Nucleotide Analogues As Antiviral Agents (Acs Symposium Series)

ACS SYMPOSIUM SERIES401 Nucleotide Analogues as Antiviral Agents John C. Martin,

EDITOR

Bristol-Myers Company

Developed from a symposium sponsored by the Division of Carbohydrate Chemistry and the Division o f Medicinal Chemistry at the 196th National Meeting of the A m e r i c a n C h e m i c a l Society, Los Angeles, California, September 2 5 - 3 0 , 1988

American Chemical Society, Washington, DC 1989

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Library of Congress Cataloging-in-Publication Data Nucleotide analogues as antiviral agents John C. Martin, editor Developed from a symposium sponsored by the Divisions of Carbohydrate Chemistry and Medicinal Chemisty at the 196th National Meeting of the American Chemical Society, Los Angeles p. cm.—(ACS Symposium Series, 0097-6156; 401). Includes bibliographies and index. ISBN 0-8412-1659-2 1. Antiviral agents—Testing—Congresses. 2. Nucleotides—-Derivatives—Therapeutic use—TestingCongresses. I. Martin, John C., 1951- . II. American Chemical Society. Division of Carbohydrate Chemistry. III. American Chemical Society. Division of Medicinal Chemistry. IV. American Chemical Society. Meeting (196th: 1988: Los Angeles, Calif.). V . Series RM411.N83 1989 616.9'25061—dc20

89-15114

CIP

Copyright ©1989 American Chemical Society All Rights Reserved. The appearance of the code at the bottom of the first page of each chapter in this volume indicates the copyright owner's consent that reprographic copies of the chapter may be made for personal or internal use or for the personal or internal use of specific clients. This consent is given on the condition, however, that the copier pay the stated per-copy fee through the Copyright Clearance Center, Inc. 27 Congress Street, Salem, M A 01970, for copying beyond that permitted by Sections 107 or 108 of the U.S. Copyright Law. This consent does not extend to copying or transmission by any means—graphic or electronic—for any other purpose, such as for general distribution, for advertising or promotional purposes, tor creating a new collective work, for resale, or for information storage and retrieval systems. The copying fee for each chapter is indicated in the code at the bottom of the first page of the chapter. The citation of trade names and/or names of manufacturers in this publication is not to be construed as an endorsement or as approval by ACS of the commercial products or services referenced herein; nor should the mere reference herein to any drawing, specification, chemical process, or other data be regarded as a license or as a conveyance of any right or permission to the holder, reader, or any other person or corporation, to manufacture, reproduce, use, or sell any patented invention or copyrighted work that may in any way be related thereto. Registered names, trademarks, etc., used in this publication, even without specific indication thereof, are not to be considered unprotected By law. PRINTED IN T H E U N T I E D STATES O F A M E R I C A

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

ACS Symposium Series M. Joan Comstock, Series Editor 1989 ACS Books Advisory Board Paul S. Anderson Merck Sharp & Dohme Research Laboratories

Mary A. Kaiser Ε. I. du Pont de Nemours and Compan

Alexis T. Bell University of California—Berkeley

Michael R. Ladisch Purdue University

Harvey W. Blanch University of California—Berkeley Malcolm H. Chisholm Indiana University Alan Elzerman Clemson University John W. Finley Nabisco Brands, Inc. Natalie Foster Lehigh University Marye Anne Fox The University of Texas—Austin G. Wayne Ivie U.S. Department of Agriculture, Agricultural Research Service

John L. Massingill Dow Chemical Company Daniel M . Quinn University of Iowa James C . Randall Exxon Chemical Company Elsa Reichmanis A T & T Bell Laboratories C. M . Roland U.S. Naval Research Laboratory Stephen A. Szabo Conoco Inc. Wendy A. Warr Imperial Chemical Industries Robert A. Weiss University of Connecticut

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Foreword The A C S S Y M P O S I U M SERIES was founded in 1974 to provide a medium for publishing symposia quickly in book form. The format of the Series parallels that of the continuing A D V A N C E S IN C H E M I S T R Y S E R I E S except that, in order to save time, the papers are not typeset but are reproduced as they are submitted by the authors in camera-read the supervision of the Editors with the assistance of the Series Advisory Board and are selected to maintain the integrity of the symposia; however, verbatim reproductions of previously pub­ lished papers are not accepted. Both reviews and reports of research are acceptable, because symposia may embrace both types of presentation.

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Preface L A R G E L Y IN RESPONSE T O T H E AIDS EPIDEMIC, the amount of research

directed toward the discovery of antiviral agents has grown dramatically over the last several years. Nucleoside analogues, such as acyclovir and zidovudine, are the most common approved drugs for the treatment of viral infections. In general, these substances have a mechanism of action that involves first phosphorylation in cells to nucleotide analogues and then inhibition of an Analogues of nucleotides (phosphorylated derivatives of nucleosides) are often negatively charged. In the past they were thought to have little potential for efficacy because of the low permeability of cells to charged molecules. Contrary to this expectation, several negatively charged nucleotide analogues have been found during the last five years to exert potent in vivo antiviral effects. This new awareness of the potential therapeutic utility of nucleotide analogues has led a number of laboratories to develop research programs on this topic. The contributors to Nucleotide Analogues as Antiviral Agents are an international group of scientists carrying out research at the forefront of nucleotide drug design. The individual chapters describe many new concepts and results, much of the information unpublished to date. The goal of this book is to provide any chemist, biochemist, or biologist who is interested or involved in antiviral research with a collection of information that will offer insight into this increasingly important research topic. The first chapter provides an overview of the field and also considerable new structure-activity data on analogues of pyrophosphate. Chapters 2-6 describe the chemistry and biological activities of a number of stable acyclic nucleotide analogues that have in vivo activity against herpesviruses and retroviruses. Progress in biochemical and molecular biological research has advanced our understanding of virus replication and resulted in the identification of new targets for inhibition, in addition to the viral D N A polymerases. Chapters 7-9 describe nucleotide analogues that inhibit viral thymidine kinase, glycosylation of viral proteins, and aminoacyl-tRNA synthetase, respectively. Chapter 10 provides, by example, means to design nucleoside analogues to be phosphorylated to nucleotides by host vii

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

enzymes, or in some cases, analogues that are resistant to this phosphorylation. Chapter 11 details the evaluation of nucleotide dimers of nucleosides active against human immunodeficiency virus. The final chapter of this book contains a discussion of the current efforts to design stable oligonucleotides as antisense inhibitors of the transcription or translation of key viral genes. To date, no nucleotide analogues have been approved for use as antiviral drugs. However, a number of the substances described in this book have the potential to be developed as antiviral therapies. As our knowledge of the biological properties (potency, mechanism of action, toxicity, pharmacokinetics, and metabolism) of nucleotide analogues increases, the rational design of superior antiviral agents should become increasingly successful.

Acknowledgments I am very appreciative of the excellent and timely contributions made by authors of chapters in this book. I am also grateful to the many experts in the field of antiviral research who contributed their time to the evaluation of the individual chapters in order to make valuable suggestions for improvements and to Cheryl Shanks of the ACS Books Department. David C. Baker of the Division of Carbohydrate Chemistry and William T. Comer of the Division of Medicinal Chemistry of the American Chemical Society were instrumental in arranging for the financial support of their respective divisions. Also, David C. Baker was especially helpful in his support and advice concerning the organization of the symposium and this book. J O H N C. M A R T I N

Bristol-Myers Company Wallingford, C T 06492-7660 May 18, 1989

viii

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Chapter 1

Inhibitors of Viral Nucleic Acid Polymerases Pyrophosphate

Analogues

Charles E. McKenna , Jeffrey N. Levy , Leslie A Khawli , Vahak Harutunian , Ting-Gao Ye , Milbrey C. Starnes , Ashok Bapat , and Yung-Chi Cheng 1

2

1

1,3

1,4

2

2,5

2,6

Department of Chemistry, University of Southern California, Los Angeles, CA 90089-0744 Department of Pharmacolog of North Carolina-Chapel Hill, Chapel Hill, NC 27514 1

2

Virus-specific enzymes essential for viral nucleic acid replication or related functions are targets for inhibition by substrate or product nucleotide analogues i n which one or more P-O bonds are replaced by a P-C bond. The simplest examples of these are PFA (phosphonoformic acid ) and PAA (phosphonoacetic acid), representing analogues of 'pyrophosphate' moieties i n nucleotides. The synthesis of a series of α-halogenated and α-οxο PAA and MDP (methanediphosphonate) derivatives i s described and structure/activity relationships in their i n h i b i t i o n of several human (α,β,γ) and viral (HSV, EBV, HIV) DNA polymerases are present­ ed. Inhibition of HIV RNA-directed DNA polymerase (reverse transcriptase) by PFA, α-oxophosphonoacetate and α-oxomethanediphosphonate is shown to be pH- and template-dependent. Combination of phosphonoacetate derivatives and a n t i - v i r a l nucleo­ sides into 'hybrid' nucleotide analogues is brief­ ly discussed. Viruses, i n f e c t i n g and reproducing w i t h i n host c e l l s , have long been an e l u s i v e t a r g e t f o r chemotherapy. However, recent advances i n 3

Current address: Department of Pathology, School of Medicine, University of Southern California, Los Angeles, CA 90033 Current address: Wenzhou Institute of Pesticide Research, Huiqiaopu, Wenzhou, Zhejiang, China Current address: Wyeth Laboratories, P.O. Box 8299, Philadelphia, PA 19101-8299 Current address: Department of Pharmacology, School of Medicine, Yale University, New Haven, CT 06510 0097-6156/89/0401-0001$06.00/0 o 1989 American Chemical Society

4

5 6

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

2

NUCLEOTIDE ANALOGUES

molecular v i r o l o g y have increased optimism about the f e a s i b i l i t y of c r e a t i n g r a t i o n a l l y designed, e f f e c t i v e and non-toxic a n t i - v i r a l agents (1). Virus-encoded gene products required f o r DNA r e p l i c a ­ t i o n can s i g n i f i c a n t l y d i f f e r i n substrate s p e c i f i c i t y from normal DNA polymerases involved i n host c e l l reproduction. For example, Herpes simplex v i r u s e s 1 and 2 (HSV-1, HSV-2) induce synthesis of DNA p o l y m e r a s e s h a v i n g Km v a l u e s f o r d e o x y n u c l e o s i d e 5'triphosphate substrates (dNTP's) that are smaller (~10" M) than the Km values of any known mammalian DNA polymerase (2). This confers an advantage to the v i r u s i n competing f o r i n t r a c e l l u l a r s u b s t r a t e s , but a l s o renders i t vulnerable to s e l e c t i v e i n h i b i t i o n . A s i m i l a r r a t i o n a l e underlies ongoing e f f o r t s to develop i n h i b i t o r s f o r other types of v i r u s - s p e c i f i c n u c l e i c acids polymerases, such as the RNA polymerase of influenza viruses and the RNA-dependent DNA polymerase (reverse t r a n s c r i p t a s e ) of Human Immunodeficiency V i r u s (HIV). V i r u s - s p e c i f i c enzymes involved i n r e l a t e d f u n c t i o n s , such as nu­ cleoside kinases and integrases V i r u s - s p e c i f i c DNA condensation of a nucleoside 5'-triphosphate with a growing polynu­ c l e o t i d e strand, e l i m i n a t i n g pyrophosphate (Scheme 1). dNTP sub­ s t r a t e s , and also the pyrophosphate byproduct, are recognized s t a r t ­ ing points f o r i n h i b i t o r design. M o d i f i c a t i o n of mononucleotide may focus on the purine or pyrimidine base, the sugar, the triphosphate, or s e v e r a l m o i e t i e s t o g e t h e r . The r e s u l t i n g analogue c o u l d be intended to i n t e r a c t r e v e r s i b l y or i r r e v e r s i b l y w i t h the targeted v i r a l DNA polymerase. Nucleotides l a c k i n g a 3' h y d r o x y l group can cause chain termination, r e s u l t i n g i n product i n h i b i t i o n . In addi­ t i o n to i n h i b i t o r potency, p r o p e r t i e s important f o r u s e f u l a n t i ­ v i r a l a c t i v i t y include s e l e c t i v i t y r e l a t i v e to s i m i l a r host enzymes, and a b i l i t y to penetrate c e l l membranes. 7

Triphosphate

Base

ι

1 0

0

Pu/Py

Pu/Py

0

" ^ - o J - o J - o - T ι Her

viral

OH

PolyNu—0—[>—0| OH

| ^0^

polymerase HO

H

HO

I

I

H I

Sugar +

0

PolyNu-3'-0H I

0

HCT

DNA Strand

Chain Termination X)H

Pyrophosphate SCHEME 1

Pyrophosphate analogues might also be thought of as fragmentary nucleotides, with only the oligophosphate moiety mimicked. Perhaps the s t r u c t u r a l l y simplest compound discovered to have s i g n i f i c a n t a n t i - v i r a l a c t i v i t y , phosphonoformic a c i d (PFA, 1), belongs to t h i s category. PFA i s b e l i e v e d to i n t e r a c t d i r e c t l y w i t h v i r a l polymer-

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

1.

McKENNA E T AL.

Pyrophosphate Analogues

3

ases, i n t e r f e r i n g with substrate binding and thus b l o c k i n g r e p l i c a t i o n of DNA. Phosphonate analogues of pyrophosphate, which c o n t a i n P-C bonds i n place of P-0 bonds, w i l l be introduced i n more d e t a i l following t h i s background section. More recently discovered nucleoside a n t i - v i r a l agents such as A c y c l o v i r (ACV, 9-(2-hydroxyethoxymethyl)guanine ; a c t i v e against HSV) and AZT (3'-azido-2',3'-dideoxythymidine ; a c t i v e against HIV) r e q u i r e c o n v e r s i o n to n u c l e o s i d e t r i p h o s p h a t e s by v i r a l and/or c e l l u l a r k i n a s e s f o r a c t i v i t y . These drugs are thus l i p o s o l u b l e precursors of corresponding nucleotide analogues and are believed to i n h i b i t the v i r a l polymerases by competing w i t h n a t u r a l dNTP subs t r a t e s f o r a common b i n d i n g s i t e . ACV and AZT triphosphate a l s o exert a n t i - v i r a l a c t i v i t y by f u n c t i o n i n g as v i r a l DNA polymerase mononucleotide substrates, l e a d i n g to t h e i r i n c o r p o r a t i o n i n t o the bound product DNA strand where they act as chain terminators. This dual i n h i b i t i o n mechanism i s o u t l i n e d f o r ACV i n Scheme 2 where ACV-MP and ACV-TP are AC DP represents a Herpes simplex the product DNA strand terminated by ACV-MP, which has no 3' hydroxy l group. The employment of a nucleoside prodrug circumvents two disadvantages inherent i n d i r e c t use of the corresponding nucleoside triphosphate: i t s a n i o n i c charge, which l i m i t s c e l l t r a n s p o r t ; and the s u s c e p t i b i l i t y of i t s triphosphate group to enzymatic hydrolys i s . An a c t i v a t i o n process s o l e l y dependent on host c e l l u l a r enzymes i s indicated f o r AZT (3), but a v i r a l thymidine kinase (TK) has been i m p l i c a t e d i n the i n i t i a l phosphorylation of ACV to ACV-MP O b l i g a t o r y a c t i v a t i o n by another v i r a l enzyme a m p l i f i e s the s e l e c t i v i t y of the i n h i b i t o r (and presents an a d d i t i o n a l a n t i - v i r a l drug target ( 6 ) ) . However, nucleoside analogues phosphorylated s e l e c t i v e l y by v i r a l TK on the path to t h e i r a c t i v e triphosphate forms may have a r e s t r i c t e d spectrum of a c t i v i t y due to virus-dependent v a r i a t i o n i n TK s p e c i f i c i t y (7). ACV-resistant HSV i s o l a t e s o f t e n have a TK" phenotype, whereas mutants with a l t e r a t i o n s i n DNA polymerase appear to a r i s e with lower frequency (8).

D N A - A C V - M P (terminated c h a i n )

HSV ACV

JK

Cellular —

ACV-MP

Kinases

AcV-TP

H S V - D P (inhibition) SCHEME 2

Nucleotide a n t i - v i r a l analogues would be presumed to a f f e c t the target polymerase d i r e c t l y , bypassing a c t i v a t i o n or, i n the case of

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

4

NUCLEOTIDE ANALOGUES

nucleoside monophosphate analogues, r e q u i r i n g only c e l l u l a r kinases f o r f u r t h e r phosphorylation (9). V i r u s - i n f e c t e d c e l l s are o f t e n more permeable to n u c l e o t i d e s than normal c e l l s , which might m i t i ­ gate the p o t e n t i a l problem of l i m i t e d c e l l transport due to i o n i c charge, and n u c l e o t i d e analogues i n which l a b i l e P-0 bonds a r e replaced by P-C or other bonds r e s i s t a n t to enzymatic hydrolysis may be l e s s t o x i c and teratogenic than corresponding nucleosides (10). In support of t h i s idea, a phosphonate analogue o f DHPG (9-[(1,3dihydroxy-2-propoxy)methyl]guanine) monophosphate was found t o e x h i b i t s u b s t a n t i a l a c t i v i t y a g a i n s t Cytomegalovirus (CMV) w i t h s i g n i f i c a n t l y lower t o x i c i t y than corresponding mono- and diphos­ phate DHPG i n h i b i t o r s (11). Active i n t e r e s t i n nucleotides as a n t i ­ v i r a l agents i s r e l a t i v e l y recent, although other uses o f phosphonates as biophosphate analogues have been known f o r some time (12). Oligonucleotide analogues c o n s t i t u t e a f o u r t h category o f nu­ c l e o t i d e a n t i - v i r a l agent, designed e.g. to block v i r a l gene expres­ sion a t the l e v e l o f t r a n s c r i p t i o s p e c i f i c n u c l e i c a c i d segment with complementary v i r a l template ("anti-sense" i n h i b i t i o n ) , and generally have modified 3',5' phosphate linkages to improve s t a b i l i ­ ty to nucleases and enhance transport (13). The v i r a l i n h i b i t o r design s t r a t e g i e s summarized above a r e r e f l e c t e d i n the d i f f e r e n t contributions comprising t h i s volume. Our paper w i l l focus p r i m a r i l y on pyrophosphate analogues as such, and ( b r i e f l y ) as components of 'hybrid' nucleotide analogues. Pyrophosphate

Analogues

The e a r l i e s t pyrophosphate analogue found t o possess a n t i - v i r a l a c t i v i t y was phosphonoacetic a c i d (PAA, 2a) (14) ( f o r convenience, s t r u c t u r a l references f o r the analogues o f t h i s type are given i n f u l l y protonated forms (Scheme 3); the actual i n h i b i t o r s are assumed to be corresponding anionic forms). Both PFA and PAA i n h i b i t r e p l i ­ cation of HSV-1 and HSV-2 and suppress i n i t i a l herpes lesions when applied t o p i c a l l y (14.15). PFA i s more potent than PAA against HSV although IC50 values against HSV-induced DNA polymerases are s i m i l a r f o r the two compounds, suggesting that multiple factors are involved i n o v e r a l l drug effectiveness. Modifications i n the phosphonate/carboxylate groups o f PAA and PFA by e s t e r i f i c a t i o n w i t h simple a l k y l or a r y l groups or by r e ­ placement w i t h other combinations o f a c i d i c f u n c t i o n a l groups have g e n e r a l l y r e s u l t e d i n l e s s a c t i v e compounds (15.) , although excep­ t i o n s have been reported (16.17). P r i o r s t u d i e s o f PAA-like com­ pounds (15-20) provide evidence that one phosphonate group, i n close p r o x i m i t y ( p r e f e r a b l y one o r zero i n t e r v e n i n g atoms) t o a carboxyl a t e , i s a s s o c i a t e d w i t h a c t i v i t y . Replacement o f the carboxylate group i n 2a by a phosphonate group (methanediphosphonic acid/methylene b i s [phosphonic a c i d ] , MDP, 3a) r e s u l t e d i n l o s s o f a c t i v i t y (18) . From the p e r s p e c t i v e o f p r o v i d i n g access t o d e r i v a t i v e s u s e f u l f o r probing s t r u c t u r e - f u n c t i o n r e l a t i o n s h i p s , the methylene group present i n PAA o f f e r s s u b s t i t u t i o n a l l a t i t u d e not a v a i l a b l e i n PFA, b u t α-substitution o f PAA has u s u a l l y decreased a c t i v i t y (15.17-18). Apart from t h i s apparent s t e r i c e f f e c t , and the termi­ nal-group c o r r e l a t i o n s reviewed above, 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 ­ ships f o r pyrophosphate analogues are not w e l l understood.

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

1.

McKENNAETAL.

0 HO.

Pyrophosphate Analogues

0

0

Il

II

HO

;:P—c—OH HO^

X

||

^P HO^

0

0

I

II

H0

— C — C — O H

OH

N

HO'

I

V

Y PFA,

1

PAA,

MDP,

2a

(X.Y =

I

II

ll/OH

HO.

0

0

I II

II

=> — C —

HO

COPAA,

COMDP, 4

SCHEME

3a

(X.Y =

H)

0 HO.

0H

Y

H)

C—OH

5

3

a-Halogenated Pyrophosphate Analogues. Fluorophosphonoacetic and difluorophosphonoacetic acids (2b, 2c) were previously discussed as p o s s i b l e new v i r a l i n h i b i t o r s (21). The p o l a r -CHF- and -CF2groups i n 2b and 2c more c l o s e l y mimic the anhydride oxygen i n P^-OP i than does the -CH2- group of PAA, at a cost of r e l a t i v e l y small s t e r i c perturbation, but the net e f f e c t of such m o d i f i c a t i o n was not c l e a r l y p r e d i c t a b l e . An a n a l y t i c a l advantage of α-fluoromethylene analogues i s the presence of the l ^ F nucleus as a p o t e n t i a l l y useful NMR probe. To e s t a b l i s h s y s t e m a t i c a l l y t h e e f f e c t o f h a l o g e n as u b s t i t u t i o n on the a c t i v i t y of PAA towards p a r t i c u l a r v i r a l poly­ merases, we prepared an i n t e g r a l set of α-halo analogues: XYPAA, X,Y - H,F ( 2 b ) ; F,F ( 2 c ) ; H,Cl ( 2 d ) ; C l , C l ( 2 e ) ; H,Br ( 2 f ) ; Br,Br ( 2 g ) ; F,Cl ( 2 h ) ; F,Br ( 2 1 ) ; Cl,Br ( 2 j ) ; CH ,F ( 2 1 ) ; CH ,C1 (2m); CH3,Br ( 2 n ) . Reported i n h i b i t i o n of an RNA v i r u s polymerase by C1MDP ( 3 d ) , C1 MDP (3e) and Br MDP ( 3 g ) , but not by MDP i t s e l f (22.23) prompted us to include i n our i n h i b i t i o n studies a compara­ ble set of α-halo methanediphosphonates (XYMDP, 3 b - 3 j ) . 3

2

3

2

a-0xo Pyrophosphate Analogues. PAA, i n c o n t r a s t to i t s a b i l i t y to i n h i b i t v i r a l DNA polymerases, was recently found to be i n e f f e c t i v e as an i n h i b i t o r of HIV-1 reverse t r a n s c r i p t a s e , whereas PFA was a potent i n h i b i t o r of t h i s enzyme (24). I t was a l s o shown that aoxomethanediphosphonate (carbonyldiphosphonate, COMDP, 4) i s a moderately good i n h i b i t o r of r e v e r s e t r a n s c r i p t a s e , whereas the corresponding methylene compound 3a i s i n a c t i v e ( 2 4 ) . I n these s t u d i e s a r t i f i c i a l homonucleotide templates were used i n reverse t r a n s c r i p t a s e i n h i b i t i o n assays, and considerable v a r i a t i o n i n the

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

NUCLEOTIDE

6

ANALOGUES

i n h i b i t o r y a c t i v i t y o f i n d i v i d u a l compounds was observed w i t h d i f ­ ferent templates. The existence of a ketone-hydrate equilibrium f o r 4 i n aqueous s o l u t i o n , p o t e n t i a l l y c o m p l i c a t i n g i d e n t i f i c a t i o n o f the actual i n h i b i t o r y species, was not addressed. Commonality o f an cr-carbonyl group i n the reverse t r a n s c r i p ­ tase i n h i b i t o r s 1 and 4 l e d us t o s y n t h e s i z e and determine the reverse transcriptase i n h i b i t i o n a c t i v i t y of the α-keto analogue of PAA, α-oxophosphonoacetic a c i d (phosphonoglyoxalic a c i d , COPAA, 5 ) . The i n f l u e n c e o f assay template on observed i n h i b i t o r a c t i v i t y was examined f o r 1 and 4, and the e f f e c t of pH on reverse transcriptase i n h i b i t i o n by 1, 4 and 5 was also investigated. Synthetic Aspects. α-Halo Phosphonates. The 9 t r i e t h y l e s t e r s (6b-j) were prepared from a s i n g l e p r e c u r s o r , t r i e t h y l p h o s p h o n o a c e t a t e (6a) (21.25 ; McKenna, C E . et a l . , J d i c h l o r o and dibromo ester (NaOCl or NaOH/Br2) » d i f l u o r o e s t e r 6c was obtained w i t h the monofluoro product 6b by treatment of 6a with tBuOK followed by FCIO3. Reduction o f 6e (Na2S03) and 6g (Sn^+) provided the corre­ sponding monochloro and monobromo esters 6d and 6f. o f

6 a

OCIO

O H O

6 e

6 d

r

fi? n

6

0 X 0

O H O

*MU-C-OEt •

j

EtO^

J*

"MUJcUoEt EtO^

O H O 6b EtO^

I

0

H

F

0

EtO^

j

6c

Br 0 Et

6 g

6I

NU_y_oEt

E t o /

I

6h

X = Br;

0 X 0 E

Et0

X - CI;

*NU-C-OEt

6α

0

I

in

O H O

X = H;

6k

MLU_

X = F;

6 1

X = CI;

6m

X = Br;

6 η

Et0/

L

0Et

6 f

SCHEME 4

S i m i l a r hypohalogenation procedures y i e l d e d the mixed d i h a l o esters 6h and 61 from 6b, and 6j from 6d. α-Methyl α-halo e s t e r s 61-6n were synthesized by analogous methods from t r i e t h y l 2-phosphonopropionate 6k.

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

1. M c K E N N A ET AL.

7

Pyrophosphate Analogues

The c o r r e s p o n d i n g a c i d s 2b-2j and 21-2n were p r e p a r e d by r e f l u x i n g the e s t e r s i n cone. HC1 f o r 6 h and were i s o l a t e d as dicyclohexylamine (DCHA) or pyridine (Pyr) s a l t s ( 2 1 ; McKenna, C.E. et a l . . J. Fluorine Chem., i n press; (26). We f i n d that an improved y i e l d o f BrPAA (2f) free from traces o f C1PAA can be obtained by replacing the HC1 by aqueous HBr (25*, r e f l u x , 45 min); a l s o , y i e l d s of d i h a l o acids 2g and 2 j can be optimized by a d j u s t i n g the r e f l u x time i n HC1 (25% recommended) to the minimum necessary (ca. 1/2 h) (Ye, T.-G., unpublished). I n v e s t i g a t i o n o f tetramethyl, t e t r a e t h y l and t e t r a i s o p r o p y l MDP as a common s y n t h e t i c o r i g i n f o r a l l nine XYMDP d e r i v a t i v e s revealed that t e t r a i s o p r o p y l MDP i s p r e f e r a b l e f o r t h i s purpose. Synthetic routes s i m i l a r to those o u t l i n e d above f o r 6b-6j were used to prepare the set of t e t r a i s o p r o p y l esters corresponding to α-halo methanediphosphonic acids 3b-3j, which were then obtained by r e f l u x ­ ing the appropriate ester i n HC1 ( 2 2 ) . α-Keto Phosphonates. Our s y n t h e t i c studies of 5 and i t s t r i e t h y l e s t e r 7 were r e c e n t l y communicated (28). A purported preparation o f e s t e r 7 via Michael i s - A r b u z o v r e a c t i o n between e t h y l o x a l y l c h l o r i d e and t r i e t h y l phosphite (29) i n our hands l e d to other f i n a l products. Attempted a l k a l i n e hydrolysis of CI2PAA (2e) to 5, by analogy to the prepara­ t i o n of carbonyldiphosphonate 4 from 3e (30), l e d to decomposition; methods used to oxidize d i e t h y l malonate to d i e t h y l oxomalonate (31) could not be r e a d i l y extended to 6a. A l t e r n a t i v e oxidative pathways s t a r t i n g from t r i e t h y l phosphonoacrylate using ozonolysis, Ru04/I04~ and RUO4/CIO" were a l s o explored (Levy, J.N.; McKenna, C.E., Phos­ phorus Sulfur, i n p r e s s ) . Our p r e f e r r e d route to 5 e x p l o i t s carbene-mediated oxygen t r a n s f e r chemistry o r i g i n a l l y developed f o r deprotection of alkenes protected as epoxides (32): thermal decom­ p o s i t i o n o f t r i e t h y l diazophosphonoacetate (33.) 8 c a t a l y z e d by rhodium ( I I ) acetate i n the presence o f propylene oxide gives 7 i n good y i e l d . However, we were unable to convert 7 to 5 by d i r e c t hydrolysis with HC1 or HBr, owing to the r e a c t i v i t y o f i t s α-carbonyl group, which a l s o q u a n t i t a t i v e l y adds H2O. S i l y l d e a l k y l a t i o n u s i n g c h l o r o - , bromo- or i o d o t r i m e t h y l s i l a n e a l s o proved u n s a t i s f a c t o r y . We circumvented t h i s problem using a s y n t h e t i c sequence i n which P-OR s i l y l d e a l k y l a t i o n precedes the oxytranfer step, followed by s e l f - c a t a l y z e d a c i d hydrolysis of the remaining (carboxylate) ester group, and i s o l a t i o n o f the ketone 5 as an amine s a l t (Scheme 5 ) . Thus, the f r e e a c i d was prepared by s i l y l d e a l k y l a t i o n o f 8 w i t h bromotrimethyIsilane (34) to e t h y l P , P - b i s ( t r i m e t h y l s i l y l ) diazo­ phosphonoacetate 9 , oxygenation as described above to the oxophosphonoacetate mixed e s t e r 10 and treatment w i t h water a t 25 °C to selectively hydrolyze the t r i m e t h y l s i l y l groups (11 and i t s hy­ drate) . Heating the r e s u l t i n g s o l u t i o n to 56 C f o r 26 h removed the carboxyl e t h y l group, g i v i n g the ketone 5 i n e q u i l i b r i u m w i t h i t s hydrate 12. The phosphonic monoacid o f 5 was recovered as the bis(dicyclohexylammonium) s a l t . Sodium carbonyldiphosphonate 4 was prepared by a s l i g h t modi­ f i c a t i o n of a published method (30). e

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

NUCLEOTIDE ANALOGUES

8

0 N

Eto. H

0

N

TMSO^II

H

0

2

il ll

^;P—c—c—OEt —

H

^P—C—C—OEt —

0

0

0 0 0

TMSO^II

10

9 0

0

-P-U-

0

0

II II II

HO.

OEt

S J _ H _ B - O H

ι—

M M

/Ρ—C—C—OEt TMSO^

Β

HO

0

TMSO^

EtO^

H0

2

HO^

5

11

0 HO. H

H HO^

OH

0

| Il

—C—C—OH

12

| OH

SCHEME 5 Ketone-Hydrate E q u i l i b r i an e q u i l i b r i u m with i t (21; C.E.; Levy, J.N., i n preparation). In the t r i a n i o n , negative charge s t a b i l i z e s the ketone form, which predominates (> 99X) i n H2O. P r o t o n a t i o n s s u c c e s s i v e l y lower the c h a r g e , a c t i v a t i n g the ocarbonyl and thus producing more hydrate. At pH 7-8, the a-carbonyl r e a c t i v i t y i s h i g h e r ( s i g n i f i c a n t f r a c t i o n o f ketone p r e s e n t as d i a n i o n ) , but not s u f f i c i e n t l y to favor the hydrate (present as a few percent at room temperature). Below pH 6, the hydrate becomes the major species. o-Oxomethanediphosphonate 4 d i s p l a y s s i m i l a r behavior but with r e l a t i v e l y greater preference f o r the keto form at a given pH. Biochemical Aspects. P r e p a r a t i o n o f DNA Polymerases. A c t i v a t e d c a l f thymus DNA, DNA polymerases from HSV-1 and HSV-2, EBV ( E p s t e i n - B a r r v i r u s ) , and p e r i p h e r a l b l a s t s from chronic lymphocytic leucophoresed p a t i e n t s undergoing b l a s t c r i s i s were prepared by previously published meth­ ods (35-38) . Generally, DNA polymerases were p u r i f i e d by sequen­ t i a l chromatography on DEAE-cellulose, phosphocellulose, and s i n g l e or double-stranded-DNA c e l l u l o s e . The p u r i f i e d enzymes were d i a lyzed against and stored i n 50 mM Tris-HCl (pH 7.5) containing 1 mM each of DTT, EDTA and PMSF, p l u s 30* g l y c e r o l . P u r i f i c a t i o n of r e v e r s e t r a n s c r i p t a s e from HIV-1 w i l l be d e s c r i b e d elsewhere (Stames, M.C. ; Cheng, Y.-C, J . Biol. Chem. , i n press). Herpesvirus and Human DNA Polymerase Assays. Standard v i r a l HSV and EBV polymerase r e a c t i o n mixtures contained the f o l l o w i n g : 50 mM T r i s - H C l , pH 8.0; 4 mM MgCl ; 0.5 mM d i t h i o t h r e i t o l (DTT); 0.2 mg/ml bovine serum albumin; 0.15 M KC1; 0.25 mg/ml a c t i v a t e d c a l f thymus DNA; 0.1 mM each of dATP, dCTP and dGTP; and 10 μΗ [ H]TTP i n a f i n a l r e a c t i o n volume of 50 μΐ. The r e a c t i o n was s t a r t e d by adding the enzyme to the reaction mixture and allowed to proceed f o r 20 min at 37°C. Samples were spotted onto 2.1 cm GF/A f i l t e r d i s c s and processed to determine t r i c h l o r o a c e t i c a c i d i n s o l u b l e , f i l t e r bound r a d i o a c t i v i t y . When determining the i n h i b i t o r y a c t i o n of PAA analogs, the r e a c t i o n mixture c o n t a i n i n g the appropriate amount of 2

3

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

1.

Pyrophosphate Analogues

McKENNA ET AL.

9

analogue was kept on i c e before i n i t i a t i o n . Assays w i t h human DNA polymerase α were done s i m i l a r l y except that the pH of the reaction mixture was 7.5 and contained no KC1. For β and 7 DNA polymerases the reaction mixture included 100 mM KC1. HIV-1 Reverse Transcriptase Assays. Standard assays were run a t 37°C and contained: 50 mM T r i s , pH 8.0, 0.5 mM DTT, 8 mM MgCl2, 100 μg/mL BSA, 150 μg/mL gapped c a l f thymus DNA, 100 μΜ each dATP, dCTP, dGTP, 10 μΜ [ H]-dTTP, and 1-5 /iL enzyme i n a f i n a l volume of 50 /iL. Modified assays f o r pH dependence i n h i b i t i o n studies w i t h PFA (1) and α-οχο phosphonates (4 and 5) contained 50 mM Hepes, pH 8.2 6.5, 8 mM MgCl2, 100 mM KC1, 100 Mg/ml BSA, 0.5 A26O u n i t s / m l o f p o l y ( r A ) · (dT)io, 100 μΜ [ H]dTTP, and 1-5 μL enzyme i n a f i n a l volume of 50 μL. Samples were processed as described above. 3

3

Herpesvirus DNA Polymerase I n h i b i t i o n Studies α-Halo Phosphonates. Compound i n h i b i t o r s of HSV-1, HSV-2, and EBV DNA polymerases vs. human α, β and 7 DNA polymerases. IC50 values ( i n h i b i t o r concentrations g i v i n g 50% i n h i b i t i o n ) , i n c l u d i n g data f o r PAA and PFA c o n t r o l s , were p r e v i o u s l y r e p o r t e d i n a p r e l i m i n a r y communication (26.) and are presented i n Table I. S i g n i f i c a n t i n h i b i t i o n of h e r p e s v i r a l polymer­ ases was observed w i t h FPAA (IC50 2-4 μΜ (HSV-1, HSV-2); 14 μΜ (EBV)), C1PAA ( I C 3-5 μΜ (HSV-1, HSV-2); 15 μΜ (EBV)), and BrPAA (IC50 5-6 μΜ (HSV-1, HSV-2); 25 μΜ (EBV)). The more active compounds had s l i g h t l y l e s s a b s o l u t e potency than PAA or PFA (IC50 1-2 μΜ (HSV-1, HSV-2, EBV)) but showed better s e l e c t i v i t y r e l a t i v e to human α-DNA polymerase. None of the compounds tested s i g n i f i c a n t l y inhib­ i t e d human β and 7 DNA polymerases. EBV DNA polymerase was somewhat less s e n s i t i v e to 2b, 2d and 2f than the HSV enzymes, i n contrast to the parent compound (PAA) and PFA, f o r which a l l three v i r a l poly­ merases had s i m i l a r IC50 values. The corresponding d i h a l o PAA group 2c, 2e and 2g showed sub­ s t a n t i a l l y less i n h i b i t o r y a c t i v i t y (IC50 > 100 μΜ), the most active member of the group being F2PAA which was 5-10x less e f f e c t i v e than FPAA. Among the three mixed d i h a l o PAA compounds (2h-2j), and the three α-halo, α-methyl PAA compounds (21-2n), only FBrPAA had IC50 values < 100 μΜ f o r the v i r a l enzymes (IC50 37-94 μΜ). As expected, the 12 t r i e t h y l esters of the compounds tested showed no i n h i b i t o r y a c t i v i t y i n the v a r i o u s polymerase s c r e e n s ( d a t a not shown i n Table). I n h i b i t i o n data f o r XYPAA samples t e s t e d at a s i n g l e concen­ t r a t i o n of 100 μΜ d i f f e r e n t i a t e d some o f the compounds having an IC50 > 100 μΜ (26). The r e s u l t s i n d i c a t e that EBV DNA polymerase, but not the HSV-1 and HSV-2 enzymes, had some s e n s i t i v i t y to the amethyl α-halo PAA d e r i v a t i v e s 21-2m (40-50% i n h i b i t i o n ) and t o C1 PAA, Br2PAA and ClBrPAA (30-40% i n h i b i t i o n ) (Table I I ) . None of the corresponding XYMDP s a l t s (3b-3j) had IC50 < 100 μΜ w i t h the three v i r a l enzymes t e s t e d . The compounds CI2MDP, Br2MDP, and C1MDP were reported to show a c t i v i t y against RNA poly­ merase from influenza v i r u s A, while PAA was a less e f f e c t i v e inhib­ i t o r (22.23). 5 0

2

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

10

NUCLEOTIDE ANALOGUES

Table I . IC50 values (μΜ) f o r DNA Polymerase I n h i b i t o r s Viral 5

Compound* 1 PFA, PAA, 2a FPAA, 2b C1PAA, 2d BrPAA, 2f F2PAA, 2c CI2PAA, 2e Br2PAA, 2g FC1PAA, 2h FBrPAA, 2i CIBrPAA, 2j CH3FPAA, 21 CH3CIPAA, 2m CH BrPAA, 2n XYMDP. 3b-31 3

a

HSV-•1 1.1 1. 3 2. 5 3.2 6 18 >100 >100 >100 37 >100 >10 >10 >100 >100

a

Human

HSV- 2 1.1 1.2 3.8 5.,4 5.,4 30 >100 >100 >100 70 >100

EBV 1.2 1.6 14 14.5 25 65 >100 >100 >100 94 >100

α 15 21 >100 80 >100 >100 >100 >100 >100 >100 >100

>300 >300 >300 >300 >300 >300 >300 >300 >300 >300 >300

>100 >100

>100 >100

>100 >100

>300 >300

R e p r o d u c e d with permission from réf. 26. Copyright 1987 Pergamon.

b D C H A salts.

Table I I .

Percent I n h i b i t i o n of DNA Polymerases by α-Halo Phosphonoacetates (100 /iM) a

Viral Compound HSV-1 FPAA, 2b 100 2d 96 C1PAA, BrPAA, 2f 87 F PAA, 2c 62 CI2PAA, 2e 0 Br2PAA, 4.5 2g FBrPAA, 21 71 0 CIBrPAA, 2j 0 CH3FPAA, 21 CH3CIPAA, 2m 0 CH^BrPAA. 2n 0 R e s u l t s f o r compounds t e s t e d as averaged. 2

a

HSV-2 100 96 88 65 0 4 70 0 0 0 0 both DCHA and

Human EBV 74 66 85 77 32 34 43 33 42 44 50 Pyr s a l t s

α 11 45 38 16 0 10 40 16 2 2 9 are

V i r a l I n h i b i t i o n R e s u l t s . Table I I I presents i n v i t r o data (IC50 f o r plaque reduction, and V i r a l Ratings) f o r s e v e r a l XYPAA d e r i v a ­ t i v e s , PAA and PFA as i n h i b i t o r s of HSV-1 (F) and two HSV-2 variants (G and Lovelace). Also included are data f o r an RNA v i r u s (Influen­ za A/Japan). Data are omitted f o r the remaining compounds from Table I , a l l o f which had V i r a l R a t i n g s < 0.4-0.5 f o r a l l v i r u s r e f e r r e d to i n the Table. The HSV r e s u l t s manifest an approximate c o r r e l a t i o n with the HSV DNA polymerase a c t i v i t i e s (Table I ) . FPAA (data f o r two d i f f e r e n t samples) i s seen to be midway between PFA and PAA i n e f f e c t i v e n e s s , although another group found i t t o be somewhat l e s s a c t i v e than PAA i n an HSV-2 plaque r e d u c t i o n assay (12).

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

1. M c K E N N A E T AL.

11

Pyrophosphate Analogues

Enantiomers of drugs may d i f f e r i n t h e i r metabolism, pharma­ c o k i n e t i c s and t o x i c i t y . Although PFA and PAA are a c h i r a l mole­ c u l e s , α-monosubstituted PAA d e r i v a t i v e s , and PAA d e r i v a t i v e s ad i s u b s t i t u t e d w i t h u n l i k e s u b s t i t u e n t s , possess a c h i r a l center. However, a l l our current data are f o r the racemates. I n some i n ­ stances (Tables I , I I I ) , racemic FPAA compares favorably i n potency with PAA. I t would be of i n t e r e s t to determine the r e l a t i v e c o n t r i ­ butions made by the d i f f e r e n t FPAA enantiomers t o , e.g., HSV v s . human α DNA polymerase i n h i b i t i o n , or to the d i f f e r e n t parameters (39) which enter into c a l c u l a t i o n of the V i r a l Rating. Table I I I .

A n t i - V i r a l A c t i v i t i e s of Some α-Halo Phosphonoacetates i n v i t r o VR/IC o ' 5

Compound

b

a

c

Influenz A/Japan

(G) (Lovelace) (F) PAA, 2a 0.8/190 μΜ 0.5/190 μΜ 0.6/190 μΜ PFA, 1 0.6/52 μΜ 0.8/165 μΜ 0.8/165 μΜ 2d C1PAA, 0.4/320 μΜ 2b FPAA, 0.6/130 μΜ 0.5/130 μΜ 0.8/150 μΜ CI2PAA, 2e 0.4/320 μΜ 2f BrPAA, 0.4/280 μΜ 0.6/890 μΜ CIBrPAA. ?1 0.4/790 uM 0.4/790 uM D a t a obtained at Department of A n t i m i c r o b i a l Research, Syntex Research, Mountain View, CA. W R , the V i r a l Rating index f o r the compound, i s defined as: < 0.5, i n s i g n i f i c a n t a c t i v i t y ; 0.5-0.9, marginal-moderate a c t i v i t y . IC50 i s here d e f i n e d as i n h i b i t o r concentration g i v i n g 50% plaque reduction. For d e t a i l s , and i n f o r ­ mation on assay protocols, see r e f . 39. Data f o r 2b are averages f o r separate experiments with two d i f f e r e n t s a l t s : IC50 values are ± 20 μΜ, VR values are ±0.1. a

c

HIV-1 Reverse Transcriptase I n h i b i t i o n Studies. E f f e c t of Template. The template dependence of HIV-1 reverse trans­ c r i p t a s e i n h i b i t i o n ( H-dGTP or H-TTP i n c o r p o r a t i o n ) by PFA and PAA was determined using poly rA and poly rC. The r e s u l t s are given i n Table IV. The IC50 v a l u e s f o r PFA c o n f i r m i t t o be an a c t i v e i n h i b i t o r , but d i f f e r e d by a f a c t o r of f i v e depending on the tem­ p l a t e used i n the assay, p o l y rA producing the lower o f the two values. The same pattern was observed f o r PAA: with poly rA an IC50 of 240 μΜ corresponding to weak i n h i b i t i o n was found, whereas w i t h poly rC the IC50 was a t l e a s t 60% l a r g e r (too large to be measured e x a c t l y under the conditions used). Comparison w i t h the data ob­ tained f o r PFA and PAA i n h i b i t i o n using an a c t i v a t e d DNA template (Table V ) , i n d i c a t e s that the poly rC and ' n a t u r a l ' templates give s i m i l a r IC50 values. 3

3

q-Oxo Phosphonates. I n h i b i t i o n assay r e s u l t s f o r PFA, PAA, 4, 5 and pyrophosphate as i n h i b i t o r s of human α, β and 7 DNA polymerases, and of HIV-1 reverse transcriptase are summarized i n Table V. Activated DNA template was used i n a l l experiments reported i n t h i s Table.

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

NUCLEOTIDE ANALOGUES

12

Pyrophosphate ( P P i ) , the nominal reference s t r u c t u r e f o r the analogues, was not i n h i b i t o r y (IC50 < 400 μΜ) . PFA s e l e c t i v e l y i n h i b i t e d HIV-1 reverse t r a n s c r i p t a s e w i t h an IC50 value (0.6 μΜ), almost 50x lower than i t s IC50 f o r human α DNA polymerase, and a t l e a s t 103 times lower than i t s IC50 values f o r human β and 7 DNA polymerase. The α-οχο phosphonate 4 had an IC50 o f about 20 μΜ under the same conditions. When tested under the same standard assay c o n d i t i o n s , ' 5 ' showed an apparent IC50 about 6x l e s s than that of 4. However, 5 was found to react with one or more assay components. These were i d e n t i f i e d i n P NMR s t u d i e s as T r i s b u f f e r and d i t h i o t h r e i t o l (DTT) (Levy, J.N.; unpublished). Under the same condi­ t i o n s , s i m i l a r reactions of 4 were not detected. 3 1

Table IV.

Template E f f e c t on HIV-1 Reverse Transcriptase I n h i b i t i o n a

D

oolv r C Compound polv r A 0.4 PFA 0.08 >400 PAA 240 Assay conditions as described i n Biochem­ i c a l Aspects s e c t i o n on standard Reverse Transcriptase assays, except f o r template and [MgCl2] which here - 6 mM. 0 . 5 A26O units/mL. D

a

b

Table V. I n h i b i t i o n of HIV-1 Reverse Transcriptase by Pyrophosphate Analogues a

Compound PPi

PFA,

1

PAA,

2a

COMDP, 4

'COPAA, 5 '

Polymerase α β 7 HIV α β 7 HIV α β 7 HIV α β 7 HIV α β 7 HIV

IC^o (uH) > 400 > 400 > 400 > 400 25 + 3 > 400 > 400 0.55 + 0.07 19.6 ± 6 > 400 > 400 > 400 > 400 > 400 > 400 20.1 ± 12 210 ± 33 > 400 > 400 130 ± 50

%

I n h i b i t i o n at 400 ι*Μ N.D. N.D. N.D. N.D. N.D. 15 ± 7 40 ± 3 N.D. N.D. 42 ± 9 36 ± 4 43 ± 5 21.5 ± 9. 3 38.4 ± 5.4 32.5 ± 3. 3 N.D. 71 ± 2 25 ± 10 5.2 ± 3. 7 66 ± 0. 7

a

S t a n d a r d assay c o n d i t i o n s as described i n Biochemical Aspects section.

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

1. M c K E N N A E T A L .

13

Pyrophosphate Analogues

The assay mixture was then modified to replace the T r i s with a demonstrably nonreactive b u f f e r (Hepes), w i t h DTT omitted (Bio­ chemical Aspects s e c t i o n ) . This assay system was used to determine IC50 values f o r 1, 4 and 5 over a pH range of 6.5-8.2 (Table V I ) . In these experiments poly rA was used as template to maximize s e n s i t i v ­ i t y . I n t e r p o l a t i o n o f the data f o r 1 i n Table VI allows comparison of i t s IC50 value i n the modified and standard assay mixtures a t pH 8.0 with i d e n t i c a l template (poly rA). The difference approaches the l i m i t o f e r r o r i n the two experiments, i n d i c a t i n g that the b u f f e r s u b s t i t u t i o n and omission o f DTT d i d not have a d r a s t i c e f f e c t on the observed i n h i b i t i o n at t h i s pH. COMDP 4 i s estimated to be about 20x more a c t i v e under the modified c o n d i t i o n s (Table VI) than when assayed w i t h the standard mixture using an a c t i v a t e d DNA template (Table V). Due to i t s r e a c t i v i t y i n the standard assay, the same comparison cannot be made f o r COPAA 5, but i t i s seen to be moder­ a t e l y active at pH 8.0, a l b e i t considerably less active than 1 or 4 The data i n Tabl pH, with a l l three i n h i b i t o r The v a r i a t i o n i n IC50 ranges from 16-17x f o r the t r i b a s i c 1 and 5, to 200x f o r the t e t r a b a s i c 4. An obvious i n f e r e n c e from these r e s u l t s i s the need f o r caution i n extrapolating reverse t r a n s c r i p ­ tase i n h i b i t i o n assay data (pH 8) to p h y s i o l o g i c a l conditions (pH 7) for i o n i c i n h i b i t o r s whose charge varies with pH. A d e t a i l e d discussion of the ketone/hydrate and anionic acidbase e q u i l i b r i a relevant to 4 and 5 i n aqueous solutions w i l l not be attempted here, but q u a l i t a t i v e l y the p a r a l l e l a c t i v i t y trends f o r 1, 4, and 5 appear inconsistent with the hydrate form of 5 (or of 4) being the most important i n h i b i t o r y species over the pH i n t e r v a l examined. Table VI. pH Dependence of HIV-1 Reverse Transcriptase I n h i b i t i o n by Pyrophosphate Analogues a

IC pH 6.,50 6.,83 7.,26 7.,74 8..20

PFA. 1 2.,15 + 0,.14 0.,616 + 0,.013 0.,303 + 0,.079 0.,207 + 0,.037 0.,130 + 0,.072

5 0

(μΜ)

COPAA. 5 510 + 91 154 + 39 74.5 + 9.9 32.4 + 3.8 32.5 + 4.2

COMDP. + 144 34.0 + 10.3 + 1.97 + 0.719 +

4 35 1.9 0.79 1.2 0.021

a

M o d i f i e d assay conditions as described i n Biochemical Aspects section. Nucleotide Halophosphonate

Analogues

The mechanism o f PFA and PAA r e s i s t a n c e by HSV-1 i s r e l a t e d to i n d u c t i o n o f a l t e r e d HSV-1 DNA polymerases i n i n f e c t e d c e l l s (36)· I t has been suggested that f o r future treatment of herpes-associated d i s e a s e s , agents e x e r t i n g t h e i r a c t i o n s independently should be developed and used i n combination t o circumvent drug r e s i s t a n c e (36) . We have r e c e n t l y prepared examples o f s e v e r a l n u c l e o t i d e analogues c o n t a i n i n g phosphonoacetate moieties (e.g. 13, 14), an

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N U C L E O T I D E ANALOGUES

anti-herpetic nucleoside (ACV as monophosphate) and an a n t i - h e r p e t i c phosphonoacetate (McKenna, C E . ; Harutunian, V., i n press) (Scheme 6). The concept underlying "hybrid" drugs i s that a s y n e r g i s t i c or other enhanced e f f e c t might be o b t a i n e d by combining a P A A - l i k e i n h i b i t o r and a nucleoside drug such as ACV or AZT i n t o a s i n g l e n u c l e o t i d e analogue. Such combinations y i e l d a matrix l a r g e r than the sum o f i t s components, e.g., 3 PAA analogues and 3 nucleosides provide 9 hybrid p a i r s . The examples given here were synthesized i n adequate y i e l d u s i n g a m o d i f i e d d i c y c l o h e x y l c a r b o d i i m i d e (DCC) coupling method. While t h i s work was i n progress, synthesis and a n t i - v i r a l data f o r a series of 2'-deoxyuridine and r e l a t e d pyrimidine n u c l e o s i d e and a c y c l o n u c l e o s i d e e s t e r s o f PFA and PAA were reported (40-41). These d e r i v a t i v e s , r e f e r r e d to as 'combined pro­ drugs', are diphosphate r a t h e r than triphosphate n u c l e o t i d e ana­ logues. E v a l u a t i o n of t h e i r a n t i v i r a l a c t i v i t y w i t h s e v e r a l her­ pesviruses produced no evidence f o r a s y n e r g i s t i c a c t i o n o f t h e i r combined drug moieties.

