e

the

suggestion

compounds,

biological

be

of No.l

P-

From

lead c o m p o u n d

in T a b l e

improvement

log of

In

0

"accumulate"

form.

could

for

s = 0.09

is o p e r a t i v e

The

the

data

obtained:

support

rophobicity of

the

factor

~---~-

may

active

indices

new

significant

cognition

therefore, the

= 0.81(0.11)log

to

uncom-

OH

~"~'/\ C O O H

in p a r e n t h e s e s

r = 0.95

garded

-

CHO

between

activity the

I/ED50

n = 14

..

rectifying

included

equation

..

as

log(i/ED50 ) values a

_

No.4,

receptor

(No.6),

the

H

0

R

is,

and

(ED50

the

is

(No.4)

a

equilibration:

'"'OH

The

that body.

acid,

of

a matter synthetic

e.,

the

syn-

S-form

i.

H

with

As

suggested

chiral

the h e m i a c e t a l - l a c t o n e

the

S-form,

stereoselectively

no

compounds

resolved.

behave

discrimination.

The

of the c o r r e s p o n d i n g

mmol/kg).

active

to

not

I as the

than that

more

owing

and

of c o m p o u n d

is l o w e r

R-form

tiomeric

were

3-position

activity

(No.6,

pounds

following

study

at the

= 0.93 m m o l / k g ) natural

the

with

the

the

anticon-

parabolically

optimal

value

at

304 log on

P = 2.70, the

which

central

teraction

falls

nervous

plays

an

in t h e u s u a l

system

important

/ED50

(3).

role

range

for c o m p o u n d s

Furthermore,

in t h e

the

acting

chiral

in-

activity.

Bu _.ez

3.4

e ) ? . . P ~ c -Hex

(M

! "/:"

3.2

.\o-.o.

io7

5.0

\ .oo,

2.8 2.6

R

/Ureido

2.4

I[0 Figure

i.

Diagram

and

log

In

order

to

activity,

a

sized, of

and

further series

the

3 and

I/ED50

n = 25 log

according

the

explore

I/ED50

relationship

and

between

the

activity

2.

the

listed

effects

of

substituents

phthalides

in T a b l e

2.

For

3,6-di-substituted

on

were

the

synthe-

whole

series

phthalides,

equa-

P-

s - 0.28

= 0.84(0.21)iog

0.18(0.05)(log

F2,22

p) 2 + 2 . 0 8 ( 0 . 2 5 ) [3]

= 14.54

P - 0.14(0.04)(log

p) 2 + 0 . 3 6 ( 0 . 1 2 ) M R 6

+ 2.16(0.22) n = 25 In

r = 0.84

equation

tuent

at

parabolic variance calculated

4,

terms in

the from

3,6-disubstituted

[4]

s = 0.24 MR 6

position

the

obtained-

= 1.02(0.24)iog

r = 0.75

logP

' 4.0

3,6-disubstituted

are

3-mono-

4 were

' 3.0

to equation

of

data

twenty-five

tions log

P,

showing

210

is

the

6 scaled of

log

activity equation

F3,21

= 16.23

molar

refractivity

by

P

0.i.

can (r 2 3,

phthalides

As

only =

the was

log

seen

for

Examining

experimental higher

of

from

account

0.56).

P(opt)

than

= 3.00

the

R3

equation about the

activity that

substi3, t h e

half

the

residuals of

calculated.

most

305 Table

2

Structure,

R
activity,

and

phthalides.

properties

of

3,6-disubstituted

RI

0

No.

R1

R2

15 16 17 18 19 20 21 22 23 24 25

n-Bu n-Bu n-Bu n-Bu n-Bu n-Bu n-Bu n-Bu Et n-Hex Me

H H H H H H H H H H Me

This

suggests

ring

tional R3

the

defining

significance

MR6,

equation

ance.

The

plies

the

steric

is

polarity

exhibits

an

of MR

be

The from

equation

2 and

Figure

improved

log

for

of t h e

~ log

70

to

to

be

aromatic use

effects P,

of

r

that

p) 2,

0.91

and

the

vari-

in e q u a t i o n

4 im-

operative, by

since

the

a

negative

3-n-butyl-6-amino-phthalide

(No.19)

activity

between is

as

the

compared

measured

large

compound the

as

as

an

to

value

0.82

log

outlier

correlation

of t h e

= 0.74(0.13)iog =

the

improve

the and

other

conge-

that

unit,

as

calcuseen

as

variance P-

from

shown

in t h e

the

analysis,

in

in e q u a t i o n

s

=

0.15

we

5, w h i c h

activity-

0 . 1 3 ( 0 . 0 3 ) (log

p) 2 + 0 . 3 5 ( 0 . 0 8 ) M R 6

+ 2.23(0.14) n = 24

addiof

to

(log

percent

usually

0.74 0.60 0.80 0.29 0.54 1.49 0.98 0.63 0.54 0.54 1.56

the

failed

MR 6 term

verified

MR 6

2.

this

83 p e r c e n t

I/ED50

electronic With

account

is

into

Attempts

Hammett

C6-substituents

4

materially

describes

to

high

difference

Dropping

and

P

term.

unusually

lated

potency.

E s and

log 2.92 3.53 4.24 3.12 2.86 2.77 2.25 2.39 2.29 3.65 2.42

substituents

correlation.

noted

ners. Table

the

effect

of t h e

should

3.65 3.54 3.41 3.48 3.58 3.91 3.67 3.57 3.52 3.50 3.91

coefficient

hindrance

It

3.69 3.77 3.53 3.56 4.40 4.01 3.71 3.59 3.41 3.17 3.50

steric

Taft

able

positive

coefficient

as

of

4

c a l c d . (Eq. 4)

introducing

such

I / E D 5 0 (mol/kg)

obsd.

anticonvulsive

parameters

substituent,

the

NO 2 C1 Br OH NH 2 NHAc CONH 2 CN NH 2 NH 2 NMe 2

that

augments

log

R3

[5] F3,20

=

33.12

log

P(opt)

=

2.85

306 Although activity group

no d i r e c t

of c o m p o u n d

is b e n e f i c i a l

nisms

(e.g.

animals) and/or

its

or

No.19

is a v a i l a b l e ,

m a y be

for a c t i v e

due

the

pK a

is

value

the u n u s u a l l y

to the

absorption

bioavailability

that

binding

evidence

81

is

fact

and/or

that

the

transport

percent

in

favorable

high amino

mecha-

experimental

for

the

delivery

processes.

LOG ED 50

4.

,

3-n-Bu-6-NHz ( No. 19 )

3

i.1

.,,

-.:~---~ _~

I-."F'"I'.! . . . . .""~-.-L|,! J-.i..*'

m'~:'~: ~c_......;.:-:~-~.... ~ . ' , 1 " . . .

!",..,21

z.6 l ~:.~.,!:",.::<~-.-.I:~.;:::,~,.-!..';~ ~...~- ~

..~.<.'.. . / . . . ~

~.".:." , 7 7 "

2", .2/

,I

,

,

'

,~

I

I

'

!

J._--J:,'~- ~ . - ' ~ . - - . ~ i!

I

~

"....'" . -." ~,~ /

9

.. i

,:

~

LOG P

/

!

'

/

I

/

i I

MR-6

Figure

2.

Diagram

a n d log P, a s Using

the

phthalides regression the b e s t

I/ED50

=-

wherein

FO 1

sities

(HOMO)

6-position,

and

orbital we

with

that

the

for

was

with

standard

electronic

similar the

activity

4.

CNDO/2,

the

the

log

activity

P vs

indices

values. the

of

F2,11

FC 6 w e r e

defined

the

carbonyl

densities

frontier

In

suggest at

electron

of From

electronic

computed-

s = 0.27

respectively.

electron

approach,

closely

analyses

between

to e q u a t i o n

calculated

equation

coefficients

tier

relationship

2 . 8 2 6 F O 1 + 5 . 7 4 6 F C 6 + 3.398

r = 0.84

of two

according

(4),

indices,

n =14

as MR6,

molecular

multiple

log

the

well

parameterizations fourteen

showing

[6]

= 12.76 as

the

frontier

oxygen

and

the

this

equation,

opposing

effects

these

two

sites.

theory

has

been

electron

carbon the

by the

is

developed

well to

den-

at

opposite

exerted It

atom

the

signs fronknown

explain

307 the

difference

molecule

(5).

position tals.

with

in

reactivity

at

The

reaction

should

the

Thus,

largest

there

are

for the b i n d i n g

tions

of the p h t h a l i d e s .

ever,

the

=

values

The

electronic

for the s t r o n g tive

sign)

molecule the sive

being

that

the

where

the

the

§

-

i,,..........~ ,

Figure The

~

', "

6-amino

to

a

as i l l u s t r a t e d

the

and

low

candidates

for

Meanwhile,

resolved

to

that

the

each

R-form

appro-

space part

sign) space

as

for

6-substituted

remaining

area,

FC 6

rationalization

of the

the

values

(negaof

except is

shown

so for

the for

extenthe

6-

~

+s

§

",~

X

++

+4. ++ .......

9 ~§ +,~L' "

, "ill

'h

. 2 o , ;:''

i

~~h.+'ll,,.,'""

~ C ; ~+~i: § ~ ~ O ..... '~<'.';i":',"'"'"'"' ,,,,t' / ~ 5 .... 111111" 4-4.+-Jr F{'I""~ t llllllll till'

of

IIi'i,, ,~++++ '""'I"I'"~ ,l ~ , i;'.I, i ! I, '" ,t, tI! i ''''l,"|'l,f'",l ,,,, ,|,

No.19,

smaller

however, region,

in Figure

adverse

trials.

further

(positive

the the

with has

treatment

of

its pure

is

more

of

electron-

the

lactone

anticonvulsive

selected

epileptics

mixture than

high

been

enantiomers.

active

the

from

4.

response, racemic

localizes

isolated

3-(RS)-n-Butyl-6-amino-phthalide, activity

possesses

at

How-

c o n t o u r m a p of 3 - n - b u t y l - 6 - c h l o r o - p h t h a l i d e .

compound

area

density

different

For m o s t

~.++,.,,'~'i,,'"lil,u

litI lllqliI lilt il I ",,,,t|,,,,,t l' ,,i,"

3. E l e c t r o n i c

repelling space,

:

two posi-

3).

~ . :+~. ~+++-c~ .................++ ..

orbi-

require-

potency.

electron-repelling

lactone

+.4.~++++++

~++r

the

~++ + + + +++. ++ ++ +I+ ~++ + 4-++ t ~ ~/.,+ ++ § +

-§ ++-I" +

No.19

quite

moiety,

(Figure

these

the

electron-repelling

with

of No.16

with

at

frontier

in the e l e c t r o n

provided

a large

lactone

aromatic

at O 1 and C 6 (FO 1 = 0.322,

have

map

an occur

electronic

increase

electron-withdrawing

it m e r g e s

analog

to

of No.19.

exists

around

6-position,

chloro

contour

in

in the

receptor

that c o m p o u n d

compounds

activity

there

density

increase

O 1 seem

of d e n s i t i e s

other

the two atoms.

phthalides,

The

at

position

preferentially different

of the

look r e v e a l e d

equal

0.324). The

sites

decrease

a closer

ximately

electron

presumably

ments C 6 and

each

the

in

of No.19

was

Animal

tests

RS-racemate,

as

one

of

clinical chemically indicated which

in

308 turn

shows

with

the

stronger potency than the S-form.

above

antiepileptic

discussion

activity

and

offers

of phthalides

This

additional

is consistent

proof

is dependent

that

upon

the

the con-

figuration. +. ...... +: ++/ ++

~++.-+$. ++§ +

.+

%

*~,+

+

: "r "'" F . ~ , : ~ ~<;r,"::,,.',~',:,i'" ",~~,;~,,,;,'.',':,,.,, ".~,+ +./ ~ /..r.. ..... ' ,I' .#* ' % ,I ',

~+ I|1

""

III

,

§ .@~ ~'-~ *j. )4= Iill lITI ~ t.~ .4

, I i I '~+ - .

ii,.,Ii,!

r . i - )~ +

§ 9

~ **

~'~

I "11 O II I IIII

.,,..I,1 "" | I'1 ' I ~!

I

--

~"* I~ I,I I

'"

Figure 4. Electronic contour map of 3-n-butyl-6-amino-phthalide.

3.

ANTIPEPTIC

ULCER

HETEROCYCLIC

SEMICARBAZONES

Medical doctors may accidentally discover unexpected and new actions

of

diseases. is

drugs

generally

tivity. the

for

clinical

suitable

this

kind

medicinal

uncovered

practice

application

not

But,

vistas

in

Direct

of

because

One

to

of

(II), which was serendipitously

and duodenal

of

the

to the

the

fortuitous

chemists

activity.

during

of the drug

treatment

new

inherently

finding usually

design

the

novel

examples

low

selec-

opens

structures

was

of

indication

new

with

furazolidone

found to be effective

for gastric

diseases when it was used in China for the treatment

of infections.

Afterwards, investigated healing

a

percent)

year

higher

54.7%

cases

the

period.

cer models

(33.3

than

and

gastric

study,

treated

percent)

in

Pharmacological

only

(6,7).

duodenal

The

ulcer

two weeks was

cimetidine

cases, the

clinically and

p.o.)for

with

for cimetidine,

follow-up

furazolidone

one

treating

(0.053 mmol/kg/day,

against 2-4

effect of furazolidone has been

experimentally

after

significantly

lidone In

both

rate

furazolidone be

the antiulcerous

found to

for

furazo-

(0.048 mmol/kg/day,

p.o.)].

four

out

while

control

experiments

[71.4%

with

of

seven

group,

fifty-two out

of

relapsed

(7.7

twentyduring

showed that gastric ul-

in rats induced by indomethacin,

pyloric

ligation,

and

309 acetic

acid are s i g n i f i c a n t l y

protected

by the c o m p o u n d

II.

. / • •CH=NN------!

O"J~O,,'J(]I)

OzN" The

well-known

severely

would

limited

be

derive

its use

highly

a

arrive

new

at

some

the u n i q u e

side

effects to

skeleton

is the

the

substructure

nitrofuran

pyrrole, the

furan

structural

II

is

rings,

essential

done moiety,

as a cyclic urethan,

and

or by

tures,

and the oxygen atoms were

nitrogen

aromatic

thiosemicarbazides, then

condensed

formula

it was

action,

nitrobenzene,

examined

whether

The

oxazoli-

to linear struc-

r e p l a c e d by sulfur

The r e s u l t i n g and

to

retain

5-nitrofurfural

antiulcer

was s i m p l i f i e d

heterocyclic

and

the p h a r m a c o p h o r e

activity.

isosterically

aminoguanidines,

with

of

benzene,

and

still

it

to

lower toxicity.

for the by

to the

rings.

Therefore,

prototype,

which

product

replaced

thiophene

part

yet with

responsible

was

however,

modifications,

In order to a s c e r t a i n

system and

nitrofuran

of

II,

II as the

by o p t i m i z a t i o n s

condensation

with N - a m i n o o x a z o l i d o n e . or

compound

with

of the prototype,

Furazolidone

the

start

by

new c o m p o u n d s

actions

the

in g a s t r i c u l c e r therapy.

desirable

lead

of

acyl

semicarbazides,

hydrazides

carboxaldehydes,

as

were

shown

in

III:

X=O, S, NH, -CH=CH~X H . L CH=NNC-R u (111") Y The a n t i u l c e r o u s was

evaluated

A fixed

dose

rats

along

Half

an

with

hour

4.5

the a

activity

were

given

was

group

ulcerogenic

by

induced

compounds

and

the

which degree

the

(No. Er)

or by a s c o r i n g m e t h o d

(7).

tion

ulcer

formation

for

each

.~ synthesized

ulcer

given

agents

counting

of

hours

NH

of the newly

indomethacin

control

later,

S,

R=NH?_ _ ~ R

of the test

deoxycholate) after

by

Y=O,

The

group

models

orally

received of

no

lesions of

of

protection. and

was

erosions

for the

evaluated

sodium

assessed

gastric

indices were

in rats.

to groups

(indomethacin

number

compounds

inhibi-

according

310 to e q u a t i o n s

7 and 8.

Inhibition

Index

(No. Er)

=

Inhibition

Index

(score)

=

Toxicities

of c o m p o u n d s

relative

t o x i c i t y was

Relative

toxicity =

The

structure

bition

listed

in Table

Score (control)

in

the

effect,

ting

is

nitro not

different

The

increase

compounds,

The benzene

3, we can see that,

the o-, m-, vity, the

toxicity

between

separation

of

and

between

50%)

when

for

No.

the ~ furan

[9].

[9]

parameters,

and more

of the b e n z e n e goes

down.

toxicity

seems

for the activity.

was

almost no change

the

This

indicated

antibacterial

the

two

seems

toxicity. is

sugges-

activities. due

to

the

replaced No.

than d o u b l e d of a nitro

2,

by

with

toxicity group

at

ring raises the acti-

against The

if n i t r o f u r a n

compound

Introduction

competitive

are

activity,

1

inhi-

and t o x i c i t y

ring

especially

of these two responses.

be i n d i s p e n s a b l e

to

antiulcerous

in a c t i v i t y

distinctly

furazolidone.

critical

action

benzene,

not

(No.l),

a strong systemic

furazolidone.

still

activity

the

(ca.

decreases

or p - p o s i t i o n

although

is

for

that causes

decrease with

from

which

toxicity

substituted

compared

furan ring

place

mechanisms

activity or

a seven-fold as

group,

their physical

to that of furazolidone,

in Table

took

high s o l u b i l i t y

a c c o r d i n g to e q u a t i o n

The

LD50 of test c o m p o u n d ( m m o l / k g )

necessary

in

in mice.

LD50 of f u r a z o l i d o n e ( m m o l / k g )

3 (8).

activity

the

[8]

Score(control)

r e p l a c e d by the u n s u b s t i t u t e d that

[7]

- Score (test)

were e x p r e s s e d by LD50

relative

From the data

- No. Er(test)

No. Er (control)

calculated

of the

indices

No. Er(control)

furazolidone.

absence

to p r o v i d e

of

But,

parallelism

a possibility

The furan ring is u n l i k e l y

for to

311 Table

3

activity,

Structure,

and

toxicity

~(CH=CH)-CH=NN

of

X

Y

II

1 O 2 CH=CH 3 CH=CH,o-NO 4 CH=CH,m-NO 5 CH=CH,p-NO 6 NMe 7 S 8 NH 9 O i0 O ii NH 12 NMe 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 a) b) c) d)

O S O NH S S 0 NH S O NH O O 0 NH NMe

NH 2 NH 2 NH 2 NH 2 NH 2 NH 2 Ph Ph Ph 3-NO2Ph 3-NO2Ph 4-Pyr d 3-NHAcPh OBz OBz OBz

effects

activity

existence mental

of

of

1.08 0.14 0.64 0.91 0.73 0.83 _ b 1.00 1.09 0.93 0 93 0 96

4.44 2.89 9.36 12.71 21.28 3.12 8.16 2.63 0.61 4.33 1.81 1.72

1.50 2.30 0.71 0.52 0.31 2.04 0.78 2.43 10.46 1.52 3.65 3.87

_ b _ b

1.12 3.55

_ b

1.19 i . 22 0.55 2.45 2.82 15.22 7.72 7.72 4.65 3.69 5.12 4.12 2.92

5.95 1.88 13.06 5.60 ii. 70 2.78 2.78 2.36 0.44 0.86 0.86 1.37 1.80 1.30 1.61 2.27

_

b

_

b

are a

similar

heteroatom

prerequisite

for

to

that

0.51

_ b -0.45 0.45 0.45 -0.40 -0.09 -0.70 -1.65 0.75 0.88 1.06

thiophene of

Rel. Tox.

b _ b _ b _ b _ b _ b _ b _ b _ b _ b _ b _ b _

1.26 1.14 1.42 1.37 1.18 1.44 -0.98 0.62 0.70 -0.76 -0.08 -0.96 -2.84 0.44 0.94 1.03

pyrrole,

LD50 (mmol/kg)

Score

The value relative to the inhibition of A negative value means that the erosion the control group. Not tested. Piperidinylmethyl. 4-Pyridinyl. The

the

0 0 S S S NH 0 O O O O O O O O O

Index a

No. E r

0 H 0 H 0 H 0 H 0 H 0 H 0 H 0 H 1 H 0 CH2N(CH2)5c c 0 CH2N(CH2) 5 0 CH2N(CH 2)5c

2 2 2

(No. 13-28) Y

Inhibition

R

compounds.

H CH=NNC-R

i R

(No I~2Y) 0 ~ 0 j

No

twenty-eight

furazolidone is g i v e n . is m o r e s e v e r e t h a n in

and furan,

N-methyl

pyrrole

indicating

within

the

aromatic

producing

the

antiulcer

ring

is

action.

that a

on the

fundaInser-

312 tion

of

a

toxicity

vinyl

oxazolidone cule,

fragment

(No.9).

but

into

Introduction

raises with

the

no

furfural

causes

an

increase

of a p i p e r i d i n y l m e t h y l

basicity

and n u c l e o p h i l i c i t y

significant

change

in

both

group

of onto

of the mole-

the

activity

and

isosterically

changing

the

toxicity. Opening oxygen several

of

with of

(No.18)

of

heterocyclic

to

that

No.

26-28

carbonyl in

No.

what

(Table group

cou l d

chain

Table

can

seen

3,

it

aldehydes parent

be

still

not s i g n i f i c a n t l y

If

one

of

the

is s u b s t i t u t e d

by a phenyl

the

are

compounds

these

devoid

the

following

atoms

com-

of

these

Compounds also

show

flanking

or p y r i d i n y l

of

on the a r o m a t i c

results

activity

toxicity

hydrazones,

hetero

From

semicarba-

decrease.

the b e n z y l o x y c a r b o n y l

with

and a m i n o g u a n i d e

retain The

carbon,

moiety.

that

(No.15-17),

compound.

and

synthesized

cyclic

does

3),

were

of the

of s u b s t i t u e n t s

From

side

nitrogen

significance

the

activity.

19-25,

kind

of

however,

reasonable

sulfur,

thiosemicarbazones

parable

substances,

and

like

a linear

the

data

(No.13,14),

ring

elements

deciding

biological

zones

oxazolidone

into

compounds

a view the

the

atoms

activity,

group,

no

the

as

matter

ring. preliminary

conclusions

be drawn: i. The cyclic moiety, activity

group

and

its c o u n t e r p a r t s

vity.

Electron

2. The p r e s e n c e

atoms

tant 3.

oxazolidone,

antiulcer

of h e t e r o a t o m s flow

to the C=O,

factor

stituent The g e n e r a l

from the

C=S or C = N H

or b e n z e n e

are crucial

formula

on both

sides

lone pairs group

ring

seems

for the

of the carbonyl

is a p r e r e q u i s i t e

for the activity.

A heteroaromatic

is not crucial

of furazolidone.

for acti-

on these

hetero-

to be an impor-

carrying

a polar

sub-

to the activity.

IV r e p r e s e n t s

the situation,

Y IV) where

X,

indicated The

Y,

R

strategy

concomitant the

and

positions

stand for

optimization

pharmacokinetic

for

changeable

of the skeleton. the

modification

atoms is

to

or

have,

of both the p h a r m a c o d y n a m i c

property.

In

reality,

groups

at

the

hopefully, behavior

a compromise

and

between

313 the

two

has

geners

were

4.

being

the

tition

The

and

42) of

the

carbazones

(Y=O),

Using tative

of

measured

hydrophobic variable

R as

was

zero

are

as

log

Ascor e = 0.05(0.05)Iog 15

log

Ascor e = 0.48(0.14)iog

n

15

=

r

=

0.72

s = 0.23

s

=

0.17

log

Ascor e = 0.54(0.13)iog

n -

15

-

r =

s =

0.16

- 0.32(0.13) ~ 15

r = 0.87

Similarly,

for

the

the

are

by

log

multiplied

by

compounds were

from

for

relationships

The

par-

ii,

method

in

the

using

the

~ Leo

series

the

constant (i0); of

furan

the

quanti-

(thio)semicar-

[10]

P - 0.21(0.17) F1 PF2

P-

13 = 0 . 9 4 0.06(0.02)(log 12

=

p) 2 _ 0 . 8 9 ( 0 . 2 4 )

6.30

0.07(0.02)

[11]

(log

p) 2 +

0.16(0.09)I

log

Ascor e = 0.30(0.10)log

r = 0.48

[12] F3,11 P-

s =

0.ii

= 6.03

0 . 0 7 ( 0 . 0 1 ) (log

P-

= 7.92

s - 0.ii

+ 0.19(0.08)I

P-

[13] log

0.03(0.01)(log

F = 2,12

P(opt)

0.03(0.01)(log

= 3.43

p) 2 _ 0 . 4 3 ( 0 . 2 4 )

= 1.79

-0.75(0.21) r = 0.76

p)2

-0.93(0.23)

series-

15

an

semi-

follows:

pyrrole

Ascor e = 0.22(0.12)iog

16,

(Y=S).

analyses,

for

in

Ascor e

i,

and the

thiosemicarbazones regression

shown

i00.

(No.

Hammett

Hansch

unity

acti-

Ascore,

calculated

the

con-

partition

structure,

shaking-bottle

as

F4,10

log

15

and

parameters

(i0);

s = 0.13

n =

n =

The

here

others

quoted for

forty-five

1.13(0.26)

0.79

log Ascor e = 0.48(0.ii)iog n =

by

developing

n =

r = 0.26

(9).

parent

assigned

structure-activity 1-15)

(score) six

constant

was

stepwise

(No.

tested

So,

electronic

represented

index

n-octanol/water,

substituent

evaluation.

different

and

is

(P)

was

the

physico-chemical

activity

indicator

bazones

with

inhibition

substituent of

in

IV

coefficient

31,

system

made

designed

toxicity,

Table

26,

be

formula

characters vity,

to

of

[14] p) 2 +

0.19(0.06)I [15]

F3,11

= 5.05

C~. i~ . 0

O

O~

D~

0

gt~-

~m I-'-Q ~tO ~ 0 0

v v

~

O

0

I

I

I

O

O

O

O

O

O

O

I

0

0

I

0

I

I

I

O

I

I

0

I

0

I

r~ O

0

I

I

I

I

--.1 ~.) 00 O

I-~ O

0

I

I

I

00

~)

I

I

I~

I

t~

9

0

0

9

0

0

I--' [k) [k) 0 t"O -.,1 (.,.) O

9

9

9

9

0

(-,.) '~

9

U'I ,~::~, t ~

9

0 0 0 0 0

~

--

9

9

9

I

9

I

9

9

9

t~

9

9

9

0 0 0 0 0 0 0 0 0

9

9

U'I (..,.) O~ ~D - q

9

9

9

I

9

~D I~

9

r,.o 0

I-~ l-J I-~ l-J I-~ I-~ I-~ l-J I-~ I-~

I--'~ (.~) ".1 b.) (J1 IX.) tk) tX.) 0 lO"t~ IX.) 0~ t ~ -.~1 (..,.) (..,.) - . . 1 0

I

9

O"LD

I

I-~ O~

O"(.rl O L D

I

I

0 0 0

I

(..O (..,.) (..,.) (.O (..O U'I , ~ (.,,.) t ~ I~

(-.) t',.) I--' I-,, t'O I-~ b.) ,;~ U'I tk) I--' I--' Co O~ O~ I--' LYl [%.) 0

I

I

0

O,~

I

I

-~1 IX.) I ~

0

O

00 O~ - q

I

O

(,0 L.O (.,.) ~ LD 00 ~ 1 0 ~

~ I I I I ~,o I b.) 0 ~ 0 [,~.,~.,~'.~ I ~ I (~0('~01 I t ~(~ ~ OOO ~ , ~ t~(~ O ~

O

(..,.)t~ I ~

0

CO,l~ I I ~ O 0 1 ~ ~(~

~

~ 4~, ~t:~ ~::~ ~:~ , ~ L~ ~,, (..O ~ I ~ O

~

I

~

O

0

o

o

I

I

o

O

0

I

0

I

o

9

9

9

o

I

o

o

I-, ...1

I

o

0

9

O

o

ko

O

0

0

O

I

O

I

0

o

0

I

0

I~ t~ O

0

I

[~) O O~ O

o

~

0

o

O

I~ .~

9

9

o

9

9

9

-.1 s

9

0

I

0

I

0

O'~ I ~

9

0

0

0

9

t',O

0

0

0

0

0

0

0

I

0

0

I

0

0

0

I

I-~ I-~ i-~ i-~ ~-~ I-~ I-~ I-~ I-~ I-~

(.~ I--, (.,,) ...l U.) ( . ; I [ k ) [~0 t%) 0 -..1 t%) t%) O0 t',O -..1 Co (...) -.] 0

0

~:~, (..~ - q ~:~ I ~ 00 I.~ ~::, t~O ..q ~:~. O~

I-, (.0 [k) t',O 0 t'~O t',O -..1 (...) 0

0

I~

O

~) ~)

I.-~ O

O

O O

(1)

t"O -.11--'

9

t ~ (.,.) U'I . ~ I ~ ~D I-~ O

0

t',.) , ~ O0 O0 U 1 0 0

9

i~

O

r~) 0'~ (..O ~:::, ( ~ (..O [~) O~ ~ ) ~ . I ~ r~O (..0 t ~

O0 ,4:~ , ~

I

0

I~ I~ O

0

L.O .,..] ~:~. O

I.~ t~O O

0

O

,;~ ,;:~ ~ ,;~ I I I I

O

I

o

O

L~)~ I I O ~ i ~ O 0 ~ ~ ('~ 0 ( ~ 0("~ 0 I I I ~ o o ~ ~ ~0(~ 0 ~

O

I

I

O

O O O O O I~ O ~D O'~ ~::~. I ~ ~:~, (..,.) O

o

I ('~ ~ ~

O

~-~ r ~ O O~ I ~ O

I

o

(1) Ix.)

II

I ~ 0 Ot~ ~ ~(~(I)

0,.~ bJ('~ I ~

~

(..,.) L~) t ~ t ~ t~) t ~ t'~) [~) t~O [~.) t ~ I ~ t ~ I ~ I ~ O ~D 00 ..~10~ U'I ,1:~. L,.) L~ I ~ O ~:) 00 - . . 1 0 ~

~

~

I

~

0

O

0

0

O

0

O

0

O

0

O

0

~ ~

O

0

0

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0

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0

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0

O

0

~D 00 -.,10'~ U~I , ~

0

0

I

I

0

0

0

0 O I~

0

0

I

0

I

0

I~

9

O

9

9

9

[,O I~

O

9

9

I-~

I-~ 0

O

t~

O~ [~O O

9

I

O

9

9

9

I~ 9

9

(J11-~ I-~ -~I O 0 0 ~

I~

0

O

9

I~ 9

O~ [~)

I~

I ~ I~ (..O O~

I~

O~ ~ , I-~ (..O t ~

O

-q

..~10

O~ ~::~ O

~,

t~

0

0

0

0

0

t~ t~ (~ -..l 0

[~O

[~O

~-~ I-~ I-~ I-~ I-~ I-~ I-~ I-~ i-~ I-~

-..l t~ [~) t~ --.l O0 (.~) b~ --.l 0

I

00 t~O O

(..,.) (..O t ~

I I I I 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

I~

(..O (..,~1 (.O (.,.) (..,.) (.,.) t ~

4:~ bJ- U'I ~:~, h.) h.) L.O L.O kD -..] ~::~ O0 O0 L.,'Ih.) U'I 0'~ I.~ U-I ~I:~ O0 I--~ kO O0 CO h.) -..I I--~ O0 O0

9

O

-~I -~I 0

I~

t~) I ~

~) 0

~

I

O O'~ ~ O O (..O ...,1U~I (.,.) O

0

I I I I I I I 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

(..,,) O O O O I ~ I ~ (.yl ~ ) 00 LD . ~

I

O O

0

0

0

I

(1) bo

I

(.~) t ~

IL~ I I I 1 0 0 0 ~ I I I O O O 0 ~ O O O O 0 ~ O 0 ~ ~ ~ ~ o o ~ O~ ~

0 (~

0

0

I ~ I ~ I.~ I-~ I ~ I~ (31 ,~::~ L.O ~ ) I ~ O

11)

0

0

ft"

g

(1) rl" (1)

"el

0 I

I-,.

m'

~

~-~

0

I~

315 log A s c o r e = 0 . 2 7 ( 0 . i 0 ) i o g n = 15

r = 0.79

furan

and

have

the

antiulcerous

ted

to

the

activity

P,

R groups

consistent carbonyl

group

indicator

carbazones Only

than

data

pectively.

and

of

For lation

than

the

made

equation

analogous

to

also

the

structure-toxicity

pounds less

with

toxic;

portant

effects

Among dehyde

these

in

relationship

of

all of As

absorption

leads

to

con-

In a d d i t i o n , restricts

no

eva-

of

these

the

vari-

significant

the

however, for

the

for

the

coefficient steric

corre-

variations

in the

revealed

furan

correlation

examinations

and

res-

response. the

activity

This

describing

observed

partition

16,

range.

quantitative

electronic

in the expeand

activity

inevitably

pyrroles,

of

of

in semi-

administration

of m a x i m u m

indices.

examination,

Qualitative

lower the

N-methyl

is

(thio)

coefficient

13

of

after

antiulcerous dose.

which

the

thiosemicarbazones.

differences

activity

fixed

that

significant

congeners.

on

nega-

electron-

activities

equations

compounds the

capable

was five

a

of

16

rela-

The

activity,

measurement

to a n a r r o w

Qualitative no

test of

of

was

by

was

and

increasing

that

positive

higher

at the m o m e n t

in the at

series

the

neglect

the

activity

for

density

out at a fixed t i m e

comparison

was

activity. roughly

because

deviations

in the

of the

the

are d i f f e r e n t .

indicates

corresponding for

13

respectively).

The

for

parabolically

(i00 x r 2) of the v a r i a t i o n s

the of

ones

improvement

required

electronic

accounted

rather

fact,

and

compounds ations

62 %

is

term

signifies

of the

be is

distribution

siderable

4.50,

above.

term

carried

compounds

luation

presumed

can

was

matter

necessary

those

This

compounds a

as

No

being

electronic

variable

76 % and

rimental

test

the

best

= 4.50

Equations

series

favor the e n h a n c e m e n t

with

the

the c o e f f i c i e n t s

both

= 3.43,

the

are

parameters.

hydrophobicity

of

log P(opt)

respectively.

of

(log P(opt)

coefficient

donating

the

high

16

although

activity

[16]

= 4.22

and

other

same p a r a m e t e r s

The

tive

13

series,

introducing

log

F4,10

equations

pyrrole

by

- 0.65(0.22)

s = 0.I0

Obviously, reached

P - 0.03(0.01) (log p) 2 + 0 . 2 0 ( 0 . 0 6 ) I

0.12(0.11)0-

-

whole

series. to

generally do

trend There

account

set

indicated

factors

a

of

forty-

that

appear not

for com-

to

exert

be im-

on the t o x i c i t y . compounds,

two

substances,

N4-(p-methoxy-phenyl)-semicarbazone

pyrrole-2-carboxal(No.17)

and

-thiose-

316 micarbazone than

the

are

in

owes

(No.28)

others.

the

including

variable

of f u r t h e r 4.

the

judgement

discovery

research compounds

a tonic

in Chinese

Fructus

capable symptoms

hepatitis the

tion

with

less

toxic

studies

substitution

electron-donating compared

Although

lower

shown

its

than

by the

the

the

sign

low t o x i c i t y

to

other

thio-se

correspon-

of the

is still

indiworthy

from

new

things,

in disguise"

of

viral

of

especially damage

of

were

has

B.

carried tests C

been

out

Schizandra

develop

new

principles seven

indicated possess

that

as

It was

alleviating

and

the

prescribed time.

of

To

is

is the

anti-

from

dibenzo-

and c h a r a c t e r i z e d .

(V),

by carbon

in the h i s t o r y

for a long and

or

gratifying

examples

on the active

isolated

schizandrin

to

activity.

kernels

hepatitis

biological

caused

the

the

functions

studies were

deduction

lead

of

Baill.

medicine

liver

systematic

lignans

One

chinensis

Schizandra

results

incorrect

is not unusual

liver-protective

the

chronic

an

serendipitously

administration

drugs,

against

COMPOUNDS

traditional

improving

cyclooctadiene lignans,

group.

development.

Schizandrae

that of

kernels The

of could

and

biphenyl

reported

with

BIPHENYL

" A blessing

drug

strongly

activity as

p-methoxy

property

m-methoxy an

No.28

sometimes

results.

the

and

and p r e c l i n i c a l

the

its

active

investigations.

HEPATOPROTECTIVE

In

that

to

in general

term,

be more

partition

shows

semicarbazones

cat o r

obvious

proper

series

to

pharmacological

is

activity

the

substituents micarbazone

of

It

favorable and

found

Their

progress.

character

ding

were

some

of

a protective

the

ac-

tetrachloride.

H&

Schizandrin

C promotes

of

and

proteins

crosome

hepatic

glycogen.

cytochrome

P-450

It and

anabolism, induces alters

the

as

the

activation

such

of

liver

of

the

profiles

synthesis mi-

meta-

317 bolism and the DNA-binding In the

tioning

was

early

enedioxy.

assigned

as

Therefore,

not present dicated

of

structural

benzopyrene.

determination,

of 6 , 6 ' - d i m e t h o x y - 4 , 5 , 4 ' , 5 ' - b i s m e t h y l e n e d i o x y

mistakenly

product

of carcinogenic

phase

an

that

its

one

attempt

intermediates of the

groups

to

synthesize

Mass

the

screening

of the

for h e p a t o p r o t e c t i v e

intermediates,

in the protection

tetrachloride,

mice and rats,

thioacetamide,

against

dimethyl

toxicities

D-galactosamine

superior to schizandrin

CH3

<

C (ii).

<

rectification

synthetic

activity

that

the

of

activity

the

structure

4,4'-dimethoxy-

derivative

were b i o l o g i c a l l y

of dimethyl

The regioisomer

(IX),

This

fact

Institute

and

prompted

not,

why

the

activity, and

differs

but

its

Comparing

<

a great

activity

synthetic

evaluated.

It was

was

surpri-

the 4,6'-dimethoxy

~-OCH3 HsC

them

the

H3C0/-',L...~.,uC-OCH3

interest

to explore

simplified

substantially

in

activity.

(VIII)

attracted

V,

(VIII)

of VII and VIII,

~-OCH3

.d

induced by carbon

4,5,4',5'-bismethylenedioxy-

shows an intermediate

H~

(VII) was

and p r e d n i s o l o n e

as

6,6 ' - d i m e t h o x y - b i p h e n y l - 2 , 2 '-dicarboxylate

singly weak.

in-

~CH3 (VII)

sample and its intermediates found

VI

?CH3

3 (VI) After

in V

compound

5,6,5',6'-bismethylenedioxy-biphenyl-2,2'-dicarboxylate

effective

posi-

4,4'-dimethoxy-5,6,5',6'-bismethyl-

in Nature was made.

and

the

of

the

of scientists compound

regiomers

from each other.

the wavelengths

why

ring-cleaved

of maximum

(I X)

substance VII,

absorption

in this

V shows VIII

VIII,

the

does

and

IX

of compounds

318 VII,

VIII,

that

of

well

known

extent

and IX

of

the

IX,

related

(2k max 276,

conjugation

which

is

269, and 273 nm, respectively),

in VII

in

turn

was

than

the

coplanarity

of

the

the

in ultraviolet

coplanarity

absorption

and

VII.

Interestingly,

this

two

hepatoprotective

order

severe

VIII

rings

out

could

importance

between

only

be

groups

and

the

the

with

the

angle;

in

However,

the

schizandrin

twist

angle

X-ray

pounds

V,

pectively.

the

it is understood that

is

C

groups

the

steric (V)

primarily

is

two

of

com-

benzene

interference only

of

determined

of

a minor by

the

Obviously,

compound

structural

moieties

compound

crystallographic The

VII,

of

ring.

whereas

argument.

that

5,6,5',6'-bismethylenedioxy

interference. the

to

twisting

which reduce the repulsion to some extent, twist

is

from VIII to IX,

parallel

by

constraint of the fused cyclooctadiene VII,

is

which

In this case,

6,6'-dimethoxy

relieved

coplanarity.

6,6'-dimethoxy

is

potency.

congestion

of

(12).

than

It

systems

rings,

increases

is

higher

of biphenyl

From the viewpoint of their structure,

pound

be

of VIII.

benzene

spectra

between the two rings

to

that

that the extent of conjugation

to

reflected

the

thought

higher

the

angles

VIII,

data

IX are

From these findings,

i. The extents

of the twist

possesses the smallest

has

provided

between

and

IX

an

intermediate

further

the two benzene

620,

560,

780,

evidence rings

and

for

in com-

60.40,

res-

two conclusions were drawnangle of compounds VII,

VIII,

and IX are in accordance with the order of the wavelength of UV absorption maximum

mutual

, and also with the extents

repulsion of sterically vicinal methoxy groups.

2. The h e p a t o p r o t e c t i v e

potency

of these

correlate to the size of biphenyl greater VIII,

the angle,

compounds

twist angle:

the lower the activity.

the two phenyl

of

seems to the

In compound

rings are almost p e r p e n d i c u l a r

to

each other. The phy rise

conformation

is shown to

two

below

of VII

(X).

revealed by the X-ray

Restricted

atropisomers.

In

rotational

fact,

(+)-VII

crystallogra-

orientation and

gives

(-)-VII

were

obtained by resolution of the free acid and reesterification. Animal

experiments

between

the

racemate

indicated

that

a

large

and the two stereoisomers:

as active as the racemate,

and

difference (+)-VII

(-)-VII is inactive

(13).

exists is twice

319

0, OCH3, . . C H 3 /'-"-" ~ '3. C_O ~

5.

CONCLUSION

In

medicinal

finding

pectedly leads.

outlier(s)

to reveal

of

this

between

Without

using

QSAR

the

The of

an

antiinfectious

drug.

semicarbazones

were

because chemical

biphenyl

deduction

phically,

the

clinical The

identified

p-methoxy-phenyl

characters.

derivatives of

the

was

active

the c o n t i n g e n c y

ACKNOWLEDGEMENT:

for his

The

important

by

the

group

The

in a n t i c o n v u l s i v e

ac-

series

"chiral"

would

of

principle

of

QSAR

an

a

seems to reside is g r e a t l y

comment.

was

not

deduced

compounds. have

compounds

activity

analyses, optimum

of

for

an

(thio)

obviously physico-

hepatoprotective

erroneous

folk

been

started

p-methoxy-phenyl

development upon

author

the

possesses

based

as to prethe

of

active

new

shows a

illustrated

phthalide

discovery

unex-

in

information

highly

an

with

developing

as well

as

The study of a n t i p e p t i c - u l c e r

accidental

provide

and

of action

activity

this

along

the QSAR p r o c e d u r e

difference the

abnormal

QSAR,

often

compounds,

article.

the

serendipity

designing

phase,

untested

unexpectedly

and

analysis

for

the m e c h a n i s m

of

enantiomers

found so easily.

Liang

the

chance

information

activity

tivity

from

in

important

power the

examples from

chemistry,

In the lead o p t i m i z a t i o n

great dict

(x)

medicine.

structural Phyloso-

in the necessity.

indebted

to P r o f e s s o r

X.

320 REFERENCES

1

S.

2

S. Yu and S. You, Acta P h a r m a c e u t i c a Sinica, 19 (1984) 566570. E.J. Lien, J. Med. Chem., 13 (1970) 1189-1191. Z. Guo, u n p u b l i s h e d data. N. G. Richards, Q u a n t u m Pharmacology, 2 nd, Butterworths, London, 1983, p.153. Z. Zheng, Z. Wang, Y. Chu, Y. Li, Q. Li, S. Lin and Z. Xu, in" P r o c e e d i n g s of the Second N a t i o n a l C o n f e r e n c e on Enterology, Chinese Medical A s s o c i a t i o n , Oct 1983, Nanjing, pp. 48-50. S. Zhang, J. Shao and Y. Yu, Acta P h a r m a c e u t i c a Sinica, 19 (1984) 5-11. Z. Guo, G. Yang, F. Chu, G. Xu, J. Zhang, S. Zhang and Y. Yu, Acta P h a r m a c e u t i c a Sinica, 24 (1989) 737-743. Z. Guo, G. Yang, F. Chu, S. Zhang and Y. Yu, Acta P h a r m a c e u t i c a Sinica, 24 (1989) 822-832. C. H a n s c h and A. Leo, S u b s t i t u e n t C o n s t a n t s for C o r r e l a t i o n A n a l y s i s in C h e m i s t r y and Biology, John W i l e y & Sons, New York, 1979, pp. 49-52. J. Xie, J. Zhou, J. Yang, H. Jin and X. Chen, Acta P h a r m a c e u t i c a Sinica, 17 (1982) 23-27. C. Rao, U l t r a - v i o l e t and V i s i b l e Spectroscopy, Butterworths, London, 1961, pp. 82-84. C. Zhang, S. Zhan and J. Xie, Science Bull., 1987, 72.

3 4 5 6

7 8 9 I0 ii 12 13

Yu,

(1984)

S.

You,

486-490.

and H.

Chen,

Acta

Pharmaceutica

Sinica,

19

QSAR and Drug Design - New Developments and Applications T. Fujita, editor 9 1995 Elsevier Science B.V. All rights reserved

CHEMICAL MODIFICATION STUDIES OF PIPERINE DEVELOPMENT FROM FOLK

321

AND STRUCTURE-ACTIVITY AND ITS ANALOGS: AN MEDICINE

RELATIONSHIP EXAMPLE OF DRUG

REN-LI LI and SHU-YU WANG School of Pharmaceutical Sciences Beijing Medical University Beijing 100083 China ABSTRACT:

A folk medicine, the c o n s t i t u e n t s of w h i c h are w h i t e pepper and radish powders, has been used in the northern part of China in the treatment of epilepsy for m a n y years. Piperine(I) was shown to be the active ingredient of this recipe. Structure m o d i f i c a t i o n of p i p e r i n e r e s u l t e d in N - ( 3 , 4 - m e t h y l e n e d i o x y c i n n a m o y l ) - p i p e r i d i n e w h i c h was then used in clinics u n d e r the n a m e of a n t i e p i l e p s i r i n e ( I I ) . For the improvement of its a n t i c o n v u l s a n t a c t i v i t y , f u r t h e r s t r u c t u r a l m o d i f i c a t i o n s of (II) h a v e b e e n e x t e n s i v e l y s t u d i e d f o r e a c h of s t r u c t u r a l moieties in t h i s m o l e c u l e . Q S A R a n a l y s e s of N - c i n n a m o y l p i p e r i d i n e s and c i n n a m a m i d e s s h o w e d that +~, +~ s u b s t i t u e n t s w i t h s m a l l b u l k on the b e n z e n e r i n g f a v o r the a n t i c o n v u l s a n t activity. S u b s t i t u t i o n on the v i n y l e n e l i n k a g e d e m o n s t r a t e d that the s p e c i f i c m o l e c u l a r c o n f i g u r a t i o n and c o n f o r m a t i o n are crucial for the anticonvulsant activity. On the basis of these s t r u c t u r e - a c t i v i t y r e l a t i o n s h i p studies, the m o d e of r e c e p t o r binding of this kind of compound was suggested. i.

INTRODUCTION

Discovery especially the m a i n

structure

us with a novel

active

approaches of the

to

lead

active

drug on a large paradigm

sources.

ingredients

plants,

lead coming

an optimized

brilliant

natural

of

from h i g h e r

treatment

from natural

compound which

scale.

and

In this chapter,

The

folk medicines

natural

the

used

to

is of great

of

could be supplied of p r o c a i n e

lead

generated

in u s i n g h e r b a l m e d i c i n e s approach

one

lead

of

the

could provide

as

is a from

in the

generation

importance

from

in China.

we will present our research work as an example

of d e v e l o p i n g a novel drug from one of the Chinese recipes.

sources, as

Modifications

sources

The d i s c o v e r y

of how to o p t i m i z e

of d i s e a s e s .

from

been w i d e l y

generation.

China has a long h i s t o r y traditional

has

folk medicine

322 2.

ANTIEPILEPTIC

Dry

powder

medicine

of w h i t e

recipe

University, recipe

FOLK MEDICINE

kept

which

was

pepper

by

in the n o r t h e r n

part

handed

on

of C h i n a

presumed

to be safe subjected

to h i m

was

of

from

a secret

Beijing

his

body,

In 1970,

this

recipe was

effects were also studied with animals.

prescriptions

with

designed results

and

tested

showed

that,

of a n t i e p i l e p s y the

most

prescription

with

in

of w h i t e

with

dose,

(2) .