13c : X « H ; Y = Cl SCHEME 6

Conclusion α-Halogenation creates a s e t o f PAA congeners o f v a r y i n g p o l a r i t y with some s t e r i c p e r t u r b a t i o n . These compounds d i s p l a y a s i g n i f i ­ cant range o f a c t i v i t y as i n h i b i t o r s o f HSV and EBV DNA polymerases. FPAA i s the most a c t i v e i n h i b i t o r i n t h i s group and a l s o has the highest a c t i v i t y against HSV plaque formation i n v i t r o . Correspond­ ing α-halo MDP d e r i v a t i v e s are uniformly i n a c t i v e as HSV and EBV DNA polymerase i n h i b i t o r s . Although PFA and PAA have s i m i l a r IC50 values f o r the herpesvirus DNA polymerases tested, only PFA s i g n i f i ­ cantly i n h i b i t s HIV-1 reverse t r a n s c r i p t a s e . The presence of an ooxo group i n PAA or MDP c o r r e l a t e s with HIV-1 reverse t r a n s c r i p t a s e i n h i b i t i o n a c t i v i t y , the MDP d e r i v a t i v e 4 having the lower IC50. Hydration of the α-keto groups i n 4 and 5 i s pH dependent, increas­ ing at lower pH, whereas HIV-1 reverse t r a n s c r i p t a s e i n h i b i t i o n by 4 and 5 decreases s i g n i f i c a n t l y as the assay pH i s lowered from 8.2 to 6.5. The pH dependence of HIV-1 reverse t r a n s c r i p t a s e i n h i b i t i o n by PFA shows a s i m i l a r trend. This r e s u l t may be s i g n i f i c a n t i n as­ sessing d i f f e r e n c e s between i n h i b i t i o n assays o f i s o l a t e d reverse transcriptase and of v i r a l r e p l i c a t i o n , performed a t d i s s i m i l a r pH. The s p e c i f i c i t y differences observed f o r PFA, PAA, MDP and t h e i r ohalo and α-οχο d e r i v a t i v e s (and f o r nucleotide i n h i b i t o r s generally, see elsewhere i n t h i s volume and, e.g. (42.)) a r e l a r g e l y unex­ p l a i n e d pending d e t a i l e d molecular information on substrate and product b i n d i n g s i t e s of v i r a l polymerases. As progress i n t h i s

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

1. M c K E N N A E T A L .

Pyrophosphate Analogues

15

area begins to be made (43.44), a d e t a i l e d basis f o r t r u l y r a t i o n a l design of a n t i - v i r a l agents i s l i k e l y to emerge. Acknowledgments We thank Drs. Anne Bodner and Robert C.Y. Ting (BioTech Research L a b o r a t o r i e s ) f o r HIV-1 r e v e r s e t r a n s c r i p t a s e and Dr. Thomas Matthews (Syntex Research) f o r supplying the i n v i t r o v i r u s i n h i b i ­ t i o n data presented i n Table I I I . Several α-halo methanediphosphonates were prepared a t USC by J.-P. Bongartz and P. Pham. This r e s e a r c h was supported by NIH grants AI-21871, AI-25697 and CA44358. J . N. Levy was a 1987-89 U n i v e r s i t y of C a l i f o r n i a U n i v e r s i t y wide Taskforce on AIDS Postdoctoral Fellow. Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

Reines, E.D.; Gross 715. Cheng, Y.-C.; Ostrander, M.; Derse, D.; Chen, J.-X. Nucleoside Analogs 1979, 20, 319-335. Cheng,Y.-C.;Dutschman G. E.; Bastow, K. F.; Sarngadharan, M. G.; Ting, R.Y.C. J. Biol. Chem. 1987, 262, 2187-2189. Derse D.; Cheng, Y.-C.; Furman, P.A.; St. Clair, M.H.; Elion, G.B. J. Biol. Chem. 1981, 256, 11447-11451. St. Clair, M.H.; Miller, W.H.; Miller, R.L.; Lambe, C.U.; Furman, P.A. Antimicrob. Agents Chemother. 1984, 25, 191-194. Bone, R.; Cheng, Y.-C.; Wolfenden, R. J . Biol. Chem. 1986, 261, 16410-16413. Wahren, B.; Larsson, Α.; Ruden, U.; Sundqvist, Α.; Solver, E. Antimicrob. Agents Chemother. 1987, 31, 317-320. Christophers, J.; Sutton, R.N. Antimicrob. Agents Chemother. 1987, 2 0 , 389. Lin, J.-C.; DeClercq, E.; Pagano, J.S. Antimicrob. Agents Chemother. 1987, 31, 1431-1433. Robins, R.K. Pharm. Res. 1984, 11-18. Prisbe, E.J.; Martin, J.C.; McGee, D.P.F.; Barker, M.F.; Smee, D.F.; Duke, A.E.; Matthews, T.R.; Verheyden, J.P.H. J. Med. Chem. 1986, 2 9 , 671-675. Blackburn, G.M.; Perree, T.D.; Rashid, Α.; B i s b a l , C.; Lebleu, B. Chemica Scripta 1986, 26, 21-24. Smith, C.C.; Aurelian, L.; Reddy, M.P.; Miller, P.S.; Ts'o, P.O.P. Proc. Nat. Acad. Sci. USA 1986, 8 3 , 2787-2791. Boezi, J.A. Pharmac. Ther. 1979, 4, 231-243. Oberg, B. Pharmac. Ther. 1983, 19, 387-415. Herrin, T.R.; Fairgrieve, J.S.; Bower, R.R.; Shipkowitz, N.L.; Mao, J.C.-H. J. Med. Chem. 1971, 2 0 , 660-663. Noren, J.O.; Helgstrand, E.; Johansson, N.G.; Misiorny, Α.; Stening, G. J. Med. Chem. 1983, 26, 264-270. Eriksson, B.; Larsson, Α.; Helgstrand, E.; Johansson, N.-G.; Oberg, B. Biochim. Biophys. Acta 1980, 607, 53-64. Eriksson, P.; Oberg, B.; Wahren, B. Biochim. Biophys. Acta 1982, 696, 115-123. Mao, J.C.-H.; Otis, E.; Von Esch, A.M.; Herrin, T.R.; Fairgrieve, J.S.; Shipkowitz, N.L.; Duff, R.G. Antimicrob. Agents Chemother. 1985, 27, 197-202.

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16 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44.

McKenna, C.E.; Khawli, L.A. Phosphorus Sulfur 1984, 18, 483. Hutchinson, D.W.; Naylor, M. IRCS Med. Sci. 1985, 13, 1023. Hutchinson, D.W.; Naylor, M.; Semple, G. Chemica Scripta 1986, 26, 91-95. Vrang, L.; Oberg, B. Antimicrob. Agents Chemother. 1986, 29, 867-872. McKenna, C.E.; Khawli, L.A. J. Org. Chem. 1986, 51, 5467-5471. McKenna, C.E.; Khawli, L.A.; Bapat, Α.; Harutunian, V.; Cheng, Y.-C. Biochem. Pharm. 1987, 36, 3103-3106. McKenna, C.E.; Khawli, L.A.; Bongartz, J.P.; Pham, P.; Ahmad, W.Y.; Phosphorus Sulfur 1988, 37, 1-12. McKenna, C.E.; Levy, J.N. J.C.S. Chem. Com. 1989, 246. Kreutzkamp, N.; Mengel, W. Arch. Pharm. 1967, 300, 389-392. Quimby, O.T.; Prentice, J.B.; Nicholson, D.A. J. Org. Chem. 1967, 32, 4111-4114. Dox, A.W. Org. Synth. Coll. 1944, 1, 266-269. Martin, M.G.; Ganem Regitz, M.; Anschutz 101, 3734-3743. McKenna, C.E.; Schmidhauser, J . J. Chem. Soc. Chem. Comm. 1979, 739. B a r i l , Ε.; Mitchner, J.; Lee, L.; B a r i l , Β. Nucleic Acids Res. 1977, 4, 2641-2656. Derse, D.; Barstow, K.F.; Cheng, Y.-C. J. Biol. Chem. 1982, 257, 10251-10260. Tan, R.S.; Datta, A.K.; Cheng, Y.-C. J. Virol. 1982, 44, 893899. Fisher, P.A.; Wang, T.F.-S.; Korn, D. J. Biol. Chem. 1979, 254, 6128-6133. Sidwell, R.W.; Huffman, J.H. App. Microbiol. 1971, 22, 797801. Griengl, H.; Hayden, W.; Penn, G.; De Clercq, E.; Rosenwirth, B. J. Med. Chem. 1988, 31, 1831-1839. Lambert, R.W.; Martin, J.Α.; Thomas, G.J.; Duncan, I.B.; Hall, M.J.; Heimer, E.P. J. Med. Chem. 1989, 32, 367-374. De Clercq, E. Biochem. Pharm. 1988, 37, 1789-1790. Wang, S.-F.; Wong, S.W.; Korn, D. FASEB J. 1989, 3, 14-21. Gibbs, J.S.; Chiou, H.C.; Bastow J.D.; Cheng, Y.-C.; Corn, D.M. Proc. Natl. Acad. Sci. USA 1988, 85, 6672-6676.

RECEIVED June 15, 1989

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Chapter 2

Synthesis of Acyclonucleoside Phosphonates for Evaluation as Antiviral Agents E. J . Reist , P. A. Sturm , R. Y. Pong , M. J . Tanga , and R. W. Sidwell 1

1

1

1

1

2

Life Sciences Division, SRI International, Menlo Park, CA 94025 Utah State University, Logan, UT 84322-0300 2

Isosteric phosphonat and ganciclovir have been prepared and found to have significant activity against human and murine cytomegalovirus in vitro. The mono ethyl esters also are active, possibly serving as prodrugs for the diphosphonic acids. Higher homologs of the isosteris resulted in significant loss of a n t i v i r a l activity. The phosphonates appear to have low toxicity and some of them are promising candidates for c l i n i c a l evaluation.

Chemotherapy o f v i r a l i n f e c t i o n s has lagged s i g n i f i c a n t l y behind chemotherapy o f b a c t e r i a l and p a r a s i t i c i n f e c t i o n s . One reason f o r t h i s i s t h a t many o f the metabolic processes t h a t c o n t r o l the reproduction and growth o f b a c t e r i a and p a r a s i t e s a r e unique t o the invading organisms and thus o f f e r a means o f a t t a c k on the invaders by b l o c k i n g a metabolic t r a n s f o r m a t i o n t h a t has no c l o s e p a r a l l e l i n the metabolism o f the host. V i r a l metabolic processes on the other hand resemble the host processes t o a greater extent and a v i r u s i n f a c t w i l l f r e q u e n t l y u t i l i z e host enzymes t o meet i t s metabolic needs. The net r e s u l t i s that i d e n t i f y i n g a nontoxic a n t i v i r a l agent i s much more c h a l l e n g i n g than i d e n t i f y i n g an e f f e c t i v e nontoxic a n t i b a c t e r i a l o r a n t i p a r a s i t i c agent. To t h i s time, there have been only seven compounds that have been l i c e n s e d by the FDA f o r treatment o f v i r a l diseases ( F i g u r e 1). Nucleosides predominate. This i s probably a r e s u l t o f f a l l o u t from the NCI program o f the l a s t 30 years aimed a t the s y n t h e s i s of a n t i c a n c e r agents. There was a heavy n u c l e o s i d e s y n t h e s i s component t o t h i s program and many o f them were a v a i l a b l e f o r e v a l u a t i o n i n a n t i v i r a l screens. IUDR ( 1 ) , Q ) , T r i f l u o r o t h y m i d i n e (2),(2) Ara A (3),(3) and azidothymidime (AZT)(4) were a l l o r i g i n a l l y s y n t h e s i z e d on the NCI program. R i b a v i r i n (5) i s a 0097-6156/89/0401-0017$06.00/0 ο 1989 A m e r i c a n C h e m i c a l Society

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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NUCLEOTCDE ANALOGUES

Figure 1. A n t i v i r a l agents approved by the F o o d and D r u g Administration.

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

2.

REIST ET

AL.

Synthesis of Acyclonucleoside Phosphonates

19

broad spectrum a n t i v i r a l that i s a c t i v e against a number of RNA and DNA v i r u s e s . ( 5 ) T r i f l u o r o t h y m i d i n e , ( 6 ) IUDR,(6,7) and Ara A(7) have a much narrower spectrum of a c t i v i t y and t h e i r main u t i l i t y i s against the herpes family of v i r u s e s , e s p e c i a l l y herpes simplex v i r u s (HSV). AZT has good a c t i v i t y against human immunodeficiency v i r u s (HIV).(8,9) A l l f i v e of agents have s i g n i f i c a n t t o x i c i t y which l i m i t s t h e i r o v e r a l l u t i l i t y i n the clinic.(6,7) A c y c l o v i r ( 6 ) , the newest member on the l i s t was prepared by Burroughs Wellcome and was found to have outstanding a c t i v i t y against some of the herpes v i r u s e s ( I O ) — n a m e l y herpes simplex 1 and 2 and v a r i c e l l a z o s t e r . To date i t has shown minimal s i d e e f f e c t s at high concentration i n animal s t u d i e s . The only nonnucleoside on the l i s t — a m a n t a d i n e ( 7 ) — i s one of the o l d e s t a n t i v i r a l s and was prepared by DuPont about 25 years ago and was shown to have a c t i v i t y agains its antiviral activity, Parkinson's disease by a f f e c t i n g dopamine metabolism.(V3) This has the unfortunate r e s u l t that amantadine has s i d e e f f e c t s due to CNS involvement—insommia, d i z z i n e s s , mood swings, etc.(14) Thus from t h i s short l i s t of approved compounds, only o n e — a c y c l o v i r — s h o w s good a n t i v i r a l a c t i v i t y with no s i g n i f i c a n t side effects. The mechanism by which a c y c l o v i r expresses i t s a c t i v i t y i s o f i n t e r e s t . (V5,_16) The herpes v i r u s has a number of unique enzyme systems, among them a v i r a l thymidine kinase (TK). The f u n c t i o n of thymidine kinase i s to phosphorylate the deoxynucleoside thymidine (8) to give thymidine monophosphate ( t h y m i d y l i c a c i d ) (9) (Scheme 1). Thymidylic a c i d i s phosphorylated by a thymidylate kinase to give the diphosphate and t h i s i s f u r t h e r phosphorylated by another kinase to y i e l d thymidine triphosphate (10) which i s one of the substrates used by DNA polymerase f o r the s y n t h e s i s of DNA. The nucleoside kinases normally have great substrate s p e c i f i c i t y . Thymidine kinase w i l l only phosphorylate thymidine. Deoxyguanosine kinase w i l l only phosphorylate deoxyguanosine, e t c . In the case of the herpes v i r u s thymidine kinase, the substrate s p e c i f i c i t y i s not so great and i t can accept a c y c l o v i r , a guanine analog, as a substrate to be phosphorylated to the n u c l e o t i d e (11).(7) Subsequently, c e l l u l a r kinases phosphorylate the a c y c l o v i r phosphate (11) to the triphosphate (12). A c y c l o v i r triphosphate then serves as a substrate f o r the v i r a l DNA polymerase but i s a chain terminator s i n c e i t does not have the b i f u n c t i o n a l i t y i n the s i d e chain that i s necessary f o r DNA chain extension (see 13). A c y c l o v i r triphosphate i s not a substrate f o r host DNA polymerase, so i t has no e f f e c t on the host DNA s y n t h e s i s . Thus a c y c l o v i r manifests i t s s e l e c t i v i t y towards HSV i n two ways: (1) I t i s a substrate f o r v i r a l thymidine kinase but not for host thymidine kinase. (2) As the triphosphate, i t i s a substrate f o r v i r a l DNA polymerase but not f o r host DNA polymerase. A change i n the v i r a l TK can r e s u l t i n r e s i s t a n c e of

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N U C L E C m D E ANALOGUES

Scheme 1

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

2. REIST ET AL.

Synthesis ofAcyclonucleoside Phosphonates

21

the herpes v i r u s to a c y c l o v i r . This has been demonstrated i n the laboratory and herpes v i r u s that does not have i t s own thymidine kinase i s indeed r e s i s t a n t to a c y c l o v i r . Fortunately thymidine kinase negative herpes has not developed i n t o a c l i n i c a l problem at t h i s time, although i t i s always a t h r e a t . An analog o f a c y c l o v i r , g a n c i c l o v i r ( 1M) (Figure 2) a l s o has outstanding a c t i v i t y against HSV-1, HSV-2 and v a r i c e l l a zoster.(_18) In a d d i t i o n to these v i r u s e s , g a n c i c l o v i r has i n vivo a c t i v i t y against cytomegalovirus (CMV), a member o f the herpes family that does not have a s p e c i f i c v i r a l thymidine kinase.(19) I t i s probable that g a n c i c l o v i r i s phosphorylated by the v i r a l thymidine kinase of herpes simplex to the triphosphate. However, due to the e x t r a f u n c t i o n a l i t y , i t i s not n e c e s s a r i l y a chain terminator, so i t s a n t i v i r a l e f f e c t must be somewhat d i f f e r e n t from that o f a c y l o v i r . The a c t i v i t y o f g a n c i c l o v i r against CMV i s a l s o not f u l l y understood kinase the mechanism by obvious. I t has been speculated that the CMV i s able to s t i m u l a t e the host thymidine kinase to excessive phosphorylation.(20) P o s s i b l y i n the process, the s e l e c t i v i t y o f the c e l l u l a r thymidine kinase i s somewhat compromised. I t i s i n t e r e s t i n g to note that a c y c l o v i r with i t s monofunctional character must be a DNA chain terminator, has minimal s i d e e f f e c t s and i s e s s e n t i a l l y nontoxic. G a n c i c l o v i r , a c l o s e s t r u c t u r a l r e l a t i v e with i t s b i f u n c t i o n a l character can get incorporated i n t o DNA and has many t o x i c s i d e e f f e c t s — n e u t r o p e n i a , leukopenia, t e s t i c u l a r atrophy, azospermia, atrophy o f the G.I. mucosa, e t c . ( 2 j 0 Although g a n c i c l o v i r i s the only drug to show adequate i n v i v o a c t i v i t y against CMV to date, i t has not y e t been c l e a r e d by the FDA f o r t h i s purpose because o f concern over these s i d e e f f e c t s . The a c y c l o v i r / g a n c i c l o v i r s t o r y o f f e r e d an unusual opportunity f o r analog development to prepare new compounds that maintain the desired a n t i v i r a l a c t i v i t y while a t the same time, the s i d e e f f e c t s observed f o r g a n c i c l o v i r could p o s s i b l y be decreased or e l i m i n a t e d . The preparation o f i s o s t e r i c phosphonates o f ACV and g a n c i c l o v i r was an e s p e c i a l l y a t t r a c t i v e t a r g e t f o r analog development. As can be seen, a c y c l o v i r phosphate (11) and the i s o s t e r i c phosphonate (15) are q u i t e s i m i l a r . Space f i l l i n g models i n d i c a t e that both are o f very s i m i l a r bulk. Since a c y c l o v i r phosphate can be phosphorylated to the triphosphate (12) by c e l l u l a r kinases, i t could be hoped that the i s o s t e r i c phosphonate (15) could a l s o be a substrate f o r these same kinases to give an analogous triphosphate (16) (Scheme 2 ) . Generally speaking, nucleoside phosphates have d i f f i c u l t y c r o s s i n g the c e l l membrane to enter a c e l l because the h i g h l y polar nature o f the phosphate moiety i s not compatible with the l i p o p h i l i c character o f the c e l l membrane. However, i t has been demonstrated(22) that c e l l s that have been i n f e c t e d by v i r u s have a modified c e l l membrane that i s more permeable to a p o l a r molecule, such as a phosphate. In a d d i t i o n , a phosphonate i s somewhat l e s s polar than phosphate, so i t i s p o s s i b l e that a

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

N U C L E O T I D E ANALOGUES

Ο

Ο HNf

3JC.> A

A HN

Ν

2

HN

Ν

2

HOCH

2

CH

HOCH

2

CH

2

2

HC 2

HC

I

DHPG, 2'-NDG BW759U, Biolf 62 CH OH

Acyclovir

Figure Against

2.

Acyclic

the

Nucleosides

Herpes

Family

Active

of

Viruses

ο

HN 2

Ο Ο Ο

Il II II (HO) POCH 2

(HO) POPOPOCH 2

2

I I

2

CH

HC 2

HO CH H C^

2

2

11

CH

2

CH

2

12 Ο

Ο HN^

cellular kinases

Ν

Η Ν'

HN

2

2

Ο Ο Ο

Il II II (HO) PCH CH 2

2

(HO) POPOPCH CH 2

2

2

2

HO CH HC 2

CH

H C 2

2

US

J_5 Scheme 2

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

2. REIST E T AL.

Synthesis ofAcyclonucleoside Phosphonates

23

nucleoside phosphonate such as shown here could have some degree of s e l e c t i v e absorption i n t o an i n f e c t e d c e l l and thus be concentrated i n the c e l l s that need i t . With t h i s as the r a t i o n a l e , a number o f acyclonucleoside phosphonates have been prepared f o r e v a l u a t i o n as a n t i v i r a l s . The syntheses s t a r t e d as o u t l i n e d i n Scheme 3 with the r e a c t i o n o f t r i e t h y l p h o s p h i t e (17) with 1,3-dibromopropane (18) t o give d i e t h y l 3-bromopropyl phosphonate (19) i n 71? y i e l d . Replacement of the bromide by acetate was accomplished using sodium acetate i n DMF. Acid catalyzed h y d r o l y s i s o f the O-acetate (20) using Dowex 50 (H ) gave the 3-hydroxypropyl phosphonate (21) i n 50? o v e r a l l y i e l d from the bromide. Chloromethylation was accomplished using paraformaldehyde and hydrogen c h l o r i d e to give the 3-chloromethoxy propyl phosphonate (22) s u i t a b l e f o r coupling with an appropriate purine. A number o f approache couple the chloromethyl suga convenient from the standpoint o f ease o f handling, s o l u b i l i t y c h a r a c t e r i s t i c s , e t c . u t i l i z e d 2-amino-6-chloropurine (23) as s t a r t i n g m a t e r i a l . Treatment o f t h i s purine with hexamethyld i s i l a z a n e gave a d i - t r i m e t h y l s i l y l d e r i v a t i v e (24) which was condensed with the chloromethyl ether (22) to give a 47? y i e l d o f c r y s t a l l i n e 2-amino-6-chloropurine nucleoside phosphonate as the d i e t h y l e s t e r . Treatment o f the blocked acyclonucleoside with tetraethylammonium hydroxide and trimethylamine h y d r o l i z e d the 6-chloro group and gave a 72? y i e l d o f c r y s t a l l i n e guanine nucleoside phosphonate (28) as the d i e t h y l e s t e r . Heating the d i e t h y l e s t e r (28) with cone, ammonium hydroxide gave a 65? y i e l d of the monoethyl e s t e r (30) that was homogeous on TLC and HPLC with s a t i s f a c t o r y UV spectrum f o r a 9-substituted guanine. An a l t e r n a t i v e synthesis was developed, s t a r t i n g from guanine (26). S i l a t i o n of (26) was accomplished by standard procedures t o y i e l d a very l a b i l e d i s i l y l guanine d e r i v a t i v e (27) that was a l k y l a t e d d i r e c t l y with the chloromethyl ether (22). When 1 mole o f mercuric cyanide was present,a y i e l d o f 35? o f 9-substituted guanine (28) was obtained with small amounts (*2?) o f 7s u b s t i t u t e d isomer (29). I f mercuric cyanide was omitted during the condensation, y i e l d s up to 55? were obtained, however the product obtained contained 10? o f (29) which could be separated by reverse phase chromatography o f the monoesters (30 and 32). +

Complete d e e s t e r i f i c a t i o n o f the phosphonate (30) was r e a d i l y accomplished by treatment o f the mono e t h y l e s t e r with bromot r i m e t h y l s i l a n e to y i e l d the d i a c i d (3D i n good y i e l d a f t e r purification. By a s i m i l a r sequence, s t a r t i n g from 1,7-dibromo heptane (33), a h e p t y l analog (35) was prepared (Scheme 5 ) . The r a t i o n a l e t o prepare such a compound was based on the idea that such a molecule i s very s i m i l a r i n chain length t o the t r i p h o s p h o r y l a t e d s i d e chain o f a c y c l o v i r that i s b e l i e v e d to be the u l t i m a t e a c t i v e a n t i v i r a l , although c e r t a i n l y the h e p t y l phosphonate i s s i g n i f i c a n t l y d i f f e r e n t i n polar character.

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

24

N U C L E O T I D E ANALOGUES

(C H 0) P 2

5

3

•

(C H 0) PCH CH CH Br

BrCH CH CH2Br 2

12

2

2

5

2

2

2

2

11

13.

Ο

II (C H O) PCH CH CH OH

(C H 0) PCH CH CH OCH CI 2

5

2

2

2

2

2

2

12

s

2

2

2

(C H 0) PCH CH CH OAc

2

2

6

2

2

2_Q

2_L

Scheme 3

2_3

IA

Ο

II (EtO) P(CH )30CH 2

U

2

2

I

Scheme 4

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

2

2

2. R E I S T E T AL.

Synthesis of Acyclonucleoside Phosphonates

25

The preparation o f the next higher homolog (39) o f a c y c l o v i r phosphonate was accomplished by the sequence o f r e a c t i o n s i n Scheme 6. Hydroboration o f d i e t h y l 3-butene phosphonate (36) gave the 4-hydroxybutyl phosphonate (37). Chloromethylation followed by r e a c t i o n with s i l a t e d 2-amino-6-chloropurine gave the expected 2-amino-6-chloropurine butyloxymethyl phosphonate (38) as the d i e t h y l e s t e r . S e l e c t i v e h y d r o l y s i s gave the monoester (39). Bromination o f the monoester (30) gave the 8-bromo d e r i v a t i v e (40) i n good y i e l d (Scheme 7 ) . Hydrogenolysis o f the 2-amino-6-chloropurine d i e t h y l e s t e r (25) gave the 2-aminopurine d i e t h y l ester which was then s e l e c t i v e l y h y d r o l i z e d to give the mono ester phosphonate o f 2-aminopurine (42) (Scheme 7). The r a t i o n a l e f o r the preparation o f t h i s compound i s based on the observation that the 2-aminopurine analog of a c y c l o v i r i s an e x c e l l e n f i c a n t l y higher o r a l b i o a v a i l a b i l i t r e a d i l y f u n c t i o n a l i z e d i n v i v o by xanthine oxidase t o produce a c y c l o v i r i n s i t u . However, the 2-aminopurine analog o f a c y c l o v i r i s s i g n i f i c a n t l y more t o x i c than a c y c l o v i r , p o s s i b l y due t o cleavage i n v i v o t o 2-aminopurine which i s i t s e l f q u i t e t o x i c . In the above syntheses, the end products are g e n e r a l l y the monoethyl e s t e r s rather than the d i a c i d s . We have observed that the monoethyl ester appeared to have comparable or even better a c t i v i t y than the d i a c i d and seemed to have superior s o l u b i l i t y c h a r a c t e r i s t i c s . A monoester would a l s o be l e s s p o l a r than the d i a c i d hence could p o s s i b l y penetrate the c e l l membrane more e a s i l y and could conceivably be h y d r o l i z e d to the d i a c i d by esterases w i t h i n the c e l l . Thus i t seemed reasonable t o evaluate the e f f e c t o f higher e s t e r s and the mono b u t y l (50) e s t e r was prepared by an i d e n t i c a l r e a c t i o n sequence as described f o r the preparation o f the e t h y l ester (Scheme 8 ) . Phosphonate d e r i v a t i v e s o f g a n c i c l o v i r were prepared by a sequence of r e a c t i o n s s t a r t i n g from d i e t h y l methylphosphonate (51) (Scheme 9). Preparation o f the l i t h i u m s a l t , using b u t y l lithium/cuprous i o d i d e , followed by r e a c t i o n with a l l y l bromide gave an 80? y i e l d of d i e t h y l 3-butenyl phosphonate (36). Epoxidation o f the o l e f i n with meta chloroperbenzoic a c i d gave a 75? y i e l d o f the epoxide (52). Acid catalyzed cleavage o f the epoxide with g l a c i a l a c e t i c a c i d gave a 50? y i e l d o f d i e t h y l 4-acetoxy-3-hydroxy butylphosphonate (53). The M/S cracking pattern showed the presence o f CH 0Ac, i n d i c a t i n g that the desired isomer was formed. 2

Chloromethylation gave the desired chloromethyl ether (54) which was condensed with the di(TMS) d e r i v a t i v e o f 2-amino-6chloropurine t o give the blocked nucleoside. Treatment with aqueous 1N NaOH gave the monoethyl phosphonate (55) i n 21? o v e r a l l y i e l d . The mono ester was a l s o completely deblocked by treatment with bromotrimethy1 s i l a n e followed by water t o give the phosphonic d i a c i d (56). C y c l i z a t i o n o f the d i a c i d using DCC i n

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

26

N U C L E O T I D E ANALOGUES

Br(CH ) Br

(EtO) P(CH ) OH

13

1A

2

2

7

2

7

HN

Ν

2

EtOP(CH )sCH 2

I

2

I I ^ O

HO

v

*CH

Scheme 5

(EtO) PCH CH CH=CH 2

2

2

(EtO) PCH CH CH CH OH

2

2

2

2

11

2

2

1Z CI TMS Ν

Λ

TMSNH

Ν

14

HN

Ν

2

Ο EtO-P-CH CH CH 2

CH

2

HC

II (EtO) PCH CH CH

2

2

CH

2

2

2

I

2

2

11

CH

HC 2

Scheme 6

11

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

2

2

2. REIST E T AL.

Synthesis of Acyclonucleoside Phosphonates

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

28

N U C L E C m D E ANALOGUES

(BuO) P 3

4-2

•

Br(CH2) Br

Ο

Ο

Il

II

(BuO)2PCH CH2CH Br

3

2

(BuO)2PCH CH2CH OAc

2

2

2

±

LA

Ο

Ο

Il

II

(BuO) PCH CH CH OCH CI 2

2

2

2

(BuO) PCH CH CH OH

2

2

4-8

2

2

AI

Cl

HN 2

Ο

Ο

II

BuOPCH CH 2

(BuO) PCH CH 2

2

2

HO H C ^ 2

HC 2

2

CH

*CH

2

51

A3. Scheme 8

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

2

2

Synthesis of Acyclonucleoside Phosphonates

2. R E I S T E T A L .

ο

ο

Il

II

(EtO) PCH 2

ρ

II

(EtO)2PCH CH CH=CH2

3

29

2

_

(EtO) PCH CH CH

2

2

2

CH

2

2

Ο

11

1&

11

II

(EtO) P(CH ) CHCH OAc 2

H

2

N ^ N ^

2

2

(EtO) P(CH ) CHCH OAc

2

2

I

N

2

2

OCH CI

CH

2

Ο EtOPCH CH 2

ι HO

11

1A 2

ι I / O

v

"CH

HC

2

I O^OH

Ο ΗΝ

Λ

HN 2

HN

Ν

2

Ο

II

CH ÇH 2

(HO) PCH CH 2

2

2

CH

2

Ρ HC

*CH

»'\

2

CH

2

I Ο — CH

2

CH OH 2

11

il

2

I

Scheme 9

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

30

N U C L E O T I D E ANALOGUES

p y r i d i n e gave the c y c l i c phostonate (57) i n reasonable y i e l d . The HPLC behavior of the monoester was i n t e r e s t i n g . I t c o n s i s t a n t l y showed a double peak that was not adequately resolved f o r separation. The u l t r a v i o l e t spectrum and a n a l y s i s were as expected f o r the assigned product. On complete d e e s t e r i f i c a t i o n , t h i s behavior disappeared and the m a t e r i a l was homogeneous i n HPLC, UV and a n a l y s i s were as expected. On preparation of the phostonate, two peaks again appeared, again with s a t i s f a c t o r y UV and elemental a n a l y s i s . We assume t h i s i s due to 2 isomeric p a i r s due to 2 asymétrie centers. The branched methyl analog o f g a n c i c l o v i r phosphonate (60) (Scheme 10) was prepared f o r two reasons: (1) The analogous d e r i v a t i v e o f g a n c i c l o v i r showed a n t i v i r a l a c t i v i t y comparable to g a n c i c l o v i r f o r r a t e of phosphorylation by HSV-1 thymidine kinase(24) thus showing that the methyl group was compatible with the HSV enzyme system. (2) I f i t showe i n t e r e s t i n g monofunctiona propagate the DNA chain. Opening the epoxide (52) using borohydride gave the expected secondary a l c o h o l (58). Chloromethylation followed by coupling w i t h 2-amino-6-chloropurine gave a f t e r deblocking the deoxygancic l o v i r phosphonic a c i d , mono e t h y l e s t e r (60). Some of the b i o l o g i c a l a c t i v i t y that we have obtained i s shown i r 1. A c y c l o v i r phosphonate (3D and i t s monoethyl e s t e r (32) both show moderate a c t i v i t y against HSV-1 although n e i t h e r as a c t i v e as a c y c l o v i r or g a n c i c l o v i r . S u r p r i s i n g l y , (3D was i n a c t i v e against murine cytomegalovirus a t doses up to 1000 yg/rr although the mono ester showed good a c t i v i t y and a very good therapeutic index.

Table

The h e p t y l analog (35) showed low a c t i v i t y against both HSV-1 c human cytomegalovirus. Likewise the d i e t h y l e s t e r (28) and 8bromo analog (40) were e s s e n t i a l l y i n a c t i v e . The r e s u l t s o b t a i n s f o r the monobutyl ester (50) were somewhat s u r p r i s i n g . There was no a c t i v i t y against HSV-1. Although there was a c t i v i t y against HCMV, i t was s i g n i f i c a n t l y lower than that observed f o r the mono e t h y l e s t e r (32). The a c t i v i t y of the g a n c i c l o v i r analogs prepared so f a r i s c o n s i s t e n t l y low a g a i n s t HSV-1. However some o f them show good a c t i v i t y against human cytomegalovirus. The d i a c i d (56) shows a t h e r a p e u t i c index o f 500 with an E D of 2 yg/mL, comparable to g a n c i c l o v i r i n a c t i v i t y . The monoethyl e s t e r (55) i s n e a r l y as e f f e c t i v e with a t h e r a p e u t i c index of 258 and an E D o f 5.8 yg/mL. The c y c l i c phostonate (57) i s l e s s a c t i v e w i t h a t h e r a p e u t i c index of 64 and an E D of 75 yg/mL, while the deoxy analog (60) has only low a c t i v i t y w i t h a t h e r a p e u t i c index o f 5 and an E D of 400 yg/mL. 50

50

50

50

We have done some s t u d i e s on the t o x i c i t y of the monoethyl e s t e r (30) o f ACV phosphonate i n the mouse. An acute dose of 2000 mg/kg i n the t a i l v e i n o f the mouse gave no s i g n i f i c a n t adverse r e a c t i o n . On chronic dosage once d a i l y over 5 days a t 2000 mg/kg

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

2.

REIST E T AL.

(EtO) PCH CH CH — C H 2

2

12

2

31

Synthesis of Acyclonucleoside Phosphonates

2

-

(EtO) PCH CH CHCH 2

2

2

3

(EtO) PCH CH CHCH 2

12

12

12 Scheme

2

10

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

2

3

NUCLECOTDE ANALOGUES

32

Table 1.

A n t i v i r a l A c t i v i t y o f A c y c l i c Phosphonates Against Herpes Viruses

ο χ

JL^"

î

x

I

ROPtCHi^CHOCHt

I

I

ED ue/ml T . I . 50

Cpd

X

11

Η

R

R"

R* H

H

Η

η

V.R. »

2

0.6

100

2

EDso UK/ml

T.I.

110

10

C H 2

5

H

H

2

19

Η

C H 2

5

H

H

3

35

Η

C H

5

H

H

6

0.1

>1000-

28

Η

C H 2

5

C H

H

2

0.1

1000 ·

40

Br

C H

H

H

2

0

<500

>2

5

2

0

100

15

2

0.3

1

5.8

258 5

50

Η

55

Η

2

C H 2

5

2

5

H

H

H

CH 0H 2

1000*

1000* 1000

u

CH

3

2

0.1

320

0.3

H

CH 0H

2

0.3

320

3

2

500

2

0.1

320

0.1

5

64

Acyclovir

1.8

4

375

25

60

Ganciclovir

1.8

2

500

2

500

6

20

Η

C H

Η

H

57

Η

H

2

5

2

CH

2

DHPG C y c l i c Phosphate

10-20

300

1.3

700

1.6

75

1

H

60

10

N. A. 3

400

56

2

N.A.3

>320

4

1.6

94

16

Η

2

EDso yg/ml T . I .

0

>10

10

0.9

V.R.»

2

5

V. R. i s virus rating and represents a measure of a n t i v i r a l a c t i v i t y . ( 2 5 ) A r a t i n g between 0 and 0.5 i s low a c t i v i t y . A r a t i n g between 0.5 and 1.0 i s moderate a c t i v i t y . A rating greater than 1.0 i s strong a c t i v i t y . T . I . i s therapeutic index and i s the r a t i o o f cytotoxic dose ( C D ) to minimum e f f e c t i v e dose ( E D ) . N.A. i s not a c t i v e . ••Top dose tested i s 1000 yg/mL. Data from ref. 26. 1

2

50

5 0

3

5

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

5

5

2.

REIST ET AL.

33

Synthesis ofAcyclonucleoside Phosphonates

and a l s o as 1000 mg/kg, there was 1 death each i n 5 mice. mg/kg there were no t o x i c e f f e c t s .

At 500

The d i a c i d o f g a n c i c l o v i r phosphonate (56) has a l s o been prepared by P r i s b e , e t a l . ( 2 6 ) They a l s o observed that the herpes a c t i v i t y was minimal but that there was good a c t i v i t y a g a i n s t HCMV i n v i t r o . 56 a l s o showed e x c e l l e n t a c t i v i t y when a p p l i e d subcutaneously against MCMV. They reported low t o x i c i t y f o r the compound and b e l i e v e i t i s a good candidate f o r c l i n i c a l e v a l u a t i o n against HCMV. Acknowledgment This work was supported i n part by c o n t r a c t N01-AI-72643 from the N a t i o n a l I n s t i t u t e o f A l l e r g y and I n f e c t i o u s Diseases. References 1. 2.

3.

4. 5. 6. 7. 8. 9. 10. 11.

12. 13. 14.

15. 16. 17.

Prusoff, W. H. Biochim. Biphys. Acta. 1959, 32, 295. a) Heidelberger, C.; Parsons D.; Remy, D. C. J. Am. Chem. Soc. 1962, 84, 3597. b) Ryan, K. J.; Acton, Ε. M.; Goodman, L. J. Org. Chem. 1966, 31, 1181. a) Lee, W. W.; Benitez, Α.; Goodman, L.; Baker, B. R. J. Am. Chem. Soc. 1960, 82, 2648. b) Reist, E. J.; Benitez, Α.; Goodman, L.; Baker, B. R.; Lee, W. W. J. Org. Chem. 1962, 27, 3274. Horowitz, J. P.; Chua, J.; Noel, M. J. Org. Chem. 1964, 29, 2076. Sidwell, R. W.; Huffman, J. H.; Khare, G. P.; Allen, L. B.; Witkowski, J. T.; Robins, R. K. Science 1972, 177, 705. Nicholson, K. G. Lancet II 1984, 617. Nicholson, K. G. Lancet II 1984, 503. Mitsuya, H., et a l . Proc. Nat. Acad. Sci. USA 1985, 82, 7096. Fischl, Μ. Α., et al. New Eng. J. Med. 1987, 317(4), 185. Collins, P.; Bauer, D. J. J. Antimicrob. Chemoth. 1979, 5, 431. a) Galbraith, A. W.; Oxford, J. S.; Schild, G. C.; Watson, G. I. Lancet 1969, 1026. b) Bull. WHO 1969, 41, 677. Nicholson, K. G. Lancet II 1984, 562. Allen, R. M., et a l . Clin. Neuropharmacol. 1983, 6, (Suppl. 1), S64. a) Bryson, Y. J.; Monahan, C.; Pollack, M.; Shields, W. D. J. Infec. Dis. 1980, 141, 543. b) Flaherty, J. Α.; Bellur, S. N. J. Clin. Psychiat. 1981, 42, 344. Elion, G. B.; Furman, P. A.; Fyfe, J. Α.; de Miranda, P.; Beauchamp, L.; Schaeffer, H. J. Proc. Nat. Acad. Sci USA 1977, 74, 5716. Furman, P. Α.; de Miranda, P.; St. Clair, M. H.; Elion, G. B. Antimicrob. Agts. Chemother. 1981, 20, 518. Elion, G. B. Amer. J. Med. 1982, 73, 7.

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

34 18.

19. 20. 21. 22. 23.

24. 25. 26.

RECEIVED

NUCLE(mDE

ANALOGUES

a) Smith, K. O.; Galloway, K. S.; Kendall, W. L.; Ogilvie, K. K.; Radatus, Β. K. Antimicrob. Agts. Chemother. 1982, 22, 55. b) Martin, J. C.; Dvorak, C. Α.; Smee, D. F.; Matthews, T. R.; Verheyden, J. P. H. J. Med. Chem. 1983, 26, 759. Mar, E.-C.; Cheng, Y.-C.; Huang, E.-S. Antimicrob. Agts. Chemother. 1983, 24, 518. Estes, J. E.; and Herang, E.-S. J. Virol. 1977, 24, 3. Koretz, S. H., et a l . New Eng. J. Med. 1986, 314, 801. Carrasco, L. Nature 1978, 272, 694. Krenitsky, T. Α.; Hall, W. W.; deMiranda, P.; Beauchamp, L. M.; Schaeffer, H. J.; and Whiteman, P. D. Proc. Nat. Acad. Sci, USA 1984, 81, 3209. Fyfe, J. Α.; McKee, S. Α.; and Keller, P. M. Mol. Pharmacol. 1983, 24, 316. Sidwell, R. W. Vira Systems; In "Chemotherap Gadebusch, ed.) pp. 31-54, CRC Press, Cleveland. Prisbe, E. J.; Martin, J. C.; McGee, D. P. C.; Barker, M. F.; Smee, D. F.; Duke, A. E.; Matthews, T. R.; Verheyden, J. P. H. J. Med. Chem. 1986, 29, 671. February 22, 1989

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Chapter 3

Structural Requirements for Enzymatic Activation of Acyclonucleotide Analogues Relationship to Their Mode of Antiherpetic

Action

Richard L. Tolman Merck Sharp & Dohme Research Laboratories, Box 2000, Rahway, NJ 07065 The structure-substrat thymidine kinase (TK enzymes ( G M P kinase) have been studiea and relevant acyclonucleoside conformations amenable to phosphorylation have been proposed based o n molecular modelling. The modes of action of other acyclonucleoside antiviral agents, which are not viral DNA polymerase inhibitors, and therefore TK-independent, e.g. 2'nor-cGMP, have also been discussed. The most safe and effective agents of the present generation of antivirals for members of the group of herpes viruses are guanine acyclonucleosides. These agents have been shownQL,2) to ultimately inhibit the viral replication process as their triphosphate derivatives.

Ganciclovir

Thymidine

It is a viral-specified enzyme, thymidine kinase (TK), which accomplishes the first phosphorylation® of guanine acyclonucleosides, such as acyclovir (ACV), to monophosphate tnereby accounting for the great selectivity of their antiviral action. Our initial interest i n this area was i n understanding the manner of this remarkable enzymatic event i n accepting a guanine derivative as a surrogate for the natural substrate thymidine. The structuresubstrate relationships for acyclonucleoside derivatives were examined for herpes (HSV-1) thymidine kinase as well as for the host enzyme G M P kinase, which converts monophosphate to diphosphate(4). There are many 0097-6156/89/0401-0035$06.00/0 © 1989 A m e r i c a n Chemical Society

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

NUCLEOTIDE

36

ANALOGUES

enzymes® which convert acyclonucleoside diphosphate to triphosphate, the final agent which inhibits viral D N A polymerase and thereby viral replication (Fig. 1). Molecular modelling was found to be a source of stimulation toward the synthesis of new chemical entities and to provide plausible scenarios for enzymatic phosphorylation. Molecular modelling studies are arguably less relevant in their application to substrate studies as there are two components in substrate activity, binding and efficacy. It is the binding component which is more easily dealt with in the absence of an X-ray structure ot the enzyme. H S V Thymidine Kinase Keller et Λ/.(6) have examined in a cursory fashion the substrate requirements of herpes thymidine kinase (HSV-TK) particularly with regard to guanine derivatives. The structural requirements of pyrimidine nucleoside analogs have been examined(7-10) for substrate activity. It has been demonstrated that 5-substitution of the pyrimidin Thymidine is a much bette 5-substituent. The ability of other bulkier and more hydrophobic 5substituents to function even more effectively than thymidine as substrates has led to the thesis that the binding site of T K which accommodates the 5methyl of thymidine is in fact a long cylindrical hydrophobic pocket©. 2'Deoxy-cytidme is also a quite a good substrate(one-twentieth as effectively phosphorylated as thymidine, Π) despite the fact that it has hydrogen at C5. The phosphorylation of ganciclovir (GCV) by H S V - T K has been shown to be stereospecific for the pro-S hydroxyl(12) as evidenced by the fact that enzymaticallv-prepared G C V - M P is rapidly converted by G M P kinase to G C V - D P , wnereas the chemically-prepared M P (racemic) is only 50% converted to D P under the same conditions. Molecular Models. A statement of Garland Marshall's(13) describes the promise of molecular modelling for studies of the type of the thymidine kinase structure-substrate study: "Without detailed information about the three-dimensional nature of the receptor, conventional physicochemically based approaches are not possible. One can only attempt to deduce an operational model of the receptor that gives a consistent explanation of the known data and, ideally, provides predictive value when considering new compounds for synthesis and biological testing." U s i n g a modified M M 2 force field(14), tnree dimensional energy maps were generated for thymidine and ganciclovir (Figs 2 & 3) using two sidechain dihedral angles(15). The conformationally-constrained thymidine was seen to have a number of low energy wells corresponding to anti-conformers with gauche- or gauche+ 5'-hydroxyls, whereas permutations of G C V ' s side chain position produced little variation i n energy (total energy range = 10 kcal). The C 8 - N 9 - C l ' - 0 torsion angles from the X-ray structures of A C V Q 6 18) and GCV(19,15) (-91 and -110° respectively) fell i n a low energy portion of the contour plot, but were not the lowest energy structures i n that energy well. The key to understanding how H S V - T K could accept a guanine acyclonucleoside as a substrate seemed to be i n the positioning of the purine on the active site of the enzyme. The effect of some purine substituent changes upon T K substrate ability have been examined by others(20,6). Table I shows the relative substrate ability of various substituted guanine derivatives within any of three side-chain series. A n y change of the 6-oxo function abolished substrate activity [the substrate activity of 2'β

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Structural Requirements for Enzymatic Activation

3. T O L M A N

Ο

Ganciclovir (GCV)

OH

OH HSV Thymidine Kinase

GMP kinase (host)

/

HN

II

Ν

Μ

[Ι

cr

o-

2

OH

host enzymes Ν

HN 2

}

if

ο

?

Î

o'NHS^Nr o- o- oOH

Figure 1. Viral Activation of Acyclonucleosides

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

37

38

N U C L E O T I D E ANALOGUES

Thymidine

Energy -30.00

-0

90

180

270

Figure 2. Energy Contour Plot for Thymidine (X-ray Structure) for Two Torsions, θΐ (C8-N9-Cl'-0) and Θ2 (0-C4'-C5'-05')

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

360

3.

TOLMAN

39

Structural Requirements for Enzymatic Activation

Ganciclovir

Energy ι

-0

90

180 θ

270

36

2

Figure 3. Energy Contour Plot for Gandclovir (X-ray Structure) for T w o Torsions, θΐ (C8-N9-Cl'-0) and Θ2 ( Ο Ζ ^ ' ^ ' - Ο δ ' )

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

NUCLEOTIDE ANALOGUES

40

deoxycytidine might have predicted the 6-amino derivative to be a substrate]. Similarly, changes were not tolerated at the purine-2-position of guanine, except for the 2-methylamino(6) which retained weak substrate activity. However, hydrophobic substituents at the 8-position (particularly halogen or alkyl) enhanced substrate activity, while hydrophilic substituents were not substrates. A likely explanation for the substrate activity of 8-substituteduanines is that the hydrophobic 8-bromo or -methyl fits into the postulated ydrophobic pocket which exists i n our model to account for the enhanced substrate activity of 5-substituted thymidines. Table I. Structure-Substrate Relationship for Modified Guanines

R = C H 2 O C H 2 C H 2 O H (+) R = C H 2 0 C H ( C H 2 0 H ) 2 (+++) R = C H 2 C H 2 C H 2 C H 2 O H (++)

N O T E :

A = OH(-), H(-), NHMe(+) NHCH2Ph(-), N H N H 2 ( - ) , Cl(-) Β = H , SH, NH2, N H M e , OMe, N H n P r , N H C H 2 P h , all(-) C = OH(-),NH2(-),SH(-) 8-aza(+), Br(++), Me(+++)

- , not a substrate; +, poor substrate; + + ,

good substrate; and + + + ,

excellent

substrate. Assay protocol, ref. 21.

In order to test the hypothesis by superposition of molecular models, randomly generated ganciclovir conformational structures were optimized and grouped into families when atoms differed i n position by <0.l Â. The lowest energy family (A141, -42 kcal) when rigidly superimposed upon the X-ray structure of thymidine produced 'scenario A ' (Fig. 4), which could account for many of the observed substrate requirements. Five probable points of interaction between the substrate (thymidine) and enzyme were postulated based on this scenario: (a) the hydrophobic pocket at C5, (b) the 3'- and 5'-hydroxyls, (c) the furanose ring oxygen, and (d) the 4-oxo group. In each case the low energy ganciclovir conformer possessed a binding counterpart. Distances between corresponding binding functions of the two molecules i n the superposition were generally small (<1.0 Â). The largest displacements of these binding counterparts were (a) the pyrimidine-40x0//the guanine-6-oxo (this disparity could perhaps be accommodated if the interacting group on the enzyme were the terminal carboxyl of A s p or Glu), and (b) the 3'-hydroxyl//pro-R hydroxyl (rotation of the G C V pro-R hydroxymethyl 60° produced a virtual overlap with the thymidine counterpart). In this binding scenario, the two heteroaromatic systems are nearly coplanar. M o v i n g the purine out of this plane does not markedly effect any of the hypothetical oinding interactions except the positioning of the purine C8 substituent into the thymidine C 5 hydrophobic pocket.

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

3.

TOLMAN

Structural Requirements for Enzymatic Activation

41

Figure 4. Binding Scenario A : Rigid Superposition of a Low-energy Ganciclovir Conformation (dotted) and Thymidine X-ray Structure (solid)Front and Top Stereo-Views

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

NUCLEOTIDE ANALOGUES

42

Taking note that the Ν2-3Ό distance is 4.5 Â i n the superposition suggests that, if that space were a cavitv i n the enzyme, an N2-substituent bearing a hydrophilic terminus and reacning into the postulated 3'-hydroxyl binding site mignt pick up substrate activity. The extended length of the 2-hydroxyethyl group is close to 4.5 Â whereas the 2,3-dihydroxypropyl derivative, which was also prepared, is somewhat longer and may not have the proper geometry for good binding. To check that the N2-hydroxyalkyl was not being phosphorylated by HS VTK, the two methylated side-chains were prepared. Table II shows that N 2 (2-methoxy)ethyl was still a substrate while the 9-(2-methoxy)ethoxymethylderivative was not a substrate, lending additional credence to 'binding scenario A ' as a predictive model. These compounds were shown not to be converted to higher phosphates in HSV-infected cells. Table II. N2-Hydroxyalkyl-guanines as H S V - T K Substrates

Purine 2-substituent

9-Substituent

Substrate

NÎÏ2 NHCH2CH2OH NHCH2CHOHCH2OH NHCH2CH2OCH3 NHCH2CH2OH

CH2OCH2CH2OH CH2OCH2CH2OH CH2OCH2CH2OH CH2OCH2CH2OH CH2OCH2CH2OCH3

+(ACV) +++ + ++ -

N O T E : - , not a substrate; +,

poor substrate; + +,

good substrate; and + + + ,

excellent

substrate. Assay protocol, ref. 21.

Other superpositions did not explain the data as well, but 'binding scenario B' (Fig. 5) received serious consideration because it was able to accommodate the fact that 2'-deoxycytidine was a good substrate for the enzyme. In this superposition there is excellent overlap of the 2-amino of G C V with the 4-oxo of thymidine as well as good positional agreement between the 3' and 5' hydroxyls of each of the two structures. Although the ' f i f of the two structures is not as eood and this is a higher energy conformer of G C V , this superposition clearly predicts that 8-hydroxyalkyl guanines should be substrates. However, when 8-(3-hydroxypropyl)-9-methylguanine(22) and 8-(2-aminoethylamino)-GCV(23) were prepared, they were found not to be substrates although molecular models of these structures were well accommodated i n binding scenario B'. This scenario also could not account for the fact that an 8-(methyl or bromo) substituent enhanced substrate activity. Acyclosugar Structure Substrate Activity. A great number of 9-side-chain variants were made by us and others(25^27). It is obvious from Figure 6 that the best substrate activity is achieved when the potential substrate has a In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

3. T O L M A N

Structural Requirements for Enzymatic Activation

Figure 5. Binding Scenario B: Rigid Superposition of a Ganciclovir Conformation (dotted) and Thymidine X-ray Structure (solid)-Front and Top Stereo-Views

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

43

N U C L E O T I D E ANALOGUES

44

G

G

R R = CI (+)

F(++) N (++) 3

OCH (+) 3

SCH

3

SCH

2

(-) Ph (-)

SCH CH 2

2

X«2(+++)

O(-)

X = 3( )

SCH CH C0 H(-) 2

2

+

2

G

G

G

G = Guanine

Figure 6. Structure Substrate Relationship for the 9-Side-chain of Guanine Acyclonucleosides

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

3.

TOLMAN

Structural Requirements for Enzymatic Activation

45

binding functionality i n both the 5' and 3' hydroxy binding sites for thymidine. Modified G C V s lose significant substrate activity wnen one of the hydroxyl's is replaced with a large or less hydrophilic group. Substitution of the Γ-position is not tolerated for substrate activity® and surprisingly the 2'-carba derivatives are better substrates than tneir oxa counterparts. The m i n i m u m side-chain length for substrate activity is 5 atoms (excluding hydrogen), the A C V side-chain length; longer side-chains are even better substrates for H S V - T K , but are more poorly converted to higher phosphates. There appears to be a chiral preference of the enzyme for the [SI configuration at the 4'-position(26) and considerable bulk tolerance. It has been shown that there is poor bulk tolerance on the carbon bearing the hydroxyl which is phosphorylated(26). Some conformationally-constrained acyclonucleosides(28) have been shown to have enhanced substrate activity, but the conformationally-restricted derivatives i n which a phenyl ring(23) is employed to give directionality to the side chain do not serve as substrates, possibly due to their excessive bulk. It should be noted that since 'substrate ability is a measure not onl this receptor, that these poo lack the proper geometry for efficient phosphorylation. 7

G M P Kinase This enzyme which converts the H S V - T K product (monophosphate) to diphosphate(DP) has itself been shown to be very selective® i n choice of substrates. The chiral G C V - M P ' s have both been shown to be substrates for G M P kinase but at vastly different rates(26). The [S] enantiomer, the one most analogous to the natural configuration, is the better substrate by two orders of magnitude. Figure 7 shows the substrate ability of various monophosphate derivatives. A s in the case of thymidine kinase, branching of the acyclo side-chain at 1' abolished any substrate activity. Unlike TK, which preferred longer side-chains as substrates (8 > 7 > 6 > 5), G M P kinase phosphorylates the shorter side-chains best and 8-membered side-chains not at all. Therefore, the monophosphates of the best T K substrates (with respect to side-chain length) are not substrates for G M P kinase. The 2'-oxa substitution increases K m and retards Vmax- It is not surprising that [S] chirality at position 4' favors phosphorylation by the enzyme since this w o u l d aid in the recognition of tne natural substrate. A hydroxyl in the region of the 3'-hydroxyl of G M P enhances substrate activity, but phosphorylation of the hydroxyl at this position (3') abolishes substrate activity. Conversion to Triphosphate Use of the 'staggered assay'(21) permits the comparison of the relative phosphorylation rates of various acyclonucleosides. Although all compounds shown i n Table III are converted to triphosphate, they differ i n the facility with which this occurs. It should also be noted is that not all of the triphosphates (TP) inhibit H S V D N A polymerase and have antiherpes activity. In particular, the unsaturated acyclonucleoside in line 3 is relatively poorly converted to a TP which does not inhibit the viral D N A polymerase, out the compound nonetheless has antiherpes activity (the acyclonucleoside may be an SA H hydrolase inhibitor). It is also interesting that the trishydroxymethyl acyclonucleoside and the cyclopropyl compound (lines 4 & 5, respectively) are equally well converted to TP, are also equally devoid of viral polymerase inhibition, and yet only one of the materials (the latter one) still has antiherpes activity by some as yet unidentified mechanism. In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

m

V ax (nmol/min) (μΜ)

m

Figure 7. Kinetic Data for Phosphorylation b y G M P Kinase

K Vmax (nmol/min)

87.5 43

(μΜ) m

K 5-GMP

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

3.

Structural Requirements for

TOLMAN

47

Enzyme

It is more desirable for an acyclonucleoside to be poorly converted to a triphosphate which is an efficient inhibitor of viral replication (e.g. A C V ) , than one which is efficiently converted to triphosphate. Efficient phosphorylation by viral-specified kinases w i l l undoubtedly mean more phosphorylation (although orders of magnitude less) by host cell kinases i n an uninfected cell. A n y significant level of phosphorylated acyclonucleoside i n uninfected cells can disturb the balance of nucleotide pool sizes(30) and lead to side effects i n one of the myriad of various cell types and their metabolic systems for regulation of nucleic acid synthesis. Table ΠΙ. Relative Phosphorylation Rates of Acyclonucleosides by H S V - T K and HSV-Infected Cell Extracts and H S V D N A Polymerase Inhibition HSV-TK

% Inh'n

ED50

DNApol

HSV-1(29)

79 22

3 0.16

3 3

9 60 10 64

0

9

25

50

51

0

8

86

>100

45

0

0

76

80

0

5

82

17 90

ACV GCV

%

OH OH

|\—OH ^OH

>100

2'Nor-cGMP Still another type of antiherpetic mode of action employing phosphorylated acyclonucleosides is typified by 2'nor-cGMP(31-33). This antiviral agent has potent activity against members of the herpes group and other D N A viruses, including cytomegalovirus(34,35), adenovirus(36), papilloma virus(31), varicella-zoster(31), and herpes keratitis(37). Cell culture studies and animal studies have shown that 2'nor-cGMP is equally effective against TK-deficient (or minus) H S V strains, indicating that this activity i n independent of viral thymidine kinase. Although some hydrolysis of the cyclic pnosphate occurs intracellularly in vivo to produce levels of G C V - M P (and therefore GCV-TP),

American Chemical Society Library

1155as 16th S t Agents; . N.W.Martin, J.; In Nucleotide Analogues Antiviral Washington, C 20036 ACS Symposium Series; American Chemical D Society: Washington, DC, 1989.

N U C L E O T I D E ANALOGUES

48

the amount of T P produced is not enough to account for the antiviral activity observed(38).

Unlike G C V - M P , 2'nor-cGMP is taken up intact from the gut [10% of a labelled dose (10mg/kg p.o., rats) is recovered i n the urineQS)] and is relatively stable i n plasma (80% of drug recovered from urine is intact cyclic phosphate). The mechanism of antiherpetic action of 2'nor-cGMP remains unknown, since the cyclic phosphate directly inhibits neither HSV-1 D N A polymerase nor C M V polymerase Summary Molecular modelling has proven to be a positive factor i n the development of structure-activity relationships as a source of stimulation and predictive models. Although the means is available to design acyclonucleoside substrates which w i l l be phosphorylated i n herpes-infected cals, there is no assurance that the resultant monophosphates w i l l be converted to higher phosphates by G M P and other kinases. It is also not possible to predict a rion whether these triphosphates w i l l inhibit viral polymerases and/or ave antiherpetic activity. Other acyclonucleoside or acyclonucleotides have antiviral activity whicn is independent of D N A polymerase or viral activation by viral thymidine kinase or both.

K

Acknowledgments The author is very grateful to the many contributors to this work, especially to Dr. Malcolm MacCoss, Dr. Wallace T. Ashton, and Dr. John D. Karkas who helped to shape and guide this work and to Dr. Dennis Underwood who helped to solve the molecular modelling problems. Literature Cited Elion, G. B. Am. J. Med. 1982, 73, 7 (Acyclovir Symposium). Elion, G. B.; Furman, P. Α.; Fyfe, J. Α.; de Miranda, P.; Beauchamp, L.; Schaeffer, H. J. Proc. Natl. Acad. Sci. USA 1977, 74, 5716. 3. Fyfe, J. Α.; Keller, P. M.; Furman, P. Α.; Miller, R. L.; Elion, G. B.J.Biol. Chem. 1978, 253, 8721. 4. Miller, W. H.; Miller, R. L.J.Biol. Chem. 1980, 255, 7204. 5. Miller, W. H.; Miller, R. L. Biochem. Pharmacol. 1982, 31, 3879. 6. Keller, P. M.; Fyfe, J. Α.; Beauchamp, L.; Lubbers, C. M.; Furman, P. Α.; Schaeffer, H. J.; Elion, G. B. Biochem. Pharmacol. 1981, 30, 3071. 7. Schildkraut, I.; Cooper, G. M.; Greer, S. Mol Pharmacol. 1975 11, 153. 8. Cheng, Y.-C.; Dutschman, G.; De Clercq, E.; Jones, A. S.; Rahim, S. G.; Verhelst, G.; Walker, R. T. Mol. Pharmacol. 1981, 20, 230. 9. Sim, I. S.; Goodchild, J.; Meredith, D. M.; Porter, R. Α.; Raper, R. H.; Viney, J.; Wadsworth, H. J. Antimicrob. Ag. Chemother. 1983, 23, 416. 10. Cheng, Y.-C. Ann. Ν. Y. Acad. Sci. 1977, 284, 594. 1. 2.

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

3.

11. 12. 13. 14.

15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34.

TOLMAN

Structural Requirements for Enzymatic Activation

Cheng, Y.-C.; Ostrander, M. J. Biol. Chem. 1976, 251, 2605. Karkas, J. D.; Germershausen, J. G.; Tolman, R. L.; M . MacCoss; Wagner, A . F.; Liou, R.; Bostedor, R. Biochem. Biophys. Acta 1987, 911, 127. Marshall, G.; Trends Biol. Sci. 1988, 236. The MM2-extended force field (MM2-X), developed internally at Merck, shares many parameters with M M 2 ; it differs principally i n that lone pairs on neteroatoms are not used (compensating changes have been made in torsional parameters and charge distributions), and that electrostatic interactions take place between atom-centered charges, thus allowing proper treatment of charged systems: M M 2 - X has been parameterized for a wide ranee of systems. Birnbaum, G . I.; Shugar, D. i n Nucleic A c i d Structure, Part 3, Topics i n Molecular and Structural Biology, ed. S. Neidle, M a c M i l l a n (London, 1987) p. 1. Birnbaum, G . I.; Cygler, M . ; Kusmierek, J. T.; Shugar, D. Biochem. Biophys. Res. Commun. Birnbaum, G. I.; Cygler, M . ; Shugar, D. Can. J. Chem. 1984, 62, 2646. Birnbaum, G . I.; Johansson, N. G.; Shugar, D. Nucleosides & Nucleotides 1987, 6(4), 775. Stolarski, R.; Lassota, P.; Kazimierczuk, Z.; Shugar, D. Z. Naturforsch 1988, 43c, 231. Beauchamp, L. M . ; Dolmatch, B. L.; Schaeffer, H . J.; Collins, P.; Bauer, D. J.; Keller, P. M . ; Fyfe, J. A . J. Med. Chem. 1985, 28, 982. Ashton, W. T.; Canning, L. F.; Reynolds, G. F.; Tolman, R. L.; Karkas, J. D.; L i o u , R.; Davies, M.-E. M . ; DeWitt, C. M . ; Perry, H . C.; Field, A. K. J. Med. Chem. 1985, 28, 926. Stein, J. M . ; Stoeckler, J. D.; Li, S.-Y.; Tolman, R. L.; MacCoss, M . ; Chen, Α.; Karkas, J. D.; Ashton, W. T.; and Parks, R. E., Jr. Biochem. Pharmacol. 1987, 36, 1237. Tolman, R. L.; Ashton, W. T.; MacCoss, M . ; Karkas, J. D.; Underwood, D.; Meurer, L. C.; Cantone, C. C.; Johnston, D. B. R.; Hannah, J.; Liou, R. J. Med. Chem. 1989, submitted. Martin, J. C.; McGee, D. P.C.; Jeffrey, G. Α.; Hobbs, D. W.; Smee, D. F.; Matthews, T. R.; Verheyden, J. P. H. J. Med. Chem. 1986, 29, 1384. McGee, D. P. C.; Martin, J. C.; Smee, D. F.; Matthews, T. R.; Verheyden, J. P. H . J. Med. Chem. 1985, 28, 1242. Karkas, J. D.; Ashton, W. T.; Canning, L. F.; Liou, R.; Germershausen, J.; Bostedor, R.; Arison, B.; Field, A . K.; Tolman, R. L. J. Med. Chem. 1986, 29, 842. Larsson, Α.; Tao, Pei-zhen Antimicrob. Ag. Chemother. 1984, 25, 524. Ashton, W. T.; Meurer, L. C.; Cantone, C. L.; Field, A . K.; Hannah, J.; Karkas, J. D.; L i o u , R.; Patel, G. F.; Perry, H . C.; Wagner, A . F.; Walton, E.; Tolman, R. L. J. Med. Chem. 1988, 31, 2304. Field, A . K.; Perry, H . C. personal communication. Bradley, M. O.; Sharkey, N. A . Nature 1978, 274, 608. Tolman, R. L.; Field, A. K.; Karkas, J. D.; Wagner, A . F.; Germershausen, J.; Crumpacker, C.; Scolnick, Ε. M. Biochem. Biophys. Res. Comm. 1985, 128, 1329. Field, A. K.; Davies, M.-E.; DeWitt, C. M . ; Perry, H . C.; Schofield, T. L.; Karkas, J. D.; Germershausen, J . ; Wagner, A . F.; Cantone, C. L.; MacCoss, M . ; Tolman, R. L. Antiviral Res. 1986, 6, 329. Prisbe, E. J . ; Martin, J. C.; Baker, M . F.; Smee, D. F.; Duke, A . E.; Matthews, T. R.; Verheyden, J. P. H. J. Med. Chem. 1986, 29, 671. Duke, A . E.; Smee, D. F.; Chernow, M . ; Boehme, R.; Matthews, T. R. Antiviral Res. 1986, 6, 299. In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

49

50 35. 36. 37. 38.

39.

N U C L E O T I D E ANALOGUES

Yang, Ζ. H . ; Lucia, H. L.; Hsiung, G. D.; Tolman, R. L.; Colonno, R. J. Antimicrob. Ag. Chemother. 1989 submitted. Baba, M . ; M o r i , S.; Shigeta, S.; DeClercq, Ε. Antimicrob. Ag. Chemo. 1987, 31, 337. Gordon, Y. J.; Capone, Α.; Sheppard, J.; Gordon, Α.; Romanowski, E.; Araullo-Cruz, Τ Current Eye Res. 1987, 6, 247. Germershausen, J. G.; L i o u , R.; Field, A . K.; Wagner, A . F.; MacCoss, M . ; Tolman, R. L.; Karkas, J. D. Antimiaob. Ag. Chemother. 1986, 29, 1025. Hucker, H . ; Germershausen, J. personal communication.

R E C E I V E D June 9,

1989

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Chapter 4

Phosphonylmethyl Ethers of Nucleosides and Their Acyclic Analogues Antonin Holý , Erik De Clercq , and Ivan Votruba 1

2

1

Institute of Organic Chemistry and Biochemistry, Czechoslovak Academy of Sciences, 166 10 Praha 6, Czechoslovakia Rega Institute, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium

1

2

Structure-activit of a c y c l i c nucleotide analogs bearing a modified phosphoric acid residue at the s i d e - c h a i n revealed two novel classes of a n t i v i r a l s : N-(3-hydroxy-2-phosphonylmethoxypropyl)(HPMP-) and N-(2-phosphonylmethoxyethyl) (PME-) d e r i v a t i v e s of h e t e r o c y c l i c bases. Adenine, guanine, 2-aminoadenine and ( i n the HPMP-series) c y t o sine d e r i v a t i v e s act s p e c i f i c a l l y against DNA viruses (herpes viruses,adenoviruses,poxviruses). The PME-compounds are also active against retroviruses (MSV,HIV)and exhibit a c y t o s t a t i c effect on L-1210 mouse leukemia cells.The drugs are converted by the action of c e l l u l a r nucleotide kinases into t h e i r diphosphates and i n h i bit v i r a l and, to a lesser extent, c e l l u l a r DNA synthesis. These metabolites exert a comparatively low i n h i b i t o r y effect on v i r a l (HSV-1) DNA polymerase. The diphosphates derived from PME-compounds strongly i n h i b i t v i r a l (HSV-1) r i b o nucleotide reductase and AMV reverse t r a n s c r i p tase. The

m a j o r i t y

exert

t h e i r

d i r e c t b r i n g

b i o l o g i c a l l y

f o r

apparent

c e r t a i n e t c . )

membranes these

cases than

i s

d e p h o s p h o r y l a t i o n s vent

t h e

which

and

i n

c a t a b o l i c t o

t h e

e a s i l y

l a t t e r i n

which

t h e

parent i n

analogs However,

does

t h e

t h e

by

many o f

h a s

( i n c r e a s e d (1_>-

r a p i d

serum,

not

a c t i v i t y

n u c l e o t i d e

n u c l e o s i d e

c y t o p l a s m . I n

analogs

o f

parameters

e x p l a i n e d

r e a c t i o n s ,

s y n t h e s i z e

compounds

improvement

occur

t h e

n u c l e o s i d e

5 * - n u c l e o t i d e s .

pharmacological

paradox

undertaken

o f

a c t i v e

t h e i r

any s u b s t a n t i a l

favorable

s o l u b i l i t y ,

l u l a r

v i a

a p p l i c a t i o n about

except more

o f

e f f e c t s

i n

order

T h i s

enzymatic t h e

t o

attempts

n u c l e o t i d e s

c e l -

c i r c u m -

have with

0097-6156/89/0401-0051$06.25/0 o 1989 A m e r i c a n C h e m i c a l Society

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

been mo-

52

N U C L E O T I D E ANALOGUES

d i f i e d both

p h o s p h o r i c chemical

e x h a u s t i v e s u l t e d d i e d

the

many

l o g i c a l r e a c t i o n s

i n

i n t e r e s t i n g a n t i v i r a l , of

d e s i g n

important s t e r i c

the

same

bond

s i m i l a r i t y ,

o f

o n l y

c o m p l e t e l y

(e.g.

to

i n

the

type

d i r e c t l y a

for

o f

to

of

the

above

a

sugar

R - P ( 0 ) ( 0 H )

2

major

a d d i t i o n

always

have

c o u n t e r p a r t s .

a n a l o g s

ETHER o f

the

portance

the

on

the

(3) to

p o l a r

i s o s t e r i c

n e a r l y

p a r t s .

These

i c a l l y

r é s i s t e n t means

a d a p t a t i o n

d e r i v a t i v e s e t h e r

sugar of

of

changing e t h e r

r e s i d u e

a

of

R - 0 C H

2

e v i -

r e s i d u e

n u c l e o s i d e

P ( 0 ) ( O H )

2

2

r e s i d u e )

i n a c t i v i t y

led

i n

to

of

c l a s s

analogs

which

are

(type with

C33)

c o n t a i n

j o i n i n g the

the

u n i t .

m o l e c u l a r

c o n f o r m a t i o n

the

pK

of

the

with

an

T h i s

phosi s o -

c o u n t e r enzymatof

a

group-

e n a b l e s

not

r e s u l t i n g

the

im-

phosphoof

a c i d

u n i t

o f

the

h y d r o x y l

p h o s p h o r i c

methylene

our

both

phosphate

a

value

r e a l i z e

v i c i n i t y

t h e i r

phosas

requirements

novel

with

comparison

the

well

a

linkage

moiety

us

the

o f

C23)as

the i n

are

a c i d

s t r u c t u r a l

atom

n u c l e o t i d e

n u c l e o s i d e

c o n d i t i o n s

p h o s p h o r i c

i n v e s t i g a t e

p h o r u s - m o d i f i e d and

above

t h i s

analogous

cleavage,

(type

which

oxygen us

o f

are

DERIVATIVES.

n u c l e o t i d e s

enzymes

of

which

3

b i o l o g i c a l

experience

s t i m u l a t e d

component

v a r i a t i o n

2

n u c l e o l y t i c

methyl

the In

linkage-

mentioned

p r e c e d i n g

a l l y

o f

a l c o h o l

enzymatic

b e a r i n g the

C-P

PHOSPHONYLMETHYL

by

i n v e s t i g a t i o n

phosphomonoester

must

c h e m i c a l

( R = 5 ' - d e o x y n u c l e o s i d e - 5 ' - y 1

ing

e f f e c t s .

phosphate

f u l f i l l

the

1

rus

i s o l a t e d

s i g n i f i c a n t

f i e l d :

compounds

CI 3,

compounds

C23)by

phonate

e x h i b i t e d

some

one

a n a l o g s

t h e i r

b i o -

a n a l o g s

d e t a i l e d as

s t u -

enzymatic

posses

t h i s

were t h e i r

o f

s e l e c t e d

nature

f i e l d

r e s i s t

RO-P(O)(OH)

The

o f

s p l i t t i n the

narrow

The

l i n k e d

the

An r e -

occurenc

capable very

d e n t l y

by

r e s i s t (2.)·

decades

which

b i o l o g i c a l

a c t i v e as

two

against

isopolarity

phosphomonoesters

and

o f

of

f e a t u r e s

d i s s o c i a b i 1 i t y

l i n k a g e . to

s p e c i f i c i t i e s

past

a n a l o g s

found

o t h e r

would

i n h i b i t i o n

some

complemented

d i s r e g a r d i n g

leaves

as

was

or

which

h y d r o l y s i s

e v a l u a t i o n

a c t i v i t i e s

uncovered

wide

enzymes

well

them

s t r u c t u r a l

to

numerous

Though

a n t i c a n c e r

h y d r o l a s e s ,

the

i n c l u d i n g

as

o f

s t u d i e s

s t r u c t u r a l

The

o f

v i t r o .

l i n k a g e

enzymatic

d u r i n g

i n h i b i t o r y

(1_),none

These

as

r e s p e c t s

a c t i v i t i e s

enzymes

e s t e r

well

i n v e s t i g a t i o n

i n

i n

a c i d

as

an

s u b s t a n t i p h o s p h o n y l -

c o r r e s p o n d i n g

phos-

phomonoester . 5

1

-O-Phosphony1methyl

a n a l o g s

i n v e s t i g a t e d

pounds by

an

d e r i v e d

s y n t h e s i s

was

a l k o x i d e

a n i o n

with

from

e t h e r i f i c a t i o n

d i a l k y l

N u c l e o s i d e s . i n

t h i s

n a t u r a l l y of

t h e i r

s e r i e s

The

f i r s t

group

encompasses

o c c u r i n g

r i b o n u c l e o s i d e s

5 ' - h y d r o x y l

a c c o m p l i s h e d

by

treatment

(generated

by

sodium

o f

com-

of

group. a

h y d r i d e

T h e i r

n u c l e o s i d e r e a c t i o n )

p - t o l u e n e s u l f o n y l o x y m e t h y l p h o s p h o n a t e

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

C43-

4.

53

Phosphonylmethyl Ethers of Nucleosides

HOLY E T AL.

The intermediary d i e s t e r i s then cleaved by b r o m o t r i m e t h y l s i l a n e t r e a t m e n t f o l l o w e d by a c i d h y d r o l y s i s a n d RO

" + TsOCH P(0) COAlk> 2

2

** C R O C H P < 0 ) ( A 1 k ) 3 2

2

*-C33

4 (R-ribonucleosid-5'-yl

residue)

Scheme 1 removal o f t h e p r o t e c t i n g g r o u p (Scheme 1) ( 4 ) . T h e s e compounds r e s i s t t h e a c t i o n o f ρ h ο s ρ h ο mο η ο e s t e r a s e s ( 3 . ) ; also the internuc1eotidic linkage containing this group­ ing SD as well as the cyclic esters of this type (6) are s t a b l e a g a i n s t t h e r e l e v a n t ribonuc1 eases and phos­ phodiesterases. The s u b s t r a t e and i n h i b i t o r y a c t i v i t y o f ribonucleoside 5 *-triphosphate a n a l o g s c o n t a i n i n g modi­ f i e d α-phosphorus atom, a s o b s e r v e d w i t h u r i d i n e kinase Z» Q · , P y n u c leot· i d e p h o s p h o r y l a . s e ( 9 ) a n d E > N A - d e p e n d e n t RNA polymerase < 1Q. might r e p l a c e t h e 5'-phosphate cer nucleoside analogs at their target enzymes and i n ­ t e r f e r e with metabolic pathways o f n u c l e o t i d e s . The de­ termination of a n t i v i r a l a c t i v i t y against both RNA a n d DNA v i r u s e s d i s p r o v e d t h i s e x p e c t a t i o n f o r 5 ' - O - p h o s p h o nylmethyl r i b o n u c l e o s i d e s (E.De C l e r c q and A-Holy, un­ published data). We h a v e a l s o prepared t h e 5'-0-phosphonylmethyl ethers o f those sugar-modified nucleoside analogs which p e r se e x h i b i t an outstanding antiviral and/or c y t o s t a t i c effect: 6-azauridine, cytosine arabinoside, adenine arabinoside, thymine arabinoside, adeni­ ne x y l o s i d e and R i b a v i r i n (J.Brokes, A.Holy, J . Z a j i c e k , C o l l e c t • C z e c h . Chem•Commun.. i n p r e s s ) b y t h e a b o v e m e t ­ hod. However, a l l o f them a r e d e v o i d o f a n t i v i r a l activ­ ity i n vitro against a l l v i r u s e s t e s t e d (E-De C l e r c q : unpublished data). The reason f o rthis failure remains to be e l u c i d a t e d ; s i n c e a l l t h e above compounds e x e r t only marginal ( i f any) c y t o t o x i c i t y on t h e host cell lines, i t might be a n t i c i p a t e d t h a t they a r e unable t o penetrate the cell membrane t o t h e e x t e n t that i s requi­ red t o cause a b i o l o g i c a l effect. (

(

}

o

1

Phosphonylmethyl Ethers o f A c y c l i c Nucleotides. Acyclic nucleoside analogs i n which t h e carbohydrate moiety i s r e p l a c e d by an open c h a i n m i m i c k i n g t h e p a r t o f t h epentafuranose r i n g , have a t t r a c t e d a major i n t e r e s t i n many laboratories including oursThe development i n this f i e l d was l a r g e l y s t i m u l a t e d by t h e a n t i v i r a l activity found f o r numerous r e p r e s e n t a t i v e s o f t h i s group. In t h e guanine s e r i e s , a c y c l o v i r C53and i t s congeners (gan­ c i c l o v i r C63, buciclovir C 73)are active against herpes v i r u s e s , whereas t h e adenine d e r i v a t i v e s 9 - ( S ) - ( 2 , 3 - d i hydroxypropyl)adenine (DHPA) C 8 3 a n d 3 - ( a d e n i n - 9 - y 1 ) - 2 -hydroxypropanoic acid and i t s e s t e r s C93 i n h i b i t t h e multiplication of certain DNA a n d RNA v i r u s e s with a preference f o r (-)-stranded and (+)-stranded RNA v i r u s e s (12-16). The a c t i v i t y o f t h e former group o f a n t i v i r a l s , i.e. acyclovir and a l l o f i t s analogs,depends on t h e i r

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

54

NUCLECmDE

p h o s p h o r y l a t i o n f u r t h e r h i b i t i o n

of

(1_2_).

by

An

of

v i r u s - s p e c i f i c

circumvent

of

s t a b l e )

the

loss

the

of o f

the

i n ­ (TK)

phosphate

these

group

equivalent

drugs

high

i s

k i n a s e "

s t r u c t u r a l

s p e c i f i c i t y

with

undergo

i n

p h o s p h o r y l a t i o n

"thymidine

i n t r o d u c t i o n

the

a l b e i t ,

then

u l t i m a t e l y

s y n t h e s i s . T h i s

( e n z y m a t i c a l l y

v i r u s e s , act

a

which

r e s u l t i n g

DNA

a r t i f i c i a l

i t s

might

monophosphates

v i r a l

c a t a l y z e d or

to

t r a n s f o r m a t i o n s

ANALOGUES

for

T K

s e l e c t i v i t y

+

of

ion. G

HOCH

2

0

χ

^ C H

G H0CH

I ^ C H

2

/ Ο

2

CH

2

G HOCH

I C H

^

2

^ C H

2

C H

|

2

^ C H

2

2

HOCHg 5

The

6

1 , 3 - c y c l i c

l o v i r

phosphat

C9-(1,3-dihydroxy-2-propoxymethy1)guanine3

p l a y s

s i g n i f i c a n t

other

DNA

i n h i b i t o r y

v i r u s e s

( 1_7) .

the

above

h y p o t h e s i s .

i t y

which

has

been

l e o t i d e s

with

linkage a l s o

being

might

t a i n i n g t h e i r

exert

any

or

t h e i r

shown

c o n s i d e r e d

cannot mic a

it

1,which

mixture

a l l y

of

from

against any

RNA

i n t e r e s t of

DHPA

v i r u s e s towards

of

T h i s

CH

2

I I OH

0-phoswhich

the

r a c e -

( 22_)

obtained

effect

(vide

was

those

an

a

I ^ C H R00C-CH

OH

I OH 9

r a -

e t h e r s

e x c e p t i o n ­

s p e c i f i c a l l y

observed

with

by

a f f o r d s

0-phosphonylmethyl

p a r t i c u l a r

the

d e s c r i b e d

d i o l s

d i r e c t e d

No

the

analogs

method we

(21)

context,

from

e x h i b i t e d

CH

8

the

v i v o

t h i s

as

on are

mediate

i n v e s t i g a t e

product

(23.). DHPA

In

a c y c l i c

the

against greatest

s u p r a " ) ·

A

2

of

depend

i n

not

a c t i v i t y

A ' ^ C H

c o n ­

analogs

or

do

isomers,

isomeric

of

not

These

(20)

DHPA)

v i c i n a l

p o s i t i o n

two

do

i o n . S t a r t i n g

case

v i r u s e s , i n

s e n s i t i v i t y

(19.)-

to

the

a n t i v i r a l

DNA

e f f e c t s

o b s t a c l e

a c t i v i t i e s

enantiomers

i n

two

nuc­

phosphate

phosphonate

compound.

u t i l i z i n g

DHPA.

high

parent

and

of

of

and

u l t i m a t e

v i t r o

C83 the

mixture

d e r i v e d

the of

the

adenine

from

dephosphory1at

compound

cemic

i n

(e.g.

e t h e r s

undergo

Scheme

by

of

the

from

e i t h e r

phosphates

phonylmethyl

be

p h o s p h o r y l a t i o n

phosphorylated

analogs

moiety

b i o l o g i c a l

d e r i v e d

and

w - h y d r o x y a l k y l

that

a c t i ν i t i e s . T h e s e

p o l a r i t y not

a c t i v ­

9-(w-phospho-

a c t i o n .

other

not

a c t i v i t y

the

and

analogs

preceding

carbohydrate

might

p e n e t r a t i o n

" p a r e n t "

b i o l o g i c a l

that

A n t i v i r a l

(the

and

supports

a n t i v i r a l

v a r i o u s

suggests

CWV

f u l l y

marked

for

d i s ­

against

d i s c o v e r y

the

(1_8)

i n a c t i v e )

molecule

n u c l e o s i d e

T h i s

Also,

m o d i f i e d

i n d i c a t e

a c t i v i t y

d e s c r i b e d

n y l a l k y l )hypoxanthines compounds

we

7

G

2

0

/0-CH2 Ρ

/ HO

N

\

CH /

0 - C H

II

^ 0

2

10

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

^

C H

2

Phosphonylmethyl Ethers ofNucleosides

4. H O L Y E T A L

Structure-activity Investigatign

55

qf Acyclic Nucleotide

Analogs. T h i s

d i s c o v e r y

a d d i t i o n a l modified

prompted

a c y c l i c phosphate

a c y c l i c

s i d e

groups:

c h a i n (a)

to

by

other

i d e n t i f i c a t i o n

a n t i v i r a l

a c t i v i t y

(b)

changes

of

the

s i d e - c h a i n

t e m a t i c a l l y

i n

the

s e r i e s

v a t i v e s

( c ) v a r i a t i o n

and of

ional

of

of

which

might

i n t o

isomer(s) were

the

r e s p o n s i b l e

from

e t h e r s , s y s -

adenine

h e t e r o c y c l i c

a r i s e

The three

i n t r o d u c e d

d e r i v a t i v e s

a

the

linkage.

d i v i d e d

9 - s u b s t i t u t e d

of

to

phosphonylmethyl

which

2,3-dihydroxypropy1

s e r i e s

of

DHPA

d e t a i l c o n t a i n

l i n k e d

e s t e r

was

i n

which

group

than

i n v e s t i g a t i o n

for

s e r i e s

i n v e s t i g a t e analogs

(phosphonate)

s t r u c t u r e - a c t i v i t y main

us

n u c l e o t i d e

d e r i -

base

and

i n

any

the

a d d i t -

the

s i d e - c h a i n

v a r i -

r e a c t i o n

o r i g i n a l l y

deve-

ât i o n . Phosphonylmethyl n i n e .

The

t i t l e

E t h e r

s y n t h e s i

compound

loped

for

the

makes

r i b o n u c l e o s i d e s Scheme

2

treatment a

isomers

with

aqueous T h i s

o f

can

tography

(22)·

s t a r t s

mixture

and

a of

2 ' ( 3 ' ) - O - p h o s p h o n y l m e t h y l

r e a c t i o n

(S)-

or

sequence

(R ) - D H P A

chloromethylphosphonyl separated f u r t h e r to

by

i s

r a c e m i z a t i o n

proceeds not

which

ion

exchange

by

h e a t i n g e t h e r s

v i a

an

accompanied

+

2

C1CH

2

P(0)C1

These chroma-

with

i n t r a m o l e c u l a r by

any

i s o m e r i -

2

8 f o

'

» A-CH

2

I

9

CH-CH

2

I

0P(0)CH C1

0-P(0)CH

C1

2

I

OH

9

OH

I OH 11

.

12

I A-CH

2

CH-CH

2

OCH

2

P(0)(0H)

A~CH CH-CH 0H

2

2

OH

2

0CHP(0)(0H) 13

14 A

=

a d e n i n - 9 - y l Scheme

an

C13,143-

OH

A-CH CH-CH 0H

by on

a f f o r d s

C11,123-

(25.):

A-CH CH-CH 0H 2

or

d e s c r i b e d

(2j4)

d i c h l o r i d e

phosphonylmethyl

which

mechanism

HPLC

transformed

the

t r a n s f o r m a t i o n or

the

from

a l k a l i

s a t i o n

of

chloromethylphosphonates

be

c y c l i s a t i o n

use

p r e p a r a t i o n

(HPMPA)

r e s i d u e

2

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

2

NUCLEOTIDE ANALOGUES

56 The

e v a l u a t i o n

reoisomers C143

i . e - 9 - (

adenine v i t y ,

i s

The

that

the

are

compound

V a r i a t i o n

o f

the

of

o f

s i m p l i c i t y

nine

which

ion

i t s

a l l

four

s t e ­

( S ) - s e r i e s

The

parent

at

two

the

s t r a t e g y )

or,

a c t i ­

two

(R)-

e f f i c i e n c y

HPMPA.

m o d i f i c a t i o n s C143concerned F o r

d e r i v e d

p o s i t i o n

N9.

g r o u p i n g

the

from

approaches

( u s i n g

(23.).

as

f e a t u r e s .

were

general

backbone

the

c h e m i c a l

p h o s p h o r u s - c o n t a i n i n g

9 - a l k y l a d e n i n e

as

s t r u c t u r e

s t r u c t u r a l

s u b s t i t u t e d

a n t i v i r a l

a b b r e v i a t e d

compounds

o f

the well

a n t i v i r a l

C h a i n .

important

was

f o r as

c u r r e n t l y

the

c o n s i s t e d a

the

any

i s

Side

o f

o f

o f

(S)-C143

sake

ponding

the

o f

r e s p o n s i b l e

d e v o i d

s e v e r a l

t i o n

o f

2 ' - i s o m e r

( S ) - 3 ' - i s o m e r

s i d e - c h a i n

s y n t h e s e s

a c t i v i t i e s

the

S ) - ( 3 - h y d r o x y - 2 - p h o s p h o n y l m e t h o x y p r o p y l ) -

whereas

the

a n t i v i r a l

e n t i r e l y

enantiomers

of

o f

r e v e a l e d

ade­

Chemical : i n t r o d u c ­

i n t o

c o r r e s ­

s u i t a b l e

p r o t e c t ­

a l k y l a t i o

p h o s p h o r u s - c o n t a i n i n group

( a l k y l

s y n t h o n s ) .

halogenide

These

paper

C263-

bear

the

well

as

or

T h i s

s e r i e s

of

a d d i t i o n a l d e r i v a t i v e s the

o f

(vide

i t s the

the

replacement l o s s

o f

methyl

e t h e r

C273,

C32,333

HPMPA

which

part

i n o f

apart

the

from

the

s i d e

the

2 ' -

or

3 ' - p o s i t i o n

ted

i n v a r i a b l y Though

the

importance of

of

i n

above the

the

HPMPA

(23).

and

i t s

can

be

the

base

C183

h o x y a l k y l C20-223 ion

m o d i f i e d as

hydroxymethy1

m e t h y l -

o f

nor

the

o f

a c t i v i t y

analog

C

a

methylene

s t r e s s e s

o f

phosphonate u n i t

at

C23-253

i n

de­

homolog

a n t i v i r a l

group

203,

1 ' - C - a l k y l

the

r e s u l ­

a c t i v i t y .

the

a b s o l u t e

HPMPA,

the

a c t i v i t y

o f

which

i n

i s

s t u ­

a

with

r e f e r r e d

congeners

as

P M E - d e r i v a t i v e s .

s i m p l i f i e d

a n a l o g the

o f

a c t i v i t y atom

HPMPA

by

o f

PMEA PMEA

l a c k i n g

w-phosphonylmet-

d e r i v a t i v e s

whatsoever,

c h a i n

as

phosphonylmethoxy-

s t r a i g h t - c h a i η p e n t y l )

t o

q u a l i ­ that

be

b u t y l ,

two-carbon

r e s p e c t s

comparable

g r o u p . N e i t h e r

any

9-(2-phosphony1methomany

w i l l

o t h e r

( p r o p y l ,

e x h i b i t

l a c k

s u b s t i t u t i o n

o f

o f

u l t i m a t e

the

by

o f

C15,163)

o f

with

s k e l e t o n

l o s s

com­

9-(tt-phosphony1methoxyalkyl)adenines

compound

regarded

the

d i s t a n c e

the

HPMPA

q u a n t i t a t i v e l y

T h i s

these

by

any

i n t e r p r e t a t i o n

C193,

and

s h i f t e d

hydroxymethy1

a n t i v i r a l

x y e t h y l ) a d e n i n e t a t i v e l y

the

i n

i s

and

A l s o ,

complete

homologous

u n v e i l e d

28-303

g r o u p i n g i s

as phos-

C 1 7 3 ) r e s u l t e d

deoxy

e v i d e n t l y

C263-

i n

the

but

o f a

by i t s

C

whole

o f

base

homologs most

and

atom

r e l a t i v e

the

o n l y

C133,

1)

e t h e r i f i c a t i o n

(as

the

from

not

HPMPA

oxygen

an

group

group

which

(Table

HPWPA(compounds

A l s o

c h a i n

base

i n

type

o r i g i n a l

compounds

a c t i v i t y

that

methyl

witnessed

r i v a t i v e s

dy

group

by

o f

the

2).

a n t i v i r a l

a c t i v i t y .

3 ' - i s o m e r

the

(Table

r e v e a l e d

phosphonylmethoxy the

o f

i n

r e s i d u e

l a c k i n g

the

h y d r o x y l

importance,as of

of

i n f r a )

primary

in

composed e t h e r

phosphorus

E v a l u a t i o n

or

i s

p - t o l u e n e s u l f o n a t e

d e s c r i b e d

p h o s p h o n y l a l k o x y a l k y l -

v i c i n i t y

the

are

phosphonylmethyl

p h o n y l a l k y l

pounds

a l k y l

s y n t h e s e s

and

of

adenine

s u b s t i t u t ­

hydroxymethy1

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

group,

4.

Phosphonylmethyl Ethers ofNucleosides

H O L Y ET AL. which

i n

t a l l y

i n e f f e c t i v e

p o s i t i o n

However, mutual not

2

o f

i n

the

s u f f i c i e n t The C343

butyl)adenine Table

1.

o f

with

t o

compounds.

c h a i n

o f

t o

PMEA, carba

exert

t o

HPMPA,

(compound

σ-phosphonyl

a n t i v i r a l

and t h e

C383

1

respect

cause

isomer

leads

p o s i t i o n

presence

o r i e n t a t i o n

d e r i v a t i v e

t h e

t h e

57

no

t h e

t o ­

group

and

adenine

base

a c t i v i t y

i n

t h e

i t s i s

above

2-phosphonylethoxymethyl analog,

9-(4-phosphony1-

a n t i v i r a l

a c t i v i t y .

Nor

was

9-(Phosphonylmethoxyalky1)adenines Side

No.

Chain

15

-CH CH(0CH P(0)(OH)2 > CH 0CH

16

-CH CH(0CH P(0)(OH) )CH 0C H ^

1 7

-CH CH(0CH P(0)(OH)2)CH

18

-CH 0CH -CH CH 0CH P(0)(OH)

2

2

2

2

2

2

2

3

8

7

2

2

19

2

20 21 22

-CH2CH2CH2OCH2P(0)(OH)2 - C H

2

( C H

> CH 0CH P(0)(OH)2

2

2

2

2

23

-CH (CH ) CH 0CH P(0)(OH)2 -CH CH(0CH P(0)(OH)2 >CH(OH)CH 0H

24

-CH C(CH )(0CH P(0)(OH) )CH 0H

25

-CH CH(0CH P(0)(OH) )CH(OH)CH

26

-CH CH CH(0CH P(0)(OH) )CH 0H

27

-CH CH(0CH )CH 0CH P(0)(OH)

28

-CH CH CH(OH)CH 0CH P(0)(OH)

29

-CH^CH(OH)CH(OH)CH 0CH P(0)(OH)

30

-CH2CH(0H)CH(CH )0CH P(0)(0H)

31

-CH(CH 0H)CH 0CH P(0)(OH)

32

-CH(CH )CH(OH)CH 0CH P(0)(OH)

33

- C H ( C

34

-CH2OCH2CH2OCH2P(0)(OH)2

2

2

3

2

2

2

2

2

2

3

2

2

2

2

2

2

2

3

2

2

2

2

2

2

2

f i e d

any

can

be

an

concluded

methoxy

group

a c t i v i t y

o f

presence rus

i s

o f

not

compounds The

2

plays

and o f

d e r i v a t i v e s base

on

to

from

the

t h e

C34,383-

atom

o f

r o l e

n u c l e o t i d e

atom as

i n

t h e

the

homologs

c h a i n

oxygen

2

with

t h e i r

determining

at i n

the i n

t h e

t h e

o r

Thus,

i t

a n t i v i r a l

analogs. by

s i m p l i ­ with

phosphonyl-

v i c i n i t y

witnessed

l i m i t e d

the t h e

our

s i d e - c h a i n design

o f

subsequent

base-modified

The o f

sole

phospho­

i n a c t i v i t y

o f

analogs

defined a c y c l i c

s t u d i e s o f

t o

HPMPA

and

a

narrow

n u c l e o t i d e t h e

inves­

PMEA.

N-(3-Hydroxy-2-phosphonylmethoxypropyl)

o f

h e t e r o c y c l i c

i n v e s t i g a t e

the

d e r i v a t i v e s

2

2

C 413-

margin

Base-modi f i e d order

a

oxygen and

2

observed

with

i n

a c y c l i c

m o d i f i c a t i o n

t i g a t i o n

o r

t h e

s u f f i c i e n t , C403

2

2

2

2

a c t i v i t y

that

2

)CH(0H)CH 0CH P(0)(0H)

λ

linkage

t h e t h e

s t r u c t u r a l analogs

1

C36,373

ether

2

2

a n t i v i r a l

molecules

without

H

2

2

3

3

3

2

2

2

6

2

2

2

2

there

In

i s

C313).

a n t i v i r a l o f

racemic

t h e

type

the

bases

(HPMP-derivatives)-

effect

a c t i v i t y , C143 were

o f

purine

t h e

h e t e r o c y c l i c

and

s y n t h e s i z e d

N-(2,3-dihydroxypropy1)

p y r i m i d i n e at

f i r s t

d e r i v a t i v e s

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

58

NUCLEOTIDE

Table

2.

9-(Phosphony1alky1)adenines

No.

Side

35

- C H

0 C H

2

Chain

C H

2

2

P ( 0 ) ( 0 H )

2

36

- C H

2

P ( 0 ) ( O H )

37

- C H

2

C H

38

- C H

39

~CH CH(0H)CH(0H)CH P(0)(0H)

40

"CH

41

)

2

2

2

P ( 0 > ( 0 H )

2

2

CH(0H)P(0)(0H)

2

2

a c c e s s i b l e

(24).

bed

by

The Scheme

2

by

the used

2.

those

In

i n v e s t i g a t i o n

confirme

ty,

2 ' - i s o m e r

the a

v i a

pure

route

which

a f f o r d s

s p e c i f i c a l l y B-CH

2

followed

2

cases

of

h e t e r o c y c l i c

e x a c t l y where

u n e q u i v o c a l l y

p r o t e c t e d

-CH-CH

2

a l k y l a t i o n

method

2

2

-CH CH CH(OH)Ρ(0)(OH)

e a s i l y

by

P ( 0 ) ( 0 H )

2

( C H

2

2

2

ses

ANALOGUES

the

the

i n t e r m e d i a t e s

0H

»

that

ba­

d e s c r i ­

p r e l i m i n a r y

s i n g l e

isomer

(Scheme

3)«.

B-CH -CH-CH 0DWTr 2

2

ι

OH

. OH

B-CH CH-CH 0H 2

«

2

«

B-CH CH-CH OH 9

I

2

I

0 C H

2

P ( 0 ) ( 0 H )

OBz

2

43

42

Scheme 3 ( B z . . . b e n z o y l , T h i s

method

use

of

group

a

at

and

developed

s p e c i f i c the

zoylated)

d e t r i t y l a t i o n

r i d e und the

a f f o r d

by

T h i s

r e a c t i o n

purine,

guanine c y t o s i n e

of

r e q u i r e d cause

to

was

e l i m i n a t e

the

to

d e r i v a t i v e s ,

u r a c i l

I.,

Dvorékovâ,

H .

in

The

compounds

C433

p r e s s ) . are

Another bed

in

the

- d i t r i t y l phosphorus a f f o r d s

l i s t e d

in

s t r a t e g y

for

l i t e r a t u r e d e r i v a t i v e synthon

HPMPA

on

the

C43

:

compo­

a p p l i e d

whereas

t h i s

for

2 , 6 - d i a m i n o i n

the

procedure

treatment

which

d e r i v a t i v e

prepared

was would

(Holy,

Α.,

by

the

above

pro

3.

p r e p a r a t i o n t h i s

(S)-DHPA a c c o r d i n g

subsequent

a f f o r d s

Collect•Czech•Chem.Commun.

Table

(27.)· of

of

on

d i c h l o -

a l k a l i

2-aminopurine,

Rosenberg, cedures

which

r e q u i r e d

s u c c e s s f u l l y

a l k a l i n e

the

the

(ben-

b e n z o y l a t i o n C423

aqueous

m o d i f i c a t i o n

makes

b a s e - p r o t e c t e d

subsequent

with

other

a

deamination

a

(250

d i m e t h o x y t r i t y l

2-0-benzoate

adenine,

and

HPMPA

the

debenzoy1 at ion)

of

case

of

group)

chloromethylphosphony1

r e a c t i o n

p r e p a r a t i o n

for

by

The

the

with

simultaneous

C433-

hydroxyl

m a t e r i a l .

treatment

followed

(under

o r i g i n a l l y

p r o t e c t i o n

primary

s t a r t i n g

s u c c e s s i v e

D W T r . . . d i m e t h o x y t r i t y l

of

HPMPA

route which to

treatment

was

s t a r t s

d e s c r i -

from

r e a c t s

the of

Scheme the

N

with 1

6

, 0

3

the and

p r o t e c t e d

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

-

Phosphonylmethyl Ethers ofNucleosides

4. HOLY ET AL.

59

i n t e r m e d i a t e w i t h b r o m o t r i m e t h y l s i l a n e and aqueous a c i d S i m i l a r method makes use o f N - b e n z o y 1 - 3 * - O - t r i t y 1 - ( S ) -DHPA a s a s t a r t i n g m a t e r i a l ( H o l y , Α., R o s e n b e r g , I . , Dvorâkovâ, H. Co11ect.Czech.Chem•Commun - i n p r e s s ) . An a l t e r n a t i v e g e n e r a l p r o c e d u r e f o r t h e s y n t h e s i s o f H P M P - d e r i v a t i v e s i s b a s e d on a l k y l a t i o n o f t h e h e t e r o c y c l i c bases w i t h c h i r a l synthons which possess a preformed s t r u c t u r e o f t h e s i d e - c h a i n . Such a method was u s e d f o r t h e s y n t h e s i s o f HPMPC; t h e key-compound, t o s y l a t e (or mesylate) of d i e t h y l (2S)-(3-benzyloxy-1-hydr o x y - 2 - p r o p o x y ) m e t h y l p h o s p h o n a t e was p r e p a r e d f r o m ( R ) -glycerol a c e t o n i d e and s y n t h o n C43 by a multi-step p r o c e d u r e (28)· 6

:

N-(2-Phosphony1methoxvethvl) D e r i v a t i v e s o f H e t e r o c y c l i c Bases ( P M E - D e r i v a t i v e s ) The o r i g i n a l p r o c e d u r e f o r t h e p r e p a r a t i o n o f compoun the treatment o f 9-(2-hydroxyethyl)adenin t h o n C43 and s u b s e q u e n t h y d r o l y s i s ( 2 9 ) was r e p l a c e d by an a l t e r n a t i v e p r o c e d u r e r e p r e s e n t e d i n Scheme 4. C1CH CH 0CH C1 2

2

m» C 1 C H C H 0 C H P ( 0 ) ( 0 C H )

2

2

2

2

2

5

2

44

B-CH CH OCH P(0)(0H) 46 2

2

2

2

~+

B-CH CH 0CH P(0)(0C H ) 45 2

2

2

2

5

2

Scheme 4

The c h l o r o m e t h y l a t i o n o f 2 - c h l o r o e t h a n o l by 1 , 3 , 5 - t r i oxane/HCl a f f o r d s an i n t e r m e d i a t e w h i c h i n an A r b u z o v reaction with t r i e t h y l phosphite unequivocally gives the s t a b l e phosphorus-synthon C443 ( t h i s s y n t h e s i s i s more f a v o r a b l e t h a n t h e a l t e r n a t i v e r o u t e s t a r t i n g from d i e t hyl 2 - h y d r o x y e t h o x y m e t h y l p h o s p h o n a t e (29.))Alkylation o f t h e h e t e r o c y l i c b a s e ( p r e f e r a b l y i t s s o d i u m s a l t ) by t h i s r e a g e n t a f f o r d s compound C453 w h i c h i s e a s i l y c o n v e r t e d t o t h e f i n a l p r o d u c t C463by b r o m o t r i m e t h y l s i l a n e t r e a t m e n t (Holy,Α., R o s e n b e r g , I . , Dvofâkovâ,H.: C o l l e c t . Czech.Chem.Commun.. i n p r e s s ) . The b a s e - m o d i f i e d PME-der i v a t i v e s C463which were o b t a i n e d by t h i s p r o c e d u r e a r e l i s t e d i n T a b l e 3. Anti v i r al

acti v ity.

HPMPA i s e f f e c t i v e a g a i n s t a l l m a j o r herpesviruses at c o n c e n t r a t i o n s which a r e f a r below t h e t o x i c i t y f o r t h e host c e l l s (23.). I t s potent a c t i v i t y was d e m o n s t r a t e d not o n l y w i t h HSV-1 and HSV-2; a l s o a n i m a l h e r p e s v i r u s e s , e . g . s u i d , b o v i d and e q u i d h e r p e s v i r u s e s t y p e 1 ( 2 3 ) as w e l l as s e a l herpes v i r u s (30) a r e s e n s i t i v e towards the a c t i o n o f t h i s a n a l o g . E p s t e i n - B a r r v i r u s ( 3j_) and all strains o f v a r i c e l l a - z o s t e r v i r u s e s and c y t o m e g a l o viruses t e s t e d s o f a r were a l s o effectively inhibited (23.32.33).Most importantly,HPMPA i s equally active against w i l d - t y p e i T K * ) a n d TK" mutant s t r a i n s o f herpes

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

60

NUCUXmDE

v i r u s e s kedly

In

that

d i f f e r

r e s p e c t ,

from

a c t i v i t y

of

encoded)

thymidine

which

Poxviruses fever

pond

very

(32,)

a

congeners

anti-HSV

23.),

and

to

HPMPA ASFV

was

mar­

agents by

the

( v i r u s -

i r i d o v i r u s e s

adenoviruses

(35.)

treatment.

(34.)

a

In

HPMP-

formula

r e s ­

some

s e l e c t i v i t y

and

B-CH

2

cases,

index

P M E - d e r i v a t i v e s

CH(R)OCH

2

P(0)(OH)

of

4

of

the

2

Abbrev.

C46 3

Abbrev.

( R =H)

(R= C H 0 H )

)

( A f r i c a n a l s o

achieved.

C433 Β

i t s

of

p h o s p h o r y l a t i o n

v i r u s ,

Base-modified general

on

34.) or

magnitude

3.

and

k i n a s e .

v i r u s ,

VZV

of

Table

depends

markedly

with

orders

HPMPA

majority

( v a c c i n i a

swine e.g.

the

ANALOGUES

2

Adenine 2-Aminoadenine

43b

2-Methyladenine

43c

2 - M e t h y l t h i o a d e n i n e

43d

N 6 - D i met h y 1 a d e n i ne

43e

6 - H y d r a z i n o p u r i n e

43f

6-Hydroxylaminopurine

43g

PMEDAP

46b

HPMPDAP

46d 46f 46h

6 - M e t h y l t h i o p u r i n e Hypoxanthine

43i

HPMPHx

46i

PMEHx

2-Aminopurine

43j

HPMPMAP

46j

PMEMAP

Guanine

43k

HPMPG

46k

PMEG

Isoguanine

431

461

U r a c i l

43m

HPMPU

46m

PMEU

Thymi ne

43n

HPMPT

46n

PMET

C y t o s i n e

43o

HPMPC

46o

PMEC

5 - M e t h y l c y t o s i n e

43p

5-F1uorourac

43q

a

^

In

a d d i t i o n

2,

same

to

C43k3

potent

and

against

these

purine

C43g3

- 3 - y l

or

s t i t u t e d were

ve

with

C43p3-

C43n3

v i r u s e s

was

the

of

herpes

the

v a c c i n i a

of

a n t i v i r a l

6

the

as

in

43f 3 of

well

analogs

HPMPA as

i t s

p y r i m i d i n e

HPMPC

i n a c t i v i t y

of

5 - m e t h y l c y t o s i n e

d e r i v a t i v e s

are

C

4 3 i 3 ,

v i r t u a l l y

2-subC43e3

u r a c i l

C43o3 C

s h a r p l y

43m3

s t r i c t

s e r i e s

of

or

of

(adenin-

d e r i v a t i v e

a c t i v i t y

Hypoxanthine

v i r u s .

a c t i v i t y

a c t i v i t y . S i m i l a r

the

1

The

6-hydroxylamino­

C

- s u b s t i t u t e d

encountered

4)

a n t i v i r a l

by

C43J3)

N

type

against

isomers

or

v i r u s e s (Table

marked

43b3,

emerged

HSV-1(32)

shown

hand,

C

d e r i v a t i v e s

6 - h y d r a z i n o p u r i n e

devoid

marked

2-aminoadenine

43o3

a c t i v e

s t i l l

C43c,d,13

the

t r a s t s

a l s o

other

i s

C

against

2-aminopurin-9-y1

s p e c i f i c i t y

i t s

s t r a i n

but

and

the

t o t a l l y

which

are

l e s s ,

On

C43a3,

agents

res i due.

p u r i n - 9 - y l

c y t o s i n e

TK"mutant

compounds

HPMPA.