Thus,

effect

as

white

white

pepper

Piperine(I) extract, results

was

hence

low t o x i c i t y .

so

made

identified

synthetic Its

65.36

(2).

ED50

that

and

DISCOVERY

plentiful, could

Similar

it was

tablets in

piperine

value

and

crude

clinical

was

tested

electroshock

its LD50 was

(LD50/ED50)

the

source

of

and in addition,

be i s o l a t e d

complicated bonds

results

that

was

and

the

were

the

showed the same

as the m a j o r c o m p o n e n t

mg/kg

possesses

obvious

of

effect

pepper,

extract

trials

of

(3).

of this crude in

rats.

The

a c t i v i t y with

seizure, 348.6

MES)

in

(+ 49.65)

5.42.

2

(I)

OF A N T I E P I L E P S I R I N E

considered.

double

therapeutic

in this recipe.

used

(maximum

(+- 14.76)

The P.I.

Because

was

The

radish

CH=CH--CH=CH -C--N 3.

tests.

showed that it shows potent a n t i c o n v u l s a n t

rats was mg/kg

were

animal

extract of w h i t e p e p p e r

pepper,

were

Several

pepper

of w h i t e

(i).

the

white

the

without

effect

r a d i s h p o w d e r was only an e x c i p i e n t The crude a l c o h o l i c

and

the content

pepper

tests

of

clinics

therapeutic

in a n i m a l

ratios

an equal

increased

significant

obtained

both

radish

both are

In the m e a n w h i l e ,

pharmacological

different

it was

Because

so that

trials.

This

of e p i l e p s y

is a kind of seasoning,

clinical

folk

Medical

family.

in the t r e a t m e n t

for m a n y years.

for the human

to

radish

member

to the University.

and white pepper

directly

and

family

by the staff m e m b e r

is a v e g e t a b l e

PIPERINE

a staff

had been used by his

presented

AND

white

from w h i t e But,

and thus

the

pepper,

the

synthesis

it w o u l d

is e l i m i n a t e d

pepper

in

China

only 20 % of the content synthesis

of

piperine

be e x p e n s i v e .

from its

structure,

is

not

of piperine of p i p e r i n e is

rather

If one of the two synthesis

of the

323 resulting

molecule

vinylogy,

this

anticonvulsant

prediction. named

was

would

be

simple.

simplified

activity.

Pharmacological

antiepilepsirine,

(• 12.3) mg/kg

(mice)

did exhibit

tests

The

P.I.

(• 14.3) mg/kg

(• 12.1) mg/kg values

! I

A After systematic

subjected

1.5

clinical

broad

B tests

observations

effective

and

showed

spectrum

for

experiments

the

that

Compared

treatment

antiepileptic

clinics,

potent

MODIFICATION

activity,

the

(rats)

and acute and of

was

epilepsy.

an

antiepilepsirine

is

and

its

side

have given various

OF ANTIEPILEPSIRINE

the anticonvulsant For

1.8

is

agent,

At present,

to the other antiepileptic

enough.

(• 19) mg/kg

antiepilepsirine

of its therapeutic effect.

STRUCTURAL

The

antiepilepsirine(II)

still used in clinics in China but physicians 4.

(4).

(II)

(4),

trials

but

88.5

C

effects are relatively low (5). evaluations

this

activity

(rats)

(mice)

-I I I

pharmacological

toxicological to

verified

(mice) and 177

were

CH=CH

Clinical

of

possess

(MES) values were

which were not as high as those of piperine. O t i

chronic

principle

also

anticonvulsant

Its ED50

and 98.6

LD50 doses were 132.6 (4).

the

could

N-(3,4-methylenedioxy-cinnamoyl)-piperidine(II),

less potent than piperine.

(rats)

From

molecule

agents usually used in

activity of antiepilepsirine

improvement

structural modifications

of

its

is not

anticonvulsant

have been extensively studied

according to the A, B, and C moieties of its structure(II). 4.1 Modifications

in the Aromatic Ring Moiety

4.1.1 V a r i a t i o n s

substituted

piperidine

benzene

piperidine(III)

markedly decreased

(III)

R i n g System:

by the propenyl

group,

ring

is displaced

resulted.

in

(ED50:200

o

(A in II)

of the A r o m a t i c

W h e n the

N-(3,4-methylenedioxy-cinnamoyl)Its

N-(hexadienoyl)-

anticonvulsant

mg/kg)

(6).

activity

9 (IV)

/-A

was

324 If

the

benzene

ring

was

replaced

furanacryloylpiperidine(IV), and all m i c e

Z(cis)

have

indicated

form

show

a CNS

form exerts

verified due

to

Tung

whether its

(9)

protons

have

NMR be

shown are

spectrum

of

to

cinnamic

hence

the

cinnamamides

effect

of

whereas

the

It r e m a i n e d

to be

of

IV is

the

Rappe

(8)

coupling acids

than

IV, it

the

13,

derivatives

the

with

does

and

Speziale

E

of

while than

hold

for

the

Z

coupling

was

(6).

found

Thus,

the c o n f i g u r a t i o n

not

two

the

From the

constant form

of

the

13.

and

the

cinnamamides

coupling

takes

compound

constants

and

are all g r e a t e r

of the a c t i v i t y

acid

resulted

activity

smaller

compound

CNS

the

effect.

E forms

15,

relationship

(7) that

forming

stimulating

cinnamic all

of

ring

stimulating

that

of

of their

close

CNS

furan

depressing

configuration.

CH=CH

configuration constants

a CNS

the

Z(cis)

on

(6).

et al.

E(trans)

the

a stimulation

died of c o n v u l s i o n

Balsamo

the

by

IH to the

observed

for

furanacryloyl

analogs. 4.1.2

Variations

of

Substituents

Cinnamoylpiperidines

with

various

ring w e r e

and

evaluated

results

synthesized

indicated

influenced

Table

these E.

TABLE

1 (6).

1

The c o u p l i n g were

X

all

0.342 0.437 0.502 0.248

the p u r p o s e

anticonvulsant

MES

is ring

the

benzene

to

15,

their

the

Ring: benzene

test.

The

significantly

of the two vinyl

as

shown

in

protons

of

comparison

of

configuration

being

cinnamoylpiperidines

ED50 (MES) mmol/kg

3,4-0CH20H 4-0Me 4-CI

Benzene on

activity

on

constants

close

the

by the m o u s e

anticonvulsant

substituent

Substituted

For the

the

compounds

No. i 2 3 4

by

that

on

substituents

No. 5 6 7 8

of rational activity

of

ED50 (MES) mmol/kg

X 4-Br 4-N02 3,4,5-(0Me) 3 4-0H, 3-0Me drug

0.481 1.250 0.306 1.812

design,

compounds

of

the

Table

1 was

made

325 first

by

reference, benzene

the

ring

substituted

tree, be

Topliss

(10).

was

by

substituted

chlorine.

Taking

activity by

synthesized, 1

(ii).

but

was

Thus,

as

found the

compound

decreased

methoxy,

According

3,4-dichlorocinnamoylpiperidine(V)

Table and

method

the a n t i c o n v u l s a n t

less

to

but

the

was

potent

second

the

Compound

They were VII

piperidines

is

all more potent

the m o s t

potent

was

synthesized

the s t r u c t u r e - a c t i v i t y

relationships

Modifications

The

moiety

C of

ethoxycarbonyl acid

ethyl

group

amide

group

of

b i n d i n g with

among

in the A m i d e M o i e t y resulting

(6).

This

cinnamamides

in

synthesized

of

with

is

isopropylamine

and

Cinnamoylpiperidines O

o_%/?

We

result

led

would

be

to

4.3).

cinnaclarify

to the

us

to

inactive

believe

indispensable

aliphatic

found

changed

as

an

that

the

probably

for

receptor.

(VIII)

and

that

sec-butylamine

and

4-chlorocinnam-

cyclic

cinnamamides are

exhibit m o d e r a t e

c. c.-c-.

(IX)

cinnamoyl-

0.282 0.217 0.120

was

almost

3,4-methylenedioxy-(IX)

various

(6).

in order

i.

3,4-methylenedioxy-cinnamic

which

the a n t i c o n v u l s a n t

series

the

(C in II)

antiepilepsirine(II)

O

A

synthesized

substituted

X : 3,4-CI2 X : 2,4-CI 2 X : 4-CF 3

II 0 ~ ~ " / - CH=CH---C-OEt

amides(X)

4 of

4 of Table

(See section

V VI Vll

ester(VIII),

anticonvulsant

to

ED50 (MES) mmol/kg

O

4.2

choice

compound

were

Meanwhile, extensively

when

decision

next

compound

molecule

so far s y n t h e s i z e d .

moylpiperidines

than

the

2,4-dichloro-(VI),

4-trifluoromethylcinnamoylpiperidine(VII)

(12,13).

increased

Topliss

than

choice,

2 as

if 4-H on the

among

the

amines

formed

most

were

with

potent.

activity.

R: NH 2, NHPr, NHPr(i), NHBu, NHBu(i), NHBu(s), NHPent, NHPent(i), NHHept, N(Me) 2, N(Et)2, N(Pr)2, N(i-Bu) 2, NHCH (CH2 )4' NHPh, NHPh (p-Me ), NHCH2Ph, N(CH2)4, N(CH2CH2)20, N(CH2)5

326 oII

CI

R: N. 2, NePr

CH=CH--C--R (X)

the

'

NHPr(i)

Each of the p - c h l o r o c i n n a m a m i d e s ( X )

corresponding

obvious

that

NHBu

'

N.Bu(i)

'

'

NHBu(s),NHPent, NHHex, N(Me)2, N(Et)2, N(Bu)2, NHCH(CH 2)4, NHCH(CH 2)5, N(CH 2)4, N(CH2) 5 was m o r e p o t e n t

3,4-methylenedioxycinnamamide(IX).

the

anticonvulsant

activity

of

than

It was

cinnamamides

is

influenced by substituents not only on the benzene ring but also

at the amide moiety. 4.3

Quantitative S t r u c t u r e - A c t i v i t y A n a l y s i s

Derivatives

amine,

of c i n n a m o y l p i p e r i d i n e ,

three

ring w e r e

series

tested

(12,13).

of c i n n a m a m i d e s ,

quantitative

structure-activity

the H a n s c h

The QSAR of cinnamoylpiperidines(No. studied

equation

(14).

1, w h e r e

mice.

log I/C = -0.248 n = 19, series

value

of

(log p)2 + 1.885

1 shows

that

compounds

(P b e i n g

of

equivalent

r = 0.875,

The electronic 0.i)

C is

the

l-octanol/water

contributions

an e l e c t r o n

the a c t i v i t y

to variations

withdrawing

One

(No.

related

ring

and a small

of c i n n a m o y l p i p e r i d i n e .

ring s u b s t i t u e n t s

compound

activity

in the activity.

of ZMR 2 3 4 shows the existence

benzene

was

of

this

with of

make

in

as

in

[i]

was 6-fold more potent than the predicted value.

P

significant

Substituents

The n e g a t i v e

did not

log

compounds.

(MR scaled by

of steric hindrance

2) w h i c h

limited

the

size will

and the c o r r e s p o n d i n g

9 of Table

studies

represented

c oefficient)

benzene

effect

in

ED50(MES ) mmol/kg

(~) and steric parameter the

used

(log P)opt = 3.80

anticonvulsant

on

of t h e s e

(log P) + 0.150 Za

s = 0.158,

parameter

(QSAR)

was

1 - 20 of Table 2) was

with

is p a r a b o l i c a l l y

substituents

approach

correlation

0.285 EMR2,3, 4 - 2.664

-

Equation

The best

substituents on

On the b a s i s

relationship

order to further optimize the structure.

C i n n a m a m i d e s

cinnamoylisopropyl-

and cinnamoyl-sec-butylamine with various

the b e n z e n e

first

of

with

increase

coefficient between

receptor

the

site.

fit e q u a t i o n

i,

327 TABLE

2

Parameters

used

in

--

No

X

1 2 3 4 5 6 7

Ra

3-Ci 3-F 4-F 4-Br 2,4-Ci 2 3,4-Ci 2 4-Ci 3,4-OCH20d 3,4,5-(0Me)3 e 4-NO 2 3-NO 2 3-CF 3 2-CF 3 4-CF 3 3-OH,4-OMe 4-OMe 3-I 4-OEt 4-OPr(n) 4-OBu(n) 3-CI 3-F 4-F 4-Br 2,4-Ci 2 3,4-Ci 2 4-CI 4-CF 3 3-CF 3 3-C~ 3-F 4-F 4-Br 2,4-CI 2 4-CI 3,4-Ci 2 4-CF 3 3-CF 3

8

9 i0 ii 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37

38

a. c. d. e.

--CH=CH--

obsd

N(CH2) 5 N(CH2) 5 N(CH2) 5 N(CH2) 5 N(CH2) 5 N(CH2) 5 N(CH2) 5 N(CH2) 5 N(CH2) 5 N(CH2) 5 N(CH2) 5 N(CH2) 5 N(CH2) 5 N(CH2) 5 N(CH2) 5 N(CH2) 5 N(CH2) 5 N(CH2) 5 N(CH2) 5 N(CH2) 5 NHBu(s) NHBu(s) NHBu(s) NHBu(s) NHBu(s) NHBu(s) NHBu(s) NHBu(s) NHBu(s) NHPr(i) NHPr(i) NHPr(i) NHPr(i) NHPr(i) NHPr(i) NHPr(i) NHPr(i) NHPr(i)

derivation

0.788 0.578 0.458 0.314 0.664 0.550 0.606 0.564 0.793 0.268 0.324 0.921 0.723 0.921 -0.272 0.218 0.320 0.500 0.290 0.180 0.410 0.495 0.495 0.540 0.735 0.977 0.714 0.772 0.989 0.620 0.301 0.288 0.580 0.600 0.801 0.498 0.899 0.924

log I/C

calcd b

0.658 0.572 0.499 0.582 0.673 0.709 0.621 0.116 0.037 0.292 0.274 0.744 0.772

0.772

-0.263 0.152 0.525 0.252 0.285 0.196 0.717 0.674 0.602 0.633 0.680 0.716 0.681 0.819 0.790 0.628 0.674 0.452 0.559 0.665 0.592 0.701 0.747

0.719

of

equations

2 and

cinnamoylisopropylamines

included

in

the

analysis,

and an

3

% R

IAI

log P

0.130 0.006 0.041 0.268 0.009 0.159 0.015 0.448 0.756 0.024 0.050 0.177 0.049 0.149 0.009 0.066 0.205 0.248 0.005 0.016 0.307 0.179 0.107 0.093 0.055 0.211 0.033 0.047 0.199 0.008 0.373 0.164 0.021 0.065 0.209 0.203 0.152 0.205

3.42 2.86 2.86 3.57

4.14 4.14 3.43 2.66 2.66 2.44 2.44 3.60 3.60 3.60 2.05 2.70 3.84 3.19 3.77 4.27 3.67 3.10 3.10 3.82 4.38 4.38 3.67 3.84 3.84 3.33 3.10 2.76 3.48 4.04 3.33 4.04 3.50 3.50

EMR2,3,4 c

Z~ 0.37 0.34 0.06 0.23 0.46 0.60 0.23 -0.32 0.07 0.78 0.71 0.43 0.54 0.54 -0.15 -0.27 0.35 -0.24 -0.25 -0.32 0.37 0.34 0.06 0.23 0.46 0.60 0.23 0.54 0.43 0.37 0.34 0.06 0.23 0.46 0.23 0.60 0.54 0.43

0.80 0.29 0.29 1.09 1.30 1.30 0.80 1.00 1.68 0.94 0.94 0.70 0.70 0.70 1.17 0.99 1.60 1.45 1.91 2.37 0.80 0.29 0.29 1.09 1.30 1.30 0.80 0.70 0.70 0.80 0.29 0.29 1.09 1.30 0.80 1.30 0.70 0.70

N(CH2)5: piperidinyl, b. Calculated using eq. 2. MR(H): 0.I0 was accounted. Not used in the derivation of eq. 2. Not used in the derivation of eqs. i and 2. When

were

the

cinnamoyl-sec-butylamines

almost

identical

correlation

328 was o b t a i n e d

as shown in equation

log i/C = -0.155 n = 35, The

that

(log p)2 + 1.305 log P + 0.257

0.264

-

ZMR2,3, 4 - 2.032

r = 0.844,

optimum

value)

binding

obtained

except

for

2

Compounds compound

are

did

not

8 and

31 w a s

to five

and

QSAR

of

were The

of

(MSA)

log P - 0.301

and

of

In e q u a t i o n lengths,

r = 0.917, 3, A is

0 and N of the a m i d e

while

the

reason

compound

structure

volume

(16).

(Vo), and

using

3 as of the best quality.

[3]

of the t r i a n g l e

These

bond

by the MO that

of c i n n a m a m i d e s

is p a r a b o l i c a l l y

the o v e r l a p p i n g

area

(So)

the

(So)opt = 33.67

of

3 indicates

these

(log p)2 + 0.202 So

product

optimized

Equation

next

for c o m p u t i n g

the area

group.

31 of

(15) w a s

analysis

(log P)opt = 3.82,

the

reflecting

was

in the

dropped.

potent,

chosen

overlapping

quantitative

This

9,

predicted,

series

effect

in equation

were

analysis

this

P.

8,

hence

arbitrarily

gave equation

log

Topliss

The

variations

more

surface area,

(So).

term

than

the

reflected

5.7-fold

potent

shape

to

potent results

0.003 So 2 + 9.222A - 22.218

n = 25,

the

less

the

volume, area

log I/C = 2.298

4.4.2).

2.8-

with

4.1.2).

the

optimum

generate

not

and

with

the

ring

Compounds

2

to and the

accord

according

than

The role

ring

(with

structural

enough.

molecular

shape p a r a m e t e r s

by

in

the

the

equation

cinnamamides

the m o l e c u l a r

bond

broad

9 were

examine

overlapping

-

fit

+~

(see Section in

fact that

1/2.4

Hopfinger's

Twenty

is

syntheses

included

not

benzene

that

This

[2]

higher

large.

of compounds

on the b e n z e n e

mentioned

that

somewhat

is not

substituted 2 shows

in the amide m o i e t y was

b e i n g obscure. applied

the

design

to the

moiety

Table

the

as p r e v i o u s l y

attributable amide

of

activity.

from

2 is

the transport

+~ s u b s t i t u e n t s

of v a r i a t i o n s 2,

equation

Equation

anticonvulsant scheme

includes

site.

and

of

Z~

(log P)opt = 4.35

i, but the d i f f e r e n c e

of log P p r o b a b l y receptor

s = 0.156,

log P v a l u e

of e q u a t i o n

hydrophobic

2 (14).

values

of

lengths

C=O

were

calculation

related

with and

C-N

by C,

estimated

(see

the a n t i c o n v u l s a n t

of the m o l e c u l e

and

defined

Section activity

log P as well linearly

as

related

329 with

A.

The

to t h a t amide

are

ring

reflection shown

bond

ring

the

C=O

character

closer

aspects:

with

an

the

one

ethylene

or

substitution 4.4.1 Methylene

of m o i e t y

of a c a r b o n

unit

diverse

results TABLE

3

are

of

linkage

Anticonvulsant

the

1 2 3 4 5 6

R H H H 3,4-0CH20 3,4-0CH20 3,4-0CH20

Except

X

to

donating

increase

the

bond

length.

effect

the

of

N-substituents

(B in II) includes

the v i n y l e n e

and

the

Vinylene

with

of the d o u b l e

in

the

other

moiety

3 and Table

activity

ED50(MES) mmol/kg

CH=CH 0.437 (CH2) 2 0.747 CH 2 >0.985 CH=CH 0.342 (CH2)2 0.237 CH 2 inactive

for the u n s u b s t i t u t e d

an

bond

moiety is

the

Ethylene

or

and

deletion

of c i n n a m a m i d e s respectively.

anticonvulsant

activity.

result They The

4 (17).

of p h e n y l a c y l p i p e r i d i n e s

o

No.

of

and p h e n y l a c e t a m i d e s ,

in Table

bond

electron

since

Structure

activity

double

B of a n t i e p i l e p s i r i n e

from the v i n y l e n e

variations shown

the

the

the the

Electron

bond.

Saturation

in p h e n y l p r o p i o n a m i d e s gave

for

overweighs

replacement

methylene

Replacement

Linkage:

the

moiety

C=O group.

is the

on the d o u b l e

that

group

in the L i n k a g e

A is a c t u a l l y

lower

is r e a s o n a b l e

to the amide

Modifications

to

of the the

enhance

amide

C=O

This

C-N

on

favorable

mean

the

and

is close

substituents

substituents.

ring

leading of

of C=O in

probably

could

3.82 w h i c h

so that

these

the

of the

The m o d i f i c a t i o n two

of

bond

A term

lengths

nitrogen,

on

N-substituents

3 is

variations

2 would

substituents.

are m u c h 4.4

amide

bond

effects

positive

of

single

the

the

the

equation

of

the

The with

substituents

in

character effect

2.

varied

and of

withdrawing

Thus,

log P of e q u a t i o n

of e q u a t i o n

group

benzene

as

optimum

r-x

No. 7 8 9 i0 ii 12

R 4-Ci 4-CI 4-CI 4-NO 2 4-NO 2 4-NO 2

X

ED50(MES) mmol/kg

CH=CH 0.248 (CH2)2 0.234 CH 2 0.638 CH=CH 1.250 (CH2)2 0.901 CH 2 inactive

derivative(compound

2) in Table

330 3,

the

was

activity

close

However, less

in

of

to that

all

substituted

of t h e i r

the s u b s t i t u t e d

potent

Table

4.

than

For

their the

indicate

than

that

their

activity

the removal

is,

unsaturated

double

bond

seems

TABLE 4

Anticonvulsant

1 2 3 4 5 6

X

H H H 3,4-0CH20 3,4-0CH20 3,4-0CH20

the

to

ring

EDs0 (MES) mmol /kg

No. 7 8 9 I0 ii 12

postulation

bond b e t w e e n

the

group.

But,

postulate.

position

could

effect the

The

anticonvulsant

anticonvulsant respectively,

be tenable, of the ring

results

activity

pionylpiperidine

These

not

results

group

necessarily

of

The

indis-

EDs0 (MES) mmol /kg

X

4-ci 4-Ci 4-Ci 4-N02 4-N02 4-N02

CH=CH (CH2)2 CH 2 CH=CH (CH2)2 CH 2

0.158 0.546 0.441 0.359 inactive inactive

is to e l e c t r o n i c a l l y

carbonyl

of

introduction

of the b e n z e n e

but

less

to the a c t i v i t y .

group

of

the

amide

saturation

Table

3 did

1.76-fold,

activity

was

by i n t r o d u c t i o n

the

whereas

activity

enhanced

not atom

interrupt

support into

that

enhanced

into

and

of a m e t h y l e n e d i o x y

this

the p a r a -

the

phenylpro-

3.19-fold.

1.28-

If

on the c a r b o n y l

ring of c i n n a m o y l p i p e r i d i n e

enhanced

function, the double

ring will

substituents

of a c h l o r i n e

of

on

influence

a c t i v i t y of compounds.

the

the amide group and the b e n z e n e

the e l e c t r o n i c

all

were

from the vinyl

R

and hence to alter the a n t i c o n v u l s a n t this

shown

that the role of s u b s t i t u e n t s

of c i n n a m a m i d e s of

4),

as

a c t i v i t y of p h e n y l a c y l i s o p r o p y l a m i n e s

we p o s t u l a t e d

polarization

be

activity.

CH=CH 0.760 (CH2) 2 0.977 CH 2 0.590 CH=CH 0.265 (CH2)2 0.602 CH 2 >1.449

Originally,

the b e n z e n e

Table

cinnamamides.

unfavorable

to the a n t i c o n v u l s a n t

R

3 of

were all

analogs

phenylacetamides,

of one carbon

in general,

pensable

No.

of

cinnamoyl

(compound

corresponding

cinnamamides

cinnamoylpiperidines.

phenylpropionylisopropylamines

corresponding

phenylacetylisopropylamine potent

phenylpropionylpiperidines

corresponding

The

3.15-fold,

group into the

331 3,4-position

of

the

benzene

ring

These

results

propionylpiperidines. substituents effect

on

on

interaction

then

the

amide

the b i n d i n g

the vinyl Me,

this

group

CI,

larger

OMe,

and

the

(18).

convulsants

Double

or less

TABLE 5 Anticonvulsant sec-butylamines

the

was

located

s-hydrogen

of as

whereas

Also,

influenced

the

where

on o p p o s i t e and the

linkage

substitutents

increased,

compounds

the

anticonvulsant

the

activity.

drastically are

in

Cinnamoyl-sec-

small

the

that

electronic

(17).

Bond:

When

activity

anticonvulsant

potent

an

on the v i n y l e n e

such

Z configuration moiety

exert

5 shows

by

phenyl-

to m e a n

participate

compounds.

the

compounds

bond were

only

also

Table

of

decreased

the

amide

of the d o u b l e

on t h e

SMe,

and

believed

site of the r e c e p t o r

substituted

and

of

activities:

ring

was

not

but

cinnamoyl-

were

s-substitutions

series

substituents

configuration CNS

various

synthesized of

ring

group

s-Substitution with

activities F,

benzene

with

4.4.2 butylamines were

the

of

the

the

type

of

benzene

sides(trans)

E(cis)

forms w e r e

anticonvulsants.

activities

of s - s u b s t i t u t e d

cinnamoyl-

NH --sec-Bu

No. i 2 3 4 5 6 7 8 9 i0 a. After of

the

number

Me Et Bu-n PhCH 2 Ph F CI(E) CI(Z) Br(E) Br(Z)

that

0.314 1.054 0.950 >1.365 0.461 0.289 a 0.472 a 1.003

realizing bond

Z(trans)

bromo-cinnamamides show

No. ii 12 13 14 15 16 17 18 19

ED50 (MES) mmol/kg

R OMe(E) OMe(Z) OEt SMe SEt SPh NHCOMe NHCOPh H

0.662 0.348 0.525 0.219 1.073 0.536 0.419 1.755 0.621

convulsant effect.

double of

ED50 (MES) mmol/kg

R

that

of

small

substituents

cinnamamides

and were

E(cis)

may

isomeric

synthesized

on

enhance pairs

(19

the

s-position activity,

of ~ - c h l o r o

- 21).

N-(~-halo-cinnamoyl)-sec-butylamines

the

Tables are more

a

and ~6 and

7

potent

332 than

the

cinnamamides

substituent

on

the

formed

benzene

with

ring.

other For

chloro-cinnamoyl-sec-butylamines, or

bromo

the

atom

into

anticonvulsant

derivatives, decreased note

more

to

in

Tables the

for

(X)-cinnamamides TABLE

6

X

1

7

8 9

i0 ii 12 13 14 15

16 17 18 19 20 21 22

2,4-C12 2,4-C12 2,4-C12

3,4-CI 2

3,4-Cl 2

3,4-Ci 2

3,4-C12 2-Cl 2-Cl

MES

s-Bu s-Bu i-Pr i-Pr s-Bu s-Bu i-Pr i-Pr s-Bu s-Bu i-Pr i-Pr s-Bu s-Bu i-Pr i-Pr s-Bu s-Bu i-Pr i-Pr i-Pr i-Pr

of

Z E Z E Z E Z E Z E Z E Z E Z E Z E Z E Z E

is

Z(trans)

why

X

27

8.23 7.06 8.22 7.06 8.25 7.07 8.25 7.08 8.23 7.06 8.23 7.08 8.33 7.47 8.32 7.45 8.20 7.01 8.19 7.02 8.38 7.36

of

activity

ED50 (MES) mmol/kg 0.076 0.517 0.234 0.485 0.098 0.606 0.180 1.178 0.195 0.195 0.814 0.857 0.202 0.463 1.332 0.417 0.311 0.351 0.636 1.461 1.135 0.576

u-halo

(22).

Br

6.56 5.54 6.55 5.56 6.56 5.62 6.59 5.62 6.62 5.56 6.60 5.49 6.60 5.60 6.55 5.64 6.63 5.53 6.59 5.67 6.63 5.54

are

Speculation

CONHR

NH

to of

activity, isomers

E-isomer

8ppm

atom

pairs

\c=c' / \

UV IHNMR MeOH X max(nm) ~-H

other

interesting

of ~ - b r o m o c i n n a m a m i d e s

H

282.5 267.5 283.0 268.0 281.5 264.5 281.5 266.5 272.5 257.0 272.5 257.5 273.0 268.0 273.0 268.5 278.0 267.5 278.5 267.5 265.0 258.0

for

bromo

isomers. the

3-

chloro

increased

anticonvulsant the

same

and a

But, or

the

anticonvulsant

~ONHR

Configuration

It

E(cis) of

activity

Br

R 4-Br 4-Br 4-Br 4-Br 4-CI 4-CI 4-CI 4-CI 3-CI 3-Ci 3-C1 3-Ci 2,4-Ci 2

possesses

\c=c" I \

H

2 3 4 5 6

Most

of

linkage

fold.

all

the

4-bromo,

chloro

of

7 exhibit

explanation

and

a

degrees.

corresponding

also

vinylene

1.2-1.6

isomers

with

introduction

of

various E

6 and

the

Structure

No.

about

extents.

than

made

to

and

various

potent been

activity Z

cinnamamides

of

introduction

both

4-chloro,

the

u-position

activity

the

the

that

though has

the

amines

333 The

dipole

the

role

some

of of

~-C-X

C=O

of

in

the

E-isomer

Z-s-halo

E-~-halo-cinnamamides

position The

of

steric

dihedral side

the

hindrance angle

with

benzene

ring

of

between

are

ortho the

could

probably

cinnamamides.

More

substituents

more

potent

on

than

substituents

supplement interesting,

would benzene

the

the

ortho

Z-isomers.

influence

planes

of

the

ring

activity

of

~-chlorocinnamamides

and

chain.

TABLE

7

Structure

and

X

C!

1 2 3 4 5 6 7 8 9 i0 Ii 12 13 14 15 16 17 18 19 20 21

R

22

23 24 25 26 27 28

29 30 31 32

4-CI 4-CI 4-Ci 4-CI 4-Ci 4-Ci 4-Br 4-Br 4-Br 4-Br 3-CI 3-CI 3-Ci 3-Ci 3-Ci 3-Ci

3,4-Cl 2 3,4-CI 2 3,4-CI 2

3,4-C12

2-Ci 2-CI 2-Ci 2-Ci 2-Ci 2-CI

2,4-Cl 2 2,4-Cl 2

2,4-Ci 2

2,4-C12 2,4-C12 2,4-C12

X

\c=c' H'

No.

MES

s-Bu s-Bu i-Pr i-Pr CH(CH2) A ~ CH(CH2) & ~ s-Bu s-Bu i-Pr i-Pr s-Bu s-Bu i-Pr i-Pr CH(CH2) A ~ CH(CH2) n ~ i-Pr i-Pr CH(CH2)&i CH(CH2) & i s-Bu s-Bu i-Pr i-Pr CH(CH2) ~ CH(CH2) & ~ s-Bu s-Bu i-Pr i-Pr CH(CH2) n ~ CH(CH2)._.~

\CONHR

CONHR

\c=c' /

n

Configuration

UV MeOH I max(nm)

Z E Z E Z E Z E Z E Z E Z E Z E Z E Z E Z E Z E Z E Z E Z E Z E

280.0 264.0 281.0 265.0 281.0 265.0 282.0 266.5 282.5 266.5 272.0 259.5 272.0 259.5 272.0 259.0 277.5 265.5 278.5 265.5 267.5 256.5 268.0 258.0 268.0 256.5 274.5 264.5 274.0 265.5 275.0 264.0

\Cl

IHNMR ~-H 7.96 6.93 7.96 6.93 7.96 6.93 7.95 6.92 7.94 6.91 7.95 6.88 7.94 6.89 7.95 6.90 7.91 6.87 7.90 6.88 8.19 7.12 8.19 7.13 8.20 7.27 8.14 7.07 8.14 7.08 8.13 7.08

8ppm

EDs0 (MES)

NH

mmol/kg

6.51 5.76 6.60 5.75 6.74 5.85 6.61 5.73 6.68 5.96 6.52 5.73 6.58 5.76 6.74 5.81 6.63 5.83 6.65 5.86 6.50 5.86 6.53 5.74 6.67 5.79 6.46 5.87 6.58 5.83 6.61 5.95

0.113 0.358 0.294 0.488 0.887 0.222 0.079 0.251 0.152 0.431 0. 211 0.845 0.775 0.435 0.887 0.534 0.684 0.171 0.249 0.440 0.735 0.825 1.550 1.096 1.226 0.704 0.518 0.416 1.352 0.609 1.056 0.528

the the

334

The

configurations

assigned and of

by the

the m o l a r

steric

and

the

the

of

distortion, ring

the

substituted shift

of

amido form

absorptivity

6ma x is

benzene

side

of

and

the

the

shows

and

in Tables

listed of

E and

isopropylamine also

81,

by

between

of the E isomer was

on

6 and

and

the

are all w i t h i n (23).

of

where

located

is

on

the

by

shifts

in the

same

of

~-

chemical

nitrogen

shifts

longer

the

the

7, the ~ - p r o t o n

of

the

of the E Z form

is

of the NH proton

5.49-5.95

and those

IH NMR s p e c t r o s c o p y

crystallography

the b e n z e n e

46.4 ~ , w h i l e

TABLE 8 Anticonvulsant cinnamoyl-sec-butylamines

isomer

of Z

2,4-dichloro-~-bromocinnamoyl-

by the X max and

X-ray

grades

configuration

The chemical

6.46-6.74

assigned

are

were (Xmax)

lower

isomer

E(cis)

distinguished

proton

6.88-7.47

Z isomers

confirmed

angle,

the

of 7.91-8.38.

are w i t h i n

The

also

~-proton

the

The

maximum

of the

Z(trans)

of

group

(23).

was

of E - ~ - h a l o - c i n n a m a m i d e s isomers

of the

amido

cinnamamides

absorption

Because

those

As shifts

in the range

Xmax

than

bond

cinnamamides

of the

(6max).

the

greater

double

group.

s-substituted

UV w a v e l e n g t h

ring

(23).

and v i n y l e n e

that of the

activity

of

The

linkage

Z isomer was

~-substituted

were

dihedral planes

27.8 ~ .

4-chloro-

R

HI

No.

on

ED50(MES) mmol/kg

No.

0.240 0.073 0.070 0.655 0.348 0.144 0.217

8 9 i0 ii 12 13 14

1 2 3 4 5 6 7

Me Et n-Pr i-Pr Ph CI Br

a.

With convulsant effect.

4.4.3

~-Substitution

smaller

substituents

the

that

R

double

bond

NH --sec-Bu

gave

ED50(MES) mmol/kg

R

NH 2 0.244 NHMe 0.713 NHEt 0.635 NHPr(i) 0.524 NHPr 1.358 a NHBu(i) >1.314 NHBu >1.314

on the D o u b l e

results

similar

enhance

the

Bond:

~-Substitution

to s - s u b s t i t u t i o n ,

anticonvulsant

so

activity

335 and larger substituents

Table

It is interesting

of compound only

group

n-propyl

compound

benzene

4 (24)

drastic

of compound of

distortion

of

4.4.4

Table

are crucial

torsion

chemical

(81)

angle

and

m i n i m u m potential As m e n t i o n e d

energy above

cinnamamides,

to be g e n e r a l l y the

torsion

anticonvulsant E(trans) 30 ~ a n d

Table the

9

fall

plane

of

angles,

activity

amide

if

82 is not w i t h i n

linkage.

fit the requirement

activities

of compounds

seem

do

not

for compounds

data and

and

next and

on c o m p o u n d

vinyl

group

vinyl

group

with

activity

is a l m o s t

to

additional

be

9.

crucial

In spite

the

show

range

to

Z(cis)

of

the

the

between

anticonvulsant In this

perpendicular

The conformations

3 and 5 of the

of

have

factors

2, 4, 6, 8, 10 and

of the receptor.

the

of the double bond.

from -90 ~ to -115 ~ .

group

were

conformation

such

in Table

of

a c t i v i t y of this

group

and

cinnamamides

Because

by the EHMO method.

(trans)

however,

why

3 for X-

ring and the amide m o i e t y

82,

of

it was

configuration

for anticonvulsant

shown

of

of the

bond on the

compounds

81

as

in the range

plane of vinylene do not

Bond:

ring

(25).

82 values

the

of c o m p o u n d

the

amide

side

than

is p r o b a b l y

on the d o u b l e

benzene

(4.1.1),

analogs,

configuration, The

crystal

were m a d e

the

on the opposite

-64 ~ , t h e

activity.

This

for the stable

the benzene

For ~ , ~ - d i s u b s t i t u t e d

as

between

estimated

hindrance

Unfortunately,

From the c r y s t a l l o g r a p h y

that

(82) were

ring.

that

the

The

the

The existence

for the anticonvulsant

between

3.

that

the planes

80 ~ .

~,~-disubstituted

calculations

activity

crystallography

on the Double

9 shows

is

(see

of the vinyl m o i e t y caused

decreased.

a suitable

activity,

steric

X-ray

of s u b s t i t u e n t s

of compounds.

6, q u a n t u m

benzene

study.

effect

(22).

conformation

simple

3.

the angle between

~,~-Disubstitution

anticonvulsant studied

the

to o b t a i n

the i m p o r t a n t

planes

that

compounds

4 has greater

dramatically

ray crystallographic

planes

two

group at the ~-position

not p o s s i b l e

The

these

compound

showed

its a c t i v i t y was

(24)

i/i0 that of compound

ring and the side chain is about

the isopropyl

class

8 is about

between

group

the a c t i v i t y

to note that the anticonvulsant

4 of Table

difference

isopropyl

the

m a r k e d l y decrease

8).

16 in range,

with

the

of these compounds The anticonvulsant configuration

are

336 probably these

to

a

molecules

dipole role

of of

Z(cis) only the

due

and

the the

similarity

amide

difference

about

of

but

between

42 ~ , which

ii

and

bond

carbonyl

8 I.

cyano

compounds

s-C-halogen

configuration,

value

of

should

the

cyano take

Compound

7

not

to

be

be

and a

a

is

and

the

of

the

in

the

part also

compounds

for

In

activity.

critical

exceeded

groups. group

convulsant

compound

seems

carbonyl

probably

possesses

this

There

13,

could

group. it

and

3

and

value

the

The

for

5

is

81

of

anticonvulsant

activity. TABLE 9 Configurations, torsion activities of ~,~-disubstituted butylamines

01

angles, and anticonvulsant 4-chlorocinnamoyl-sec-

02

D

No.

RI

R2

i 2 3 4 5 6 7

H Me Me Et Et n-Pr n-Pr i-Pr i-Pr Br Br Cl Cl Me Me n-Pr Et Me

CN CN CN CN CN CN CN CN CN Br Br Cl C1 Et Et Et Me Me

8

9 I0 ii 12 13 14 15 16 17 18

a. In Balsamo

and

activities show

CNS

Configuration

to

the

coworkers

Torsion Angle 8 8 1 2

E(trans) E(trans) Z(cis) E(trans) Z(cis) E(trans) Z(cis) E(trans) Z(cis) E(trans) Z(cis) E(trans) Z(cis) E(trans) Z(cis) E(trans) E(trans) E(trans)

6.08 5.46 6.00 5.36 6.01 5.36 6.04 5.38 5.75 5.34 5.90 5.70 5.50 4.72

With convulsive

addition

dimethyl

IHNMR NH(ppm)

14 ~ 21 ~ 17 ~ 40 ~ 34 ~ 47 ~ 44 ~ 54 ~ 52 ~ 20 ~ 20 ~ 17 ~ 17 ~ 9~ i0 ~ 52 ~ 42 ~ ii ~

the

depressant

ED50(MES) mmol/kg

- 31 ~ -105 ~ 103 ~ -107 ~ i00 ~ -105 ~ 105 ~ -115 ~ i00 ~ _ 90 ~ 81 ~ - 64 ~ 104 ~ - 31 ~ 30 ~ _ 95 ~ - 45 ~ 34 ~

0.417 0.362 a 0.148 a 0.344 a 0.188 0.082 a 1.314 a 0.146 a 1.314 a 0.253 a 0.146 0.223 0.532 0.506 0.716 a 0.593 a 0.197 0.113

action. results

(7)

have

described

central

that

display

nervous

activity,

above

indicated

cinnamoyl-monoalkylamines on

u

system

whereas

the

(Section the

E-

quite

(CNS). Z

isomers

4.1.1),

and

Z-~,~-

different The

E

cause

forms CNS

337 stimulation.

are c o n s i s t e n t

not.

Results with

The convulsant

drastic

The

isomer

of

the

field,

benzene

for c o m p o u n d

ring

and

amide

group

linkage.

in Table

9 (22).

X-ray

showed

5.

that

the

RECEPTOR

is in the

crystallography

In

summary

of

conclusions: The

adjusted

by

nitrogen. (2) The bulk,

and

the

we

should

substituents

product

moiety

activity.

amide

This

come

possess

on

the

The

opposite least

of the values

distorted On

receptor

too much basis

for

and

of the double

carbon

the

possessing

atoms.

benzene

and

ring

of

the

amide

the

above

interaction this

ring

that

binds,

the

from the hydrophobic

be small

almost

sites.

available

space

be

in in of

the

must

group

on

be separated

the

by at

not

linkage.

be

hypothetical

a planar

structure

There is a hydrophobic which might also exert

with the substituted area,

lengths

the amide

should

conclusions,

hydrophobic

amide

anticonvulsant

moieties

to the benzene moiety,

In the

of

the

group

two

has

high

can be

and/or

should

bond and should

These

compounds

a charge-transfer benzene

the

following

electron-withdrawing

influences

at least three binding

region

ring

from the plane of the vinylene

these

area c o r r e s p o n d i n g

Apart

on

activity

the

comparatively

to the consideration

ring

to

of C=O and C-N bond

significantly

benzene

sides

two

are

Their l i p o p h i l i c i t y

on the b e n z e n e

hydrophobic

leads

can

binds with the receptor. (4)

group

(25).

structure-anticonvulsant

(log P = 3.82 ~ 4.35).

comparatively

amide

the

linkage

cinnamamides,

substituents

(3) The

the

of

compounds

lipophilicity

nature.

ring

also

MAPPING

relationships (i)

benzene

sides of the vinylene

was

That of the E

of E - N - ( ~ - c y a n o - ~ - n - p r o p y l - 4 - c h l o r o - c i n n a m o y l ) - s - b u t y l a m i n e opposite

planes

cinnamamides

shifts of the NH proton.

field and that of the Z isomer

as shown

9

16 is

16 is p r o b a b l y due to the

of ~ , ~ - d i s u b s t i t u t e d

by the chemical

is in the lower

higher

but that

and ethyl groups on the vinylene

configuration

assigned

14, 15, 17 and 18 in Table

results,

effect of compound

distorsion

caused by propyl

for compounds

their

where is

benzene

the

somewhat

area at a proper distance,

ring.

substituted narrow.

there is a

338 binding

carbonyl

there

site w h i c h

is

hydrogen

group

of the

a site

The

a dipole-dipole

ligand.

which

bonding.

Fig.

exerts

can

group

atom

of

(22).

CONCLUSION

was

is probably

not known

fact

that

piperine

before

cinnamamides

the active

without

knowing

piperidine

were started.

modification

studies

The m o d i f i c a t i o n

cinnamoyl)-piperidine

than

them

the

i.

probably

E-s-halo-

anticonvulsant

activity

of this

antiepileptic

activity of

of piperine

Among

12,

14,

6, and

26,

is n o w

under

the

structural

of N - ( 3 , 4 - m e t h y l e n e d i o x y has

approach, the

than and

200

ring.

preclinical

studies

studies. have

reached

effect

of

on the

on the a n t i c o n v u l s a n t synthesized,

2, c o m p o u n d s

7 in T a b l e

From the economic

extensively

Substitutions

cinnamamides

38 in Table

1 and

been

we quickly

crucial

and c o n f o r m a t i o n

29,

of

N-(3,4-methylenedioxycinnamoyl)-

revealed

compounds

relationship

just b e f o r e

for the benzene

more

activity

to

was

We found this out only during a

(antiepilepsirine)

configuration

can

in place of the

anticonvulsant

of the structure

linkage

antiepilepsirine.

activity

in Fig.

interaction

The m o d i f i c a t i o n

literature

of

substituent

vinylene

in T a b l e

region,

the reason why

component

By the aid of the Hansch

the optimum

compounds

that

had been reported.

r e v i e w of the

activity.

by

but the anticonvulsant

had been reported.

systematic

molecular

nitrogen

N-(3,4-methylenedioxy-cinnamoyl)-piperidine

cinnamamides

the

the

activity.

possesses

folk m e d i c i n e was discovered,

studied.

are illustrated

E-~-halo-cinnamamides

This

with

dipolar

amide

region of the receptor

show anticonvulsant

6.

designed

the

Model of c i n n a m a m i d e - r e c e p t o r

halogen

generate

to this

with

dipol~

I.

cinnamamides The

Close

These situations

interact with the dipolar carbonyl

bind

interaction

7 are m o r e

point

of view,

Moreoever,

provided

1 and 5 potent

one of

structure-

us w i t h

a better

339

understanding receptor.

REFERENCES

of the interaction

between cinnamamide

and its

1 Epilepsy Clinics, People's Hospital. Beijing Medical College Affiliated Hospital, Journal of Beijing Medical College, 1974, 6: 214. 2 Faculty of Pharmacology, School of Basic Medicine, J. Beijing Medical College, 1974, 6; 217. 3 Pharmaceutical Factory of Beijing Medical College, J. Beijing Medical College, 1974, 6: 221. 4 Yin-quan Pei, Jia-shan Li, Zhi-ji Cai, Bao-heng Zhang, Cheng Tao, Bao-shan Ku, J. Beijing Medical College, 1977, 9: 234. 5 Epilepsy Clinics, People's Hospital, Beijing Medical College Affiliated Hospital, Chinese J. Internal Medicine, 1977 (new), 2: 321. 6 Xiao-hui Zhang, Ren-li Li, Meng-shen Cai, J. Beijing Medical College, 1980, 12: 83. 7 A. Balsamo, P.L. Barili, P. Crotti, B. Macchia, F. Macchia, A. Pecchia, A. Cuttica, and N. Passerini, J. Med. Chem., 1975, 18: 843. 8 C. Rappe, Acta Chem. Scand. 1964, 18: 818. 9 A.J. Speziale and C.C. Tung, J. Org. Chem., 1963, 28: 1353. I0 J.G. Topliss, J. Med. Chem., 1972, 15: 1007. ii Shu-yu Wang, and Ji-cang Zhou, J. Beijing Medical College, 1982, 14: 65. 12 Shu-yu Wang, Ren-li Li, Wei-qin Liu, Ping Liu, Jing-mei Song, Yin-quan Pei, Hai-yan Yao, Xue-min Gao, Acta Pharmaceutica Sinica, 1986, 21: 542. 13 An-liang Li, Wei-qin Liu, Yin-quan Pei, Shu-rong Zhang, and Chen Xu, Acta Pharmaceutica Sinica, 1984, 19: 888. 14 Ren-li Li, and Shu-yu Wang, Acta Pharmaceutica Sinica, 1986, 21: 580. 15 A.J. Hopfinger, J. Am. Chem. Soc., 1980, 102: 7196. 16 Shu-yu Wang, Ren-li Li, Wei-qin Liu, Xiao-jie Xu, Yue Guan, Chinese J. Org. Chem., 1988, 8: 217. 17 Xiao-hui Zhang and Ren-li Li, J. Beijing Medical College, 1985, 17: 225. 18 Wei-qin Liu, Ji-cang Zhou, An-liang Li, Cheng Bi, J. Beijing Medical College, 1984, 16: 62. 19 Shu-yu Wang, Gui-qi Wang, Gao-yun Hu, Song-jing Cai, Jingmei Song, Wei-chin Liu, Cheng Tao, Acta Pharm. Sinica, 1987, 22: 420. 20 Shu-yu Wang, Wei-qin Liu, Ren-li Li, Chemical J. Chinese University, 1987, 8: 813. 2 1 S h u - y u Wang, Wei-qin Liu, Yin-fen Wang, Jing-min Song, Cheng Tao, Xiang-fang Zhou, J. Beijing Medical University, 1988, 20: 297. 22 Shi-qi Peng, Wei-qin Liu, Yin-quan Pei, Su-ming Chen and Fang Guo, Acta Pharm. Sinica, 1986, 21: 20. 23 Shu-yu Wang, Dong Wang, Liang Qiao, Ying-fen wang, Ji-hai Pang, Wei-qin Liu, Ren-li Li, Chinese J. of Microwave and Radiofrequency Spectroscopy, 1987, 4: 355. 24 Wei-qin Liu, Ji-cang Zhou, and Xiao-ping Lei, Acta. Pharm. Sinica, 1983, 18: 912. 25 Ji-cang Zhou, Shi-qi Peng, and Wei-qin Liu, J. Mol. Sci. (Wuhan, China), 1986, 4: 187.