HPMPA and

a n t i v i r a l

and

S l i g h t l y

or

p y r i m i d i n -1 - y l

B a s e . . .

guanine as

i1

46p

i n

c o n ­

d e r i v a t i ­ thymine

i n a c t i v e -

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Phosphonylmethyl Ethers ofNucleosides

4. H O L Y E T A L . Table

4.

A n t i v i r a l ves

o f

HPMP-

and

w i c

b

P M E - d e r i v a t i -

(33)

A b b r e v .

Compound

a c t i v i t y

61

a

)

43a

HPMPA HPMPDAP

)

0

( U g / m l ) W

HSV-2

HSV-1

43b

5

2

4

10

20

HSV-1

T K "

2

0.7

10

2

43c

>400

>400

>400

>400

43d

>400

>400

>400

>400

43e

>400

>400

>400

>400

43f

70

70

20

40

43g

10

20

7

7

>400

>400

>400

>400

100

200

150

>400

43i

HPMPHx

43j

HPMPMAP

43k

HPMPG

431 43m

HPMPU

>400

>400

>400

>400

43n

HPMPT

70

70

300

>400

43o

HPMPC

4

10

4

2

43p

>400

>400

>400

>400

43q

300

300

>400

>300

150

7

46a

PMEA

7

7

46b

PMEDAP

2

0 ., 7

1

>400

>400

>400

46d

>400

46f

>400

>400

>400

>400

46h

>400

>400

>400

>400

>400

>400

>400

>400

70

10

>200

150

46i

PMEHx

46j

PMEMAP

46k

PMEG

c

)

0.2

0.2

0. 2

7

7

70

10

461

1

46m

PMEU

>400

>400

>400

>400

46n

PMET

>400

>400

>400

>400

46o

PMEC

>400

>400

>400

>400

>300

>400

>400

>300

46p a

20

^ A b b r e v . ,

r e q u i r e d primary lues

see to

rabbit

for

(KOS,

Lyons)

and

v a r i a n t s

The

same

nine,

two

the four to

5

to

other

A l l

the

Minimum

i n h i b .

induced

c y t o p a t h o g e n i c i t y

c u l t u r e s

c e l l s

kinase

at

W

DNA

compounds

ous

s t r a i n s

and

adenoviruses.

Average

type

1

s t r a i n s

d e f i c i e n t

i . e .

i n v a -

(HSV-1) (G,

(TK")

v i r u s .

adenine,

d e r i v a t i v e s ,

t h e i r

The

196, HSV-1

^ C y t o -

d e r i v a t i v e s

data l i s t e d

are o f

proved

i n -

summarized with i n

i n h i b i t o r y

v i r u s ,

comparison

2-aminoade-

a l s o

s u p e r i o r i t y

mentioned

v a r i c e l l a - z o s t e r The

50%.

v a c c i n i a

:

v i r u s e s .

demonstrate

HSV-2

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

>4ug/ml.

HPMP-compounds, c y t o s i n e

by

v i r u s

three

VMW-1837).

other

o f

)

simplex

thymidine

base-modified

four

c e l l

herpes

and

(33.)

D

M c l n t y r e ) ,

host

guanine

h i b i t o r y Table

F,

(B2006,

for

3; v i r u s

kidney

three

s t r a i n s

t o x i c

Table

reduce

i n

respect Table to

3.

v a r i -

cytomegaloviruses

s e l e c t i v i t y

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

indexes

62

NUCLEOTIDE

is

c l e a r l y

HPMPC

i s

C43k3

favor

though

pound A

i n

s u p e r i o r

of

very

t h i s

in

out

the

others

a c t i v e ,

was

VZV

i n

CMV

the

s t r u c t u r a l

the

to

i n a c t i v e

be

c y t o s i n e

C46a3,

C46k3

d e r i v a t i v e s

about

e q u a l l y

and

whereas HPMPG

c y t o t o x i c

com-

i n

d e r i v a t i v e

as

an

emerged

as

of

the

the

(PMEC)

agent.

C

46b3

potent

and

t u r -

Thus,

and

only

guanine

a n t i v i r a l

HSV-1

a n t i -

P M E - s e r i e s :

C46o3

a n t i v i r a l

against

(Table

agents,

HSV-2

as

t h e i r

4).

a c t i v i t y

i v i r a l

dependence a l s o

of

HPMP-

PME - d é r i v â t

and

ives

Abbrev.

Compound

v z v

c

CMV

)

43m

HPMPU

0.33

15

C1673

2

2.8

C >1003

C453

100

>125

>400 C1 3

HPMPG

)

1

HPMPHx

43k

e

C >200 3

HPMPDAP

43 i

A V

)

C1333

C10003 43b

d

0.

0.02

HPMPA

43a

0.07 M C>1

C>4 3

C1 1

0.

2.6

15

43 3

C673

C2.73

300

25

>125

.33

C163 100

C1 3 >125

43n

HPMPT

70 C>6 3

C>4 3

C1 3

43o

HPMPC

0.25

0.08

3.4

C6253

C>373 >125

C200 3

46b

PMEDAP

46j

PMEMAP PMEG

46k

)

i n

t i o

10

25

C103

C43

2

10

>125

C203

C43

C<0.53

150

200

>125

C >2 3

C >2 3

M

0.05

0.25

2.7

C503

C103

CO.933

PMEA

46a

a

AV,

i n h i b i t i o n .

most

2-aminoadenine

a c t i v e

HPMP-counterparts

5. Ant

i s

for

encountered

case,

adenine

Table

HPMPA

the

s i m i l a r

a c t i v i t y

t h i s

ned

of

s e r i e s .

s t r i k i n g l y

v i r a l

to

ANALOGUES

human of

v i r a l

embryonic

minimum

lung

c y t o t o x i c

c o n c e n t r a t i o n ;

c e l l a - z o s t e r s t r a i n s

v i r u s

(07-1,

σ

(CMV)

three

adenovirus

s t r a i n s (AV)

d

)

b

)

s e l e c t i v i t y

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

a v e r a g e

value

( V Z V ) s t r a i n s

YSR);

v i r u s

c e l l s ;

a v e r a g e

(Davis, types

value

AD169); and

to

for

(YS,0ka)

(2,3

C1 3

for e

)

i n d e x

minimum two

and

TK* two

two

a v e r a g e

:

3

r a -

a n t i v a r i ­

TK"

VZV

cytomegalo­ value

4).

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

for

4.

In

t h i s

s e r i e s ,

a l s o

d e r i v a t i v e

C4613

whereas

isomers

were to

the

much be

i s

less v i r u s

against

Another

by

foci of

at

and

i n

host

low

i v i t y

at

ably

as

r i v a t i v e against

i n f e r i o r

a c t i v i t y

^ - d e r i v a t i v e turned

(Table

5)

a c t i v i t y

o f

Also

to

that

PME-compounds

C463

i s

and

i t s

reduced

t h e i r

t h e i r

number

The

c l e a r l y against

c e l l s

i n f e c t e d

2-aminoadenine

the

of

d e r i v a t i v e

M S V - i n f e c t e d

s e l e c t i v i t y

guanine

indexes

d e r i v a t i v e

thes None

l i s t e d

of

i n

up

HPMPA

a c t i v e

the

as

C43b3

were

3

to

and

against

well

a d d i t i o n a l

Table

showed

at

In

MSV:

C43k3

or

the

least

10

times

than

m o d i f i e d

anti-MSV

c o n t r a s t

congeners

Moloney

HPMPG

base

any

200μΜ.

i t s

6 . A n t i - H I V

t h e i r

effect

a c t i v i t y on

of

were

the

parent

l e s s

and

c e l l

5

P M E - d e r i v a t i v e s

transformat (

0

M )

U

a

'

b

ion(34)

)

Abbrev. c

MSV

)

d

)

HPMPA

>125

C<13

>8

C<53

>125

C<13

6

C333

43 i

HPMPHx

>125

C<13

>200

43k

HPMPG

>25

C<13

46a

PMEA

2.0

C33 3

46d

PMEDAP

1.0

C183

0.6

46 i

PMEHx

>125

C<53

>200

46j

PMEMAP

4 5 . 0 0 2 8 3

46k

PMEG

3.8 0.47

ddCyd

:

the

c e l l s

v i a b i l i t y

dose against

r e q u i r e d HIV

determined

number

s e l e c t i v i t y C3H

de­

PME-counterparts

HPMPDAP

the

com­

e f f i c i e n t

43a

0

the

c o n s i d e r ­

43b

5

a c t -

to

2-aminopurine

t h e i r

HPMP-

MSV-induced

HIV

E D

PMEG

analo

E D

;

PMEG

6).

Compound

the

out and

t h e i r

of

a c t i v i t y

(C3H)

i o n s , w i t h

MSV-transformation

(Table

apparent

f i b r o b l a s t

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

less

Table

the

concentrât

P M E - d e r i v a t i v e s , pound,

Ν

c y t o t o x i c i t y ) .

i s

r e s p e c t i v e l y .

(36.).

PME-compounds

and

PMEA

However,

c e l l s

(the

CMV

which

2-aminopurine

e f f e c t s .

the

P M E - d e r i v a t i v e s

high

murine

MSV,

67,

and

adenoviruses

HPMP-series,

In

very and

The

4)

and

s i g n i f i c a n t l y

200

C46J3

a n t i v i r a l

s.

the

Moloney

C46b3

PMEA

i t s

(2-hydroxyadenine)

marked

against

VZV

item

from

r e t r o v i r u s e s . with

of

(Table

HPMP-count erpart d i f f e r

isoguanine

e f f i c i e n t .

c o u n t e r a c t e d

potency

the

d i s p l a y e d

i n e f f e c t i v e

v a c c i n i a

63

Phosphonylmethyl Ethers ofNucleosides

H O L Y ET AL.

of

5

days

MSV-induced

index

C33

to

CSI3(see

by

Table

1

C200 3 C673 C13

0.12C2.53 29

achieve

a f t e r

f o c i

C6.73

1.21C1.33

C1703

i n f e c t i o n

C13

6

50%

(as

C>6.93 p r o t e c t i o n

monitored

i n f e c t i o n ) o r 50%. 4 ) .

c

D

> i n

^ i n

by to

of

c e l l reduce

parentheses,

MT-4

c e l l s ;

c e l l s .

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

d

)

i n

64

NUCLEOTIDE

PMEA,

PMEG,

b i t o r s

of

PMEDAP

HIV

Complete

p r o t e c t i o n

PMEDAP.

The

in

c e l l s

ATH8

ive

same

was

MT-4

the

with

group

those

in

w i l l

be

v i v o

l e s s

than

important

to

at

for

apt

the

to

undergo

n u c l e o s i d e

f u r t h e r

pursue

most

5

a c t ­

value

0

of

and/or

e.g.

i t s

d é g r a d â t i v e as

are

a n t i v i r a i s ,

PMEA

a n t i v i r a l s , them

and

observed

P M E - d e r i v a t i v e s

other

Since

was E D

(36)•

PMEA

the

an

i n h i ­

6)

with

was

with

s e l e c t e d

reported

potent

(Table

p a t t e r n

:

of

be

5μΜ

HIV PMEDAP t e s t e d ,

2 ' , 3 ' - d i d e o x y r i b o n u c l e o s i d e s . logs

to

c e l l s

achieved

p o t e n c i e s

to

proved

i n

s t r u c t u r e - a c t i v i t y

of

(36).The

comparable

PMEMAP

i n f e c t e d

a n t i v i r a l

1.5μΜ

and

r e p l i c a t i o n

ANALOGUES

ana­

processes

it

might

p o t e n t i a l

seem

a n t i - H I V

agents. The

a n t i v i r a l

pounds

was

potency

f u r t h e r

i n

models:

HSV-1

k e r a t i t i s

(33.37).

HSV-1

i n f e c t i o

treatment), (38)

and

ments

HSV-1

demonstrate f e s t a t i o n s E

D

5 0

o

t i o n

in

50

AZT

Thus,

it

the

PMEG

mg/kg

( i . ρ . ) a g a i n s t

(compared mice (40.)

c e l l s ,

the μΜ

ne

the

the

of

of

The

(S)-C

1

HSV-2

have

i n f e c ­

a c y c l o v i r ) ( 3 9 ) . and

by

a s s o c i a t e d

90-100%

at

20-

of

PMEA

a n t i r e t r o v i r a l

f u r t h e r

in

of

explore

the

a n t i v i r a l s

for

humans.

was

HPMPA

c e l l χ

k i n a s e s . from

of

18 of

T h i s HPMPA

a f t e r in

L-1210

the

5x

HPMPA was

h

higher 6

c e l l

the

HEL

l i n e on in

i s o l a t e d with

15

mock-

used

(41).

the HEL

c e l l

l i ­

than

Vero

c e l l s . by

a l s o

confirmed

by

leukemia

of

c e l l s :

than

c a t a l y z e d

presence

pool

extent

i s

the

phospho­

treatment

c e l l

was mock-

5 ' - t r i p h o s ­

diphosphate

depended

pmole/10

mouse

i s

v i r u s - i n f e c t e d

of

It

m o c k - i n f e c t e d 6

and HSV-1

into

it

between

and

i n

with

(41.42)-

where

of

(HEL)

a c i d - s o l u b l e

mono-

pool

approximately

of

c e l l s

c o n c e n t r a t i o n to

a c t i o n

penetrates

and

higher

of c e l l s

i n f e c t e d

C3HPMPA

v i r u s -

i r r e s p e c t i v e

amounted

p h o s p h o r y l a t i o n

4

and

d i f f e r e n c e

i n

mode

embryonic

( r i b o n u c l e o s i d e

a n a l y s i s

p h o s p h o r y l a t i o n

pernatant

to

compound

some

2-2-5

c e l l s

n u c l e o t i d e

mani-

to

combination

c l a s s

and

human

diphosphate

i n t r a c e l l u l a r

The

for

g r e a t e r

i n f e c t i o n s

the

labeled

was

and

the

r e p o r t e d

alone.

new

v i r u s - i n f e c t e d

i t s

a c i d - s o l u b l e

c e l l s

t h i s

i n

p h o s p h o r y l a t i o n

it

a

metabolism

use

i n d i c a t e d

used;

i n

e x p e r i c l e a r l y

against i s

MSV

the

drugs

n o n - i n f e c t e d

that

a n a l o g ) .

HPMPA

with

ingl y,

The

(23.33)

systemic

mg/kg

mandatory of

v i r a l

both

and to

- i n f e c t e d The

seem

on

with

sum

from

the

performed

demonstrated

HPMPA

com-

animal

Studi es

Vero

f u r t h e r

of

treatment)

mice.

formation

r e s u l t s

of

p o t e n t i a l

were

phate

. Interest

of

50

tumor

i n o c u l a t e d

e i t h e r

HPMPA

- i n f e c t e d

with

suppressed

s t u d i e s

r y l a t e d

i n HPMPA

e f f e c t

B i ochemi c a l

(K0S),

with

i n f e c t i o n .

treatment

The

( t o p i c a l

p r o t e c t i v e

would

t e r a p e u t i c

i n f e c t i o n

performed

( R e t r o v i r )

than

r a b b i t s

v i r a l

i n

effect

s e r i e s several

marked

mg/kg/day

with

both with

a

PMEA

m o r t a l i t y

of v i v o

of

mice

Also,

i n

v i r u s

were

0.125

f

i n

an

v a c c i n i a

which

v i t r o

confirmed

of

c e l l s .

c e l l u l a r i n

100000 T h i s

v i t r o g

s u ­

phospho-

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

4.

r y l a t i o n

r e q u i r e s

s t i m u l a t e d

by

OTP

and

t h i s

r e a c t i o n

the

the

CTP

presence

being

l e s s

(41).

PMEA

c o n d i t i o n s ;however,the was

approximately i n h i b i t s

The

e f f e c t

i n c o r p o r a t i o n

o f

HSV-1- i n f e c t e d

i f

HPMPA

was

same

HPMPA.

A

a n t i v i r a l

i n h i b i t e d

that

r e q u i r e d

o n l y

porated drug DNA

a

DNA

DNA

Vero

pmole/10 r e f l e c t s

an

n a t i o n . HPMPA a l s o

The

The

l a b e l l e d L-1210

as

The

DNA

i s

low

as

suggests

a l s o

57μΜ

c y t o s t a t i c

a c t i v i t y

HSV-1

novo

Polymerase.

diphosphate proven

pted

to

phate me

with

used

that

r e f e r

the

the

by

The

the

c e l l s .

The

i n h i b i t o r y

s t r u c t u r a l l y

based

it

t e r m i ­ of

s y n t h e s i s

upon by

r a d i o ­

the

15.5μΜ

for

at

than

1

and CD-

4

c e l l

I C

5

on v a l ­

0

PMEA,

c e l ­

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

PMEA(43.)in

C

compounds

Whilst 50%

HPMPA of

leukemia

both

T h i s

the

d i s c r e p ­

case

of

i n h i b i t i o n

i n v o l v e d ,

at

t r a n s f o r m a t i o n o f

analog)

v i r a l

DNA

i n t e r a c t i o n

polymerase

purpose

DNA

mouse

s t r i k i n g

t r i p h o s p h a t e

DNA

0.5

whether

least

PMEA

of

DNA

i n

the

drugs.

i n h i b i t i o n

HSV-1

for

Hela

and

the

i n v e s t i g a t e

i n f e c t e d to

of

and

250μΜ

be

DNA

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

s y n t h e s i s

o f

o t h e r

might

(a

mentally us

and

or

exceed

c h a i n

v i r a l

L-1210

HPMPA

v i r a l

c e l l u l a r

not

DNA

of

c e l l u l a r equivalent

i n c o r p o r a t i o n

reduced

mechanisms

de

DNA

i n

the

d e t e c t i o n .

DNA

the

by

i n c o r ­

with

the

i n t o

or

i n ­

i n h i b i t i o n

d i s c e r n

i n h i b i t

e f f e c t s

for

HPMPA

to

of

f u l l y

into

does

determined.

was

s y n t h e s i s

i t s

than

(41)•

i n h i b i t e d

under

HPMPA and

process

c e l l u l a r by

i s

50%

c e l l s

of

l e v e l

p a r t i c u l a r l y

that

of

c e l l s

lower

treatment

i n c o r p o r a t i o n

the

o f

were

i s

HEL

c e l l s

i s

i n t r a c e l l u l a r

c y t o s t a t i c

185μΜ

0·5μΜ

terms

i n

i n c o r p o r a t i o n HEL

to

HPMPA

s y n t h e s i s

which

i n

i n f l u e n c e

d i f f i c u l t

followed

to

whereas at

s i g n i f i c a n t l y

24h

low

2 ' - d e o x y t h y m i d i n e

c e l l s

high

ancy

It

why

amounted

l u l a r

no

novo

i s

not

i n

extremely

i n h i b i t i o n

c u l t u r e .

μΜ

1000x

causes

i n c o r p o r a t i o n

below

a l s o

DNA

suppressed

only

Vero

used.

v i r a l

500

s y n t h e s i s

a f t e r

does

s u f f i c i e n t

f a l l s

was

(and

extremely i s

l i n e

into

of

i n

de

which

found

i s

e x p l a i n s

PMEA

DNA

i n t e r n u c l e o t i d i c

that

a c t i v i t y

ues

The

DNA

HPMPA

However,

c e l l s .

6

v i r a l

c e l l s ,

was

c e l l s

c e l l

r e p l i c a t i o n

s y n t h e s i s

c e l l u l a r

s y n t h e s i s ) .

s y n ­

c e l l s

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

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

c o n d i t i o n s . in

DNA

s y n t h e s i s

c e l l u l a r

(43)

(HSV-1)DNA

a l s

Vero

i n t o

at

v i r a l

In

HPMPA

observed

i n h i b i t i o n

where

c e l l u l a r

50%.

HEL

was

HSV-1

HPMPA

c o n d i t i o n s

h i b i t e d ,

i n

i n

under

t r a n s f o r m ­

of

completely

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

v i r u s

experiments

e n t a l

a

the

at

host

was

d i f f e r e n c e

for

v i r a l

the

achieved

a c t i v i t y :

was Our

at

donors

t h i s

that

2

was

s i m i l a r

of

than

C^ P3-orthophosphate c e l l s

i s GTP,

phosphorylated

e f f i c i e n t l y

Vero

r e a c t i o n

phosphate

e f f i c a c y

on

the

system(41.43)

was

lower

depends of

added

e f f e c t

5x

very

The

the

ATP;

e f f i c i e n t

A l s o ,

a t i o n

t h e s i s .

o f

A T P - r e g e n e r a t i n g

these

HPMPA

65

Phosphonylmethyl Ethers ofNucleosides

HOLY ET AL.

was

i n

r e s u l t s a c t i v i t i e s

r e l a t e d

(44.)from

HPMPA

diphosphates

o f

into

e x p e r i ­

t h i s

presented of

HPMPA the

s y n t h e s i s of

v i t r o

p u r i f i e d

of

and

prom­ d i p h o s ­

The

enzy­

HSV-1

(KOS)

in

Table

7

diphosphate N - ( 2 - p h o s -

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

66

N U C L E O T I D E ANALOGUES

phonylmethoxyethyl) ne bases· These

r e s u l t s

demonstrate

c o m p a r a t i v e l y (

m

K

/

K

i

HSV-1

weak

from

the

t h e i r

r e l a t i v e

^m'^i

v a l u e s

h i g h e r

enzyme

ever,

HPMPA

e x p l a i n e d

by

ion

to PMEA

Table

most

i s

the

7.

o f

a

more

o f

for

HPMPA

e f f i c i e n t T h i s

d i f f e r e n c e

i n

corresponded

The

i n d i c a t e d

against

the

diphosphate.

How­

of

c o u l d

the

extent

i s

h i g h e r

which

to

HSV-1.

s e r i e s

i n h i b i t o r

d i s c r e p a n c y

for

diphosphates

against

t h i s

a

a c t i v i t y

i n h i b i t o r s

the

i s

polymerase

the

C473

o f

p y r i m i d i -

DNA

i n h i b i t o r y

s e r i e s

these

noted

and

diphosphate

v i r a l

e f f i c i e n c y

o f

diphosphate

(vide

HPMPA

of

compounds

PMEA. a

purine

P M E - d e r i v a t i v e s

than

than

the

a n t i v i r a l

o f

p l i c a t i o n

order

i n

e f f i c i e n c y

v i r a l

for

The

polymerase

of

that

i n h i b i t o r

v a l u e : 0 - 6 3 ) DNA

d e r i v e d

a

d e r i v a t i v e s

HSV-1

r e ­

p o s s i b l y

of

be

t r a n s f o r m a t ­

for

HPMPA

than

supra)•

I n h i b i t i o

n u c l e o s i d e

t r i p h o s p h a t e

analogs

Com-

C o m p e t i t i v e

pound*)

Substrate

C47,483 K

Ki

m

(μΜ)

/K

^m i

(μΜ)

PMEApp

dATP

0 .. 7 3

o..

PMEGpp

dGTP

1 .. 1 2

0. 090

PMECpp

dCTP

0 .. 9 0

1 . 27

PMETpp

dTTP

1 .. 2 5

1 . 01

1 .. 2 4

PMEUpp

dTTP

1 .. 2 5

5.. 9 0

PMEDAPpp

dATP

o..

o.

o.. 2 1

HPMPApp

dATP

0 .. 7 3

AZT-TP

dTTP

1 .. 2 5

3 2 7 ..0

ddTTP

dTTP

1 ,. 2 5

21 . 1

)

A b b r e v i a t i o n s ,

see

Table

R i b o n u c l e o t i d e

Reductase.

which

p l a y s

important

c y c l e

and

s p e c i f i c

an

a f f e c t s

i t s

s t e r i c a l l y

r e g u l a t e d

(dATP,

and

by

ATP

c o m p e t i t i o n

t i c

s i t e .

s i b l e

It

t a r g e t s

HPMP-

and

the

Table

8

zyme

for

by

REF

(46.).

or

demonstrate mono-

and

P M E - d e r i v a t i v e s

most

free

P a r t i a l l y of

was

s i g n i f i c a n t

diphosphates C473-

The

r e s i d u e

i s

HSV-1 ( r i b o enzyme

not

a l l o -

5 ' - t r i p h o s p h a t e s probably the

one

of

data

pos­

of

v i r a l

the r i b o ­

(according from

HSV-1

summarized

i n h i b i t i o n i s

c a t a l y ­ the

analogs

enzyme

d e r i v e d

r e g u l a t e d

common

i s o l a t e d

enzyme

the

v i r a l

it

p u r i f i e d

The

enzyme

r e p l i c a t i o n i s

The

as

c e l l u l a r

c e l l s .

o.. 0 0 4 o.. 0 6

reductase

n u c l e o t i d e

c r i t e r i a ) Vero

for

regarded

a c y c l i c

reductase

i n f e c t e d

but

t h e r e f o r e

immunological

(K0S)

and

was

s y n t h e s i s

s u b s t r a t e s

o.. 5 1

v i r u s

n u c l e o s i d e

(45),

the

PME-type

n u c l e o t i d e to

dTTP) of

the

c o u n t e r p a r t :

by

2 5 .. 1

029

v i r u s - i n d u c e d

i n

r e d u c t a s e ) .

e u k a r y o t i c

1 .. 4 1

diphosphate

r i b o n u c l e o t i d e

5 ' - d i p h o s p h a t e

from

p p . . .

DNA

1 2 .. 4

1 .. 4 2

Another r o l e

v i r a l

( v i r u s - e n c o d e d )

n u c l e o s i d e d i f f e r s

the

3;

73

6 .. 9 5

105

o f

t h i s

i n e n ­

from

HPMP-C48D

not

modulated

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

4.

by

n a t u r a l

t e s

and

2 ' - d e o x y not

(data

67

Phosphonylmethyl Ethers ofNucleosides

H O L Y E T AL.

5

r i b o n u c l e o s i d e

1

- t r i p h o s p h a

shown)· B-CH

2

CH

2

OCH

2

0

0

0

P-0 OH

0-P-OH OH OH

47 0

0

Q

0-P-OH

B-CH ÇH-OCH P-0 2

2

CH 0H

Table

8.

OH

OH

OH 48

2

encoded

r i b o n u c l e o t i d e

In h i b i t i o n

of

HSV-1

r e d u c t i o n

of

r i b o n u c l eot ide

reductase

IC 50

Compound * 0

5

- d i p h o s p h a t e s

1

3

tuM]

CD PMEA

96

2 6 . 0

4. 6

PMEApp PMEGp

2 5 . 7

)

ND

ND

1460

ND

ND

180

ND

ND

ND

ND

>2000

ND

ND

1600

ND

ND

>2000

ND

ND

PMEGpp

19. 2

PMEDAPpp PMECpp PMETp

σ

>2000

PMEDAPp PMECp

ND 86*>

ND<*> 320C)

>2000

PMEAp

ND

ND

PMEUp

>2000

ND

ND

PMEUpp

>2000

ND

PMETpp

1 1

480^ )

HPMPAp

a

)

CSD=16

phate, not

μΜ;

D

Reverse

T r a n s c r i p t a s e .

tase

-

a

The

assay

key

the for

as

depended

the

and

3;

C S 3

p . . .

=

22

)

monophos­ μΜ;

upon

order >

of

u r a c i l was

ddTTP

=

while

The

d

)

N D . . .

the

compounds.

c h a r a c t e r

of

thymine.

>

The

than

adenine

diphosphates reverse

are

we

have C473

t r a n s c r i p ­

m u l t i p l i c a t i o n reverse 2

- 1 8

a c t i v i t y

the

[463

Therefore

(47)•

d e t e r g e n t - d i s r u p t e d

compared

i n h i b i t

the

the

o l i g o ( d T ) ^

was

potent

o f

towards

o f

2-aminoadenine

more

P M E - d e r i v a t i v e s

with

source

r e f e r e n c e C473

C473-

)

r e t r o v i r u s

the

diphosphates

ddTTP

for

r e a c t i o n .

diphosphates

s i n e

G

c

0.5C )

)

(MSV,HIV).

a f f i n i t y

performed

as

template

Several

compounds

enzyme

was

t r a n s c r i p t i o n t i o n e d

the

these

r e t r o v i r i o n s

the

C

Table

r e s i d u e ;

r e t r o v i r u s e s

i n v e s t i g a t e d from

and

A b b r e v i a t i o n s , s e e

against

d e r i v e d

and

5

1 8 . 0

determined.

a c t i v e a l s o

c

p p . . - d i p h o s p h a t e

)

ND 3 7 0

>2000 >

0 . 9c )

HPMPApp

C

-primed

of

with It

the

was

the

found

and >

AZT

d e r i v a t i v e

that

decreased

guanine 5

the

i n h i b i t i o n

2-aminoadenine

e i t h e r

men­

t r i p h o s p h a t e

the

base

adenine

reverse

above

AZT

enzyme;

AMV

t r a n s c r i p t a s e

>

in

c y t o ­

d e r i v a t i v e

' - t r i p h o s p h a t e

(PMEA

diphospha-

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

NUCLEOTIDE

68 te)

h a d approximately

rence

compounds

i n h i b i t e d t i o n s

both

Table

9 .

9 ) .

potency

by

r e f e ­

compound r e a c ­

t r a n s c r i p t a s e . t r a n s c r i p t a s e

reverse

o f AMV

5 ' - t r i p h o s p h a t e

n u c l e o s i d e

o f

t h e two

mentioned

a n d DNA-dependent

t h e reverse

I n h i b i t i o n

analogs

a s

The last

t h e RNA-dependent

c a t a l y z e d

a c y c l i c

t h e same

(Table

ANALOGUES

Compound *) 3

I

( μ Μ )

5 0

C

min

3

ο

by

C483

)

5 m i n 0 . 18

PMEDAPpp

o. 2 3

PMEApp

1 .. 3 5

1 .00

PMEGpp

2 .50

2. 10

PWETpp

3 .60

3.25

AZT-TP

1 .05

1 . 13

ddTTP

1 .50

1 .00

PHEUpp

a

)

A b b r e v i a t i o n s , s e e

c e n t r a t i o n p o r a t i o n

Footnote

c a u s i n g

i n t o

i n c u b a t i o n

o f

Table

50% d e p r e s s i o n

t h e growing

8 .

o f

D

)

I n h i b i t o r

l a b e l l e d

DNA c h a i n ,

a f t e r

NTP

a

3

c o n ­ i n c o r ­

o r

5 m i n

time.

Conclusion Two

groups

analogs

o f

b i o l o g i c a l l y

which

r e s u l t e d

i n v e s t i g a t i o n general

c a n f o r m a l l y

formula B - C H

2

a c t i v e

from be

a c y c l i c

o u r

n u c l e o t i d e

s t r u c t u r e - a c t i v i t y

r e p r e s e n t e d

by

a

s i n g l e

C493:

C H ( R ) - 0 C H

2

P ( 0 ) ( 0 H )

C463 R C433 R

2

H (

S)-CH 0H 2

C493 Nevertheless, t h e i r as

well

a s

systems,

o n

i n c l u d i n g and

t h e i r

The

o f

t h e understanding

o f

i n

may

analogs,

enzyme

d i f f e r e n t

which

excludes

s t r u c t u r e s , t o

these

t h e i m ­

molecules

T h e c o m p a r a t i v e l y bases

p r o t o n a t i o n o f

i s o l a t e d

p o i n t s

o f

c o n t a i n i n g

might

t h e i r

o n

a c t i v i t y ,

a c t by

margin

f e a t u r e s

arrangement.

capable

a v a i l a b l e

t h e parent

h e t e r o c y c l i c

lead i c a l

o f

a n d carba

mutual f o r

f a r

a n t i v i r a l

e f f e c t s

s t r u c t u r a l

group(s) f o r

s o

groups

v a r i a t i o n s

t h e s t r u c t u r a l

t h e i r

s e l e c t i v i t y

both

narrow

i s o s t e r s o f

a r e

i n v i t r o

that

t h e smallest

portance

which

a n d i n p a r t i c u l a r

suggest

mechanisms. even

t h e data

b i o l o g i c a l ,

be

a n

behavior

high amino

important i n

b i o l o g ­

systems. P e n e t r a t i o n

l i m i t e d . T h i s between c e l l

these perhaps

t h e a c t i v i t i e s

systems.

prodrugs, i t a t e d

o f

fact

F u r t h e r

i n p a r t i c u l a r

t r a n s p o r t ,

might

compounds e x p l a i n s i n

i n t o

i s o l a t e d

development with lead

new

c e l l s

i s

d i s c r e p a n c i e s

enzyme i n t h e

p r o t r a c t e d t o

l i v i n g

c e r t a i n

a n d

i n t a c t

d i r e c t i o n

a c t i o n

avenues

o f

o r

f a c i l ­

f o r

t h e r a -

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

4.

p e u t i c point

a p p l i c a t i o n . t o

s e r i e s

a

HPMPG),

a n

analogs

might

q u i t e

HPMPA),

i n c r e a s e d well

be

t h e animal

lower

than

o f

t o x i c i t y

i n t r a c e l l u l a r a

e x h a u s t i v e .

- c h a i n

m o d i f i e d o r

n u c l e o t i d e

developments

i n t h i s

adenine (PMEG,

c o n c e n t r a t i o n were

p o s s i b l e

that

n u c l e o t i d e

p r o p e r t i e s .

mode

t h e

s e r i e s

o f t h e

disadvantage.

i s

a c y c l i c

o t h e r

b i o c h e m i c a l

a c y c l i c

It

experiments o f

t h e guanine

s t r u c t u r e - a c t i v i t y s t u d i e s

a n t i v i r a l the

Although

c o m p a r a t i v e l y

(PMEA,

Our

69

Phosphonylmethyl Ethers of Nucleosides

H O L Y ET AL.

o f

a c t i o n

analogs new

A

might

n e c e s s a r i l y a d d i t i o n a l

analogs

b e t t e r o f

might

e x h i b i t

u n d e r s t a n d i n g

t h e two

h e l p

not

s i d e -

t o

c l a s s e s

f o s t e r

o f o f

f u r t h e r

f i e l d .

Literature cited 1. Robins,R.K. Pharm.Res- 1984, 11-18. 2. Scheit,K.H. Nucleotid New York, 1980 3. Holý,A.in Phosphorus Chemistry D i r e c t e d Towards B i o logy; Stec,W.J.,Ed.;Pergamon Press-Oxford,1980,pp. 53-64. 4. Holý, Α., Rosenberg,I. Collect.Czech.Chem.Commun. 1982, 47, 3447-3463. 5. Rosenberg,I., Holý,A. Collect.Czech.Chem.Commun. 1987, 52, 2572-2588. 6. Rosenberg,I., Holý,A. Collect·Czech.Chem.Commun. 1985, 50, 1507-1513. 7. Vesely,J.,Rosenberg,I.,Holý,A. Collect.Czech.Chem. Commun. 1982, 47, 3464-3469. 8. Vesely,J.,Rosenberg,I.,Holý,A. Collect.Czech.Chem. Commun. 1983, 48, 1783-1787. 9. Holý,A.,Nishizawa,M.,Rosenberg,I.,Votruba,I. C o l lect.Czech.Chem.Commun. 1987, 52, 3042-3057. 10. Horskà,Κ.,Rosenberg,I.,Holý,A.,šebesta,Κ. Collect. Czech.Chem.Commun. 1983, 48, 1352-1357. 11. Horskà, K., Cvekl,A.,šebesta,K.,Rosenberg,I., Holý,A. Abstr.14th IUB Congress. F r 355; Prague,1988. 12. Shannon,w.m. i n A n t i v i r a l Agents and V i r a l Diseases of Han: Galasso,G.J., Merigan,Τ.C., Buchanan,R.A., Eds.; Raven Press: New York, 1984; pp.55-121. 13. Robins,R.K., Revankar,G.R. i n Antiviral Drug Deve­ lopment. A M u l t i d i s c i p l i n a r y Approach; De C l e r c q , E . , Walker,R.T.,Eds.; Plenum Press: New York, 1988; pp. 11-36. 14. Holý,A. i n Approaches t o A n t i v i r a l Agents: Harnden, M.R., Ed.; Macmillan Press: Basingstoke, 1985; pp. 101-134. 15. De Clercq,Ε.,Holý,A. J.Med.Chem. 1979, 22, 510-513. 16. De C l e r c q , E . Nucleosides&Nucleotides 1985, 4, 3-11. 17. Tolman,R.L., Field,A.K., Karkas,J.D., Wagner,A.F., Germershausen,J., Crumpacker,C., Scolnick,Ε.M. Bio­ chem.Biophys.Res.Commun. 1985, 128, 1329-1335. 18. Hutchinson,D.W., Naylor,M. N u c l e i c Acids Res. 1985, 13, 8519-8530. 19. Holý,A. Chemica S c r i p t a 1986, 26, 83-89.

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

70

NUCLEOTIDE

20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43.

ANALOGUES

Votruba.I., Η ο l ý , Α . , De Clercq.E. Acta V i r o l . 1983, 27, 273-276. H o l y , Α . , Čihák,A. Biochem.Pharmacol. 1981, 30, 2359 -2361. Rosenberg,I., Holý,A. Collect.Czech.Chem.Commun. 1883, 48, 778-789. DeClercq,E., Holý,Α., Rosenberg,I., Sakuma,T., Balzarini,J., Maudgal,P.C. Nature 1986, 323, 464-467, Holy,A. Collect.Czech.Chem.Commun. 1978, 43, 3103-3117. Holý,Α., Rosenberg,I. Collect.Czech.Chem.Commun. 1987, 52, 2775-2791. Rosenberg,I., Holý,A. Collect.Czech.Chem.Commun. 1988, 53, 2753-2777. Webb,R.R., M a r t i n , J . C . Tetrahedron Letters 1987, 28, 4963-4964. Webb,R.R., Wos,J.A. Tetrahedron Letter Holý,Α., Rosenberg,I. Collect.Czech.Chem.Commun. 1987, 52, 2801-2809. O s t e r h a u s , A . D . Μ . Ε . , G r o e n , J . , D e C l e r c q , Ε . Antiviral Res. 1987, 7, 221-226. Lin,J.C., De Clercq, Ε., Pagano,J.S. Antimicrob.Ag. Chemother. 1987, 31, 1431-1433. Baba,M., Κοnnο,Κ., Shigeta,S., De C l e r c q , Ε . E u r . J . C l i n . M i c r o b i o l . 1987, 6, 158-160. De C l e r c q , Ε . , Sakuma,T., Baba,M., Pauwels,R.,Balzarini.J., Rosenberg,I., Holý,A. Antiviral Res. 1987, 8, 261-272. Gil-Fernandez,C., De C l e r c q , Ε . Antiviral Res. 1987, 7, 151-160. B a b a , Μ . , M o r i , S . , S h i g e t a , S . , D e C l e r c q , Ε . Antimicrob. Ag.Chemother. 1987, 31, 337-339. Pauwels,R.,Ba1zarini,J.,Schols,D.,Baba,M.,Desmyter, J., Rosenberg,I., Η ο Ι ý , Α . , De C l e r c q , Ε . Antimicrob. Ag.Chemother. 1988, 32, 1025-1030. Maudgal,P.C.,De C l e r c q , Ε . , H u y g h e , Ρ . Invest.Ophtal. mol.Vis.Sci. 1987, 28, 243-248. Hitchcock,M.J.M.,Ghazzouli,I., T s a i , Y . H . , B a r t e l l i , C . A . , Webb,R.R., Martin,J.C. Abstr.2nd Int.Conf.on Antiviral Research. Williamsburg (USA): 1988. Bronson,J.J.,Kim,C.U.,Ghazzouli,I.,Hitchcock,M.J.M. Martin,J.C.Abstr.2nd Int.Conf.on Antiviral Research Williamsburg (USA) 1988. B a l z a r i n i , J . , Naesens,L., Rosenberg,I., Holý,Α., De Clercq.E.Abstr.Int.Symp.on AIDS,San Marino:1988. Votruba,I., Bernaerts,R., Sakuma,T., De C l e r c q , Ε . , Merta,A.,Rosenberg,I., Holý,A. Mol.Pharmacol. 1987, 32, 524-529. Sakuma,T., De C l e r c q , Ε . , Bernaerts,R., Votruba,I., Holý,A. in Frontiers in Microbiology, Martinus N i j hoff Publishers; Dordrecht: 1987, pp.300-304. Veselý,J.,Merta,A.,Votruba,I.,Rosenberg,I., Holý,A. Abstr.14th IUB Congress, Tu 431; Prague,1988.

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

4.

H O L Y ET AL.

44. 45. 46. 47.

Phosphonylmethyl Ethers of Nucleosides

71

Merta,A.,Votruba,I.,Rosenberg,I.,Otmar,Μ., Holý,A. A b s t r . 1 4 t h IUB Congress, Tu 433; Prague,1988. Averett,D.R., Lubbers,C., E l i o n , G.B., Spector,T. J . B i o l . C h e m . 1983, 258, 9831-9838. Černý,J., Vonka,V.,Votruba,I., Rosenberg,I.,Holý,A. Abstr.14thIUB Congress, Tu 621; Prague,1988. V o t r u b a , I . , T r á v n i č e k , M . , R o s e n b e r g , I . , Otmar,M., H o l ý . A . A b s t r . 1 4 t h I U B Congress. Tu 432; Prague,1988.

RECEIVED February 14, 1989

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Chapter 5

Synthesis and Antiviral Activity of Phosphonylmethoxyethyl Derivatives of Purine and Pyrimidine Bases Joanne J . Bronson , Choung Un Kim , Ismail Ghazzouli , Michael J . M. Hitchcock , Earl R. Kern , and John C. Martin 1

1

1

1

2

1

1

Bristol-Myers Company, Pharmaceutical Research and Development Division, 5 Research Parkway, Wallingford, CT 06492-7660 University of Alabama, Birmingham, AL 35294 2

Acyclic nucleotid phonylmethoxy)ethyl (PME) side chain attached to a purine or pyrimidine base were prepared and selected for in vivo studies based on in vitro antiviral activity against retroviruses and herpesviruses. The adenine derivative PMEA (2) showed good in vitro activity against human immunodeficiency virus (HIV) and Rauscher murine leukemia virus (R-MuLV), and was more potent in vivo than 3'-azido-3'-deoxythymidine (AZT) in the treatment of R-MuLV infection in mice. PMEA also had a significant antiviral effect in vivo against murine cytomegalovirus. The guanine derivative PMEG (10) was exceptionally potent in vitro against herpesviruses. In vivo, PMEG was >50-fold more potent than acyclovir against HSV 1 infection in mice. A number of analogues of PMEG were also prepared, but these derivatives were less potent than PMEG or devoid of antiviral activity. The report by De Clercq and Holy (J.) on the broad-spectrum a n t i v i r a l a c t i v i t y of ( S ) - 9 - ( ( 3-hydroxy-2-phosphonylmethoxy)propy1)adenine (HPMPA, 1) has prompted i n t e r e s t i n the synthesis and evaluation of related a c y c l i c nucleotide analogues. Replacement of the adenine base i n HPMPA with d i f f e r e n t purine and pyrimidine bases has provided other HPMP-derivatives with potent and s e l e c t i v e anti-DNA v i r u s a c t i v i t y (2). A series of phosphonate d e r i v a t i v e s s i m i l a r t o HPMPA, but lacking the hydroxymethy1 appendage present on the HPMP side chain, have also shown s i g n i f i c a n t a n t i v i r a l a c t i v i t y . The adenine d e r i v a t i v e i n t h i s s e r i e s , 9-((2-phosphonylmethoxy)ethyl)adenine (PMEA, 2) (3), was f i r s t reported t o have i n v i t r o a c t i v i t y against a murine r e t r o v i r u s (1). Further studies have shown that PMEA and related PME-dérivâtives are e f f e c t i v e i n h i b i t o r s of human immunodeficiency v i r u s (HIV), the human r e t r o v i r u s which causes acquired immunodeficiency syndrome (AIDS) (4). In addition, members of the PME-series have shown i n v i t r o potency s i m i l a r t o the HPMPd e r i v a t i v e s against DNA viruses such as herpes simplex viruses (2). 0097-6156/89/0401-0072$06.00/0 o 1989 American Chemical Society

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

5.

BRONSON ET

AL.

Phosphonylmethoxyethyl Derivatives ofPurine

73

A further aspect of the a n t i v i r a l a c t i v i t y of HPMPA and r e l a t e d phosphonate derivatives i s t h e i r i n h i b i t o r y e f f e c t on DNA viruses that lack v i r a l thymidine kinase (TK) a c t i v i t y , including TK-deficient s t r a i n s of herpes simplex v i r u s (HSV), as w e l l as viruses that do not encod A c t i v i t y against CMV i s p a r t i c u l a r l one of the leading cause opportunisti patients, and there i s c u r r e n t l y no therapy approved i n the U.S. f o r treatment of CMV i n f e c t i o n . These types of viruses are generally r e s i s t a n t t o nucleoside analogues such as a c y c l o v i r (ACV) that are dependent on i n i t i a l phosphorylation by v i r a l TK i n order t o exert an a n t i v i r a l e f f e c t . For example, the mechanism of action of ACV against HSV 1 involves p r e f e r e n t i a l monophosphorylation by HSVencoded TK and then further phosphorylation by c e l l u l a r kinases t o a triphosphate analogue. I t i s the triphosphate metabolite of ACV which acts as an i n h i b i t o r of v i r a l DNA polymerase and consequently v i r a l r e p l i c a t i o n (5). By contrast, the potent a n t i v i r a l a c t i v i t y of HPMP- and PME- derivatives against CMV and TK" s t r a i n s of HSV (2) demonstrates that t h e i r mode of action i s not dependent on v i r u s s p e c i f i e d TK. These phosphonate derivatives act as stable monophosphate equivalents and can therefore bypass the virus-dependent phosphorylation step. In biochemical studies on the mechanism of action of these nucleotide analogues, HPMPA was shown to be converted d i r e c t l y by c e l l u l a r kinases t o d i - and triphosphate analogues (6). In addition, HPMPA was shown t o i n h i b i t v i r a l DNA synthesis at a concentration much lower than required for i n h i b i t i o n of c e l l u l a r UNA synthesis (6). While s u b s t i t u t i o n of a phosphonic acid group for a phosphate monoester i s a common strategy i n designing metabolically stable phosphate equivalents (2), HPMPA and r e l a t e d compounds are unique nucleotide analogues i n that they are phosphonylmethyl ether derivatives [O-CH -P(0)(OH) ] rather than simple a l k y l phosphonates [R-CH -P(0)(OH) J. The electron-withdrawing oxygen substituent adjacent t o the phosphonate group may serve an important r o l e by providing a s i t e for binding t o target enzymes and i n f l u e n c i n g the e l e c t r o n i c nature of the phosphorous moiety. For example, the e f f e c t of the electron-withdrawing substituent on the degree of d i s s o c i a t i o n can be seen by comparison of the second pK values for d i f f e r e n t phosphorous derivatives (7,8). For the phospfionylmethyl ether d e r i v a t i v e PMEA, pK i s 6.8, c l o s e l y resembling that of phosphate esters [R-O-P^KOH^] which are i n the range of 6.5 7.0; a l k y l phosphonates are less a c i d i c with p K ^ values of 7.7 8.2. The i s o e l e c t r o n i c nature of the phosphorous moiety may be an 2

2

9

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

74

N U C L E O T I D E ANALOGUES

important

factor

demonstrated

Following the

in

Our

focus

this

of

because

of

potent of

order

to

on

antiviral

Synthetic

quantities

various

of

along

derivative agent in

in

vivo

murine

the

PMEG, w h i c h

the

evaluation infection

Synthesis

was

PME-series.

of

of

are

synthesis

which

Synthesis process with

phosphite overall

the

ethanol),

key

the

at

°C

using with

sodium

of

deprotection carried

mesylate

5a

the of out

and

the

mesylate ester

phosphite

salt

of

in

of

in

the

the by

also

observed;

with

was

in

a

the

4a

100

mesyl

chloride

over

90% y i e l d

are

without

used

in

prepared Arbuzov

in

R =

a

similar

reaction. provided

the

b,

R =

PMEA

5 with

access

the

sodium

to

provide

the

(2) The

salt

ethyl

was

to

of

3

the

0

as

competitive ester

c,

R =

Me

achieved followed

alkylation of

diethyl

9-ethyladenine from

i-Pr;

adenine

moiety.

of

phosphonate

manner

Reaction

Oflf

Et;

derivative

arises

from

purification.

5

derivative

product

was

with

0

°C

60% group

treated

phosphite

Formation of

in

acetate

aqueous

5a

acid

Arbuzov

acid,

OAc

phosphonic

treatment (10).

hydrochloric

5c.

adenine

a

triethyl

ether

of

base with four-step

by

3 with

For

employed group.

chloride

ether

derivative

treatment

this

zinc

was

the

Derivatives.

4

side-chain

55% y i e l d . salt

extended

derivatives

ether

mesylate

5 b was in

dimethyl

ester

i n DMF a t

(6a)

adenine

are

antiviral

neterocyclic

achieved

of

alcohol

OAc

Synthesis

was

certain

guanine

and

ether

1,3-dioxolane

(cone,

a,

of

potent

approach

Removal

3

coupling

and

the

activity

phosphonylmethyl

furnish

methyl

^

q

5 was

chloromethyl

conditions

phosphonate

corresponding

o—\

of

Pyrimidine

general

presence

resulting to

triisopropyl the

the

multigram

derivatives

phosphonylmethyl

opening

the

and

a

distillation.

alcohol

Diisopropyl

most

appropriate

the

ring in

acidic the

the

provided

after

and

of

resulting

100

under

the

the

is

interest

retroviruses

providing

phosphonylmethyl

intermediate

with

triethylamine both

particular

analogues

be

Purine

bearing

chloride

yield

effected

4;

of

of

of

antiviral

analogues

presented.

(PME)

coupling

fragment

acetyl

of

antiviral

PME-dérivâtives,

starting

reaction

and

of

involves

side-chain

continued ether

Phosphonylmethy

Phosphonylmethoxyethyl the

of

against

to

vitro

selected

models

are

and p y r i m i d i n e

found In

have

as

nucleotide

capable

preparation

we

potential

of

activity

(9),

activity

analogues.

phosphonylmethyl

compounds

routes

antiviral

nucleotide

i n HPMPA

their

PME p u r i n e

with

of

related

PME-series These

DNA v i r u s e s . described

of

determine

the

chapter.

their

class

interest

evaluation

work

and b r o a d - s p e c t r u m

this

initial

and

agents.

the

members our

synthesis

derivatives

in

by

adenine ester

by by

reaction with

of

PMEA

a byproduct reaction

a n d was

of

separated

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

was the from

5.

B R O N S O N E T AL.

Phosphonylmethoxyethyl Derivatives ofPurine

75

6a by c a r e f u l chromatography. On larger scales, i t was more conven­ ient to use the iso-propvl phosphonate d e r i v a t i v e 5b i n the coupling reaction, since formation of an a l k y l a t e d adenine byproduct i s minimized and p u r i f i c a t i o n i s simpler i n the absence of the comigrating byproduct; a s i m i l a r y i e l d of the coupled product 6b was obtained. Reaction of 6a or 6b with bromotrimethy l s i lane (JL1) i n a c e t o n i t r i l e , followed by concentration and then aqueous hydrolysis of the r e s u l t i n g s i l y l a t e d intermediate, afforded PMEA as a z w i t t e r i o n i c c r y s t a l l i n e s o l i d i n 90-95% y i e l d .

a, R = Et; b, R = i - P r Formation of regioisomeric N-9 and N-7 s u b s t i t u t i o n products i s t y p i c a l l y observed i n a l k y l a t i o n reactions of guanine and r e l a t e d d e r i v a t i v e s . In i n i t i a l e f f o r t s d i r e c t e d toward the synthesis of t j e guanine d e r i v a t i v e PMEG (10), reaction of mesylate 5a with Ν -acetyl guanine was found t o give a 2:1 mixture of a l k y l a t e d products with the N-7 isomer predominating. The desired N-9 a l k y l a t e d product was i s o l a t e d i n only 15% y i e l d . Improved r e s u l t s were obtained when 6-0-(2-methoxyethyl)guanine or 6-0-benzylguanine was employed i n the coupling reaction: the r a t i o of N-9/N-7 isomers was 1-2:1, and the N-9 isomer could be i s o l a t e d i n 30-45% y i e l d . The high degree of r e g i o s e l e c t i v i t y (up t o 15:1 r a t i o s of N-9/N-7 isomers) reported for a l k y l a t i o n of these 6-0-protected guanine derivatives with 4-bromobutyl acetate (.12) was not observed f o r coupling with the mesylate 5a. Further i n v e s t i g a t i o n showed 2-amino-6-chloropurine t o be more u s e f u l i n our a l k y l a t i o n procedure (13). Reaction of t h i s guanine synthon with sodium hydride and then 5a gave a >6:1 r a t i o of the a l k y l a t e d products 7 and 8, with the desired N-9 isomer 7 i s o l a t e d as the major product i n 55-65% y i e l d .

The structure assignments f o r 7 and 8 were based on Η and C NMR spectroscopic data (14) and confirmed by a 2D NMR experiment: for the N-9 isomer 7, a three-bond coupling i n t e r a c t i o n was observed between C-4 of the purine base and the protons at C - l on the side chain, while t h i s long range coupling was seen between C-5 and H-l* 1

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

76

NUCLEOTIDE ANALOGUES

for the N-7 a l k y l a t e d product 8. Conversion of 7 to PMEG (10) was achieved i n 70-80% o v e r a l l y i e l d by d e e s t e r i f i c a t i o n with bromotrimethy l s i lane i n DMF to provide the phosphonic acid 9, followed by hydrolysis of the chloro group with aqueous a c i d at r e f l u x .