This Page Intentionally Left Blank

QSAR and Drug Design - New Developments and Applications T. Fujita, editor 9 1995 Elsevier Science B.V. All rights reserved

STRUCTURAL ANTAGONISTS

REQUIREMENTS

341

OF L E U K O T R I E N E

HIROSHI TERADA, SATORU GOTO, HITOSHI HORIt and ZENEI TAIRA$

Faculty of Pharmaceutical Sciences, University of Tokushima, Shomachi-1, Tokushima 770, Faculty of Engeneering, University of Tokushima, Minami-josanjima, Tokushima 770, :~Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashirocho, Tokushima 770, Japan

A B S T R A C T : The structural characteristics of benzanilide derivatives having alkyl or alkoxyl side chains on the benzoyl benzene ring and tetrazolyl oxygen-heterocycles condensed with the anilide benzene ring as leukotriene antagonists were examined in comparison with those of leukotrienes. The three-dimensional arrangements of the conjugated benzamide moiety, tetrazole and benzopyranone or benzodioxane ring, similar to those of the triene moiety, peptide carboxylic acid and cysteine residue of leukotrienes, respectively, were very important for the anti-leukotriene activity. Effective lengths of alkyl or alkoxyl chains in dynamic conformational equilibria were estimated by taking all possible conformations of these flexible chains into consideration. The effective lengths and conformations which are similar to those of the w-chain of leukotrienes were required for the maximal antagonist activity, other things being equal. The terminal aliphatic carboxylic acid of leukotrienes was suggested to be essential for the agonistic activity. 1.

INTRODUCTION

In development of potent bioactive compounds, it is very important to consider both conformational characteristics and their physicochemical properties. Usually structural similarities of compounds are discussed in terms of their stable conformations in their minimum energy states, and the molecular design of more potent compounds is sometimes based on fixing their flexible conformations in their stable conformations. For instance, some platelet-activating-factor (PAF) antagonists were designed from the conformation of PAF by fixing its flexible moieties as shown in Fig. 1.(1,2). However, the flexibilities of bioactive compounds are sometimes very important for their biological activities, as exemplified by those of weakly acidic uncouplers of oxidative phosphorylation. Uncouplers inhibit ATP synthesis by oxidative phosphorylation in energy transducing membranes such as mitochondria and chloroplasts. Most

342

R

H-(~-OCH2"CH2-CnHm CH3-CO-O-(~H CH2O-[PC]

Fig. 1. Designof PAF-antagonists by fixing the flexible structures of PAF and 1S-methyl-PAF. Two possible conformations are illusA c - O X ~'-"CnHm trated. The left one is based CHs-Q',[pc] on the structure of dioxanone derived from the conformation of PAF fixed as a "decaline". This compound was inactive. The right one is CH2O~ based on an assumed active O=~-Ox conformation including a fuHO ICH2)2 ran ring. This compound was t~SS~ active as the PAF-antagonist. [PC] 9phosphocholine.

RfH, PAF

R = CH3, LS-methyl-PAF

CnHmX.........-r~

o

g

C13He7~o '

uncouplers are hydrophobic weak acids, and they act as protonophores carrying H + from outside to inside the H+-impermeable mitochondrial m e m b r a n e and from inside to outside chloroplasts, in which the orientation of membrane proteins is the reverse of that of mitochondria. Thus, uncouplers make the H+-imperlneable membranes leaky to H + by their protonophoric action, and dissipate the H+-motive force that supports ATP synthesis by oxidative phosphorylation, as shown diagrammatically in Fig. 2(3,4).

I

H*-circuit

O~ 0---

Respiratory Chain ~iiiiiiiiiiiiii!iii~

~.0 .,,, 0

U'~-- U" ~ uncoupler ~--_

H§

_

)

~ H§

ATP

H+

--~H

+

9

Fo-F~-ATPase

UH -~.UH

Fig. 2. Oxidative phosphorylation and uncoupling (/eft), and uncoupling caused by a weakly acidic protonophore (right). The uncoupler is shown as U, and U- and UH indicating its anionic and neutral forms, respectively.

343 The protonophoric action of uncouplers is thought to be achieved by the shuttle mechanism shown in Fig. 2, in which the anionic form of the uncoupler U- traps H + at the membrane surface, becoming the neutral form UH, which is far more hydrophobic than U-. At the other membrane surface, UH releases H +, and becomes U- again, which returns to the original membrane surface. This cycle of the uncoupler in the membrane causes transport of H + across the membrane, the efficiency of the cycle being governed by the stability of U- in the hydrophobic biomembrane. Electron delocalization of the polar ionic charge of the U- is expected to be favorable for stabilization of U- in the membrane(3,4). In the case of a very powerful weakly acidic uncoupler SF6847 (3,5di-tert-butyl-4-hydroxybenzylidenemalononitrile) and its homologs, NMR spectra and molecular orbital calculations have indicated that the malononitrile moiety shows restricted rotation relative to the phenyl ring, and that this rotation regulates the electron-withdrawing ability of the malononitrile moiety, i.e., the coplanar conformation of the two is favorable for delocalization of the ionic charge (Fig. 3). Furthermore, alkyl groups ortho to the phenolic OH were found to regulate the rotation of the malononitrile moiety, bulkier groups restricting the rotation of the malononitrile moiety more and causing a more planar conformation, resulting in increase in the electronwithdrawing ability of the malononitrile moiety. This effect is directly related with the uncoupling activity, because the activation energy of the rotation of the malononitrile moiety is linearly correlated with the uncoupling activity(5,6). Thus, "flexibility" of the malononitrile moiety is very important for exhibition of uncoupling activity. From the above examples, we became interested in the role of "flexibility" of certain substituents and substructures in the biological activity. In this article, we examine the structural characteristics of benzamide type leukotriene(LT)-antagonists with special attention of their flexibility, and attempt to deduce the structural requirements of LT-antagonists.

cN

Fig. 3. Intramolecular rotation of the malononitrile moiety relative to the phenyl ring of a potent uncoupler SF6847 and its "diortho" homologs.

344 2.

DEVELOPMENT

OF

LT-ANTAGONISTS

Peptido-LTs such as LTC4, LTD4 and LTE4 (for chemical structures, see Fig. 4) have various biological effects, such as bronchoconstriction, increase of microvascular permeability, ileum contraction, and tracheal edema, and possibly mediation of asthma(7). As their antagonists should be therapeutically effective in treatments of allergic asthma and other hypersensitivity diseases, for many years, attempts have been made to develop their potent antagonists(8-22). The slow-reacting substance of anaphylaxis (SRS-A) has been found to be a mixture of LTs, so effective LT-antagonists are developed by modifications of typical SRS-A antagonistic hydroxyacetophenones (HAP) leading to FPL-55712(8,9) and others. Among them, the most commonly used antagonist is probably LY-171883(10), while L-649923 is under clinical trial(ll). Another approach in developing LT-antagonists is modifications of the chemical structures of LTs(12-15). SK&F-101132(12), SK&F-104353(13) and U19052(14) are representative examples of LT-antagonists in this category. There are other effective antagonists that contain neither the HAP moiety nor structural features similar to LTs. Examples are quinoline derivatives (L-660711)(16-18) and ICI-198615(19,20). These compounds are classified as the third category of LT-antagonists. The chemical structures of representative LT-antagonists are shown in Fig. 5. Trials have also been made to fit antagonists into the LT receptor(s) by introduction of a partial structure of LTs into antagonistic quinoline derivatives(17) and other compounds belonging to the third category(19,21). Toda, Nakai and coworkers(22,23) found that benzamide derivatives such as ONO-1078 (cf. Fig. 5) show anti-LT activities. The structures of these compounds have no superficial similarity with those of LTs, although 11

cj

~ii~i~i~i~!~i~iii~i~!~i~ii~i~i~i~i~!:i!i~i~i~!i~i~i~i~:!~!~i!i~i~i~i~i~i]

7]-iii! ~ H

:CO0~ i !]

~'@~:,II~ [,,~i

~.~Nh.'....

~!~~:~

~..',~.!E.,',.~: ,~

i:'.:~.9.'.:~'~.,,.,.'~..~.~,~}. :~...~.:~i:.~:,t~

hvdro hilic re ion

y Glu I

R = Cys-Gly

LmC4

:

LTD4

: R = Cys-Gly

LTE4

: R = Cys

hydrophobic region

Fig. 4. Peptidoleukotrienes (LTs). R: peptide moiety.

345 0

nPr

OH

CIy~'~ N ~ S ~ N ( C H a ~ S-Cys-Gly

nPr

FPL-55712

SK&F 101132

HO~.~.O~/~S~ .P,

CS~

~

~

1_,-649923

L-660711

~"

~ o

H

~I._~

CH30

~Lcoo H

SK&F 104353

ICI 198615

OH

LY171883

U-19052

COOH

N=N

ONO-1078

Fig. 5. Representative LT-antagonists. HAP-derivatives are shown in the left column, LT-analogs in the middle column, and compounds of the third category in the right column. Nakai et al.(23) designed these compounds to contain similar hydrophobic and hydrophilic regions to those of LTs. These compounds also belong to the third category of LT-antagonists. We were interested in the structural requirements for the anti-LT activities of benzamide type LT-antagonists, because, like LT(s), they contain a flexible alkyl chain. Thus, we believed that the structural features of benzamides including the molecular flexibility are very important to rationalize why they show antagonist activities. We analyzed benzamides, such as N-(4'-substituted benzoyl)- 8-amino2-(tetrazol-5"-yl)-benzodioxane (BDs, c/. Fig. 6), and N-(4'-substituted benzo-

BDs

o / ~ ,

/

~

BPs

N

O

o~ N

O

R~ N'-N

BDMs R9 BDOs " R -

CnH2n+l OCnH2n+l

I.

i I N=N

BPMs "R BPOs "R -

CnH2n+l OCnH2n+l

Fig. 6. Chemical structures of benzamide type LT-antagonists.

346 yl)- 8-amino-2-(tetrazol-5"-yl)-4H-l-benzopyran-4-one (BPs, cf. Fig. 6). In these derivatives various alkyl or alkoxyl chains are introduced at the C(4~) position of the benzoyl ring; for simplicity, we refer to their alkyl derivatives as BDMs and BPMs, and to their alkoxyl derivatives as BDOs and BPOs. The structures of these derivatives are shown by the number of C atoms (n) of the alkyl (-C,H2n+I in the BDM and B P M series) or alkoxyl (-OC,H2.+1 in the BDO and BPO series) group at the end of BDM, BDO, B P M or BPO; e.g., BPM3 represents B P M with a propyl chain. The length of the alkyl or alkoxyl group is also shown by the number of C atoms, such as C3 for the propyl group, and OC3 for the propoxy group. The inhibitory activities of these compounds, determined as their 50% inhibitory values (P/D0) against guinea pig ileum contraction induced by LTD4 determined by Nakai et al.(23), are summarized in Table 1. Table 1. p Is0 Values and physicochemical parameters of benzamide type LTantagonists. Antagonist BDM6 BDM7 BDM8 BDM9 BDM10 BDO5 BDO6 BDO7 BDO8 BDO9 BPM6 BPM7 BPM8 BPM9 BPO6 BPO7 BPO8 BPO9

pls0 ~ 6.68 7.15 7.00 6.80 6.30 7.00 7.89 8.05 7.85 7.70 7.82 8.38 8.26 8.15 9.07 9.30 8.26 8.13

Lb 12.44 13.38 14.49 15.43 16.54 12.33 13.27 14.40 15.34 16.46 12.44 13.38 14.49 15.43 13.27 14.40 15.34 16.46

B4 r 5.87 6.39 7.33 7.85 8.79 5.73 6.23 7.16 7.66 8.60 5.87 6.39 7.33 7.85 6.23 7.16 7.66 8.60

7r d

DL ~

5.26 5.80 6.34 6.88 7.42 4.25 4.79 5.33 5.87 6.41 5.26 5.80 6.34 6.88 4.79 5.33 5.87 6.41

-1.290 0.252 0.241 -0.447 -0.929 -1.363 0.313 0.178 -0.466 -0.951 -1.290 0.252 0.241 -0.447 0.313 0.178 ,0.466 -0.951

a: The pls0 values were means for several runs, and their deviations were about +0.04(10%) (T. Miyamoto, personal communication), b: STERIMOL parameter representing the chain length(24), c: STERIMOL parameter representing the maximum width of the chained substructure(24), d: Hydrophobic substituent constant, e: DL is defined with eqs. 4-7(see text).

347

3.

Q S A R A N A L Y S E S OF B E N Z A M I D E NISTS

TYPE

LT-ANTAGO-

We first performed the Hansch-Fujita analysis for the pI50 values of BDs and BPs, and found that the correlation in terms of the Verloop's STEPdMOL parameter L(24) was most significant. p l h 0 - -25.945 0.163 L 2 + 4.650 L + 0.770 Io + 1.081 /BP (• (+0.087) (J:2.522) (4-0.291) (:t:0.295)

(1)

(n - 18, r - 0.952, s - 0.283)

In eq. 1, Io is an indicator variable taking a value of 1 with alkoxyl groups and 0 with alkyl groups, and IBp, another indicator variable, is 1 with benzopyranone derivatives (BPs) and 0 with benzodioxanes (BDs). Values in parentheses represent 95% confidence intervals, and n, r and s are the number of antagonists used in the analysis, the correlation coefficient and the standard deviation, respectively. Eq. 1 shows that pI50 depends on the length of the alkyl or alkoxyl chain in a parabolic manner. The optimum value of the chain length L of the alkyl or alkoxyl group was 14.24, corresponding to C8 for the alkyl group and to OC7 for the alkoxyl group. The STERIMOL parameter B4(24) gave the same correlation as that with L because of the colinearity between L and B4. The anti-LT activities were analyzed in terms of the hydrophobic substituent constant 7r of the alkyl groups (BPMs and BDMs) and the alkoxyl groups (BPOs and BDOs). A significant correlation, but slightly poorer than that in eq.1, was obtained as shown in eq. 2. p I 5 0 - -0.574 0.243 7r2 2.732 7r + 0.669 Io + 1.079 /BP (+4.031) (+0.119) (+1.395) (+0.198) (+0.182)

(2) (n -- 18, r -- 0.916, s = 0.370)

Because of a high colinearity (r = 0.809) also between ~ and L or B4, it was difficult to decide which of hydrophobic or steric effects of side chains is really significant in governing the variations in the pI50 value. In eqs. 1 and 2, however, the value of the coefficient with the Io indicates that an alkoxyl group is about 5 times more favorable than an alkyl group of the same chain length for high activity. Furthermore, the value of the coefficient with IBp

348 shows that the benzopyranone ring is more than 10 times as effective as the benzodioxane ring. As the van der Waals volumes of these two rings are about the same, some other factor(s) seems important. Thus, it was necessary to clarify why the alkoxyl groups are more favorable than the corresponding alkyl groups, why the benzopyranone ring is more effective than the benzodioxane ring, and the role of alkyl and alkoxyl chains in the LT-antagonist activities of benzamides. 4.

STABLE

CONFORMATIONS A N D LTS

OF

BENZAMIDE

TYPE

ANTAGONISTS

To obtain more detailed information about structural requirements for the anti-LT activities of the benzamides, the most stable conformations of these antagonists were compared with that of LTE4. We used LTE4 as a representative LT because of its structural simplicity. The initial arbitrary configurations were generated from the partial structures of antagonists and LTE4 determined by X-ray crystallography taken from the Cambridge Structural Database of the Computer Center, in the University of Tokyo. The most stable conformations were first estimated by optimizing the conformations that showed the lowest van der Waals potential by the Giglio function(25) among conformers generated randomly by rotating all their single bonds. Energy minimization of these conformers was performed by molecular mechanics with MM2PRIME(26-28).Molecular superimpositions on LTE4 of the antagonists in their stable conformations were performed with the molecular modeling program FREE-WHEEL(29). Ball and stick models of the stable conformers of BPO7 and LTE4 are compared in Fig. 7(30). The atoms O(1), C(8), amide carbonyl-C, C(2') and O(a) of BPO7 were found to correspond to the S atom of the sulfide bond, C(7), C(9) and C(ll) of the conjugated triene moiety, and the C(14) atom of LTE4, respectively. Namely, the conjugated benzamide moiety seemed to be equivalent to the conjugated triene moiety, and O(1) in the benzopyranone ring and the C-NH moiety of the tetrazole ring of the antagonist superimposed well on the S atom and the COOH group of the peptide moiety (peptide COOH) in LTE4. However, there was no structural moiety in BPO7 that fitted the terminal aliphatic COOH group of LTE4. Furthermore, the conformation of the alkoxyl group at the 4~-position of the benzoyl ring of BPO7 was quite different from that of the w-chain of LTE4. These features are summarized two-dimensionally in Fig. 8. Similar structural characteristics to those of BPO7 were also observed in BDMS, BDO7 and BPMS.

349

aliphatic terminal COOH

conjd, polyene Q

13 ~r 14

~(

d~

o/ u

/ "~ co-chain

%

peptide-COOH

~ ~@~r~ol]@

~Okogy$ro~

Fig. 7. S u p e r i m p o s i t i o n of B P O 7 on LTE4. Closed and open circles r e p r e s e n t LTE4 and B P O 7 molecules, respectively. The n a m e s of moieties of B P O 7 are shown in outline-font .

conjd, triene

~~"

terminal COOH

, ,~.~::::::~ sulfur l

co-chain

HOUN("

~=N

peptide-COOH

Fig. 8. Two-dimensional comparison of the s t r u c t u r e of B P O 7 with t h a t of LTE4. Broken and solid lines represent the s t r u c t u r e s of LTE4 and B P O 7 , respectively, and open arrows indicate moieties or a t o m s of LTE4 t h a t fit those of BPO7. Closed arrows show moieties of LTE4 t h a t do not fit those of BPOT. R e p r o d u c e d from S. Goto, Z. R. Guo, Y. Futatsuishi, H. Hori, Z. Taira and H. Terada, J. Med. Chem. 35(1992)24402445, by permission of t h e A m e r i c a n Chemical Society.

350 5.

CONFORMATIONS OF BENZOPYRANONE ZODIOXANE RINGS OF BENZAMIDE ANTAGONISTS

AND TYPE

BENLT-

To understand why the antagonists that contain a benzopyranone ring have 10 times higher activity than those that contain a benzodioxane ring in spite of their similar hydrophobicities and the van der Waals volumes, we examined the conformations of these rings. 8-Amino-2-(tetrazol-5 ~yl)-derivatives of benzopyranone and benzodioxane were used as models of BPs and BDs, respectively. We determined the most stable conformations of these compounds, taking into consideration their conjugated structures, by molecular orbital calculation using the semiempirical AM1 method with MOPAC(31). Fig. 9 shows dot surface models of the stable conformations of these compounds(30). Interestingly, the tetrazole and benzopyranone rings are almost coplanar (A), but the tetrazole ring is not coplanar with the benzodioxane ring (B). Thus, the conformational coplanarity of the former two rings may be favorable for the C-NH moiety of the tetrazole ring to be located in a similar position to that of the peptide COOH of LTs. This ,~.-~. ,. ,~i-::~.:..'::;:~:.~::+*: ~i!~:. :.

.,,.~.~-~.~....

~;.-'.~:.'.:',':.:.:~::~':+:..

9 - .......

....~(.:: . . : : . -::.~ ~:!4~... " : : 9":..:::::: . . . . :"::.:::::':::::"~'

;ii:/i::~.i;i:::/•

.:-~:::,:-""- .:,.'~,."..~.,:~:..~i!.!~:~ :'"-'::/." ":-:':::::~!!:-."!~".~;':i~:'.,

..... ! : : : : ! ~ ; . ~

!i~-!~',~%

9........

, ....

.;.~".~.-::.-:2.2,..::i::-,.zi:. ...r

:

A.'..-" 9 :. : ,".' .'. .....

...<,;. ............

!~:~".!-%":~.".'::'.':'~.:!'~:~:!.'.':i:~:!:~i~.'~.. . .~i..`.!.;i..$!~.~..*.:...`;.~!~!i...*..i!~!i.*...iii~i~...*i..:!.....*..~.~..:.

..----~

~..*.~.~~'::'.:.-.--...~.~e..,.

~

....:.:........:.:.:......

:;;

:i~i~..:~:~*.:~_~i..'.:~/.~

~.~i :: :~,.~:::!~i~::::::::i~:: . . . : ~ : ~ ~ 9

.

.

i"*:~;~~:~i~

9 9 ." : -" 9 ..'.....~... ". 9 9 "." ..'" .'.",

~:. . : .-,. ~ . . /

'. . . . . . . . . .

.:,,~

9..

~"~'2~.:;~".":'::

--.:::;i"'.:.:"'..g~::".":: :: .:~~ :J:::":!i! : / . ~ . : : : ::: : " : : : : : i ! : : : : ~ ~ " : : ~ ' ~

i.,..::~.:::.~!.~!$~,..

.......

,.

~":"" :"" ' " " : "

""""

~ .....

~ii~ii~- 9~. / ~ i i ~ ! ~

":-~'/.!-.(~

4"i.

"."

. . . . .

".17.".

"""

~

".'..'.':~',:':-.,,',.~':"

Fig. 9. Dot surface models of the stable conformer of 8-amino-2-(tetrazol-5'-yl) derivatives of benzopyranone (A) and benzodioxane (B). Upper models show sideview conformations, and lower models top-view conformations. Shaded moieties represent the tetrazole ring.

351 conformational difference could be one reason why antagonistic activities than the corresponding BDs. as the carbonyl group at C(4) and the double bond in the benzopyranone ring, being able to conjugate could be favorable for the coplanarity in BPs.

0

BPs showed higher LTSuch structural factors between C(2) and C(3) with the tetrazole ring,

ROTATIONAL FREEDOMS OF ALKYL CHAINS OF BENZAMIDES

AND

ALKOXYL

Eq. 1 shows that an alkoxyl chain is more effective than the corresponding alkyl chain. The difference between these chains could be rationalized by their conformations. We determined the conformational distributions of the alkyl and alkoxyl chains as functions of the torsional rotation angle 0 about the C(4')-C(c 0 bond of the alkyl chain and about the C(4')-O(c 0 bond of the alkoxyl chain. We defined 0 as 0 ~ and 4-180 ~ when the benzoyl benzene ring and the C(a)-C(/3) or the O(a)-C(/3) bond were in the same plane, and as +90 ~ when they were perpendicular to one another. The steric energy ei of the i-th conformation was determined by the empirical molecular mechanical program MM2PRIME(26-28), and the probability of occurrence of the conformation P / w a s calculated by eq. 3 according to Boltzmann statistics(32).

Pi-

exp(-ei/RT)

N

(3)

Z exp(-ei/RT) i=l

In eq. 3, R is the gas constant and N is the number of conformational states generated. The distribution maps of various conformations were drawn by plotting their probabilities as functions of the torsional rotation angle 0 of the C(4')-C(c~) bond or C(4')-O(a) bond of the antagonist. Fig. 10 shows the distribution maps of various conformations of BPM8 and BPO7 as functions of 0. The conformational distribution for BPM8 was almost symmetric with minima at 0 - 0 ~ and 4-180 ~ and maxima at 0 - -t-90~ but interestingly the distribution for BPO7 was just the opposite with minima at about 0 - 5=90~ and maxima at 0 - 0 ~ and =t=180~ These results indicate that the C(a)-C(/3) bond is perpendicular to the benzoyl ring, whereas the O ( a ) - C ( ~ ) bond is in the same plane as the benzoyl ring. Other compounds with alkyl and alkoxyl chains of different lengths gave similar results. No conformational difference was observed between alkyl and alkoxyl chains other than C(c~)-C(/3) and O(c~)-C(/~) bonds. Thus, the conforma-

352

BPM8

BP07

o N.I I

H

~ ~ ~ ~ ~ 0

6"

4-

V

2-

H

.,

.

A

O~ N/i o

~;\

!

./j,~

~1i1 i I! I! t i s i !~ it iiIiY !Yi~, 0 100 200 0 (deg)

-200 -100

J t 1' ~l i til

A

o.

-i~

t"

! I T"

-200 -100 0 100 200 0 (deg)

Fig. 10. Conformational probabilities about the C(4')-C(c~) a n d C(4')-O(c~) bonds of B P M 8 and BPO7, respectively. Pi : probability of the generated conformation i of these bonds, 0 9 dihedral angle. Reproduced from S. Goto, Z. R. Guo, Y. Futatsuishi, H. Hori, Z. Taira and H. Terada, J. Med. Chem. 35(1992)2440-2445, by permission of the American Chemical Society.

tions of these chains were governed by those of the bond between C(4') of the benzoyl benzene ring and the a-atoms. Accordingly, it can be concluded that the a-atoms of alkyl and alkoxyl groups, working as hinges, regulate the rotations of these groups. Possibly, the coplanar conformation allows the alkoxyl chain to take similar conformations to those of the w-chains of LTs more easily than the perpendicular conformation of the alkyl chain. This could explain why BPOs and BDOs have higher anti-LT activities than BPMs and BDMs with corresponding chain lengths. 0

EFFECTIVE L E N G T H S OF F L E X I B L E A L K Y L AND A L K O X Y L G R O U P S OF B E N Z A M I D E A N T A G O N I S T S R E L A T I V E T O T H E w - C H A I N OF LTE4

The result in eq. 1 could show that a certain chain length of the alkyl or alkoxyl group is required for potent anti-LT activity, although the structures of stable conformers of the antagonists are quite different from that of the w-chain of LTE4. As the alkyl and Mkoxyl chains of antago-

353

a

O

Fig. 11. Possible conformations of alkoxyl chains attached to the 4'position of the benzoyl ring of LTantagonists. Reproduced from S. Goto, Z. R. Guo, Y. Futatsuishi, H. Hori, Z. Taira and H. Terada, J. Med. Chem. 35(1992)2440-2445, by permission of the American Chemical Society.

0

nists and the w-chain of LTE4 are flexible, we thought that alkyl and alkoxyl chains may have chances to take an effective length similar to that of the w-chain. To examine this possibility, we determined the probabilities of occurrence of various conformations of alkyl and alkoxyl groups as functions of their chain lengths. The conformational distribution of antagonists was analyzed statistically with 104 conformations, in which the torsional angles of all the rotatable single bonds were varied randomly at 30 ~ increments and their energies were calculated with MM2PRIME(26-28). The conformations in which at least one of the interatomic distances became less than 0.8 I in the computation was omitted because of a possible steric repulsion. Their thermal fluctuations were analyzed at an absolute temperature T of 3,000 K by the Monte Carlo approach. The conformations of the alkyl or alkoxyl chains generated were assumed to be rigid ellipsoids, and their longest axis was taken as their effective length. Some of the possible conformations of the alkoxyl chain are shown in Fig. 11. In the computation, the covariance value A for the i-th conformation generated was defined by eq. 4. m

~ i - Y~luij- < u > 12 j=l

(4)

In eq. 4, vector uij is the coordinate of the j-th atom of these chains on the moment of inertia, vector < u > is the coordinate of the gravity center of these chains (c]. Fig. 12), and m is the total number of O and C atoms in the chain. The distributions of alkoxyl chain lengths are shown in Fig. 13. Then, the effective lengths(RL) of the alkyl and alkoxyl chains were expressed relative to that of the w-chain of LTE4 defined by eq. 5.

354

.~ :--~-x

I

\.,.2~/

,;

7

/

,'

,' , ; ,,

,u2

~

"

v , ~, : '

.

~

~

~

-i,,.'

t ,,~

"~/,.



u5 u6

U'_/

/~ Moment

z

Gravity center

i'.~.

~"~,

, ,'

Fig. 12. Moments of inertia and gravity center < u > of alkoxyl

~.

of i n e r t i a

chains.

(5)

RL = )~LT

In eq. 5, )~LT is the covariance value of the w-chain of LTE4 in its most stable conformation. We took the length of the w-chain in its most stable conformation as the reference, because this chain can be assumed to take the most extended conformation when it binds to its receptor(38). A value of RL - 1 indicates an identical effective length to t h a t of the w-chain at certain 0

9

,

9

,

9

,

3

o~

, k

20 i

i

t

f

10

=:" / -/

CnH2n,1-0

L)

5

:,~ 6 :~: '7 "~......;, !..i i

8

9

10

hi'...

X" - ' ",,.,t-.,".'$,,";>"7%-,: A'Ja-". t ' """ " " " ""-" .t-

..

~::

::"

:: .... "';

20

40

""..:i'"

.... ...

60

Z# Fig. 13. Distribution probability (Pi) of i-th conformation of the alkoxyl chain length as a function of the covariance value ()q). Numbers besides traces represent chain lengths n.

356 1.0

i

0.5

0.0 ,,J

-0.5 -1.0

-2.0

J!

qp

-1.5

i

f 4

5

6

7

8

9

10

Fig. 15. C h a n g e of DL with length of the alkoxyl chain of B P O s . n: C n u m b e r of the chain.

lengths with the w-chain length: the highest similarity corresponds to Fs 1.0 and D L = oe, and 50% similarity to Fs = 0.5 and D L = O. Values of D L are summarized in Table 1. The D L value of the alkyl group of C, was about the same as that of the alkoxyl group of OC,_I, indicating that the feasibilities of these corresponding chains to have an effective length similar to the w-chain are very close. The highest D L values were observed with chains of C7-C8 and OC6-0C7. Fig. 15 shows the change in D L with the alkyl chain length n of BPOs. The D L value was maximal between n - 6 and 7, at which maximal activity was observed. It is noteworthy that the curve was not symmetric, change of D L with n being very steep in the region of less than n - 6 and relatively less in that of more than n - 7, indicating that the flexibility of an alkoxyl chain (and alkyl chain) becomes greater with increase in its chain length.

0

EFFECTS OF EFFECTIVE LENGTHS OF ALKYL AND ALKOXYL CHAINS OF BENZAMIDES ON ANTI-LT ACTIVITIES

It was of interest to know whether antagonists with chains of similar effective lengths to that of the w-chain exhibit potent anti-LT activities. Thus, we analyzed the antagonist activities of these benzamides in terms of DL, and obtained the significant correlation shown by eq. 8. plso-

7.088 + 0.478 D L + 0.735 Io + 1.096 IBp (+0.240) (+0.215) (+0.261) (• (n -- 18, r -- 0.957, s = 0.259)

(s)

355

BPMs

I0.0 7.5

o~

~

__~

--~i

7.5

.o_

-T"I

5.0

BPOs

I0.0

::::::::::::::::::::::::::

..

~.~

. ..

g:::,.:.....::l r ~..::~.~-~:~ ::.. 9 ::.~

~ 5 - 0 F.:i:!:::::~::"

2.5

2.5

-

i !

0

ii i

U,~I ~:~:~:~i.a . . . . . . . . .

0.4

/

i::..

.~.:.. :::~ !

0.8

!

12

!

.6

0 2

0

0

0.4

0.

O

!.2

1.5

2.0

RL

RL

Fig. 14. Conformation probability (P~) of alkyl chain (left) and alkoxyl chain (right) of B P s as a function of their effective lengths relative to that of the w-chain of LTE4. N u m b e r s besides traces are C numbers.

conformations of alkyl and alkoxyl moieties. Some of the results with BPMs and BPOs are shown in Fig. 14. Binomial distributions of conformations were observed, the distributions being the same for BPMs and BPOs with the same chain lengths. To quantify the similarities in the effective lengths of alkyl and alkoxyl groups to that of the w-chain of LTs, we estimated the feasibilities of these chains to take a certain range of lengths similar to the length of the w-chain of LTE4. The feasibility is expressible by summation of the probability of occurrence (P) of conformations in the RL range of 0.8 to 1.2, shown by the shaded area in Fig. 14. The sum of the areas under the distribution curves in this range is referred to as Fs as shown in eq. 6.

(i "0.8 _< RL <_ 1.2)

Fs = E P~ i

(6)

We further defined DL by eq. 7.

Fs

D L - log 1 - - ~ s

(7)

Namely, F s / ( 1 - Fs) was assumed to correspond with a "conformationM equilibrium constant" for the alkyl and alkoxyl chains to adopt a range of effective length similar to that of the w-chain. A greater Fs and a more positive DL are measures of closer similarity of alkyl and alkoxyl effective

357 The positive sign of the coefficient with DL in eq. 8 indicates that the antagonist activity increases as DL increases: similar effective lengths of alkyl and alkoxyl groups to that of the w-chain of LTs are important for their activities. Thus, the feasibility of benzamide LT-antagonist to take a similar effective length to that of the w-chain is concluded to be very important for potent antagonistic activity. It is noteworthy that t h e coefficients with indicator variables Io and/BP in eq. 8 are very similar to those in eq. 1. DL correlated with the STERIMOL parameter L in a parabolic manner with relatively high significance, as shown in eq. 9. DL=-65.140(+20.633)

0.316L 2 + 9.091L (+0.100) (+2.879)

(9)

(n = 18, r = 0.868, s = 0.333)

The correlation of DL with the hydrophobic substituent ~ was poorer. Moreover, the ~ and ~2 terms in eq. 2 were successfully replaced with a single term of DL in eq. 8. Thus, the significance of the DL term is regarded as representing that the dynamic steric effect is of prime importance in governing the variations in the activity. As the conformations of these chains were geometrically quite different from that of LTs (cf. Fig. 7), the roles of these chains in induction of antagonist activities cannot be understood by the use of either singular L or ~ term alone. The present results indicate that, for induction of biological activity of flexible compounds, the stable conformations, in which steric energies are minimal, are not always most effective, and that conformational fluctuation is important for the activities of compounds with flexible groups such as alkyl and alkoxyl chains. 0

STRUCTURAL REQUIREMENTS AND LT-AGONISTS

OF L T - A N T A G O N I S T S

The results in Sections 4 to 8 indicated that the conjugated benzamide moiety, the O(1) in benzopyranone and benzodioxane rings, the C-NH moiety of the tetrazole ring, and the alkyl and alkoxyl side chains of benzamides correspond to the triene moiety, the S atom of the sulfide bond, the peptide COOH and the w-chain of LTs, respectively. Corey's group(33,34) considered the structure of LTs as being composed of hydrophilic and hydrophobic regions, whereas, as shown in Fig. 4, we considered that the structure of LTs involes hydrophobic and hydrophilic regions, and a connecting

358 conjugated triene moiety. The roles of these three structural regions in LTs, and their antagonists and agonists, are discussed below. 9. 1.

Role o f t h e c o n j u g a t e d

triene moiety

The triene moiety of LTs consists of a conjugated system of a C6 unit (Fig. 4). Modifications of the triene moiety of LTs are reported to affect the LT-activity, whereas those at the isolated double bond position are not in LTC4(35). The saturation of one of the three conjugated double bonds had no significant effect on the agonistic activity unless each of the two double bonds left is isolated. The entire saturation of the three conjugated double bonds resulted in almost complete loss of activity. Furthermore, the benzene ring serves as if it is the two conjugated double bonds(36,37). These results suggest that the existence of a conjugated system, either two or three conjugated double bonds, is important for high LT-agonist activity. Structural analysis of LTD4 showed that the length of the conjugated planar triene moiety is 6/~ (38). Nakai et al.(23) reported the activities of cinnamamide type LTantagonists. As summarized in Table 2, the LT-antagonistic activities of the cinnamamides and benzamides are dependent on the position of the substitution of the pentyl (-C5Hl1) and pentoxy (-OC5Hl1) chains introduced into the cinnamoyl and benzoyl benzene rings. The antagonistic activities of compounds with substitutions in the ortho-position (I, III, V and VIII) axe low. In contrast, the activities of those with para-substituents (IV, VII and X) are very high, except that of the p-pentylbenzamide derivative (II). The activity of the m-pentoxycinnamamide derivative (IX) is similar to that of the p-isomer (X), but that of the m-pentoxybenzamide (VI) is about 1/50 Table 2.

P/so Values of benzamide and c i n n a m a m i d e derivatives. 0

R

Tet

Benzarnide (m = O) ortho

-CsH,,

(I):4.40

meta -

para

(II):5.59

-OC5HI, (V):5.03 (VI):5.30 (VII):7.00

Cinnamamide (m = 1) ortho

(III):5.67

meta

-

para

(IV):6.43

(VIII):5.64 (IX):6.82 (X):6.92

359

O ^ ~ H --- COOH O ...--, x" " ..,i~H PiJ.~,F "" "~~'~'''~:)'~ H """ F ~ NF' ~'v'''~'~'-~OH ,'~,]/ ,,,COOH ^

12

F

~

j

L

"]

N

N=N

l. m p

/

9w e a k l y active " active

N

~1=1~

~

[

m : active P

: active

Fig. 16. Comparison of structures of benzoyl and cinnamoyl type LT-antagonists with that of LTE4. For styles of structural formulas, see Fig. 8. of that of the corresponding p-isomer (VII). In general, the activities of the cinnamamides are higher than those of the corresponding benzamides. The comparison of the chemical structures of cinnamamide type antagonists with that of LTE4 in Fig. 16 shows that the benzodioxane ring and the amide conjugated moiety of the antagonists well fit with the polar region and conjugated triene moiety, respectively, of LTE4. Interestingly, the meta-alkyl and alkoxyl chains of cinnamamide type antagonists were found to take similar orientations to those of the w-chain of LTE4, although the para-substituents were better oriented in benzamide type antagonists. This could be one of the reasonings why the antagonistic activities of the meta-substituted cinnamamides shown in Table 2 are higher than those of corresponding benzamides. Furthermore, the conjugated moiety of cinnamamides longer than that of benzamides should afford more flexible adjustment of the cinnamamide antagonists to the LT receptor(s). The antagonistic activities of the benzamides were highly dependent on the position of the substituent on the benzoyl benzene ring. This should be due to the shorter and more fixed conjugated system in the benzamides. Thus, we conclude that the conjugated triene moiety or its similar structure is necessary for both antagonist and agonist activities, and that a longer connecting moiety can afford a more flexible alignment of the terminal alkyl and alkoxyl chains even at the LT receptor sites. 9.2.

Role of h y d r o p h i l i c r e g i o n

The coincidences of the geometric positions of the 0(1) in both the benzopyranone and benzodioxane rings, and the acidic C-NH moiety of the tetrazole ring of benzamide type antagonists with those of the S atom and COOH group of the peptide moiety of LTs, respectively, were shown to be

360

Table 3.

Structures and antagonistic activities of c i n n a m a m i d e type LTantagonists modified in the hydrophilic region (23). Tet: tetrazol-5'-yl. O

~ .~

coo."

Nt~

.~

oo." ooo.

P15o

,ot

. ooo.

0 ooo.

(XI)

(XII)

(XIII)

(XIV)

(XV)

(XVI)

4.85

4.70

5.05

5.52

6.08

7.00

~ Tet

P15o

.

Tet

9 "let

~ Tet

0

9 Tet

Tet

(XVII)

(XVIII)

(XIX)

(XX)

(XXI)

(XXII)

5.16

5.51

6.52

6.00

6.43

7.52

important for antagonist activity in Section 4. These findings are supported by the results of modification of the aromatic amine moiety of cinnamamides (23). As shown in Table 3, introduction of an O(c~) atom at the ortho-position of the anilide ring increased the antagonist activity (compare compounds XI and XII with compounds other than XIX and XX). Fixation of this O(c~) atom by cyclization, the 6-membered ring being more preferable than the 5-membered ring, seems more effective, because this enables the O atom to take the right position relative to that of the S atom of LTs. The dioxane or pyranone ring (XV, XVI, XXI and XXII) can be replaced by the benzene ring (XIX), but its replacement by the pyridine ring is less effective (compare XIX, XXI and XXII with XX), the reason being not clear at present. The conformations of the 0(1) of the benzodioxane and benzopyranone rings and the N(1) of the quinoline ring are similar to that of the aromatic C(1) of the naphthalene ring. Furthermore, the electron density (Muliken charge) of the O(1) and N(1) atoms in these rings are highly negative, but that of the naphthalene C(1) is almost null, as shown in Fig. 17. Rigorous examinations

361 -0.02

o.o,A. ~A.o.oo ",~'~Y "~' ~,

~.o,

\ / N--N -0.03 -0.04

(XXIII)

-0.01

-0.02

-0.07

o.o,A ~ A . ~ o.,A ~o.o, o.o,~, H2N ~-0.01"~ "O/ -O~6 , ~ f o.,, u ~, "~'o.,~'T "n o.,, ~., __., 9

~.,, p ~ , \ / N~----'N -0.03 -0.04

(XXIV)

0.,20 ]

~.,, ~ . o ,

.

i~.,, ~ , ~ _~

.o.,.j ~~

\ / N-~N -0.03 -0.03

\ t N'--N -0.02 -0.04

(XXV)

(XXVI)

Fig. 17. Electron densities (Muliken charge) of 8-amino-2-(tetrazol-5"-yl)naphthalene (XXIII) and heterocycles XXIV, XXV and XXVI as model compounds of the hydrophilic regions of compounds XIX, XX, XXI and XXII, respectively (cf. Table 3). of the characteristics of these atoms are necessary, as we did for the difference of O(1) of the benzodioxane ring and the benzopyranone rings as described in Section 5. The increase in antagonist activity by introduction of the tetrazole ring (XIV, XXI and XXII) is probably due to its C-NH moiety corresponding to the peptide COOH of LTs, as described above. As the peptide COOH is thought to interact with the LT receptor(s) in the deprotonated COOform(34,35), the ionized C - N - of the tetrazole ring should be responsible for the interaction of the benzamide type antagonists with the LT receptor(s). Conceivably, the delocalized electronic structure of the ionized tetrazole ring is important for its interaction with the peptide COO- site of the LT receptor(s), because the ionized tetrazole ring can be a resonance hybrid of various electronic structures. This possibility should also be examined. 9. 3.

Role of the h y d r o p h o b i c region

An alkyl or alkoxyl chain of similar effective length and conformation to those of the w-chain of LTs was found to be favorable for potent LTantagonist activity. As indicated in Section 6, the O(c~)-C(~) bond is more coplanar than the C(c~)-C(~) bond. Thus, an alkoxyl chain is favorable to an alkyl chain for adaptation of a similar conformation to that of the wchain of LTs. The anti-LT activities of benzamide derivatives in which a benzene ring is introduced at the end of the alkoxyl chain are also reported to depend on the chain length, the activities of both BD and BP with C4 (abbreviated as BDOC4Ph and BPOC4Ph) being highest(23). As shown in Table 4, BPOC4Ph is more active than BDOC4Ph, as observed with other BDOs and BPOs, and their anti-LT activities are higher than 10 times those

362 Table 4. pls0 Values of BDs and BPs in w h i c h a b e n z e n e ring (Ph) is i n t r o d u c e d at t h e end of the alkoxyl chain (23).

R -C7 -C8 -OC7 -OC4Ph

BDs 7.15 7.00 8.05 9.43

plso BPs 8.38 8.26 9.30 11.40

BDs Tet

BPs R

Tet

of benzamides of similar alkoxyl chain length (BDO7 and BPO7). Thus, the phenyl ring at the end of the alkoxyl chain is favorable to the corresponding alkyl chain, probably because the aromatic ring can bind more tightly to the LT receptor(s) than the alkyl chain, if the length of the phenylalkoxyl chain is similar to that of the w-chain of LTs. 9. 4.

S t r u c t u r a l f e a t u r e s n e c e s s a r y for L T - a n t a g o n i s t a n d L T - a g o n i s t activities

From these results, the following structural features seem to be required for potent anti-LT activity of benzamides of BP and BD types: 1. A conjugated moiety of similar length to the triene moiety of LTs. 2. An O(1) atom in the benzopyranone and benzodioxane rings allowing a similar conformation to that of the S atom of the peptide moiety of LTs. 3. An acidic tetrazole C-NH moiety allowing a similar conformation to that of the peptide COOH of LTs. 4. A similar conformation of the ionized tetrazole ring to that of the ionized peptide COOH of LTs. 5. A similar effective length and conformation of the alkoxyl group to that of the w-chain of LTs. It can be concluded that these characteristics of the moieties of LTantagonists support the binding to the LT receptor(s), but do not induce the activities of LTs. It is noteworthy that the conformational and electronic structure of the terminal aliphatic COOH group of LTs are decisive for their agonist activities, because no moiety in the benzamide type LTantagonists fits the terminal aliphatic COOH group of LTs. This prediction

363

o

o2;J'

~ S

~

OOH

COOH

[I .~

YM-17690

COOH

HN"1 COOH

F

Fig. 18. Chemical structures of LT-agonist YM-17690 and the LT-antagonist SK&:F-101132 (nor-LTD1).

is supported by the following examples. A derivative of benzamide, YM17690 (Fig. 18), was first discovered as a non-LT type agonist. Its LT-agonist activity was considered to be due to its binding to the same binding site as LTs in the LT receptor(s)(20,39), although its chemical structure is similar to those of benzamide type antagonists including the carboxymethoxy residue (-OCH2COOH) at the ortho-position of the aniline ring. As described above, this residue seems important for binding to the LT receptor(s). The agonist activity is apparently due to the propionic acid moiety (-CH2CH2COOH) introduced at the meta-position of the aniline ring. The conformation of this residue could be similar to that of the terminal aliphatic COOH group of LTs. To certify this possibility, the most stable conformation of YM17690 was compared with that of LTE4 by the method described for superimposition of benzamide type antagonists (Section 4). However, the conformation of the propionic acid moiety of YM17690 did not fit that of the terminal aliphatic COOH group of LTE4 at the most stable conformation. We examined the most similar conformations of these compounds, and found that the conformations shown in Fig. 19 gave the best results, the conformation of LTE4 superimposing well on the most stable confomation of the LT-agonist YM17690 except for its OC4Ph moiety. The value of E[total] of this conformation of LTE4 was 13.90 kcal/mol calculated from the molecular mechanics of MM2, and that of the most stable one was 10.59 kcal/mol. The difference of about 3 kcal/mol between these E[tota~ values was due to the difference in E[vdw]. This difference can be regarded as small, because some energy is consumed by binding of the ligand to its receptor. The terminal Miphatic

364

a-COOH

conjd,polyene

m:l J

%

~.j\.,I..A~r li([~

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(o.po,ion'~'~ (~I?[~~[K~[~|

C O O H group of the a-chain in LTs appears to be decisively i m p o r t a n t for induction of their agonistic activity. A compound, in which the propionic acid residue is introduced at the meta-position of the aniline ring of 0 N 0 - 1 0 7 8

biological activity terminal ...-~. .:;-.:.~-.~" ..,. aliphatic-COOH~;!;;:... i COOH ~ if conjugated triene

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Fig. 20. A possible model of the binding of LTE4 with LT receptor. Shaded areas represent the sites of interaction.

365 (Fig. 5) is predicted to be a very potent LT-agonist. Furthermore, the importance of the terminal aliphatic COOH group of c~-chain for LT-agonistic activity is demonstrated with an LT-antagonist, nor-LT (SK&F-101132, see Fig. 18)(12). Its antagonist activity can be attributed to the c~-chain which is one carbon shorter than that of LTs, other structural features being similar to those of LTs. It is possible that the terminal aliphatic COOH group acts as a trigger to induce LT-agonist activity. To summarize, the hydrophobic interaction of the w-chain, electronic natures of the triene moiety and the S atom of the sulfide bond, and electrostatic interaction of the anionic peptide C O 0 - group support the binding of LTs to their receptor(s), and the interaction of the aliphatic COOH with the receptor(s) triggers LT-agonist activity. The conformation of the LT receptor(s) can be characterized as being able to accomodate: 1) terminal aliphatic and peptide COOH groups being about 10/~ apart, 2) a conjugated triene moiety, and 3) a flexible hairpin conformation of the w-chain. These features are depicted schematically in Fig. 20. REFERENCES

AND

NOTES

1. M. Ohno, S. Kobayashi, M. Shiraiwa, H. Yoshiwara and Y. Eguchi, in: Z. Yoshida, T. Shiba, and Y. Ohshiro (Eds.), New Aspects of Organic Chemistry I, Kodansha Ltd, Tokyo, 1989, pp. 549-560. 2. R. Yanoshita, I. Kudo, K. Ikizawa, H. W. Chang, S. Kobayashi, M. Ohno, S. Nojima and K. Inoue, J. Biochem. (Tokyo) 103 (1988) 815819. 3. H. Terada, Biochim. Biophys. Acta 639 (1981) 225-242. 4. H. Terada, Environ. Health Perspect. 87 (1990) 213-218. 5. K. Yoshikawa and H. Terada, d. Am. Chem. Soc. 104 (1982) 76447646. 6. H. Terada, N. Kumazawa, J. Ju-ichi and K. Yoshikawa, Biochim. Biophys. Acta 767 (1984) 192-199. 7. B. Samuelsson, Science (Wash. D. C.) 220 (1983) 568-575. 8. J. Augstein, J. B. Farmer, T. B. Lee, P. Sheard and M. L. Tattersall, Nature (London) New Biol. 245 (1973) 215-217. 9. R. A. Appleton, J. R. Bantick, T. R. Chamberlain, D. N. Hardern, T. B. Lee and A. D. Pratt, J. Med. Chem. 20 (1977) 371-379. 10. J. H. Fleisch, L. E. Rinkema, K. D. Haisch, D. Swanson-Bean, T. Goodson, P. P. K. Ho and W. S. Marshall, J. Pharmacol. Exptl. Ther. 233 (1985) 148-157. 11. T. R. Jones, R. Young, E. Champion, L. Charette, D. Denis, A. W. Ford-Hutchinson, R. Frenette, J. Y. Gauthier, Y. Guindon, M.

366

12.

13.

14. 15. 16. 17.

18.

19. 20. 21. 22.

23.

24. 25.

Kakushima, P. Masson, C. McFarlane, H. Piechuta, J. Rokach and R. Zamboni, Can. J. Physiol. Pharmacol. 64 (1986) 1068-1075. J. G. Gleason, T. W. Ku, M. E. McCarthy, B. M. Weichman, D. Holden, R. R. Osborn, B. Zabko-Potapovich, B. Berkowitz and A. Wasserman, Biochem. Biophys. Res. Commun. 17 (1983)732-739. C. D. Perchonock, I. Uzinskas, M. E. McCarthy, K. F. Erhard, J. G. Gleason, M. A. Wasserman, R. M. Muccitelli, J. F. DeVan, S. S. Tucker, L. M. Vickery, T. Kirchner, B. M. Weichman, S. M. Miller, M. O. Scott, G. Chi-Rosso, H. L. Wu, S. T. Crooke and J. F. Newton, Y. Med. Chem. 29 (1986) 1442-1452. P. R. Bernstein, E. P. Vacek, E. J. Adams, D. W. Snyder and R. D. Krell, J. Med. Chem. 31 (1988)692-696. T. H. Gieske, J. S. Sabol and R. Raddatz, J. Pharmacol. Exptl. Ther. 254 (1990) 192-197. J. H. Musser, D. M. Kubrak, J. Chang and A. J. Lewis, J. Med. Chem. 29 (1986) 1429-1435. R. A. Galemmo, Jr., W. H. Johnson, Jr., K. S. Learn, T. D. Y. Lee, F. C. Huang, H. F. Campbell, R. Youssefyeh, S. V. O~Rourke, G. Schuessler, D. M. Sweeney, J. J. Travis, C. A. Sutherland, G. W. Nuss, G. W., Carnathan and R. G. Van Inwegen, J. Med. Chem. 33 (1990) 2828-2841. J. Y. Gauthier, T. J. E. Champion, L. Charette, R. Dehaven, A. W. Ford-Hutchinson, K. Hoogsteen, A. Lord, P. Masson, H. Piechuta, S. S. Pong, J. P. Springer, M. Therien, R. Zamboni and R. N. Young, J. Med. Chem. 33 (1990) 2841-2845. D. W. Snyder, R. E. Giles, R. A. Keith, Y. K. Yee and R. D. Krell, J. Pharmacol. Exptl. Ther. 243 (1987) 548-556. R. D. Krell, R. E. Giles, Y. K. Yee and D. W. Snyder, J. Pharmacol. Exptl. Ther. 243 (1987) 557-564. C. A. Catanese, R. C. Falcon and D. Aharony, J. Pharmacol. Exptl. Ther. 251 (1989) 846-851. M. Toda, H. Nakai, S. Kosuge, M. Konno, Y. Arai, T. Miyamoto, T. Obata, A. Katsube and A. Kawasaki, Adv. Prostaglandin Thromboxane Leukotriene Res. 15 (1985) 307-308. H. Nakai, M. Konno, S. Kosuge, S. Sakuyama, M. Toda, Y. Arai, T. Obata, N. Katsube, T. Miyamoto, T. Okegawa and A. Kawasaki, J. Med. Chem. 31 (1988)84-91. A. Verloop, W. Hoogenstraaten and J. Tipker, in: E. J. Ari~ns, (Eds.), Drug Design, Vol. 7, Academic Press, New York, 1976, pp. 165-207. A. J. Stuper, T. M. Dyott and G. S. Zander, in: E. C. Olson and R. E. Christoffersen (Eds.), Computer-Assisted Drug Design, Amer.

367

26. 27. 28.

29. 30.

Chem. Soc., Washington. D. C., 1979, pp. 383-414. N. L. Allinger, J. Am. Chem. Soc. 99 (1977) 8127-8134. E. Osawa, C. Jaime, T. Fujiyoshi, H. Goto and K. Imai, JCPE P015 (1989); Revised by the present authors for FACOM M760. J. Burkert and N. L. Allinger, Molecular Mechanics, Amer. Chem. Soc., Washington. D. C., 1982; S. Wolfe, D. F. Weaver and K. Yang, Can. J. Chem. 66 (1988)2687-2702. Molecular superimositions were performed with the molecular modeling program FREE-WHEEL, (NAA00705 NIFTY-Serve, 1992). Ball and stick models were drawn by the molecular modeling program 0RTEP-II; Revised by J. Toyoda for NEC PC-9801 microprocessor as ORTEPC.

31. Ver.5, J. J. P. Stewart, QCPE Bull. 9 (1989) 10; Revised by T. Hirano, University of Tokyo for HITAC and UNIX machines; Further revised by the present authors for FACOM M760. 32. R. L. Lopetz de Compadre, R. A. Pearlstein, A. J. Hopfinger and J. K. Seydel, J. Med. Chem. 30 (1987) 900-906. 33. J. M. Drazen, R. A. Lewis, K. F. Austen, M. Toda, F. Brion, A. Marfat and E. J. Corey, Proc. Natl. Acad. Sci. U. S. A., 78 (1981) 3195-3198. 34. R. A. Lewis, J. M. Drazen, K. F. Austen, M. Toda, F. Brion, A. Marfat and E. J. Corey, Proc. Natl. Acad. Sci. U. S. A., 78 (1981) 4579-4583. 35. S. Okuyama, S. Miyamoto, K. Shimoji, Y. Konishi, D. Fukushima, H. Niwa, Y. Arai, M. Toda and M. Hayashi, Chem. Pharm. Bull. 30 (1982) 2453-2462. 36. a) P. R. Bernstein, D. W. Snyder, E. J. Adams, R. D. Krell, E. P. Vacek and A. K. Willard, J. Med. Chem. 29 (1986) 2477-2483; b) D. W. Snyder and P. R. Bernstein, Eur. J. Pharmacol. 138 (1987) 397-405. n. Yoshio Z. Shudo of B oac37. N. tire Molecules, Soft Science Publications, Tokyo, 1986, pp. 298-325. 38. R. W. Harper, D. K. Herron, N. G. Bollinger, J. S. Sawyer, R. F. Baldwin, C. R. Roman, L. E. Rinkema and J. H. Fleisch, J. Med. Chem. 35 (1992) 1191-1200. 39. K. Tomioka, T. Yamada, K. Teramura, M. Terai, K. Hidaka, T. Mase, H. Hara and K. Murase, J. Pharm. Pharmacol. 39 (1987) 819-824.

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as

For the sake

values

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to

that

these

of

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steric

A MR(X)

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= MR(X)

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A B5(X ) = B5(X ) - B5(H ).

Table 3 Ca-antagonistic activity and physicochemical parameters of R3-substituted compounds (II) Me0,

CN

Me

Me0~C-(CH

2 )3N (CH2) 3 0 0 ~

MeOr--- R3

Me

PA2 Compd. No.

R3

~

a)

) AMRb) AB5C

11-5 H 0.00 0.00 11-6 Me 0.54 0.46 11-7 Et 1.08 0.93 11-8 n-Pr 1.62 1.39 11-4 iso-Pr 1.49 1.40 11-9 n-Bu 2.16 1.86,, 11-10 iso-Bu 2.03 1.86~! II-11 n-Hex 3.24 2.79~! 11-12 , n-Oct 4.32[! 3.72!! II-13 g) n-dodecyl 6.48t) 5.58t) 11-14 benzyl 2.22 2.90 II-15 (CHg)~OMe-0.32~! 1 57f) 11-16 (CH~i~OEt 0.50t) 2 03f) a) b) c) d) e) f) g)

0.00 1.04 2.17 2.49 2.17 3.54 3.45 4.96 6.39 9.27 5.02 3.49 3.81

A c) Obsd.d) Eq. 1 B1 Calcd.(A )e) 0.00 0.52 0.52 0.52 0.90 0.52 0.52 0.52 0.52 0.52 0.52 0.52 0.52

Eq.3

Eq.2 Calcd.(A )e)

5 . 5 6 6.28(-0.72) 6.76 6.91(-0.15) 7.44 7.33 (0.11) 7.79 7.52 (0.27) 8.05 7.49 (0.56) 7 . 2 1 7.50(-0.29) 7.53 7.52 (0.01) 7.46 6.79 (0.67) 5 . 0 6 5.21(-0.15) 5.33 -0.80 6.48 7.48(-1.00) 6.80 6.22 (0.58) 6.68 6.56 (0.12)

Calcd. ( A )e) 5.76 (-0.20) 6.77(-0.01) 7.38 (0.06) 7.46 (0.33) 7.38 (0.67) 7.43(-0.22) 7.45 (0.08) 6.68 (0.78) 5.10(-0.04) -0.49 6.63(-0.15) 7.44(-0.64) 7.35(-0.67)

5.83(-0.27) 6.61 (0.15) 7.15 (0.29) 7.43 (0.36) 7.43 (0.62) 7.47(-0.26) 7.47 (0.06) 6.79 (0.67) 5.11(-0.05) -1.28 6.64(-0.16) 7.48(-0.68) 7.42(-0.74)

From ref. i i unless otherwise noted. Scaled by 0.i and from ref. 12 unless otherwise noted. Calculated from the values cited from a brochure given by Dr. A. Verloop. pA9 values in the KCl-depolarized guinea-pig taenia coli. A~ the difference between observed and calculated values. Estimated from those of closely related substituents, see ref. I0 for the detail. Omitted from the correlation.

In Eqs. because reason

of was

another length

]-3 compound its not

quality

in T a b l e

especially

large

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in terms

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in Eqs.

2 and

from

extra have

correlation

particularly 3,

an

might

of the

was omitted

deviation

but

site

of the R 3 chain.

satisfactory, shown

clear,

receptor

The

(II-]3)

pronounced

for

from the c a l c u l a t i o n

the

correlations.

binding

arisen

interaction

due

of Eqs.

to

the

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of the standard alkoxyalkyl

3. We o r i g i n a l l y

The with

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379 Table 5 Ca-antagonistic a c t i v i t y and physicochemical parameters of R4-substituted compounds ( I I ) MeO.

7N

Me I

MeO~C-(CH2)3N(CH2)30~ MeO~ - ~ I iso-Pr

R4 PA2

Compd. No.

R4

11-17 11-18 11-19 11-4 11-20 11-21 11-22 11-23 11-24 11-25 11-26 11-27 11-28 11-29 11-30 11-31 11-32 11-33 11-34 11-35 11-36 11-37 11-38 11-39 11-40 11-41 11-42 11-43

H o-Me o-n-Pr o-OMe o-OEt o-F o-Cl o-NO^ o-NH~ m-Me m-t-Bu m-OMe m-F m-Cl m-NO^ m-NH~ m-CF~ m-CH~OH p-Me p-n-Pr p-t-Bu p-OMe p-F p-Cl p-NO; p-NH$ p-CNp-CH20H

11-44 11-45 11-46

2,3-(0Me) 2 2,4-Me^ 2,5-Me~

11-47. 2,6-(0Me)2 II-48!)3,4-(0Me)2 II-49])3,5-(0Me)2 II-501)3 5-Me2

d) AB papa 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1.04 2.49 2.17 2.07 0.35 0.80 1.44 0.97 0.60 1.70

a) ~ para 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.52 1.43 1.82 -0.08 0.21 0.77 0.03 -1.35 -0.30 -I.00

-O.05h ) -O. zoh ) 0.26 1.04~ ) -0.24~ ) -0.04 1.06 n) -0.19 n) -0.04

0.00 1.04 0.00

0.00 0.52 0.00

-0.16h ) -0.32h ) -O.05n ) -O. lOn) O.06n) 0.12n ) 1.08 n) -0.14 n)

0.52 h) 0.00 0.00 0.00

2.07 0.00 0.00

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0.00

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~

a)

0.00 0.52 1.43 -0.08 0.30 0.21 0.77 0.03 -1.35 0.54 1.85 0.03 0.26 0.79 -0.03 -1.14 0.98 -0.91 0.52 1.43 1.82 -0.08 0.21 0.77 0.03 -1.35 -0.30 -1.00

o

0 b)

0.00 -0.12 -0.13 g) -0.16 -0.14 0.17 0.27 0.82 -0.38 -0.07 -0.07 0.06 0.35 0.37 0.70 -0.14 0.47 0 O0g) -0.12 -0 13g) -0.17 -0.16 0.17 0.27 0.82 -0.38 0.69 0.05 g)

II-51i)3,4,5-(0Me)3 -0.02 h) -0.04 h) a) b) c) d) e) f) g) h) i) j)

c) F ortho 0.00 -0.04 -0 06 0.26 0.22 0.43 0.41 0.67 0.02 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

Obsd.

e)

Eq.7

8.19 8.31 7.79 8.05 8.10 7.95 7.69 7.26 8.15 8.48 6.88 9.00 8.29 8.36 8.17 8.13 8.06 8.11 7.61 6.88 6.88 7.64 8.02 7.73 7.39 6.50 7.64 6.56

Calcd.(A )f) 8.50(-0.31) 8.50(-0.19) 7.83(-0.04) 8.16(-0.11) 8.18(-0.08) 7.78 (0.17) 7.56 (0.13) 7.20 (0.06) 7.89 (0.26) 8.41 (0.07) 7.17(-0.29) 8.48 (0.52) 8.35(-0.06) 8.12 (0.24) 8.25(-0.08) 8.04 (0.09) 7.96 (0. i0) 8.17(-0.06) 8.05(-0.44) 6.90(-0.02) 6.79 (0.09) 7.27 (0.37) 8.31(-0.29) 8.04(-0.31) 7.36 (0.03) 6.71(-0.21) 7.72(-0.08) 6.60(-0.04)

8.23 7.97 8.68

8.14 (0.09) 7.83 (0.14) 8.18 (0.50)

7.57 8.35. 10 20@1 9 53J

7.82(-0.25) 7.26 8.46 8.09

9.32 j ) 7.24

Estimated from Eq. 5 taken from ref. 14. Taken from ref. 15 unless otherwise noted. Taken from ref. 16. Calculated from the B5 values presented in a brochure given by Dr. A. Verloop. pA2 values in KCl-depolarized guinea-pig taenia coli unless otherwise noted. A , the difference between observed and calculated values. Estimated from the values for related substituents. Values are the sum of the values for substituents. Omitted from the correlation. pA2 values in KCl-depolarized rabbit thoracic aorta.

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388

Table 7 Ca-antagonistic a c t i v i t y

and physicochemical parameters of R2-substituted

compounds ( I I )

CN

Me

- (CH2)3N(CH2)30 Me

R2 i so-Pr

PA2 Compd. No. 11-52 11-53 11-54 11-55 11-56 11-57 11-58 11-59 11-60 11-61 I I-62 11-63 11-64 11-65 11-66 11-67 11-68 11-69 11-70 II-71 II-72 11-73 11-74 11-75 11-76

R2

H o-Me o-OMe o-OEt o-F o-Cl o-Br m-Me m-OMe m-OEt m-F m-Cl m-CONMe 2 p-Me p-iso-Pr p-OMe p-OEt p-F p-Cl p-Br p-NOp p-NH~ p-NHAc p-CONMep 3,4-(OMe)~

Jr

a)

) A MRa meta

b) AL para

Obsd.

c)

Eq. 10 Cal cd. ( A

)d)

0.00 0.56 -0.02 0.38 0.14 0.71 0.86 0.56 -0.02 0.38 O. 14 O.7~e -i.0o ) 0.56 1.53 -0.02 0.38 0.14 0.71 0.86 -0.28 -1.23

0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.46 0.68 1.14 -0.01 0.50 1 83 e) 0.00 0.00 0.00 0.00 0.00 0.00 0.00 O.O0 0.00

0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 O. O0 0.00 0.00 0.81 2.05 1.92 2.74 0.59 1.46 1.76 1.38 0.72

7.59 7.25 7.60 7.53 7.00 7.40 7.60 7.80 8.20 7.84 7.35 7.50 6.64 7.71 7.26 7.64 7.32 8.25 8.04 7.80 7.87 7.15

7.50 (0.09) 7.42(-0.17) 7.50 (0. i0) 7.46 (0.07) 7.50(-0.50) 7.37 (0.03) 7.30 (0.30) 7.77 (0.03) 7.91 (0.29) 7.74 (0. I0) 7.48 ( -0.13 ) 7.73(-0.23) 6.67(-0.03) 7.82(-0.11) 7.13 (0.13) 7.81(-0.17) 7.24 (0.08) 7.83 (0.42) 7.80 (0.24) 7.67 (0.13) 7.93(-0.06) 7.47(-0.32)

-0.97 ~ - I 05e{ -0 04e)

0.00 0.00 0.68

3.03 2.71 1.92

6.76 6.93 7.99

6.74 (0.02) 7.01(-0.08) 8.22(-0.23)

II-77 f) 3,5-(0Me)2

-0.04 e)

0.68

0.00

8.88

8.31 (0.57) g)

II-27 f) 3,4,5-(0Me)3

-0.06 e)

0.68

1.92

9.00

8.61 (0.39) g)

a) b) c) d) e) f) g)

Taken from ref. 12 unless otherwise noted. Taken from a brochure given by Dr. A. Verloop. pA~ values in KCl-depolarized guinea-pig taenia coli. A ~ the d i f f e r e n c e between observed and calculated values. Estimated from those of c l o s e l y related substituents, see ref. 18 f o r the d e t a i l . Omitted from the c o r r e l a t i o n . 2 + 0 . 73A Lpara Estimated from the equation pA2 = - 0.27~ 2 - O.30A Lpara + 2 ( - 0.77A MR~eta+ 1.12A MRmeta) + 7.50.

~

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I

O

II

II

(30 --.1

9

~3 II o

II ~o <]-I

~

Cg(D

-.I-

:m.

O3

II DO

h3 O~

~3

IX.)

<71

II


9

c)

II

~

II O0 PO

~

~

o',o

--3

--~ <j3

003 " "

+

133

~ ~

C) C)

-4-

o-

~

CDCD 9 9 --~ (Y~ m=CD

O

ct

~3

ct

~el"

c-~

0~

O

I

C~

09

SO

0

ct

(1)

ct

O

on CD

~

003 ~ q c)

r',o -'~ (DO 9 9

C)(D

I

II

~

L~

~"

n:J

w

(I)

c-~

t~

~

m

i--~.

~.

9

~

w

(1)

(1)

~

c:~

"5

o

~

r~

9

9

-n

c)-

---

...1.

r-t-

9

cn

~

r

c+

N

--.-

o

:::r

~

m

~o

-5

~

~.~g.

cn

~

~

c-t"

~

c-t

c't

m

}m

9

_..s

m

~

C~

.

=~- oo

0

"--J .

P

P

r-'-

t>r"-

bd

P

P

r--

t> r---

I>

. ".4. r~)

0

XD

bd

0

P

P

. k~). -.j

0

X~)

bd

,, ~,,

.C~ t'o

P

P

I>"

X=)

bl

.--J

~,,

P

P

X=)

bd

,,

--J

P

P

XD

bl

(.~

,~,

P

P

bl

m

O -h

~F

F~z

CD

-q

=~

.mII hO

CD

9

II


9 Oo

II

>

9

+

9

+

+

--~

ho

c=zn ---,

CDLn

.

CD--~ . . . O~ ~ --~WI

P~

~ O O

~13 ~

.

I

[-~

~-j

~U ---

c-~ O

~ O

LIl - q
CD--~

~)

"

+

-~

O0-q

~ OCD "

I

R:~ Ix.)

--tO O~O

--~ tJl -q
LIl

OC9

l

CDC9

I

9

--~

+

~>

m~

(I) ct

Ln/z: tx) W l

O

I

~U (]) c-~ P] ~

O O

C.~
9

~ CDC)

bd

CDtO 00--~

(DO

h3

O

-.q

II Ix.)

CD 07 Co

9

II

--~


9

CD

II

~

9

+

9

+

9

9

--~

LIltO L~)O0

(DLn

~=)

--~

~ >

C D --~ . . . O~ C~ 0(3",

P~

~, O O

UU ~

LFI

CD(D

.

"

"

I --~

~O

O uu

c-~

~-]

O~ ~ O0--~

O

fl)

t-~

CO--J

~ CDO

+

"J

~C~ h 0

~ CD(D

I

~U

+

~>

(1) c-t ~) ,~,

:z~

~

~U ~ (1) C-~

CD--~

~

O

-~

hO
wbl

9

~

~ C) CD 9

II

bd

~ (DC9

PO

__~ 9

d)

Cr

~

F-~"

~" O~ ct Cb

F-~

C~

O

O

O

O

CI)

~D ~, r~

c-t (I) CZL

p]

(]) 9

~) Cr

bJ

393 substituents on the A ring (A), B ring (B) and quaternary carbon atom (Q), respectively. In these equations, the electronic u o t e r m , w h i c h w a s p r e v i o u s l y s i g n i f i c a n t in t h e a n a l y s i s f o r substituents on the B ring (Eq. 7), was insignificant. Since the u o term in Eq. 7 was the least significant judging from the value of the 95% confidence intervals, it seemed reasonable that this term disappeared for the larger set of compounds where more than half the number of derivatives have OMe, the g o values of which are close to zero, at either the ortho- or meta-positions of the B ring. If the variations of the substituents could be made greater in terms of their u0 value, the significance of the u o term would be increased again.

Table 10 Correlation coefficient matrix for the parameters of Eq. 12

z7l (En)

1.00

x7I

0.93

1.00

AFR:(A)

0.08

0.14

1.00

A F R (A)

0.02

0.09

0.86

1.00

A L (A) P A L (A) P

0.00

0.09

0.01

0.18

2m

1.00

0.04

0.06

0.02

0.26

0.96

1.00

0.10

0.13

0.06

0.19

0.14

0.18

1.00

A B ( 6 ) 0.15 5P 7rp(B) 0.39

0.11

0.08

0.19

0.16

0.20

0.23

1.00

0.41

0.02

0.05

0.04

0.05

0.07

0.39

1.00

AB,(Q)

0.16

0.09

0.20

0.17

0.21

0.42

0.14

0.04

Fo(B)

0.01

1.00

The possible significance of an indicator variable term I(A) for the effect of the 5-OMe group on the A ring in derivatives where R2=3,5-(OMe)2 and 3,4,5-(OMe)3 was considered because the effect of the 5-OMe group of these compounds was not taken into c o n s i d e r a t i o n i n E q . 1 0 . T h e I(A) t e r m , h o w e v e r , w a s insignificant. It seemed that the effect of the 5-OMe group in the 3,5-(OMe)2 and 3,4,5-(OMe)3 derivatives not incorporated in Eq. 10 was accounted for by the A MRmeta(A) term in Eqs. 1 1 and 12 s i n c e t h e e f f e c t of t h e 5 - O M e g r o u p w a s c o n s i d e r e d t o be equivalent to that of the 3-OMe group, and also because the number

394 Table I i Observed and calculated Ca-antagonistic a c t i v i t i e s for the whole set of verapamil analogs ( I I ) CN Me ~-(CH2)3N(CH2)30 R

I

R3

R4 PA2

Compd. No.

R2

R3

R4

Obsd.

Eq. 11

Eq. 12

Calcd. ( A ) a )

Calcd. ( A ) a )

11-52 11-53 11-54 11-55 11-56 11-57 11-58 11-59 11-60 11-61 11-62 11-63 11-64 11-65 11-66 11-67 11-68 11-69 11-70 11-71 11-72 11-73

H o-Me o-OMe o-OEt o-F o-Cl o-Br m-Me m-OMe m-OEt m-F m-Cl m-CONMe 2 p-Me p-iso-Pr p-OMe p-OEt p-F p-Cl p-Br p-N09 p-NH~

iso-Pr iso-Pr iso-Pr iso-Pr iso-Pr iso-Pr iso-Pr iso-Pr iso-Pr iso-Pr iso-Pr iso-Pr iso-Pr iso-Pr iso-Pr iso-Pr iso-Pr iso-Pr iso-Pr iso-Pr iso-Pr iso-Pr

m-OMe m-OMe m-OMe m-OMe m-OMe m-OMe m-OMe m-OMe m-OMe m-OMe m-OMe m-OMe m-OMe m-OMe m-OMe m-OMe m-OMe m-OMe m-OMe m-OMe m-OMe m-OMe

7.59 7.25 7.60 7.53 7.00 7.40 7.60 7.80 8.20 7.84 7.35 7.50 6.64 7.71 7.26 7.64 7.32 8.25 8.04 7.80 7.87 7.15

7.53 (0.06) 7.43(-0.18) 7.53 (0.07) 7.48 (0.05) 7.52(-0.52) 7.37 (0.03) 7.30 (0.30) 7.92(-0.12) 8.11 (0.09) 7.93(-0.09) 7.50(-0.15) 7.89(-0.39) 6.64 (0.00) 7.81(-0.10) 7.04 (0.22) 7.82(-0.18) 7.26 (0.06) 7.84 (0.41) 7.79 (0.25) 7.65 (0.15) 7.93(-0.06) 7.43(-0.28)

7.48 (0.11) 7.39(-0.14) 7.48 (0.12) 7.44 (0.09) 7.48(-0.48) 7.34 (0.06) 7.27 (0.33) 7.86(-0.06) 8.03 (0.17) 7.87(-0.03) 7.46(-0.11) 7.83(-0.33) 6.65(-0.01) 7.82(-0.11) 7.08 (0.18) 7.84(-0.20) 7.27 (0.05) 7.83 (0.42) 7.81 (0.23) 7.68 (0.12) 7.94(-0.07) 7.40(-0.25)

11-74 11-75

p-NHAc p-CONMe2

11-76 11-77 11-27 11-17 11-18 11-19 11-20 11-21 11-22 11-23 11-24 11-25 11-26 11-28 11-29 11-30 11-31 11-32 11-33

3,4-(0Me)2 3,5-(0Me)2 3,4,5-(0Me)3 3,4,5-(0Me)3 3,4,5-(0Me)3 3,4,5-(0Me)3 3,4,5-(0Me)3 3,4,5-(0Me)3 3,4,5-(0Me)3 3,4,5-(0Me) 3 3,4,5-(0Me)3 3,4,5-(0Me)3 3,4,5-(0Me)3 3,4,5-(0Me)3 3,4,5-(0Me)3 3,4,5-(0Me)3 3,4,5-(0Me)3 3,4,5-(0Me)3 3,4,5-(0Me)3

iso-Pr iso-Pr iso-Pr iso-Pr

m-OMe m-OMe m-OMe m-OMe m-OMe H o-Me o-n-Pr o-OEt o-F o-Cl o-NO^ o-NH~ m-Me m-t-Bu m-F m-Cl m-NO^ m-NH~ m-CF~ m-CH~OH

6.74 (0.02) 6.99(-0.06) 8.40(-0.41) . 8 11. (0 77) . 8.40 (0.60) 8.40(-0.21) 8.40(-0.09) 7.93(-0.14) 8.08 (0.02) 7.79 (0.16) 7.68 (0.01) 7.46(-0.20) 7.74 (0.41) 8.34 (0.14) 7.44(-0.56) 8.40(-0.11) 8.25 (0.11) 8.40(-0.23) 7.94 (0.19) 8.16(-0.10) 8.10 (0.01)

6.72 (0.04) 6.98(-0.05) 8.39(-0.40) 8.03 (0 85)

iso-Pr iso-Pr iso-Pr iso-Pr iso-Pr iso-Pr iso-Pr iso-Pr iso-Pr iso-Pr iso-Pr iso-Pr iso-Pr iso-Pr iso-Pr iso-Pr iso-Pr

6.76 6.93 7.99 8 . 88 9.00 8.19 8.31 7.79 8.10 7.95 7.69 7.26 8.15 8.48 6.88 8.29 8.36 8.17 8.13 8.06 8.11

8.39 (0.61) 8.39(-0.20) 8.41(-0.10) 7.96(-0.17) 8.00 (0.10) 7.64 (0.31) 7.54 (0.15) 7.22 (0.04) 7.70 (0.45) 8.33 (0.15) 7.46(-0.58) 8.38(-0.09) 8.25 (0.11) 8.38(-0.21) 7.91 (0.22) 8.16(-0.10) 8.07 (0.04)

395 Table 11. (Continued) 11-34 3,4,5-(OMe)3 11-35 3,4,5-( OMe ) 11-36 3,4,5-(OMe)3 11-37 3,4,5-(OMe)3 11-38 3,4,5-( OMe )3 11-39 3,4,5-( OMe ) 11-40 3,4,5-( OMe) 11-41 3,4,5-( OMe)3 11-42 3,4,5-(OMe)3 11-43 3,4,5-(OMe)3 11-44 3,4,5-(OMe)3 11-45 3,4,5-( OMe ) 11-46 3,4,5-( OMe ) 11-47 3,4,5-(OMe)3 11-4 3,4,5-( OMe ) 11-5 3,4,5-( OMe ) 11-6 3,4,5-(0Me)3 11-7 3,4,5-(OMe)3 11-8 3,4,5-( OMe)3 11-9 3,4,5-( OMe) 11-10 3,4,5-( OMe ) 11-11 3,4,5-(OMe)3 11-12 3,4,5-(OMe)3 11-14 3,4,5-(OMe)3 11-15 3,4,5-(OMe ) 11-16 3,4,5-(OMe)3 H 11-78 11-79 o-OMe 11-80 m-OMe 11-81 p-OMe 11-82 3,4-(OMe)2 11-83 2,3,4-( OMe ),

iso-Pr iso-Pr iso-Pr iso-Pr iso-Pr iso-Pr iso-Pr iso-Pr iso-Pr

iso-Pr

p-Me p-n-Pr p-t-Bu p-OMe

P-F p-c 1 P-NO~ P-NH~ p-CN

p-CH 0H 2,3-(OMe)2 2,4-Me2 2,5-Me2 2,6-(OMe ) o-OMe H o-OMe Me o-OMe Et o-OMe n-Pr o-OMe n-Bu o-OMe iso-Bu o-OMe n-Hex o-OMe n-0ct o-OMe benzyl o-OMe (CH2)20Me o-OMe (CH2 I2OEt o-OMe iso-Pr o-OMe o-OMe iso-Pr o-OMe iso-Pr iso-Pr o-OMe iso-Pr o-OMe iso-Pr o-OMe

iso-Pr iso-Pr iso-Pr iso-Pr iso-Pr

7.61 6.88 6.88 7.64 8.02 7.73 7.39 6.50 7.64 6.56 8.23 7.97 8.68 7.57 8.05 5.56 6.76 7.44 7.79 7.21 7.53 7.46 5.06 6.48 6.80 6.68 6.83 6.87 7.29 7.46 7.41 7.17

7.92(-0.31) 6.89(-0.01) 6.86 (0.02) 7.12 (0.52) 8.27(-0.25) 8.07(-0.34) 7.55(-0.16) 6.67(-0.17) 7.88(-0.24) 6.63(-0.07) 8.03 (0.20) 7.75 (0.22) 8.17 (0.51) 7.65(-0.08) 8.03 (0.02) 5.70(-0.14) 7.02(-0.26) 7.30 (0.14) 7.41 (0.38) 7.33(-0.12) 7.36 (0.17) 6.63 (0.83) 5.Z-0.16) 7.31(-0.83) 6.52 (0.28) 6.77(-0.09)

7.91(-0.30) 6.89(-0.01) 6.86 (0.02) 7.12 (0.52) 8.25(-0.23) 8.06(-0.33) 7.54(-0.15) 6.67(-0.17) 7.87(-0.23) 6.64(-0.08) 7.93 (0.30) 7.76 (0.21) 8.18 (0.50) 7.46 (0.11) 7.93 (0.12) 5.77(-0.21) 6.99(-0.23) 7.28 (0.16) 7.39 (0.40) 7.32(-0.11) 7.35 (0.18) 6.64 (0.82) 5.25(-0.19) 7.30(-0.82) 6.48 (0.32) 6.73(-0.05) 7.03(-0.20) 7.03(-0.16) 7.58(-0.29) 7.38 (0.08) 7.93(-0.52) 7.93(-0.76)

a) A , the difference between observed and calculated values.

of derivatives with R2=3,4,5-(OMe)3 wds very large. In E q s . 1 1 and 1 2 , t h e AMRmeta ( A ) value f o r R2=3,5-(OMe), and 3,4,5-(OMe)3 g r o u p s w a s not t h e sum of the A M R values of t h e t w o m-OMe g r o u p s , but rather that of the single m-OMe group. In Eqs. 1 1 and 12, A B 1 (19) (the STERIMOL width parameter) was shown to describe the steric effect of substituents on the quaternary carbon atom. The A B 1 was insignificant in the analysis for the effect of only an R3-substituent (see section 3 . 1 ) . B1 r e p r e s e n t s t h e minimum width o f s u b s t i t u e n t s f r o m t h e a x i s connecting the a -atom to the rest of the molecule. The effect of R3-substituents was then shown to be rationalized by their steric s i z e in addition to t h e II value as a fraction of the hydrohobicity of the entire molecule. The pA2 value for derivative (11-4) of R3=iso-Pr with a relatively large A B l value (0.90)

396

was observed to be higher than that calculated from Eq. 1 . For o t h e r R 3 - s u b s t i t u e n t s o f t h e c o m p o u n d s in T a b l e 3 , A B 1 is located at either 0 or 0.52. In Eqs. 1 1 and 12, including a large number of derivatives with R3=iso-Pr, the significance of the B1 t e r m was r e v e a l e d , a l t h o u g h it l o o k s l i k e j u s t an i n d i c a t o r variable for the iso-Pr substituent. Thus, no definite conclusion could be made about the effect of the R3-substituent. However, Eqs. 1 1 and 12 may indicate that the greater the minimum width of the R3-substituent, the higher the activity. This suggests that more s y m m e t r i c and bulkier s u b s t i t u e n t s s u c h a s t e r t - B u a r e favorable for activity. Since tert-Bu derivatives were almost impossible to synthesize, the iso-Pr group could be practically the best as the R3-substituent in terms of the steric effect. E q u a t i o n 12 g a v e t h e o p t i m u m C II - 1 . 5 3 i n d i c a t i n g t h e existence of an optimum in the total molecular hydrophobicity, presumably in connection with t h e transport process. I t a l s o indicates t h a t optimum s t e r i c c o n d i t i o n s f o r meta- and p a r a s u b s t i t u e n t s on t h e A r i n g , the i m p o r l a n c e of t h e e l e c t r o n releasing effect of ortho-substituents as well as smaller and yet hydrophobic para-substituents on the B ring, and symmetric bulky substituents on t h e quaternary carbon atom were favorable f o r activity. T h e present quantitative analyses of the effects of s u b s t i t u e n t s on t h e A and B r i n g s and quaternary carbon atom seemed adequate to allow us to identify the most effective structural features for Ca-antagonistic activity. The compound series (I) where R,=H showed a certain degree of C a - a n t a g o n i s t i c a c t i v i t y a s s h o w n in T a b l e s 1 , 2 , 4 and 6 . Although the effect of the R3-substituent (Table 2) was linearly related to that in compounds where R 1 = M e (r=0.89), no reasonably significant relationships were observed directly between the two series for the effects of substituents on the two ring systems. It could be possible to apply quantitative procedures similar to those for compounds of R,=Me. However, the potency o f R 1 = H c o m p o u n d s was generally lower than t h a t of t h e corresponding tertiary amines on average by about 1/20. Moreover, the range of potency variations in secondary a m i n e s is narrower than t h a t found in tertiary amines. Since it was thought that the accuracy of t h e activity data was poorer for t h e less active compound series, and also since the effect of structural variations was

397

l e s s s i g n i f i c a n t l y r e f l e c t e d in t h e p o t e n c y v a r i a t i o n s , quantitative structure-activity analyses were not performed for the secondary amino compound series.

QUANTITATIVE STRUCTURE-ACTIVITY ANALYSES OF a -BLOCKING ACTIVITY The effects of structural modifications of I on a -blocking activity were examined under the structural conditions of m=3, n=2 and R,=H. We attempted to correlate the activity of compounds in which various R2-substituents were introduced into the A ring ( I 84-105), alkyl and alkoxyalkyl R3-substituents were placed on the quaternary carbon atom (I-106-113), and various R4-substituents were attached t o t h e B ring (1-114-151) with substituent parameters (20). 4.

CN

H

(1-84-105): R 3=iso-Pr,,R4=o-OMe (1-106-113): R2=3,4,5-(OMe) 3’R4=o-OMe (1-1 14-151 ) : R2=3,4 ,5- (OMe)3,R3=iso-Pr 4.1 E f f e c t s o f R i n g S u b s t i t u e n t s o f t h e P h e n y l a c e t o n i t r i l e M o i e t y F i r s t , the effect of the R2-substituent on the A ring was examined while the R 3 - and R4-substituents were fixed a s iso-Pr and o-OMe, respectively. Among the compounds mono-substituted at the ortho-, meta- or para-position on the A ring listed in Table 12 (I-84-104), t h e p-OMe (1-97) and p-N02 (1-101) derivatives showed activities close to pA2=8. The 3,4-(OMe)2 derivative (I105) e x h i b i t e d a n a c t i v i t y h i g h e r t h a n p A 2 = 8 , a l t h o u g h i t s activity was lower than that of the 3,4,5-(OMe)3 derivative (1-3) which is close to that of prazosin. Among the quantitative correlations of t h e p A 2 values of unsubstituted (1-84) and mono-substituted derivatives (1-85-104) using single parameters, that with a parabolic function of the hydrophobic parameter A , as shown i n Eq. 13, was found to be of satisfactory quality. The A value used here was that for monos u b s t i t u t e d b e n z e n e s l i s t e d in T a b l e 13. T h e s i t u a t i o n is illustrated i n Fig. 8.

398 Table 12 a -Blocking activity o f R -substituted a -isopropyl-a -[3-[2-(2-rnethoxy2 phenoxy)ethylarnino]propyl]phenylacetonitriles (I)

Compd. No. 1-84 1-85 1-86 1-87 1-88 1-89 1-90 1-91 1-92 1-93 1-94 1-95

pA2

= -

R2

PA2

Compd. No.

H

7.75 7.54 7.65 7.42 7.11 7.24

1-96 1-97 1-98 1-99 1-100 1-101 1-102 1-103 1-104 1-105 1-3

o-Me o-OMe 0-F 0-c1

o-Br rn-Me rn-OMe

7.47

rn-F

rn-C1 rn-CONMe2 p-Me

0.23~

(0.14)

-

7.87 7.90 7.66 7.60 7.32

R2

p-iso-Pr p-OMe P-F p-c1 p-Br P-NO2 P-NH* p-NHAc p-CONMe2 3,4-(OMe)2 3,4,5-(OMe)3 prazosin

0 . 3 0 ~ + 7.73

(0.11) (0.11) (n=21 , r=O.84, s=O.1 8 , F 2 , 8=20.7 5 )

PA2 6.87 8.02 7.57 7.35 7.28 8.04 7.85 7.68 7.88 8.25 8.42 8.63

[I31

In Eq. 13, the 3,4-(OMe), (1-105) and 3,4,5-(OMe)3 (1-3) derivatives were not included. The activities of these compounds were higher than expected from the parabola (Fig. 8). The higher activities of the 3,4-(OMe), and 3,4,5-(OMe)3 derivatives were consistent with the fact that the activities of m-OMe and p-OMe analogs also deviated upward to some extent, although the reason for this was not clear. Equation 13 shows that ii values between -0.6 and -0.7 were favorable for activity. The fact that the p-NO, derivative (I101) was the most effective among the mono-substituted compounds could be rationalized by the fact that the x value of p-NO2 is closest to the optimum. 4.2 E f f e c t s of S u b s t i t u e n t s o n t h e Q u a t e r n a r y C a r b o n A t o m

Compounds where the R,- and R4-substituents were fixed a s 3,4,5-(OMe)3 and o-OMe, respectively, and the R3-substituent was

Table 13

a -Blocking activity and physicochemical parameters of R2-substituted compounds (I) H I

R 2 .iso-Pr

Eq. 13

Compd.

No. ~

1-84 1-85 1-86 1-87 1-88 1-89 1-90 1-91 1-92 1-93 1-94 1-95 1-96 1-97 1-98 1-99 1-100 1-101 1-102 1-103 1-104

Me0

n

R2 ~

a)

Obsd. Calcd. ( A )b)

~

H

I-105d)

P-NH,L p-NHAc p-CONMe2 3,4-(OMe)2

-0.04')

7.75 7.54 7.65 7.42 7.11 7.24 7.47 7.87 7.90 7.66 7.60 7.32 6.87 8.02 7.57 7.35 7.28 8.04 7.85 7.68 7.88 8.25

I-3d)

3,4,5-(OMe)3

-O.0bc)

8.42

o-Me o-OMe 0-F 0-c1

o-Br m-Me m-OMe

m-F

m-C1 m-CONMe2 p-Me p- i so-Pr p-OMe

P-F

p-c1 p-Br

P-NOz

0.00 0.56 -0.02 0.14 0.71 0.86 0.56 -0.02 0.14 0.71 -1.05') 0.56 1.53 -0.02 0.14 0.71 0.86 -0.28 -1.23 -0.97 -1.05')

7.73 (0.02) 7.50 (0.04) 7.74(-0.09) 7.69(-0.27) 7.41(-0.30) 7.31(-0.07) 7.50(-0.03) 7.74 (0.13) 7.69 (0.21) 7.41 i o . 25 j 7.80 -0.20) 7.50 -0.18) 6.75 (0.12) 7.74 (0.28) 7.69 -0.12) 7.41 -0.06) 7.31 -0.03) 7.80 (0.24) 7.76 (0.09) 7.81(-0.13) 7.80 (0.08) 7.74 7.74

a) 7z value for mono-substituted benzene series. b) A , the difference between observed and calculated values. c) Estimated from those of closely related substituents, see ref. 20 for the detail. d ) Omitted from the correlation.

varied within alkyls (1-107-111) exhibited potencies higher than pA,=8 (Table 14). The unsubstituted (1-106) and alkoxyalkyl ( I 112, 113) analogs showed lower activity. Since the activity was highest in the Et derivative, an optimum hydrophobicity seemed to also exist for R,. Unfortunately, quantitative analyses o f the

400

effect of R g gave no significant correlation since the number of compounds in the set used was too small.

K 91 8-

.3,4,5-OMe3 .3,4-OMe2

NH CONMe, 0,0

0 CI

7-

6 ,

I

I

I

Fig. 8. R e l a t i o n s h i p of a - b l o c k i n g a c t i v i t y w i t h t h e h y d r o p h o b i c 0 : ortho-substituted derivatives, A : metaparameter. substituted derivatives, 0 : para-substituted derivatives, 0 : d i - s u b s t i t u t e d d e r i v a t i v e s . ( R e p r o d u c e d f r o m ref. 20 by p e r m i s s i o n of the Pharmaceutical Society of Japan.) 4.3 E f f e c t s o f R i n g S u b s t i t u e n t s of t h e P h e n o x y M o i e t y Since the 3,4,5-(OMe)3 substitution o n the A ring in the

phenylacetonitrile moiety was the most favorable for activity (see section 4 . 1 ) and also because there was no particular advantage associated with changing the substituent at the quaternary carbon atom from iso-Pr (section 4 . 2 ) , these substitution patterns were maintained as shown in Table 15. Among the derivatives mono-substituted on the B ring, orthosubstituted derivatives such as the o-OMe (1-3) and o-OEt (1-118) analogs exhibited higher potency. Bulkier alkoxy groups decreased the activity. Di-substituted analogs with the o-OMe group were synthesized because the o-OMe g r o u p was thought to be favorable for activity among various substituents. The activity of all d i substituted derivatives including the 2,6-(OMe)2 analog (I-149),

40 1

Table 14

a -Blocking activity o f a -R3-substituted a -[3-[2-(2-methoxyphenoxy)ethylamino]propyl lphenyl acetonitriles (I)

Me0 Compd. No. 1-106 1-107 1-108 1-109 1-110

R3

PA2

Compd. No.

R3

PA2

H Me Et

7.83 8.40 8.79 8.46 8.57

1-111 1-112 1-113 1-3

iso-Bu (CH2)20Me (CH2)20Et iso-Pr

8.67 7.76 7.46 8.42

n-Pr

n-Bu

Table 15 a -Blocking activity o f R4-substituted a -isopropyl-a-[3-(2-phenoxyethylamino)propyl]phenylacetonitriles (I) H

R4

iso-Pr Compd. No. 1-114

1-115

1-116 1-117 1-118 1-119 1-120 1-121 1-122 1-123 1-124 1-125 1-126 1-127 1-128 1-129 1-130 1-131 1-132 1-133

R4

PA2

Compd. No.

R4

PA2

H

7.37 6.57

1-134 1-135 1-136 1-137 1-138 1-139 1-140 1-141 1-142 1-143 1-144 1-145 1-146 1-147 1-148 1-149 1-150 1-151 1-3

m-NH m-CN2 m-CF p-Me3 p-n-Pr p-t-Bu p-OMe

5.94 6.11 5.48 6.31 5.23 5.24 5.86 6.36 5.84 5.42 6.26 5.64 5.71 6.04 6.81 6.09 7.34 8.69 8.42

o-Me o-n-Pr O-t-Bu

0-OEt 0-F

0-c1 o-N02 o-NH2 0-CN o-OPr 0-OBU o-OCH2Ph 0-OH m-Me

m-t-Bu m-OMe m-F

m-C1 m-NO,

6.30

5.57 8.48 7.45 6.91 7.01 6.53 7.44 7.57 7.37 6.90 7.55 6.30 5.37 6.34 7.35 6.63 6.30

P-F

p-c1

P-NOz

P-NH2 p-CN 2,3-(OMe)2 2,4-(OMe)2 2,5-(OMe)2 2,6-(OMe)2 2-OMe,5-Me 2-OMe,5-F o-OMe

402 which possessed two ortho OMe groups, was low, except for the 2 OMe-5-F derivative (1-151) which exhibited an activity higher than that of the o-OMe derivative and close to that of prazosin. 9

< 8

0 OH

OF

0

CN

AF

0 OPr

0 OBu

7

6

5

\

I

-1

0

1

2 T

Fig. 9. R e l a t i o n s h i p of a - b l o c k i n g a c t i v i t y w i t h t h e h y d r o p h o b i c 0 : ortho-substituted derivatives, A : metaparameter. substituted derivatives, 0 : para-substituted derivatives. The s o l i d l i n e w a s d r a w n a s t h a t o f t h e best fit. ( R e p r o d u c e d f r o m ref. 20 by p e r m i s s i o n o f t h e P h a r m a c e u t i c a l S o c i e t y of J a p a n . ) Quantitative analysis for unsuhstituted (1-114) and monosubstituted derivatives (1-3, 115-145) using single parameters showed that a quadratic equation of the hydrophobic parameter B , a s shown by Eq. 14, was the best, although the quality of t h e correlation was not a t all satisfactory. The 71 value used here was that for substituted anisoles estimated according t o E q . 5. The correlation is illustrated in Fig. 9. In this figure, it was noticed t h a t the a c t i v i t i e s of o-alkoxy derivatives deviated markedly upward from t h e parabola. Figure 10 was prepared by

403

-b Lo

0%

Q!