9

10

The 2-amino-6-chloropurine intermediate 7 proved u s e f u l for the synthesis of other PME-purine d e r i v a t i v e s . For example, removal of the chloro group v i a t r a n s f e r hydrogénation (20% Pd(0H)« on carbon, cyclohexene/ethanol) an phonate ester provided th 80% o v e r a l l y i e l d . The 2,6-diaminopurine d e r i v a t i v e 12 (PMEDAP) was prepared v i a displacement of the chloro group with sodium azide, followed by reduction of the azido group and d e e s t e r i f i c a t i o n of the phosphonate ester. In t h i s sequence, use of the d i i s o p r o p y l ester d e r i v a t i v e of 7 was found to give better y i e l d s i n the reaction with sodium azide, probably because of decreased side reactions at the phosphonate ester moiety. The o v e r a l l y i e l d for PMEDAP (12) was 50% from the 2-amino-6-chloropurine intermediate 7. NH

11

t

12

For synthesis of the pyrimidine d e r i v a t i v e PMEC (15), coupling of cytosine with the side-chain mesylate 5 was required. This transformation was c a r r i e d out by treatment of a mixture of 5a and cytosine with potassium carbonate i n DMF to a f f o r d a 4:1 r a t i o of Ν and O-alkylated products 13 and 14. The N-l isomer 13 was i s o l a t e d i n 45% y i e l d a f t e r separation from the less polar isomeric product by chromatography. Conversion of 13 to PMEC was achieved i n 80% y i e l d by deprotection using bromotrimethylsilane.

13

14

15

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

5.

B R O N S O N ET AL.

Phosphonylmethoxyethyl Derivatives ofPurine

77

Structure assignments f o r 13 and 14 were based on comparison of t h e i r NMR spectra with those f o r known cytosine d e r i v a t i v e s . For example, the chemical s h i f t s of the carbons on the pyrimidine r i n g are nearly i d e n t i c a l f o r 13 and c y t i d i n e (Table I ) , while for the O-alkylated isomer 14, the pattern of the aromatic carbon peaks i s s u b s t a n t i a l l y d i f f e r e n t . For the product assigned as the N-isomer 13, the chemical s h i f t of C - l ' i s also u p f i e l d r e l a t i v e t o that f o r the 0-isomer 14 (48 vs. 66 ppm). Furthermore, a 2D NMR experiment showed three-bond coupling between C - l and the proton at C-6 of the cytosine base for the N-alkylated isomer 13 but not f o r the isomeric product 14. f

Table I.

C NMR Data f o r Cytosine Derivatives Chemical S h i f t (δ)

Compound 13 (N-l isomer) 14 (0-2 isomer) cytidine

93 100 95

166 165 166

156 165 157

146 157 143

Analogues of 9-(2-Phosphonylmethoxy)ethylguanine (PMEG). The f i n d i n g that the guanine d e r i v a t i v e PMEG (10) i s an exceptionally potent antiherpesvirus agent (vide i n f r a ) has prompted e f f o r t s t o synthesize a number of guanine d e r i v a t i v e s r e l a t e d t o t h i s lead compound. The synthesis of monoesters of PMEG was of i n t e r e s t t o determine the e f f e c t of changing the i o n i c character of the phos­ phorous moiety. These d e r i v a t i v e s might exert a d i r e c t a n t i v i r a l e f f e c t or be metabolized t o PMEG. Preparation of the monoethyl ester of PMEG (16) was accomplished i n 75% y i e l d by treatment of 2-amino-6-chloropurine d e r i v a t i v e 7 with sodium hydroxide s o l u t i o n at r e f l u x for 2 hours t o e f f e c t cleavage of one of the ester groups and hydrolysis of chloro group on the purine base. Under these conditions, formation of the d i a c i d occurred only t o a small extent (<0.2% by HPLC), although up t o 1% PMEG was obtained a f t e r prolonged heating (>8 hours) of the reaction mixture. The monoisopropyl and monomethyl esters of PMEG (17 and 18) were prepared from the corresponding d i e s t e r d e r i v a t i v e s analogous t o 7. A l t e r n a t i v e l y , d i e s t e r s of PMEG served as precursors t o the desired monoesters. The required d i e s t e r s were prepared by coupling of the sodium s a l t 0 16:

UJ

R = Et

17 : R = i - P r -OR

1 8 : R = Me

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

78

NUCLEOTIDE ANALOGUES

of 6-0-benzylguanine with mesylate 5, followed by removal of the benzyl group under t r a n s f e r hydrogénation conditions. The desired PMEG monoester derivatives were then obtained upon treatment of the d i e s t e r with 1 Ν sodium hydroxide at room temperature f o r 1 h. Analogues of PMEG with longer a c y c l i c side chains (15) are of i n t e r e s t because of t h e i r s i m i l a r i t y t o phosphorylated metabolites of a c y c l o v i r . For example, 9-(3-phosphonylmethoxy)propylguanine (PMPG, 22) can be considered an i s o s t e r i c analogue of ACV mono­ phosphate since the same number of atoms separate the guanine base and the phosphorous group. The synthesis of PMPG i s representative of the procedure used t o prepare the longer chain ( b u t y l , pentyl, hexyl, and heptyl) d e r i v a t i v e s . The side chain required f o r PMPG was prepared by chloromethylation [paraformaldehyde, hydrogen chloride (g)] of 3-bromopropanol (19), followed by reaction with the sodium s a l t of d i e t h y l phosphite t o a f f o r d bromide 20 i n 75% y i e l d . Coupling of 6-0-benzylguanine with 20 gave a 2:1 mixture of N-9/N-7 isomeric products; reactio cleavage of the phosphonat PMPG (22) i n 30% o v e r a l l y i e l d from bromide 20. OR

Br l

Br

\ ^ 0 H

— *

^

K

^(K^y<0Et>

H

H> > ^

Q

r

e

*

H

ο

J%J^7

<0R')»

19

ao 21 : R = CHgPh, R

1

= Et

2 2 : R = R' = H To determine the e f f e c t of increased s t e r i c bulk adjacent t o the phosphorous group, α-methyl and a,α-dimethyl d e r i v a t i v e s of PMEG (23 and 24) were prepared. The synthetic approach t o these compounds i s based on the a b i l i t y of the phosphonate group t o s t a b i l i z e an α-carbanion which can then undergo s u b s t i t u t i o n reactions with e l e c t r o p h i l e s such as methyl iodide. The key 0

Η,ϋ^Λ^

|°| P<0H>,

substrate f o r the a l k y l a t i o n reaction, the s i l y l ether 25b, was prepared by treatment of alcohol 25a with t e r t - b u t y l d i m e t h y l s i l y l chloride and t r i e t h y l amine. Note that 25a i s an intermediate i n the PME side-chain synthesis. Reaction of 25b with 1.2 equivalents of seç-BuLi (16) and then methyl iodide at -78 °C gave the a l k y l a t e d

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

5.

product the

26b

85-90% y i e l d .

Repetition of

dimethylated phosphonate

silyl

protecting

mesylation chain was of

in

of

group

the

derivative

obtained α-methyl

using

the

in

in

26b

resulting 28

a

in

in

(acetic

acid,

alcohol,

yield

by

aq

provided the

the

process

THF), the

for

the by

side-

mesylate The then

completed of

PMEG.

0

OR

28 Antiviral In

A c t i v i t y of

Vitro

report

and

by

In

De

Vivo

on

HPMPA

included

a

of

of

PMEA o n

a murine

a

subsequent

activity than

PMEA w a s

DNA v i r u s e s

(2).

More

significant

antiviral

least

of

The

toxic

vivo

to

be

using

more

caused

by

Our

effect

of

of

in vivo

examined

herpes

simplex

as

demonstrated

by

it

was

against

ganciclovir

its

potent

in vitro

(HIV),

(HCMV).

potent

sarcoma

virus

(HSV)

inhibitory

(DHPG),

simplex

but

showed

types good

virus

potent

and

to

the

(4). further

PMEA was

found

formation

(17). evaluation

II)

in vitro The

against leukemia

1 and

2,

results

human virus

and

human

anti-retroviral o n HIV

to

antiviral

activity

and R-MuLV,

both

viruses.

PMEA w a s

than

acyclovir

(ACV)

a c t i v i t y to

DHPG

similar

be

activity

using

murine

effect

t h a n AZT a g a i n s t

herpes

(Table

In

have

tumor

experiments.

Rauscher

PMEA s h o w e d

HIV,

further

agent,

less

to

demonstrated of

effect (_1).

antiviral

i n mice:

virus

on

(MSV)

was

anti-HIV

suppression

focused

it

shown

been

virus

inhibitory

virus

against

PMEA h a s

original

anti-DNA

in vitro

been

having

selection

cytomegalovirus

have

in vitro

in

The

the

although

antiviral

virus

of

sarcoma

PMEA h a s

Moloney

i n PMEA h a s

PMEA w a s

less

well,

an

immunodeficiency (R-MuLV),

to

u t i l i t y as

for

PMEA.

i n f e c t i o n model

t h a n AZT

infection with

guide

as

a c t i v i t y of

effective

its

shown

activity

retrovirus

interest

determine a

a

Moloney

recently,

Derivatives

broad-spectrum

description

PME-dérivâtives

anti-retroviral

in

as

the

the

retrovirus,

report,

against

HPMPA

A n t i v i r a l A c t i v i t y of and H o l y

properties

29

Phosphonylmethyl Ether

Clercq

29

syntheses

synthesis

0

OR

required

were

the

of

followed

sequence.

PMEG ( 2 4 )

described

provided

Removal

a,α-dimethyl

same

and a , α - d i m e t h y l

0

this

90-95% y i e l d .

80-85% y i e l d ;

similar

PMEG ( 2 3 )

27b

chemistry previously

OR

79

Piwsphonylmethoxyethyl Derivatives ofPurine

BRONSON ET AL.

although less

or against

HCMV. The the

Rauscher

antiviral

infection

in vivo

was

by

shown

murine

a c t i v i t y of (Table

significant

leukemia

virus

PMEA c o m p a r e d III).

The

model with

antiviral

i n h i b i t i o n of

the

was

chosen

AZT a g a i n s t effect

of

splenomegaly

to

study

retrovirus each

drug

caused

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

by

80

NUCLEOTIDE ANALOGUES

Table

II.

In

Vitro

Antiviral

A c t i v i t y of

Retroviruses

and

ID Compound

HIV

R-MuLV

PMEA

57Ô

0.05

(2)

ACV

-

DHPG

-

AZT -

0.1-0.5

The by

50% i n h i b i t o r y reduction

assay

(18)

ation

in

cells

and

was

used

cells

virus At

a

AZT

of

0.3

effect

as

lower the

compared

in

of

vitro

against

seen

than

dose

per

-

0.23

0.94

this

AZT

with

40

in HIV-infected

(enzyme

capture

MuLV.

CEM

assay).

50% i n h i b i t i o n o f In

data

In

placebo-treated administration

the AZT

development at

this

in preventing

mg/kg

per

which

murine

III.

with

cells

T h e XC

syncitia

form­

HSV-infected

vero

day.

Vivo

Antiviral Rauscher

Dose

Efficacy

Spleen

0.14

80

0.18

40

0.36

100

0.22

80

0.58

40

Control

Mice were

infected was

30

Wt.

Control

i.p.

initiated

consecutive

days.

with

PMEA

A was

the

disease

are

in

at

contrast

effective

than

ρ

AZT

% Inhibition

0.03

99*

0.11

96*

0.14

85*

0.12

94*

0.64

± ± ±

1.65

±

0.80

0.12

±

0.02

value

0.53

71*

0.47

67*

murine <

of

Splenomegaly

± ± ±

Rauscher =

PMEA a n d

Administration)

(g)

4 h post-infection *

of

Ave.

100

Treatment

less

(I.P.

PMEA

Uninfected

results be

in

splenomegaly.

although

MuLV

(mg/kg/day)

Placebo

dose,

PMEA t o

Compound

AZT

of

control.

PMEA r e s u l t e d

induction of These

showed

infected

of

retrovirus.

against

for

1.8

-

Rauscher

day,

inhibition of was

effective

Table

-

38

assays

100 m g / k g

complete

similar

with

2/7

9Λ

0.5

determine

HCMV

HCMV-infecte

infection

almost

the

HSV 2

21.0

determined

with

1

AD-169.

dose

more

to

HSV

-

protein

infected

ρlaque-reduction HCMV,

gag

Against

(pg/mL)

5 0

0.002

d o s e was

i n p24

PMEA

Herpesviruses

leukemia

and

virus.

continued

BID

0.05.

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

5.

Evaluation CMV was

also

infection used

in

model was is

these

for

by from

per

day

this

significant antiviral with

Table

(Table

IV)

similar

of

the

a

dose

effect of

of

especially

excitin

IV.

In

Vivo

in

up

For

Antiviral

Cytomegalovirus

at

Placebo

The

HIV

of

good

these

PMEA o r

Infection

(I.P.

Treatment

Delay:

6

-

DHPG a g a i n s t

Survival 24

h

27

20

13

0

-

7

-

33

33.3

93*

67*

33

11.2

67*

80*

73*

53

13

0

20

-

27 i.p.

a n d was

with

given

MCMV. BID

Treatment

for

PME-Derivatives

and p y r i m i d i n e activity

deficient

strain

in Table as

replication. antiviral

h

5

initiated

consecutive

at

days.

*

the =

ρ

0.05.

antiviral

expressed

48

h

67*

A c t i v i t y of

summarized

Murine

60*

inoculated

kinase

make

47*

times

vitro

coupled

infections,

67*

indicated

PME-purine

vitro

93*

Control

Antiviral

similar

in

Control

<

with

a

20

Mice were value

resulted

100

3.8 Placebo

almost 100

Administration)

Control

Untreated

data

of

a n d human CMV,

of

Control

DHPG

dose

DHPG p r o v i d e d

4

Untreated

compound

regimen

%

(mg/kg/day)

PMEA

a

was

compared

comparison,

Dose Compound

the

as

PMEA p r o v i d e d this

day.

both

viral

model

E v e n when t r e a t m e n t

models

Efficacy

against

animal

h post-infection,

per

i n murine

no of

time

DHPG.

observed.

mg/kg

is

effect

survival

48

PMEA

infection

administration

to

of

there

antiviral

using

PMEA a g a i n s t

potency

since

animals.

was

33.3

in vivo

CMV (MCMV)

6 h post-infection:

infected delayed

antiviral

activity

common o p p o r t u n i s t i c

controls.

systemic

when g i v e n d o s e was

at

The

increase

experiment

upon

a

(19),

and p l a c e b o - t r e a t e d a

activity

its

an

CMV i s

A murine

significant

protection

protection

it

a

93% s u r v i v a l

PMEA a t

antiviral

since

patients.

studies

untreated

shown

fcAs

in

in vivo

h u m a n CMV i n f e c t i o n .

complete m

the

interest

i n AIDS

shown

with

of

of

81

Phosphonylmethoxyethyl Derivatives ofPurine

BRONSON ET A L

the

derivatives against

of

HSV

Against were

HSV t y p e s

1,

and

Herpesviruses.

evaluated 1 and

h u m a n CMV.

V show t h e

in vitro

potency

concentration

required

for

The

activity

guanine against

derivative each

virus

2,

for a

The of

results compound

50% i n h i b i t i o n o f

a n d was

the

in

thymidine

each

PMEG e x h i b i t e d

The

their

viral

significant

most

potent

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

of

the

NUCLEOTIDE ANALOGUES

82

phosphonate antiviral

derivatives

effect

substantially

than

more

thymidine

kinase

antiviral

activity

HCMV,

PMEG was

derivative was PMEC

of

in

agreement

these

strain

PMEG i s more

also

less

PMEG e x e r t e d

DHPG a g a i n s t

than

deficient

20-fold

exhibited

relative

potent

PMEDAP

somewhat

tested.

ACV o r

not

than

the

antiviral

effect

activities

the

V.

results

Comparison

of

Compound

HSV

by

the

In

HSV

(11)

>100

PMEDAP

(12)

2.4

De

Clercq

purine

although PMEMAP

HSV t y p e the

1 and

and

2.

Holy

it

and The

PME-dérivâtives

Antiviral

1 (TK

are

HCMV

2

0.09

0.06

-

>100

5.0

3.2 >100

-

ACV

0.5

4.3

0.3

38

DHPG

0.23

>10

0.94

1.8

-

The in

50% i n h i b i t o r y HSV-infected

strains:

HSV

Based for

on

further

1,

i n mice effect

mice

(Table

similar

while

response.

At

day.

its

efficacy

although

The

at

a

dose

systemic model,

a

PMEG was

>40-fold i n mice at

a

was

potent

a dose

Administration

of

and

where

of

of

at

doses

As

in

than

lower

PMEG e v e n

at

mg/kg for

a

was HSV

and dose

5

mg/kg by

antiviral observed. 1

of

HSV

2

infection

exhibited

than where a

in

upon

demonstrated

treatment the

acyclovir

0.125

above

toxicity

VII).

effect

required

substantial the

as

complete

clearly

in

The

infection

only

A C V was

effect

10-50-fold

time

antiviral

dose

its

selected

significant

1 systemic

PMEG i s

AD-169.

virus

a

survival

of

Virus

acyclovir. by

PMEG a f f o r d e d

than

(Table

a

observed

doses

lower

at

HCMV,

PMEG was

with

HSV

day

G;

assays

cells.

simplex

significant

utility of

2,

indicated

per

reduction

MRC-5

potency,

i n mean

doses,

antiviral

much more

activity

observed.

range

HSV

herpes

Against

10 m g / k g

toxicity

similar

infection

antiviral was

a

increase

higher

plaque

treatment was

administration of

therapeutic over

PMEG h a d

to

control.

(i.p.)

vitro

compound

and

Z826;

against

PMEG e x h i b i t e d dose

per

in

compared

by

HCMV-infected

1 (ΤΚ'),

in vivo

each

a

protection,

effect

and

placebo

VI),

determined and

exceptional

in mortality with

day,

HSV

of

intraperitoneal per

BW ; S

its

infection

compared

cells

evaluation

antiviral reduction

d o s e was

vero

in

(2).

Activity

HSV

)

-

>100

(15)

activity,

a

the Against

The

0.04

0.08

(10)

that

TK.

compounds.

of

was against

PME-Dérivâtives

1

PMEMAP

analogues

on v i r a l

Vitro

greater

but

ganciclovir.

against

reported

2,

indicating

antiviral

antiviral

with

1,

control

no

of

PMEC

than

vitro

Table

PMEG

HSV

dependent

showed good

potent

slightly

1 and

nucleoside

of

potent

a

HSV

of

toxicity 0.125

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

mg/kg

5.

BRONSON ET AL.

Phosphonylmethoxyethyl Derivatives oj Purine

83

per day resulted i n a s u b s t a n t i a l reduction i n mortality. The greater i n vivo potency of PMEG compared with a c y c l o v i r i s i n contrast with the i n v i t r o data which show PMEG t o be only s l i g h t l y more potent against both HSV 1 and 2.

Table VI. In Vivo A n t i v i r a l E f f i c a c y of PMEG against HSV 1 Systemic Infection i n Mice (I.P. Administration) Dose (mg/kg/day)

PMEG % survival

MST

10 5 1.25 0.50 0.25 0.125 0.031

9 100* 100* 100*

10.8 21.0* 21.0* 21.0*

10

8.9

0

7.3

Placebo Control

Acyclovir % survival MST 50* 17 0

14.1* 11.3* 7.8

- Mice were inoculated i.p. with HSV 1 (HL-34 s t r a i n ) . Treatment was i n i t i a t e d 3 h p o s t - i n f e c t i o n and continued BID f o r f i v e consecutive days. MST = mean s u r v i v a l time; experiment was terminated at day 21. * = ρ value < 0.05.

Table VII.

Dose (mg/kg/day) 50 25 10 5 1 0.25 0.125 0.031 Placebo Control

In Vivo A n t i v i r a l E f f i c a c y of PMEG against HSV 2 Systemic Infection i n Mice (I.P. Administration) PMEG % survival

MST

Acyclovir % survival MST 40 20

0 92* 100* 90* 60* 20

7.6 20.1* 21.0* 20.3* 17.2* 12.1

10

9.0

13.0* 13.0*

Mice were inoculated i.p. with HSV 2 (G s t r a i n ) . Treatment was i n i t i a t e d 3 h p o s t - i n f e c t i o n BID f o r 5 days. MST = mean s u r v i v a l time; experiment was terminated at day 21. * = ρ value < 0.05.

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

84

NUCLE(mDE ANALOGUES

Antiviral

Activity

monoesters, and

ester

effect

(18)

were

showed

herpes

less

Overall,

activity

with

increasing

ester

PMEG ( 1 6 )

of

showed o n l y

weak

modifications antiviral additional analogue herpes chain

in

the

carbon

of

the

tolerated:

HSV

2 and

the

a

of

PMEG, of

activity.

PME-backbone with

alkyl

PMEG.

side

similar

α

and

to

were

the

the

monoethyl

also in

(22),

and

and

antiviral

iso-propyl

resulted is

PMEG

showed a

(17)

that

decrease

which an

vitro The

monomethyl

acyclovir

results

showed no

results

Substitution

to

in

VIII).

although

PMPG

chain

their

(Table

group:

18,

The

for

PMEG

analogues,

decreasing

ester

than

PMEG

evaluated viruses

trend

monophosphate,

viruses;

Herpesviruses.

comparable

the

potent

acyclic

in

derivatives.

not

size

compared

acyclovir

simplex

was

less

antiviral

activity

than

activity

there

was

were

simplex

potent

antiviral

ganciclovir.

Against

phosphonylmethoxy(alkyl)

PMEG d e r i v a t i v e s against

derivatives

PMEG

PMEG A n a l o g u e s

chain

α-substituted

antiviral

of

longer

has

in

an

isosteric

activity

obtained

phosphonate

against

for

the

longer

g r o u p was

also

α-methyl α,α-dimethy

activity.

Table

VIII.

Comparison

of of

the

In

Vitro

Analogues

of

Antiviral

ID Compound .

Ester

HSV

Derivatives

Monoethyl

Chain

PM(Propyl)G PM(Butyl, or .

5

PMEG ( 1 7 )

1.2

75

35

>100

>100

>100

>100

Derivatives

Pentyl,

Hexyl,

PMEG

Derivatives

PMEG ( 2 3 ) PMEG ( 2 4 )

>100

48

>100

>100

PMEG

0.08

0.06

ACV

0.5

0.3

DHPG

0.23

0.94

The

50% i n h i b i t o r y HSV-infected

The

ester

evaluation In

2

Heptyl)G

a-diMe

in

HSV

1.6

(22)

α-Substituted α-Me

1

5

PMEG ( 1 8 )

Monoisopropyl

(vg/mL)

5 0

PMEG

PMEG ( 1 6 )

Monomethyl

Longer

of

Activity

PMEG

cells.

derivatives

based

preliminary

d o s e was

vero

on

their

studies

16 in

determined Virus

and

18 w e r e

vitro

comparing

by

HSV

reduction 1,

considered

activity

the

plaque

strains:

against

toxicity

of

the

BW

for HSV

;

assays

HSV

2,

further 1 and

ester

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

2.

G.

5.

BRONSON ET

derivatives, toxicity

to

determine with

an

IX).

As

phosphonate prodrug

results toxic

in

of

derivative

IX.

In

a

ethyl

that of

was

in vivo to

an

ester

and

than

16 w a s

antiviral of

PMEG

efficacy

for

mice provided

day,

at

protection of

from

Whether

obtained

in

per

complete

effect

ongoing

To

selected

infection

indicated

acyclovir.

similar

c o u l d be

monoethyl

the

85

toxic.

100 m g / k g

before

studies,

had

less

advantage

while

50

seen

greater

subject

Vivo

16 was

HSV 2 s y s t e m i c

doses

was

16

without

was

this its

in

vitro

acts

as

a

cleavage

of

studies.

Antiviral Efficacy

of

Monoethyl

PMEG

against

2 Systemi

Dose

Monoethyl %

(mg/kg/day)

PMEG

Acyclovir %

MST

survival

MST

survival

200

84*

19. 9 *

80*

2 0 . 1*

100

92*

2 0 ., 6 *

67*

19. 2 *

50

80*

1 9 ., 8 *

38*

1 7 .. 1 *

25

46*

1 5 ., 8 *

17

1 3 ., 8 *

12.5

25

13. 4 *

13

11. 8 *

10

1 0 .. 2

6.25 Placebo -

of

showed

at

exerts

is

HSV

PMEG,

relative

PMEG o r group

of

ester

therapeutic

effect

other

m o n o m e t h y l PMEG (18)

ethyl

treatment

activity

ester

Table

a

that

the

in vivo

protection

doses,

antiviral

an

the The

achieved.

found

derivative

in

substantial higher

was

whether

evaluation

the

it

PMEG, w h i l e

ester

(Table

Phosphonylmethoxyethyl Derivatives ofPurine

Al»

Control

Mice were

inoculated i.p.

initiated

3 h post-infection

days. 21.

MST = *

=

mean

ρ value

with

survival <

HSV

and

time;

(G

2

Treatment

strain)

c o n t i n u e d BID experiment

for

was

5

was

consecutive

terminated

at

day

0.05.

Summary

from the

The

phosphonate

our

studies

treatment

particular HIV,

the

cause

of

as

of

derivatives promising

retrovirus

interest

because

human r e t r o v i r u s opportunistic

effect

of

i n f e c t i o n models.

PMEA w a s of

PMEA a g a i n s t less

PMEA a t

murine the

dose

leukemia

same

murine

a

potent

dose.

CMV,

of

virus

giving

of

its

good

responsible

these

for

viruses

was

40 mg/kg provided

per

showed

significant

day

greater good

to

AIDS,

and

CMV, The

demonstrated results

than AZT, mice

at

a

common

antiviral

in vivo

using that

administration with

Rauscher

t h a n AZT g i v e n

activity a

dose

of

against

indicated

infected

in vivo

in

PMEA w a s

activity

protection

protection

identified

evaluation

infections.

patients.

in vitro

retroviruses

been

further

in vitro

i n AIDS

Although

against

PMEA a l s o

PMEG h a v e for

and h e r p e s v i r u s

infection

murine

PMEA a n d

candidates

of

at

against 100 m g / k g

day.

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

per

86

N U C L E O T I D E ANALOGUES

The

guanine

phosphonates herpesviruses. against

a dose

guanine

The

HSV t y p e

significantly at

derivative

evaluated

high

1 and

more

>40-fold

for

potency

2

lower

than

modifications

of

the

complete

of

antiviral

loss

derivatives activity the

of

less

potent

herpes

ester

against

improvement

over

will

subject

the

be

phosphonate

being AIDS

the

the

was

some

infection

therapeutic further

derivatives

a particularly

as

i n both

generally

seen.

led

having

types

than

i n mice

index

of

antiviral

A number

a

decrease

good

in

2.

Both

into

agents,

of

the

with

or ester

vitro

In it

and p r o v i d e d

investigations

was

activity

and monoethyl

however,

PMEG.

vivo PMEG

however,

to

1 and

PMEG;

the

in

cases,

substantial

Monomethyl

virus

toxic

all

against

demonstrated

showed

promise,

of

activity

t o x i c i t y was

activity.

less

potent

PMEG w e r e p r e p a r e d ;

simplex

HSV 2 of

PMEG w a s

ACV a n d

PMEG s k e l e t o n

PMEG s h o w e d

against

monoethyl

to

most

i n mice:

t h a n where

related

the

antiviral

of

infection

potent

derivatives

PMEG was in vitro

vivo, was

only much

no

PMEA a n d

PMEG

potential

of

PMEA

promisin

patients.

Literature Cited J.; J.;

1.

De Clercq, E . ; Holy, Α.; Rosenberg, I.; Sakuma, T . ; Balzarini, Maudgal, P. C. Nature 1986, 323, 464-467. 2. De Clercq, E . ; Sakuma, T . ; Baba, M.; Pauwels, R.; Balzarini, Rosenberg, I.; Holy, A. Antiviral Res. 1987, 8, 261-272. 3. Holy, Α.; Rosenberg, I. Collect. Czech. Chem. Commun. 1987, 52, 2801-2809. 4. Pauwels, R.; Balzarini, J.; Schols, D.; Baba, M.; Desmyter, J.; Rosenberg, I.; Holy, Α.; De Clercq, Ε. Antimicrob. Agents Chemother. 1988, 32, 1025-1030. 5. Elion, G. B.; Furman, P. Α.; Fyfe, J . A.; de Miranda, P.; Beauchamp, L.; Schaeffer, H. J . Proc. Natl. Acad. Sci. USA 1977, 74, 5716-5720. 6. Votruba, I.; Bernaerts, R.; Sakuma, T . ; De Clercq, Ε.; Merta, Α.; Rosenberg, I.; Holy, A. Mol. Pharmacol. 1987, 32, 524-529. 7. Engel, R. Chem. Rev. 1977, 77, 349-367. 8. Blackburn, G. M.; Eckstein, F . ; Kent, D. E . ; Perree, T. D. Nucleosides Nucleotides 1985, 4, 165-167. 9. Webb, R. R.; Martin, J . C. Tetrahedron Lett. 1987, 28, 4963-4964. 10. Bailey, W. F.; Rivera, A. D. J . Org. Chem. 1984, 49, 4958-4964. 11. McKenna, C. E . ; Schmidhauser, J . J . Chem. Soc., Chem. Commun. 1979, 739. 12. Kjellberg, J.; Liljenberg, M.; Johansson, N. G. Tetrahedron Lett. 1986, 27, 877-890. 13. For a related example, see: Harnden, M. R.; Jarvest, R. L.; Bacon, T. H.; Boyd, M. R. J . Med. Chem. 1987, 30, 1636-1642. 14. Kjellberg, J.; Johansson, N. G. Tetrahedron 1986, 42, 6541-6544.

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

5.

15.

16. 17. 18. 19.

B

R

O

N

S

O

N

E

T

A L .

Phosphonylmethoxyethyl Derivatives ofPurine

87

Kim, C. U.; Luh, Β. Y.; Misco, P. F . ; Bronson, J.; Hitchcock, M. J. M.; Ghazzouli, I.; Martin, J. C. 8th International Round Table Meeting on Nucleosides, Nucleotides, and their Biological Applications, Orange Beach, AL, October 1988, Abstract No. 41. Binder, J.; Zbiral, E. Tetrahedron Lett. 1986, 27, 5829-5832. Balzarini, J.; Naesens, L.; Herdewijn, P.; Rosenberg, I.; Holy, Α.; Pauwels, R.; Baba, M.; Johns, D. G.; De Clercq, E. Proc. Natl. Acad. Sci. USA 1989, 86, 332-336. Lin, T-S.; Chen, M. S.; McLaren, C.; Gao, Y-S.; Ghazzouli, I.; Prusoff, W. H. J. Med. Chem. 1987, 30, 440-444. Kelsey, D. K.; Kern, E. R.; Overall, J . C., J r . ; Glasgow, L. A. Antimicrob. Agents Chemother. 1976, 9, 458-464.

RECEIVED February 23, 1989

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Chapter 6

Synthesis and Antiviral Activity of Nucleotide Analogues Bearing the (S)-(3-Hydroxy-2-phosphonylmethoxy)propyl Moiety Attached to Adenine, Guanine, and Cytosine Joanne J . Bronson , Ismail Ghazzouli , Michael J . M. Hitchcock , Robert R. Webb II , Earl R. Kern , and John C. Martin 1

1

1

1

1

2

1

Bristol-Myers Company, Pharmaceutical Research and Development Division, 5 Research Parkway, Wallingford, CT 06492-7660 University of Alabama, Birmingham, AL 35294 2

The nucleotide analogue methoxy)propyl)adenine (HPMPA, 1), (S)-9-((3-hydroxy-2phosphonylmethoxy)propyl)guanine (HPMPG, 3) and (S)-1((3-hydroxy-2-phosphonylmethoxy)propyl)cytosine (HPMPC, 4) have been synthesized on a multigram scale and evaluated in vitro and in vivo for antiviral efficacy against herpesviruses. HPMPC emerged as the most selective antiherpes agent of this series. It showed a much greater in vivo efficacy than acyclovir against herpes simplex virus types 1 and 2 and also was more potent than ganciclovir in a murine cytomegalovirus infection model. Much o f t h e r e s e a r c h antiviral

activity

nucleoside acyclovir that

analogues.

involves

which

and thus

are

nucleotide

called

analogues,

virus

which

been

reported

discovery

b y De C l e r c q

antiviral

analogues acyclic

structurally

nucleoside

additional variety

studies,

1 and 2 ) ,

(5),

Epstein-Barr

of

(HPMPA,

1)

(Maloney

sarcoma

type

virus

(6),

virus).

a n d DHPG

(S)-9-((3-hydroxy-2as a p o t e n t , of

broad

nucleotide ethers

In that

and i n

publication

simplex

in vitro virus

varicella-zoster

adenovirus

In vivo

species

nucleotide

o f A C V (J.)

t o be a c t i v e

(CMV),

Ultimately,

s p e c i f i e d DNA

as p h o s p h o n y l m e t h y l

i n c l u d i n g herpes

1 was d e m o n s t r a t e d

administration

(4).

action

mono­

activity.

a new c l a s s

characterized

cytomegalovirus

stable

and Holy

HPMPA w a s f o u n d

a

(Τ),

activity

with

of

as

of

The phosphorylated

antiviral

has defined

derivatives

o f DNA v i r u s e s

(HSV

virus

agent

give

the virus

derivatives

phosphonylmethoxy)propyl)adenine spectrum

to first

a n d some

t o have

such

a mechanism

to a diphosphate.

replication.

analogues,

o f compounds

and e v a l u a t i o n

analogues,

have

inhibits

i n c l u d i n g phosphonate

have

these

(DHPG),

i s converted

formed

polymerase

The

cases,

phosphorylations

i n turn is

the discovery

on the synthesis

I n most

enzymatic

triphosphate

(2,3),

towards

(ACV) and g a n c i c l o v i r

phosphate the

directed

has focused

and a against

of

against

types virus

(VZV)

retrovirus herpes

by t h e i . p . and t o p i c a l

a

1 and 2

simplex

routes

(4). 0097-6156/89/0401-0088$06.00/0 ο 1989 A m e r i c a n C h e m i c a l Society

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

of

6.

HPMPA

(1)

has

2 -deoxyadenylic involves then to

two

that

HPMPA at

this

must

out

acyclovir

against These HPMPA

is

to

deficient,

analogue

equivalent.

lack In

against

have

acyclovir

the

of

for

is

action

which

bypassing

this

activity

resistant

In

(4).

strain

of

is

1 in

The the

structural

provides active

chirality isomer,

form as

is

The have

been

this

been

kinase

rabbit

model

of

2

analogy

rationale of

HPMPA

phosphate

of

for

HPMPA t o the

This

(4). The

2.

fact

2 -deoxyadenylic the

S

S enantiomer

opposite

acid

!

that

isomer has

enantiomer

as

the

of

(2)

drawn

is

same

HPMPA,

the

R

inactive.

guanine,

cytidine,

described

DNA v i r u s e s , synthesis

a

of

(JjO).

1

also

mutants

activation,

HPMPA h a s

a

is

active

substrate. of

fact,

result

not

deficient step

whereas,

efficiently

is

a thymidine HSV

that

the

a

which

similar

selectivity

only

and

it

first

against

is

is

derives

However,

(9).

that

monophosphate;

thymidine kinase

such viruses

in vivo

keratitis

enzymes

a kinase

CMV a n d

virus.

The d i f f e r e n c e to

phosphorylation

l i m i t e d spectrum

that

mechanism

Acyclovir

has

a

This

(8).

specified

active

reported

action

a triphosphate

acyclovir.

the

of

give

phosphorylated

in that

include

simplex

herpes

for

be

an analogue

a mechanism o f

virus

viruses

viruses

herpes

above

step

to

be

have

DNA p o l y m e r a s e s

first

by

to

and t o

a monophosphate first

carried that

viral

described

is

proposed

(2)

phosphorylations

inhibits

acyclovir

been

acid

1

89

Adenine, Guanine, and Cytosine

BRONSON E T AL.

but

and

series,

HPMPT ( 5 )

antiviral HPMPA,

3

and

thymidine

HPMPG ( 3 )

(5).

is

not.

comparison

HPMPG,

and

analogues

a n d HPMPC In of

this the

(4)

chapter,

three

related

are

to

active we

active

describe members

HPMPC.

4

HPMPA against

5

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

of

the

90

NUCLEOTIDE ANALOGUES

Chemistry The p r e p a r a t i o n requires to

the

bases

the

well to

the

side

chains.

should

hydroxy1

on the

secondary Two

1.

In

to 7,

strategy

A

used

the

be

used

used

A second

issue

protected

analogues

in

addition

coupling

of

nucleoside

only

active

the

starting is

so

S

material

that

that

introduced

X,

for

with

the

for

terminal

the

selectively

on

the

for

to

this

terminal

hydroxyl

phosphonylmethyl via

an

series

labeled

are A,

represented

a

preformed

protected

ether

electrophilic

with

P,

functionality. side

is The

chain

alkylat base for

the ideal

the

to

analysis

contain provides

the

preparation is

the

the

proceeds

the

best

approaches

retrosynthetic

6,

(B)

of

is

alternative was

that the

be

nucleotide

the

therefore,

chiral.

synthetic the

which

group

introduction

can

is

of

complications

with

requirement

c h a i n must

introduce

strategy

derivative

for

series

synthetic

associated

One

be

this

f u n c t i o n a l i t y may b e

nucleoside

leaving

in two

prepared:

side

possible

alkylated second

of

alcohol.

Scheme

acyclic

be

c h a i n must

phosphonylmethyl

in

compounds

known p r o b l e m s

side

enantiomer

of

recognition

of

HPMPA.

alkylation of for

the

synthesis

more

synthesis

of

crystalline

of

a

Since

specific the

intermediates, compound

preformed

many h e t e r o c y c l e s ,

preparation HPMPG a n d

of

a number

the of

and

second analogues

HPMPC.

Base

OH

X

•OP

Scheme 1

was

electrophile

7

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

and

HPMPA S y n t h e s i s . appeared

Our

(JJL).

The

account

chiral

dihydroxypropyl)adenine adenine

and

chloride

and

to

then

diethyl

protected then

cleavage ten

as

reaction

the

in

the

all

multigram

quantities

was

the

prepared

procedure

worked

(13)

(15).

to

the

to

HPMPA

removal

(90

of

of

all

treatment groups

acetone

were

was

11

(JL4)

caused

NaH

and

fully removed

(85

%),

by

and

accomplished i n DMF a t

The workup the

hydroxy

with

give

11

volatiles

of

trityl

secondary

furnish

%).

in hydrolysis

exchange HPMPA

in

well

reverse

36 % o v e r a l l

prepared

side

on of

4-dimethylaminopyridine,

by a 13

for

by

of

room

this

evaporation.

intermediate

silyl

precipitation of

acid large with

and

phase

the

yield

by

HPMPA

transfer

general

benzylation

to

give

the

of

approach

B.

functionality R-glycerol

literature

(S)-1-0-benzylglycerol

monomethoxytrityl

triethylamine

of

adenine.

more

by

This

preparation

a phosphonate

hydrolysis scale

purifications.

efficient from

the

chain bearing

Phase

3).

followed

Reaction

or

allowed

HPMPG was

(Scheme

(12)

trityl

acid

with

from

was

(8)

nucleotid

appropriate

acetonide

The

i n DMF t o

functionalities

furnish

ion

approach

HPMPG S y n t h e s i s .

The

(13)

has

synthesized

DHPA

reaction 2).

HPMPA

zwitterion the

tedious

readily

successive

acetic

then d i l u t i o n with

for

by

of

(S)-9-(2,3-

Briefly,

t r i m e t h y l s i l y l bromide

resulted

synthetic

First,

ester of

involved

crystalline

avoids

by

90 % y i e l d .

5 h to

water

and

effective and

of

for

of

(12).

65 % y i e l d

alkylated

was

w h i c h was

8),

i n DMF ( S c h e m e

80 % a q u e o u s

equivalents

Addition esters,

9 was 10

with

temperature

in

9

synthesis

material

tosyloxymethylphosphonate

with final

of

HPMPA

hydrolysis

afford

a multigram

acetonide

triethylamine

functionality

of

starting

(DHPA,

(S)-glycerol

bis-protected

91

Adenine, Guanine, and Cytosine

6. BRONSON E T AL.

chloride,

i n dichloromethane

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

gave

92 14

NUCLEOTIDE ANALOGUES

In

NaH (0 (55

quantitative

i n THF at °C

to

y i e l d without

reflux

then

room t e m p e r a t u r e ,

% yield

from

13,

acetate/hexanes). (steam bath, 16 h )

and

purification.

diethyl

14 h )

furnished

chromatographed,

Detritylation

20 m i n )

of

Amberlyst-15

86

-

afforded

16

in

acetate/hexane

to

8 % ethanol/ethyl

tosyl

in pyridine

chloride

chromatography. mesylate the

18

chloride

in

synthesis

and

quantitative of

18 m a k e s

after

reaction

of

an

without

in

compound in

o

•

n

T

16

superior

to

to

acetate)

a

byproduct the (18). to

with ester

0 R

OBn

•

OBn

17, R=Ts; 18, R=Ms

17

i n DMF a t

80

°C

was

2-amino-6-benzyloxypurine

give

was

after

workup

and

of

19

as

identified

as

the

20

were

of

first

19

and

step

of

hydrogénation a

ο

0 H

•

59 % y i e l d

The

afford

of

of

structures

transfer

of

17.

3

mixture

carbonate

ease

14

OBn

A solution solid

gave

The

1—OBn

16

Scheme

with

after

with

purification.

OBn

15

% ethyl

OflTr

ο

r

15

acid

16

dichloromethane

13

o

(75 of

91 % y i e l d

OH

12

(13)

ether

temperature,

Treatment

intermediate

•

with

acetic

(room

chromatography

triethylamine

it

80 % a q u e o u s

acetate). 17

yield

14

75 % e t h y l

i n methanol

tosylate

Alternatively,

methanesulfonyl

50 % t o

90 % y i e l d gave

of

phosphonylmethyl

15 w i t h

or

Treatment

tosyloxymethylphosphonate

90 % y i e l d

the

(20 of

an

N-7 as

based

deprotection 2

as

a

foam

10 % m e t h a n o 1 / d i c h l o r o m e t h a n e ) . 21 w i t h

a

ten

room t e m p e r a t u r e recrystallization

for

fold

excess

5 h gave

from

of

HPMPG

(5

4).

20,

one

and

t

(Scheme

isomer

in

(16 17)

chromatography oil

% Pd(0H) /C, 21

treated

and

the

polar

of

19 w a s

achieved

treatment

68 % y i e l d

of

of

data

by

ethanol,

(chromatographically Finally,

a

minor

assignments

o n NMR s p e c t r o s c o p i c

cyclohexene,

in

with

% methanol/ethyl

A more

bromotrimethylsilane (3)

batch

cesium

reflux) purified

diethyl

i n DMF following

ethanol/water.

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

at

6.

B

R

O

N

S

O

N

E

T

A

93

Adenine, Guanine, and Cytosine

L

OBn

HPMPC

Synthesis.

Mesylate

HPMPC

(Scheme

(19).

5)

and

cesium carbonate

for

2.5

h.

After

chromatography desired (23

The

(5

next

on

repeated

for

the

by

conversion

the

were

converted

to

was give

water

(20

adjusted the

to

to

the

mL p e r

(7.5

heated

was

with

soluble

sodium and

on

23

C-13

isomer

4 h

and

22

then a

forms

treated

with

solutions

to

foam

minimize

yield

material (4) of

as

after a white

these

highly

powder

nucleotide analogues

substances 1.00

partial bromide

solution.

nucleotide

were

a

was

trimethylsilyl

HPMPC

The

tic

reduction,

the

aqueous

other

white,

the

byproduct

HPMPC 2 4 a s

over

that

salts.

resulting as

the

for

of

fresh

of

°C

by

10 % m e t h a n o l / d i c h l o r o -

typical an

mmol)

90

% palladium

reflux

from

found

from

give

The d e s i r e d

ester to

at

23 were b a s e d (20

of

(29

purified

to

monitored by

95 % y i e l d

analogues

and

ethanol,

the

highly

gram)

The

22

cytidine.

we

HPMPC a n d

soluble

nucleotide

cytosine

an O - a l k y l a t e d

resulting

ethanol

studies,

of

catalyst a

and

carefully

Finally,

needed.

7.

preparation

DMF w a s

product

diethyl

Also,

the

afforded

more

the

mmol),

hydrogénation

byproduct

product.

in vivo

%)

chromatography

replacing

analogues with

to

precipitation with For

(66

transfer

derivative.

to

crude

assignments

r e a c t i o n was

deesterification after

22

comparison to

after

dihydrouracil

for

(24

10 % m e t h a n o l / d i c h l o r o m e t h a n e )

furnish

of

18

5 0 mL o f

the

cyclohexene,

This

of

in

8 h)

formation

maximized

utilized

carbon,

70 % y i e l d

methane).

mmol)

isomer

in

subjected

hydroxide in

-

structural

NMR s p e c t r o s c o p y was

(49

evaporation,

N-alkylated

%).

18 w a s

A mixture

were

Ν NaOH u n t i l then

soluble

the

lyophilized powders.

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

were

mixed pH to

94

In

NUCLEOTIDE ANALOGUES

Vitro

Data

The

50 %

HSV

1 and

closely these

inhibitory parallel (ACV)

potent

strain

of

analogues

to

do

1, not

which rely

experimental three that

phosphonates of

DHPG,

therapeutic

index

against

animal

model

to

have

is on

for

an

this

infection these

in vitro

Clercq ten

2. a

for

in

vitro

experiments

compounds

the

with

against

by

the

to

HPMPA which

i n mice. human

than

phosphonates

these

are

deficient

nucleotide

pathogen this

potencies shown

(21).

showing

potent

Ganciclovir

caused

recently

series

(4)

against data

activation.

patients.

infections

These

thymidine kinase

cytomegalovirus,

activity

less

an o p p o r t u n i s t i c

AIDS

in vitro

I).

and H o l y

fold

However,

kinase

is

HPMPC

(Table

indication that

exhibited of

yg/ml

De

1 and

this

(CMV)

murine

by

against

a n d HPMPC was

active

phosphonates,

HSV

i n many

therapy

HPMPG a n d

25.2

approximately

acyclovir

infections

HPMPA, to

reported

be

Cytomegalovirus severe

of

2.3

against

than

HSV

doses from

that

substances

acyclovir more

2 ranged

HPMP

causing

(DHPG)

virus

slightly

have

the

and

HPMPC

the

In

contrast

side

are

virus

in

to

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

in

other not

virus

(22-24).

to

vitro

also

used

c h a i n were

immunodeficiency

an All

superior

best

is

is

(20).

found

6.

Adenine, Guanine, and Cytosine

BRONSON ET A L

Table

I.

Antiviral

Activity

I D

Virus HSV

1

(BWS)

HSV

2

(G)

HSV

1

(TK-)

In

Vitro

ug/ml

50>

HPMPA

HPMPG

HPMPC

ACV

DHPG 0.2

9.3

2.3

5.4

0.5

25.2

7.3

2.3

0.3

3.2

0.7

0.7

4.3

0.9 >10

HCMV

(AD169)

0.27

0.39

0.22

1.8

MCMV

(Smith)

0.02

--

0.02

0.06

In

vitro

relative

antiviral

in vivo

potency

comparable

viruses

in vitro

a number

of

antiviral studies Toxicity To

set

were

to

only

for

for

route,

the

at

of

HPMPA

rather

Table

two

orders In

of

reliable an

predictor

in

magnitude

order

nucleotide

studies, oral

route the

were

where

mice

to

of

vitro

more

assay

analogues,

for

potent

in

the

true

in

vivo

extended

even

the

toxicities

animals

given

twice

a

the

animals toxic,

at

the

toxicity i.p.

route

Presumably slightly

II.

the

i.p.

Toxicity

route.

of

were

low

dose

but

because

less

five

days

dosed

once

a

HPMPG,

by

and

by

of

10

the

day

for

i.p.

for

all

at

fewer given

the

mice

the days

by

least

HPMPC t o %

to

mg/kg/day.

toxic

when

far

the

death

not of

toxic

and

assigned

for

in

was

phosphonates

(i.p.), were

especially

HPMPC was

HPMPA ,

day

resulting

10 m g / k g / d a y .

than

the

Three

and

HPMPG w e r e

of

intraperitoneal

II).

highly

intermediate by

the

(p.o.),

(Table

HPMPG was

all

an

by

100 m g / k g / d a y

level

these

routes

i.v.

killing

dosed

i.v.

of

to

(25,26).

Compounds

days.

showed

dosing,

one

in vivo

(i.v.)

HPMPA dose

a

DHPG h a s

tha is

i n mice

group.

three

not

instance,

Mice

intravenous except

sometimes

initiated.

doses

dose

is For

models

potential

assessed

each

to

but

in vivo

were

data

potencies.

of

the

toxic

Mice

Survival HPMPG

HPMPC

Route

HPMPA

200

p.o.

100

33

100

100

p.o.

100

100

100

10

p.o.

100

100

100

200

i.p.

0

0

100

100

i.p.

0

0

100

10

i.p.

100

0

100

200

i.v.

0

0

100

100

i.v.

100

0

100

10

i.v.

100

100

100

Dose,

mg/kg/day

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

96

NUCLEOTIDE ANALOGUES

and

showed no

three

effect

on

the

HPMPA P h a r m a c o k i n e t i c s In a

order study

to of

100 m g / k g 5 % by

evaluate HPMPA

of

oral

recovery

indicate

of

that

the

to

200

mg/kg/day

of

a phosphonate

are

dosing

after

one

m i c e was

of

which

by

all

time

indicating

A possible

half-life

is

that

the

cells

is

only

slowly

enhanced

in vivo

Table

of

this the

or

The

i.v.

routes.

as

duplicate

the

data

and

use

i n Table

i.p.

of

routes

the

more

be

dose

of

i.v.

to

that

III of

easily

substance

for

this

nucleotide

released.

persistence

this

explanation

phosphonate

The

i n plasma

analogue

slow

which

has

release

could

a

long does of

result

the in

an

effect.

III.

Pharmacokinet i c s of Plasma

Time,

the

supports

terminal

lead

for

the

Because

i.p.

mice.

of

to

evaluations

HPMPA w e r e m e a s u r e d three

with

determined

point

half-life.

compound w o u l d

the

analogue, dosed

percent

averaged.

by

was

fo

dramatically

but

was

the

efficacy

out

from

get

into

i n which

initial

exposure

similar

hour

slows

pooled

Each animal

bioavailability

carried

overall

i.p.

terminal

the

concentrations plasma

administration the

oral

HPLC a s s a y

were

performed plasma

undertaken.

individual

analogues

assays

long

up

pharmacokinetics

First,

five

The plasma

the

dosed

Mice

bioavailability,

nucleotide HPLC

the

HPMPA.

from

in

i n m i c e was

a urinary

recovered low

mice

routes.

min

HPMPA

Concentration

Mice of

HPMPA,

ug/ml p.o.

i.p.

i.v.

5

in

0.40

117

196

0.63

15

63.4

30

25.5

52.6

0.63

45

10.6

27.5

0.78

110

60

4.54

8.65

0.86

90

3.45

5.76

0.56

120

3.94

5.11

150

3.50

3.32

---

Systemic

Treatment

HPMPA,

HPMPG,

herpes

simplex

to

acyclovir

survival

virus

in

infected

i.p. by

Herpes

a n d HPMPC w e r e

since

analogue

of

an

types

with

the

of

virus,

and

then

routes

indicated

tables

significance

of

<0.05

model,

by

of

were twice the

Infections

i n mice

These

die

Animals

the

2.

mice

post-infection. in

Virus

evaluated

1 and

encephalitis

untreated

various

Simplex

and

the

for

efficacy

with

a

for

day

Fisher

half

the

5 days.

exact

were

is

infection.

treatment

Mice

activity

substances

a d m i n i s t r a t i o n was dosed

in

with

measured

Animals a

total

as

were

nucleotide

initiated A *

against compared

3

h

daily

indicates

test.

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

dose a

%

6.

Adenine, Guanine, and Cytosine

BRONSON E T AL.

HPMPA a n d HPMPG A g a i n s t in

mouse

and t h e s e

compounds

HPMPG i s t h e m o r e exaggerated

started

route.

complete

An important

who w e r e

able

systemic

mouse

toxicity

o f HPMPG i s s i m i l a r

al.

to achieve model

f o r r a c e m i c HPMPG

Table

IV.

point

but poor

by the

to that

both

c o u l d be

somewhat HPMPA o r

substances

achieved. index.

The

b y De C l e r c q

and Holy

protection, i n a

of the higher recently

is

less

therapeutic

but not complete,

The r e s u l t

manner.

neither

doses

reported

IV),

dependent

i s that

At higher

that

efficacious (Table

the difference

100 % s u r v i v a l

with

some,

(4).

i n a dose

although

protection.

consistent

infections

HPMPA w a s g i v e n

compound h a d a s i m i l a r

f o r HPMPA i s

simplex

t h e mice

analogue

IV b e c a u s e

t o show t o x i c i t y b e f o r e

each

data

potent

oral

HPMPG p r o v i d e d

HPMPA a n d HPMPG w e r e b o t h

o f herpes

did protect

i n Table

bioavailable

Thus,

HSV.

m o r t a l i t y models

97

potency and

described

by T e r r y

et

(27).

In Vivo

Efficac an % Survival

Dose,

mg/kg/day

HPMPA,

p.o.

ACV,

300

40*

90*

200

70*

67*

100

59*

50*

50

25*

34*

25

17

10

-----

----

1 0.1 0.05 Placebo

provided

HSV.

nucleotide

analogue

what was o b s e r v e d toxicity

to that

selected

gave

survived

when

dosed

Acyclovir This for

given

by the i . p . route at the high

o f HPMPC w a s

superior

less

in this

to that

of

---

dose

than

of at

index

since

A l l of

evidence

efficacious

a

potency

In the f i r s t

V.

over

HPMPC s h o w e d

o f HPMPA a n d y e t a HPMPG.