P

PA2

cE OMe OH 0

=

-

~o0Et 0 AF

0 . 3 5 ~ + 6.83 (0.27) (0.37) (n=33, r=0.43, s=0.80, F 1 ,31=6.Y2)

1141

s u b s t r a c t i n g a n a p p r o p r i a t e i n c r e m e n t o n l y for t h e a l k o x y derivatives from the pA, value on the ordinate, This is equivalent t o c o n s i d e r i n g a n i n d i c a t o r v a r i a b l e Iortho f o r o - a l k o x y derivatives. The coefficient, -0.95, of the Iortho term added to pA, w a s s e l e c t e d so t h a t a t l e a s t t h e p l o t f o r t h e o r t h o derivatives was aligned as nicely a s possible, a s shown by the solid line. It is noticed i n Fig. 10 that most of the meta- and para-substituted derivatives deviated downward from the parabola, regardless of the electronic properties of the substituent, the

404

deviations f o r t h e para-substituted derivatives being mostly larger. T h e s e f a c t s s u g g e s t e d t h a t p o s i t i o n - s p e c i f i c s t e r i c effects may be significant at the meta- and para-positions. By considering the position-specific steric parameters, A B5 for the meta-substituents and A L for the para-substituents, in addition t o x 2 and the indicator variable I o r t h o , E q . 15 was finally derived, where the linear term of x was insignificant. The physicochemical parameters of each substituent used in this analysis are listed in Table 16.

pA2

=

-

+

0.307~2 (0.14)

7.03 (0.24)

ortho tho

-

0.478 B5 (0.25)

-

0.57A Lpara (0.20)

[151 (n=33, r=0.90, s=0.40, F4,28=30.91)

pA2

=

- 0.54A B5 - 0.66A Lpara 0.28n 2 (0.14) +(::::fortho (0.21) (0.19) + 7.08 (0.25) (n=38, r=0.90, s=O.43, F4,33=34.01) -

[I61

The 'ortho parameter probably accounts for a stereospecific hydrogen-bond accepting effect of the o-alkoxy groups. This type of hydrogen-bonding effect was s h o w n t o be expressible by a n indicator variable (21). The hydrogen-bond formation might be either with an acidic group on the receptor or intramolecularly with the NH hydrogen. In E q . 16 and Fig. 1 1 , five di-substituted c o m p o u n d s in w h i c h o n e o f t h e s u b s t i t u e n t s w a s o - O M e w e r e included, except for the 2,6-(OMe)2 derivative (1-149). The Iortho was not assigned to the o-alkoxy substituent, which might n o t work a s a hydrogen-bond acceptor. Thus, 'ortho = 1 was applied to the 2,4-(OMe)2 (I-147), 2,5-(OMe)2 (I-148), 2-OMe-5-Me (1-150) and 2 - O M e - 5 - F (1-151) d e r i v a t i v e s , but not t o t h e 2,3-(OMel2 analog (1-146). Being sandwiched by the oxyalkylene bridge and t h e 3 - O M e , t h e o - O M e g r o u p in 1 - 1 4 6 c o u l d n o t t a k e s u c h a conformation for intra- or intermolecular hydrogen-bond formation as that in 1-147-151. The activity of the 2,6-(OMe)2 compound (I149) w a s much lower than that even expected for Iortho=O. T h e r e a s o n w a s n o t c l e a r , but a buttressing e f f e c t o f v i c i n a l l y

405 Table 16 a -Blocking a c t i v i t y and physicochemical parameters of R4-substituted compounds ( I ) MeO. MeO

CN

H

- (CH 2) 3N(CH 2) 2 0

MeO~so Pr

R4 PA2

Compd. No. 1-114 1-115 1-116 1-117 1-3 1-118 1-119 1-120 1-121 1-122 1-123 1-124 1-125 1-126 1-127 1-128 1-129 1-130 1-131 1-132 1-133 1-134 1-135 1-136 1-137 1-138 1-139 1-140 1-141 1-142 1-143 1-144 1-145 1-146 1-147 1-148~, 1-149 T) 1-150 1-151 a) b) c) d) e) f)

R4 H o-Me o-n-Pr o-t-Bu o-OMe o-OEt o-F o-Cl o-NO^ o-NH~ o-CNz o-OPr o-OBu o-OCH^Ph o-OH z m-Me m-t-Bu m-OMe m-F m-Cl m-NO^ m-NH~ m-CNz m-CF3 p-Me p-n-Pr p-t-Bu p-OMe p-F p-Cl p-NO9 p-NH~ p-CN2,3-(OMe)~ 2,4-(OMe)~ 2,5-(OMe)~ 2,6-(OMe)~ 2-OMe,5-M~ 2-OMe,5-F

~

a)

0.00 0.52 1.43 1.82 -0.08 0.30 0.21 0.77 0.03 -1.35 -0.30 0.92 1.38 1.48 -0.80 0.54 1.85 0.03 0.26 0.79 -0.03 -1.14 -0.32 0.98 0.52 1.43 1.82 -0.08 0.21 0.77 0.03 -1.35 -0.30 ~ -O.05e! -0.16 e ) -0.05 e) -0.16 e) 0.46 e) 0.18 e)

I b) ortho 0.00 0.00 0.00 0.00 1.00 1.00 0.00 0.00 0.00 0.00 0.00 1.00 1.00 1.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1.00 1.00 0.00 1.00 1.00

A LC) para 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.81 2.86 2.05 1.92 0.59 1.46 1.38 0.72 2.17 0.00 1.92 0.00 0.00 0.00 0.00

A _meta c) 55 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1.04 2.17 2.07 0.35 0.80 1.44 0.97 0.60 1.61 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 2.07 0.00 2.07 0.00 1.04 0.35

Obsd.

Eq.16 Calcd. ( A )

7.37 6.57 6.30 5.57 8.42 8.48 7.45 6.91 7.01 6.53 7.44 7.57 7.37 6.90 7.55 6.30 5.37 6.34 7.35 6.63 6.30 5.94 6.11 5.48 6.31 5.23 5.24 5.86 6.36 5.84 5.42 6.26 5.64 5.71 6.04 6.81 6.09 7.34 8.69

d)

7.08 (0.29) 7.01(-0.44) 6.51(-0.21) 6.15(-0.58) 8.03 (0.39) 8.00 (0.48) 7.07 (0.38) 6.92(-0.01) 7.08(-0.07) 6.57(-0.04) 7.06 (0.38) 7.79(-0.22) 7.49(-0.12) 7.41(-0.51) 6.90 (0.65) 6.44(-0.14) 4.95 (0.42) 5.96 (0.38) 6.87 (0.48) 6.47 (0.16) 6.30 (0.00) 6.19(-0.25) 6.73(-0.62) 5.94(-0.46) 6.47(-0.16) 4.62 (0.61) 4.80 (0.44) 5.81 (0.05) 6.68(-0.32) 5.95(-0.11) 6.17(-0.75) 6.10 (0.16) 5.62 (0.02) 5.96(-0.25) 6.75(-0.71) 6.91(-0.10) 7.07 7.41(-0.07) 7.83 (0.86)

~ values for mono-substituted anisoles. Indicator variable which takes the value of one for o-alkoxy groups and zero for others. Taken from a brochure given by Dr. A. Verloop. A , the difference between observed and calculated values. Values are the sum of the values for substituents. Omitted from the correlation.

406 located 6-OMe and oxyalkylene groups might be operative on the conformation of the 2-OMe group. T h e hydrogen-bond f o r m a t i o n , being a short-range stereosensitive interaction, might be highly dependent on the stereochemistry of the hydrogen acceptor substituents. The fact that the o-NO2 and o-CN compounds d i d not require the Iortho parameter might be in accord with this property of hydrogen-bonding. The locations of the hydrogen accepting site for the o-NO, and o-CN groups differ from that in the case of the alkoxy substituents.

a-

02-OMe,5-F

m c

0

E,

m

0 OH

a,

In

?

CNoM$"z

1

CNoM$NOz 02-OMe, -5-r$e 2,5-OMez0 2,3-OMez0 0 F

,

7-

A NH,

4

g

F OMe OMe &OEt CNO OgHOF

$7

CnN

2,4-OMeb 0 NO2

wPr wPr 0 0

,cl

Me0 Me0 wPr

6-

OCH,Ph 0 t-Bu

5 1

-2

'

1

-1

0

2

1 T

Fig. 11. Relationship o f a -blocking activity with the hydrophobic parameter, steric parameters and the indicator 0 : ortho-substituted derivatives, A : met& variable. substituted derivatives, 0 : para-substituted derivatives, 0 : d i - s u b s t i t u t e d d e r i v a t i v e s . ( R e p r o d u c e d f r o m ref. 20 by p e r m i s s i o n of t h e Pharmaceutical Society of Japan.) Equation 16 shows that substituents with a il value close t o z e r o are favorable for activity. I t also indicates that an alkoxy substituent a t the ortho-position and small substituents a t the meta- and para-positions are necessary for high activity. The most favorable substitution seems t o be o-OMe. The smallest

407

meta-substituent F could also be favorable, as was experimentally observed in the 2-OMe-5-F derivative (1-151). 4.4 Analysis of t h e E n t i r e S e r i e s of Analogs F i n a l l y , a n a l y s i s of t h e combined s e r i e s of a n a l o g s w a s performed to give E q . 17 with the parameters used in E q s . 13 and 16 for individual s e r i e s . T h e s t e p w i s e development of E q . 17 justified statistically for fifty-nine derivatives is shown in Table 17. In Table 1 8 , the intercorrelation between independent variables was shown to be insignificant. The calculated PA, value of each compound is listed in Table 19.

Table 1 7 Development of E q . 17 parameters

r

S

Fa) 1 , n-m-1

a) Observed F value. 1: the number of additional parameter terms, m: the number of total parameter terms. Theoretical F values are: F 1 , 5 0 : a = o . o ~ = ~ . and O ~ F 2 , 5 0 : a = 0 . 0 5 =3.18.

Table 1 8 Correlation Coefficient Matrix for the Parameters of E q . 17 ( Z ii)2

(C

7c

)2

crr

c

7c

I(A)

AB5m(B)

Lp(B)

1.00

0.32

1.00

I(A)

0.12

0.13

1.00

Io(B)

0.16

0.04

0.73

1.00

A B5,,,(B) Lp(B)

0.00

0.06

0.34

0.28

1.00

0.12

0.11

0.30

0.32

0.19

1.00

408

Table 19 Observed a n d calculated a -blocking activities for t h e w h o l e s e t o f verapamil a n a l o g s ( I )

CN

Dt-

7

( c H ~3~ ) ( C H 1~2

R4

R2 iso-Pr

Compd. No.

1-84 1-85 1-86 1-87 1-88 1-89 1-90 1-91 1-92 1-93 1-94 1-95 1-96 1-97 1-98 1-99 1-100 1-101 1-102 1-103 1-104 1-3 1-114 1-115 1-116 1-117 1-118 1-119 1-120 1-121 1-122 1-123 1-124 1-125 1-126 1-127 1-128 1-129 1-130 1-131 1-132 1-133 1-134

~Q

E q . 17

R4

R2

H

o-OMe

o-Me o-OMe

o-OMe o-OMe

o-OMe

0-F

o-OMe o-OMe o-OMe o-OMe o-OMe o-OMe o-OMe o-OMe

0-c1

o-Br m-Me

m-OMe m-F

m-C1 m-CONMe2

p-Me p-iso-Pr p-OMe P-F p-c1

o-OMe

p-Br

P-NO2 P-NH2 p-NHAc p-CONMe2 3,4,5-(OMe)3 3,4,5-(OMe)3 3,4,5-(OMe)3 3,4,5-(OMe)3 3,4,5-(OMe)3 3,4,5-(OMe)3 3,4,5-(OMe)3 3,4,5-( OMe)3 3,4,5-(OMe)3 3,4,5-(OMe)3 3,4,5-(OMe)3 3,4,5-(OMe)3 3,4,5-(OMe)3 3,4,5-( OMe)3 3,4,5-(OMe)3 3,4,5-( OMe) 3 3,4,5-(OMe)3 3,4,5-( OMe)3 3,4,5-(OMe)3 3,4,5-( OMe)3 3,4,5-(OMe)3 3,4,5-( OMe) ‘1 J

o-OMe o-OMe o-OMe o-OMe o-OMe o-OMe o-OMe o-OMe o-OMe

H

o-Me

o-n-Pr 0-t-Bu 0-OEt 0-F

0-c1

o-N02 0-NH

0-CN 2

o-OPr 0-OBU

o-OCH2P h 0-OH

m-Me

m-t-Bu m-OMe

m-F m-C1

m-N02 m-NH, L

Obsd. Calcd. ( A )a) 7.75 7.54 7.65 7. 42 7.11 7. 24 7.47 7. a7 7.90 7.66 7.60 7.32 6.87 8.02 7.57 7.35 7.28 8.04 7.85 7.6 8 7.88 8.42 7.37 6. 57 6.30 5.57 8.48 7.45 6.91 7.01 6.53 7.44 7.57 7.37 6.90 7.55 6.30 5.37 6.34 7.35 6.63 6.30 5.94

7.71 (0.04 7.56(-0.02 7.71(-0.06 7.68(-0.26 7.50(-0.39 7.43(-0.19 7. 56( -0.09 7.71 (0.16 7.68 (0.22 7.50 (0.16 7.61(-0.01 7. 56( -0.24 6.99(-0.12 7.71 (0.31 7.68(-0.11 7. 50(-0.15 7.43( -0.15 7.73 (0.31 7.55 (0.30 7.64 (0.04) 7.61 (0.27) 8.06 (0.36) 7.06 (0.31) 6.92( -0.35) 6.41(-0.11) 6.08(-0.51) 7.99 (0.49) 7.02 (0.43) 6.82 (0.09) 7.05( -0.04) 6.86(-0.33) 7.08 (0.36) 7. 74(-0.17) 7.44(-0.07) 7.36( -0.46) 7.04 (0.51) 6.37(-0.07) 4.90 (0.47) 5.96 (0.38) 6.82 (0.53) 6.39 (0.24) 6.30 (0.00) 6.43( -0.49)

409

Table 19. ( C o n t i n u e d ) 1-135 1-136 1-137 1-138 1-139 1-140 1-141 1-142 1-143 1-144 1-145 1-146 1-147 1-148 1-150 1-151

m-CN m-CF 3

3,4,5-(OMe)3 3,4,5-(OMe)3 3,4,5-(OMe)3 3,4,5-(OMe)3 3,4,5-(OMe)3 3,4,5-(OMe)3 3,4,5-(OMe)3 3,4,5-(OMe)3 3,4,5-(OMe)3 3,4,5-(OMe)3 3,4,5-(OMe)3 3,4,5-(OMe)3 3,4,5-(OMe)3 3,4,5-(OMe)3 3,4,5-(OMe)3 3,4,5-(OMe),

p-Me p-n-Pr p-t-Bu p-OMe p-F p-C1 p-NO p - N H 22 p-CN

2,3-(0M~?)~ 2,4-(OMe)2 2,5-(OMe)2 2-OMe,5-Me

2-OMe,5-F

6.11 5.48 6.31 5. 23 5. 24 5. 86 6.36 5.84 5.42 6.26 5. 64 5. 71 6.04 6.81 7.34 8.69

6. 77(-0.66) 5.86( -0.38) 6.40(-0.09) 4.56 (0.67) 4.75 (0.49) 5.83 (0.03) 6.64(-0.28) 5.87(-0.03) 6 . 1 6 ( - 0 . 74) 6.40(-0.14) 5.68(-0.04) 5.97(-0.26) 6.83( -0.79) 6.97(-0.16) 7.39( -0.05) 7.84 (0.85)

a) A , t h e d i f f e r e n c e b e t w e e n observed a n d c a l c u l a t e d values. pA2 =

-

-

0.22(Z rr )2 (0.12)

-

0.17I: rr (0.13)

+ 0.35I(A)

(0.31)

+ 0.9910rtho

(0.29)

(B)

0.53A B5 meta(B) - 0.65A Lpara(B) + 6.70 (0.18) (0.16) (0.34) (n=59, r=0.92, ~ ~ 0 . 3 7F6,52=48.25) ,

In Eq. 17, A and B in parentheses mean that the parameter applies t o the corresponding rings. An indicator variable I(A) which took the value of one for the derivatives with R2=3,4,5(OMe), was newly introduced since the upward deviation in t h e activity of the 3,4,5-(OMe)3 derivative was not accounted for by Eq. 13. C il was used a s the hydrophobic parameter s i n c e t h e coefficient of the r r 2 term in Eq. 13 was close t o that in Eq. 16. Equation 17 shows that actually a single optimum value, C 7~ =0.39, existed for the total hydrophobicity of the entire molecule, which r e f l e c t s t h e requirements of t h e transport process. Compounds having various R3-substituents (1-106-113) were n o t i n c l u d e d in E q . 17, b e c a u s e s i g n i f i c a n t p h y s i c o c h e m i c a l parameters were not found. T h e pattern of t h e activity v a r i a t i o n s , s u g g e s t i n g an optimum hydrophobicity in t h i s R 3 substituted s e r i e s , however, seemed t o conform with Eq. 17 a s described above. In summary, the above quantitative analyses of the effects of substituents on t h e A and B r i n g s were considered t o have revealed the most effective structural conditions for a -

410

blocking activity in a manner similar t o those f o r C a antagonistic activity. CONCLUSION Table 20 lists the structural requirements and substituent effects favorable f o r the two types of activities exerted by the new verapamil analogs. 5.

Table 2 0 Structural and physicochemical effects favorable for the activities.

Whole Molecule Optimum Hydrophobicity Carbon Chain Length N-Substituent (R1) Ring A Substituent (R2)

Ca-antagonistic

a -Blocking

X

X

I

(R2,R3,R4)=1.5 m=n=3 Me

A MR(m-substituents)

:optimum=0.8 A L(p-substituents) :optimum=1.3

I

(R2,R4)=-0.4 m=3, n=2 H

I [ 3,4,5-( OMe ),I :slope=0.35

Quaternary Carbon Substituent (R3)

B :large 1

Lower alkyl groups

Ring B Substituent (R4)

F(o-substituents) :negative I (p-substituents) : pos it ive B5(p-substituents) : small

I(o-alkoxy) : sl ope=0.99 B5(m-substituents) : small L(p-substituents) :short

There are similarities as well as dissimilarities between the structural requirements for the two types of activities. Although physicochemical uncertainties still remain with respect to the meaning of indicator variable terms such a s I[3,4,5-(OMe)3] and I(o-alkoxy) contributing t o a -blocking p o t e n c y , s t r u c t u r e activity patterns could not be analyzed without the quantitative procedure. The series of studies presented here were, a s a matter of

41 1

f a c t , t h e f i r s t a t t e m p t by u s f o r a d o p t i n g a q u a n t i t a t i v e approach. After synthesizing and testing quite a few a n a l o g s , highly potent compounds which could be candidates for f u r t h e r developmental studies were found. However, we wanted to ascertain whether the candidate compounds really belong to the "best" ones. In the course of our analyses, we have learned how to disentangle overlapping structural effects by stepwise examinations. Although t h e n e t e f f e c t of a c e r t a i n s t r u c t u r a l m o d i f i c a t i o n d i f f e r s between different pharmacological activities, we have been able t o understand what was achieved in t h e s e r i e s of syntheses in terms of possible physicochemical factors governing the v a r i a t i o n s in t h e a c t i v i t i e s . F i n a l l y , t h e m o s t e f f e c t i v e structural f e a t u r e s f o r Ca-antagonistic and a -blocking activities were identified and the structures of the candidate compounds were confirmed to be among the best. We are convinced that the quantitative structure-activity analyses are not only powerful for the identification of candidate compounds for (pre)clinical tests but also instructive for synthetic chemists t o understand a number of "principles" which could be incorporated in their design of syntheses in lead optimization. REFERENCES

V. H. Haas and G. Hartfelder, Arzneim.-Forsch., 1 2 (1962) 549 - 5 5 8 ; V. M. Schlepper and E. Witzleb, i b i d . , 1 2 (1962) 559561; V. W. Appel, i b i d . , 1 2 (1962) 562-566. T. Takenaka, K. Honda, T. Fujikura, K. Niigata, S. Tachikawa, and N. Inukai, J . Pharm. Pharmacol., 3 6 (1984) 539-542; C. M e l c h i o r r e , L. B r a s i l i , D. G i a r d i n a , M . P i g i n i , a n d G .

Strappaghetti, J . Med. Chem., 2 7 (1984) 1535-1536. J. Augstein, W. C. Austin, R. J. Boscott, S. M. Green, and C. R. Worthing, J. Med. Chem., 8 (1965) 356-367; J. Augstein, W. C. Austin, C. A . Bartram and R. J . Boscott, ibid., 9 (1966) 812-818 . K. Mitani, T. Yoshida, K. Morikawa, Y. Iwanaga, E. Koshinaka, H. Kato, and Y. Ito, Chem. Pharm. Bull., 3 6 (1988) 367-372. T. H. Althuis and H. -J. Hess, J. Med. Chem., 20 (1977) 146149. C. Hansch and T. Fujita, J . A m . Chem. S O C . , 86 (1964) 16161626.

R. Mannhold, R . Steiner, W. H a a s , and R. Kaufmann, NaunynS c h m i e d e b e r g ' s Arch. P h a r m a c o l . , 3 0 2 (1978) 217-226; R . M a n n h o l d , P. Z i e r d e n , R . B a y e r , R . R o d e n k i r c h e n , and R. S t e i n e r , Arzneim.-Forsch., 3 1 (1981) 7 7 3 - 7 8 0 ; A. Goll, H. G l o s s m a n n , a n d R . M a n n h o l d , N a u n y n - S c h m i e d e b e r g ' s Arch. Pharmacol., 3 3 4 (1986) 3 0 3 - 3 1 2 ; R. Mannhold, R . B a y e r , M. Ronsdorf, and L. Martens, Arzneim.-Forsch., 3 7 (1987) 419-424. V . G. Grun and A. Fleckenstein, Arzneim.-Forsch., 2 2 (1972)

412

9 10

11 12

13 14 15 16

17

18 19 20 21

334-343.

K. Mitani, T. Yoshida, S. Sakurai, K. Morikawa, Y. Iwanaga, E.

Koshinaka, H. Kato, and Y. Ito, Chem. Pharm. Bull., 36 (1988) 373-385. K . Mitani, T . Yoshida, T. Suzuki, E. Koshinaka, H. Kato, Y. Ito, and T. Fujita, Chem. Pharm. Bull., 36 (1988) 776-783. C. Takayama, M. Akamatsu, and T. Fujita, Quant. Struct.-Act. Relat., 4 (1985) 149-160. C . Hansch, A. Leo, S. H. Unger, K. H. K i m , D. Nikaitani, and 0 . J. Lien, J. Med. Chem., 16 (1973) 1207-1216. A. Verloop, in: J. Miyamoto and P. C. Kearney (Eds), Pesticide C h e m i s t r y , Human Welfare and t h e Environment. Vol. 2 , Pergamon Press, Oxford, 1983, pp. 339-3114. T. Fujita, J . Pharm. Sci., 7 2 (1983) 285-289. 0 . Exner, in: N. B. Chapman and J. Shorter (Eds), Advances in Linear Free Energy Relationships, Plenum Press, London and New York, 1972, pp. 1-69. C. H a n s c h and A . J . L e o , in: S u b s t i t u e n t C o n s t a n t s f o r Correlation Analysis in Chemistry and Biology, John Wiley and Sons, New York, 1979, pp. 65-167. R . W . T a f t , Jr., J . P h y s . C h e m . , 6 4 (1960) 1805-1815; Y. Yukawa, Y. Tsuno, and M. Sawada, Bull. Chem. S o c . Jpn., 39 (1966) 2 2 7 4 - 2 2 8 6 ; i d e m , ibid., 45 (1972) 1198-1205; i d e m , ibid., 45 (1972) 1210-1216. K. Mitani, S . Sakurai, T. Suzuki, K. Morikawa, E. Koshinaka, H. Kato, Y. I t o , a n d T. Fujita, Chem. Pharm. Bull., 36 (1988) 4103-4120. A . Verloop, W. Hoogenstraaten, and J. Tipker, in: E. J. Ariens (Ed.), Drug Design. V o l . VII, Academic Press, New York, 1976, pp. 165-207. K. Mitani, S. Sakurai, T. Suzuki, K. Morikawa, E. Koshinaka, H. Kato, Y. Ito, and T. Fujita, Chem. Pharm. Bull., 36 (1988) 41 2 1-41 35. T. Fujita, T. Nishioka, and M. Nakajima, J . Med. C h e m . , 20 (1977) 1071-1081.

QSAR and Drug Design - New Developments and Applications T. Fujita, editor 9 1995 Elsevier Science B.V. All rights reserved

413

A P P L I C A T I O N S OF QUANTITATIVE S T R U C T U R E - A C T I V I T Y R E L A T I O N S H I P S TO D R U G D E S I G N OF PIPERAZINE DERIVATIVES Hiroshi OHTAKA

New Drug Research Laboratories, Kanebo Ltd., 1-5-90 Tomobuchi-cho, Miyakojima-ku, Osaka 534, J a p a n ABSTRACT: Applications of quantitative structure-activity relationship (QSAR) procedures to our own practical drug research are reviewed. A benzylpiperazine cerebral vasodilator (KB-2796) and a piperazine-acetate antiulcer agent (KB-5492) were successfully optimized by use of QSAR information. In these cases, appropriate strategies of the synthetic research were devised and QSAR analyses were performed repeatedly in each step during the research. In the last example, the selection of a 2-homopiperazinylbenzimidazole (KG-2413) as a candidate for antihistaminics was confirmed to be valid by QSAR analysis after the synthetic research project was over. The antiulcer and cerebral vasodilative agents are now under extensive clinical trials, while the antihistaminic agent has been used clinically. 1.

INTRODUCTION

A considerable number of 1,4-disubstituted piperazines have been found to possess interesting pharmacological properties and some of them have been used clinically. Syntheses of novel 1,4-disubstituted piperazine derivatives have been performed with the aim of finding new drugs. Piperazine is still a very important starting material in the pharmaceutical industry. The drug research and development project involves a number of steps and requires exhaustive effort and considerable expenditure. Medicinal chemists contribute mainly to the earliest stages of the project wherein new drug candidates are synthesized and selected. Such chemists must not only design novel chemical structures and perform syntheses, but also arrange the huge amount of structureactivity data in a meaningful order. However, since the majority of

414 newly synthesized compounds are abandoned in the earlier stages of the project, chemists are eager to discover or apply more efficient drug design methods than ever which reduce trial and error to a minimum. Quantitative structure-activity relationship (QSAR) analysis introduced by Hansch and Fujita (1) about 30 years ago, is still used widely as an effective and rational drug design approach. In this approach, the potency variations in a series of bioactive compounds are considered to be determined by several physicochemical properties. The q u a n t i t a t i v e relationship is analyzed, for example, by use of the following equation, log 1/C = kl~ + k2a + k3E s + .... + const.

[1]

where C is the equieffective dose or concentration, which should be properly expressed on a molar basis, and ~, ~ and E s represent hydrophobic, electronic and steric factors, respectively. When the activity of a compound is represented by potency rating (++, +, -, etc.), one of the modifications of the Hansch method, an adaptive least squares (ALS) method developed by Moriguchi and co-workers (2), is t h o u g h t to be appropriate. There have been m a n y successful applications of these QSAR methods to practical problems (3-7). In this article, our own examples in designing three types of drugs derivatized from piperazine are reviewed. 2. BENZYLPIPERAZINE CEREBRAL VASODH ATOR Cerebrovascular disorders were the most common cause of death in J a p a n up to 1981. Although the mortality attributable to these disorders has been decreasing recently, the number of patients showing after-effects has been increasing. Cerebrovascular disorders can roughly be classified into i n t r a c r a n i a l hemorrhage and cerebral infarction. In these disorders, infarction of the brain parenchyma occurs by h e m o r r h a g e , t h r o m b u s or embolus, leading to an insufficiency of glucose and oxygen, which supply the energy necessary for n e u r o n a l activity. This results in functional and organic disturbances in the ischemic area. Accordingly, drugs which facilitate

415

the supply of glucose or oxygen to the ischemic area by increasing the cerebral blood flow are effective for the t r e a t m e n t and prevention of these disorders. The potency, duration of action and cerebrovascular specificity are considered to be i m p o r t a n t properties for cerebral vasodilators (8). From these points of view, however, such c u r r e n t drugs as papaverine hydrochloride (9) and cinnarizine (10), are not necessarily satisfactory. 2 . 1 Design of the Possible ~ d S t r u c ~ In the course of our search for novel cerebral vasodilators, we have noticed publications showing t h a t 1-(2,3,4-trimethoxybenzyl)piperazine (I: trimetazidine) dihydrochloride, a coronary vasodilator, is not only distributed to the brain and to the heart of mice (11) but also relaxes dog basilar arteries more effectively t h a n coronary arteries after contraction with prostaglandin F 2 a (12). Trimetazidine is one of a few monosubstituted piperazines used clinically and seems to be much more hydrophilic t h a n cinnarizine (II). Therefore, we t h o u g h t it appropriate to modify the structure of trimetazidine to more lipophilic 1,4-disubstituted piperazine derivatives with the aim of finding a new cerebral vasodilator, and designed c i n n a m y l - ( I I I a ) and diphenylmethyl-trimetazidines (1Va).

OMe

I: Trimetazidine (CoronaryVasodilator)

I1: Cinnarizine Q (CerebralVasodilator)

I

MeO

OMe

Ilia

e ~

416

Cinnamyl-trimetazidine and Related Compounds A series of 1-benzyl-4-cinnamylpiperazine derivatives (III) was prepared and tested for cerebral vasodilative potency, which was evaluated from the response in dogs anesthetized with pentobarbital. The response was measured in terms of the ratio of the maximum change in blood flow in vertebral a r t e r i e s after i n t r a v e n o u s administration of the test compound (1 m g / k g ) t o that produced by papaverine (1 mg/kg). Although compounds III showed negligible activity, the synthetic intermediates (V) exhibited considerable activity (13). Qualitative analysis according to the Topliss scheme (14) suggested that the activity was positively dependent on the lipophilicity of the substituent X. Further analogs of V were therefore synthesized and this tendency was confirmed. Then, in order to make the molecule more lipophilic and to study the effects of the number and location of methoxy groups, the derivatives of V represented by the general formula (VI)were prepared and tested (15). 2.2

~ . . N ~ ~x OMe

V

OH

Vl

OH

For the QSAR analysis, the potency of compounds should be appropriately expressed on the molar basis. In this research, the response was observed to increase almost linearly with the log(dose) value within a certain range of concentrations where the response was from about 1/2 to 3/2 of t h a t of papaverine (1 mg/kg) for four derivatives. The log(dose)-response relationships for the four derivatives were almost parallel, the slope being estimated as 1.25 + 0.01 (n = 4), taking the log(dose) as the independent variable. For the rest of compounds, the response was evaluated at a single dose (1 mg/kg). Therefore, the log(dose)-response relationships for these compounds were assumed to have identical slopes. This assumption can be expressed by Eq. 2.

417 response = 1.25 log(dose) + C

[2]

Equation 2 can be rewritten as Eq. 2' by converting the log(dose) into the dependent variable. log(dose) = 0.8 response + C'

[2']

In these equations, C and C' are intercepts with the respective ordinates. The biological activity index of interest for structure-activity analysis is the negative l o g a r i t h m of the dose, D, required to produce a given response, and so the "response" in Eq. 2' is set as a certain value. At the given response, Alog D - AC' among derivatives. Since AC' - Alog(dose) 0.8 Aresponse, where the "response" is not set as a c o n s t a n t but variable and t h a t induced by the dose applied, the biological activity in terms of log(l/D) of a derivative A is represented as Eq. 3 where S stands -

for a reference compound. log(1/D)A - log(1/D)s + log(dose) s - log(dose)A 0.8 (response s - response A) -

[3]

C o m p o u n d Vie was t a k e n as a reference, since the dose (mol/kg) r e q u i r e d for 100% increase in blood flow in v e r t e b r a l a r t e r i e s was m e a s u r e d so t h a t log(l/D) S = 6.30. By converting the log(dose) value (dose = 1 mg/kg) into the log value on a molar basis for each compound and introducing the "response" values in terms of the ratio to t h a t of p a p a v e r i n e (1 mg/kg) into Eq. 3, the log(l/D) v a l u e s for the 35 compounds listed in Table 1 were estimated. The D (mol/kg) value is t h a t required for 100% increase in blood flow for each compound and is a p p r o x i m a t e l y equivalent with t h a t inducing 2/3 of the effect of the s t a n d a r d compound, papaverine (1 mg/kg). QSAR analysis for these derivatives was performed to yield Eq. 4 (16), log(l/D) - - 0.686(+0.129)~R 2 + 1.361(+0.274)~ R + 0.342(+0.128)~ x [4] + 0.288(+0.091 )Im + 0.207(+0.171 )Ip + 4.292 n-35, r-0.934, s-0.132, F=39.70

418 TABLE 1. S t r u c t u r a l F e a t u r e s a n d Cerebral Vasodilative Activities of 1-Benzyl-4(3-hydroxy-3-phenylpropyl)piperazine Dihydrochlorides (V a n d VI)

Compd.

log(l/D)

No.

Y

R

X

Ip

Im

~R

XX

Obs.

Calcd. (Eq.4)

Va

2,3,4-(OMe) 3 2,3,4-(OMe) 3 2,3,4-(OMe) 3

H H H

H 4-Me 4-C1

1 1 1

3 3 3

0.00 0.00 0.00

0.00 0.56 0.71

5.37 5.51 5.66

H H H Me Me Me Me Me

3,4-C12 3,4-Me 2 3,4-(CH) 4 H 4-Me 4-OMe 4-C1 3,4-C12

1 1 1 1 1 1 1 1

3 3 3 3 3 3 3 3

0.00 0.00 0.00 0.50 0.50 0.50 0.50 0.50

1.25 0.99 1.32 0.00 0.56 0.56 0.71 1.25

5.84 5.67 0.59 5.87 6.03 6.03 6.06 6.30

5.3C~ 5.56 5.61 5.79 5.70 5.82

VIk VI1 Vim VIn VIo VIp VIq

2,3,4-(OMe) 3 2,3,4-(OMe) 3 2,3,4-(OMe) 3 2,3,4-(0Me) 3 2,3,4-(OMe) 3 2,3,4-(OMe) 3 3,4,5-(OMe) 3 2,4,6-(OMe) 3 2,4-(OMe) 2 3,4-(OMe) 2 3,5-(OMe) 2

Me Me Et Et Pr Bu Bz Me Me Me Me Me

3,4-(CH) 4 2,4-C12 3,4-C12 2,4-C12 3,4-C12 3,4-C12 3,4-C12 3,4-C12 3,4-C12 3,4-C12 3,4-C12 3,4-C12

1 1 1 1 1 1 1 1 1 1 1 0

3 3 3 3 3 3 3 3 3 2 2 2

0.50 0.50 1.00 1.00 1.50 2.00 2.01 0.50 0.50 0.50 0.50 0.50

1.32 1.42 1.25 1.42 1.25 1.25 1.25 1.25 1.25 1.25 1.25 1.25

6.15 6.38 6.64 6.54 6.33 5.91 5.74 6.47 6.22 6.26 6.06 5.51

6.32 6.36 6.47 6.53 6.29 5.77 5.76 6.30 6.30 6.01 6.01

VIr

2,3-(OMe) 2

Me

3,4-C12

0

2

0.50

1.25

5.75

5.81

VIs

2-OMe

Me

3,4-C12

0

1

0.50

1.25

5.55

5.52

VIt

4-OMe

Me

3,4-C12

1

1

0.50

1.25

5.61

5.72

VIu

3,4,5-(OMe) 3

Me

2,4-C12

1

3

0.50

1.42

6.34

6.36

VIv

3,4,5-(OMe) 3

Et

3,4-C12

1

3

1.00

1.25

6.50

6.47

VIw VIx

3,4,5(OMe) 3 3,4,5-(OMe) 3

Et Pr

2,4-C12 3,4-C12

1 1

3 3

1.00 1.50

1.42 1.25

6.30 6.37

6.53 6.29

Vb Vc Vd Ve Vf Via VIb a

2,3,4-(OMe) 3 2,3,4-(OMe) 3 2,3,4-(OMe) 3 2,3,4-(OMe) 3 2,3,4-(OMe) 3 VIc a 2,3,4-(OMe) 3 V I d a 2,3,4-(OMe) 3 Vie 2,3,4-(OMe) 3 VIf 2,3,4-(OMe) 3 VIg VIh Vii

vij

5.87 6.07 6.07 6.12 6.30

5.81

419 TABLE 1. C o n t i n u e d

Compd. No.

log(l/D) Y

VIy VIz

3,4,5-(OMe)3 3,4,5-(OMe)3 V I a a 3,4,5-(OMe)3 Vlbb 3,5-(OMe)2 Vice 4-OMe

R

X

Ip

Im

XR

XX

Pr Bu Bu Et Et

2,4-C12 3,4-C12 2,4-C12 3,4-C12 3,4-C12

1 1 1 0 1

3 3 3 2 1

1.50 2.00 2.00 1.00 1.00

1.42 1.25 1.42 1.25 1.25

Obs. Calcd. (Eq.4) 6.20 5.64 5.81 6.29 5.84

6.35 5.77 5.83 5.97 5.89

a) Dimaleate.

where the figures in parentheses are the 95% confidence intervals, n is the n u m b e r of data points used in deriving the equation, r is the correlation coefficient, s is the s t a n d a r d deviation, and F is the ratio between regression and residual variances. In Eq. 4, ~X is the lipophilicity of substituent X of the phenyl moiety, ~R is the lipophilicity of substituent R at the benzylic position, I m represents the number of methoxy groups on the benzyl moiety and Ip is an indicator variable for the presence (Ip = 1) or absence (Ip - 0) of the para-methoxy group. F r o m Eq. 4, the optimum ~R was calculated to be 0.993 and an ethyl group was confirmed as being the best R substituent. Many kinds of drug activity have been found to depend upon lipophilicity, which is one of the most f u n d a m e n t a l c h a r a c t e r i s t i c s of d r u g s t r u c t u r e s determining biological activity. In the present case, Eq. 4 indicates that the local lipophilicity around the a s y m m e t r i c carbon atom is of importance. This may be the case, because intravenous administration of drugs does not involve the absorption process or first-pass effect, and so the interaction of drugs with the active site is the most critical step. The most favorable s u b s t i t u t i o n p a t t e r n of the benzyl moiety is suggested to be 2,3,4,5,6-pentamethoxy, but this is not practical. E q u a t i o n 4 also suggests t h a t the introduction of more lipophilic s u b s t i t u e n t s for the group X would make the compound more active. The most active of those prepared, however, exhibited no cerebral vasodilative activity when administered intraduodenally, although they

420

were more potent t h a n cinnarizine when applied intravenously. Therefore, our search for new cerebral vasodilators in this series was terminated. Diphenylmethyl-trimetazidine a n d Related C o m p o u n d s Unlike the series of compounds described in the preceding section, d i p h e n y l m e t h y l - t r i m e t a z i d i n e (IVa) showed considerable cerebral vasodilative activity even on intraduodenal application. This compound has been described in the patent literature but nothing about its medical utility has been disclosed (17). We considered this compound to be a possible lead for new cerebral vasodilators and therefore a series of 1benzyl-4-diphenylmethylpiperazines ( I V ) w e r e p r e p a r e d and tested (18). 2.3

z

Most compounds in this series were also administered at a certain single dose, but the log(dose)-response plots of potent derivatives were examined and found to be parallel. Accordingly, the potency in terms of the log(l/D) value was estimated in a manner similar to that used in the preceding section. QSAR analyses were performed for each subseries where Y = Z = H ( 1 V a - 1Vm), Y - Z - F (Wp, l V x - lVff) and X - 2,3,4-(OMe) 3 (lVa, l V p - lVw)(19). The two compounds (lVn and 1Vo) showed much lower activity t h a n expected. They also caused acute toxicity in addition to showing activity. Since data on these compounds were thought to distort the correlation, they were not included in the analyses. Then, 35 compounds in Table 2 were subjected to analysis leading to Eq. 5, log(l/D) = - 0.839(+0.258)Z~ - 0.075(+0.038)MR + 5.582 n = 3 5 , r=0.788, s=0.231, F=26.18

[5]

421 TABLE 2. S t r u c t u r a l F e a t u r e s a n d C e r e b r a l Vasodilative Activities of 1-Benzyl-4d i p h e n y l m e t h y l p i p e r a z i n e D i h y d r o c h l o r i d e s (IV)

Compd. No.

log(l/D) X

Y

Z

Z~

MR

Obs.

Calcd. (Eq.5)

Duration Obs.Calcd. (Eq.6)

IVa

2,3,4-(OMe) 3

H

H

-0.42

1.03

6.23

5.86

0

1

IVb a

4-OAc

H

H

0.31

1.03

5.45

5.25

1

0

IVc

4-C1

H

H

0.23

1.03

5.34

5.31

0

0

IVd a

4-F

H

H

0.06

1.03

5.40

5.46

0

0

IVe ivf b

H

H

H

0.00

1.03

5.39

5.51

0

0

4-NHAc

H

H

0.00

1.03

5.44

5.51

0

0

IVg

3,4,5-(OMe) 3

H

H

-0.03

1.03

5.48

5.53

0

0

IVh

3,4-(OMe) 2

H

H

-0.15

1.03

5.43

5.63

0

0

IVi

4-Me

H

H

-0.17

1.03

5.70

5.65

1

0

ivj

3,4-OCH20-

H

H

-0.32

1.03

5.58

5.77

0

0

IVk

4-OH

H

H

-0.37

1.03

5.92

5.82

0

0

IVl a

2,4-(OMe) 2

H

H

-0.54

1.03

5.97

5.96

1

1

IVm a

2,4,6-(OMe) 3

H

H

-0.81

1.03

6.58

6.19

0

d

IVn a

4-NH 2

H

H

-0.66

1.03

5.70

c

0

1

IVo a

4-NMe 2

H

H

-0.83

1.03

5.55

c

1

1

IVp

2,3,4-(OMe) 3

F

F

-0.42

0.92

6.32

5.87

0

0

IVq

2,3,4-(OMe) 3

Me

Me

-0.42

5.65

6.08

5.51

0

0

IVr

2,3,4-(OMe) 3

C1

C1

-0.42

6.03

5.57

5.48

0

0

IVs a

2,3,4-(OMe) 3

OMe OMe

-0.42

7.87

5.22

5.35

0

0

IVt a

2,3,4-(OMe) 3

F

H

-0.42

1.03

6.08

5.86

1

1

IVu a

2,3,4-(OMe) 3

Me

H

-0.42

5.65

5.32

5.51

0

0

IVv a

2,3,4-(OMe) 3

C1

H

-0.42

6.03

5.64

5.48

1

0

IVw a

2,3,4-(OMe) 3

OMe H

-0.42

7.87

5.04

5.33

0

0

IVx

3,4,5-(OMe) 3

F

-0.03

0.92

5.47

5.54

0

0

IVy

2,4,6-(OMe) 3

F

F

-0.81

0.92

6.09

6.19

0

d

IVz

2,4-(OMe) 2

F

F

-0.32

0.92

5.57

5.78

0

0

IVaa

3,4-OCH20-

F

F

-0.32

0.92

5.57

5.78

0

0

lVbb

4-OH

F

F

-0.37

0.92

5.70

5.82

1

1

IVcc

H

F

F

0.00

0.92

5.37

5.51

0

0

IVdd

4-Me

F

F

-0.15

0.92

5.41

5.66

1

0

IVee a

4-NMe 2

F

F

-0.83

0.92

6.22

6.21

1

1

lVff a

4-OAc

F

F

0.31

0.92

5.51

5.25

0

0

IVgg a

2,4,6-(OMe) 3

F

H

-0.81

1.03

5.91

6.19

1

d

F

422

TABLE 2. C o n t i n u e d

Compd. No.

IVhh a IVii IVjj IVkk a

X 2,4-(OMe)2 3,4,5-(OMe) 3 3,4-OCH204-NMe 2

Y

Z

Zo

F F F F

H H H H

-0.54 -0.03 -0.32 -0.83

log(l/D) Duration MR Obs. Calcd. Obs.Calcd. (Eq.5) (Eq.6) 1.03 1.03 1.03 1.03

5.95 5.52 5.38 6.04

5.96 5.53 5.77 6.20

1 0 0 1

1 0 0 1

a) Fumarate. b) Maleate. c) Not included in the correlation, d) Not included in the analysis.

where Z(~ represents the electronic effect of substituent X on the benzyl moiety and MR r e p r e s e n t s the molar refractivity value for the larger s u b s t i t u e n t of Y and Z. The correlation coefficient of Eq. 5 is not so high as one would like. However, the correlation is highly significant and is not inconsistent with those for the above mentioned subseries where much b e t t e r correlations were observed. Equation 5 suggests t h a t the e l e c t r o n - d o n a t i n g effect of the s u b s t i t u e n t on the benzyl moiety is i m p o r t a n t for the p o t e n t cerebral vasodilative activity. Since the coefficients of Zc are close to unity, protonation at the benzylic nitrogen atom seems to play a significant role. Although lipophilicity itself is a very i m p o r t a n t factor for these c o m p o u n d s to e x e r t a c t i v i t y as e x p e c t e d for t h e h y d r o p h o b i c trimetazidines, the term for the lipophilicity was not significant in Eq. 5. This may be t a k e n to indicate t h a t the molecular lipophilicity of these compounds is sufficiently high and t h a t r a t h e r small differences in lipophilicity are not critical in governing the variations in activity. The electronic and steric interactions of the drugs with the active site is more critical. B u l k y s u b s t i t u e n t s at t h e para-position of the diphenylmethyl moiety are not favorable for potency. Aromatic fluorine is the s m a l l e s t group in t e r m s of MR, and t h u s fluorine is the best substituent for both Y and Z. Next, the r e l a t i o n s h i p b e t w e e n the s t r u c t u r e and d u r a t i o n of action was examined. Compounds were judged to be long-acting when their durations of action were greater t h a n 20 times t h a t of papaverine.

423

From the pharmacological data, compounds of higher potency also seemed to be long-acting. ALS analysis for the duration of action with Z(~ and MR appearing in Eq. 6, gave a significant result omitting three 2,4,6-trimethoxybenzyl derivatives (IVm, IVy and lVgg). Y = - 1.774 Za - 0.128 MR - 0.302 n = 3 4 , Rs=0.620, nmi s - 6 , t - 4 . 4 7 ,

[6] p < 0.001

In Eq. 6, Y is the rating score taking either unity or zero depending upon the judgment for long-acting or not. The Y value ultimately calculated by Eq. 6 was categorized into integers corresponding to the rating score according to certain rules (2). In Eq. 6, n represents the number of compounds used to derive the equation, nmi s is the number of compounds where observed and calculated rating scores do not match, Rs is the Spearman rank correlation coefficient, t is Student's t value calculated by t - Rs[(n - 2)/(1 - Rs2)] 1/2, and p is the level of significance. One possible justification for the exclusion of the above three compounds is that their conformation may be different from the others because of their di-ortho-substitution on the benzyl moiety. The duration of action of drugs depends upon various factors such as its elimination, distribution, metabolic transformation, and, in some cases, the biological activity of the metabolites. However, Eqs. 5 and 6 suggest t h a t introduction of electron-donating substituents at the benzyl-benzene ring and sterically small substituents at the paraposition on the diphenylmethyl moiety bring about strong interaction of the compounds with the active site, resulting in high potency as well as prolonged action. Compounds with high cerebral vasodilative activity would have high affinity for the active site and may bind too tightly to allow easy washing out by the blood flow, giving a long-lasting action, whereas the opposite situation would apply to compounds with ow activity. Generally, substituents containing such hetero atoms as O and N are electron-donating. However, compounds bearing 4-NH 2 (lVn) or 4 - N M e 2 (lVo and 1Vkk) are acutely toxic. Therefore, the substitution on the benzyl moiety has to be a combination of alkoxy groups. From the results obtained with compounds having various numbers and

424 locations of methoxy groups, the 2,3,4-trimethoxy seemed to be the best substitution pattern. Therefore, 1-[bis(4-fluorophenyl)methyl]-4-(2,3,4trimethoxybenzyl)piperazine was thought to be the best compound with respect to potency and duration. The dihydrochloride of this compound (IVp : KB-2796) was selected as a candidate for the development of a cerebral vasodilator, and is now under extensive clinical evaluation.