HPMPC

ether

therapeutic

In fact,

Table

i n vivo

a l l the doses t h e HPMPC

o f drug

treated

toxicity

mg/kg/day.

and gave

at best

a 75 %

200 mg/kg/day. lower

The data

doses

t o determine

indicate

1 mg/kg/day

experiment

5

HSV w i t h

a t 2 0 0 , 1 0 0 , a n d 10

less

m o d e l was r e p e a t e d

orally

active

protection,

t h e a c t i v i t y o f HPMPC.

dose

an improved

a n HSV 1 i n f e c t i o n w i t h o u t

was c o n s i d e r a b l y

protection

20

a phosphonylmethyl

o f HPMPC w a s u n d e r e s t i m a t e d

complete

60* 50* 5

than that

o f t h e more

the potency

mice

less

---

30

f o r HPMPA a n d HPMPG.

substantially

comparable study,

c o u l d have

70* 50*

10

against

i.p.

75*

--

results

indication that

ACV,

0

0

The i n v i v o

the f i r s t

i.p.

----

--

0

HPMPC A g a i n s t

HPMPG,

p.o.

(Table

and found

that VI).

t o have

an end p o i n t

t h e 50 %

effective

HPMPC w a s

a potency

also

slightly

acyclovir.

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

98

NUCLEOTIDE ANALOGUES

Table

V.

In Vivo

Efficacy

o f HPMPC

(i.p.) %

Dose,

mg/kg/day

HPMPC,

Against

HSV 1

Survival ACV,

i.p.

75*

200

100*

100

100*

70*

10

100*

20

Placebo

Table

5

5

VI.

In Vivo

Efficacy

o f HPMPC

(i.p. %

Dose,

mg/kg/day

and p . o . )

Against

HSV 1

Survival

HPMPC

200

78*

70*

60*

100

----

90*

40

0

23

--

16

---

16

10

100*

1

90*

0.1

50

Placebo

16

The

data

infection

obtained

with

of

2 virus

was a c h i e v e d

found

at t h e dose

again

Table

from

more

VII.

at

level

(Table

VII)

Significant

0 . 1 mg/kg/day, of

10.

Although

efficacious

In Vivo

--

than

Efficacy

mg/kg/day

HPMPC,

i.p.

were

very

protection

and complete

of

potent

systemic

similar

T h e i . p . HPMPC d a t a less

to the

against

the

protection is

by t h e o r a l

a

type

was

combination

route,

HPMPC

acyclovir.

HPMPC %

Dose,

16

t h e u s e o f HPMPC a g a i n s t

t h e HSV 1 s t u d y .

two e x p e r i m e n t s .

was

(p=0.08)

HSV 2 i n m i c e

results

of

i.p.

(i.p.

and p . o . )

Against

HSV 2

Survival

ACV, i . p .

HPMPC,

p.o.

ACV, p . o .

200

100*

100*

90*

50*

100

100*

20

70*

10

10

100*

10

30

0

1

60*

0.1

40*

Placebo

0

0

5

5

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

6.

Topical

Efficacy

Acyclovir

has

clinical

of

on

HPMPC was

compared

in

pigs

guinea

infected

on

in

was

5 days.

The

commercial

lesion,

and

disease.

topical

not

of

of

a

to

the

In

reduce

this

and

the

of

formulation

day

for

1 through

lesion

score

overall

of

a

treated

a

of

was

A score

commercial

provided

a

VIII.

benefit

Topical

even

in

Efficacy

a

of

dilute

Lesion 0.8*

% HPMPC

1.0*

0.1

% HPMPC

1.4*

5.0

% ACV

2.4

Control

disease

model

with

the

started The than

data

whereas,

4

or

and

at 5.

this

the

to

48 time

h

after point

apparent

these

due

already

to

at with

also

a

acyclovir

of

0.1

%.

1

Score

was

of

24, is

efficacy BID and

i.p. 48

about

low

which

mice

are for

at

drug

but

for HPMPC

a

the

and infected was

more

given

1.2

potent dose.

only

sick

highest

at

and

by

the

the

11.2

initiation regimen die

doses

48 h p o s t - i n f e c t i o n toxicity

For

mg/kg/day;

challenging

quite

use

human

post-infection.

fold

achieved a

the

the

when t r e a t m e n t is

Mice

treatment

at of

in

were

hours

ten

dose

DHPG was

of

Animals

survivors

efficacy the

predictive

IX).

then

the

initiated of

be

the

of

infection,

lack

Cytomegalovirus

efficacious

untreated

enhancement

sick

6,

HPMPC

number

effect

DHPG w h e n t r e a t m e n t

probably

that

HPMPC was

and

of

protective

significant

The

HSV

cytomegalovirus,

to

(Table

route

the

human

model,

times

demonstrate

Murine

thought

mouse

i.p.

increasing

that

for

is

compared

indicated

dose.

delayed

since

available

were

HPMPC was a

mg/kg/day

HPMPC A g a i n s t

i n mice

In by

the

DHPG i n

instance,

was

is

(DHPG)

virus at

of

virus

(3,31).

ganciclovir

indicates of

3.0

Efficacy

mouse

(*

2.8

Control

in vivo

no

severe

derived

HPMPC A g a i n s t

% HPMPC

the

indicates

formulation

1.0

of

The

0

disease

5.0

No

used.

5 % formulation

Mean

Systemic

day

vehicle.

highl

substantial

Compound

Placebo

a

80/PEG

statisticall

HPMPC was

Virus

were

topical

lesions.

increasingly

the

pigs

with twice

of

statistically of

model

the

tween

acyclovir

severity

The

was

in (28).

guinea

continued

represent

is

infection

severity and

route

herpes

study,

then

severity. 4

topical

genital

cutaneous

regions

formulation

each

the

However,

Table

in

by

primary

VIII).

six

the

<0.01).

provide

of

3 h post-infection

HPMPC,

T h e mean

significance

(Table in

1

efficacy

acyclovir

attempt

scores

HSV

treatment

backs

scored

representation did

to

an

For

were

limited

started

for

lesions

a the

(29.30)

their

formulations Treatment

HPMPC A g a i n s t

shown

trials

99

Adenine, Guanine, and Cytosine

BRONSON ET A L

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

day

HPMPC

was

disease

animals.

on

of

in

100

NUCLEOTIDE ANALOGUES

Table

IX.

In

Vivo

Efficacy

Murine

of

(i.p.)

HPMPC

% Dose,

mg/kg/day

Treatment

Against

Cytomegalovirus

Start,

h

Survival

HPMPC

DHPG

33.3

6

100*

93*

33.3

24

87*

67*

33.3

48

33

33

11.2

6

100*

67*

11.2

24

93*

80*

11.2

48

67*

3.8

6

100*

53 13

73*

3.8

24

53

3.8

48

67*

48

60

24

20

20

27

27

0

1.2 1.2 1.2 Placebo Control

20

(p=.06)

Summary Although

possessing

HPMPC e m e r g e d in

vivo

against

demonstrated mg/kg/day in m

g/kg/day,

acyclovir

which

In

a murine

significantly

limited

since

commercial

of

therapeutic

index

of

infections

agent

dramatically only

index

0.1

(32),

than

more formulation

HPMPC w a s s h o w n

Ganciclovir

t o b e n o more cells

t o be

potent

by p r o v i d i n g

CMV i n f e c t i o n s

for possible

f o r HPMPC.

pigs.

model,

ganciclovir

Also

found

more

in a topical

i n guinea

is

this

therapy

i n immunocompromised

currently (20).

toxic

to

protection

i n the c l i n i c

due t o m y e l o s u p p r e s s i o n

marrow

acyclovir,

up t o 200

HPMPC w a s

0 . 1 % HPMPC w a s

infection

reported

evaluation

at doses

therapeutic

5 % acyclovir

than

of

b u t i t was s t i l l

1.2 mg/kg/day.

t o human b o n e

cytomegalovirus

low i . p . dose

high

infection

potent

HPMPC w a s r e c e n t l y further

o f HPMPC w a s

bioavailability,

cytomegalovirus

to

antiviral

o f HSV 1 a n d 2 i n f e c t i o n s .

In addition,

for the treatment

ganciclovir merits

down t o a models

relative

efficacious

The potency

administration,

more

potency

a highly

i n a very

low o r a l

route.

low dose

evaluation

as

t o x i c i t y was a p p a r e n t

results

by o r a l than

of

a n HSV 1 c u t a n e o u s

down t o a a

mortality

of

by t h i s

efficacious

be

by i t s a c t i v i t y

because

active

in vitro

study

herpesviruses.

no evidence

Probably

against

moderate this

i n mouse

mice,

less

from

under

but has

Especially

than

nucleotide

analogue

against patients.

Literature Cited 1.

Sidwell, R. W.; Huffman, J . H.; Barnard, D. L.; Reist, E. J. 8th International Round Table Meeting on Nucleosides, Nucleotides and Their Biological Applications Abstract 14, Orange Beach, AL, October 3, 1988.

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6.

BRONSON ET A L

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.

Adenine, Guanine, and Cytosine

101

Prisbe, E. J.; Martin, J . C.; McGee, D. P. C.; Barker, M. F.; Smee, D. F.; Duke, A. E . ; Matthews, T. R.; Verheyden, J . P. H. J . Med. Chem. 1986, 29, 671-675. Duke, A. E . ; Smee, D. F.; Chernow, M.; Boehme, R.; Matthews, T. R. Antiviral Research 1986, 6, 299-308. De Clercq, E . ; Holy, Α.; Rosenberg, I.; Sakuma, T.; Balzarini, J.; Maudgal, P. C. Nature 1986, 323, 464-467. De Clercq, E . ; Sakuma, T.; Baba, M.; Pauwels, R.; Balzarini, J.; Rosenberg, I.; Holy, A. Antiviral Research 1987, 8, 261-272. Lin, J-C.; De Clercq, E . ; Pagano, J . S. Antimicrob. Agents Chemother. 1987, 31, 1431-1433. Baba, M.; Mori, S.; Shigeta, S.; De Clercq, Ε. Antimicrob. Agents Chemother. 1987, 31, 337-339. Votruba, I.; Bernaerts, R.; Sakuma, T . ; De Clercq, Ε.; Merta, Α.; Rosenberg, I.; Holy, A. Mol. Pharmacol. 1987, 32, 524-529. Elion, G. B.; Furman, P. Α.; Fyfe, J . Α.; de Miranda, P.; Beauchamp, L.; Schaeffer 74, 5716-5720. Maudgal, P. C.; De Clercq, E . ; Huyghe, P. Invest. Ophthamol. Vis. Sci. 1987, 28, 243-248. Webb, R. R.; Martin, J . C. Tetrahedron Lett. 1987, 28, 4963-4964. Holy, A. Collect. Czech. Chem. Commun. 1975, 40, 187-214. Holy, Α.; Rosenberg, I. Collect. Czech. Chem. Commun. 1982, 47, 3447-3463. McKenna, C. E . ; Schmidhauser, J . J . Chem. Soc. Chem. Commun. 1979, 739. Golding, B. T.; Ioannou, P. V. Synthesis 1977, 423-424. MacCoss, M.; Chen, Α.; Tolman, R. L. Tetrahedron Lett. 1985, 26, 1815-1818. Robins, M. J.; Robins, R. K. J . Org. Chem. 1969, 34, 2160-2163. Kjellberg, J.; Johansson, N. G. Tetrahedron 1986, 42, 6541-6544. Webb, R. R.; Wos, J . Α.; Bronson, J . J.; Martin, J . C. Tetrahedron Lett. 1988, 29, 5475-5478. Collaborative DHPG Treatment Study Group, N. Engl. J . Med. 1986, 314. 801-805. Snoeck, R.; Sakuma, T.; De Clercq, Ε.; Rosenberg, I.; Holy, A. Antimicrob. Agents Chemother. 1988, 32, 1839-1844. Pauwels, R.; Balzarini, J.; Schols, D.; Baba, M.; Desmyter, J.; Rosenberg, I.; Holy, Α.; De Clercq, Ε. Antimicrob. Agents Chemother. 1988, 32, 1025-1030. Balzarini, J.; Naesens, L.; Herdewijn, P.; Rosenberg, I.; Holy, Α.; Pauwels, R.; Baba, M.; Johns, D. G.; De Clercq, E. Proc. Natl. Acad. Sci. USA 1989, 86, 332-336. Ghazzouli, I.; Bronson, J . J.; Russell, J . W.; Klunk, L. J.; Bartelli, C. Α.; Franco, C.; Hitchcock, M. J . M.; Martin, J . C. J . Cell. Biochem. 1989, S13B. 300. Martin, J . C.; Dvorak, C. Α.; Smee, D. F.; Matthews, T. R.; Verheyden, J . P. H. J . Med. Chem. 1983, 26, 759-761. Smee, D. F,; Martin, J . C.; Verheyden, J . P. H.; Matthews, T. R. Antimicrob. Agents Chemother. 1983, 23, 676-682. Terry, B. J.; Mazina, Κ. E . ; Tuomari, Α. V.; Haffey, M. L.; Hagen, M.; Feldman, A.; Slusarchyk, W. A.; Young, M. G.; Zahler, R.; Field, A. K. Antiviral Research 1988, 10, 235-252.

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

102

N U C L E O T I D E ANALOGUES

28. Corey, L.; Nahmias, A. J.; Guinan, M. E . ; Benedetti, J . K.; Critchlow, C. W.; Holmes, Κ. Κ. N. Engl. J . Med. 1982, 306, 1313-1319. 29. Mansuri, M. M.; Ghazzouli, I.; Chen, M. S.; Howell, H. G.; Brodfuehrer, P. R.; Benigni, D. Α.; Martin, J . C. J . Med. Chem. 1987, 30, 867-871. 30. Alenius, S.; Oberg, B. Arch. Virol. 1978, 58, 277. 31. Kelsey, D. K.; Kern, E. R.; Overall, J . C., J r . ; Glasgow, L. A. Antimicrob. Agents Chemother. 1976, 9, 458-464. 32. Snoeck, R.; Lagneaux, L.; Bron, D.; De Clercq, E. 28th Interscience Conference on Antimicrobial Agents and Chemotherapy Abstract 739, Los Angeles, CA, October 25, 1988. RECEIVED February 23, 1989

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Chapter 7

Design of Inhibitors of Herpes Simplex Virus Thymidine Kinase J. A. Martin, I. B. Duncan, and G. J . Thomas Research Division, Roche Products Limited, Ρ.Ο. Box 8, Welwyn Garden City, Hertfordshire AL7 3AY, England The role of herpe the pathogenesis of infection, i t s mechanism of action and the current status with regard to the design of inhibitors are reviewed. Examples of inhibitors based on substrate and product analogues are described, some of which are very potent and highly selective for the viral enzymes. One class of inhibitors with high in vitro potency and selectivity has shown a protective effect in mice under certain conditions and i t is possible that compounds of this type may have potential as antiviral agents in man. Herpes simplex v i r u s (HSV) t y p e s 1 and 2 c a u s e m i l d t o s e v e r e d i s e a s e i n man, p r i m a r i l y o r o f a c i a l and g e n i t a l i n f e c t i o n s . A f e a t u r e o f t h e s e v i r u s e s i s t h e i r a b i l i t y t o become l a t e n t i n neuronal g a n g l i a , from whence r e - e x p r e s s i o n c a u s e s recurrent c l i n i c a l episodes. Thus f a r , t h e d e s i g n o f a n t i v i r a l agents a g a i n s t HSV i n f e c t i o n s has c e n t e r e d on compounds t h a t i n h i b i t major v i r a l enzymes i n t h e l y t i c c y c l e , whereas p r o c e s s e s t h a t c o n t r o l l a t e n c y and r e a c t i v a t i o n have been r e l a t i v e l y n e g l e c t e d t a r g e t s . The

R o l e o f Thymidine K i n a s e i n Herpes Simplex

Virus Infections

Under normal growth conditions, eukaryotic cells obtain the t h y m i d i n e n u c l e o t i d e s r e q u i r e d f o r DNA s y n t h e s i s t h r o u g h a ds. novo pathway, i n w h i c h t h y m i d i n e monophosphate i s s y n t h e s i s e d f r o m d e o x y u r i d i n e monophosphate. Thus, t h y m i d i n e k i n a s e (TK) i s not r e q u i r e d f o r normal c e l l growth but t h e s y n t h e s i s o f a new s p e c i e s o f TK by HSV i s p r e s u m a b l y n e c e s s a r y t o accommodate t h e i n c r e a s e d demand f o r t h y m i d i n e t r i p h o s p h a t e t o f u e l v i r a l DNA s y n t h e s i s . It is more t h a n twenty-five years s i n c e the first report (D e s t a b l i s h i n g t h e p r e s e n c e o f a v i r u s - e n c o d e d TK i n H S V - i n f e c t e d mouse f i b r o b l a s t s . The r e l e v a n c e o f t h i s v i r u s - i n d u c e d enzyme t o p r o d u c t i v e and l a t e n t i n f e c t i o n b o t h i n c e l l c u l t u r e and i n v i v o has been d e s c r i b e d 11-4). 0097-6156/89A)401-0103$06.00A) ο 1989 A m e r i c a n C h e m i c a l Society

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

104

NUCLEOTIDE

ANALOGUES

V i r u s m u t a n t s have b e e n i s o l a t e d w h i c h do n o t p r o d u c e a t h y m i d i n e k i n a s e (TK~) but r e t a i n an u n i m p a i r e d a b i l i t y t o r e p l i c a t e i n c e l l c u l t u r e (υ). S i n c e TK~ mutants have r e d u c e d p a t h o g e n i c i t y in experimental animal models (6-11) t h e e x p r e s s i o n o f TK i s p r o b a b l y an i m p o r t a n t d e t e r m i n a n t of v i r u l e n c e i n v i v o . These f i n d i n g s t o g e t h e r w i t h i d e n t i f i a b l e d i f f e r e n c e s between t h e v i r a l enzyme and i t s c e l l u l a r c o u n t e r p a r t w i t h r e g a r d t o i m m u n o g e n i c i t y , molecular weight, t h e r m o s t a b i l i t y and particularly substrate specificity (12-16) make t h i s f u n c t i o n an a t t r a c t i v e t a r g e t f o r a n t i v i r a l chemotherapy. W h i l s t i t has been s u g g e s t e d ( 17.18) t h a t s p e c i f i c i n h i b i t o r s o f v i r a l TK may s u p p r e s s one o r more a s p e c t s o f HSV d i s e a s e c o m p l e x , t h e r e have b e e n r e m a r k a b l y few reported a t t e m p t s t o d e v e l o p p o t e n t and s p e c i f i c i n h i b i t o r s o f t h i s v i r a l enzyme. S e v e r a l s t u d i e s have d e s c r i b e d compounds t h a t i n h i b i t TK f r o m E s c h e r i c h i a c o l i (12) , Walker 256 c a r c i n o m a 120.21), Yoshida sarcoma (22. 23) and L1210 cells (2_£) D i f f e r e n c e s have been identified between th s u r p r i s i n g l y these studie

Mechanism of Action Thymidine k i n a s e c a t a l y s e s the p h o s p h o r y l a t i o n of thymidine i n the presence o f d i v a l e n t c a t i o n s s u c h as magnesium w i t h adenosine triphosphate (ATP) as t h e c o n v e n t i o n a l p h o s p h a t e d o n o r . The phosphoryl t r a n s f e r occurs with i n v e r s i o n of c o n f i g u r a t i o n at p h o s p h o r u s , which has been e x p l a i n e d (26) by a s i n g l e i n - l i n e g r o u p t r a n s f e r between ATP and t h y m i d i n e w i t h i n t h e enzyme complex. A more e x t e n s i v e s y s t e m has been d e s c r i b e d (22) i n w h i c h ADP o r AMP may a l s o a c t as t h e p h o s p h a t e d o n o r i n 5 - p h o s p h o t r a n s f e r a s e r e a c t i o n s t o t h y m i d i n e and i n s u p p o r t o f t h i s t h e r e i s e v i d e n c e (2&) t h a t HSV-1 TK i s t r a n s l a t e d as t h r e e p o l y p e p t i d e s t h a t may e x i s t as a heteromultimer with a p o t e n t i a l m u l t i p l i c i t y of s u b s t r a t e b i n d i n g sites. Earlier studies (2 9-31 ) h a d been r e p o r t e d i n which p h o s p h o r y l a t e d s p e c i e s o f t h y m i d i n e a f f e c t e d TK c a t a l y s e d r e a c t i o n s b u t t h i s work u s e d c r u d e enzyme p r e p a r a t i o n s and t h e p r e s e n c e o f i n t e r f e r i n g components cannot be r u l e d o u t . However, now t h a t t h e n u c l e o t i d e and p r i m a r y amino a c i d s e q u e n c e s o f v i r a l TK a r e known ( 3 2 - 3 4 ) , t h e enzyme has been c l o n e d and e x p r e s s e d i n p r o k a r y o t e s (35 36), and d i r e c t e d m u t a g e n e s i s s t u d i e s have begun (37.38), t h e r e i s t h e p r o s p e c t t h a t a f u l l s t r u c t u r a l a n a l y s i s o f t h e enzyme may be a v a i l a b l e soon. E v i d e n c e c o u l d t h e n be o b t a i n e d t o e x p l a i n t h e s e e a r l i e r o b s e r v a t i o n s and e n a b l e t h e d e s i g n o f b e t t e r i n h i b i t o r s f r o m molecular modelling studies. Thus f a r , t h e d e s i g n o f i n h i b i t o r s has been based i n s t e a d on either an understanding of the enzyme-catalysed c h e m i c a l t r a n s f o r m a t i o n s t h a t o c c u r o r on the b i o c h e m i c a l p r o p e r t i e s o f t h e enzyme i t s e l f . Future s y n t h e t i c programmes c o u l d be b a s e d on s u b s t r a t e a n a l o g u e s , p r o d u c t analogues, metal c h e l a t i o n or a l l o s t e r i c i n h i b i t i o n . 1

r

S u b s t r a t e A n a l o g u e s as

Inhibitors

W h i l s t t h e mammalian TK has a v e r y s t r i n g e n t s u b s t r a t e the v i r a l enzyme i s c o n s i d e r a b l y more t o l e r a n t of analogues ( ϋ ) . More p r e c i s e l y , t h e c e l l u l a r enzyme can

specificity nucleoside process

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

7. MARTIN E T AL.

Inhibitors of Herpes Simplex Virus

105

o n l y t h y m i d i n e and a few c l o s e a n a l o g u e s , whereas t h e v i r a l enzyme has an a b i l i t y t o p h o s p h o r y l a t e a wide range o f p y r i m i d i n e s and even some p u r i n e n u c l e o s i d e s . T h i s marked d i f f e r e n c e has been e x p l o i t e d by many r e s e a r c h g r o u p s i n t h e s e a r c h f o r new a n t i v i r a l agents, r e s u l t i n g n o t a b l y i n t h e d i s c o v e r y o f a c y c l o v i r (40-42) . Such compounds depend on s e l e c t i v e a c t i v a t i o n by v i r a l TK and c e l l u l a r k i n a s e s t o t h e i r t r i p h o s p h a t e s , which t h e n i n h i b i t t h e v i r a l DNA p o l y m e r a s e . C l e a r l y t h e mode o f a c t i o n o f such a n t i v i r a l n u c l e o s i d e s i s not i n h i b i t i o n o f TK p e r s e . a l t h o u g h t h e y do compete w i t h t h e n a t u r a l s u b s t r a t e , t h y m i d i n e , f o r p h o s p h o r y l a t i o n and w i l l t h e r e f o r e d e p l e t e the p o o l of thymidine n u c l e o t i d e s i n i n f e c t e d c e l l s .

1 A n a l o g u e s o f a c y c l o v i r have been s y n t h e s i s e d w i t h m o d i f i c a t i o n s i n t h e g u a n i n e and s i d e c h a i n m o i e t i e s (43 4 4 ) . These compounds had e i t h e r weak o r no a c t i v i t y a g a i n s t HSV-1 i n a plaque reduction assay. In an attempt t o r a t i o n a l i s e t h e l a c k o f a n t i v i r a l a c t i v i t y t h e a c c e p t a b i l i t y o f t h e s e compounds as s u b s t r a t e s f o r v i r a l TK was measured. As m i g h t have been e x p e c t e d none o f t h e compounds, i n c l u d i n g t h e a c y c l i c a n a l o g u e s 1 and 2, were p h o s p h o r y l a t e d . Both 1 and 2 i n h i b i t e d t h e p h o s p h o r y l a t i o n o f a c y c l o v i r , i n d i c a t i n g t h a t t h e y d i d b i n d t o t h e enzyme. The development o f more p o t e n t and s e l e c t i v e i n h i b i t o r s b a s e d on t h e s e l e a d s t r u c t u r e s has n o t been reported. A s e r i e s of N - p h e n y l s u b s t i t u t e d guanine d e r i v a t i v e s was s c r e e n e d a g a i n s t HSV-1 TK i n o r d e r t o i d e n t i f y l e a d s t r u c t u r e s t h a t c o u l d be m o d i f i e d t o g i v e p o t e n t and s e l e c t i v e i n h i b i t o r s (45. 46) . From t h e r a n g e o f g u a n i n e s s c r e e n e d ( T a b l e I) i t i s c l e a r t h a t substitution i n t h e N 2 - p h e n y l m o i e t y has a marked e f f e c t on potency; f o r example, an e l e c t r o n - w i t h d r a w i n g group i n e i t h e r the meta o r p a r a p o s i t i o n e n h a n c e d p o t e n c y , whereas a l k y l g r o u p s s u c h as m e t h y l o r b u t y l i n t h e p a r a p o s i t i o n r e d u c e d a c t i v i t y . However, a m e d i u m - s i z e d a l k y l r e s i d u e s u c h as e t h y l was t o l e r a t e d i n t h e meta p o s i t i o n . G l y c o s y l a t i o n o f t h e u n s u b s t i t u t e d g u a n i n e 3 gave t h e c o r r e s p o n d i n g 2'-deoxyguanosine d e r i v a t i v e 9 which was t h e most a c t i v e compound r e p o r t e d . D e t a i l e d s t u d i e s have shown 9 t o be s e l e c t i v e f o r t h e i s o l a t e d v i r a l enzyme and i t a l s o i n h i b i t e d TK i n i n f e c t e d c e l l s as m e a s u r e d by t h e e n t r a p m e n t o f r a d i o l a b e l l e d t h y m i d i n e as nucleotides. At h i g h c o n c e n t r a t i o n and with p r o l o n g e d e x p o s u r e 9 d i d e x e r t some t o x i c i t y a g a i n s t t h e mammalian h o s t c e l l s b u t t h e l e a d s g e n e r a t e d i n t h i s s t u d y may provide a s t a r t i n g p o i n t f o r t h e d e v e l o p m e n t o f more p o t e n t and selective inhibitors. P

2

W i g d a h l and P a r k h u r s t (Al) have shown 5 - t r i f l u o r o t h y m i d i n e t o be a c o m p e t i t i v e i n h i b i t o r o f t h y m i d i n e p h o s p h o r y l a t i o n by b o t h c e l l u l a r and more e f f e c t i v e l y HSV-1 TK. I t has a l s o been p r o p o s e d t h a t the t r i p h o s p h a t e of t h i s compound m i g h t m i m i c thymidine t r i p h o s p h a t e f e e d b a c k r e g u l a t i o n o f b o t h enzymes. I t has been

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

106

ΝϋίΧΕΟΉΌΕ ANALOGUES

I . I n h i b i t i o n o f H S V - 1 Thymidine K i n a s e by Derivatives

Table

R

N2-Phenylguanine

3

IC50

[μΜ]

HSV-1

Rl

R2

R3

3

Η

Η

Η

8

4 5 6

Me n-Bu

Η

Η

50

Η

Η

50

7

Br

8 9

Η

Cl

Η

Η

Compound

Η 1.3

Η

2-deoxy-fi-Dribofuranosyl

0.3

shown (A3.) t h a t 5 - t r i f l u o r o t h y m i d i n e i s a s u b s t r a t e f o r b o t h v i r a l and host cell TK, and a l s o t h a t 5·'-amino-5 -deoxythymidine s e l e c t i v e l y i n h i b i t s i t s p h o s p h o r y l a t i o n by t h e c e l l u l a r r a t h e r t h a n t h e v i r a l enzyme, so i t i s p o s s i b l e t h a t c l o s e l y related a n a l o g u e s may s e l e c t i v e l y i n h i b i t t h e v i r a l enzyme. 1

Product

A n a l o g u e s as I n h i b i t o r s

D e r i v a t i v e s of 2'-Deoxy-5-iodouridine. I n an attempt t o improve the a n t i v i r a l e f f i c a c y o f 5 ' - a m i n o - 2 ' , 5 - d i d e o x y - 5 - i o d o u r i d i n e a s e r i e s o f N - a c y l d e r i v a t i v e s was p r e p a r e d as p o t e n t i a l prodrugs (AiL) . W h i l s t none o f t h e compounds showed a n t i v i r a l a c t i v i t y i n t i s s u e c u l t u r e , some were f o u n d t o be i n h i b i t o r s o f H S V - 1 TK ( T a b l e II) . R e c e n t l y , a d d i t i o n a l d a t a have b e e n r e p o r t e d (5SL) w h i c h i n c l u d e a c t i v i t y a g a i n s t t h e t y p e 2 enzyme. 1

A l l o f t h e compounds shown i n T a b l e I I were more a c t i v e a g a i n s t H S V - 2 TK t h a n a g a i n s t t h e t y p e 1 enzyme w h i c h may be a p r o p e r t y o f t h e 5 - i o d o s u b s t i t u e n t as was o b s e r v e d w i t h t h e p a r e n t 5'-amino n u c l e o s i d e . I n h i b i t o r y a c t i v i t y against both viral enzymes a p p e a r e d t o c o r r e l a t e r e a s o n a b l y w e l l w i t h c h a n g e s i n l i p o p h i l i c i t y a r i s i n g from m o d i f i c a t i o n s i n t h e 5 ' - s u b s t i t u e n t . The most a c t i v e compound i n t h e s e r i e s , 14, c o n t a i n e d t h e v a l e r y l s i d e c h a i n and was a p p r o x i m a t e l y twenty t i m e s more a c t i v e a g a i n s t t h e t y p e 2 enzyme. Interestingly, the separation i n a c t i v i t y between t h e two v i r a l enzymes i n c r e a s e d w i t h i n c r e a s i n g o v e r a l l potency of these i n h i b i t o r s . The m e t h a n e s u l p h o n a m i d e 16 was a p p r o x i m a t e l y t w i c e as a c t i v e as t h e c o r r e s p o n d i n g a c e t a m i d e 10 a g a i n s t H S V - 2 TK b u t from t h i s s i n g l e example i t i s i m p o s s i b l e t o p r e d i c t whether s u l p h o n a m i d e s a r e i n g e n e r a l more p o t e n t t h a n t h e c o r r e s p o n d i n g amides. In a l l c a s e s a c y l a t i o n o f t h e 3 - h y d r o x y 1 1

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

7. MARTIN E T AJL

107

Inhibitors of Herpes Simplex Virus

f u n c t i o n markedly reduced potency. None o f t h e amides i n T a b l e I I showed a n t i v i r a l a c t i v i t y i n c e l l c u l t u r e and t h e s e compounds were i n a c t i v e a g a i n s t an HSV-1 i n f e c t i o n i n vivo· Table

II.

I n h i b i t i o n o f Thymidine K i n a s e by N - A c y l D e r i v a t i v e s 5'-Amino-2 ,5 -dideoxy-5-iodouridine 1

IC50

Compound

[μΜ]

R HSV-1

10 11 12 13 14 15 16

of

1

HSV-2

CH3CO

>200

78

C2H5CO

159

21

(CH3)2CHCO

55

7

(CH3)2CHCH2CO

68

8

CH3(CH2)3CO

38

2

PhCO

85 >200

16 35

CH3SO2

The 5 ' - a z i d o d e r i v a t i v e 17 has been r e p o r t e d (4_&) t o be an i n h i b i t o r o f HSV-1 TK ( I C 5 0 280 μ Μ ) . A l t h o u g h 17 was a n t i v i r a l i n v i v o i t was n o t a c t i v e i n c e l l c u l t u r e . Mechanism o f a c t i o n s t u d i e s have n o t been r e p o r t e d , and so t h e r e i s no e v i d e n c e t o a s s o c i a t e t h e o b s e r v e d a n t i v i r a l a c t i v i t y w i t h i n h i b i t i o n o f TK.

HO 17

D e r i v a t i v e s of Thymidine. S'-amino-S'-deoxythymidine

A s e r i e s of sulphonamide d e r i v a t i v e s of ( T a b l e I I I ) have been shown t o i n h i b i t

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

108

NUCLEOTIDE ANALOGUES

HSV TK (5JL) a n d have b e e n compared described i n the previous section. Table I I I .

with

t h e amide

I n h i b i t i o n o f Thymidine K i n a s e by Sulphonamide D e r i v a t i v e s of 5 -amino-5 -deoxythymidine 1

1

IC50

Compound

[μΜ]

R HSV-1

18 19 20 21 22 23 24

derivatives

HSV-2

P-CH3C6H4SO2

>200

88

P-CH3OC6H4SO2

>200

116

P-NO2C6H4SO2

127

21

p-BrCgH4S02

171

13

CF3SO2

>200

>200

P-HOSO2C6H4SO2

>200

>200

HOS02(CH2)3S02

>200

>200

In common w i t h amide d e r i v a t i v e s ( T a b l e I I ) a l l o f t h e a c t i v e s u l p h o n a m i d e s were s i g n i f i c a n t l y more p o t e n t a g a i n s t t h e t y p e - 2 enzyme. In the case of the benzenesulphonamides an e l e c t r o n - w i t h d r a w i n g group i n t h e benzene r i n g m a r k e d l y enhanced t h e potency, p a r t i c u l a r l y against t h e t y p e - 2 enzyme. Compounds c o n t a i n i n g an a c i d i c m o i e t y i n t h e 5 ' - s i d e c h a i n were t o t a l l y i n a c t i v e a g a i n s t b o t h enzymes. An e v a l u a t i o n o f t h e a n t i v i r a l e f f i c a c y o f t h e s e compounds has n o t been r e p o r t e d .

1

I t i s p o s s i b l e t h a t 5 - e t h y n y l t h y m i d i n e 26 (ILL) was d e v e l o p e d f r o m t h e i n h i b i t o r s 17 and 25 (4_£) , and i t i s n o t e w o r t h y t h a t 2 6 has a t o t a l l y d i f f e r e n t s p e c t r u m o f a c t i v i t y compared w i t h t h e

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

7.

109

Inhibitors of Herpes Simplex Virus

MARTIN E T AL.

i n h i b i t o r s d e s c r i b e d t h u s f a r , b e i n g more p o t e n t a g a i n s t t h e t y p e 1 ( K i 0.09 μΜ) t h a n t h e t y p e 2 enzyme ( K i 0.38 μΜ) . S i n c e 26 d i d n o t i n h i b i t human c y t o s o l i c TK, i t must a l s o be s e e n as h i g h l y s e l e c t i v e f o r t h e v i r a l enzymes. I t was n o t c y t o t o x i c i n c e l l c u l t u r e and d i d not i n h i b i t h o s t c e l l u l a r DNA s y n t h e s i s . Thymidine kinases with altered substrate specificity isolated from bromovinyldeoxyuridine (BVDU) and a c y c l o v i r - r e s i s t a n t v i r u s s t r a i n s were a l s o i n h i b i t e d by t h i s compound. Mechanism o f a c t i o n s t u d i e s i n c e l l c u l t u r e showed t h a t t h e p o o l s i z e o f t h y m i d i n e t r i p h o s p h a t e was r e d u c e d but not t h e c o r r e s p o n d i n g p o o l s o f t r i p h o s p h a t e s d e r i v e d f r o m a d e n o s i n e , g u a n o s i n e and c y t o s i n e r e s p e c t i v e l y . As e x p e c t e d c o m p o u n d 26 was not a n t i v i r a l i n v i t r o . i n agreement w i t h the o b s e r v a t i o n t h a t v i r a l TK i s not e s s e n t i a l f o r HSV r e p l i c a t i o n i n c e l l culture. However, 26 d i d r e v e r s e t h e a n t i v i r a l e f f e c t o f a c y c l o v i r , DHPG, FIAC, BVDU and 5 - a m i n o - 5 ' - d e o x y t h y m i d i n e , a l l o f which r e q u i r e an i n i t i a of a c t i v i t y . The e v a l u a t i o so f a r . 1

1

D e r i v a t i v e s of 2 -Deoxy-5-ethyluridine. A r a t i o n a l approach t o the d e s i g n o f p o t e n t and s e l e c t i v e i n h i b i t o r s o f HSV TK has been d e s c r i b e d r e c e n t l y (52-56). The d i s s o c i a t i o n c o n s t a n t s o f a s e r i e s o f 5 - s u b s t i t u t e d u r i d i n e a n a l o g u e s a g a i n s t HSV and c e l l u l a r TK (ϋ2) were examined (Table IV) i n o r d e r t o i d e n t i f y t h e n u c l e o s i d e m o i e t y most l i k e l y t o c o n f e r s e l e c t i v i t y f o r t h e v i r a l enzyme. Table

IV.

D i s s o c i a t i o n Constants

of Nucleoside D e r i v a t i v e s

D i s s o c i a t i o n Constant Compound

R

Viral HSV-1

27 28

I CF

29 30 31

TK HSV-2

[μΜ]

Cellular Cytosol 7.4 4.2

TK

Mitochondria 8 2 30

0 6 0 4

0 3 0 5

C2H5 n-C3H

0 7

0 3

82

30

0 6

0 7

21

18

n-C4H9

1 6

4 0

100

40

32

CH=CH2

0 5

0 5

35

33

CH=CHBr

0 4

3 0

3

7

>100

1 7 0 9

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

N U C L E O T I D E ANALOGUES

110

The h i g h a f f i n i t y o f i d o x u r i d i n e 27 and t r i f l u o r o t h y m i d i n e 28 f o r t h e c e l l u l a r c y t o s o l i c enzyme a n d t h e s t r o n g a f f i n i t y o f vinyldeoxyuridine 32 a n d b r o m o v i n y l d e o x y u r i d i n e 33 f o r t h e m i t o c h o n d r i a l enzyme was i n d i c a t i v e o f p o o r s e l e c t i v i t y . The e t h y l , p r o p y l a n d b u t y l d e r i v a t i v e s , 2 9 , 30 and 31 r e s p e c t i v e l y , were more s e l e c t i v e f o r t h e v i r a l enzymes and i t was r e a s o n e d t h a t i n h i b i t o r s b a s e d on 29 c o u l d be e x p e c t e d t o show t h e h i g h e s t p o t e n c y and s e l e c t i v i t y f o r HSV TK. T a b l e V.

Product

Analogues

HO

IC50

Compound

[μΜ]

R HSV-1

HSV-2 40

34 35 36 37

i-PrOP(0)(OH)O MeP(0)(0H)0 PhP(O) (0H)0 MeS020

208 320 72.6 8.1

38

p-MeC6H4S020

12.7

4.1

39

MeS02NH

6.0

7.5

40

PhS02NH

15.2

4.6

41 42 43

MeCONH PhCONH PhCH2C0NH

15.8 3.1 1.0

4.7 3.2 0.3

44

Ph0CH2C0NH

0.7

0.3

-

20.3 4.8

Having i d e n t i f i e d 29 as t h e p r e f e r r e d n u c l e o s i d e m o i e t y , d e r i v a t i v e s c o n t a i n i n g f u n c t i o n a l groups i s o s t e r i c and i s o e l e c t r o n i c w i t h t h e p h o s p h a t e r e s i d u e were p r e p a r e d ( T a b l e V) . The p h o s p h a t e 34 and p h o s p h o n a t e s 35 and 36 were r a t h e r p o o r i n h i b i t o r s , whereas several other s t r u c t u r a l classes showed s i g n i f i c a n t l y better inhibition o f b o t h t h e HSV-1 a n d HSV-2 enzyme. In g e n e r a l , s u l p h o n a t e s and s u l p h o n a m i d e s were more p o t e n t a g a i n s t t h e t y p e 2 t h a n t h e t y p e 1 enzyme, w h i l e t h e benzamide 42 was i d e n t i f i e d as a p o t e n t i n h i b i t o r o f b o t h enzymes w i t h scope f o r e a s y m a n i p u l a t i o n t o a f f o r d a n a l o g u e s w i t h enhanced p o t e n c y . I t was n o t e d d u r i n g t h e s e s t u d i e s t h a t h o m o l o g a t i o n o f t h e benzamide 42 i n c r e a s e d p o t e n c y , suggesting the presence of a hydrophobic i n t e r a c t i o n i n the v i c i n i t y of the i n h i b i t o r b i n d i n g s i t e . I n t h i s s e r i e s 43 and 44 w e r e i d e n t i f i e d as good i n h i b i t o r s , e s p e c i a l l y a g a i n s t t h e t y p e 2 enzyme.

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

7. MARTIN ET A K

111

Inhibitors of Herpes Simplex Virus

H a v i n g o p t i m i s e d t h e l e n g t h o f t h e l i n k a g e between t h e a r y l r e s i d u e and t h e n u c l e o s i d e r e s i d u e i n 4 3 , additional analogues were p r e p a r e d t h a t c o n t a i n e d s p a c e r groups t h a t were i s o s t e r i c w i t h t h e acetamide moiety. Whereas c a r b a m a t e (OCONH NHCOO) and u r e a (NHCONH) d e r i v a t i v e s were l e s s a c t i v e , amine ( C H 2 C H 2 N H ) , ketone ( C H 2 C O C H 2 ) and a l k a n e ( C H 2 C H 2 C H 2 ) a n a l o g u e s had s i m i l a r p o t e n c y t o the amide 4 3 , s u g g e s t i n g t h a t t h e s p a c e r group i s n o t i n v o l v e d i n i n t e r a c t i o n s w i t h t h e enzyme b u t s e r v e s o n l y t o l o c a t e t h e a r y l r e s i d u e i n t h e optimum p o s i t i o n . f

A number o f a n a l o g u e s o f t h e p h e n y l a c e t a m i d e 43 w h i c h were s u b s t i t u t e d e i t h e r i n t h e p h e n y l r i n g , o r on t h e α - c a r b o n atom were prepared. I t was f o u n d t h a t a medium s i z e d s u b s t i t u e n t i n t h e o r t h o p o s i t i o n o f t h e p h e n y l r i n g i n c r e a s e d p o t e n c y by as much as t e n - f o l d , as d i d a m e t h y l o r e t h y l g r o u p on t h e α - c a r b o n . In c o n t r a s t , s u b s t i t u t i o n on t h e α - c a r b o n w i t h p o l a r g r o u p s s u c h as h y d r o x y l o r amino l e d t were e x t e n d e d t o p o l y - s u b s t i t u t e 2, β - d i s u b s t i t u t e d analogues were particularly active, the 2 , 6 - d i c h l o r o and 2 , 6 - d i m e t h y l d e r i v a t i v e s , 45 and 4 6 , had IC50 v a l u e s o f 0.003 μΜ and 0.008 μΜ r e s p e c t i v e l y a g a i n s t t h e t y p e 2 enzyme. As had been o b s e r v e d w i t h t h e p h e n y l a c e t a m i d e 43 t h e c o r r e s p o n d i n g amine, k e t o n e and a l k a n e d e r i v a t i v e s a l s o showed enhanced p o t e n c y when 2 , 6 - d i c h l o r o o r 2 , 6 - d i m e t h y l s u b s t i t u t i o n was i n t r o d u c e d i n t o the phenyl r i n g . I n f a c t , compound 47 i s one o f the most p o t e n t i n h i b i t o r s o f HSV-2 TK known, w i t h an I C 5 0 o f 0.0024 μΜ.

A similar series of substituted analogues of the phenoxyacetamide 44 was also studied. As i n t h e c a s e o f p h e n y l a c e t a m i d e s , s u b s t i t u t i o n i n t h e s i d e c h a i n by an a l k y l group gave an i n c r e a s e i n p o t e n c y o f a l m o s t t w e n t y - f o l d . I n c o n t r a s t t o the p h e n y l a c e t a m i d e s , however, o p t i m a l a c t i v i t y was o b s e r v e d w i t h a 2 , 4 - d i s u b s t i t u t e d phenyl residue.

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112

NUCLEOTIDE ANALOGUES

Replacement o f t h e e t h e r l i n k a g e by a s u l p h i d e , s u l p h o x i d e , s u l p h o n e o r m e t h y l e n e group gave analogues with s i g n i f i c a n t l y reduced activity. One o f t h e more p o t e n t compounds i n t h e p h e n o x y a c e t a m i d e s e r i e s was 48 w i t h an I C 5 0 o f 0.004 μΜ a g a i n s t t h e t y p e 2 enzyme. The p o t e n t i n h i b i t o r s 4 5 , 47 and 48 were e v a l u a t e d a g a i n s t c y t o p l a s m i c TK d e r i v e d from two mammalian c e l l l i n e s ( T a b l e V I ) , and a h i g h d e g r e e o f s e l e c t i v i t y f o r t h e v i r a l enzyme was o b s e r v e d i n each c a s e . Table VI.

I n h i b i t i o n of V i r a l

IC

5 0

and C e l l u l a r Thymidine

[μΜ] Selectivity

Compound

45 47 48

Kinase

HSV-2

Hela

Vero

0.003 0.0024 0.004

>200

>200

>54,000

A s t u d y o f t h e k i n e t i c s o f i n h i b i t i o n o f HSV-2 TK by t h e amide 48 showed i t t o be c o m p e t i t i v e w i t h r e s p e c t t o t h y m i d i n e , a n d n o n - c o m p e t i t i v e w i t h r e s p e c t t o ATP. I t was c o n c l u d e d t h a t t h i s compound l o c a t e d , as e x p e c t e d , a t t h e t h y m i d i n e b i n d i n g s i t e o f t h e enzyme, and that the additional binding a f f o r d e d by t h e 2,4-dichlorophenoxypropionamide r e s i d u e , which c o n t r i b u t e d t o t h e marked p o t e n c y o f t h i s i n h i b i t o r , d i d n o t i n v o l v e t h e ATP b i n d i n g site. As e x p e c t e d , none o f t h e s e p o t e n t i n h i b i t o r s showed a n t i v i r a l a c t i v i t y i n t i s s u e c u l t u r e , b u t compound 48 d i d show a m a r k e d antagonism of the a n t i v i r a l a c t i v i t y of a c y c l o v i r i n a plaque reduction assay, which p r o b a b l y resulted from i n h i b i t i o n of i n t r a c e l l u l a r v i r a l TK. Thymidine, a t t h e same c o n c e n t r a t i o n , e x h i b i t e d a s i m i l a r antagonism o f t h e a n t i v i r a l e f f e c t o f a c y c l o v i r . Compound 48 d i d p r o d u c e a p r o t e c t i v e e f f e c t i n mice i n f e c t e d w i t h HSV-2, b u t t h e e f f e c t was v a r i a b l e and p a r t i c u l a r l y s e n s i t i v e t o t h e s t r a i n o f mouse, s i z e o f v i r u s inoculum, formulation of test compound a n d r o u t e o f a d m i n i s t r a t i o n . This i n v i v o antiviral a c t i v i t y has not as y e t been c o n c l u s i v e l y a s c r i b e d t o i n h i b i t i o n o f v i r a l TK. Conclusions I t i s e v i d e n t from d a t a p r e s e n t e d i n t h i s r e v i e w t h a t a number o f r e s e a r c h groups have p r e p a r e d p o t e n t and s e l e c t i v e i n h i b i t o r s o f HSV TK. Thus f a r , i n h i b i t o r s have been d e s i g n e d a n d d e v e l o p e d from e i t h e r s u b s t r a t e o r product analogues but a l t e r n a t i v e approaches could i n v o l v e metal c h e l a t i o n , a l l o s t e r i c i n h i b i t i o n o r b i s u b s t r a t e mechanisms. Indeed, the b i s u b s t r a t e approach (58 59) has b e e n successful i n the design of i n h i b i t o r s of b a c t e r i a l (£_Q_) a n d mammalian d e o x y n u c l e o s i d e k i n a s e s (61-63) . I n h i b i t o r s o f HSV TK t h a t a r e a v a i l a b l e now may p r o v e t o be u s e f u l b i o c h e m i c a l t o o l s t o probe t h e s t r u c t u r e and f u n c t i o n o f t h e a c t i v e s i t e o f t h e v i r a l enzymes. Improved knowledge o f t h e enzyme a c t i v e s i t e t h r o u g h r

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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f u r t h e r b i o c h e m i s t r y and m o l e c u l a r b i o l o g y s h o u l d p r o v i d e a b e t t e r understanding of the molecular i n t e r a c t i o n s t h a t occur with both s u b s t r a t e s and i n h i b i t o r s . Meanwhile, t h e c u r r e n t g e n e r a t i o n o f i n h i b i t o r s may p r o v i d e a means o f s t u d y i n g t h e r o l e o f TK i n t h e development o f HSV i n f e c t i o n i n a n i m a l models, and may f i n d a p l a c e i n t h e management o f herpes i n f e c t i o n s i n man.

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7.

M

51. 52.

53.

54. 55. 56. 57. 58. 59. 60. 61. 62. 63.

A

R

T

I

N

E T

A

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115

Nutter, L. M.; G r i l l , S. P.; Dutschman, G. E.; Sharma, R. A.; Bobek, M.; Cheng, Y.-C. Antimicrob. Agents Chemother. 1987, 31, 368. Martin, J. Α.; Duncan, I. B.; Hall, M. J.; Lambert, R. W.; Thomas, G. J.; Wong-Kai-In, P. Second International Conference on Antiviral Research. Williamsburg, Virginia, U. S. Α., 10-14 April 1988; Antiviral Res. 1988, 9, 84. Martin, J. A. Second Symposium on Medicinal Chemistry in Eastern England. Hatfield, Hertfordshire, England, 21 April 1988. Lambert, R. W.; Martin, J. Α.; Thomas, G. J. E. P. 257378. Lambert, R. W.; Martin, J. Α.; Thomas, G. J. E. P. 256400. Lambert, R. W.; Martin, J. Α.; Thomas, G. J. E. P. 255894. Cheng, Y.-C. In Antimetabolites in Biochemistry, Biology and Medicine; Skoda, J., Langen, P., Eds.; Pergamon Press: London, 1979, p.263 Wolfenden, R. Accts. Lienhard, G. E. Annu. Ikeda, S.; Chakravarty, R.; Ives, D. H. J. Biol. Chem. 1986 261, 15836. Bone, R.; Cheng,Y.-C.;Wolfenden, R. J. Biol. Chem. 1986, 261, 5731. Davies, L. C.; Stock, J. Α.; Barrie, S. E.; Orr, M.; Harrap, K. R. J. Med. Chem. 1988, 31, 1305. Hampton, Α.; Hai, T. T.; Kappler, F.; Chawla, R. R. J. Med. Chem. 1982, 25, 801.

RECEIVED January 4, 1989

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Chapter 8

Nucleotides as Herpesvirus-Specific Inhibitors of Protein Glycosylation Roelf Datema and Sigvard Olofsson 1

2

Bristol-Myers Company, Pharmaceutical Research and Development Division, 5 Research Parkway, Wallingford, CT 06492-7660 Göteborgs Universitet, Department of Clinical Virology, Guldhedsgatan 10B, S-41346 Göteborg, Sweden 1

2

We describe a strategy to obtain herpesvirus-specific glycosylation inhibitors. This involves selective phosphorylation in infected cells of a nucleoside analog to a 5'-monophosphate, which inhibits translocation of sugar nucleotides from the cytoplasm into the Golgi-compartment where the terminal glycosyltransferases are located. Such an inhibitor may be useful in antiviral chemotherapy, or could serve as a tool to study the role of terminal glycosylation of viral glycoproteins in the intact host organism. V i r a l p r o t e i n s a r e N - g l y c o s y l a t e d i n i t i a l l y by t h e t r a n s f e r en b l o c o f a g l u c o s y l a t e d high-mannose t y p e o l i g o s a c c h a r i d e f r o m t h e l i p i d d o l i c h o l - d i p h o s p h a t e u s u a l l y t o a n a s c e n t p r o t e i n (1). T r a n s f e r o f n o n - g l u c o s y l a t e d o l i g o s a c c h a r i d e s has been o b s e r v e d ( 2 ) , b u t , as y e t , n o t w i t h v i r a l p r o t e i n s . The N - l i n k e d o l i g o s a c c h a r i d e s a r e p r o c e s s e d by g l y c o s i d a s e s , and some o f t h i s p r o c e s s i n g o c c u r s i n t h e rough e n d o p l a s m i c r e t i c u l u m ( 3 ) . The e x t e n t o f p r o c e s s i n g i s dependent on a v a r i e t y o f c o n d i t i o n s , which may i n c l u d e t h e l o c a t i o n o f a p a r t i c u l a r o l i g o s a c c h a r i d e on the p r o t e i n ( 4 ) , b u t t h e removal o f g l u c o s e r e s i d u e s a p p e a r s t o be u b i q u i t o u s . The t r i m m i n g o f s u g a r r e s i d u e s and t h e subsequent a d d i t i o n o f p e r i p h e r a l sugars, t o r e s u l t i n s o - c a l l e d hybrid-type o r complex-type o l i g o s a c c h a r i d e s , o c c u r s by c o n c e r t e d a c t i o n o f g l y c o s i d a s e s and g l y c o s y l t r a n s f e r a s e s i n t h e G o l g i a p p a r a t u s ( 9 ) , and c a n r e s u l t i n a p l e t h o r a o f o l i g o s a c c h a r i d e s . (See F i g u r e 1 f o r one example.) Some v i r a l g l y c o p r o t e i n s c o n t a i n 0 - l i n k e d o l i g o s a c c h a r i d e s , u s u a l l y i n a d d i t i o n t o N - l i n k e d o l i g o s a c c h a r i d e s ( 5 ) . The 0 - l i n k e d o l i g o s a c c h a r i d e s a r e assembled by s t e p w i s e a d d i t i o n o f sugar r e s i d u e s o n t o a p r o t e i n i n t r a n s i t t h r o u g h t h e G o l g i a p p a r a t u s ( 6 ) . Thus, t h e s y n t h e s i s o f 0 - l i n k e d o l i g o s a c c h a r i d e s occurs simultaneously with the a d d i t i o n o f p e r i p h e r a l sugars t o 0097-45156/89/0401-0116S06.00/0 o 1989 A m e r i c a n C h e m i c a l Society

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

8.