2HCI

KB-2796 F

2.4

A p p l i c a t i o n to ~

Evolution

From the slope and sign of the Z~ term in Eq. 5, it was suggested t h a t an increase of electron density on the benzylic nitrogen atom is much more important t h a n t h a t on the other nitrogen for enhancement of the potency. Since factors for the t r a n s p o r t process were assumed not to be critical, and considering the steric effect of p a r a substituents of the diphenylmethyl moiety, a model for the interaction of these compounds with the active site is proposed, as shown in Fig. 1.

~

.,y

Anionic

Site

iuClsion

Fig. 1 Model of the Active Site (Reproduced from ref. 19 by permission of the

Pharmaceutical Society of Japan).

425

The putative active site may consist of a hydrophobic pocket, bearing an anionic site which interacts electrostatically with the positively charged benzylic nitrogen atom. The hydrophobic effect of the diphenylmethyl moiety does not appear explicitly in the equations, but its introduction resulted in much higher activity than that of trimetazidine. Thus, there may be a strong hydrophobic interaction between the diphenylmethyl moiety and the wall of the pocket. The depth of the pocket from the anionic site is limited so the steric repulsion of bulky Y and Z substituents lowers the binding interaction of the molecule. The above results prompted us to a t t e m p t further structural modifications for new leads. Since the electron density on one of the two nitrogen atoms of the piperazine ring is important, we first designed the vinylog, the structure with a double bond between the phenyl moiety and the methylene bridge, to t r a n s m i t the electronic effect of substituents on the benzyl-benzene ring through the double bond to the nitrogen atom.

x

~, x

IV ~ z

VII

Y

Namely, 1-(substituted cinnamyl)-4-diphenylmethylpiperazines (VII), i.e., substituted analogs of cinnarizine (II), were synthesized and their activities were tested (20). As expected, compounds bearing electron-donating substituents on the cinnamyl moiety showed potent activity (Table 3). The 4-NMe 2 derivative (VIIe) was one of the most potent compounds, but its acute toxicity was high, as in the case of the benzyl analog. Among the compounds p r e p a r e d , l-[bis(4-fluorophenyl)methyl]-4-(2,3,4-trimethoxycinnamyl)piperazine dihydrochloride (VIIa, KB-3512) was selected for further studies. Many substituted cinnarizine derivatives have been reported (21, 22), but there seems to be no compound so far which is more potent than c i n n a r i z i n e (VII :X - Y = H) and f l u n a r i z i n e (VII :X - H , Y - F),

426

TABLE 3. Structural Features and Cerebral Vasodilative Activities of 1Cinnamyl-4-diphenylmethylpiperazines (VH)

Compd. No.

X

2,3,4-(OMe) 3 2,3,4-(OMe)~ VIIc d 2,4-(OMe) 2 VIId d 2,4-(OMe) 2 VIIe d 4-NMe 2 Cinnarizine H Flunarizine" 2HC1 H VIIa b

VIIb b

e.j

Y

Activitya

F H F H F H F

1.25c 0.98 1.25 0.99 1.65 0.71 0.79

Duration 1 1 1 1 1 0 1

a) Activity is expressed in terms of increase of maximun blood flow relative to that of papaverine ata dose of 1 mg/kg, i.v. b) Dihydrochloride. c) The dose of this compound was 0.3 mg/kg, i.v. d) Fumarate.

b e c a u s e s o m e h a v e a n e l e c t r o n - w i t h d r a w i n g g r o u p on t h e c i n n a m y l moiety a n d some h a v e a b u l k y s u b s t i t u e n t on the d i p h e n y l m e t h y l group. T h u s , KB-3512 (VIIa) is t h o u g h t to be a m o n g the most p o t e n t analogs of cinnarizine. Recently, a series of vinylogs of s u b s t i t u t e d c i n n a r i z i n e s (VIII) h a s b e e n r e p o r t e d to h a v e p o t e n t v a s o d i l a t i v e action (23). A m o n g t h e m , a vinylog of KB-3512 ( V I I I a most potent compounds.

9X = 2,3,4-(OMe)3, Y = F) w a s one of the It is of i n t e r e s t t h a t t h e s t r u c t u r e - a c t i v i t y

r e l a t i o n s h i p of this series of c o m p o u n d s s e e m s to be s i m i l a r to t h o s e of t h e i r benzyl (IV) a n d c i n n a m y l (VII) analogs.

•

Q.~N VIII Y

The

benzylic

nitrogen

atom

p i p e r a z i n e d e r i v a t i v e s (lV) p l a y s v a s o d i l a t i v e activity.

of 1 - b e n z y l - 4 - d i p h e n y l m e t h y l a significant

role in c e r e b r a l

T h e r e f o r e , t h r e e t y p e s of p i p e r i d i n e d e r i v a t i v e s

427 (IX - X I) w e r e p r e p a r e d in o r d e r to i n v e s t i g a t e t h e role of t h e n i t r o g e n a t o m a t t a c h e d to t h e d i p h e n y l m e t h y l m o i e t y (24). The compounds

substituted

with electron-donating

X groups

e x h i b i t e d p o t e n t a c t i v i t y as well as a long d u r a t i o n of t h e action, w h e r e a s u n s u b s t i t u t e d d e r i v a t i v e s w e r e less a c t i v e a n d did n o t s h o w a n y longl a s t i n g a c t i o n (Table 4).

A l t h o u g h t h e p o t e n c y w a s v a r i e d d e p e n d i n g on

Y

X

x OH

TABLE 4. Structural Features and Cerebral Vasodilative Activities of 1-Benzylpiperidines (IX, X and XI) Compd. No.

X

Zo

Activity a

Duration

lXa

H

0.00

N.T. b

N.T.

IXb IXc Xa c

2,3,4-(OMe) 3 2,4-(OMe) 2 H

-0.42 -0.54 0.00

1.00 1.37 0.78

0 1 0

Xb c Xc c XIa d XIb c XIc c XId e

2,3,4-(0Me) 3 2,4-(OMe) 2 H 2,3,4-(OMe) 3 2,4-(0Me)2 4-NMe 2

-0.42 -0.54 0.00 -0.42 -0.54 -0.83

1.05 0.96 0.67 1.00 1.10 1.08

1 1 0 1 1 1

a) See Footnote of Table 3. b) N.T. 9not tested, c) Hydrochloride. d) Fumarate. e) Dihydrochloride.

428 the type of X substituent in a manner similar to that observed in the original series, the mode of connection between the piperidine ring and the diphenylmethyl moiety showed little effect in these modified series. When the substituent on the benzyl moiety was equal, the piperidine analog (X I) was almost equipotent to the corresponding piperazine (IV). These results suggest that the nitrogen atom attached to the d i p h e n y l m e t h y l moiety in 1-benzyl-4-diphenylmethylpiperazine derivatives (IV) plays no special role in manifestation of the activity and is exchangeable for the carbon atom, and that the piperidine derivatives interact with the active site in a manner very similar to piperazines. Unfortunately, these compounds were observed to lack cerebrovascular specificity. F u r t h e r evaluations of these derivatives as cerebral vasodilators were terminated. 3.

ANTIULCERATlVE PIPERAZINEACETATES

Peptic ulcers are classified into duodenal and gastric types based on the region affected. These ulcers are considered to be due to imbalances between aggressive factors such as acid and pepsin and the resistance of gastrointestinal mucosa against them. Acid secretion is critical for production of duodenal ulcers, whereas gastric ulcers are mainly induced by weakening of the defensive factors. Thus, antiulcer agents are generally classified into two categories, antisecretory agents, which suppress the aggressive factors, and cytoprotective agents, which strengthen the defensive mechanisms of the gastrointestinal mucosa. For the purpose of treatment and prophylaxis of peptic ulcers, however, the cytoprotective effect is thought to be more important. 3 . 1 Strategy for ~ Identification and Optimization Various antiulcer agents exert cytoprotective activity. Examples are cetraxate hydrochloride (XII) (25), sucralfate (XIII) (26) and teprenone (XIV) (27). The structures of these known agents are quite diverse. Since these compounds do not have sufficient activity, we thought it unlikely to be fruitful to derivatize them to develop novel antiulcer agents with higher cytoprotective activity.

429

NH2CH 2 9

,, ICO0

HCI

CH2CH2COOH

H

0

Ik~R Xll

9Cetraxate Hydrochloride

R ( ~ H

H

H/ ~0 OR

-J ~ LOR OR H

R = S03[AI2(OH)5] XlV"

Teprenone

XlII

9Sucralfate

Therefore, we used a random screening procedure to find a novel lead structure. After the lead compound was found, a series of congeners was synthesized and tested by the indomethacin-induced ulcer model using rats. Compounds which caused a statistically significant decrease in the ulcer index defined by the size of the ulcer from the control at a dose of 200 mg/kg were judged to be active. Oral toxicity (LD50) was examined in mice. Then, QSAR analyses (ALS method) were performed for activity ratings (1 for active and 0 for inactive compounds) to obtain a p r i m a r y clue to s t r u c t u r a l requirements for the activity. For active compounds with low toxicity, the antiulcer activity was measured in terms of ED50 and the QSAR was performed (Hansch-Fujita method) for more accurate analysis of factors enhancing the activity. The combined results of two QSAR analyses were used to predict the optimal structure. 1 - B e n z y l - 4 - p i p e r a z i n e a c e ~ d e Analogs During the course of general screening of benzylpiperazine derivatives, which were originally synthesized as possible cerebral vasodilators but found to have only low activity, 1-(pyrrolidinocarbonylmethyl)-4-(2,3,4-trimethoxybenzyl)piperazine dimaleate (XVa) was found to possess potent antiulcer activity without any antisecretory activity. This prompted us to synthesize and test various analogs of this compound (28). In the first attempt, compounds with various N-substituents in place of the entire 2,3,4-trimethoxybenzyl moiety of XVa were synthesized and tested. We found that the 2,3,4-trimethoxybenzyl moiety of XVa was not replaceable by a simple alkyl or acyl moiety without significant loss of the activity. 3.2

430

MeO

"v~

OMe

N

"

~COOH XV

XVa

Compounds X V a - X V i shown in Table 5, in which the substitution p a t t e r n X in the s t r u c t u r e X V was fixed to 2,3,4-trimethoxy, were synthesized and the effects of modifications in the amide moiety on antiulcer activity were analyzed by the ALS method to obtain Eq. 7 (29), Y = - 2.474 Vw + 2.293 n-9,

Rs=0.800,

[7]

nmi s - l ,

t = 3.52,

p < 0.01

where Y is the activity rating and Vw is the van der Waal's volume in ~3 scaled by 1/100 (30) of the NRR' moiety. Equation 7 suggests t h a t compounds with the less bulky amide group were favorable for activity. Considering the data together with toxicity data, the pyrrolidino moiety seemed to be the most suitable. Next, the effects of s u b s t i t u e n t X on the benzyl moiety were i n v e s t i g a t e d fixing the amide moiety as pyrrolidinocarbonyl with compounds XVj - X V u . In preliminary experiments, we observed that substitution at both the 3 and 4 positions was necessary for the activity. Thus, we examined various physicochemical p a r a m e t e r s of 3- and 4substituents. Among compounds with various substitution patterns of methoxy groups, only two were active: the 2,3,4- and 3,4,5-trimethoxy derivatives. The unhindered methoxy group is thought to be coplanar

~ f-~

a

CH

b

Fig. 2 Copl~n~r conformation (a; conjugated) and the out.of-plane conformation (b ; non-conjugated) (Reproduced from res 29 by p e ~ i . ~ i o n of the Pharmaceutical Society of Japan).

431 TABLE

5.

S t r u c t u r a l F e a t u r e s a n d Antiulcer Activities of 1-(Aminocarbonylalkyl)~-benzylpipernzlne Dimaleates (XV)

Compd. No.

Activity X

R

R'

n

Vw

H

-(CH2) 4H

1 1

0.705 0.177

1.35 1.35

1.90 1.90

XVc

2,3,4-(OMe) 3 2,3,4-(OMe) 3 2,3,4-(OMe) 3

Et

Et

1

0.809

1.35

1.90

1

1

XVd

2,3,4-(0Me) 3

Pr

Pr

1

1.117

1.35

1.90

0

0

XVe

2,3,4-(OMe) 3

1

0.859

1.35

1.90

0

1

XVf

1

1.005

1.35

1.90

0

0

Ph

1

0.879

1.35

1.90

1

1

CH2Ph Ph

1

1.033

1.35

1.90

0

0

1

1.041

1.35

1.90

0

0

-(CH2) 4-

1

0.705

1.00

1.00

0

0

1

0.705

1.00

1.52

0

0

1 1

0.705 0.705

1.00 1.00

1.80 1.35

0 0

0 0

XVa

XVb

2,3,4-(OMe) 3

-(CH2) 5H c-Hex

XVg

2,3,4-(0Me) 3

H

XVh

2,3,4-(0Me) 3

XVi

2,3,4-(0Me) 3 H

H Me

XVj XVk

T3

T4

Obs. Calcd. (Eq.8) 1 1

1 1

XVI

4-Me 4-C1

XVm

4-OMe

-(CH2) 4-(CH2) 4-(CH2) 4-

XVn

3,4-C12

-(CH2) 4-

1

0.705

1.80

1.80

1

1

XVo

2,4-C12 2-OMe

-(CH2) 4-

1

0.705

1.00

1.80

0

0

-(CH2) 4-

1

0.705

1.00

1.00

0

0

0.705

1.35

1.00

0

0

XVp XVq

3-OMe

-(CH2) 4-

1

XVr

2,4-(OMe) 2 3,4-(OMe) 2

-(CH2) 4-(CH2) 4-

1

0.705

1.00

1.35

0

0

XVs

1

0.705

1.35

1.35

0

0

XVt XVu

3,4,5-(OMe) 3 2,4,6-(OMe) 3

-(CH2) 4-(CH2) 4-

1 1

0.705 0.705

1.90 1.00

1.35 1.35

1 0

1 0

XVv

2,3,4-(OMe) 3

-(CH2) 4-

2

0.705

1.35

1.90

1

1

XVw

2,3,4-(0Me) 3

-(CH2) 4-

3

0.705

1.35

1.90

0

0

XVx

XVy

2,3,4-(OMe) 3 3,4,5-(OMe) 3

-(CH2) 4-(CH2) 4-

4 2

0.705 0.705

1.35 1.90

1.90 1.35

0 1

0 1

XVz

3,4,5-(0Me) 3

-(CH2) 4-

3

0.705

1.90

1.35

0

0

XVaa

3,4,5-(0Me) 3

-(CH2) 4-

4

0.705

1.90

1.35

0

0

432 with the benzene ring owing to the conjugation of the lone pair electrons with the aromatic ring. On the other hand, the central methoxy group of the 2,3,4- and 3,4,5-trimethoxyphenyl moiety may be forced out of and nearly perpendicular to the plane of the aromatic ring (31). The rotational barrier of Me around the Ar-O bond of substituted anisoles is reported to be about 3-6 kcal/mol (32), so the methoxy group could be relatively easy to rotate. For the hindered perpendicular methoxy group, the smaller value of the two thickness values for directions above and below the ring plane is represented by the Verloop's STERIMOL p a r a m e t e r B 1 which is attributed mostly to the radius of the oxygen atom (33). For the unhindered coplanar methoxy group, the smaller thickness is expressible by the STERIMOL B 2. These situations are depicted in Fig. 2. With these conformational features in mind, all analogs shown in Table 5 were subjected to analysis using an additional indicator variable, n, which is the number of methylene groups between the carbonyl group and piperazine, to obtain Eq. 8, Y = - 2.631 Vw + 1.960 T 3 + 1.449 T 4 - 0.482 n - 2.166 n - 2 7 , Rs-0.918, nmi s - l , t-11.57, p<0.001

[8]

where T is the smaller of the two thickness values for each substituent above and below the ring plane. The subscript represents the substituent position. The T value of each substituent is equivalent with the STERIMOL B 1 except for the unhindered methoxy group where it is taken as the B 2 parameter. Equation 8 shows that (a) a bulky amide moiety is disadvantageous, (b) the number of methylene units should be low, and (c) the "minimum" thickness of 3- and 4-substituents on the benzyl-benzene ring should be large for high antiulcer activity. Several compounds exhibited an antiulcer activity superior to that of the reference compounds, but, because of their subacute toxicity, they were not acceptable. 3.3

Esters of 1-Benzyl-4-piperazinealkanoic acids If just small size is among the most important conditions for the potent antiulcer activity, then the NRR' moiety could be replaceable

433 with other small groups. Thus, we expected their ester analogs to be active and found t h a t some of t h e m were indeed active as shown in Table 6 (34).

x~N/"~

0 L~,~/N- (CH2)n,,~OR XMI

P r e l i m i n a r y examinations showed t h a t the van der Waal's volume, Vw, of the OR group is a significant factor governing the potency rating, as expected. Among derivatives judged as active, the phenyl ester (XVIi) was moderately potent and least toxic as summarized in Table 7. Therefore, the s u b s t i t u t e d phenyl ester derivatives were synthesized and tested. The ALS analysis of these derivatives (Table 6, XVI1 - XVIq) gave Eq. 9, [9]

Y = - 4.591~ - 0.459 n-6,

Rs-l.000,

nmi s = 0

w h e r e ~ is the H a m m e t t constant and the ~p value was used for the ortho substituent. In Eq. 9, Vw was not significant, because variations in the Vwvalue were not so large within these substituted phenyl s u b s t i t u e n t s . The negative o t e r m of Eq. 9 s u g g e s t s t h a t electrond o n a t i n g s u b s t i t u e n t s m a k e the e s t e r s stable a g a i n s t hydrolytic transformation. H y d r o l y s i s prior to r e a c h i n g the action site(s) decreases the antiulcer activity, because the corresponding carboxylic acids are inactive. Next, all of the ester compounds listed in Table 6 (XVIa - X V I a a ) were analyzed together and Eq. 10 was obtained. Y - - 2.624 Vw + 1.779 T 3 + 1.649 T 4 - 4.109 u - 1.621 n - 1.562 n-27,

Rs-l.000,

[10]

nmi s - 0

E q u a t i o n 10 shows s t r u c t u r a l r e q u i r e m e n t s for activity of esters essentially the same as Eq. 8 for those of amides, in addition to the requirement for the phenyl moiety to possess electron-donating groups.

434 TABLE 6. S t r u c t u r a l F e a t u r e s a n d A n t i u l c e r Activities of 1-Benzyl~l-piper~_ zinea l k a n o i c Acid E s t e r D i m a l e a t e s (XVI) Compd.

Activity

No.

X

R

n

Vw

~

T3

T4

Obs. Calcd. (Eq.10)

XVIa

2,3,4-(OMe) 3

Me

1

0.304

0.00

1.35

1.90

1

1

XVIb

2,3,4-(OMe) 3

Et

1

0.458

0.00

1.35

1.90

1

1

XVIc

2,3,4-(OMe) 3

Pr

1

0.612

0.00

1.35

1.90

1

1

XVId

2,3,4-(0Me) 3

Bu

1

0.766

0.00

1.35

1.90

1

1

XVIe

2,3,4-(OMe) 3

Am

1

0.920

0.00

1.35

1.90

0

0

XVIf

2,3,4-(0Me) 3

Hex

1

1.074

0.00

1.35

1.90

0

0

XVIg

2,3,4-(0Me) 3

Hep

1

1.228

0.00

1.35

1.90

0

0

XVIh

2,3,4-(0Me) 3

iso-Pr

1

0.607

0.00

1.35

1.90

1

1

XVIi a

2,3,4-(OMe) 3

Ph

1

0.844

0.00

1.35

1.90

1

1

XVIj a

2,3,4-(0Me) 3

CH2Ph

1

0.998

0.00

1.35

1.90

0

0

XVIk

2,3,4-(OMe) 3

(CH2)2Ph

1

1.152

0.00

1.35

1.90

0

0

XVI1 a

2,3,4-(0Me) 3

4-Me-Ph

1

0.998

-0.17

1.35

1.90

1

1

X V I m a 2,3,4-(0Me) 3

4-C1-Ph

1

1.009

0.23

1.35

1.90

0

0

XVIn a

2,3,4-(OMe) 3

4-MeO-Ph

1

1.079

-0.27

1.35

1.90

1

1

XVIo a

2,3,4-(OMe) 3

3-MeO-Ph

1

1.079

0.12

1.35

1.90

0

0

XVIp a

2,3,4-(0Me) 3

2-MeO-Ph

1

1.079

-0.27

1.35

1.90

1

1

XVIq a

2,3,4-(OMe) 3

4-EtO-Ph

1

1.233

-0.24

1.35

1.90

1

1

XVIr a

H

4-MeO-Ph

1

1.079

-0.27

1.00

1.00

0

0

XVIs a

4-Me

4-MeO-Ph

1

1.079

-0.27

1.00

1.52

0

0

XVIt a

4-C1

4-MeO-Ph

1

1.079

-0.27

1.00

1.80

0

0

XVIu a

4-OMe

4-MeO-Ph

1

1.079

-0.27

1.00

1.35

0

0

XVIv a

3,4-C12

4-MeO-Ph

1

1.079

-0.27

1.80

1.80

1

1

XVIw a

3,4-(OMe) 2

4-MeO-Ph

1

1.079

-0.27

1.35

1.35

0

0

XVIx a

3,4-0CH20-

4-MeO-Ph

1

1.079

-0.27

1.90

1.90

1

1

XVIy b

3,4,5-(0Me) 3

4-MeO-Ph

1

1.079

-0.27

1.90

1.35

1

1

XVIz a

2,3,4-(0Me) 3

4-MeO-Ph

2

1.079

-0.27

1.35

1.90

0

0

X V I a a a 3,4,5-(0Me) 3

4-MeO-Ph

2

1.079

-0.27

1.90

1.35

0

0

a) Dihydrochloride. b) Difumarate.

435 Although s t r u c t u r a l modifications conforming to the above r e q u i r e m e n t s could be made, Eq. 10 only predicts w h e t h e r the compound is active or not. The prediction of compounds more potent than other potent compounds is beyond the ability of Eq. 10, when every compound belongs to the same category. At this point, we used the Hansch-Fujita analysis for eleven compounds listed in Table 7, five amides and six esters, selected in terms of the low acute toxicity so that the LD50 is higher than 2 g/kg, p.o. Compound XVIn is the most active with an ED50 against the indomethacin-induced ulcers of 10 mg/kg, p.o., whereas compound XVa is the least active with an ED50 of 164 mg/kg, p.o. For 11 compounds, Eq. 11 was derived, log(l/C) = 1.253(+0.392) Vw + 3.349(+0.293) n = 1 1 , r=0.923, s=0.15, F=52.13

[11]

where C is the ED 50 (mol/kg p.o.)value. Equation 11 indicates that the larger the Vw value of the OR and NRR' group, the more potent is the antiulcer activity. The number of methylene units in the bridging moiety is not critical. This result is contrary in terms of the steric effect to the ALS results represented in Eqs. 8 and 10. The discrepancy could be attributed to the fact t h a t the ALS analyses are just to distinguish "active" compounds from a number of "inactive" compounds. The overall trend that the lower steric Vw value is more favorable may be the case. For the active compounds included in Eq. 11, which were given the rating score of one in Eqs. 8 and 10 irrespective of the potency variations in terms of log(I/C), the steric effect of NRR' and OR on the activity may well be different from that suggested by the ALS analyses. The above results suggested that even if a compound of this series were predicted to be active according to Eqs. 8 and 10, there is no need to synthesize and test it when the OR group is smaller t h a n t h a t in compounds XVIn and XVIy. Therefore, we thought it advisable to terminate any further analog synthesis from the standpoints of availability of raw materials and ease of synthesis. For the selected six ester derivatives (Table 7), antiulcer activities against other ulcer models were examined and two compounds (XVIn, XVIy) were found to possess an antiulcer activity superior to those of

436 TABLE 7.

S t r u c t u r a l Features, Antiulcer Activities a n d Toxicities of Selected 1Benzylpiperazinealkanoic acid Derivatives

Compd. No.

log (1/C) X

OR or NRR'

n

Vw

NIZ2, Pyrr D

1 1

0.177 0.705

LD50 a (mg/kg)

Obs.

Calcd. (Eq.ll)

~ 2500

3.53 4.31

3.57 4.23

XVb XVa

2,3,4-(0Me) 3 2,3,4-(OMe) 3

XVt

3,4,5-(0Me) 3

Pyrr

1

0.705

4200

4.29

4.23

XVv

2,3,4-(0Me) 3

Pyrr

2

0.705

3400

4.37

4.23

XVy

3,4,5-(0Me) 3

Pyrr

2

0.705

3600

4.34

4.23

XVIa

2,3,4-(0Me) 3

OMe

1

0.304

3120

3.87

3.73

XVIc

2,3,4-(0Me) 3

OPr

1

0.612

3930

3.80

4.11

XVId

2,3,4-(0Me) 3

OBu

1

0.776

4000

4.21

4.31

XVIi

2,3,4-(0Me) 3

OPh

1

0.844

5660

4.24

4.41

XVIn

2,3,4-(0Me) 3

OPh(4-OMe)

1

1.079

4200

4.70

4.70

XVIy

3,4,5-(0Me) 3

OPh(4-OMe)

1

1.079

4800

4.79

4.70

a) The LD50 values for the active compounds listed in Tables 5 and 6 except for those listed here are below 3000 mg/kg, b) Pyrr : pyrrolidino.

such reference compounds as XII, XIII and XIV. Since its toxicity was low and acceptable, we selected 1-(3,4,5-trimethoxybenzyl)-4-(4methoxyphenyloxycarbonylmethyl)piperazine (XVIy) as a candidate for the antiulcer drug development (KB-5492 as monofumarate).

OMe

KB-5492

The antiulcer mechanism of this novel series of compounds seems to be their cytoprotective activity, because no suppressive effect was observed against secretion of acid or pepsin. Several 1-piperazineacetamides, including esaprazole (XVII) (35), pirenzepine (XVIII) (36) and fenoverine (X IX) (37), have been used clinically as antiulcer agents.

437

XVII: Esaprazole

XVlll : Pirenzepine

X l X : Fenoverine

These compounds show antisecretory activity. It is impossible to deny that experienced medicinal chemists would easily be able to design compounds with the structure X V as possible antiulcer agents from structures of such existing drugs as shown above, but it would not be readily predictable that not only amides (XV) but also esters (XVI) of 1benzyl-4-piperazineacetic acid show antiulcer activity based on cytoprotection. Structural resemblance does not necessarily imply similarity in the mechanisms of antiulcer activity. Extrapolative application of the QSAR results of the amides series brought forth a novel lead structure (alkyl esters) and the antiulcer activity was optimized efficiently by substituent modifications aided by the QSAR. 4. ANTIHISTAMINIC 2-PIPERAZINYLBENZIMIDAZOLES In the course of search for antiinflammatory benzimidazole derivatives, 1-alkyl-2-(4-methyl-l-piperazinyl)benzimidazoles (38) were prepared. Pharmacological profiles suggested t h a t these compounds could be possible leads for Hl-antihistaminics without significant side effects. Antihistaminics are useful for treating the symptoms of allergic reactions including seasonal hay-fever, allergic rhinitis and conjunctivitis. Conventionally used antihistaminics have, however, certain drawbacks in that they frequently induce side effects such as dry mouth resulting from their anticholinergic activity, and exert central nervous system (CNS)-depressive effects such as sedation and hypnosis. Therefore, Iemura and co-workers started a project to develop compounds with not only higher antihistaminic activity but also lower anticholinergic and CNS-depressive activities. Analogs were synthesized and their antihistaminic activities (IC50 in M) were measured using isolated ileum from guinea pigs. Among these analogs, 1- (2-ethoxyethyl)-2-(4-m ethyl- 1-hom opiper az inyl) ben zimid az ole

438

(COOH N " ~ HOOC/ I CH2CH2OCH2CH3

KG-2413

,Me ~N N-R2 I i (CH2)m R1 XX

CI~

N. Me XXI: Chlorpheniramine

difumarate (XXnn : KG-2413) was selected for further study (39). Its activity in vivo was 39 times more potent than t h a t of chlorpheniramine (XX I) (40), which is one of the most potent H 1-antihistaminic agents known. QSAR analyses were performed in order to confirm the validity of the selection (41). 4 . 1 Antihistaminic Activity Because hydrophobic (n) and steric (MR) p a r a m e t e r s are linearly related to the n u m b e r of methylenes (NM) in s t r a i g h t chain alkyl groups, NM was used as a temporary makeshift. For the compounds (XX a - X X g) in Table 8 with straight alkyl chains at the 1-position of the 2-(4-methyl-l-piperazinyl)benzimidazole, a plot of antihistaminic activity (log 1/IC50) against NM of the alkyl chain as R 1 suggested a parabolic relationship. Then, compounds with various substituents at the 1-position of the 2-(4-methyl-l-piperazinyl)benzimidazole ( X X a X X d d ) were subjected to analysis (Table 8). Analysis using another variable NA for all but seven compounds ( X X h - X X k , X X p , X X s and X X c c) gave Eq. 12. logl/IC50 = - 0.079(+_0.033) NA 2 + 0.875(+0.397) NA + 5.155(+1.165) n = 23, r = 0.754, s = 0.480, F= 13.16 [12] NA is defined as the n u m b e r of atoms other t h a n h y d r o g e n in substituents at the 1-position. The addition of other physicochemical s u b s t i t u e n t p a r a m e t e r t e r m s was not significant. Although the correlation was not very good, NA seemed to rationalize the effects of R 1

439

best as a single parameter. Since the NA p a r a m e t e r was not depend on the type of atom or bond, the physicochemical meaning of NA seemed to be steric bulk. As described above, the activities of seven compounds deviated markedly from those calculated using Eq. 12. For compounds X X h X X k , the large width of the s u b s t i t u e n t s may be unfavorable for activity. The low activities of X X p and X X s might be ascribed to the folding of the R 1 substituent, as observed in 1-(3-phenoxypropyl)uracil (42). The higher activity t h a n predicted for compound XXcc seemed to be due to the fact t h a t the phenoxyethyl group fits the cavity of the receptor better t h a n expected. If these assumptions were reasonable, the variations in the activity could be analyzed by steric p a r a m e t e r s representing the width as well as the length of substituents. Thus, an analysis including these seven compounds ( X X a - X X d d ) was performed using Verloop's STERIMOL parameters (33). In e s t i m a t i n g STERIMOL p a r a m e t e r s , the R 1 chain was assumed to take the fully staggered conformation extending toward the direction p e r p e n d i c u l a r to the benzimidazole ring except in the following cases: the 1-phenyl group (XXk) is coplanar with the benzimidazole ring, the 3-(ethylthio)propyl (XXp) and 3-(methoxy)propyl (XXs) groups fold onto the benzimidazole ring, and the benzene ring not directly attached to the benzimidazole ring in compounds XXl, X X m , X X c c and X X d d is perpendicular to the benzimidazole plane. With the two types of STERIMOL parameters, a good correlation was obtained as shown in Eq. 13. logl/IC50= - 0.096(+0.025) L 2 + 1.413(+0.369) L - 1.173(+0.321) B 3 + 4.686(_+1.401) [13] n = 30, r = 0.891, s = 0.397, F = 33.32 In Eq. 13, L represents the length p a r a m e t e r for R 1 substituents along the axis connecting the 1-N atom of the benzimidazole with the a-atom of R 1 substituents considering the folding factor for compounds X X p and X X s . B 3 is the p a r a m e t e r for the second largest width of substituents. Here, it represents the larger one of the two width value

440 TABLE 8.

Structural Features azoles (XX)

a n d A n t i h i s t a m i n i c Activities of Benzimid-

Compd. No.

log 1/IC50 R1

R2

m

B3

L

I

Obs.

Calcd. (Eq.14)

XXa a XXb b

Me Pr

Me

2

1.90

3.00

0

6.50

5.75

Me

2

1.90

5.05

0

6.42

7.13

XXc a XXd c

Bu Am

Me

2

1.90

6.17

0

7.30

7.55

Me

2

1.90

7.11

0

7.72

7.71

XXe c

Hex

Me

2

1.90

8.22

0

7.59

7.67

XXf c

Hep

Me

2

1.90

9.16

0

6.82

7.46

XXg b

Dec

Me

2

1.90

12.33

0

5.38

5.47

XXh XXi b

(CH2)2CHMe 2 CHMePr

Me

2

2.76

6.17

0

5.85

6.51

Me

2

3.66

6.17

0

5.85

5.43

X~ b XXk c

CH2CHMePr Ph

Me

2

3.18

7.11

0

5.82

6.17

Me

2

3.11

6.28

0

6.12

6.12

XXl a

CH2Ph

Me

2

1.90

5.91

0

7.77

7.47

XXm c

(CH2)2Ph

Me

2

1.90

8.41

0

7.62

7.64

XXn

CH2SPr

Me

2

1.90

7.59

0

7.17

7.72

XXo

(CH2)2SEt

Me

2

1.90

7.29

0

7.72

7.72

XXp XXq c

(CH2)3SEt CH2OPr

Me

2

1.90

3.62

0

6.28

6.25

Me

2

1.90

6.95

0

7.75

7.69

XXr c

(CH2)2OEt

Me

2

1.90

6.97

0

8.00

7.69

XXs b

(CH2)3OMe

Me

2

1.90

3.62

0

6.06

6.25

XXt a

(CH2)2NHEt

Me

2

3.03

6.68

0

6.54

6.30

XXu a XXv a

(CH2)2OH (CH2)2OMe

Me

2

1.90

4.79

0

6.39

7.00

Me

2

1.90

6.03

0

7.89

7.51

XXw

(CH2)2OCH=CH 2

Me

2

1.90

7.09

0

8.00

7.70

XXx d XXy c

(CH2)20(CH2)2OH (CH2)2OPr

Me

2

1.90

7.95

0

7.37

7.70

Me

2

1.90

8.10

0

7.70

7.69

XXz c

(CH2)2OCH2CH=CH 2

Me

2

1.90

8.32

0

7.77

7.66

XXaa c

(CH2)2OCH2C-CH

Me

2

1.90

8.73

0

7.92

7.58

XXbb c

(CH2)2OBu

Me

2

1.90

9.04

0

7.42

7.49

XXcc c

(CH2)2OPh

Me

2

1.90

7.85

0

8.16

7.71

XXdd d

(CH2)2OCH2Ph

Me

2

1.90

10.33

0

6.37

6.95

XXee c

(CH2)2OEt

H

2

1.90

6.97

0

7.96

7.69

XXff e

(CH2)2OEt

Et

2

1.90

6.97

0

7.75

7.69

441 TABLE 8. Continued Compd. No.

R1

XXgga XXhh a XXii a XXjj a XXkk a XXll a XXmm c XXnn b XXoob XXpp a XXqqa XXrr a XXss a XXttb XXuu b XXvvb XXwwb XXxx b

R2

(CH2)2OEt Pr (CH2)2OEt Bu (CH2)2OEt Am (CH2)2OEt Hex (CH2)2OEt CH2Ph (CH2)2OEt (CH2)2Ph (CH2)2OEt H (CH2)2OEt Me (CH2)2OEt Et (CH2)2OEt Pr (CH2)2OEt Bu (CH2)2OEt Am (CH2)2OEt CH2Ph (CH2)2OPr Me (CH2)2OCH2CH=CH 2 Me (CH2)2OCH2C-CH Me (CH2)2OBu Me (CH2)2OPh Me

m

B3

2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3

1.90 1.90 1.90 1.90 1.90 1.90 1.90 1.90 1.90 1.90 1.90 1.90 1.90 1.90 1.90 1.90 1.90 1.90

6.97 6.97 6.97 6.97 6.97 6.97 6.97 6.97 6.97 6.97 6.97 6.97 6.97 8.10 8.32 8.73 9.04 7.85

I

log 1/IC50 Obs. Calcd. (Eq.14)

0 0 0 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1

7.80 7.82 7.52 8.06 7.57 7.51 7.60 8.21 7.80 8.08 8.08 8.13 7.82 7.80 8.00 8.00 8.00 8.04

7.69 7.69 7.69 7.69 7.69 7.69 7.99 7.99 7.99 7.99 7.99 7.99 7.99 7.99 7.96 7.88 7.79 8.01

a) Dimaleate. b) Difumarate. c) 1.5 Fumarate. d) Fumarate.

for each s u b s t i t u e n t from the L-axis in two opposite di rect i ons p a r a l l e l w i t h th e b e n z i m i d a z o l e r i ng plane. The l a r g e s t w i d t h p a r a m e t e r B 4 did not w o r k b e t t e r t h a n B 3, because of t he difference in directions defining the

B parameters

of s u b s t i t u e n t s .

The

assumptions

for

the

c o n f o r m a t i o n of t h e R 1 s u b s t i t u e n t s o t h e r t h a n those t a k e n here did not w o r k well either. Thus, Eq. 13 would indicate t h a t the lower t he l a r g e r width,

i.e.,

t h e m or e s y m m e t r i c t he w i d t h s of s u b s t i t u e n t s from t h e L-

axis in t h e direction p a r a l l e l w i t h the b e n z i m i d a z o l e ring, t h e h i g h e r is the activity.

The o p t i m u m l engt h is r e q ui red for the R 1 substituents.

N e x t , t h e c o m p o u n d s (XXee

- XXxx)

in w h i c h R 2 is e i t h e r

h y d r o g e n or a n a l k y l g r o u p o t h e r t h a n m e t h y l a n d m is t h r e e w ere

442 considered together, leading to Eq. 14, l o g l / I C 5 0 - - 0.098(+0.019) L 2 + 1.440(+0.287) L - 1.194(+_0.252) B 3 + 0.338(+0.231) I + 4.643(+_1.135) [14] n = 5 0 , r=0.912, s=0.329, F=55.63 where I is the indicator variable for homopiperazine (I = 1) derivatives. Equation 14 indicates that the substituent R 2 at the 4-N-position of piperazine and homopiperazine has essentially no effect on the activity, and t h a t homopiperazines (I = 1) are almost uniformly more active t h a n the corresponding piperazines. The positively charged homopiperazine nitrogen may be situated closer to the anionic site of the receptor than the piperazine nitrogen. Otherwise, Eq. 14 is practically equivalent with Eq. 13. The length (L - 7.3) of the R 1 substituent is optimal for activity. From the above results, a model for the receptor binding features of this type of compounds is proposed as shown in Fig. 3. In the hypothetical receptor, an anionic site is present, which interacts electronically with the positively charged piperazine or homopiperazine nitrogen atom. There is also a slit-shaped cavity perpendicular to the region where the benzimidazole ring moiety p e r h a p s binds hydrophobically, as proposed by Rekker et. al. for antihistaminic diphenhydramine derivatives (43)

t

,

~~NHR (~

AnionicSite

:t,J"....":Cavy

Fig. 3 Model of the Binding Sites of 2-Piper~zinylbenzimidazoles (Reproduced from res 41 by permission of the Pharmaceutical Society of Japan).

443

Substructural units possessing tertiary amino groups such as piperidine, ethylenediamine, piperazine and homopiperazine moieties in conventional antihistaminics are thought to be bioisosteric. Thus, the benzimidazole derivatives with possible bioisosteric substituents at the 2position ( X X I I - XXV) shown in Table 9 were synthesized and tested (44). ~~"/',N > - -~"NNH

--~N-

I

R2

,~"',,,,~.. N R2 ~!~ 2>-- NH(CH2'm'--N:R2 I

R1 XXll

R1

XXIII

N

/--k

N I

R1 XlV

I

R1 XXV

(CH2)m

The a n t i h i s t a m i n i c activities of these compounds were in good a g r e e m e n t with those expected from Eq. 14. Therefore, all 82 compounds were subjected to analysis, and Eq. 15 was obtained as the best equation. log 1/IC50- - 0.097(+0.018) L 2 + 1.458(+0.260) L - 1.202(+0.234) B 3 + 0.299(+0.202) I + 4.528(+1.081) [15] n = 8 2 , r=0.875, s=0.321, F=62.71 Equations 14 and 15 are essentially equivalent. The antihistaminic activity (in vitro) of the additionally prepared compounds was correctly predicted by Eq. 14. In the in vivo test, however, only compounds X X V showed considerably potent activity comparable to t h a t of KG-2413. These results indicate that the pharmacokinetic characteristics such as absorption, distribution and metabolism of compounds X X V and X X, are more favorable to the activity than those of other derivatives. 4 . 2 Anticholinergic Activity As described above, classical antihistaminics commonly have unfavorable side effects due to anticholinergic activities. Therefore, the anticholinergic activities of twelve compounds (X X, Table 1 0 ) t h a t

444 TABLE

9.

S t r u c t u r a l F e a t u r e s a n d A n t i h i s t a m i n i c Activities of Various Types of Benzimidazoles

Compd.

log 1/IC50

No.

R1

R2

m

B3

L

I

Obs. Calcd. (Eq.15)

XXIIa b

(CH2)2OEt

Me

-

1.90

6.97

0

7.75

7.67

XXIlb c

(CH2)2OCH2CH=CH 2

Me

-

1.90

8.32

0

7.85

7.63

XXIIc b

(CH2)2OPh

Me

-

1.90

7.85

0

6.89

7.68

XXIId b

(CH2)2OEt

CH2Ph

-

1.90

6.97

0

7.18

7.67

XXIIe b

(CH2)2OEt

H

-

1.90

6.97

0

7.70

7.67

XXIIIa a

(CH2)2OEt

Me

2

1.90

6.97

0

7.89

7.67

XXIIIb a

(CH2)2OEt

Et

2

1.90

6.97

0

7.36

7.67

XXIIIc a

(CH2)2OEt

(CH2)2 -e

2

1.90

6.97

0

8.06

7.67

XXIIId a

(CH2)2OEt

Me

3

1.90

6.97

0

7.59

7.67

XXIIIe a

(CH2)2OEt

Et

3

1.90

6.97

0

7.23

7.67

XXIVa d

(CH2)2OEt

Me

-

1.90

6.97

0

7.77

7.67

XXIVb c

(CH2)2OCH2CH=CH 2

Me

-

1.90

8.32

0

7.96

7.63

XXIVc c

(CH2)2OCH2C-CH

Me

-

1.90

8.73

0

7.92

7.55

XXIVd c

(CH2)2OPh

Me

-

1.90

7.85

0

7.72

7.68

XXIVe c

(CH2)2OEt

H

-

1.90

6.97

0

7.92

7.67

XXIVf c

(CH2)2OEt

Et

-

1.90

6.97

0

7.82

7.67

XXVa d

(CH2)2OEt

Me

2

1.90

6.97

0

7.68

7.67

XXVb

(CH2)2OCH=CH 2

Me

2

1.90

7.09

0

7.70

7.68

XXVc b

(CH2)2OPr

Me

2

1.90

8.10

0

7.80

7.66

XXVd d

(CH2)2OCH2CH=CH 2

Me

2

1.90

8.32

0

8.02

7.63

XXVe d

(CH2)2OCH2C-CH

Me

2

1.90

8.73

0

7.92

7.55

XXVf d

(CH2)2OEt

H

2

1.90

6.97

0

7.62

7.67

XXVg c

(CH2)2OEt

Et

2

1.90

6.97

0

7.68

7.67

XXVh c

(CH2)2OEt

Pr

2

1.90

6.97

0

7.47

7.67

XXVi c

(CH2)2OEt

2

1.90

6.97

0

7.08

7.67

XXVj c

(CH2)2OEt

Me

3

1.90

6.97

1

7.85

7.96

XXVk c

(CH2)2OPr

Me

3

1.90

8.10

1

7.75

7.95

XXV1 c

(CH2)2OCH2CH=CH2

Me

3

1.90

8.32

1

8.04

7.92

XXVm c

(CH2)2OCH2C-CH

Me

3

1.90

8.73

1

8.04

7.83

XXVn c

(CH2)2OPh

Me

3

1.90

7.85

1

7.70

7.97

XXVo c

(CH2)2OEt

H

3

1.90

6.97

1

7.70

7.96

XXVp c

(CH2)2OEt

Et

3

1.90

6.97

1

8.00

7.96

(CH2)2OH

a-d) See footnot of Table 8. e) pyrrolidino.

445 s h o w e d p o t e n t a n t i h i s t a m i n i c activity in vitro as well as in vivo, w e r e m e a s u r e d . The IC 50 (M) values were e v a l u a t e d u s i n g isolated ileum from g u i n e a pigs by t h e u s u a l m e t h o d . Since t h e a n t i c h o l i n e r g i c potency is about four orders of m a g n i t u d e lower t h a n the a n t i h i s t a m i n i c potency in t e r m s of 1/IC 50' the anticholinergic side effects of this series of compounds were not serious. To examine the factors p a r t i c i p a t i n g in the anticholinergic potency, analysis was performed to give Eq. 16. log 1 / I C 5 0 - 0.287(_+0.198) B 4 + 0.725(+0.405) I + 2.411(+1.103) n-12,

r-0.879,

s-0.304,

[16]

F-15.37

In Eq. 16, B 4 r e p r e s e n t s the S T E R I M O L m a x i m u m w i d t h p a r a m e t e r of t h e R 1 s u b s t i t u e n t a n d I is the i n d i c a t o r v a r i a b l e for t h e homopiperazines. E q u a t i o n 16 indicates t h a t the more s y m m e t r i c the widths of R 1 s u b s t i t u e n t s in compounds carrying the piperazine ring, the lower is the anticholinergic activity.

TABLE 10. Anticholinergic and CN~Depressive Activities of Selected Benzimidazoles

Compd. No.

B4

MR/10

I

XXr XXy XXz

4.82 5.75 5.92 4.38 7.42 4.82 4.82 4.82 5.75 5.92 4.38 7.42

2.136 2.602 2.555 2.526 3.684 2.136 2.136 2.136 2.602 2.555 2.526 3.684

0 0 0 0 0 0 0 1 1 1 1 1

XXaa

XXcc XXee

XXff XXnn XXtt XXuu

XXvv XXxx

Anticholinergic log 1/IC50 Obs. Calcd. (Eq.16) 3.59 4.76 3.99 3.82 4.35 3.67 3.60 4.51 5.12 4.60 4.32 5.26

3.80 4.06 4.11 3.67 4.54 3.80 3.80 4.52 4.79 4.84 4.40 5.27

CNS-Depressive Effect Obs. Recog. Pred. (Eq.17) 1 1 1 1 1 0 0 0 1 0 0 1

0 1 1 1 1 0 0 0 1 0 0 1

0 1 1 1 1 0 0 0 0 0 0 1

446

4.3

CNS-Depressive Effect The other type of the common side effects of antihistaminics is hypnotic-sedative ( C N S - d e p r e s s i v e ) a c t i v i t y , r e s u l t i n g in d a y t i m e drowsiness, lack of concentration, diminished mental acuity and impaired handling of machinery or driving of vehicles. We evaluated the CNS-depressive effects of twelve benzimidazole derivatives (Table 10) in terms of their potentiation of hexobarbital-induced sleep in mice. Compounds that caused statistically significant increase in the period of hexobarbital-induced sleep at a dose of 200 mg/kg p.o. were classified as "active", and others as "inactive". QSAR was analyzed by the ALS method to obtain Eq. 17, where MR is the value of the R 1 substituent. Y = 1.061 (MR/10) - 0.431 1 - 2.458 n=12,

Rs=0.845,

nmi s = l ,

[17]

t=5.00,

p < 0.001

For confirmation of the validity of the ALS result, the leave-one-out test was performed. The predictive results showed t h a t 83% of the compounds were classified correctly. Equation 17 suggests t h a t a sterically small substituent at the 1-position and the homopiperazine moiety at the 2-position decrease the extent of CNS side effects. Astemizole, 1-(4-fluorobenzyl)-2-[[1-(4-methoxyphenethyl)-4piperidyl]amino]benzimidazole (XXVI), has been used clinically as a long-acting a n t i h i s t a m i n i c agent with few CNS side effects (45). Recently, the 1-(2-ethoxyethyl) analog of XXVI was publicized in the patent form (46). The structure of XXVI is similar to that of X X I I , and if the two compounds interact with the same active site(s) in a similar m a n n e r , Eqs. 15 and 17 suggest that the 2-ethoxyethyl moiety (L = 6.97, B 3 = 1.90 and MR/10 = 2.136) would be preferable to the 4-fluorobenzyl moiety (L - 5.91, B 3 - 1.90 and MR/10 - 2.990) in terms of both antihistaminic and CNS activity.