DATEMA AND OLOFSSON

Inhibitors of Protein Glycosylation

N - l i n k e d o l i g o s a c c h a r i d e s , and t h e s e p r o c e s s e s "terminal glYCOsylation" (7).

a r e r e f e r r e d t o as

The b i o l o g i c a l r o l e o f t e r m i n a l g l y c o s y l a t i o n o f v i r a l glycoproteins S e v e r a l l o w - m o l e c u l a r w e i g h t compounds ( s u g a r a n a l o g s and a l k a l o i d s ) a r e known t o i n h i b i t d i s c r e t e s t e p s o f t h e t r i m m i n g pathway, as shown i n F i g u r e 1, and have been u s e d t o s t u d y t h e b i o l o g i c a l r o l e o f the o l i g o s a c c h a r i d e processing using v i r u s - i n f e c t e d c e l l s i n c u l t u r e . I t became r a p i d l y a p p a r e n t t h a t t h e b i o l o g i c a l e f f e c t s were s t r o n g l y dependent on t h e p a r t i c u l a r p r o t e i n o r v i r a l system ( 8 ) . Examples i n c a s e a r e two r e t r o v i r a l systems. I n b o t h Rous Sarcoma V i r u s (RSV)- and murine l e u k e m i a v i r u s (MuLV)-infected c e l l s , b l o c k i n g N - g l y c o s y l a t i o n i n t e r f e r e s with c o r r e c t p r o t e o l y t i c p r o c e s s i n g o f the envelope g l y c o p r o t e i n s t o s t a b l e e n d - p r o d u c t s an i n t o v i r i o n s (9-10). Allowin o l i g o s a c c h a r i d e t r i m m i n g by any o f t h e g l u c o s i d a s e i n h i b i t o r s o f F i g u r e 1 p r e v e n t e d t h e f o r m a t i o n o f complex-type o l i g o s a c c h a r i d e s of the envelope g l y c o p r o t e i n s , but d i d not prevent t h e formation of f u l l y i n f e c t i o n s RSV-particles (11). In c o n t r a s t , formation o f i n f e c t i o u s MuLV was i n h i b i t e d by d e o x y n o j i r i m y c i n , an i n h i b i t o r of glucosidase I (10). S i n d b i s - v i r u s i n f e c t e d c e l l s p r e s e n t e d an i n t e r e s t i n g system to study the r o l e o f p r o c e s s i n g o f N - l i n k e d o l i g o s a c c h a r i d e s . The E l and E2 g l y c o p r o t e i n s o f S i n d b i s v i r u s e a c h c o n t a i n two g l y c o s y l a t i o n s i t e s ( 1 2 ) , and one g l y c o s y l a t i o n s i t e on E l and E2 c a r r i e s e x c l u s i v e l y complex-type o l i g o s a c c h a r i d e s when t h e v i r u s i s grown i n a v i a n o r mammalian c e l l s . H s i e h e t a l . (4) c o u l d show t h a t t h e f o l d i n g o f t h e p o l y p e p t i d e c h a i n s d e t e r m i n e d t h e extent of processing. I t i s shown i n T a b l e 1 t h a t when N - g l y c o s y l a t i o n i s i n h i b i t e d by t u n i c a m y c i n , t h e p r e c u r s o r p r o t e i n o f E2 (pE2) i s n o t c l e a v e d t o E2, t h e c e l l - s u r f a c e e x p r e s s i o n o f pE2 i s i n h i b i t e d , and v i r u s b u d d i n g d e c r e a s e s ( 1 3 ) . O t h e r work has shown t h a t c l e a v a g e o f pE2 t o E2 i s r e q u i r e d f o r v i r u s release (14). Allowing g l y c o s y l a t i o n t o occur, but p r e v e n t i n g g l u c o s e - t r i m m i n g by t h e g l u c o s i d a s e I - i n h i b i t o r s N-methyl d e o x y n o j i r i m y c i n o r c a s t a n o s p e r m i n e and t h u s e q u i p p i n g the v i r a l g l y c o p r o t e i n s with the o l i g o s a c c h a r i d e s Glc^Man^ ^GlcNAc , s t i l l p r e v e n t e d c l e a v a g e o f p E ( a n d hence v i r u s r e l e a s e ) , But a l l o w e d c e l l - s u r f a c e e x p r e s s i o n o f PE2 (15-16). The same r e s u l t was o b t a i n e d e a r l i e r w i t h bromoconduritol, t h a t caused equipping the v i r a l g l y c o p r o t e i n s w i t h GlcMan^ Q G 1 C N A C ( 1 7 ) . A l l o w i n g the removal o f the glucose group ( b u t p r e v e n t i n g mannose-trimming u s i n g deoxymannojirimycin), equipping the g l y c o p r o t e i n with Man^ g G l c N A c ^ - o l i g o s a c c h a r i d e s , p e r m i t t e d c l e a v a g e o f pE2 and allowed v i r u s budding (16). Thus, t h e p r e s e n c e o f a s i n g l e g l u c o s e group p e r o l i g o s a c c h a r i d e c h a i n d e t e r m i n e d whether o r n o t t h e p r o t e i n c a n be p r o t e o l y t i c a l l y c l e a v e d . These and o t h e r s t u d i e s (5) i n d i c a t e an e s s e n t i a l r o l e o f g l u c o s e t r i m m i n g i n t h e m a t u r a t i o n o f some v i r u s e s . 2

2

S i n d b i s v i r u s c u l t u r e d i n t h e presence o f t h e mannosidase-I

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

NUCLEOTCDE ANALOGUES

I —

-

F i g u r e 1. Processing of N-linked oligosaccharides. a b b r e v i a t i o n s , see T a b l e 1. •, GlcNAc; 0, Man; X, G l c ; ·, G a l ; •, NeuAc. DIM,1,4-dideoxy-1,4-imino-D-mannitol; 2,5-dihydroxymethYl-3,4-dihydroxYpyrrolidine

For

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

8.

DATEMAANDOLOFSSON

Inhibitors of Protein Glycosylation

i n h i b i t o r d e o x y m a n n o j i r i m Y c i n buds p r e f e r e n t i a l l y from i n t e r n a l membranes, r a t h e r t h a n t h e c e l l - s u r f a c e membrane (16)· I n t h e presence o f the mannosidase-II i n h i b i t o r swainsonine (see F i g u r e 1) t h e r e l e a s e o f i n f e c t i o u s S i n d b i s v i r u s i s n o t d i f f e r e n t f r o m that of untreated c u l t u r e s . In f a c t , the maturation o f a l l e n v e l o p e d v i r u s e s s o f a r s t u d i e d seems n o t t o be a f f e c t e d by blocking oligosaccharide processing a t the l e v e l of the G o l g i enzymes N - a c e t y l g l u c o s a m i n y l t r a n s f e r a s e I o r mannosidase I I ( 5 ) . Y e t , many o l i g o s a c c h a r i d e s o f v i r a l g l y c o p r o t e i n s a r e p r o c e s s e d p a s t t h i s s t a g e t o form complex-type o l i g o s a c c h a r i d e s . What, t h e n , i s t h e b i o l o g i c a l r o l e o f t e r m i n a l g l y c o s y l a t i o n of v i r a l glycoproteins? Based on e v i d e n c e p r e s e n t e d e l s e w h e r e , we s u g g e s t e d ( 5 ) , t h a t t e r m i n a l g l y c o s y l a t i o n o f v i r a l g l y c o p r o t e i n s may p l a y a r o l e a t t h e v i r u s - h o s t l e v e l , f o r example i n v i r a l s p r e a d and p a t h o g e n e s i s . To s t u d y t h e s e phenomena i n t h e i n t a c t o r g a n i s m i n h i b i t o r s active only i n v i r u s - i n f e c t e d c e l l s ar I n h i b i t i o n o f T e r m i n a l N- and O - G l y c o s y l a t i o n Herpesvirus-Infected C e l l s

Specific for

We c h o s e t o s t u d y t h e p o s s i b i l i t y o f development o f v i r u s - s p e c i f i c i n h i b i t o r s o f g l y c o s y l a t i o n i n t h e herpes simplex v i r u s (HSV) system, b e c a u s e HSV has been w i d e l y u s e d t o s t u d y mechanisms o f s p e c i f i c a n t i v i r a l a g e n t s ( 1 8 ) , and t h e p a t h o g e n e s i s o f HSV has been s t u d i e d i n s e v e r a l a n i m a l models (19) a l l o w i n g e v a l u a t i o n i n v i v o . The HSV-1 s p e c i f i e d g l y c o p r o t e i n gC-1 i s u s e d as a m o l e c u l a r p r o b e b e c a u s e t e r m i n a l g l y c o s y l a t i o n o f gC-1 may be i m p o r t a n t i n v i v o . Evidence f o r t h i s a r e : r e m o v a l o f t e r m i n a l s u g a r s from gC-1 changes t h e a n t i g e n i c i t y o f t h e p e p t i d e p a r t o f t h e m o l e c u l e ( 2 0 ) , and t h e C 3 b - r e c e p t o r a c t i v i t y o f gC-1 i s s i a l i d a s e - d e p e n d e n t ( 2 1 ) . F u r t h e r m o r e , gC-1 c o n t a i n s N- and 0 - l i n k e d o l i g o s a c c h a r i d e s , which c a n be s t u d i e d s e p a r a t e l y s i n c e t u n i c a m y c i n - t r e a t m e n t does not j e o p a r d i z e t h e p r o t e o l y t i c s t a b i l i t y o f gC-1 ( 2 2 ) . A l s o , e x p e r i m e n t a l t o o l s t o r a p i d l y p r o b e changes i n N- and 0 - l i n k e d o l i g o s a c c h a r i d e s o f gC-1 have been d e v e l o p e d (23-24). Terminal g l y c o s y l t r a n s f e r a s e s are located i n s i d e the t r a n s - G o l g i compartment as i s t h e a c c e p t o r g l y c o p r o t e i n (_1). However, t h e s u b s t r a t e s o f t h e t r a n s f e r a s e s , t h e s u g a r n u c l e o t i d e s a r e p r e s e n t i n t h e c y t o s o l . H i r s c h b e r g and co-workers (25) c o u l d show t h a t s u g a r n u c l e o t i d e t r a n s l o c a t o r p r o t e i n s i n t h e G o l g i membranes t r a n s p o r t s u g a r n u c l e o t i d e s i n t o t h e G o l g i compartment and t h i s t r a n s l o c a t i o n i s c o u p l e d t o e x p o r t o f t h e c o r r e s p o n d i n g n u c l e o s i d e 5 *-monophosphate, as shown f o r UDP-Gal i n F i g u r e 2. The t r a n s l o c a t i o n o f s u g a r n u c l e o t i d e s i n t o t h e G o l g i compartment c a n be i n h i b i t e d by n u c l e o s i d e 5 -monophosphates ( 2 6 ) , and t h i s phenomenon r e p r e s e n t s t h e t a r g e t f o r the design of v i r u s - s p e c i f i c g l y c o s y l a t i o n i n h i b i t o r s . The i n h i b i t i o n o f s u g a r n u c l e o t i d e t r a n s l o c a t i o n l i m i t s t h e amount o f t h e s u b s t r a t e f o r t h e g l y c o s y l t r a n s f e r a s e , and c a u s e s a b l o c k i n glycosylation. H e r p e s v i r u s s p e c i f i c i t y i s o b t a i n e d by s e l e c t i v e p h o s p h o r y l a t i o n o f a n u c l e o s i d e t o an i n h i b i t o r y n u c l e o s i d e 5'-monophosphate i n i n f e c t e d c e l l s . 1

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

120

N U C L E C m D E ANALOGUES

Table 1.

Role of G l y c o s y l a t i o n i n the Sindbis Virus/BHK C e l l System

Cleavage

Cell-surface expression Virus budding PE , E

Location of block

Inhibitor used

LLO assembly

TM

No

No

Decreased

Glc trimming

Be

No

Yes

Decreased

Glc trimming

dN, cas

Man I trimming

dMM

Yes

Yes

Yes, but intracellularly

Man I I trimming

swa

Yes

Yes

Yes

P

V 2 E

2

2

Abbreviations: LLO, l i p i d - l i n k e d oligosaccharide; TM, tunicamycin; Be, bromoconduritol; dN, 1-deoxynojirimycin; cas, castanospermin; MdN, N-methyl-l-deoxynojirimYcin; dMM, l-deoxymannojirimYcin; swa, swainsonin. For references, see t e x t .

out in

Golgi vesicle Substrate + acceptor . UDP-Gal + HO-R

PORT: UDP-Gal i_

Galactosyl transferase ANTIPORT: UMP

UMP

UDP t Gal-O-P,

I I Figure

2.

J I

etc.

Schematic diagram of sugar n u c l e o t i d e

translocation.

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

8.

Inhibitors of Protein Glycosylation

DATEMA AND OLOFSSON

We have s t u d i e d (24, 27) t h e f e a s i b i l i t y o f t h i s a p p r o a c h with the n u c l e o s i d e analog (E)-5-(2-bromovinyl)-2 -deoxyuridine (BVdU). BVdU i s a s e l e c t i v e anti-HSV-1 agent i n t e r f e r i n g w i t h v i r a l DNA s y n t h e s i s ( 1 8 ) . The s e l e c t i v i t y o f BVdU i s due i n p a r t t o i t s p h o s p h o r y l a t i o n by t h e HSV-induced t h y m i d i n e k i n a s e . In a d d i t i o n t o i n t e r f e r i n g w i t h v i r a l DNA s y n t h e s i s , t h e drug a f f e c t s t h e s y n t h e s i s o f v i r a l g l y c o p r o t e i n s (24, 28-29), an e f f e c t a l s o dependent on i n d u c t i o n o f t h e v i r a l t h y m i d i n e k i n a s e . The e f f e c t on g l y c o p r o t e i n s can be s t u d i e d s e p a r a t e l y from t h e e f f e c t on DNA s y n t h e s i s by a d d i n g t h e drug a f t e r t h e o n s e t o f v i r a l DNA s y n t h e s i s ( i . e . , 6 h p . i . ) . An a n a l y s i s o f the e f f e c t on g l y c o p r o t e i n gC-1 showed t h a t i n c o r p o r a t i o n o f g a l a c t o s e and s i a l i c a c i d i n t o N - l i n k e d o l i g o s a c c h a r i d e s and i n c o r p o r a t i o n o f s i a l i c a c i d , and t o a l e s s e r e x t e n t , o f g a l a c t o s e i n t o 0 - l i n k e d o l i g o s a c c h a r i d e s was b l o c k e d ( 2 7 ) . This r e s u l t e d i n formation of normal amounts o f gC-1, b u t w i t h d i f f e r e n t o l i g o s a c c h a r i d e s i . e . , w i t h t e r m i n a l GlcNA and t e r m i n a l 0 - l i n k e d GalNAc f

T h i s i n h i b i t i o n o f t e r m i n a l g l y c o s y l a t i o n was not c a u s e d by i n h i b i t i o n o f f o r m a t i o n o f UDP-hexoses, i n h i b i t i o n o f i n t r a c e l l u l a r transport of glycoproteins i n a g a l a c t o s y l t r a n s f e r a s e - d e f i c i e n t c e l l l i n e , nor by i n h i b i t i o n o f a c c e p t o r glycoprotein synthesis. No e v i d e n c e f o r f o r m a t i o n o f a s u g a r n u c l e o t i d e a n a l o g o f BVdU was o b t a i n e d . The i n h i b i t i o n o f terminal g l y c o s y l a t i o n d i d require formation of BVdU-5 -monophosphate, and was t h e r e f o r e not o b s e r v e d i n c e l l s i n f e c t e d w i t h T K - n e g a t i v e v a r i a n t s o f HSV-1. The b l o c k d i d o c c u r a l s o i n HSV-2 i n f e c t e d c e l l s , which can p h o s p h o r y l a t e BVdU t o i t s 5'-monophosphate (BVdUMP), b u t not any f u r t h e r . In a c e l l - f r e e system BVdUMP i n h i b i t e d t h e t r a n s p o r t o f p y r i m i d i n e s u g a r n u c l e o t i d e s ( s u c h as UDP-Gal, CMP-NeuAc) a c r o s s G o l g i membranes and, as a consequence, i n h i b i t e d t h e t e r m i n a l g l y c o s y l a t i o n . No i n h i b i t i o n o f t r a n s l o c a t i o n o f p u r i n e sugar n u c l e o t i d e was o b s e r v e d , nor d i d BVdUMP i n h i b i t g a l a c t o s y l t r a n s f e r a s e . 1

Taken t o g e t h e r , t h e s e r e s u l t s (27) show t h a t i t s h o u l d be p o s s i b l e t o o b t a i n a n u c l e o s i d e s e l e c t i v e l y p h o s p h o r y l a t e d by HSV t h y m i d i n e k i n a s e t o a 5 -monophosphate which w i l l i n h i b i t s u g a r n u c l e o t i d e t r a n s l o c a t i o n , i n o t h e r words a v i r u s - s e l e c t i v e glycosylation inhibitor. F u r t h e r m o r e , i t s h o u l d be. p o s s i b l e t o obtain HSV-specific i n h i b i t o r s of f u c o s y l a t i o n or mannosylation by u s i n g g u a n o s i n e a n a l o g s s e l e c t i v e l y p h o s p h o r y l a t e d by HSV-coded t h y m i d i n e k i n a s e (17, 3 0 ) . 1

Implications With the e x c e p t i o n of c h r o n i c a d m i n i s t r a t i o n o f o r a l a c y c l o v i r , chemotherapy has not been i m p r e s s i v e l y u s e f u l i n t h e management o f r e c u r r e n t HSV i n f e c t i o n s (32)· U s i n g t h e r a p e u t i c regimens o f a c y c l o v i r , b u c i c l o v i r or foscarnet e f f e c t i v e l y blocking HSV-1 r e p l i c a t i o n and d i s e a s e development i n s y s t e m i c , c u t a n e o u s and c e n t r a l nervous s y s t e m - i n f e c t i o n i n m i c e , no c l i n i c a l b e n e f i t was o b t a i n e d when t h e v i r u s (HSV-1) i n f e c t e d t h e s k i n o f mice v i a sensory nerves ( i n z o s t e r i f o r m - s p r e a d models), d e s p i t e the p r e s e n c e o f immunity and d r u g a v a i l a b i l i t y ( 3 2 ) . Thus, i n t h e s e

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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models o f r e c r u d e s c e n t d i s e a s e , as i n c l i n i c a l t r i a l s o f a c u t e t r e a t m e n t o f r e c u r r e n t HSV i n f e c t i o n s , t h e b e n e f i t s o f p o t e n t and s e l e c t i v e i n h i b i t o r s o f HSV r e p l i c a t i o n were m i n i m a l . This i m p l i e s t h a t management o f r e c u r r e n t H S V - i n f e c t i o n s r e q u i r e s an a p p r o a c h o t h e r t h a n i n h i b i t i n g HSV DNA s y n t h e s i s . Selective i n t e r f e r e n c e with v i r a l p r o t e i n g l y c o s y l a t i o n , r e s u l t i n g i n a l t e r e d immunogenicitγ o f t h e v i r a l g l y c o p r o t e i n s may be such an approach.

Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.

Kornfeld, R.; Kornfeld, S. Annu. Rev. Biochem. 1985, 54, 631-64. Romero, P. Α.; Herscovics, A. Carbohydr. Res. 1986, 151, 21-28. Dunphy, W. G.; Rothman Hsieh, P.; Rosner, 1983, 258, 2555-61. Datema, R.; Olofsson, S.; Romero, P. A. Pharmac. Ther. 1987, 33, 221-86. Roseman, S. In Biology and Chemistry of Eucaryotic Cell Surfaces; Lee, E. Y. C.; Smith, Ε. E., Eds.; Academic: New York, 1974; pp. 317-54. Schachter, H. Biol. Cell. 1984, 51, 133-46. Schwarz, R. T.; Datema, R. Trends Biochem. Sci. 1984, 9, 32-34. Schwarz, R. T.; Rohrschneider, J. M.; Schmidt, M. F. G. J. Virol. 1976, 12, 782-91. Pinter, Α.; Honnen, W. J.; L i , J. S. Virology. 1984, 136, 196-210. Bosch, J. V.; Schwarz, R. T. Virology. 1984, 132, 95-109. Strauss, E. G.; Strausse, J. H. In The Togavividae and Flaviviridae; Schlesinger, S.; Schlesinger, M. J., Eds.; Plenum Publishing Corp.: New York, 1986; pp. 35-90. Schlesinger, M. J. In Virology; Fields, Β. N.; Knipe, D. M.; Chancok, R. M.; Melnick, J. L.; Roizman, B.; Shope, R. E., Eds.; Raven Press: New York, 1985; pp. 1021-32. Garoff, J.; Kondor-Koch, C.; Riedel H. Curr. Top. Microbiol. Immunol. 1982, 99, 1-50. Schlesinger, S.; Koyama, A. H.; Malfer, C.; Gee, S. L.; Schlesinger, M. J. Virus Res. 1985, 2, 139-49. McDowell, W.; Romero, R. Α.; Datema, R.; Schwarz, R. T. Virology. 1987, 161, 37-44. Datema, R.; Romero, P. Α.; Rott, R.; Schwarz, R. T. Arch. Virol. 1984, 81, 25-39. De Clercq, E. Biochem. J. 1982, 2051, 1-13. Stanberry, L. R. Progr. Med. Virol. 1986, 33, 61-77. Sjöblom, I.; Lundström, M.; Sjögren-Jansson, E.; Glorisoso, J. C.; Jeansson, S.; Olofsson, S. J. Gen. Virol. 1987, 60, 545-54. Smiley, M. L.; Friedman, H. M. J. Virol. 1985, 55, 857-61. Svennerholm, B.; Olofsson, S.; Lundén, R.; Vahlne, Α.; Lycke, E. J. Gen. Virol. 1982, 63, 343-49.

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

8.

DATEMA AND OLOFSSON

23. 24. 25. 26. 27. 28. 29. 30. 31.

32.

Inhibitors of Protein Glycosylation

Lundström, M.; Olofsson, S.; Jeansson, S.; Lycke, Ε.; Datema, R.; Månsson, J.-E. Virology. 1987, 161, 385-94. Olofsson, S.; Lundström, M.; Datema, R. Virology. 1985, 147, 201-05. Hirschberg, C. B.; Snider, M. D. Annu-Rev. Biochem. 1987, 56, 63-88. Capasso, J. M.; Hirschberg, C. B. Biochim. Biophys. 1984, 777, 133-239. Olofsson, S.; Milla, M.; Hirschberg, C.; De Clercq, E.; Datema, R. Virology. 1988, 166, 440-450. Misra, V.; Nelson, R. C.; Babiuk, L. A. Antimicrob. Agents Chemother. 1983, 23, 857-65. Siegel, S. Α.; Otto, M. J.; De Clercq, E.; Prusoff, W. H. Antimicrob. Agents Chemother. 1984, 25, 566-70. Datema, R.; Ericson,A.-C.;Field, J . J., Larsson, Α.; Stenberg, K. Antiviral Res 1987 7 303-16 Douglas, J. M.; Critchlow Connor, J. D.; Hintz Μ. Α.; Α.; Winter, C; Corey, L. New Engl. J. Med. 1984, 310, 1551-56. Kristofferson, Α.; Ericson,A.-C.;Sohl-Åkerlund, Α.; Datema, R. J . Gen. Virol. 1988, 69, 1157-66.

RECEIVED January 4, 1989

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

123

Chapter 9

Antiviral Activity of the Nucleotide Analogue Ribavirin 5'-Sulfamate Donald F. Smee Nucleic Acid Research Institute, 3300 Hyland Avenue, Costa Mesa, CA 92626

Ribavirin 5'-sulfamat replication in cel protecte lethal virus challenge. This activity was unexpected since ribavirin was not inhibitory to the virus. De­ tailed mode of action studies showed that the analog inhibited viral polypeptide and RNA syntheses to a greater extent than comparable host cell functions. Viral RNA synthesis inhibition was caused by the in­ hibited synthesis of the viral RNA polymerase protein. The compound's primary mode of action was on amino­ acyl-tRNA synthesis, the first step required for mRNA translation into protein. Other cellular processes affected as a result of protein synthesis inhibition included DNA and polyamine syntheses, and methylation and sulfuration reactions.

For years i n v e s t i g a t o r s have explored the p o t e n t i a l of nucleotide analogs as i n h i b i t o r s of v i r a l i n f e c t i o n s (1), with much of the e f f o r t focused on modifications at the 5'-monophosphate p o s i t i o n . To date, 5'-phosphonate d e r i v a t i v e s of e i t h e r n a t u r a l l y occurring nucleosides (2) or of nucleoside analogs (3) have not possessed the desired b i o l o g i c a l a c t i v i t y i n c e l l culture. Part of the reason f o r this lack of a c t i v i t y i s that these polar compounds do not r e a d i l y penetrate into c e l l s . One approach to reduce p o l a r i t y i s to construct 3',5'-cyclic phosphate analogs. The strategy i s for the compounds to enter c e l l s and be cleaved to 5'-monophosphate d e r i v a t i v e s by phosphodiesterases. The free 5'-monophosphates can then be further phosphorylated to nucleoside d i - and triphosphates. Some of these compounds, such as adenosine 3',5'-cyclic phosphorothioates ( 4 ) , rather than being catabolized to 5'-monophophate forms, have turned out to be i n h i b i ­ tors of nucleoside 3',5',-cyclic phosphodiesterases, or i n other ways i n h i b i t c e l l p r o l i f e r a t i o n . The charges on the 5'-phosphate moiety can also be reduced by a l k y l blocking groups. Examples of active compounds include c e r t a i n 0097-6156/89/0401-€124$06.00/0 ο 1989 A m e r i c a n C h e m i c a l Society

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

125

Antiviral Activity of Ribavirin 5'-Sulfamate

9. SMEE nucleoside

5'-phosphonoformate

2'-deoxyuridines

(5)

and

(PFA)

purines

viruses.

For

each

of

the

reported

felt

cleavage

of

the

PFA g r o u p

that

activity

was

nucleoside

due

either

analog,

to

which

its

that

a

free

5-substituted

herpes

compounds,

occurred,

to

of

inhibit

active

PFA o r

in

derivatives

(6)

and

that

combination

state

would

simplex

the

authors

the

of

antiviral

PFA and

inhibit

the

the

virus

anyway. Phosphate 5'-sulfates penetrate this

into

series

possesses of

cells.

was

The

synthesis,

makes

growth

it of

too

both

African

antiviral

a c t i v i t y when 1,

analog

the

(11);

selective herpes

The sides

it

was

hexose

diphosphate inhibited

of

in

enveloped

proteins

o n DNA

The

to

see that

antiviral

nucleotide

in

and

the

was

to

is

an

that

inhibitor

parasite

(8),

5'-sulfamate, shown

inhibit

to

a

inhibit

protein

agent

in

swine

vitro

The at

to

20

viral

The

5'-

recently

screens.

In

5'-sulfamates

were

fever,

a

also

more

the

was

and

to

a

found

lesser

(12).

preparation

synthesis

antiviral

our

uridine

African in

cytotoxicity.

tiazofurin

used of

a

active

to

100

to

link

series of

these

ug/ml

i n h i b i t i o n of

nucleo­

of

uridine structures

(14).

The

anti­

glycosylation

of

(14).

program

at

this

institution,

ribavirin

(5'-0-sulfamoyl-l-£-D-ribofuranosyl-l,2,4-triazole-3-

carboxamide; similar

the

(9)

inactive

viruses

viral

of

and

Adenosine

nucleocidin,

(13).

attributed

part

cell

considerable

the

a c t i v i t y was As

host

certain

viral

5'-sulfamate

identified

Nucleocidin

f u n c t i o n a l i t y was

analogs

and

of

viruses

moieties

sugar

many

also

inhibitors simplex

5'-sulfamate

to

the

be

as

readily

occurring antibiotic

human u s e .

had

communication,

be

(7).

antiviral

preliminary to

to

such

will

teste it

synthesized

extent,

compound

and

(10)

and

of

non-polar

naturally

trypanosomes

coli

in Table

of

for

analog

in

sulfamate

first a

5'-monophosphates

are

activity

toxic

synthesis listed

(1)

nucleocidin,

chemically-synthesized the

nucleoside

5'-sulfamates

anti-parasitic

protein

which

mimics of

and

structure used

to

a c t i v i t y and

analog

are

below)

was

synthesize mode

of

presented

synthesized

using

a

adenosine

5'-sulfamate

action

this

of

scheme (15).

interesting

new

here.

Ο

OH

HO Antiviral Initially, ral

activity

ribavirin

activity

human

in

in

a

(CPE)

culture

5'-sulfamate

primary

cytomegalo-,

cytopathology

cell

herpes in

a

screen

was

in

and

mice

evaluated

(Table

simplex, narrow

and

1).

The

in

vitro

compound

parainfluenza

concentration

range,

for

antivi­

inhibited

virus-induced but

had

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

a

more

NUCLEimDE ANALOGUES

126 pronounced

effect

against

Forest

virus-induced

μΜ

suppression

the

ribavirin

was

not

The

analog

showed

and

it

toxic

was

concentrations inhibited extent At

the

morphology some

actively

active no to

the

Vero

cells

toxic

50% a t

Table

of

1.

in

50%, Forest

influenza to

In

uninfected

cell

cell

Vero

at

and

<1000

viruses

to

a

virus

μΜ. at

lesser type

altered

monolayers, were

320

since

markedly

proliferation

cells

to

unique

these

5'-sulfamate

stationary

32

rhinoviruses,

ribavirin

A virus,

Semliki

at

virus

parainfluenza

ribavirin

1 0 μΜ,

A and

replicate

influenza and

At

whereas

a c t i v i t y was

contrast,

1-A

effects.

1-3

Semliki

used In

rhinovirus

replicating

by

cells

1 0 μΜ.

virus.

by

This

against

concentrations,

of

100%.

against

effect

Forest

inhibited

C P E was

replication

partially

growth

of

above

inhibited

antiviral

Semliki

C P E was

3. the

indicating

assays,

arrested

in

their

μΜ.

Inhibitor and

Virus Human

(Strain)

cytomegalo

Herpes

simplex

Influenza

A

Rhino

1-A

Semliki

3

(C243)

(2060) Forest(Original)

*Fifty

percent

cytopathic

ratings

of

indicate

<0.5

tivity;

1.0

The virus

viruses the

are

of

above,

the

to

The

as

a

on

the

for in

tion

was

Μ0Ι.

decreased The

10 a n d

virus

48

nucleotide

hours

virus at

even

and

50-100 at

with

analog

method

an

about

the 2

were

virus the was

Sidwell

0.6-0.9,

(17)·

Ratings

moderate

performed

is

a

ac­

many

family

the

inhibitors

that

these

effects

of

by

reduction

RNA

Toga-

diseases might

ribavirin

5'-

methods

(19).

multiplicities

refers

the

of

added >1

degree

of

be

viruses.

quantifying

assay

to

in

understanding

different

virus

to

of

cell

simultaneously

log

μΜ c o n c e n t r a t i o n s , the

by

information of

replication

(Μ0Ι

Semliki

(18).

encephalitic

thought

the

using

positive-stranded

togavirus

detail,

where

increasing

from

activity.

reduction

1 0 0 μΜ

Μ0Ι

of

compound were a

determined

(16).

activity;

plaque

used

2),

destroyed at

by

were

(Table

achieved

However,

yield

(Μ0Ι)

the

concentration,

virus

conditions

When

>1000/0.0

determined

Forest yield

monolayers

320/0.4

10/0.8

some

under

ratio).

>10/0.0

It

virus

cell

HeLa Vero

selective

experiment

virus

100/0.8

causing

extracellular infecting

10/0.4

of

world.

more

Semliki

done

32/1.2

Vero

The

This

was

>10/0.0

the

member

activity

design

next

sulfamate

>1000/0.0

MDCK

marked

cells.

responsible

compound's

>1000/0.0

investigations

i n Vero

throughout

helpful

1/0.4 10/0.4

weak a n t i v i r a l

classified

humans

by

(μΜ)

Vero

i n h i b i t i o n assay

calculated

and

rest

virus

Rib.5'-Sulf.

virus-inhibitory

effect

**Virus

Forest

(G)

(Chile)

Parainfluenza

ED50*

Line MRC-5

(AD-169)

2

/ Virus Rating** Ribavirin

Cell

of

virus

to

produc­

regardless

of

i n h i b i t i o n of

the

virus

Μ0Ι.

cell

monolayer

of

0.01.

to

6 hours

It to

in

usually

12

hours

takes

equilibrate

a

MOI

of

nucleoside

at

or

into

an

cells,

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

and

9.

S

M

E

Table

E

Antiviral Activity of Ribavirin 5" -Sulfamate

2.

Antiviral

activity

multiplicities

of

of

ribavirin

Semliki

Log

1

5'-sulfamate

Forest

Plaque

Q

127

virus

Forming

under

varying

infection

Units/ml Pre-Treatment* Actinomcyin

Inhibitor Conc.(yM)

0.01**

0.1

D

Present

(5

None

Treatment**

Simultaneous

μΜ)

1

10

10

10

0

9.3

9.3

9.7

8.3

9.4

9.5

6.25

9.5

9.0

8.6

8.6

9.4

9.0

12.5

9.6

8.9

8.6

8.3

7.7

9.1

25

9.3

8.7

8.3

7.5

6.6

6.9

50

5.6

6.8

6.7

7.3

5.6

5.9

3.5

4.6

5.7

6.3

5.2

5.3

5.8

4.7

4.0

2.0

4.2

4.2

100 Log

reduction

at

100

μΜ

•Ribavirin

5'-sulfamat

replication. **Ribavirin the

Actinomyci

5'-sulfamate

and

virus

were

added

simultaneously

to

cells.

***Multiplicity

of

virus

infection

(Μ0Ι).

S O U R C E : R e p r o d u c e d with permission from ref. 23. Copyright 1988 Elsevier. if

necessary

Μ0Ι

the

complete this, 2).

be

virus

its

way

equilibration

of

the

inhibitor

cells

were

experiment

pre-treated was

since

in

of

antiviral

mode virus

inoculation,

i n h i b i t i o n of

a

(antagonism)

25-100 rin's

μΜ.

in

In

uninfected

dividing

the

7

In

days.

daily

with

infection was loss the as

daily and

with

at

40

at

10

treated

controls. provided of

A no

effects

uridine

subsequent

and

of

at

became

and

their

appeared study

to

an

the

a

The

more less

at

ribavi­

<40

3),

due

at

most

a

doses

tested By

mg/kg

mice

lethal in

encephalitic

day to

than

effect

survivors

induced

for

treated

protective

was

severe

of

drug

appearance.

healthy

that

reverse

analog

each

i l l

overall

cause

analog

first

compound With

the

dose.

increase

20.

more not

was

of

later

(21,22)•

otherwise Less

the

showed

infected

of

to

of

treatment

tolerated

(Table

virus.

loss

mice

loss

10 mg/kg

antiviral

survived although

known

for

(Table

needed

caused

activity

maximum

before

absence

tool

starting

inhibits

infection

Forest

some

protection

By

high

compensate

or

important

5'-sulfamate

tolerated

significant.

control

weight at

mg/kg

To

the

Actinomycin D did

it

its

culture

5'-sulfamate

5'-sulfamate

ribavirin

at

presence

an

antiviral

viruses

mice

mg/kg,

and

the

studies.

determine

Semliki

mg/kg by

the

that cell

occurred.

actinomycin D is

dose, 40

in

the

ribavirin

yield.

the

experimental

uninfected

Mode

to

statistically

judged

mice

The

mice

20

of

experiments

an

achieved

s t i l l

against

animal

with

ribavirin virus

contrast,

activity In

action

means

destroy

actinomycin D is

pronounced reversal

to

conducted

D,

before

This

on

actinomycin the

(20,21).

well

the The

metabolized

was

weight

treatment toxicity, Even

the

saline-treated

below

10

mg/kg

mice.

action

ribavirin

[3-H]amino

5'-sulfamate

acids

into

on

the

incorporation

acid-precipitable

of

[3-H]-

macromolecules

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

128

ΝϋΟ,ΕΟΉΌΕ ANALOGUES

Table 3.

E f f e c t s of r i b a v i r i n 5 '-sulfamate on a l e t h a l Semliki Forest v i r u s i n f e c t i o n i n mice

T o x i c i t y Controls % Weight Difference** Survivors/ Dose* Day 14 Day 7 Total (%) (mg/kg) 0 0 0/12 (0) 0 +2 -1 7/12 (58)*** 10 -13 -6 11/12 (92)*** 20 -32 -13 10/12 (83)*** 40 *Compound or s a l i n e was administered twice a day i n a divided dose for 7 days s t a r t i n g 2 hours before virus inoculation. **The percent difference i n mouse weight between the untreated and treated uninfected mice. * * * S t a t i s t i c a l l y s i g n i f i c a n t (p<0.01), determined by the two-taile S O U R C E : Reproduced wit of uninfected and Semliki Forest v i r u s - i n f e c t e d Vero c e l l s was determined. These standard assays (19) are an i n d i c a t i o n of e f f e c t s on RNA and protein syntheses, r e s p e c t i v e l y . The i n t r a c e l l u l a r , unincorporated (acid-soluble) amounts of these and a l l subsequently used radioactive compounds were also determined (19). Unless other­ wise mentioned, treatment of c e l l s with r i b a v i r i n 5'-sulfamate did not a l t e r the uptake of these molecules i n t o acid-soluble pools r e l a t i v e to untreated c e l l s . In uninfected c e l l s , a dose-dependent i n h i b i t i o n of amino acid incorporation occurred, with >50% i n h i b i ­ tion at 6 μΜ (Table 4). The e f f e c t of r i b a v i r i n 5'-sulfamate on u r i d i n e incorporation was not dose-dependent (a 30% i n h i b i t i o n was observed at a l l concentrations tested). An o v e r a l l 30% i n h i b i t i o n i n acid-soluble counts also occurred at the same concentrations, which i n d i c a t e a moderate i n h i b i t i o n of [3-H]uridine uptake i n t o the c e l l . These r e s u l t s show that r i b a v i r i n 5'-sulfamate i n h i b i t e d c e l l ­ u l a r protein synthesis, but had only a weak e f f e c t on RNA synthesis. In v i r u s - i n f e c t e d c e l l s , the a c t i v i t y of the analog was studied i n the presence or absence of actinomycin D (Table 4). Actinomycin D was used to suppress c e l l u l a r mRNA synthesis and subsequent protein expression without i n t e r f e r i n g with the corresponding v i r a l proces­ ses. R i b a v i r i n 5'-sulfamate i n h i b i t e d amino acid incorporation i n infected c e l l s to about the same degree whether actinomycin D was present or not. This was s l i g h t l y l e s s than the degree of i n h i b i t i o n observed i n uninfected c e l l s . For this reason i t was not possible to conclude at this stage of experimentation that the analog i n h i b i t e d v i r a l protein synthesis. A d i f f e r e n t i a l e f f e c t was noted between the incorporation of u r i d i n e into infected c e l l s treated with actinomycin D compared to c e l l s r e c e i v i n g no actinomycin D (Table 4). In the absence of actinomycin D, an o v e r a l l 50% i n h i b i t i o n of incorporated uridine occurred at 6 to 100 μΜ, which was s i m i l a r i n nature to the lack of dose-responsiveness of r i b a v i r i n 5'-sulfamate i n uninfected c e l l s . However, i n infected c e l l s treated with actinomycin D where primar­ i l y v i r a l RNA synthesis took place, a marked suppression of u r i d i n e incorporation was evident at >12.5 uM. These data i n d i c a t e that r i b a v i r i n 5'-sulfamate i n t e r f e r e d with v i r a l RNA synthesis.

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

9.

S

M

E

E

Antiviral Activity of Ribavirin 5 'Sulfamate

Table 4.

129

E f f e c t s of r i b a v i r i n 5'-sulfamate on incorporation of u r i d i n e and amino acids into Semliki Forest v i r u s - i n f e c t e d and uninfected c e l l s

Percent of Untreated Control Uninfected Virus-Infected Inhibitor* Uridine Amino Amino Acids Amino +Act.D** Acids +Act.D** Cone.(μΜ) Uridine Uridine Acids 65 80 54 72 6.25 37 49 39 47 16 42 12.5 69 26 37 38 9 21 44 25 71 23 27 10 69 18 46 50 9 16 9 54 100 68 11 *Treatment with r i b a v i r i n 5'-sulfamate before and during radiol a b e l i n g (with [3-H]uridine or [3-H]amino acids) was as follows: uninfected c e l l s , 22 hrs pre-treated/2 hrs labeled; infected c e l l s , 18 hrs pre-treated/4 hr labeled. **Actinomycin D (5 μΜ) was added at the time of v i r u s i n f e c t i o n . S O U R C E : Reproduced with permission from ref. 23. Copyright 1988 Elsevier. In order to determine whether v i r a l protein synthesis was i n h i b i t e d i n treated c u l t u r e s , v i r a l and c e l l u l a r polypeptides were separated and v i s u a l i z e d by PAGE/fluorography methods (24,25). Infected c e l l s devoid of i n h i b i t o r exhibited d i s t i n c t new bands of protein (lane 2 of Figure 1) not present i n uninfected cultures (lane 1). In lanes 3-5, treatment with r i b a v i r i n 5'-sulfamate at 25-100 μΜ completely blocked the expression of these v i r a l proteins. Also, the i n t e n s i t y of c e l l u l a r proteins i n these lanes was somewhat diminished r e l a t i v e to the untreated, uninfected c o n t r o l . These r e s u l t s show that r i b a v i r i n 5'-sulfamate i n h i b i t e d v i r a l and c e l l u l a r protein synthesis, but not necessarily to the same degree under these treatment conditions. Since the above studies demonstrated that r i b a v i r i n 5'-sulfa­ mate i n h i b i t e d v i r a l and c e l l u l a r protein synthesis and v i r a l RNA synthesis, i t was hypothesized that the i n h i b i t i o n of v i r a l RNA synthesis was a consequence of the i n h i b i t e d expression of the v i r a l RNA polymerase protein. An alternate hypothesis i s that r i b a v i r i n 5'-sulfamate d i r e c t l y i n h i b i t e d the function of the v i r a l RNA polymerase. To discriminate between the two hypotheses, v i r a l RNA polymerase a c t i v i t y was p a r t i a l l y p u r i f i e d from c e l l s (26). The v i r a l polymerase located i n the c e l l u l a r P15 f r a c t i o n (mitochondria and membranes) was q u a n t i f i e d from treated and untreated infected c e l l s (Table 5). In these enzyme assays, the RNA polymerase reac­ tion mixtures (26) did not contain r i b a v i r i n 5'-sulfamate. The r e s u l t s show that the amount of enzymatic a c t i v i t y recovered from i n h i b i t o r - t r e a t e d c e l l s was a function of the concentration present during the time the v i r u s was r e p l i c a t i n g i n c e l l culture. When 1 mM r i b a v i r i n 5'-sulfamate was added d i r e c t l y to a v i r a l RNA polymerase reaction obtained from untreated, v i r u s - i n f e c t e d c e l l s , only a 27% decrease i n the rate of polymerization occurred, i n d i ­ cating that the analog was only a weak i n h i b i t o r of the enzyme. These r e s u l t s demonstrate that v i r a l RNA synthesis was i n h i b i t e d

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

130

NUCLEOTCDE ANALOGUES

Table

5.

Effects of

of

ribavirin

Semliki

Forest

recovered

5'-sulfamate

virus

from

Inhibitor*

Polymerase Activity**

on

the

polymerase

infected

Conc.(pM)

cells Percent

3,698

100

1,673

45

12.5

670

18

25

148

4

50

0

0

virus

0

0 was

present

replication

absent

••Expressed

from as

18

hours

period

(4

of

Control

6.25

100

amount

activity

0

*Inhibitor was

RNA

before

hrs)

in

and

cell

during

the

RNA

polymerase

reaction.

counts

per

minute

million

per

the

culture,

but

cells.

S O U R C E : R e p r o d u c e d with as

a

consequence

(i.e.,

the

to

inhibited

the

Inhibition A

series

site

of

mRNA

to

effect

of

of

the

RNA

translation

studies of

rabbit

was

test

analog

Puromycin

was

also

Ribavirin

was

system also

result sites

a

When

(Table from

on

the

it

a

it

similar were

(24,25).

The

5

of

hand

2)

5'-sulfamate diminished the

treated

intensity that

the

way

sulfamate

did

of

the

To

of

a

more were

drug the

from

potent

effect

(Table

positive

15-100

μΜ.

in

at

in

Puromycin

the

the

the

6).

control

inhibitor

occurred.

compounds

translating evaluate

translation

present

protein

of

the

same This

same

can

be

at

the

aminoacyl

into

the

polypeptide

result see

is

using the

the

whether

reaction could

or

different

the

many

chain of

steps.

site

incomplete

5'-sulfamate

the

show

that

presence

(lane

fragments

3)

untreated

polypeptide

puromycin

weight

were but

the in

and

of

rate

would

of

produced

the

of

(lane gel.

polypeptide

since

in

On

not

the

ribavirin

based

These do

only

(lane

synthesis 2),

each methods

caused

40,000.

puromycin

translation,

chain-terminate

be

synthesized

control

bands

5'-sulfamate

to

on

where

electrophoresis/fluorography

assay

protein

in

at

acceptor

formation

formed

polypeptides

cells to

inhibited

ribavirin

maximum m o l e c u l a r of

inhibit

not

be

polypeptide

ribavirin to

to

a

polypeptides

a

of

as

of

The

weight

relative

indicate same

(27).

sizes

capable

conducted

protein

intracellular

commercially

simultaneously

incorporated

with

system

a

vitro

to

to

visualized

a l l

specific

Using

in

interacts

effect,

molecular Figure

other

mRNA

results

the

were

manner

due

synthesis

find

inhibited

cells

complex.

fragments.

inhibitor small

is

DNA

expression

treated

translation

additive

puromycin

terminates

have

of

to

inhibitors

interaction

and

polypeptide

proved

an

and

lysate

protein

two

ribosomal

example,

ribosomes,

on

and

Translation For

mRNA).

reactions

dose-dependent

6),

an

tube

evaluated

the

its

protein from

5'-sulfamate.

5'-sulfamate

active,

reaction. mixture

in

absent

conducted

reticulocyte

protein, the

this

of

viral

was

translation

ribavirin

of

(27).

i n h i b i t i o n of

polymerase

protein

action

available

of

viral

was upon

results act

ribavirin

synthesis.

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

in 5'-

9. SMEE

Antiviral Activity of Ribavirin 5 ''Sulfamate

1

2

3

4

131

5

Figure 1. Effects of ribavirin 5'-sulfamate on viral and cellular polypeptide synthesis. Treatment with the inhibitor began 18 h before virus; cells were exposed to [3-H]amino acids 4-6 h after virus adsorption. Lane 1, uninfected untreated; lanes 2-5, infected and treated with 0, 25, 50, and 100 μΜ inhibitor, respectively. Designations at left indicate positions in lane 2 where viral-specific nonstructural (ns86 and ns97) and structural (C, capsid; E l , E2, and E3, glycoproteins; p62, precursor to E1-E3) polypeptides occur. (Reproduced with permission from ref. 23. Copyright 1988 Elsevier.)

Figure 2. Inhibition of proteins translated by a rabbit reticulocyte lysate system. Lane 1, background control (mRNA absent); lane 2, untreated control; lanes 3-4, ribavirin 5'sulfamate at 30 and 300 μΜ, respectively; lanes 5-6, puromycin at 3 and 30 μΜ, respectively. Molecular weight markers appear at left of figure. (Reproduced with permission from ref. 23. Copyright 1988 Elsevier.)

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

132

NUCLE(ynDE A N A L O G U E S

Table 6.

E f f e c t s of r i b a v i r i n 5'-sulfamate and puromycin on protein t r a n s l a t i o n * i n v i t r o (23)

Percent of Control Conc.(yM) 102 10 76 15 45 30 14 100 76 1 Puromycin 21 3 0 10 52 (58)** 15/1 Rib.5'-Sulf./Puromycin 37 (34) 30/1 *Reactions contained a rabbit r e t i c u l o c y t e lysate system, rabbit globin mRNA, and [35-S]methionine. **( ) = expected value for a d d i t i v e drug i n t e r a c t i o n . S O U R C E : Reproduced wit Inhibitor R i b a v i r i n 5'Sulfamate

Once i t was known that r i b a v i r i n 5'-sulfamate i n h i b i t e d protein t r a n s l a t i o n , i t became evident that the compound may be s i m i l a r to adenosine 5'-sulfamate i n i t s action. I n i t i a l l y i t was thought that the two compounds had d i s s i m i l a r modes of action, because adenosine 5'-sulfamate was i n a c t i v e against Semliki Forest v i r u s i n cytopathic e f f e c t i n h i b i t i o n assays. From the l i t e r a t u r e , adenosine 5'-sulfamate was shown to i n h i b i t aminoacyl-tRNA synthetase reactions (10), which are very early steps required i n protein synthesis. Aminoacyl-tRNA synthetases l i n k amino acids to respective tRNA's, which then carry the amino acids to the ribosome complex. The experiment i n Table 7 shows that r i b a v i r i n 5'-sulfamate i n h i b i t e d methioninetRNA synthetase (the only synthetase that was tested; there i s at least one aminoacyl-tRNA synthetase f o r each amino a c i d ) . The experimental method (10) used to derive the data i n Table 7 may not be obvious to the reader so w i l l be explained. The protein t r a n s l a t i o n experiment i n Table 6 reports t o t a l i n h i b i t i o n of polypeptide synthesis and of aminoacyl-tRNA synthesis. This i s because radioactive proteins and radioactive aminoacyl-tRNA's are a l l trapped onto f i l t e r s counted i n the assays. Referring to Table 7, by b o i l i n g the f i l t e r s i n hot p e r c h l o r i c acid (PCA), the aminoacyl-tRNA' s degrade and elute o f f . Thus, the difference i n counts from boiled versus unboiled f i l t e r s represents a c t i v i t y associated with aminoacyl-tRNA synthesis. R i b a v i v i n 5'-sulfamate i n h i b i t e d t o t a l synthesis, polypeptide synthesis, and aminoacyl-tRNA synthesis a l l to approximately the same degree. Adenosine 5'-sulfamate was also i n h i b i t o r y to t h i s process, and by further experimentation was shown not to i n h i b i t the elongation of polypeptides i n the presence of pre-formed aminoacyl-tRNA's (10). By inference the same may be true for r i b a v i r i n 5'-sulfamate. DNA synthesis i s a process that i s dependent upon protein synthesis (28), so one would expect r i b a v i r i n 5'-sulfamate to i n h i b i t that process. This was confirmed by the experimental r e s u l t s of Table 8, using [3-H]thymidine incorporation as an i n d i c a t o r . Some of the a n t i p r o l i f e r a t i v e and anti-DNA v i r u s a c t i v i t i e s of the analog are related to i n h i b i t i o n of DNA synthesis, but t h i s probably would not i n t e r f e r e with Semliki Forest v i r u s r e p l i c a t i o n .

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Antiviral Activity of Ribavirin 5'Sulfamate

9. S M E E Table

7.

Effects of

of

ribavirin

rabbit

globin

5'-sulfamate

mRNA a n d

Percent Cold

Cold

Hot

Cone.(μΜ)

PCA

PCA**

on

Hot

protein

synthesis

Control

Untreated

of

translation

aminoacyl-tRNA

Reaction*

Minute

60

Reaction*

10 M i n u t e Inhibitor

on

Minus

Cold

Hot

Cold

PCA

PCA

PCA

Hot

Minus PCA

30

40

50

37

30

36

28

100

13

10

14

14

17

13

8

1

9

5

7

300 *A rabbit as

reticulocyte

indicator

**Filters

for

were

the

boiled

lysate

s y s t e m was

run

using

5 [35-S]methionine

reactions. in

3.5%

perchloric

acid

(PCA)

to

degrade

the

aminoacyl-tRNA's.

Inhibition The

of

methylation

structure

naturally

of

ribavirin

occurring

methionine

(AdoMet),

adenosine,

and

play

processes

show

AdoMet which

were

effects

as

a

transfer

from

been

such

as

cellular an

methylation have

viruses

treated AdoMet by

reactions

been

shown

components

with

methylthio-

A marked to

or

(29).

or

as

a

to

that of

inhibit

reactions.

Specific

The AdoHcy,

inhibitors

antiviral

activity

methylation.

dose-dependent

macromolecules

set

accumulation of

possess require

(PAPS)

next

ribavirin-5'-sulfamate

i n h i b i t i o n of

inhibition,

and

In

Table

labeled

i n h i b i t i o n of

occurred.

This

enzymes

in

the

general

consequence

with methyl-

effect

AdoMet of

of (30),

could

pathway protein

both.

Effects of

8.

to

certain

S-adenosyl-

in.

to

caused

Table

as

5'-sulfamate

involved in

AdoHcy h y d r o l a s e ,

synthesis

ribavirin are

The

leads

[3-H-(methyl)]AdoMet. have

of

molecules

metabolism.

donor

certain

cells

the

cellular

methyl

hydrolase

because 8,

mimics

such

(AdoHcy),

AdoHcy h y d r o l a s e

inhibits

AdoHcy

in

these

serves of

S-adenosylhomocysteine

roles

that

inhibition

potentially

derivatives

3'-phosphoadenosine-5'-phosphosulfate

important

experiments

5'-sulfamate

5'-thioadenosine

ribavirin 5'-sulfamate cellular Percent

of

on

various

functions Untreated

Control

Thymidine

Sulfate

Methyl

Methionine

Incorp.

Incorp.

Transfer

Incorp.

48

35

47

33

12.5

22

16

31

21

Inhibitor* Conc.(yM) 6.25 25

15

10

24

15

50

10

6

19

12

100

8

3

13

9

5 ' -- s u l f a m a t e b e f o r e a n d d u r i n g * C e l l s were t r e a t e d w i t h r i b a v i r i n l a b e l i n g as f o l l o w s : [3-H]thymidine, 22 h r s p r e - t r e a t e d / 2 hrs labeled;

[35-S]sulfate,

(methyl)JAdoMet

and

20 h r s

pre-treated/4

[35-S]methionine,

5 hrs

hrs

labeled;

pre-treated/1

[3-Hhr

labeled.