~_

NNk'~--NH" - ~ N - CH2CH2- - ~ ~ F

OMe

XXVl " Astemizole

447

In conclusion, compounds which have a 1-(2-ethoxyethyl) and 2(1-homopiperazinyl) substitution on the benzimidazole nucleus were confirmed to have not only potent antihistaminic activity but also low anticholinergic and CNS-depressive activities from the results of QSAR. Therefore, we selected KG-2413 as a candidate compound for d e v e l o p m e n t a l trials. KG-2413 also showed antiallergic and antiasthmatic effects and has been used clinically since August 1993. 5. C O N C L U S I O N Our application of the QSAR technique has been pragmatic to disclose the optimized structure in the shortest and most efficient way. Therefore, we have restricted ourselves to the application of established methods and the use of well defined substituent constants as far as possible. As shown in the above examples, the methods and the tabulated substituent constants are thought to be sufficient for QSAR analysis in most cases. Although the precision of biological data is of primary importance for QSAR analysis, it is costly and time-consuming to establish precise dose-response relationships for every congener. Therefore, a strategy is needed to reduce the amount of biological work as well as to accelerate the project research. In the first example, we converted the fixed dose activity data so t h a t they are appropriately utilizable in regression analysis after confirming that the log(dose)-response curves of some congeners are parallel in certain concentration ranges. In the second example, compounds were classified into two groups according to the fixed dose data and a rough analysis using the ALS method was performed first to examine structural requirements for exhibition of activity. For some potent compounds, more precise dose-response data were measured and rendered to the Hansch-Fujita analysis to establish factors enhancing the potency. The combined results of these two QSAR procedures were used to predict the optimal structure. In the first two examples, we examined QSAR analyses repeatedly at each step using biological activity data for a smaller number of compounds to design additional compounds for subsequent syntheses. Such repetitions of the cycle of synthesis-biological evaluation-analysis gradually clarified the structural requirements for exhibiting potent

448

activity, and finally, structure-activity data were summarized in one equation. Moreover, insights into structure-activity relationships gained quantitatively were extrapolated and transposed successfully to determine new lead structures (lead evolution). Thus, we found two new drug candidates more quickly and efficiently than before. The compounds, KB-2796 and KB-5492, are now undergoing extensive clinical trials. In the third example, QSAR analyses were performed after the project was over. The results confirmed that the candidate selection was valid. If the QSAR procedure had been used in the course of the project research, a much smaller number of compounds would have been needed to obtain the same information. It is by no means an exaggeration to say that QSAR analyses helped us to reduce the time required as well as the cost of the new drug research by facilitating rational and speedy decision-making. ACKNOWLEDGEMENT

The author thanks Emeritus Professor Toshio Fujita of Kyoto University for critical reading of the manuscript and many helpful discussions. REFERENCES 1 C. Hansch and T. Fujita, J. Am. Chem. Soc., 86, 1616 (1964). 2 I. Moriguchi, K Komatsu and Y. Matsushita, J. Med. Chem., 23, 20 (1980). 3 T. Fujita ed., "Structure-Activity Relationship of Drugs", Nankodo, Tokyo, 1979. 4 T. F u j i t a ed., "Structure-Activity Relationship of Drugs II", Nankodo, Tokyo, 1982. 5 T. Fujita "Drug Design, Fact or Fantasy ?", ed by G. Jolles,K. R. H. Wooldridge. Academic press, London, 1984, p.19. 6 T. Fujita ed.,"Structure-Activity Relationship and Drug Design", Kagakudojin, Kyoto, 1986. 7 T. Fujita, Acta Pharm. Jugosl., 37, 43 (1987). 8 M. Miyazaki, Medicina, 10, 75 (1973). 9 B. H u n e r m a n , R. Felix,K. Wesene a n d C. Winkler, Arzneim.Forsch., 23,652 (1975). 10 T. Godfraind, G. Towse and J. M. VanNueten, Drugs of Today, 18, 27 (1982). 11 S. Naito, S. Osumi, K. Sekishiro and M. Hirose, Chem. Pharm. Bull., 20, 682 (1972).

449 12 N. Toda, H. U sui, S. Osumi, M. K a n d a and K. Kitao, Arch. Int. Pharmacodyn. Ther., 260,230 (1982). 13 H. O h t a k a , M . M iyake, T. K a n a z a w a , K. Ito and G. T s u k a m oto, Chem. Pharm. Bull., 35, 2774 (1987). 14 J. G. Topliss, J. Med. Chem.,15,1006 (1972); idem, ib/d.,20,1 (1976). 15 H. O h t a k a , Y. F u j i m oto, K. Yoshida, T. K a n a z a w a , K. Ito and G. Tsukamoto, Chem. Pharm. Bull., 35, 2782 (1987). 16 H. Ohtaka and G. Tsukamoto, Chem. Pharm. Bull., 35, 2792 (1987). 17 G. Regnier and R.Canevari, Fr. Patent 1303080 (1962) [Chem. Abstr., 60, 2965a (1964)]. 18 H. Ohtaka, T. K a n a z a w a , K . Ito and G. Tsukamoto, Chem. Pharm. Bull., 35, 3270 (1987). 19 H. Ohtaka and G. Tsukamoto, Chem. Pharm. Bull., 35, 4117 (1987). 20 H. Ohtaka, T. K a n a z a w a , K . Ito and G.Tsukamoto,Chem. Pharm. Bull., 35, 4124 ( 1987 ). 21 Laboratoria P h a r m a c e u t i c a D r . C. Janssen,Belg. Patent 556791 [Chem. Abstr., 54,590c (1960)]. 22 P.A.J. Janssen, Ger. Offen., 1929330 [Chem. A bstr., 73, 14874g (1970)]. 23 M. Takai, S. Hattori,T. W akabayashi,Y. Suwabe and S. M i y a o k a , Eur. Patent 232205 (1987) [Chem. Abstr., 108,21927w (1988)]. 24 H. Ohtaka, T. K a n a z a w a , K . Ito and G. Tsukamoto, Chem. Pharm. Bull., 35, 4637 (1987). 25 A. Ishim ori, S. Yamagata and T. Taima,Arzneim.-Forseh.,29, 1625 (1979). 26 R. Nagashima and N. Yoshida, Arzneim. Forseh.,29, 1668 (1979). 27 M. M u r a k a m i , K. Oketani, H. Fujisaki, T. W a k a b a y a s h i and T. Ohgo, Arzneim. -Forseh., 31, 799 (1981). 28 H. Ohtaka, I~ Yoshida, I~ Suzuki, I~ Shimohara, S. Tajima and I~ Ito, Chem. Pharrn. Bull., 36, 3948 (1988). 29 H. Ohtaka, K. Yoshida and K. Suzuki, Chern. Pharm. Bull., 36, 3955 (1988). 30 I. M origuehi, Y. Kanada and K. Komatsu, Chem. Pharm. BulL,24, 1799 (1976). 31 A~Makriyannis and J. J. Knittel, Tetrahedron Lett., 2753 (1979). 32 O. Hofer, Tetrahedron Lett., 3415 (1975). 33 A. Verloop, W. Hoogenstraaten and J. Tipker,"Drug Design", Vol.7, ed. by E.J. Ariens, Academic press, N e w York, 1976, pp. 165-207. 34 H. Ohtaka, g. Yoshida, I~ Suzuki, I~ Shimohara, S. Tajima and K. Ito, Chem. Pharm. Bull.,36, 4825 (1988). 35 L. deAngelis, Drugs of the Future, ll, 263 (1986). 36 H. B r u n n e r , M. Verita, P. P o l t e r a u e and G. Grabner, A r z n e i m . Forseh., 27,684 (1977). 37 R. Pierre and R. Roustan, Med. Interne, 15, 49 (1980). 38 T. Kodam a, A. Takai, M. N a k a b a y a s h i , I. Watanabe, H. Sadaki, T. Kodama, N. Abe and A. Kurokawa, Japan Kokai Patent 126682 (1975) [Chem. Abstr., 84,44060h (1976)]. 39 R. I e m u r a , T . K a w a s h i m a , T. F u k u d a , K . Ito and G. Tsukamoto,d.

450

Med. Chem.,29, 1178 (1986). 40 M. R. Silva, ed. "Handbook of E x p e r i m e n t a l P h a r m a c o l o g y : Histamine H and Anti-Histaminics", Springer Verlag, New York, 1978, Chapter 2, Section/k 41 R. Iemura and H. Ohtaka, Chem. Pharm. Bull., 37,967 (1989). 42 B. Baker, M. Kawazu, D. Santi andT. Schwan, J. Med. Chem.,lO, 304 (1967). 43 R.F. R e k k e r , H. T i m m e r m a n , A. F. H a r m s a n d W. Th. N a u t a , Arzneim. -Forsch., 21, 688 ( 1971). 44 R. I e m u r a , T. K a w a s h i m a, T. F u k u d a , K. Ito and G. Tsukam oto, J. Heterocyclic Chem., 24, 31 (1987). 45 J. Callier, R.F. Engelen, I. Ianniello, R. Olzem,M. Zeisner and W.K. Amery, Curr. Ther. Res.,29,24 (1981). 46 F.E. Janssens, G. S. M. Diels, J. L. G. T o r r e m a n s and F. M. Sommen, Eur. Patent 282133 (1988) [Chem. Abstr., 110, 75503g (1989)].

QSAR and Drug Design - New Developments and Applications T. Fujita, editor 9 1995 Elsevier Science B.V. All rights reserved

QUANTITATIVE STRUCTURE-ACTIVITY ACRYLAMIDE ANALOGS

STUDIES

451

OF

NEUROTOXIC

KAZUO HASHIMOTO 1, HIDEJI TANII1,AKIHISA HARADA 1and TOSHIO FUJITA 2 1 Department of Hygiene, School of Medicine, Kanazawa University, Kanazawa 920, Japan 2 Department of Agricultural Chemistry, Kyoto University, Kyoto 606-01, Japan Acrylamide is an important commodity chemical to produce polymers used in various forms for industrial, scientific and public purposes. The monomer has been known to cause neurotoxic symptoms in test animals and human beings under chronic exposures. We measured the neurotoxic potency for a number of structurally related, mostly, N-substituted analogs using mice. We also evaluated related bioactivities such as acute toxicity to mice, cytotoxicity to cultured cells originated from murine nervous systems, inhibitory activities to rat-brain glycolytic enzyme preparations and i n v i t r o metabolic susceptibilities to mouse-liver enzyme preparations. The variations in each toxicity or bioactivity index among analogs were quantitatively examined with the use of physicochemical molecular and submolecular parameters by regression analyses. The physical-organic reactivities of acrylamide analogs as the Michael addition substrates and quenching agents of fluorescence from the aromatic amino acids were also examined extrathermodynamically and used as molecular parameters for the quantitative analyses whenever relevant. The comparative overview of quantitative structure-activity formulations led us to hypothesize that electronic and steric properties and hydrogen-bonding factors of the amide moiety are operating together to govern variations in the neurotoxic potency. ABSTRACT:

1.

INTRODUCTION

Acrylamide is a vinyl monomer which has been used extensively in chemical industries to produce polymers in various forms, soluble and insoluble in water. The largest utilization of polyacrylamides includes that as flocculants of floating minute solids and colloidal materials in industrial and public sewage and that as fortifiers in manufacturing papers and cardboards (1). The acrylamide monomer is also widely used in biochemical/biomedical laboratories in the preparation of polyacrylamide gels for analytical and separatory procedures of biopolymers such as DNA's and proteins (2). While acrylamide is such an important chemical, the monomer has been recognized to cause a specific type of neurotoxicity following various administration routes in experimental animals and h u m a n beings (1, 3). A number of h u m a n cases of the acrylamide poisoning are mostly due to occupational chronic exposures to its monomer (1, 4-8). Typical symptoms of the poisoning are a muscle weakness of hindlimbs, a lassitude, emotional changes and an ataxic gait (3). Thus, understanding of the

452 mechanism of neurotoxic action of the acrylamide monomer has been one of the serious topics in the field of occupational hygiene (1, 3). Etiologically, the damages of neural glycolytic enzyme systems such as enolase and glyceraldehyde-3-phosphate dehydrogenase have been suggested to participate in the acrylamide neuropathy (9). Histological studies have indicated t h a t acrylamide and some of its neurotoxic analogs induce an axonal swelling with multifocal accumulations of neurofilaments (10, 11) and morphological alterations of microtubules in peripheral nervous systems (12). Although the peripheral nervous system is vulnerable to acrylamide, it has not been clarified whether its primary target is neurons, glial cells or axons or all of these. We have been examining a number of acrylamide analogs, in which the sp 2 carbons and amide nitrogen are variously substituted, for their possible neurotoxic action (13). The structure-neurotoxicity relationship has been found to be very delicate: some analogs are neurotoxic whereas others are not in spite of structures closely related to acrylamide (13). We have evaluated a number of related bioactivities such as their acute toxicity to mice (13), cytotoxicity in vitro to cell lines derived from murine nervous systems (14), and inhibitory activity to glycolytic enzyme preparations (15, 16) as well as in vitro metabolic susceptibilities (17). Being an a,B-unsaturated carbonyl compound, acrylamide is a typical substrate of the Michael-type nucleophilic addition reactions (18). Thus, reactivity indices with such a biochemical Michael reagent as glutathione have been measured chemically (19) and biochemically with a glutathione-S-transferase preparation (17) for a series of acrylamide derivatives. Another characteristic physicochemical property of acrylamide is an efficient fluorescence quenching (20). Thus, we have measured the quenching efficiency of acrylamide and analogs for the fluorescence from irradiated aromatic amino acids (15). With physicochemical molecular and substituent parameters as independent variables, we have analyzed structure-reactivity, structure-toxicity and structureactivity relationships of acrylamide and analogs, quantitatively. In this article, through comparisons of these quantitative relationships, we attempted to obtain insights into molecular mechanism of the neurotoxicity and relating biological and biochemical (re)activities of acrylamide and its analogs, the structures being shown in Tables 1 and 2.

0

P H Y S I C A L ORGANIC M E A S U R E M E N T S A N D P H Y S I C O - C H E M I C A L PARAMETERS

R e a c t i v i t y w i t h G l u t a t h i o n e (19) In most cases, equimolar amounts (about 10 2 M) of reduced glutathione and

2.1

each of acrylamide analogs were reacted in a pH 7.3 phosphate buffer (0.1 M) at 37 ~

453

Table 1. Structure of Acrylamide Analogs

R~

/R.

H~ C--C~CONR ' IR2 Compounds a

R~

R,

R1

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

H H H H H H H H H H H H H H

H H H H H H H H H H H H H H

H t-Bu H C(CH3)2CH2COCH 3 Et Et Me Me -(CH2) 4Allyl A]lyl H n-Bu H CH2OCH2 CH(CH3)2 H CH(OH)COOH H Et H CH2OH H Me H H H i-Pr

15. N-Methacryloylpyrrolidine* 16. N-Ethylmethacrylamide* 17. Methacrylamide*

H H H

Me Me Me

H H

-(CH2) 4Et H

18. Crotonamide

Me

H

H

H

N- t-Butylacrylamide Diacetone acrylamide N,N-Diethylacrylamide N,N-Dimethylacrylamide N-Acryloylpyrrolidine N,N-Diallylacrylamide N- n-Butylacrylamide N- i-Butoxymethylacrylamide Acrylamido-N-glycolic acid* N-Ethylacrylamide* N-Hydroxymethylacrylamide* N-Methylacrylamide* Acrylamide* N- i-Propylacrylamide*

R2

a Neurotoxic analogs are asterisked. in the presence of catalytic a m o u n t of KCN to prevent spontaneous oxidation of glutathione. The reaction rate was estimated by measuring the a m o u n t of the SH group in the reaction mixture at intervals with the use of the Ellman reagent (21). The analysis of the second order kinetics afforded the rate constant, k(GL) in M -1 9 min 1. The log k(GL) value is listed in Table 3. The standard deviation of log k(GL) value averaged from at least 3 repeats was lower than + 0.03.

2.2 F l u o r e s c e n c e Q u e n c h i n g of Aromatic Amino Acids (15) According to a modified Stern-Volmer equation, the drop in fluorescence intensity of fluorophores can be related to the concentration of a quencher, Q, by Eq. 1 (22). F o / F = (1 + Ksv [Q]) exp (V [Q])

[1]

In this equation, F o / F is the ratio of the fluorescence intensity between conditions

454 with absence and presence of a given concentration of the quencher, [Q], and Ksv is the Stern-Volmer collisional quenching constant. The factor, exp(V[Q]), in Eq.1 is to describe static quenching besides collisional quenching processes, V being the static quenching constant. The static quenching is due to a mechanism involving contact between the chromophore and the quencher prior to excitation to form a "dark" complex, while the collisional mechanism involves a rapid diffusion of the quencher and its interaction with fluorophore molecules (20). Because acrylamide normally quenches the fluorescence of fluorophores by the collisional mechanism (22), the exponential term could be deleted leading to the classical Stern-Volmer equation as a first approximation which is rewritten in the form ofEq. 2 (23). 1 / (F o -- F) = 1 / (Ksv F o [Q]) + 1 / F 0

[21

The value of 1 / Ksv was estimated from the slope of the plot of 1 / (F o - F), the reciprocal of the drop in the fluorescence intensity of aromatic amino acids, against the reciprocal of the concentration of acrylamide analogs, 1/[Q], and the intercept of the plot with the use of regression analysis (23). The collisional quenching constant, Ksv , is, in effect, a product of the fluorescence life time of the fluorophore in the absence of the quencher and the bimolecular rate constant for collisional encounter (22). If the fluorophore is fixed, the fluorescence life time is a constant. Thus, the Ksv (M -~) for a series of acrylamides as the quenchers with certain aromatic acids as the fluorophore is regarded as a pseudo-association constant for collisional partners. Wave lengths (m;u) of excitation used and emission observed were 285 and 355 for tryptophan, 275 and 310 for tyrosine and 265 and 287 for phenylalanine. The measurements were made at 24~ The log Ksv values are shown in Table 3. The standard deviation of log Ksv value as the mean of 3 repeats was lower than + 0.04.

2.3 P h y s i c o c h e m i c a l S u b s t i t u e n t and Molecular P a r a m e t e r s The physicochemical (sub)molecular and substituent parameters were mostly calculated by conventional methods or estimated by empirical procedures. The molecular hydrophobicity in terms of log P, P being the 1-octanol/water partition coefficient, was calculated using the CLOGP methodology (24). The calculated values for acrylamide (-0.61), the N,N-dimethyl analog (-0.57) and methacrylamide (-0.30) were in good agreement with their experimentally measured values, -0.67 (25), -0.60 (26), a n d - 0 . 2 3 (26), respectively. Even for the N-hydroxymethyl analog and diacetone a c r y l a m i d e h a v i n g s u b s t r u c t u r e s capable of h y d r o g e n - b o n d i n g intramolecularly, the calculated values without considering such an intramolecular effect, - 0 . 9 3 and 0.29, were close to their measured counterparts (26), - 1 . 0 2 and 0.23, respectively. Thus, the use of calculated log P values is believed to be reliable

455 enough and the i n t r a m o l e c u l a r hydrogen-bonding need not be considered for the series compounds. To r e p r e s e n t electronic characteristics, electronic energy p a r a m e t e r s (in eV) for the highest occupied (HOMO) and the lowest unoccupied (LUMO) molecular orbitals were calculated according to the MINDO/3 method of Dewar and Haselbach (27) and Bodor and coworkers (28). The calculations were made for conformations, in which the C = C and C = O double bonds are syn-coplanar taking into consideration the X-ray crystalographic results found for related compounds such as benzamide (29) and its N-substituted derivatives (30), but otherwise fully optimized. The only exception was the congested N-methacryloylpyrrolidine where the dihedral angle of the two double bonds was taken as perpendicular. The results showed t h a t the HOMO covers the carbony! oxygen as well as the amide nitrogen while the LUMO is located in the C = C double bond region in this series of compounds. E i t h e r s u m m a t i o n of the Taft-Kutter-Hansch E s value (31) of substituents, R 1 and R 2, on the amide nitrogen or the s u b s t i t u e n t molar refractivity value for the NR1R 2 moiety (scaled by 1 / 10 to make the value similar size to the E s value) was used for the steric p a r a m e t e r for amide substituents (32). The E s value of R1 and R 2 substituents (CR~'R2'R3') for which the Taft-Kutter-Hansch constant was not available was evaluated from the corresponding Es c value estimated by a linear combination of Es c values of RI', R2', and R 3' (33). In some analyses, the E s value of the t-Bu group was used as an "effective" E s (Es') for the acetylmethyl-dimethyl-carbinyl group in diacetone

acrylamide.

The reference point of the E s value was shifted so t h a t

Es(H) = 0. The MR value was calculated using the additivity principle from group refractivity (32). Because the substituted amino moiety of the amide structure attaches to the conjugate system of two double bonds, the MR (NR~ 1~) value was thought to be better represented by either t h a t in the aromatic system or t h a t calculated from the aromatic values of component substituents unless otherwise noted. The effects of aand 6-methyl groups in methacryl and crotyl moieties were expressed with indicator variables when t h e y were relevant. The r e l e v a n t p a r a m e t e r values are listed in Table 2. 3.

TOXICOLOGICAL AND BIOCHEMICAL MEASUREMENTS

3.1

Acute Toxicity (13)

Male mice of the ddY strain, 5 to 6 weeks of age and 29 + 2.2 g body weight, were used.

Each of the test compounds was a d m i n i s t e r e d as a solution in either 0.9 %

saline, olive oil or DMSO with an intubation needle orally. The n u m b e r of dosage levels was four and t h a t of the animals used at each level was at least four. The dosage levels were spaced so t h a t they are in a geometric progression. animals received the corresponding volume of the vehicle.

Control

Seven days after a single

456 T a b l e 2.

Acrylamide Analogs a n d Physicochemical P a r a m e t e r s

Compounds

log pa

HOMO (eV)

LUMO (eV)

F.Es b

Y.Es, b

MR c (NR~R2)

id

Acrylamide 1. t-Bu2. Diacetone 3. (Et) 24. (Me) 25. -(CH2) 46. (Allyl) 27. n-Bu8. i-BuOCH 29. -glycolic acid 10. Et11. H O C H 212. Me13. u n s u b s t i t u t e d 14. i-Pr-

0.65 0.29 0.49 -0.57 0.06 0.58 1.00 0.77 -1.19 -0.05 -0.93 -0.58 -0.61 0.26

-9.357 -9.372 -9.126 -9.222 -9.122 -9.052 -9.450 .h -9.574 -9.448 -9.554 -9.490 -9.994 -9.402

1.003 0.895 0.745 0.779 0.855 0.721 0.936 .h 0.778 0.933 0.789 0.891 0.961 0.978

-2.78 -3.59 e -2.62 -2.48 -1.94 e -3.20 -1.60 .h -1.27 e -1.31 -1.11 -1.24 0 -1.71

-2.78 -2.78 f -2.62 -2.48 -1.94 e -3.20 -1.60 .h -1.27 e -1.31 -1.11 -1.24 0 -1.71

2.43 3.36 g 2.49 1.56 2.29 3.32 g 2.43 3.07 1.80 g 1.50 1.18 1.03 0.54 1.96

0 0 0 0 0 0 0 0 0 0 0 0 0 0

Methacrylamide 15. -(CH2) 416. Et17. u n s u b s t i t u t e d

0.37 0.26 -0.30

-9.196 -9.459 -9.823

0.738 0.851 0.866

-1.94 e -1.31 0

-1.94 e -1.31 0

2.29 1.50 0.54

1 1 1

Crotonamide 18. u n s u b s t i t u t e d

-0.08

-9.920

0.739

0.54

2

0

0

a b c d

C a l c u l a t e d by CLOGP ver. 3.54. Calculated from values in ref. 31 unless noted. Cited from or calculated from values in ref. 32 unless noted. I = 2 for crotonamide assigned arbitrarily. E s t i m a t e d from t h e EsCvalue which was p r e d i c t e d by a l i n e a r c o m b i n a t i o n of Es c v a l u e s of c o m p o n e n t s u b s t i t u e n t s w i t h use of two equations, E s = Es c + 0.306 (3 nil), n Hb e i n g t h e n u m b e r of h y d r o g e n a t o m on t h e a - c a r b o n of s u b s t i t u e n t s , a n d EsC(CRI'R2'R3 ') = - 0.17 + 3.43 EsC(R1') + 1.98 EsC(R2 ') + 0.65 EsC(R3'), RI', R 2' a n d R 3' being defined as E sc(R~ ,) > EsC(R2 ,) _~ E s c(R3'), a n d EsC(H) = 0. See ref. 33. T h e Es c v a l u e s of C O C H 3 a n d C O O H were t a k e n as e q u i v a l e n t w i t h t h a t of i-Pr in t h e calculations. f T a k e n to be equal to t h a t for t-Bu. g F o r t h e c h a i n - e n d f u n c t i o n a l g r o u p s a n d double bonds, a l i p h a t i c MR v a l u e s a n d factors were used to e s t i m a t e the MR (NR~ R 2) value. h N e i t h e r calculated nor e s t i m a t e d .

e

dosing, t h e n u m b e r of t h e killed a n i m a l s was counted.

The toxicity index, LDso

(mol/kg), was e s t i m a t e d according to Weil from a set of d o s e - m o r t a l i t y d a t a (34).

The

457 95% confidence intervals of log LD~ values were mostly lower t h a n • 0.15.

All

experiments were performed at 25 ~ 3.2

C h r o n i c N e u r o t o x i c i t y (13, 35)

Groups of 5 to 7 male mice of the ddY strain were treated with each test compound. A certain dose was given twice a week at a level within a range from 1/2 to 1/5 of the LD~ value for 8 to 10 weeks with vehicles and intubation tube similar to the acute toxicity m e a s u r e m e n t .

The dose level was so chosen, by p r e l i m i n a r y experiments,

t h a t it produces general acute symptoms as negligibly as possible.

For the test

experiments, only animals selected preliminarily t h a t are able to carry out the rotarod performance were used.

As the rotarod apparatus, a roughly surfaced PVC rod of

5 cm diameter, r o t a t i n g at t h r e e revolutions per minute, was used.

The longest

performance period within 30 sec was measured among five successive trials for each mouse, and the periods in sec were averaged for mice in the test group. The rotarod experiments were performed twice a week. As an index for the neurotoxic potency, IDa, the dose required to reduce the average performance period to half the m a x i m u m was e s t i m a t e d from a plot of the period (sec) a g a i n s t the test d u r a t i o n (day) as follows: the n u m b e r of days until the half m a x i m a l inhibition is a t t a i n e d • 2/7 • single oral dose ( m o ~ ) .

The ID~ value estimated here should not be taken as being

absolute, but relative only under dosing conditions used in this study. All experiments were done at 25~

For the quantitative analysis, the neurotoxic potency was repre-

sented by a rating score (NT), either 4, 3, 2 or 1 so t h a t 4 : ID~ < 0.01, 3 : 0.01 < ID~ < 0.1, 2 : 0.1 < ID~ and 1 : no ID~ was measurable. 3.3 C y t o t o x i c i t y (14) Two clonal cell lines derived from nervous systems, mouse neuroblastoma N18TG-2 (36) and r a t Schwannoma RT4 cells (37), were used. Cells (0.5 - 1.0 • 106) plated on plastic dishes (60 mm in diameter) were cultured in a modified Eagle medium containing various concentrations of each compound at 37~ in a humidified atmosphere of 10% CO 2. Five days for the N18TG cells and 4 days for the RT4 cells after starting the culture, cells were collected by trituration in a calcium- and magnesitun-free phosphatebuffered saline. Cells were counted with a hemocytometer and t h e i r viabilitiy was m e a s u r e d by the trypan-blue dye exclusion method.

The viability m e a s u r e m e n t s

were m a d e on days w h e n the maximal cell density was a t t a i n e d for each cell line without toxicants. From the dose-viability relationship, the I~ concentration (M) was evaluated.

The cell viability value expressed as the p e r c e n t of control at each

concentration of each compound is the m e a n of three i n d e p e n d e n t determinations. The standard deviation of pI~ value was within + 0.04.

458 3.4

M e t a b o l i c T r a n s f o r m a t i o n s in Vitro (17)

Microsomal fraction was prepared from the liver homogenate of the male mice of the ddY strain. The oxidative transformation reaction of acrylamide analogs was performed by incubating the microsomal fraction with various concentrations of each analog in the presence of an NADPH-generating system at pH 7.4. Thirty minutes after the reaction was started, an aliquot of the reaction mixture was taken out and heated to stop the reaction. The amount of the substrate left non-metabolized was analyzed gaschromatographically. In p r e l i m i n a r y e x p e r i m e n t s , the r a t e of disappearance of the substrate was observed to be almost constant at least during the first 30 minutes of the incubation. From the double reciprocal plot between the "initial" rate and the concentration, the Michaelis constant, K~ (M), and the maximum rate, v ~ (nmol metabolized, mg protein -1. rain-l), for the microsomal oxidation reaction were estimated. The standard deviation among triplicate examinations was + 0.03 in both log Km and log Vm~ values which were averaged. The glutathione S-transferase preparation was made from the mice liver homogenate according to the method of Boyland and Chassaud (38). The glutathione conjugation reaction was initiated by incubating the enzyme preparation with various concentrations of acrylamide analogs in the presence of reduced glutathione. After 60 minute incubation, the reaction was stopped by addition of perchloric acid and the concentration of reduced glutathione left was measured by the Ellman method (21). The rate of this reaction was also preliminarily examined to be nearly constant during the first 60 minutes of the incubation. The reactivity was represented by v(GT) in nmol glutathione decreased 9h -1 9mg protein-1. The accuracy for the mean of duplicate measurements in log v(GT) was mostly • 0.05. All the kinetic measurements of the in vitro metabolic reactions were done at 37~

3.5 Glycolytic Enzyme Inhibition We measured I~o (M) concentration of acrylamide analogs against preparation of phosphofructokinase (PFK) (16), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (16), and enolase (15), each from rat brain homogenates. The supernatant from the homogenate of male Wistar-rat brains with a cold 0.25M sucrose was used for the measurement of the PFK activity according to the procedure of Ling and coworkers (39) as well as that of the GAPDH activity according to the method of Howland and coworkers (9) after setting up necessary reaction conditions. The supernatant from the homogenate with a cold pH 7.4 phosphate buffer (10mM) was chromatographed with a DEAE cellulose column to separate three forms of the brain enolase, i.e., neuron-specific, hybrid and non-neuronal, according to the procedure of F r a n c i s and coworkers (40). The enolase a c t i v i t y was m e a s u r e d spectrophotometrically by the method of Odelstad and coworkers (41) using 2phosphoglycerate as the substrate.

459 All e n z y m e inhibition m e a s u r e m e n t s were r e p e a t e d t h r e e times at 30~

The

s t a n d a r d deviation of the m e a n pI~ value was within + 0.03.

0

QUANTITATIVE A N A L Y S E S OF S T R U C T U R E - ( R E ) A C T I V I T Y STRUCTURE-TOXICITY RELATIONSHIPS

AND

The 18 compounds altogether were dealt with in this s t u d y as shown in Table 1. Not every compound was included in the individual analyses, however, because of the difficulty in e x p e r i m e n t a l p r o c e d u r e s being due to l i m i t e d solubilities, lack of physicochemical

parameters

as i n d e p e n d e n t

variables,

outlying behaviors

rationalizable or not a n d / or u n m e a s u r a b l y low (re)activity or toxicity data.

The

physicochemical p a r a m e t e r s in Table 2 were used for the quantitative analyses. Some of the biological p a r a m e t e r s were also used as i n d e p e n d e n t variables whenever relevant. T a b l e 3. Physical Organic Reactivities of Acrylamide Analogs Compounds

log k(GL)

log Ksv(Tr p)

log Ksv(Phe)

obsd.

calcd, a

obsd.

Acrylamide t-BuDiacetone (Et) 2(Me) 2-(CH2) 4(Allyl) 2n-Buglycolic acid EtHOCH 2Meunsubstituted i-Pr-

-1.85 -1.25 -1.24 -1.02 -0.74 0.15 e -f -0.27 -1.12 -0.04 -1.24 -0.04 -1.66

-1.98 -1.58 -0.95 -1.00 -0.99 -1.17 -1.10 -0.34 -0.94 -0.30 -0.74 -0.34 -1.32

2.24 2.20 2.77 2.61 2.95 2.66 1.72 1.51 1.46 1.38 1.25 1.34 e 1.59

1.98 1.93 2.74 2.43 2.75 2.98 1.68 1.27 1.68 1.34 1.55 -0.11 1.83

2.70 2.90 3.43 3.32 -f -f J f J 2.49 2.34 2.22 e 2.80

2.86 2.82 3.46 3.21 3.47 3.65 2.62 2.30 2.63 2.35 2.52 1.21 2.75

2.20 1.94 2.86 2.84 -f -f -f 1.73 e -f 1.52 e 1.11 1.24 e 1.52

1.92 1.85 3.11 2.62 3.13 3.49 1.45 0.81 1.46 0.91 1.24 -1.35 1.69

Methacrylamide -(CH2) tEtunsubstituted

-g -2.30 -1.85

-2.32 -2.40 -1.75

J 1.46 1.43 e

2.51 1.65 0.45

-f J 1.99 e

3.28 2.60 1.66

-f 1.36 1.35 e

2.75 1.40 -0.47

Crotonamide unsubstituted

-g

-3.05

2.60 e

0.13

2.18 e

1.40

1.38 e

-0.97

calcd, b obsd.

log Ksv(Tyr)

calcd, c obsd.

calcd, d

a By Eq. 3. b By Eq. 4. c By Eq. 5. d By Eq. 6. e Not included in the analyses, b u t compared with the calculated values, f Not m e a s u r e d b u t calculated. g U n m e a s u r a b l y low.

460

4.1

Quantitative Analyses of Physical Organic Reactivities 4.1.1

Reactivity with Glutathione

The Michael addition of SH compounds such as g l u t a t h i o n e is one of the characteristic reactions of acryloyl compounds.

Because the second-order reaction

rate constant k(GL) was expected to depend on the electrophilicity of acrylamides, the electronic parameter, LUMO, was selected as one of the i n d e p e n d e n t variables and combinations with other p a r a m e t e r terms were examined to yield Eq. 3 as of the best quality to correlate the log k(GL) value listed in Table 3. log kaL (M -1 min ~-') = - 3.683 (+ 2.468) LUMO + 0.535 (+ 0.243) ZE s'

- 1.763 (+ 0.602) I + 3.201 (+ 2.272)

[31

n = 13, s = 0.308, r = 0.926, F (3,9) = 18.07 In this and the following equations, the figures in p a r e n t h e s e s are the 95% confidence intervals, n is the n u m b e r of compounds, r is the correlation coefficient, s is the s t a n d a r d deviation and F is the ratio b e t w e e n the regression and residual variances. The negative LUMO term is highly important as expected. The lower the LUMO level, the more easily the electrons on the SH group are accepted by the substrate. The positive ZE s' term indicates t h a t the "effective" bulk of substituents on the amide nitrogen has an effect to retard the reaction rate. For the C(CH3) 2CH2COCH 3 group in diacetone acrylamide, the E s value was t a k e n to be equivalent with the value of t-Bu group, thus ZE s' in Eq.3. Steric influence of the chain-end COCH 3 group was regarded as being insignificant. The fact t h a t the rate constant of crotonamide is too low to be m e a s u r e d is well predicted by Eq. 3 with the use of I = 2. The steric inhibition of the ~ m e t h y l against the access of glutathione is at least about twice as high as t h a t of the a-methyl group in which I = 1. In Eq. 3, the diallylamide is not included. Its log k(GL) value was considerably higher t h a n t h a t predicted by Eq. 3. Because it has two extra double bonds in the amide moiety, the reaction rate probably covers t h a t for other types of nucleophilic addition reactions the mechanism of which is unknown.

4.1.2

A s s o c i a t i o n w i t h A r o m a t i c A m i n o Acids in E x c i t e d S t a t e s

Because the Stern-Volmer quenching constant, Ksv, is an index representing the stability of the collisional complex (42) with aromatic amino acids in the electronically excited state, it was expected to be related with an electronic parameter of the quenchers. The HOMO value was found to be the p a r a m e t e r to correlate the log Ksv values as shown in Eqs. 4-6. The LUMO value was examined and found to work much more poorly (results not shown).

461 With tryptophan (Trp): log Ksv (M -~) = 3.283 (+ 0.872) HOMO + 32.701 (_+8.160) n = 1 3 , s=0.235, r = 0 . 9 2 8 , F(1,11)=68.66

[4]

With phenylalanine (Phe): log Ksv (M -1) = 2.593 (• 0.999) HOMO + 27.129 (+ 9.354) n = 7 , s=0.140, r=0.948, F(1,5)=44.51 With tyrosine (Tyr): log Ksv (M --1) = 5.137 (+ 1.791) HOMO + 49.993 (+ 16.745) n = 7 , s=0.222, r=0.957, F(1,5)=54.35

[5]

[6]

In the quenching of fluorescence of various aromatic compounds, the electronic energy transfer has been postulated to occur by a charge-transfer mechanism through the collisional association complex radiationlessly. In the case of substituted benzenes as the fluorophore quenched by diolefines, the charge-transfer is proposed to occur from diolefines to aromatic molecules (42). A similar mechanism is suggested for the quenching of fluorescence of aromatic ketones as the electron acceptor by a variety of electron donors including amines (43,44). The stability of the collisional association complex could be linearly related to the free-energy change associated with the complex formation. According to the charge-transfer hypothesis, the energy of molecules in the excited state is transferred to the ground state quenchers (44). The net energy loss of the excited molecules is the sum of the potential energy difference between excited and ground states, AE, and the electron affinity, EA, leading to the corresponding anion. The transferred energy is utilized to release an electron from quenchers to give their cations, the energy required for this process being the ionization potential, IP (43). The condition necessary for the energy transfer is that the energy state to which each of the quenchers is excited is of a lower level than the excited state energy of aromatic amino acids. Thus, the free energy difference, AG, during the process is expressible as Eq. 7 (42,43), when variations in the entropy change are negligible. AG = - (AE + EA)A + IP D+ constant

[7]

For a series of electron donors (D) or quenchers with a certain electron acceptor (A), the first two terms parenthesized by A are unchanged and combined with the constant term. Thus, the stability of the quenching association complex represented by log Ksv is given by Eq. 8 (43). log Ksv ~ - AG = - IP D+ constant

[8]

462 The ionization potential of a molecule IP Dcould be linearly related with the "negative" HOMO value calculated by appropriate MO procedures for closely related series of analogs in terms of electronic structures (45) leading to Eq. 9, in which "a" is the slope of the relationship. log Ksv = aHOMO + constant

[9]

Equations 4-6 are exactly in the form of Eq. 9, indicating that the quenching complex formation of a series of acrylamides with each aromatic amino acid in the excited state is primarily governed by the charge-transfer mechanism. The higher the HOMO level, the easier the electron release from acrylamide analogs leading to corresponding cations. Although additions of other parameter terms to Eqs. 4-6 were insignificant, this was only true for the N-mono and disubstituted analogs except for analogs with h y d r o x y l a t e d N - s u b s t i t u e n t s in the tyrosine complex. Thus, acrylamide, methacrylamide and crotonamide were not accomodated in Eqs. 4-6. These compounds, without substituents on the amide nitrogen, showed a considerably higher association constant than that calculated by these equations invariably. This may be due to the reduction of steric constraints by the absence of substituents on the nitrogen. The fact that the steric parameter term of N-substituents was insignificant may indicate, however, that the acrylamides primarily approach from the carbonyl moiety to make the association complex. The carbonyl oxygen has the highest electron density on the HOMO level and is relatively free from the steric interference with the rest of the molecule. The fact that N-ethyl-methacrylamide is well accomodated in these equations would indicate that the complex formation does not seem to be affected much by the steric effect of the a-methyl group as far as N-substituted amides are concerned. The residuals of the log Ksv value for acrylamide and crotonamide are more positive than that for metacrylamide in Eqs. 4-6, however. The a-methyl group could exhibit a minor steric effect on the complex formation when there is no substituent on the amide nitrogen. Sterically unfavorable effect of substructures on the quenching complex formation site have been suggested to be important (46). The log Ksv value with tyrosine of amides in which the N-substituent carries an OH group at the a-carbon atom was higher than that expected from Eq. 6 as shown in Table 3, so the acrylamides with the HOCH 2- and HOCO(HO)CH- substituents were not included in the analysis. Even in the possible geometry where the carbonyl group faces on the aromatic ring of tyrosine, a hydrogen-bonding interaction between the side-chain OH in amides and the aromatic OH of tyrosine may significantly participate in enhancing the complex formation.

463

4.2

Quantitative Analyses of Enzymatic (Re)activities 4.2.1 Inhibition of Glycolytic E n z y m e P r e p a r a t i o n s Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a typical SH enzyme (47). Thus, the inhibition of GAPDH by acrylamide analogs was expected to correspond with their reactivity with glutathione. With the use of such an SH reactivity index as log k(GL), Eq. 10 was formulated. pI~ a~DH(M) = 1.145 (_+0.419) log k(GL) + 3.629 (_+0.507) n=8,

s=0.303,

r=0.940,

[10]

F(1,6)=45.35

Out of 9 analogs for which the pI~ value was measured, crotonamide was not included in Eq. 10, because the k(GL) value of this compound was not measurable. The GAPDH inhibitory activity parallels with non-enzymic glutathione reactivity almost in a fashion of one-to-one correspondence. The addition of hydrophobic and steric parameter terms did not improve the correlation, suggesting that the location of the SH group within the active site of GAPDH is not surrounded by a hydrophobic milieu and its steric environment is similar to that in glutathione. Against the rat brain phosphofructokinase (PFK), the inhibitory potency of 4 out of 9 compounds included in the inhibition experiments was extremely weak. We noticed that these extremely weak inhibitors were also relatively weak inhibitors against GADPH. We also recognized that a steric effect of N-substituents on the PFK inhibition may differ from that on the GAPDH inhibition. With only 5 compounds whose pI~ values were measured, it was almost impossible to make the statistically significant analysis for this structure-inhibition pattern. Thus, Eq. 11, which is significant only above the 80% level, should be taken here as a possible expression for the structure-activity pattern. PIso Pzz (M) = 0.783 (_+1.198) Plso (GAPDH) - 0.490 (_+0.832) ZE s' - 1.492 (• n = 5, s = 0.268, r = 0.895, F (2,2) = 4.04

[11]

The inhibitory activities of each of the acrylamide analogs againt the three-types of rat-brain enolase preparations, neuron-specific, hybrid and non-neuronal, were almost identical (15). The analysis was made for the pI~ value against the neuronspecific preparation. For 10 analogs, Eq. 12 was formulated. pI~ En~ (M)= - 0.271 (_+0.080) EEs + 2.371 (_+0.158) n=10,

s=0.135,

r=0.941,

[12]

F(1,8)=61.7

The meaning of Eq. 12 is rather simple, showing that the bulkier N-substituents

464 induce the more potent inhibition. Equation 12 indicates t h a t the inhibition of neuronspecific enolase is by no m e a n s specific to neurotoxic acrylamides.

The inhibitory

potency varies depending only upon the bulk of N-substituents. The correlation data for glycolytic enzyme inhibitions are summarized in Table 4. Table

4. Glycolytic Enzyme Inhibitions of Acrylamide Analogs

Compounds

pI~o (GAPDH)

pI~o (PFK)

obsd.

calcd."

obsd.

calcdJ

obsd.

calcd, c

Acrylamide t-BuDiacetone (Et)2(Me)2HOCH 2Meunsubstituted i-Pr-

~ 1.81 2.09 2.94 3.72 2.01 3.47 1.65

1.51 2.20 2.21 2.46 3.58 2.21 3.58 1.73

_d 1.20 1.40 2.27 1.72 --f 1.35 -f

__e 1.29 1.43 2.03 1.97 0.69 1.23 0.64

3.00 3.22 3.16 3.10 2.75 2.70 2.31 3.05

3.13 3.34 3.08 3.04 2.67 2.71 2.37 2.84

Methacrylamide

1.79

1.51

_r

-0.09

2.17

2.37

Crotonamide

1.98 g

e

--f

0.06

2.46

2.37

" B y Eq. 10. b By Eq. 11.

c By Eq. 12.

pI~o (enolase)

dNot measured,

e Not calculable.

f U n m e a s u r a b l y low or no inhibition, g Not included in Eq. 10. 4.2.2

Susceptibility

to Metabolic

Transformations

in V i t r o

T h e in vitro m e t a b o l i s m e x p e r i m e n t s indicated that, while acrylamide and crotonamide are susceptible only to the cytosolic glutathione S-transferase preparation in the presence of glutathione, some other analogs are susceptible to both cytosolic and microsomal enzyme preparations as shown in Table 5. For the susceptibility to the mouse hepatic microsomal enzyme preparation, Eq. 13 was formulated for 6 compounds out of 7 analogs with which the Michaelis constant, K~, was measurable. log (1 / K m) (M -1) = 0.677 (+0.775) log P + 1.254 (+1.201) MR - 0.379 (+0.295) MR 2 + 2.276 (•

n = 6, s - 0.130, r = 0.976,

[13]

F (3,2) = 13.10

By an u n k n o w n reason, the N-t-butylacrylamide was calculated to have much higher binding ability in terms of log (1 / K m) t h a n the observed value. Thus, it was not included in the correlation.

The log P term in Eq. 13 was significant only at the 94%

465 T a b l e 5. Metabolic Transformations in Vitro of Acrylamide Analogs

log (1/Km)

Compounds

log v ~ b

obsd.

calcd, a

Acrylamide t-BuDiacetone (Et)2. (Me)2" i-BuOCH 2HOCH2. Meunsubstituted i-Pr-

2.64 d 2.42 e 3.02 3.06 _e 2.63 -f 3.46

3.53 2.41 3.38 2.93 3.08 2.60 2.77 2.43 3.46

Methacryl amide

2.70 -~

Crotonamide

log v (GT) obsd.

calcd, c

0.45 0.45 e 0.47 0.34 __e 0.43 -f -0.01

0.75 1.06 0.94 1.61 a 1.63 1.43 0.40 1.49 1.23

0.85 1.04 1.09 0.96 1.68 1.32 0.86 1.39 0.85

2.64

0.44

0.79

0.65

2.79

~

1.06 d

-~

a By Eq. 13. b From ref. 17. c By Eq. 14. d Not included in the analyses, e Not measured, f Not susceptible, g Not calculable. level. The reason for the inclusion of this term was because the size of the regression coefficient is within a range of those observed in examples for microsomal oxidation of m a n y other series of organic xenobiotics (48). The log P term appears to show up not by chance.

It could be of real significance if data for some other analogs are

augmented. Acrylamide is shown to be oxidatively transformed to glycidamide in the rat body (4). The glycidamide formation is, however, dependent on the initial level of acrylamide in the body, the fraction (%) of the oxidized metabolite being insignificantly low when the initial level is higher t h a n 10 ~-3 m o ~

(4). It was not unexpected, therefore, t h a t

acrylamide was not v u l n e r a b l e in the mice microsomal oxidation e x p e r i m e n t s in which the substrate concentration was higher t h a n t h a t comparable with 10 3 m o l / ~ in spite of the difference in test animal species. We found that the microsomal metabolic reaction is not uniform among susceptible acrylamide analogs (17). For a few analogs, the structure of their major metabolite was identified.

For i n s t a n c e , N-i-propyl- and N,N-dimethylacrylamides were

metabolized to give acrylamide and the N-methyl analog, respectively (17). For other compounds, metabolic p a t h w a y s were a p p a r e n t l y different from t h e s e two and metabolites were unidentified.

In this respect, the formulation of Eq. 13 seems

reasonable without including the electronic p a r a m e t e r term reflecting the metabolic reaction mechanism.

The log Vm~ value was almost unchanged among susceptible

466 analogs with only one exception. Variations in the susceptibility to the oxidative metabolism are believed to be governed mostly by the binding process. For the susceptibility to the cytosolic glutathione S-transferase preparation, v(GT), in terms of nmol substrate reacted, h -1. mg protein -1, Eq. 14 was formulated for 9 compounds omitting crotonamide and N~N-dimethylacrylamide with the use of nonenzymatic second-order rate constant, k(GL), with glutathione. log v (GT) = 0.449 (+0.318) log k(GL) + 0.212 (+0.397) log P + 1.540 (+0.392)

[14]

n = 9, s = 0.268, r = 0.816, F (2,6) = 5.98 Crotonamide was susceptible to the glutathione S-transferase preparation, but the non-enzymic rate constant was unmeasurably low. The susceptibility of the N,Ndimethyl analog was predicted to be much lower than the observed value. No rationalization is available at the moment. The correlation quality of Eq. 14 is not so good as one would like, but it is significant above the 96% level. Because the log P term is only significant at the 76% level, it should be taken to indicate the structurehydrophobicity trend, if any. The main reason for this poor quality is the fact that the range of variations in the log v(GT) value is rather narrow. In spite of certain statistical drawbacks, Eqs. 13 and 14 seem to be reasonable and not inconsistent with the toxicity correlations shown below. Equation 13 may suggest that the most critical factors among those involved in the hepatic oxidation metabolism are steric complementarity of the NR 1R2moiety with a possible pocket of the reaction site perhaps being assisted by hydrophobic bonding regardless of differences in the metabolic reaction pathway. In Eq. 14, the significance of the log P term is just marginal. If factors would be separated more efficiently with additional compounds, a counterpart of Eq. 14 could be that showing a "direct" link between enzymic and non-enzymic reactions. The correlation results are summarized in Table 5.

4.3

Quantitative Analyses of Toxicities 4.3.1 Acute Toxicity

Preliminary examinations indicated that the acute toxicity parameter, pLD~, is correlated with a parabolic function of log P in addition to the LUMO energy level. The pLD~ values of methacrylamide derivatives and crotonamide were significantly lower than those of the N-substituted acrylamides having equivalent molecular and amide-moiety parameters.

Thus, we used an indicator variable, I, which takes a

value of unity for methacrylamides and two for crotonamide, but otherwise zero. Equation 15 was the best equation formulating the above situation with 17 compounds for which the MO parameters were calculated. The observed and calculated pLD~ values are compared in Table 6.

467 pLD~o (mol/kg) = 1.567 (+ 0.896) LUMO - 0.585 (+ 0.133) log P 0.729 (+ 0.217) (log p)2 _ 0.450 (• 0.150) I + 1.146 (+ 0.804) n = 17, s = 0.136, r = 0.967, F (4,12) = 42.78 -

[15]

Equation 15 indicates that the higher the LUMO level, the higher the acute toxicity. As shown in Eq. 3, the reactivity of acrylamides with glutathione was nicely correlated with negative LUMO value. The higher the LUMO level, the less easily would occur the nucleophilic addition of the SH compounds leading to less toxic hydrophilic conjugates. The positive sign of the LUMO term in Eq. 15 is reasonable on this basis. In Eq. 15, the N,N-diallyl analog which behaved as an outlier in Eq. 3 was well accomodated. The two allyl double bonds may not participate in critical steps in the detoxication reaction in vivo. The optimum log P value (-0.40) in Eq. 15 is much lower than those usually observed in a series of homologous toxicants applied to whole animals, 1.5~3.0 (49). For metabolic transformations of this series of compounds in vitro, the participation of hydrophobicity was not very significant as shown in the preceding section. For those in vivo, however, it could be highly significant. In general, the greater the hydrophobicity, the more susceptible are the xenobiotics to the metabolic mechanisms in liver (48). The negative optimum log P value for the acute toxicity suggests that a biphasic dependence of the overall acute toxicity to molecular hydrophobicity is such t h a t the positive phase accompanied by the general toxicity, including the ease of transport and distribution in vivo, is reversed by the negative factors with metabolic detoxication which are made rather significant in a lower log P range than usual in this series of toxicants. For the electronic p a r a m e t e r representing the susceptibility to oxidative mechanism, the HOMO parameter should be suitable. The less negative the HOMO level, the easier would be the electron loss resulting in the oxidized metabolites. The use of HOMO term in place of as well as in addition to the LUMO term in Eq. 15, however, did not improve the correlation. This indicates that the Michael-type reactions including the SH conjugation are of much greater importance than the oxidation in the whole body detoxication of this series of compounds. Even though it could occur to some extent, the critical step in the oxidative metabolism is not susceptible to the electronic factor as suggested by Eq. 13. The major metabolite of acrylamide is shown to be the N-acetyl cysteine adduct, the mercapturic acid in mammals (50). The negative indicator variable term indicates that the a- and ~-methyl groups work to reduce the acute toxicity, the effect of the ~-methyl (I-2) being about twice that of the a-methyl (I-l). The methyl groups attached to the sp 2 carbon probably offer additional sites of metabolic degradation in vivo, the sterically less hindered ~-methyl being more susceptible than the a-methyl (51).

468 T a b l e 6. Acute and Chronic Toxicities of Acrylamide Analogs

Compounds

pLDso obsd.

NT Score calcd2

obsd.

calcd, by Eqs. 16

17

18

19

Acrylamide t-BuDiacetone (Et) 2(Me) 2-(CH2) 4(Allyl)2n-Bu-glycolic acid EtHOCH 2Meunsubstituted i-Pr-

2.13 2.33 1.96 2.47 2.65 1.56 1.30 2.04 2.5O 2.26 2.51 2.82 2.43

2.03 2.32 1.85 2.46 2.45 1.69 1.30 2.03 2.64 2.30 2.64 2.74 2.48

1

1

1

1

1

2 2 3 3 4 4

2 2 3 3 4 2

2 2 3 3 4 2

2 2 3 3 4

b

2 2 3 3 4

._b

Methacrylamide -(CH2) 4Etunsubstituted

1.59 1.55 2.28

1.54 1.83 2.16

2 2 3

1 2 3

2 2 3

2 2 4

1 2 3

Crotonamide unsubstituted

1.50

1.45

1

3

_b

_b

_b

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1 1 1

2 1 1

2 1 1

2 1 1

2 1 1

a Calculated by Eq.15. b Not included in the analyses. The formulation of Eq. 15 with a very good correlation quality should be gratified for such a biological event as the acute toxicity involving very complicated processes. 4.3.2

Neurotoxicity

The neurotoxic potency of 8 out of 17 analogs was very low with the IDso value not m e a s u r a b l e by our rotarod procedure.

Thus, the neurotoxicity was inevitably

represented in the quantitative analysis by potency scores as defined in Sec. 3.2. For the analysis, the fuzzy adaptive least squares procedure (FALS 91) developed by Moriguchi and coworkers (52) was used with combinations of various physicochemical parameters as independent variable. In the adaptive least squares (ALS) methods (53), the dependent variable is originally represented as the potency scores, i.e., integers in the order of increase in the potency. First, the regular regression analysis is performed and calculated values

469 for the dependent variable are transformed to integers according to certain rules. In the fuzzy adaptive procedure (52), some of the rules are according to a m e m b e r s h i p function. Next, the analyses are repeated until original scoring integers match best those calculated and t r a n s f o r m e d .

The m e m b e r s h i p function is a function t h a t

represents the extent of fuzziness (0 to 1) in the membership for each compound to be likely a m e m b e r of a certain potency group. This function is i m p o r t a n t especially when the n o n - t r a n s f o r m e d potency value is located n e a r the b o u n d a r y between consecutive potency groups in the p a r a m e t e r space. The "fuzzy" procedure has been designed to maximize the total sum of the membership values over all compounds in the set (52). For 17 analogs in Table 6 for which molecular orbital indices were calculated, the combination of MR, HOMO and log P was best to rationalize the r a n k of the toxicity scores to give Eq. 16. NT = - 0.549 MR - 0.826 HOMO - 0.337 log P - 6.677 [0.470]

[0.218]

[16]

[0.196]

n = 17, n (mis) = 4 (2), Rs = 0.699, R s . MMG = 0.510 In this and the following FALS equations, NT is the neurotoxicity score, n(mis) is the n u m b e r of compounds m i s m a t c h e d , the n u m b e r in p a r e n t h e s e s being t h a t m i s m a t c h e d two r a n k s lower as well as higher t h a n the original score, R s is the Spearman's r a n k correlation coefficient and MMG is the average of the membership values of compounds.

The product, R s . MMG, is used as a criterion for the best

rank-correlation among various combinations of physicochemical p a r a m e t e r s . The figures in brackets are the contribution index (not normalized) showing the relative significance of the p a r a m e t e r term j u s t above the brackets (51). The poor quality of Eq. 16 m a y be due to the fact t h a t the set of compounds is not uniform. In fact, crotonamide is the only compound with the ~-Me. In spite of the original score of unity, Eq. 16 mispredicted it to be of the score 3. This may be due to an inherent effect of the ~-Me group unfavorable to the toxicity. Thus, the correlation was reexamined for the set from which crotonamide was omitted, giving Eq. 17 which was much improved without using the log P term. NT = - 0.903 MR - 0.366 HOMO - 1.733 [0.741]

[17]

[0.089]

n = 16, n (mis) = 2 (1), R s = 0.883, R s . MMG = 0.672 The compound in which the toxicity score is calculated by Eq. 17 two ranks lower t h a n the original score was N-i-propyl acrylamide. This could be due to a peculiar behavior of this compound in its metabolic transformation. The neurotoxic potency of

470 analogs is susceptible to the activation of metabolic mechanisms (13). The consecutive weekly applications of phenobarbital to mice from a week before to the last week of the dosing greatly reduce the potency of neurotoxic compounds except for the N- i-propyl analog which shows only a delay in the development of the symptom. As indicated previously, this compound is metabolized to give acrylamide as a major metabolite in vitro with hepatic microsomal enzyme preparation (17). Other neurotoxic compounds are metabolized without giving acrylamide. These observations appear to indicate t h a t the N-i-propyl compound, but not other neurotoxic analogs, is metabolized in vivo into highly neurotoxic acrylamide. Regardless of its further transformation routes in vivo, the mice treated with this compound could be intoxicated by metabolically produced acrylamide. Thus, the neurotoxicity of the N-i-propyl compound itself should be lower than that indicated by the rotarod experimental score. The misclassification of this compound with Eq. 17 can be understood on this basis. With deletion of the N-i-propyl compound, Eq. 18 was formulated showing a much improved r a n k discrimination. NT

=

-

0.956 MR - 0.449 HOMO - 2.269 [0.811]

[18]

[0.113]

n = 15, n (mis) = 2 (0), Rs = 0.939, R s . MMG = 0.755 [leave-one-out : n (mis) = 6 (0), R s = 0.728, R s . MMG = 0.434] Equation 18 was best in terms of the r a n k correlation and the leave-one-out crossvalidation as far as the set of 15 compounds is concerned. The two misclassified compounds by Eq. 18 were the N,N-dimethylacrylamide from the original score 1 to 2 and methacrylamide from the observed score 3 to 4. Because two of the three methacrylamide derivatives are well accomodated in Eq. 18, no remarkable effect of the a-Me group seems operative other than the effects contributing to the HOMO value.

MR is the bulk p a r a m e t e r only for the NR 11~

moiety of compounds. Equation 18 indicates that the smaller the NR1R 2 moiety as well as the more negative the HOMO value, the more likely is the development of the higher neurotoxicity. Eq. 16 has an almost equivalent classification quality with Eq. 18, if ipropylacrylamide and crotonamide, the positive and negative deviant, respectively, are reasonably disregarded. This might suggest a minor role of the negative log P term. A correlation, which is somewhat poorer than Eq. 18 in terms of statistical values, Rs and Rs. MMG, but shows an equivalent discrimination of compounds with Eq. 18, was formulated as Eq. 19 with a negative log P term.

471 NT = - 0.765 MR - 0.782 HOMO - 0.117 log P - 5.782 [0.649]

[0.197]

n=15,

[19]

[0.072]

n(mis)=2(0),

Rs=0.877,

Rs.MMG=0.745

The all correlation results for the neurotoxicity are summarized in Table 6. It should be noted t h a t s t r u c t u r a l variations in the set were not too drastic, because drastic variations m a y r e s u l t in compounds with different features and mechanisms of toxic action. Thus, some but not extremely high collinearity among independent variables for 15 compounds was inevitable; for instance, r (MR = 0.766, r (MR

vs.

log P) = 0.666 and r (HOMO

vs.

vs.

HOMO)

log P) = 0.510. The collinearity

problem among variables and the fact t h a t the n u m b e r of compounds of the highest r a n k is reduced to unity are probably responsible for the statistics for the cross-validation of Eq. 18 not being as good as one would expect.

4.3.3

Cytotoxicity

Acrylamide and its N-i-propyl and hydroxymethyl analogs, which were highly neurotoxic, were also highly cytotoxic. Because structure-toxicity patterns seemed to be similar b e t w e e n neuro- and cytotoxicities, we utilized the HOMO value with combination of various other parameters as the independent variables for the analysis of the cytotoxicity index, pIso. Because of experimental difficulties, the pIso value, was m e a s u r a b l e only for 7 compounds against the N18TG-2 cells and for 6 against the RT4 cells. The number of compounds may be too low to formulate significant correlations, but Eq. 20 was obtained for the N18TG-2 cytotoxicity with the use of HOMO and XE s of N - s u b s t i t u e n t s . pI~ N:ST~2 (M)= - 7.367 (2 7.993) HOMO - 2.152 (+ 2.341) ZE s - 70.07 (+ 79.08) n=7,

s=0.403,

r=0.789,

[20]

F(2,4)=3.30

In Eq. 20, both the HOMO and E s terms are significant only at the 93% level, with the most conspicuous positive deviant, N-hydroxymethyl acrylamide. Because this deviant among 7 compounds is only one which carries the hydroxyl group on the amide N-substituents with a possible effect enhancing the toxicity, we deleted it and recalculated to give Eq. 21 with a much better correlation. PIso N18TG-2(M) = - 8.178 (2 3.454) HOMO - 2.410 (2 1.013) ZE s - 78.240 (2 34.18) n = 6, s = 0.150, r - 0.970, F (2,3) = 28.78

[21]

472 For 5 compounds with which the I~o concentrations were m e a s u r a b l e a g a i n s t both cell lines, Eq. 22 was found. pI~ RT4 (M) = 2.266 (+0.581) pI~ (N18TG-2) - 4.420 (+ 1.823)

[221

n = 5, s = 0.115, r = 0.990, F (1,3) = 154.0 The correlation results are shown in Table 7.

Table 7. Cytotoxicity of Acrylamide Analogs Compounds

pI~ (N18TG-2)

pIso (RT4)

obsd.

calcd, a

obsd.

calcd, b

Acrylamide Diacetone (Et) 2(Me) 2HOCH 2Meun substituted i-Pr-

< 2.00 c 2.85 3.00 3.40 c 2.23 3.52 2.85

7.05 2.70 3.15 2.56 2.35 3.49 2.77

< 1.70 ~ 2.16 2.40 3.32 < 1.70 ~ 3.52 1.89

-d 2.04 2.38 3.28 0.63 3.55 2.04

Methacrylamide unsubstituted

2.10

2.09

< 1.70 c

0.34

Crotonamide unsubstituted

< 2.00 c

2.88

2.30 c

a d

-

d

Calculated by Eq. 21. b Calculated by Eq. 22. c Not included in the analyses. Not calculable.

Equations 21 and 22 together with the fact t h a t the N-hydroxymethyl compound is a positive outlier suggest t h a t the cytotoxicity of this series of compounds against cell lines of n e u r o n a l (N18TG-2) and glial (RT4) origins is governed by common stereoelectronic factors of the molecule and the hydroxyl group on the amide Ns u b s t i t u e n t is favorable to the cytotoxicity perhaps through its hydrogen-donating ability. E q u a t i o n 21 indicates t h a t the b u l k i e r the N - s u b s t i t u e n t s , the h i g h e r the cytotoxicity. The compound having the bulkiest substituent is diacetone acrylamide for which the cytotoxicity is below 2.0 in terms of pI~ and not included in Eq. 21. The size of N-substituents of compounds incorporated in Eq. 21. is r a t h e r small. Thus, there seems to be an optimum bulk of N-substituents for the cytotoxicity.

The

473 unmeasurably low N18TG-2-cytotoxicity of crotonamide (pI~o<2.0) could also be due to a steric inhibition effect of the ~-CH 3 group on the cytotoxic interaction.

5.

DISCUSSION 5.1

O v e r v i e w of t h e S t r u c t u r e - ( R e ) a c t i v i t y a n d S t r u c t u r e - T o x i c i t y Correlations A number of correlation equations presented above seem to be categorized at least into two groups by which of the two electronic parameters, LUMO and HOMO, are decisive in rationalizing the structure-(re)activity and structure-toxicity relationships. The LUMO- and HOMO-dependent correlations are summarized in Tables 8 and 9, respectively. Although some parameter terms are of marginal significance, and some correlations do not include enough compounds, the correlations in each of Tables 8 and 9, when they are put together, are not inconsistent with each other as will be discussed below. Besides correlations included in these Tables, the microsomal oxidation susceptibility and the neuron-specific enolase inhibition were shown to vary without significant participation of electronic parameters. 5.2 P o s s i b l e 'TIolecular" M e c h a n i s m s of T o x i c i t i e s Except for the acute toxicity, the LUMO-dependent (re)activities are such that the lower the LUMO level, the higher the (re)activity corresponding to the susceptibility to the biological Michael reagents such as glutathione as shown in Table 8. The acute toxicity is mostly dependent on the difficulty of metabolic transformation and elimination, the dominant mechanism of which is the conjugation not only with glutathione but also with other SH compounds and relevant amines as far as the N-substituted acrylamides are concerned. Thus, the sign of the LUMO term in the acute toxicity was reversed. The hepatic oxidation mechanism is certainly involved in the overall biotransformation. But, its participation in the acute toxicity is of minor importance in general and, if involved, the critical process is not governed by the electronic parameter. In the (bio)chemical Michael addition reactions (Eqs. 3, 10 and 14), the a- and ~-Me groups exhibit steric inhibition effects, but, in the acute toxicity, they could be transformed simultaneously to reduce the toxicity as suggested before. The molecular mechanism for the neurotoxicity which belongs to the HOMOdependent correlations is believed, therefore, to be entirely different from that of the acute toxicity. Interestingly, the structural factors governing the neurotoxicity nicely conform to those governing the cytotoxicity. First, both toxicities depend on the negative HOMO. The lower the HOMO level, the higher the toxicities. The second is the dependence on the bulk of N-substituents. Equation 21 shows that the more negative the ZE s value for N-substituents, i.e., the bulkier the N-substituents, the higher the cytotoxicity. As suggested before to rationalize the behavior of diacetone

474 Table 8. LUMO-Dependent (Re)activities and the Acute Toxicity Addition of Glutathione Structural Effects

Molecular Parameters LUMO log P Others "Bulk" of N-Substituents d Effect of a-Me (I = 1) [~-Me (I = 2) Equation Number

Glycolytic Enzyme Inhibition

Acute Toxicity

Chemical

Cytosolic

log koL

log VGT

PIs0 GAPDH

pLD~

negative _b _b

(negative)a positive c log kaL

(negative)~ _b log kGL

positive biphasic _b

negative

(negative) ~

(negative) a

_b

negative negative

(negative) ~ _e

(negative) ~ _e

negative negative

[3]

[14]

[10]

[15]

a Not from the correlation equation, but according to reactivity parameter used as an independent variable, b Not entered, c Only marginal, and not significant at the 95% level, a Shown by ZE s' ofR 1 and R 2. Uncertain. e

acrylamide as the negative deviant, however, there could be an optimum steric bulk for the cytotoxicity. The positive phase of the bulk effect on the cytotoxicity which is j u s t for compounds with smaller N-substituents could be smoothed out in the neurotoxicity rank discrimination including additional members which showed no neuropathic activity with bulkier N-substituents. The ZE s value worked better than MR (NR1R 2) in Eq. 21 for cytotoxicity, but the reverse was the case in Eqs. 16-19 for neurotoxicity. The ZEs value is to define steric conditions of substituents at locations close to the amide nitrogen, whereas MR describes an overall bulkiness of substituents. To "analyze" the substituent effect on the amide moiety "precisely", the ZE s was better in the cytotoxicity, but to "classify" the potency score in neuropathy, somewhat less "precise" MR (NR1,R 2) was better. Third, the hydrophilic N-HOCH~ analog was the positive outlier in Eq. 21 for the cytotoxicity. As described above, the a-OH group on the N-substituents may exert an extra effect to enhance the toxicity. This could be reflected in the neurotoxicity analysis in Eq. 19 showing a contribution of the negative log P although it was of minor importance. Being masked by additional non-neurotoxic analogs without N-a-OH substituents, the effect of log P could not be explicitly shown up in the neurotoxicity analysis. The above considerations strongly suggest a very close link in mechanisms

475 Table 9. HOMO-Dependent Reactivities and Toxicities Association with Amino Acids Structural Effects

Molecular Parameters HOMO log P Others "Bulk" of N-Substituents d Effect of ~-Me ~-Me Other Effect of N-substituents Equation Number

Cytotoxicity

ieuro-

toxicity N18TG-2

RT4

log Ksv

pI ~o

pI 5o

NT

positive _b _b

negative _b _b

(negative) a

negative negativC

negative e

biphasic f

__g

negative b -g

_b negative

positive for a-OH with tyrosine [4,5,6]

b

pitoNlsTc~2

b __g

negative b

negative mi

positive for the HOCI-I2 analog [21]

b

[22]

[ 16,18,19]

a Not from correlation equation, but according to the toxicity parameter used as the independent variable, b Not entered, c Of marginal significance, d Expressed by MR (NR1 ,R2) or ZEs (R1 ,R2), unless noted, e Expected from the fact that the N-unsubstituted compounds are positive outliers, f Suggested by the fact that a compound with the bulkiest N-substituent is the negative outlier, g Uncertain. h Relative to the effect of ~-Me. i The N-i-propyl analog is a positive outlier. between neuro- and cytotoxicities. The cytotoxicities against cell lines from neuronal and glial origins are governed by a common molecular mechanism. Although the quenching association of acrylamide analogs with aromatic amino acids in a state excited by UV-irradiation does not seem to be related directly with the biological phenomena, the analyses shown by Eqs. 4-6 could provide us with valuable information about the molecular mechanism of neurotoxic action of acrylamide analogs. The positive effect of the a-OH group on the N-substituents in the association complex with tyrosine suggests that it is not impossible for acrylamides with similar N-substituents carrying the a-OH group to interact specifically with the active sites where the mechanism for the neurotoxic action is triggered.

For the quenching

association, the positive HOMO term in Eqs. 4-6 means that the easier the electron flow from the carbamoyl moiety of acrylamide analogs to the aromatic ring of amino acids, the more stable is the complex. Thus, the negative HOMO term in cyto- and neuro-toxicity analyses could mean that the higher the electron attracting effect of

476 N-substituents to make the electron-flow of similar types difficult, the higher could be the toxicity. The situation could be depicted as shown in Fig.l, assuming that any type of the charge-transfer is not significant between the carbamoyl moiety and aliphatic domains of the receptor sites. The factors governing the transport and distribution of compounds in the mouse body otherwise represented by a parabolic or biphasic dependence on the log P do not seem to be significant to govern variations in the neurotoxic potency. This could be due to the fact that the overall chronic toxicity was measured after compound distribution reached an equilibrium state and that the hydrophobic interaction of compounds, in particular, that of the amide moiety with the site of action is not critical being overweighed by hydrogen-bonding interaction(s). It should be noted that, for 17 analogs included in Eq. 16 for the neurotoxicity analyses, the HOMO value is highy collinear with the number of hydrogen atom on the amide nitrogen (r2=0.903). With increasing in the number of NH hydrogen atoms in the order of tertiary < secondary < primary amides, the HOMO value shifts to the more negative direction rather regularly. The more negative the HOMO level, i.e., the greater the number of NH hydrogen atom, the more tightly are the electrons bound with the carbamoyl core of acrylamide leading to the easier proton migration to possible basic sites at the same time. In place of the HOMO parameter, the number of hydrogen atoms on the amide nitrogen, HB, could be used as an independent indicator variable representing the electronic structure of acrylamide analogs. Thus, another hydrogen-bond acceptor site(s) could be considered on the receptor domain accommodating the N-substituents as shown in Fig.1. The reason why we did not use the HB parameter for the analyses is that some compounds without NH hydrogen were neurotoxic. Furthermore, for analysis of the quenching association, the use of HOMO value is theoretically justifiable. 6.

CONCLUDING REMARKS

The above analyses and discussions suggested that either glial or neuronal cells of the peripheral nerves could be the site where

the neurotoxicity is initiated.

Histological as well as physiological disorders brought about in these cells no doubt lead to poisoning symptoms. A possible molecular mechanism was hypothesized so that electronic and steric properties and hydrogen-bonding factors of the amide moiety are to operate together to govern the variations in the potency of the neurotoxic effect as summarized in Fig. 1. With the use of a series of structurally related compounds, it was only possible to separate various physicochemical factors involved in the structure-activity pattern from each other and to estimate them individually. Also very important was to cover structure-activity relationships for related (bio)chemical (re)activities.

With

comparative examinations of various quantitative sturucture-(re)activity and structure-

477 Steric inhibition

"Hydrogen-Bonding Site"

/

\

/H

o

~

@B

Optimum size for the accommodation

Non-aromatic domain (Charge-transfer prohibited)

"Hydrogen-Bonding Site"

Fig. 1. Possible Receptor Binding Features of Acrylamide Analogs to Exert Neurotoxicity toxicity formulations, the physicochemical meanings of individual analyses were able to reinforce each other yielding a hypothetical model for the molecular mechanism. The present analyses indicated that no direct relationship exists between neurotoxicity and glycolytic-enzyme inhibitory activity of acrylamide analogs. There could be a possibility to find relationships between neurotoxicity and neuronal glycolytic enzyme activity prepared from chronically intoxicated test animals for a set of acrylamide-related compounds. The ethiological observations with glycolytic enzyme preparations using just acrylamide as the test compound (9) are only phenomenological and should be extended to cover related compounds to be comprehensive. We have observed that many of the neurotoxic acrylamide analogs are also toxic to testis after chronical exposures in mice (13). Unfortunately, the testicular toxicity has been represented only qualitatively but not quantitatively. There have been quite a few studies (54,55,56) suggesting possible relationships of neurotoxicity with testicular toxicity under chronic-exposure conditions for other types of chemicals. Comparative study of quantitative structure-activity relationships should be a powerful tool to disclose similarity and dissimilarity between neuro- and testicular toxicities. We have published individual analyses from time to time in separate forms (12-17, 19, 26, 35). With some physicochemical parameters and bioactivity indices

478 revised and augmented, the present analyses should be taken to supersede the published correlations. ACKNOWLEDGEMENTS. Drs. Yoshiaki Nakagawa and Miki Akamatsu of Kyoto University and Ryo Shimizu of Tanabe Seiyaku Ltd. are gratefully acknowledged for their assistance of calculations, Drs. Chikayoshi Nagata and Marvin Charton for their suggestions on the quenching mechanism, and Drs. Takaaki Nishioka and Tohru Nagasawa for their helpful comments in general. Many thanks are also due to Misses Chieko Demura and Yuko Seto for the preparation of the manuscript. REFERENCES 1) EPA, "Preliminary Assessment of Health Risks from Exposure to Acrylamide," Office of Toxic Substances, U.S.Environmental Protection Agency, Washington, D.C., 1988. 2) R.M. Myers, T. Maniatis and L. S. Lerman, Methods in Enzymol., 155, 501 (1978). 3) H.A. Tilson, Neurobehav. Toxicol. Teratol., 3, 143 (1981). 4) E. Bergmark, C. J. Calleman and L. G. Costa, Toxicol. Appl. Pharmacol., 111, 352(1991). 5) E. Bergmark, C. J. Calleman, F. He and L. G. Costa, Toxicol. Appl. Pharmacol., 120, 45 (1993). 6) P.S. Spencer and H. H. Schaumberg, Can. J. Neurol. Sci., 1,143 (1974). 7) F. He, S. Zhang, H. Wang, G. Li, Z. Zhang, F. Li, X. Dong and F. Hu, Scand. J. Work Environ. Health, 15,125 (1989). 8) J.E. Myers and I. Macun, Am. J. Ind. Med., 19,487 (1991). 9) R.D. Howland, Toxicol. Appl. Pharmacol., 60,324 (1981). 10) P.S. Spencer and H. H. Schaumberg, J. Neuropathol. Exp. Neurol., 36,276 (1977). 11) P.S. Spencer and H. H. Schaumberg, J. Neuropathol. Exp. Neurol., 36,300 (1977). 12) H. Tanii and K. Hashimoto, Arch. Toxicol., 54,203 (1983). 13) I~ Hashimoto, J. Sakamoto and H. Tanii, Arch. Toxicol., 47,179 (1981). 14) H. Tanii, N. Miki, M. Hayashi and I~ Hashimoto, Arch. Toxicol., 61, 298 (1988). 15) H. Tanii and K. Hashimoto, Experientia, 40,971 (1984). 16) H. Tanii and K. Hashimoto, Toxicol. Letters, 26, 76 (1985). 17) H. Tanii and I~ Hashimoto, Arch.Toxicol., 48,157 (1981). 18) E.D. Bergman, D. Ginsberg and R. Pappo, Org. React., 10,179 (1959). 19) I~ Hashimoto and W. N. Aldridge, Biochem. Pharmacol., 19, 2591 (1970).

479 20) 21) 22) 23) 24) 25) 26) 27) 28) 29) 30) 31) 32) 33) 34) 35) 36) 37) 38) 39) 40) 41) 42) 43) 44) 45) 46)

47) 48)

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480 49) C. Hansch and J. M. Clayton, J. Pharm. Sci., 62, 1 (1973). 50) M.J. Miller, D. E. Carter and I. G. Sipes, Toxicol. Appl. Pharmacol., 63, 36 (1982) 51) R.T. Williams "Detoxication Mechanisms", 2nd Ed., Chapman and Hall Ltd., London, 1959, p.194. 52) I. Moriguchi and S. Hirono, "QSAR - N e w Developments and Applications", T. Fujita, Ed., Elsevier, Amsterdam, 1994, p. 275 53) I. Moriguchi, K. Komatsu and Y. Matsushita, J. Med. Chem., 23, 20 (1980). 54) S.G. Somkuti, D. M. Lapadula, R. E. Chapin, J. C. Lamb and M. B. Abou-Donia, Toxicol. Lett., 37,279 (1987). 55) I~ Boekelheide, Toxicol. Appl. Pharmacol., 92, 18 (1988). 56) F.S. Messiha, Neurotoxicol., 12,559 (1991).

481

SUBJECT INDEX Acetolactate synthase inhibitors, sulfonylureas and related compounds 244 Acrylamide analogs, acute toxicity 452,455, 466, 474 cytotoxicity 452,457, 471,475 fluorescence quenching 453, 460, 475 glycolytic enzyme inhibition 458, 463 metabolism 458, 464 neurotoxicity 451,457, 468, 475 reactivity with glutathion 452, 460, 474 Active analog approach, definition 30 quinolone-3-carboxylic acid antibacterials 103-109, 114, 120-122 Active conformation 9, 103-109, 114, 120-122 Active volume 103, 105, 109, 118, 120, 121 Acute toxicity, acrylamide analogs 452, 455, 466, 474 Acute toxicity, human, miscellaneous compounds 286 Acylanilides, as anticanceragents 251 as herbicides 251 Adaptive least squares analyses (ALS), 275 FALS 89 276 FALS 91 295 Fuzzy ALS (FALS), 276 discriminant function 276, 289, 293 fussy variance 276 membership function 277, 278 Alcohol dehydrogenase, three dimensional structure 23 Aldosterone 129, 130, 139 cz-AIkyl-0~-(phenoxyalkylaminoalkyl)phenylacetonitriles, as verapamil analogs 370-410 Alprenolol 237, 255 AM80, a synthetic retinoid 13 Amino acids, structure-hydrophobicity relationships 206-211 Amino acids, aromatic, fluorescence quenching with acrylamide analogs 453, 460 Amino acid sequence 55

482

Amino acid sequence, insertion of, with geometrical method 58 of serine proteases 90 relationship with protein functions 216, 217 Amino acid sequence segments, conserved within a set of sequences 222 Anabolic effect, of androstane analogs 145 of nandrolone analogs 128 Androgen 131, 138 Androgenic effect, of testosterone analogs 128 Androstanol 131, 133, 138, 147 Angiotensin II receptor, antagonists 239, 260, 261 subtypes 239 Anti-antidiuretic effect, vasopressin antagonists 281 Antibody, hypervariable loops in 220 Anticonvulsant activity, of antiepilepsirine 323 of cinnamoylpiperidines 324 of piperine 322 of substituted cinnamides 325, 326, 329 Anticonvulsants 253 from Chinese folk medicine 299, 322 3,6-disubstituted phthalides 305 3-substituted phthalides 299 Antiepilepsirine, as anticonvulsant 323 structure modification of 323 Antihistaminic activity, 2-piperazinylbenzimidazoles 437 Antiinflammatory agents, arylalkanoic acid-type 246 structural requirements 247 Antithrombotic agents, asthrombin inhibitors 84 Antiulcerative effects, 1-benzyl-4-piperazine-acetamides and-acetates furazolidone and analogs 306, 307, 309 Anti-vasopressor effect, vasopressin antagonists 281

429

483 Aplysiatoxin 41 Aquatic toxicity, finfish, miscellaneous compounds 291 Arachidonic acid 13 Arginine derivatives, as serine protease inhibitors 84, 90, 92 as serine protease substrates 94 Arginine-vasopressin antagonists 280 Arylalkanoic acids, as antiinflammatory agents 246 as plant growth regulators/herbicides 246 Aspartate carbamyltransferase, cytidine as the inhibitor 230 Atom acceptable region 23 N-Benzoyl-8-a m ino-2-tetrazolylbenzodioxa nes, as benzamide-type LT-antagonists 345 N-Benzoyl-8-amino-2-tetrazolylbenzopyranones, as benzamide-type LT-antagonists 345 Benzoylphenylureas 246 1-Benzyl-4-diphenylmethylpiperazines, as cerebral vasodilator 414 1-Benzyl-4-piperazine-acetamides and-acetates, as antiulcerative agents 429 Bioanalogy, as a concept broader than bioisosterism 236, 237, 254-264 BIOCES[E], a system for receptor modelling 50 Biphenyl derivatives, hepatotrophic effect of 314 cz-Blocking activity of verapamil analogs 372 398, 401,403, 404, 408 Blood coagulation cascades 83 Ca-antagonistic activity of verapamil analogs 372,374, 377, 384 Cambridge crystallographic database 7 Catepsin H, human, amino acid alignment with papain 71 rat, amino acid alignment with papain 56, 57, 60 rat, binding specificity with inhibitors 73 rat, model building 70 Cerebral vasodilating activity, 1-benzyl-4-diphenylmethylpiperazines 414 Chinese folk medicine, as the origin of lead compounds 321 celery seeds 299 for hypertension diseases 299 for tonic 314

484

Chirality, recognition for drug-receptor interaction 301 Chou-Fassman, prediction of three-dimensional protein structure 50 /~-turn propensity parameter 197 Cinnamides, substituted, anticonvulsant activity 323, 329-331,334, 336 QSAR of 326, 328 structural modifications 323, 329-331,334, 336 CoMFA (comparative molecular field analysis) 132, 146 for binding affinity for androgen receptor 139, 142 for binding affinity for estrogen receptor 135, 143 Computer graphics 7, 17 Conformation search 10, 40, 105 Conformational analyses, benzamide-type LT-antagonists 348, 350, 351 quinolone-3-carboxylic acid antibacterials 101, 103, 113, 116, 120 Conformational distribution, benzamide-type LT-antagonists 351,353, 355 Conformational flexibility, benzamide-type LT-antagonists 351,352,355, 356 weakly acidic uncouplers 342 Cromakalim and analogs 237, 238, 256 Cyclic AMP 41 Cyclooxygenase 13 Cytotoxicity, acrylamide analogs 452, 457, 471,475 DDD as insecticides and anticancer agents 246 Dielectric constant 20 Diethylstilbesterol 13 Dihydrofolate, reductase, E. coli 25 L. casei 12, 34-41 reduction of 37 superimposition with methotrexate 34-41 Dihydrotestosterone 138, 139, 141, 145 Dinucleotide fold, in amino acid sequences of dehydrogenases 218, 219, 221 Diphenylurea analogs 246 Distance-geometry method 50 Docking, between receptor and ligand 16-27, 53

485 Drug design, based on active analog approach 123 based on amino acid sequences in receptor proteins based on drug-receptor interaction 3 based on precedent structural modifications 265 based on receptor modelling 61 Drug-receptor complexes 10, 11, 18 Drug-receptor interaction, with chirality recognition 301 Drug-receptor interactions 4, 7, 16, 19, 21,49, 73 drug design based on 61 electrostatic effect on 70 hydrophobic effect on 65 Monte Carlo simulation to 55, 73 of steroid hormonal drugs 135, 139, 141, 144 Electrostatic effect, on drug-receptor interactions 70 guest-on-guest potential 70 host-on-guest potential 70 Electrostatic potential, on molecular surface 17, 38 in program GREEN 20, 23 in program RECEPS 34, 38 EMIL, operation of 264 as a system for lead evolution 235-273, 236, 237 Enzyme reaction database 227 Epitiostanol 127, 139, 145 Estradiol 131, 133, 141, 142, 143, 144 Estratrien-17/~-ol 139, 141, 142 Estrogens 13, 42, 131, 133, 141, 143 Exon, shuffling 220 as a unit of protein function 221 Factor Xa, amino acid sequence of 90 inhibitors of 88, 89 Fauchere-Pliska hydrophobicity scale of amino acid side chains 207 Fibrinolysis cascades 83 Fluorescence quenching, acrylamide analogs 453, 460, 475

230

486

Folk medicine, Chinese 299, 314, 321 FRODO, a program for protein modelling 56 Fungicides, agricultural, /~-methoxyacrylates and analogs 241 Furazolidone, antiulcerative effect of 306 as chemotherapeutic agent 306 Gap, in the amino acid sequence alignment 56 introduction with geometrical method 58 search for databases 60 Glutathion, reactivity with acrylamide analogs 452,460, 474 Glutathion reductase, coenzyme specificities 217 complex with NADPH 218 homology graphing 224 site-directed mutagenesis 218 Glycolytic enzyme systems, inhibition with acrylamide analogs 458, 463 Goodness of fit, in superimposition of molecules 33, 37 GREEN program 15 Grid points 20, 31 Hepatotrophic effect, biphenyl derivatives 314 schizandrin C 314 Hill reaction inhibitors, as herbicides 251,258, 259 Histamine H2-antagonists, 248, 249 substructural transformations 257, 258 Homology, between two proteins 72, 84 Homology graphing, for analyses of relationship between amino acid sequence of enzymes and chemical structure of ligands 226 as a procedure to find functionally important amino acid sequences Hydrogen bond, display of hydrogen bonding region 24 in ligand-receptor interactions 12 potential function 19 in program GREEN 22, 25 in program RECEPS 34, 40

222

487

Hydrophobic core, distance 61 in protein modelling 56 score 51,56,71 Hydrophobic effect, correlation index 70 on drug-receptor interactions 65, 75 field effect index 69, 77 interaction energy 65, 75 Hydrophobic index 69, 70 Hydrophobicity parameter, log P (1-octanol/water), analyses, N-acetyl oligopeptide amides 204 disubstituted benzenes 154 disubstituted pyrazines 172 monosubstituted diazines 159 monosubstituted pyridines 155 oligopeptides 188, 195, 201,202 polysubstituted pyrazines 181 Hydrophobicity scales of amino acid side chains 205-211 Ibuprofen 246, 247 IDEAS 223 Imidacloprid and analogs 250, 258 Indomethacin 13, 246, 247 Ingenol ester 41 Janin-Chothia hydrophobicity scale of amino acid side chains 210 Kyte-Doolittle hydropathy scale of amino acids 210 Lead evolution, analog design, as strategy of 236 of cerebral vasodilating N,N'-disubstituted piperazines 425 as consecutive structural transformations 236 cromakalim analogs 237, 238, 255, 256 /~-methoxyacrylates and analogs 241,261 non-peptide angiotensin II receptor antagonists 239, 260 sulfonylureas and related compounds 242, 262, 263 Lead generation 3, 15 1-benzyl-4-diphenylmethylpiperazines 414 with substructure combinations 231 Lead optimization 6, 14, 236 Leave-one-out prediction 280, 290, 294 Lenard-Jones potential 19

488

Leucotrienes (LT's), agonists, benzamide-type (YM-17690) 363 antagonists, benzamide-type 344, 345 cinnamide-type 358 close analogs of leucotrienes 344, 345 condensed N-heterocycle-type 344, 345 hydroxyacetophenone-type 344, 345 nor-LT (SK&F 101132) 364 peptidoleucotrienes, LTC4, LTD4 and LTE4 344 requirements for activity 362 in the conjugate triene moiety 358 in the hydrophilic region 359 in the hydrophobic region 361 Ligand-enzyme complexes 55 Loop structure in peptide segments 220 glycin-rich loop 221 Losartan and analogs 239, 260 Lysine derivatives, as inhibitors of serine proteases 89 as substrates of serine proteases 94 Methotrexate, binding with dihydrofolate reductase 13, 54 superimposition on dihydrofolate 34-41 ~-Methoxyacrylates and analogs 241,261 Molecular dynamics 9, 55 Molecular mechanics 9, 23, 101 Molecular orbital calculations, acrylamide analogs 455, 460, 467, 471 AM1 101, 103, 113, 120 CNDO/2 101 Gaussian82 101,102 phthalides 304 Molecular shape analyses, anticonvulsive cinnamides 328 Molecular surface 17, 24, 38 multiple triangular division of 62 solvent accessible area 68 Monte Carlo simulation, of drug-receptor interactions 55 Motif, as a structural unit of peptides 221 Multiple-minima problem 55

489

Mutagenesis, site-directed 93 Nafoxidine 133 Nandrolone 128 1-Naphthoic acids, partially hydrogenated, as antiinflammatory agents 248 as plant growth regulators 248 Neurotoxicity, acrylamide analogs 451,457, 468, 475 Nicorandil and analogs 250 Nicotinic receptor agonists, as insecticides 250, 258 Nozaki-Tanford hydrophobicity scale of amino acids 207 ONO-1078, as a benzamide-type LT-antagonist 345 PAF (platelet-activati ng-factor), antagonists, design of 342 Papain, amino acid sequence alignment with catepsin H 56, 57, 60, 71 Partition coefficients (see Hydrophobic parameter) Penicillin G and analogs 251 Peptides, structu re-hydrophobicity relationship 192, 195, 201, 202, 204 Pheneturide 253 Phenobarbital 253 cz-Phenoxypropionic acids, as antiinflammatory agents 246 as herbicides 246 as hypolipidemic agents 246 N-Phenylcarbamates, as agricultural fungicides 252 as herbicides 251 N-Phenyldicarboximides, as agricultural fungicides 252, 258, 259 as anticancer agents 252, 258, 259 Phenylsemicarbazones of hetroaromatic aldehydes, anticonvulsant effects 309 N-Phenylureas, as herbicides 251 Phenytoin 253 Phosphodiesterase inhibitors 41 Phthalides, as active ingredients of anticonvulsive folk medicine 299 Pinacidil and analogs 250

490

2-Piperazinylbenzimidazoles, as antihistaminic agents 437 Piperine, as active ingredient of Chinese folk medicine 322 Plant growth regulators, structural requirements 247 Plasmin, amino acid sequence of 90 inhibitors of 88, 89 Potassium channel activators, cromakalim analogs 237, 238, 256 nicorandil and analogs 250 pinacidil and analogs 250, 257 Prazosin 373 Protein, homologous proteins, structural similarity 50 modelling of three-dimensional structures 51, 56, 58 prediction of three-dimensional structure of 50 three-dimensional structure of 50 Protein data bank (Brookhaven) 7, 60, 216 Protein engineering 51 Protein sequence database (NBRF) 216, 234 Pyrazines, substituted, structure-hydrophobicity relationships 160, 173, 178, 179, 181 Pyridazines, substituted, structure-hydrophobicity relationships 163 Pyridines, substituted, structure-hydrophobicity relationship 155 Pyrimidines, substituted, structure-hydrophobicity relationships 161-162 Pyrimidinyl(thio)salicylates 244, 263 QSAR, for acute toxicity of acrylamides 466 for adsorbability of testosterones 128 for anticonvulsive activity of cinnamides 326, 328 for anticonvulsive effect of phthalides 300 for antihistaminic activity of 2-piperazinylbenzimidazoles 438, 439, 442, 443 for antiulcerative effect of piperazine-acetamides and-acetates 430, 432, 433, 435 for antiulcerous effect of heteroaromatic aldehyde semicarbazones 311 of benzamide-type LT-antagonists 347, 356 for binding affinity for androgen receptor 138, 141 for binding affinity for estrogen receptor 134, 143 for binding affinity for glucocorticoid receptor 129

491

for binding affinity for mineralocorticoid receptor 130 for binding affinity for progesterone receptor 129, 130 for cz-blocking activity of verapamil analogs 400, 403, 404, 409 for Ca-antagonistic activity of verapamil analogs 374, 377, 384, 390 for cerebral vasodilating activity of piperazine derivatives 417, 420, 423 for complex formation of acrylamides with amino acids 460 for cytotoxicity of acrylamides 471 for glycolytic enzyme inhibitions 463 for hydrophobicity of oligopeptides 192, 195, 201,202, 204 for hydrophobicity of substituted diazines 155-182 for hydrophobicity of substituted pyridines 155 for microsomal metabolism of acrylamides 464 for neurotoxicity of acrylamides 468 for permeability of steroid hormones 127 quinolone-3-carboxylic acid antibacterials 98, 112-113, 115-116, 119 for reactivity of acrylamides with glutathion 460 for uterotropic activity of estratrienols 128 QSAR (ALS), for antiulcerative effect of benzylpiperazineacetamides 430, 432, 433 for CNS-depressive effect of 2-piperazinylbenzimidazoles 446 for duration of cerebral vasodilating effects 423 QSAR (FALS), aquatic toxicity 291 human acute toxicity 288 for neurotoxicity of acrylamide analogs 469-471 vasopressin antagonists 283 QSAR (quantitative structure-activity relationship) 6, 14, 28 Quinolone-3-carboxylic acids, 97-124 ciprofloxacin 99, 103 conformational analysis of 101, 103, 113, 116, 121 difloxacin 97 enoxacin 98 nalidixic acid 98 norfloxacin, NFLX 98, 100, 104, 118, 122 ofloxacin, 99-100, 103 oxolinicacid 101, 116 QSARof 98,112-113, 115-116, 119 receptor model (proposed) 105, 109-111, 113, 118, 122 Rank correlation coefficient, Spearman's 281 RECEPS program 27-44 Receptors, interaction with drugs 4, 7, 16, 19, 21

492

Receptor models (proposed) 39 anticonvulsive cinnamides 337 for antihistaminic 2-piperazinylbenzimidazoles 442 for N,N'-disubstituted piperazine vasodilators 424 leucotrienes and antagonists 364 for neurotoxic acrylamides 477 quinolone-3-carboxylic acid antibacterials 105, 109-111, 113, 118, 122 Retinoic acid 13 Schizandrin C, activation of P450 314 crystallographic structure 316 as an ingredient in Chinese folk medicine 314 Serine proteases, amino acid sequence of 90 complex with inhibitors 83 inhibitors of 89 Similarity search, for amino acid sequence alignment 223 Site-directed mutagenesis, glutathione reductase 218 SMILES 229 Stern-Volmer quenching constant 453, 460 Strobilurin A 241 Structural modifications, of furazolidone 306 Substituent effects, bidirectional, in structure-hydrophobicity relationships 154-158, 164-171, 175-176 Substructure of ligands, definition 229 relationship to amino acid sequence of enzymes 229 Sulfa drugs 245 Sulfonylureas and analogs, as herbicides 242 Superimposition, active conformers of quinolone-3-carboxylic acids 105-109, 118, 120-122 ligand molecules 27, 34 LTE4 and benzamide-type LT-antagonists 348 Sweeteners, artificial, cyanosuosan and analogs 250 superaspartame and analogs 251 SYBYL 101,103 Telocidin 41 Testosterone 128, 130, 131, 145

493

Tetrahydrofolate, see dihydrofolate Thiol proteases, inhibitors of 54, 73 Three-dimensional structure-activity relationships 111 Thrombin, amino acid sequence of 90 inhibitors of 83, 88, 89 Topliss tree, for ring substituents of cinnamides 325 Trypsin, amino acid sequence of 90 complex with inhibitors 84, 86 inhibitors of 89 Uncouplers, weakly acidic, conformational flexibility 342 mechanism of 342 SF6847, a very potent uncoupler 342 van der Waals energy, Lennard-Jones function in program GREEN 20, 21 Verapamil 373 Verapamil analogs, QSAR for 0~-blocking activity 400, 403, 404, 409 QSAR for Ca-antagonistic activity 374, 377, 384, 390 Wolfenden hydrophobicity scale of amino acid side chains 209 X-ray crystallography, data-base (Cambridge) 7, 348 drug-receptor complexes 10, 18 human erythrocyte glutathion reductase-NADPH complex LTE4 348 schizandrin C and analogs 316 trypsin-inhibitor complexes 84, 86-88, 91, 92

218, 224

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