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

134

NUCLE(mDE ANALOGUES

Cells with

soluble cells, this

with

which

could

possibility,

amounts

liquid of

AdoHyc

6-100

showed

radioactivity

pressure and

treated

[35-S]methionine

at

each

represent extracts

protein

[35-S]methionine same d e g r e e

AdoMet,

between

the

two

that

weakly

9.

affected

Metabolites

amounts

AdoHcy

of

9),

cysteine suggesting

hydrolase,

as

a

(measured

by

inhibited

to

the

buildup lack

nearly

reactions, of

of

reactions,

indicating

methionine

and

utilization

for

respectively.

of

inhibitors

of

AdoHcy

[35-S]methionine

ribavirin

in

cells

treated

per Minute

10^ C e l l s

per

Rib.5'--Sulf.* (177)**

16,151

AdoHcy

1,417

864

(64)

AdoMet

31,039

56,180

(181)

=

also

inhibitor

of

serves

which

the

metabolism was

inhibition

of

ribavirin

effect

5'-sulfamate

it

synthesis

of as

to

28)

before

and

1

a

at

30-100

were

inhibited was

at

try

the

hr

synthesis

active and

of

data

inhibiting

the

activity to

was

run

showing

proteins to

whereas

s a m e was affected

protein

at

of

general (Table

10).

protein 30-100

generally

that

to

that

due

indicate

indirectly

following useful

whether

inhibitory

The

by

not

or

into

synthesis,

The

was

direct

study

more

inhibited. μΜ.

a

pathway

parallel

spermidine

concentrations

to

Cells

(another

in

discriminate

due

(33).

determined

Puromycin

was

was

to

cycloheximide

[3-H]methionine

synthesis

consequence

leading

proliferation

This (34).

polyamine

equally

protein a

of

and

polyamine

5'-sulfamate

was

were

cycloheximide

inhibitors

the

inhibition,

ribavirin than

To

step cell

10).

synthesis on

At

processes

in

because

toxic.

incorporation

synthesis

5 hrs

control.

first

role

[3-H]ornithine

polyamine

synthesis

the a

(Table

inhibited 1 0 μΜ,

in

synthesis,

overtly

of

present

5'-sulfamate

syntheses

inhibitory

compound

was

untreated

play

protein

spermine an

of

ribavirin

and

μΜ)

(66)

radiolabeling. percent

polyamines, with

(6.25

(32).

5'-sulfamate

23,785

treated

The

hydrolase

13,438

AdoMet

of

synthesis

24,472

)

but

consequence

Cysteine

**(

high

elevated

Methionine

during

for

by

showed

(Table

Untreated

*Inhibitor

both

results

was

their

Counts

protein

investigate

analyzed

reactions

The

of

by

Compound

show

To

were

methyltransfe

with

of

AdoHcy.

replication

only

Table

untreated

protein

methyltransfer

i n h i b i t i o n of

virus is

and

result

acid-

to

methyltransfer

processes.

the

labeled in

compared

cells

8)

and

180% e l e v a t i o n

decreased

Table

i n h i b i t i o n of

in

inhibit

Also,

incorporation,

synthesis

and

not

the

protein

virus

did

synthesis.

probably

a

The

methyltransfer

as

were

Forest

(31).

inhibitor-treated

5'-sulfamate

5'-sulfamate

cells

and

in

affected

role

these

present

ribavirin

link

increase

methionine

generally

a

an

chromatography

that

AdoMet

ribavirin

concentration

from

more

inhibiting

μΜ

approximately

μΜ true

these

polyamine

synthesis.

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

The

Table

10.

Effects on

of

ribavirin

methionine

5'-sulfamate

incorporation Percent

Inhibitor* Ribavirin

(μΜ)

Incorp.

follows:

of

of

RNA

PAPS

61

33

89

12

9

100

8

95

6

5

10

85

91

253

141

30

56

110

42

23

107

11

is

polyamine viruses,

whether

a

5 hrs

before

and

during

pre-treated/1 hrs

hr

4

labeling

labeled;

as [3-H]-

labeled.

synthesi but

substrate

such

effects

of

probably

this

protein

compounds

as

would

could

have

have

that

not

lead

to

studies,

cells

capacity

to

8).

treated

By

of

then

decreased

in

incorporation

into

was

5'-sulfamate

sulfuration

reactions.

Table

11.

this

was

of

an

inhibition

(35),

the

as

time,

5'-sulfamate acid

of

intracellular

results

of

to

that

inhibit

the

i n h i b i t i o n of

Untreated

over

(hrs)

Incorp.

Incorp. 94

1

160

2

65

74

3

34

52

4

30

27

5

25

27

6

25 pulse-labeled

or

[3-H]amino

at

0

acids.

The

sulfate time

Acids

Sulfate

were

may

Control*

Amino

23 hourly

using

inhibitor

(25

[35-S]sulfate μΜ)

acid

suggest

Time

*Cells

treatment,

sulfuration

incorporations

Percent

decreased

[3-H]amino

non-specifically

amino

a

hour

acting

Ribavirin

present

macromolecules

the

11).

a

the

had

into

first

protein

as In

i n h i b i t i o n of

that

inhibitors as

(Table role

and

Other

well

(36).

These

know

non-selectively

5'-sulfamate

the

to

reactions,

specific

[35-S]sulfate over

structural

interest

consequences.

of

synthesis

during

intracellular other

sulfuration

reactions

effect

with

The

and

was

chlorate

ribavirin

enhanced

protein

It

activities

inorganic

parallel

for

on

sodium

protein

with

following

sulfuration

the as

sulfuration

inhibiting

certain and

virus-inhibitory

such

incorporate

rate

ribavirin

any

of

(35)

sulfate.

5'-sulfamate

reported

inhibit

of

sulfuration molecules

chondroitin

sulfurases,

consequence

for

tyrosine

ribavirin

investigators

(Table

17 inhibitor

pre-treated/12

containing

components

of

Spermine

viruses.

proteins the

to

Spermidine

Putrescine 107

6 hrs

of

Control

Metabolism

14

100 treated with

DNA

Untreated

Ornithine

synthesis

24

[3-H]methionine,

inhibition cation

cycloheximide

10

Cycloheximide

ornithine,

and

polyamine

30

5'-

Sulfamate

were

and

of

Methionine

Cone.

*Cells

135

Antiviral Activity of Ribavirin 5-Sulfamate

9. SMEE

was

added

time.

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

play

136 on

NUCLEOTIDE ANALOGUES

the

cells

replication with

Uptake The

of

last

inside taken

ribavirin questions

of up

by

lites

by

To

a

and

compound upon

an

nanomole to

the

of of

from about

formed, have

taken

generate (21). mate

is

Table

a

fully

cells

to

of to

if

completely

For

example,

Uptake

the

for

of

Ribavirin culture toxic mode

at of

6

nm)

and to

inhibited

were

the

time

it

its

active

did

took

maximal

present of

the

which

relied

not

allow

for

the

the

protein

hours

were

ribavirin

was

were

which

being

synthesis

required

would

to

fully

5'-triphosphate

in

that

5'-sulfa-

and

ribavirin

anticellular

5'-sulfamate

into

Cone.

cells

effects.

cells*

(yM) 18

at

hours

1005

using

ribavirin 0.02

analog

effect,

metabolites

inhibit

16 m i n u t e s

a

C-18

HPLC

5'-sulfamate at

1 ml/minute

M potassium

column.

(detected flow

phosphate,

by

rate.

pH

3.5.

in

cell

discussion

mice,

inhibited although

of on

or

action the

this

Semliki was that

detected

in

cellular

synthesis,

action.

provided

synthesis,

methyltransferase

its

Since

cells

Forest

considered

nucleotide

anticellular

i n h i b i t i o n of for

it

doses

of

protein

aminoacyl-tRNA

target

system,

Hours

assayed

of

was

buffer

All

syntheses,

compound

time

viral

attributed major

method,

965 were

concentrations of

assay,

were

71

in

synthesis.

the

(HPLC)

the

metabolites

233

antiviral

inhibition

inhibitor no

Intracellular

5'-sulfamate

and

of

137

and

cells metabo-

chromatographic

368

210

intra-

possible

100

Elution

Conclusions

and

analog

treated

the of

the

5'-sulfamate

300

elution

inactive.

formed

from

and

ribavirin

(yM)

extracts

UV a t

are

variability

hypothesis

1000 The

of

of

antiviral

Extracellular

*Cell

fate

12),

exert

12

metabolite

support

responsible

Cone.

any

liquid

detection

Presumably

antiviral

12.

if

inherent

bioassay

into

studies

solely

treating be

ribavirin

extracts

sensitivity

light

the

is

compound

concentrations The

by to

was

is

longer.

the

the

(Table

found

with

extent

pressure the

cells

(UV)

process

These

high

of

tested

was

quantitie

4 hours.

the

presence

the

11

deal

what

was

cells

questions,

account

compound

Table

into

it

metabolites,

these

detected.

equilibrate

was

what

same

minute

To

virus

where

addressed

phase

into

ultraviolet

detection data

the

outside

were

be

cells. and

reverse

Taking

Forest

chlorate,

answer

for

approximately inside

to

cells,

analyzed

method.

Semliki

5'-sulfamate

mammalian

cellular^? were

of

1 0 mM s o d i u m

and

on

(within

DNA

was

on

and

the

viral

mRNA

polyamine

reactions The

considered

limits

The

involved

synthesis.

metabolites the

partially

protection.

sulfuration

which

be

impacted

protein no

to

analog

which

effects

virus

to

were analog

be

the

of

the

parent

of

detectability

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

by

UV

spectroscopy),

responsible into

a

puromycin

and

RNA

of

the

class

of

protein

Forest

is

reported

translation

(18),

virus

requires

amplify,

5'-sulfamate

effects

cycloheximide

which can

ribavirin

a l l

Semliki

viruses), the

for

larger

tion.

137

Antiviral Activity of Ribavirin 5 'Sulfamate

9. SMEE

that

(and

an

initial

appears The

inhibit

other

sensitive

it

that

of to

be fits

including

virus

replica-

positive

translation

particularly

to

compound

inhibitors,

will

probably

itself here.

its

stranded

genome

these

before

types

of

inhibitors. In

a

possess to

ADP

recent

report

to

ATP and

vice

5'-sulfamate

interacts

of

Its

enzymes.

(10)

which

also

much g r e a t e r activity it

was

of

toxicity In

whereas being

the this

other

quantity

Other further

early

of the

To

been

the

arsenal

Whether

cesses

Clearly,

that

modes

viruses

interest

to

5'-sulfamate

simplex

viruses

inhibition not

a

first

of

unique

is

class

to

the

antiviral

conclusion

that

to

be

an

of

will to

rather

than Along

action of

be

of

African of

led

However,

to

the

human

conducted ribavirin analog

their

these

way

remains

swine

antiviral

ribavirin

into to

lines,

have

fever study

it

and

will

be

herpes

show

activity, a

pro-

reported

5'-sulfamate

v i r u s - i n h i b i t o r y as

be

which

cellular

recently

present

has

model.

identified

the

substan(Table

was

affect

but

against

infection

find

be

study

test,

potential

the

antiviral

was

animal

that

that

viruses,

the

This

cell

polymerase

cells.

nucleotide

virus

host

RNA

may a l s o

reported

In the

animal

on.

here

a

continually

expressed

since

the

only

translation,

favor

viral

experiment

mice.

need

that

was

was

expressed.

for

infected

low.

in

activity

results

be

fact

the

reported

clinical

synthesis to

the

unwarranted

in

synthesis

partially

positive-stranded

Otherwise,

depend

aminoacyl-tRNA

visualized.

present

inside

those

1),

action,

observation.

to was

after

manifested

mode

were

sufficiently

appears

other

The

reported

be

than

inhibitors

(12).

only

especially

inhibitors

the

be

and

increase

with

to

would

5'-sulfamates

also

protein

compound

inhibiting

any

of

learn

uridine

the

ribosomes

(Table

compounds

virus-specific

to

abundance

the

antiviral

vitro

in

relates

probably

the

nucleoside of

determined.

of

is

inactive.

evaluated

Due the

for

cytomegalovirus

was

this

5'-sulfamate

polypeptide

proteins message

can

toxicity

on

in

inhibit

greater It

substance

up

murine

5'-sulfamate not

in

viruses

overt

cytomegalovirus against

virus

compound,

this

follow

cell

proteins

investigation

tiated

leading

viral

mRNA w o u l d of

to

AMP

ribavirin

adenosine

synthetases.

effect

probably of

mRNA was

the

masked,

that

5'-sulfamate,

studies,

Competition

kinds

potential 3).

effect

viral

by

by

adenosine

of

antiviral

circumstance.

key of

inhibited

the

amount

host

site

that

synthetases

interconvert

selective

electrophoresis

produced.

mRNA i n and

more

small

to

aminoacyl-tRNA was

They

hypothesized

AMP b i n d i n g

similar

i n h i b i t e d whereas

and

is

aminoacyl-tRNA

(37).

viru

allowed

differential

finite

a

It

the

is

compound as

was

which

completely

at

action

that

the

versa.

inhibits

inactive

shown

activities

c y t o t o x i c i t y of

5'-sulfamate

This

was

phosphotransferase

that

which is

consequence

synthesis.

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

is

the of

138

NUCLEOTIDE

ANALOGUES

Literature cited 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.

Robins, R. K. Pharmaceut. Res. 1984, 1, 1-50. Martin, J. C.; Verheyden, J. P. H. Nucleosides Nucleotides 1988, 7, 365-374. Fuertes, M.; Witkowski, J. T.; Streeter, D. G.; Robins, R. K. J. Med. Chem. 1974, 17, 642-645. Pereira, M. E.; Segaloff, D. L.; Ascoli, M.; Eckstein, F. J. Biol. Chem. 1987, 262, 6093-6100. Griengl, H.; Hayden, W.; Penn, G.; De Clercq, E.; Rosenwirth, B. J. Med. Chem. 1988, 31, 1831-1839. Vaghefi, M.; McKernan, P. Α.; Robins, R. K. J. Med Chem. 1986, 29, 1389-1393. Tobie, E. J. J. Parasitol. 1957, 43, 291-293. Florini, J. R.; Bird, Η. H.; Bell, P. H. J. Biol Chem. 1966, 241, 1091-1098. Jaffee, J. J.; McCormack 1970, 28, 535-543. Bloch, Α.; Coutsogeorgopoulos, C. Biochem. 1971, 10, 4394-4398. Andres, J. I.; Garcia-Lopez, M. T.; De las Heras, F. G.; MendezCastrillon, P. P. Nucleosides Nucleotides 1986, 5, 423-429. Perez, S.; Fiandor, J.; Perez, C.; Garcia-Lopez, M. T.; Garcia-Gancedo, Α.; De las Heras, F. G.; Fernandez, C. G.; Vilas, P.; Mendez-Castrillon, P. P. VII Internatl. Congr. of Virology, 1987,p222. Camarasa, M.-J.; Fernandez-Resa, P.; Garcia-Lopez, M. T. De las Heras, F. G.; Mendez-Castrillon, P. P.; Alarcon, B.; Carrasco, L. J. Med. Chem. 1985, 28, 40-46. Gil-Fernandez, G.; Perez, S.; Vilas, P.; Perez, C.; De las Heras, F. G.; Garcia-Gancedo, A. Antiviral Res. 1987, 8, 299310. Shuman, D. Α.; Robins, R. K.; Robins, M. J. J. Am. Chem. Soc. 1969, 91, 3391-3392. Smee, D. F.; McKernan, P. Α.; Nord, L. D.; Willis, R. C.; Petrie, C. R.; Riley, T. M.; Revankar, G. R.; Robins, R. K.; Smith, R. A. Antimicrob. Agents Chemother. 1987, 31, 1535-1541. Sidwell, R. W. In Chemotherapy of Infectious Diseases; Gadebusch, H. H., Ed.; CRC: Cleveland, Ohio, 1976; pp 31-53. Schlesinger, M. J.; Kaariainen, L. In The Togaviruses; Schlesinger, R. W., Ed.; Academic: New York, 1980; pp 371-392. Smee, D. F.; Martin, J. C.; Verheyden, J. P. H.; Matthews, T. R. Antimicrob. Agents Chemother. 1983, 23, 676-682. Votruba, I.; Holy, Α.; De Clercq, E. Acta Virol. 1983, 27, 273-276. Smee, D. F.; Matthews, T. R. Antimicrob. Agents Chemother. 1986, 30, 117-121. Malinsoki, F.; Stollar, V. Virology 1980, 102, 473-476. Smee, D. F.; Alaghamandan, H. Α.; Kini, G. D.; Robins, R. K. Antiviral Res. 1988, 7 (in press) Laemmli, U. K. Nature (London) 1970, 227, 680-685. Bonner, W. M.; Lasky, R. A. Eur. J. Biochem. 1974, 46, 83-88. Ranki, M.; Kaariainen, L. Virology 1979, 98, 298-307. Nathans, D. Proc. Natl. Acad. Sci. USA 1964, 51, 585-592.

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Antiviral Activity ofRibavirin 5 -Sulfamate

139

Seki, S.; Mueller, G. C. Biochim. Biophys. Acta 1975, 378, 354-362. Glazer, R. I.; Hartman, K. D.; Knode, M. C.; Richard, M. M. Chiang, P. K.; Tseng, C. K. H.; Marquez, V. Ε. Biochem. Biophys. Res. Commun. 1986, 135, 688-694. De Clercq, Ε. Biochem. Pharmacol. 1987, 36, 2567-2575. Wagner, J.; Danzin, C.; Mamont, P. J. Chromatogr. 1982, 227, 349-368. De Clercq, E.; Montgomery, J. A. Antiviral Res. 1983, 3, 17-24. Pegg, A. E.; Coward, J. K.; Talekar, R. R.; Secrist, J. A. Biochem. 1986, 25, 4091-4097. Seiler, N.; Knodgen, B. J. Chromatogr. 1980, 221, 227-235. Baeuerle, P. Α.; Huttner, W. B. Biochem. Biophys. Res. Commum. 1986, 141, 870-877. Huttner, W. B.; Lee R W H J Cell Biol 1982 95 389a Rapaport, E.; Pemy P. C. Proc. Natl

RECEIVED January 4, 1989

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

C h a p t e r 10

Design, Synthesis, and Antiviral Activity of Nucleoside and Nucleotide Analogues Victor E. Marquez Laboratory of Medicinal Chemistry, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892

Several categorie designed by exploiting important differences in viral and cellular biochemistry, as well as differences in chemical stability and reactivity between parent "lead" molecules and their modified structures. The synthesis, antiviral activity and mechanism of action of two carbocyclic nucleoside analogues related to the natural product neplanocin A are discussed. While the cytosine carbocycle, cyclopentenyl cytosine (CPE-C, 3), requires activation to form the biologically active nucleotide 5'-triphosphate analogue, a more selective antiviral effect is realized in the purine series by the modified 3-deazaneplanocin A (6c) analogue, due to its resistance to form nucleotide metabolites. In the area of dideoxy nucleoside analogues with anti-HIV activity, dideoxy­ -fluoro-ara-adenosine (ddF-ara-A, 15) and dideoxy­ -fluoro-ara-inosine (ddF-ara-I, 16), were designed as hydrolytically stable compounds. Although the beta stereochemistry of the fluorine atom proved not to be essential for chemical stability, i t was critical for retaining the potent anti-HIV activity of the parent compounds. In the area of nucleotide analogues which incorporate in their structure an isosteric phosphate group, the complete synthesis of the phosphonate analogue of adenosine-2'-phosphate (26) is discussed. This compound was designed as a hydrolytically stable component of a 2',5'-oligoadenylate trimer analogue (17) whose synthesis is in progress. N u c l e o t i d e analogues can be d i v i d e d i n t o t h r e e c a t e g o r i e s : 1) b a s e - m o d i f i e d , 2) s u g a r - m o d i f i e d , and 3) p h o s p h a t e - m o d i f i e d . Most o f t h e commonly known a n t i v i r a l agents belong t o t h e f i r s t two g r o u p s , which a r e generated i n t r a c e l l u l a r ! y from t h e i r c o r r e s p o n d ­ ing nucleoside precursors by e i t h e r c e l l u l a r or virus-coded This chapter not subject to U.S. copyright Published 1989 A m e r i c a n C h e m i c a l Society

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

10.

MARQUEZ

Nucleoside and Nucleotide Analogues

141

enzymes. For the most p a r t , t h e s e n u c l e o t i d e s e x e r t t h e i r a n ­ t i v i r a l a c t i v i t y as the c o r r e s p o n d i n g mono-, o r t r i p h o s p h a t e forms ( e . g . , r i b a v i r i n (1) and a c y c l o v i r (2)) which i n t e r f e r e with critical metabolic steps required for v i r a l i n f e c t i v i t y (See Figure 1). However, s i n c e these p h o s p h o r y l a t e d forms are a l s o r e s p o n s i b l e f o r the t o x i c i t y observed a g a i n s t normal c e l l s , t h e i r s e l e c t i v e generation in j u s t v i r a l l y infected c e l l s , or t h e i r s p e c i f i c i n t e r a c t i o n w i t h key v i r a l p r o c e s s e s , become i m p o r t a n t f a c t o r s i n a c h i e v i n g a good t h e r a p e u t i c r a t i o . The t h i r d c a t e g o r y c o m p r i s e s t h o s e n u c l e o t i d e analogues i n which a m o d i f i e d phosphate g r o u p , o r i t s e q u i v a l e n t , i s a l r e a d y i n c o r p o r a t e d i n t o the m o l e c u l e ( e . g , DHPG phosphonate) ( 3 ) . These compounds are i n t e n d e d t o overcome r e s i s t a n c e from v i r u s e s which e i t h e r n a t u r a l l y or by m u t a t i o n do not have the c a p a c i t y to phosphorylate the nucleoside substrates. Increase in their c e l l u l a r u p t a k e , which becomes a l i m i t i n g f a c t o r , i s a c h i e v e d by the p a r t i a l o r t o t a l maskin A t the o t h e r end o n u c l e o s i d e s f o r which the d e s i r e d a n t i v i r a l e f f e c t can o n l y be uncovered when formation of the nucleotide metabolites is prevented. In t h e s e c a s e s , the a n t i v i r a l s e l e c t i v i t y i s r e l a t e d t o the s p e c i f i c i n t e r a c t i o n o f the n u c l e o s i d e , o r a non-phosphorylated metabolite of it, with a v i t a l viral process (e.g. n e p l a n o c i n A) (4). The d e s i g n , s y n t h e s i s , and a n t i v i r a l a c t i v i t y o f s e v e r a l new a n t i v i r a l agents t h a t f a l l i n t o t h e s e d i f f e r e n t c l a s s e s w i l l be discussed. Cvclopentenvl

C v t o s i n e (3,

CPE-C).

Chemistry. C y c l o p e n t e n y l c y t o s i n e (3, CPE-C, Scheme 1) i s a p y r i m i d i n e analogue o f t h e f e r m e n t a t i o n p r o d u c t , n e p l a n o c i n A. The compound was i n d e p e n d e n t l y s y n t h e s i z e d i n our l a b o r a t o r y (5,6) and t h a t o f Ohno i n Japan (7,8) by two d i s t i n c t c h e m i c a l approaches t h a t c e n t e r e d around the s t e r e o s p e c i f i c g e n e r a t i o n o f the c h i r a l c y c l o p e n t e n y l amine p r e c u r s o r 1. A simplified version o f our most r e c e n t approach t o CPE-C i s shown i n Scheme 1 ( 6 ) . Limited structure-activity studies have indicated that the i n t e g r i t y o f the c a r b o c y c l i c component o f CPE-C i s e s s e n t i a l f o r antiviral activity. For example, the c o r r e s p o n d i n g 2 ' - d e o x y and a r a - C P E - C analogues were i n e f f e c t i v e as a n t i v i r a l agents w i t h o n l y the a r a isomer showing some a c t i v i t y against influenza (9). I n t e r e s t i n g l y , both analogues were a l s o d e v o i d o f the e x c e l l e n t a n t i t u m o r p r o p e r t i e s c h a r a c t e r i s t i c o f CPE-C ( 1 0 ) . Antiviral Activity. CPE-C has demonstrated s i g n i f i c a n t a n t i v i r a l a c t i v i t y a g a i n s t s e v e r a l DNA v i r u s e s w i t h h i g h v i r u s r a t i n g s (VR = 2.7 - 4.6) a t doses r a n g i n g from 0.1 t o 2.7 jug/ml ( 6 ) . Of p a r t i c u l a r i n t e r e s t was the e f f e c t t h a t t h i s drug d i s p l a y e d a g a i n s t the t h y m i d i n e k i n a s e d e f i c i e n t s t r a i n o f HSV-1 ( 6 , 1 1 ) . The a n t i v i r a l a c t i v i t y o f CPE-C was a l s o e v i d e n c e d i n v i v o as demonstrated by the r e s u l t s i n the murine v a c c i n i a t a i l p o x model where i t s i g n i f i c a n t l y reduced the development o f v i r u s - i n d u c e d t a i l l e s i o n s ( T a b l e I) ( 1 2 ) . In t h i s a s s a y , CPE-C was e v a l u a t e d

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

NUCLEOTIDE

142

ANALOGUES

2 Bn =CH C H 2

6

5

Scheme 1.

S y n t h e s i s o f CPE-C.

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

10.

M

A

R

Q

U

E

143

Nucleoside and Nucleotide Analogues

Z

for efficacy against the IHD s t r a i n o f vaccinia c h a l l e n g e d w i t h the v i r u s by the i n t r a v e n o u s r o u t e ( t a i l

in mice, vein).

T a b l e I. A n t i v i r a l A c t i v i t y o f CPE-C (Murine V a c c i n i a T a i l p o x Model) PBS C o n t r o l

a

b c

CPE-C

Ara-A

a

Pox Count

Dose (mg/kg)

Pox C o u n t

45.8

300

2.8

b

D i l u e n t - t r e a t e d mice (phosphate the v i r u s QD 1-7 s t a r t e d on th Mean v a l u e (20 mice)

buffer

0

Dose Pox C o u n t (mg/kg) b

1.5

saline)

c

0.8

challenged with

Other DNA v i r u s e s s e n s i t i v e t o CPE-C were c y t o m e g a l o v i r u s and v a r i c e l l a - z o s t e r , as demonstrated i n t h e y i e l d r e d u c t i o n assay and plaque r e d u c t i o n a s s a y , r e s p e c t i v e l y (6). CPE-C a l s o demonstrated significant activity against a spectrum o f RNA v i r u s e s i n v i t r o ( 6 ) . V i r u s r a t i n g s g r e a t e r than two were o b t a i n e d a g a i n s t v e s i c u l a r s t o m a t i t i s , Punta Toro and Hong Kong v i r u s e s . However, the u t i l i t y o f t h e s e a c t i v i t i e s i s moderated by low t h e r a p e u t i c index v a l u e s . By c o n t r a s t , t h i s does not appear t o be the case f o r the Japanese e n c e p h a l i t i s v i r u s , where CPE-C showed a VR o f 2 . 4 , w i t h g r e a t e r potency and h i g h e r t h e r a p e u t i c index than r i b a v i r i n ( 6 ) . No a n t i r e t r o v i r a l a c t i v i t y a g a i n s t HIV was observed f o r CPE-C i n ATH8 c e l l s . Mechanism o f A c t i o n . CPE-C i s c o n v e r t e d t o the nucleotide t r i p h o s p h a t e analogue o f c y t i d i n e (CPE-CTP), and as such it behaves as a powerful i n h i b i t o r o f CTP s y n t h e t a s e i n c e l l s (13). A l t h o u g h a d i r e c t c o r r e l a t i o n between the i n h i b i t i o n o f t h i s enzyme and i t s a n t i v i r a l a c t i v i t y has not been d e m o n s t r a t e d , i t a p p e a r s , as suggested by De C l e r c q e t a l . , t h a t t h i s r a t e - l i m i t i n g enzyme i n the de novo p y r i m i d i n e pathway may be a t h e r a p e u t i c a l l y e x p l o i t a b l e t a r g e t f o r some v i r u s e s ( 1 1 ) . The r e m a r k a b l y broad spectrum o f activity observed for t h i s compound indirectly supports t h i s assumption. 3-Deazaneplanocin A

(6cK

Chemistry. T h i s compound was prepared by a s i m i l a r approach t o the s i m p l i f i e d v e r s i o n o f the s y n t h e s i s o f n e p l a n o c i n A t h a t we had e a r l i e r developed ( 1 4 ) . Thus, t h e c y c l o p e n t e n y l m e s y l a t e 4 was r e a c t e d w i t h the sodium s a l t o f 4-chloro-imidazo[4,5-ç]pyridine (6-chloro-3-deazapurine) t o g i v e a m i x t u r e o f both N - l and N-3 isomers (Scheme 2 ) . A f t e r c h r o m a t o g r a p h i c s e p a r a t i o n and removal o f the p r o t e c t i v e groups i n each i s o m e r , the major com­ ponent was i d e n t i f i e d as the d e s i r e d compound (6a) by NMR s p e c t r o s c o p y and d i a g n o s t i c NOE measurements. The c h l o r o p u r i n e

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i n t e r m e d i a t e 6a was then c o n v e r t e d i n two s t e p s t o 3 - d e a z a n e p l a n o c i n A which was i s o l a t e d as a c r y s t a l l i n e s o l i d . The m o l e c u l a r s t r u c t u r e of 3-deazaneplanocin A was a l s o c o n f i r m e d by X - r a y crystallography (15). Antiviral Activity. 3 - D e a z a n e p l a n o c i n A was f i r s t e v a l u a t e d in. v i t r o a g a i n s t v i r u s e s f o r which n e p l a n o c i n A and o t h e r adenosine analogues are known t o be p a r t i c u l a r l y e f f e c t i v e ( 1 6 , 1 7 ) . These i n c l u d e d a DNA v i r u s ( v a c c i n i a ) and two (-)RNA v i r u s e s ( p a r a i n ­ f l u e n z a and v e s i c u l a r s t o m a t i t i s ) . In T a b l e I I , t h e minimum inhibitory concentration capable of reducing virus-induced cytopathogenic e f f e c t s by 50% ( I D C Q ) , v i r u s r a t i n g (VR), and t h e r a p e u t i c index ( T I ) , are compared f o r 3 - d e a z a n e p l a n o c i n A and three positive control standards for each o f the viruses. Treatment w i t h 3 - d e a z a n e p l a n o c i n A r e s u l t e d i n a marked i n h i b i t i o n o f p a r a i n f l u e n z a type 3 v i r u s (Huebner C243 i n H.Ep-2 c e l l s ) and V e s i c u l a r s t o m a t i t i s (VS a g a i n s t VSV i s p a r t i c u l a r l structurally related standard 3-deazacarbocycl i c adenosine analogue (3-deaza-C-Ado) (18) w i t h which 3 - d e a z a n e p l a n o c i n is structurally related. With o t h e r RNA v i r u s e s , such as RSV (respiratory syncytial) and human i n f l u e n z a ( t y p e Ao/PR/8/34), however, 3 - d e a z a n e p l a n o c i n A d i d not show any p r o m i s i n g a n t i v i r a l selectivity. S i m i l a r l y , a g a i n s t a number o f p i c o r n a v i r u s e s , t h e compound was m a r g i n a l l y e f f e c t i v e . The compound r e s u l t e d i n a c t i v e a g a i n s t r h i n o v i r u s and C o x s a c k i e t y p e B l , and o n l y a m a r g i n a l e f f e c t was observed on C o x s a c k i e t y p e A 2 1 . The i n h i b i t i o n o f p o l i o v i r u s - i n d u c e d c y t o p a t h o g e n i c e f f e c t s was a l s o v a r i a b l e and not d o s e - r e s p o n s i v e . A g a i n s t a DNA v i r u s , such as v a c c i n i a , 3 d e a z a n e p l a n o c i n A demonstrated a remarkable e f f e c t . These r e s u l t s prompted the i_n v i v o study o f the drug a g a i n s t v a c c i n i a which i s summarized i n T a b l e I I I . Treatment w i t h 3 - d e a z a n e p l a n o c i n A, a d m i n i s t e r e d s u b c u t a n e o u s l y once d a i l y from day -1 through day +5, r e s u l t e d i n a s i g n i f i c a n t r e d u c t i o n i n v a c c i n i a induced t a i l p o x compared t o d i l u e n t - t r e a t e d c o n t r o l m i c e . The t h e r a p e u t i c e f f e c t was e v i d e n t at dose l e v e l s o f 8 and 4 mg/Kg/day, a l t h o u g h t h e h i g h e r dose r e s u l t e d i n w e i g h t l o s s i n the t o x i c i t y control animals. Mechanism o f A c t i o n . V a r i o u s adenine n u c l e o s i d e analogues w h i c h function as inhibitors of S-adenosylhomocysteine hydrolase (AdoHcy-ase) have been i d e n t i f i e d as a n t i v i r a l agents ( 1 9 , 2 0 ) . F u r t h e r m o r e , a r e c e n t study has r e v e a l e d the e x i s t e n c e o f a c l o s e c o r r e l a t i o n between t h e i r i n h i b i t o r y potency a g a i n s t AdoHcy-ase and t h e i r a n t i v i r a l a c t i v i t y (VSV i n v i t r o ) ( 1 7 ) . In v a c c i n i a , as w e l l as i n o t h e r s e n s i t i v e v i r u s e s t h a t r e q u i r e a m e t h y l a t e d 5 ' cap on t h e i r mRNAs, i n h i b i t i o n o f AdoHcy-ase r e s u l t s i n t h e marked e l e v a t i o n o f AdoHcy l e v e l s and the consequent feedback i n h i b i t i o n o f S - a d e n o s y l m e t h i o n i n e (AdoMet)-dependent m e t h y l a t i o n r e a c t i o n s . T h i s enzyme i s a unique t a r g e t f o r the development o f a n t i v i r a l nucleoside analogues t h a t does not r e q u i r e a n a b o l i s m o f t h e drugs

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10.

Table I I .

A n t i v i r a l A c t i v i t y of 3-Deazaneplanocin A (In V i t r o ) P o s i t i v e Control

3-DeazaneDlanocin A RNA V i r u s e s I D

5 0

iua/ml) VR T I a

b

Compound ID50 i u a / m l ) VR

TI

Parainflu­ enza type 3

0.05

3 . 6 2.0

Ribavirin

17.50

2.5

5.7

Vesicular Stomatitis

0.07

3.6 4 . 6

3-Deaza-CAdo

2.6

3.2

3.8

0.32

3.1 3.1

Ara-A

2.0

3 . 7 16.3

DNA V i r u s e s Vaccinia

a b

V i r u s r a t i n g (Shannon and A r n e t t , SoRI) T h e r a p e u t i c i n d e x : minimum t o x i c c o n c e n t r a t i o n /ID50 Table I I I .

PBS C o n t r o l Pox Count

72.7

a

b c

A n t i v i r a l A c t i v i t y of 3-Deazaneplanocin-A (Murine V a c c i n i a T a i l p o x Model) 3-Deazaneplanocin-A

Ara-A

a

Dose (mg/kg)

Pox C o u n t

300

2.1

b

c

Dose (mg/kg) b

Pox C o u n t

0

4.3

D i l u e n t - t r e a t e d mice (phosphate b u f f e r s a l i n e ) c h a l l e n g e d w i t h the v i r u s QD 1-7 s t a r t e d on t h e day p r e c e d i n g t h e c h a l l e n g e Mean v a l u e (20 mice)

to the corresponding nucleotide l e v e l . N e p l a n o c i n A, which ranked among t h e most p o t e n t i n h i b i t o r s o f AdoHcy-ase, reduced v i r a l c y t o p a t h o g e n i c i t y a g a i n s t VSV a t t h e l o w e s t dose l e v e l (ID50 0.01 jug/ml) when compared w i t h o t h e r AdoHcy-ase i n h i b i t o r s (17). However, d e s p i t e i t s s u p e r i o r potency w i t h r e s p e c t t o 3 - d e a z a - C Ado ( t h e c a r b o c y c l i c analogue o f 3 - d e a z a a d e n o s i n e ) , n e p l a n o c i n A r e s u l t e d more c y t o t o x i c ( 1 6 ) . In v a r i o u s c e l l l i n e s t h e m e t a b o l i c c o n v e r s i o n o f n e p l a n o c i n A t o t h e n u c l e o t i d e t r i p h o s p h a t e and t h e subsequent t r a n s f o r m a t i o n o f t h e l a t t e r i n t o t h e AdoMet a n a l o g u e , S - n e p l a n o c y l m e t h i o n i n e , has been demonstrated ( 2 1 ) . Moreover, t h e conversion of neplanocin A to these metabolites has been c o r r e l a t e d t o t h e i n h i b i t i o n o f RNA s y n t h e s i s and c y t o t o x i c i t y (22). Because t h e a n t i v i r a l e f f e c t s o f n e p l a n o c i n A appeared t o be mediated s o l e l y by i t s i n h i b i t i o n o f AdoHcy-ase, b l o c k a g e o f i t s a n a b o l i s m t o t h e n u c l e o t i d e l e v e l was c o n s i d e r e d t o be an a t t r a c t i v e strategy to achieve higher a n t i v i r a l s e l e c t i v i t y . To

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t h i s e n d , based on the knowledge t h a t 3-deazaadenosine n u c l e o s i d e s are very poor s u b s t r a t e s f o r adenosine k i n a s e (18) (the first enzyme i n the a c t i v a t i o n pathway t o t h e t r i p h o s p h a t e ) , the s y n t h e s i s o f 3 - d e a z a n e p l a n o c i n A was proposed ( 2 3 , 2 4 ) . T h i s new member o f the c a r b o c y c l i c n u c l e o s i d e f a m i l y r e t a i n e d the p o t e n t i n h i b i t i o n o f AdoHcy-ase t o even h i g h e r l e v e l s than t h o s e a c h i e v e d w i t h n e p l a n o c i n A ( 2 3 , 2 4 ) , and as a n t i c i p a t e d , i t showed a more s e l e c t i v e a n t i v i r a l e f f e c t both i n v i t r o and j_n v i v o ( v i d e s u p r a ) . In t h i s r e g a r d i t i s i n t e r e s t i n g t o note t h a t n e p l a n o c i n A f a i l e d t o show any a p p r e c i a b l e a n t i v i r a l e f f e c t vn v i v o where i t proved t o be e x t r e m e l y c y t o t o x i c and l e t h a l t o mice ( 1 6 ) . 2 ' , 3 ' - D i d e o x v - 2 - f 1 u o r o - a r a - A (13, 2 ' - f l u o r o - a r a - I (14. ddF-ara-I). /

ddF-ara-A)

and

2' ,3'-dideoxy-

Chemistry. T h e , chemical r a t i o n a l e f o r the s y n t h e s i s o f t h e s e compounds was based o i n s t a b i l i t y o f the p a r e n sine (ddl) nucleosides. Both ddA and d d l have h a l f l i v e s o f a p p r o x i m a t e l y h a l f a minute a t pH 1 (37°C) which makes them u n s u i t a b l e f o r o r a l use ( 2 5 ) . Based on t h e mechanism o f a c i d catalyzed cleavage of purine nucleosides (26), i t was reasoned t h a t an e l e c t r o n e g a t i v e s u b s t i t u e n t a t the 2 - p o s i t i o n , such as f l u o r i n e , would d e s t a b i l i z e the r e s u l t i n g oxonium i o n and t h u s increase the s t a b i l i t y of the g l y c o s y l i c bond ( F i g u r e 2). F l u o r i n e was a l s o c o n s i d e r e d an i d e a l isostere for hydrogen because o f i t s r e l a t i v e s m a l l s i z e . The o n l y r e m a i n i n g i s s u e was the s e l e c t i o n o f t h e s t e r e o c h e m i c a l o r i e n t a t i o n o f t h e f l u o r i n e s u b s t i t u e n t (α o r β) t h a t was r e q u i r e d t o m a i n t a i n the a n t i - H I V activity of the parent d i d e o x y n u c l e o s i d e s . Both fluorines u b s t i t u t e d isomers o f ddA were s y n t h e s i z e d and shown t o be h y d r o l y t i c a l l y v e r y s t a b l e (no d e c o m p o s i t i o n d e t e c t e d a f t e r 24 h at pH 1/37° C ) . However, o n l y the isomer w i t h the / 3 - f l u o r i n e atom (ddF-ara-A) r e t a i n e d the p o t e n t a n t i - H I V a c t i v i t y o f the p a r e n t ddA ( 2 5 ) . The i n a c t i v e α - f l u o r i n e isomer (ddF-A, 8, Scheme 3) was o b t a i n e d i n f o u r s t e p s from 3 ' - d e o x y - a r a - A ( 2 7 ) . The procedure i n v o l v e d p r o t e c t i o n o f the the 5 ' - h y d r o x y l group w i t h dimethoxyt r i t y l c h l o r i d e t o g i v e 7, a c t i v a t i o n o f the 2 ' - h y d r o x y l group v i a f o r m a t i o n o f the c o r r e s p o n d i n g t r i f l a t e d e r i v a t i v e , i n v e r s i o n o f c o n f i g u r a t i o n at t h e 2 ' - p o s i t i o n by S^2 d i s p l a c e m e n t u s i n g t e t r a n - b u t y l ammonium f l u o r i d e , and removal o f t h e d i m e t h o x y t r i t y l group with dichloroacetic acid. A similar displacement reaction s t r a t e g y employing c o r d y c e p i n ( 3 ' - d e o x y a d e n o s i n e ) f a i l e d i n our hands t o produce 15. Instead, t h e e l i m i n a t i o n p r o d u c t was isolated. R e c e n t l y , however, t h i s type o f d i s p l a c e m e n t has been r e a l i z e d by Herdewijn e t a l . , a l b e i t i n v e r y low y i e l d ( 2 8 ) . Two d i f f e r e n t s y n t h e t i c a p p r o a c h e s , both c o n v e r g i n g on the i m p o r t a n t known i n t e r m e d i a t e (6-amino-9-/3-û-2-deoxy-2-fluroarab i n o f u r a n o s y l ) - 9 H - p u r i n e (11), were developed f o r the s y n t h e s i s o f t h e a c t i v e compounds d d F - a r a - A and d d F - a r a - I . This intermediate, o r i g i n a l l y s y n t h e s i z e d by Fox and coworkers ( 2 9 ) , was p r e p a r e d u s ­ i n g the improved, g e n e r a l procedure o f Montgomery e t a l (30), which i n v o l v e d c o n d e n s a t i o n o f 6-chloropurine with 3-0-acetyl-5/

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

10.

Scheme 2.

H N 2

147

Nucleoside and Nucleotide Analogues

MARQUEZ

S y n t h e s i s o f 3 - d e a z a n e p l a n o c i n A.

H

HO

F i g u r e 2.

Mechanism o f a c i d h y d r o l y s i s o f d i d e o x y n u c l e o s i d e s and a c i d s t a b i l i z a t i o n r a t i o n a l e .

NH

NH

2

ο

I

1

>

C

W

!

2

o |

DMTO

Scheme 3.

Synthesis o f 2',3'-dideoxy-2'-fluoro-ribo-A.

American Chemical Society Library 1155 16th St.. N.W. In Nucleotide Analogues asD.C. Antiviral Agents; Martin, J.; Washington, 20036 ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

148

NUCLEOTIDE

ANALOGUES

0-benzoyl-2-deoxy-2-fluoro-D-arabinofuranosyl bromide ( 2 5 ) . The r e q u i r e d , f u n c t i o n a l i z e d haTosugar was p r e p a r e d i n e s s e n t i a l l y the same manner as d e s c r i b e d by Fox et a l . (31) and, as e x p e c t e d , f o u r isomers were o b t a i n e d from the c o n d e n s a t i o n r e a c t i o n . After s e p a r a t i o n and c h a r a c t e r i z a t i o n o f the c o r r e c t 6 - c h l o r o i s o m e r , the r e q u i r e d i n t e r m e d i a t e (11) was o b t a i n e d upon ammonolysis. All c h e m i c a l , o p t i c a l , and s p e c t r a l p r o p e r t i e s o f t h e compound matched t h o s e r e p o r t e d p r e v i o u s l y f o r 11 ( 2 5 ) . More r e c e n t l y , we have employed the more a c c e s s i b l e l,3,5-tri-(0-benzoyl)-2-fluoro-a-Da r a b i n o s e (9a) as s t a r t i n g m a t e r i a l (Scheme 4). The c o r r e s p o n d i n g bromo sugar 9b, r e a d i l y formed a f t e r t r e a t m e n t w i t h HBr i n a c e t i c a c i d , was used d i r e c t l y i n a f u s i o n r e a c t i o n (neat 8 5 ° c ) w i t h the t r i m e t h y l s i l y l ( T M S ) - p r o t e c t e d 6 - c h l o r o p u r i n e t o g i v e the d e s i r e d c o n d e n s a t i o n p r o d u c t 10 i n 25% y i e l d . Treatment o f 10 w i t h methanolic ammonia under pressure produced the identical i n t e r m e d i a t e 11. S e l e c t i v e p r o t e c t i o n o f the p r i m a r y a l c o h o l function of 11, accomplishe c h l o r i d e , gave compoun phenyl c h l o r o t h i o n o c a r b o n a t e / D M A P , a f f o r d e d the c o r r e s p o n d i n g 3 ' O-phenoxythionocarbonate 13. R e d u c t i v e d e o x y g e n a t i o n t o 14 w i t h t r i - n - b u t y l t i n hydride/AIBN i n r e f l u x i n g t o l u e n e , and removal o f the tert-butyldimethylsilyl group with .tetra-n-butylammonium f l u o r i d e i n THF, a f f o r d e d d d F - a r a - A (15). The c o r r e s p o n d i n g i n o s i n e analogue d d F - a r a - I (16) was prepared from d d F - a r a - A e i t h e r by enzymatic (adenosine deaminase) o r c h e m i c a l d e a m i n a t i o n (NaNOo/HOAc). Antiviral Activity. In ATH8 c e l l s i n f e c t e d w i t h the HIV v i r u s , both d d F - a r a - A (25) and d d F - a r a - I were a p p r o x i m a t e l y as a c t i v e and p o t e n t as AZT, ddA, and d d l , i n p r o t e c t i n g the c e l l s a g a i n s t the c y t o p a t h i c e f f e c t s o f the v i r u s . As shown i n F i g u r e 3 , d d F - a r a - I a f f o r d e d complete p r o t e c t i o n a t 10 μΜ c o n c e n t r a t i o n and beyond. S i g n i f i c a n t a n t i - H I V a c t i v i t y w i t h d d F - a r a - A was a l s o r e p o r t e d i n i n f e c t e d MT-4 c e l l s ; however, the compound proved t o be i n f e r i o r t o i t s p a r e n t ddA i n such t e s t system ( 3 1 ) . As i t i s the case w i t h ddA, adenosine deaminase r e a d i l y t r a n s f o r m e d d d F - a r a - A t o d d F - a r a - I w i t h o u t d e t r i m e n t t o the measured a n t i - H I V a c t i v i t y . It was t h e r e f o r e i m p o r t a n t t o i n v e s t i g a t e the e f f e c t s o f the f l u o r i n e s u b s t i t u e n t on a second d e g r a d a t i v e enzyme, p u r i n e n u c l e o s i d e p h o s p h o r y l a s e , which i s c a p a b l e o f r a p i d l y c l e a v i n g the g l y c o s y l i c l i n k a g e o f d d l and r e n d e r i n g the compound i n a c t i v e . Consistent with its i n c r e a s e d chemical s t a b i l i t y towards acid-catalyzed c l e a v a g e o f the g l y c o s y l i c bond, d d F - a r a - I was shown t o be t o t a l l y i n e r t towards the a c t i o n o f p u r i n e n u c l e o s i d e p h o s p h o r y l a s e (32). In o r d e r t o study the e f f e c t o f the f l u o r i n e s u b s t i t u e n t on the g a s t r i c s t a b i l i t y and o r a l b i o a v a i l a b i l i t y o f the a c t i v e ddFa r a - A , the NCI conducted c o m p a r a t i v e s t u d i e s i n dogs t r e a t e d s e p a r a t e l y w i t h ddA o r d d F - a r a - A . Under the same c o n d i t i o n s , w i t h o u t any b u f f e r i n g , d d F - a r a - A showed s u p e r i o r b i o a v a i l a b i l i t y (87%) compared t o t h a t o f ddA (30%) ( 3 3 ) . The o r a l b i o a v a i l a b i l ­ i t y o f d d F - a r a - A was even g r e a t e r than t h a t o b t a i n e d w i t h b u f f e r e d (61%), o r e n t e r i c coated (67%) ddA ( 3 3 ) .

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

S y n t h e s i s o f 2 ' , 3 ' - d i d e o x y - 2 ' - f l u o r o - a r a - A and 2',3'-dideoxy-2'-f1uoro-ara-I. Scheme 4. In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

150

NUCLEOTIDE

ANALOGUES

Mechanism o f A c t i o n . A l t h o u g h no d i r e c t m e c h a n i s t i c s t u d i e s as y e t have been performed w i t h e i t h e r d d F - a r a - A o r d d F - a r a - I , i t i s expected t h a t both compounds w i l l be a n a b o l i z e d t o t h e c o r r e s p o n d ­ i n g n u c l e o t i d e 5 ' - t r i p h o s p h a t e analogues i n a s i m i l a r f a s h i o n as t h e i r p a r e n t d i d e o x y n u c l e o s i d e s (ddA and d d l ) ( 3 4 ) . Dideoxynucl e o t i d e t r i p h o s p h a t e analogues are known t o f u n c t i o n as e f f e c t i v e i n h i b i t o r s o f HIV r e v e r s e t r a n s c r i p t a s e t h a t b l o c k DNA s y n t h e s i s by c h a i n t e r m i n a t i o n ( 3 5 ) . M e t a b o l i c a l l y i t i s p o s s i b l e , as demonstrated f o r d d l , t h a t d d F - a r a - I M P w i l l be c o n v e r t e d t o d d F ara-AMP v i a t h e same e f f i c i e n t pathway t h a t c o n v e r t s IMP t o AMP (34). 9-

(2-Deoxv-2-dihvdroxvDhosDhinvlmethv1-/?-D-ribofuranosv1)adenine

1261. Chemistry. Compound 26 c o r r e s p o n d s t o u n i t s one and two o f a proposed m o d i f i e d 2 ' - 5 ' - l i n k e Figure 4), which due phosphorous l i n k a g e , i s expected t o be s t a b l e towards d e g r a d a t i o n by the interferon (IF)-induced 2'-phoshodiesterase (2'-PD). P r e v i o u s s t u d i e s o f s y n t h e t i c analogues o f the t r i m e r "core", A 2 ' p 5 ' A 2 ' p 5 ' A ( l a c k i n g the t r i p h o s p h a t e group a t t h e 5 ' - e n d ) , which i n c l u d e d m o d i f i c a t i o n s on t h e sugar moiety d e s i g n e d t o make the m o l e c u l e s more s t a b l e towards 2 ' - P D , showed s i g n i f i c a n t l y b e t t e r a n t i p r o l i f e r a t i v e and a n t i v i r a l a c t i v i t y t h a n t h e u n s t a b l e parent "core" (36). However, i t was l a t e r demonstrated t h a t a l l o f t h e b i o l o g i c a l a c t i v i t i e s o f t h e so c a l l e d " s t a b l e c o r e s " were due t o the monomeric components which were r e l e a s e d from t h e t r i m e r s by n o n s p e c i f i c serum enzymes ( 3 6 b , e ) . T h u s , the s e a r c h f o r s t a b l e analogues which would not y i e l d a c t i v e a n t i m e t a b o l i t e s upon n o n s p e c i f i c h y d r o l y s i s c o n t i n u e s ( 3 7 ) . The f i r s t o b j e c t i v e o f t h i s p r o j e c t , which was t h e s y n t h e s i s and b i o l o g i c a l t e s t i n g o f the phosphonate monomer 26, has been a c h i e v e d , and the assembly o f the r e q u i r e d t r i m e r 17 i s i n p r o g r e s s . We have a c c o m p l i s h e d t h e s y n t h e s i s o f 26 as shown i n Scheme 5. S t a r t i n g w i t h the versatile intermediate l,2:5,6-di-0isopropylidene-a-û-glucofuranose, the phosphonate 18 was p r e p a r e d essentially i n the same manner as r e p o r t e d by M o f f a t t and coworkers ( 3 8 ) . S e l e c t i v e h y d r o l y s i s of the 5 , 6 - 0 - i s o p r o p y l i d e n e f u n c t i o n , f o l l o w e d by p r o t e c t i o n o f t h e r e s u l t i n g p r i m a r y a l c o h o ­ l i c group o f t h e d i o l w i t h the benzoyl g r o u p , a f f o r d e d compound 20. Removal o f the 1 , - 2 - 0 - i s o p r o p y l i d e n e group gave the i n t e r ­ mediate p y r a n o s i d e 21, which was s u b j e c t e d t o p e r i o d a t e o x i d a t i o n t o g i v e the d e s i r e d phosphonate f u r a n o s i d e 22. As r e p o r t e d i n s i m i l a r r i n g c o n t r a c t i o n s , t h e e x c i s e d carbon atom ends up as a 3 ' - 0 - f o r m y l moiety ( 2 9 ) . S e l e c t i v e removal o f t h i s formate g r o u p , and r e a c y l a t i o n o f both t h e Γ - and t h e 3 ' - h y d r o x y l s w i t h a c e t i c a n h y d r i d e , s e t the stage f o r t h e c o n d e n s a t i o n o f t h e phosphonate sugar 24 w i t h p e r s i l y l a t e d 6 - c h l o r o p u r i n e , which was performed under Lewis a c i d c a t a l y s i s ( t r i m e t h y l s i l y l t r i f l a t e ) . The conden­ s a t i o n r e a c t i o n gave a r e m a r k a b l y good y i e l d (82%) o f t h e d e s i r e d / M s o m e r 25, which upon t r e a t m e n t w i t h s a t u r a t e d methanol i c ammonia, gave t h e d e s i r e d t a r g e t compound 26. The structural

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

10. M A R Q U E Z

151

Nucleoside and Nucleotide Analogues

assignment o f 26 r e s t s on s o l i d e v i d e n c e e x p e r i m e n t s as i l l u s t r a t e d i n F i g u r e 5 .

from

UV, CD, and NOE

Antiviral Activity. The monomeric s t r u c t u r e 26 showed n e i t h e r antitumor nor a n t i v i r a l a c t i v i t y . Upon i n v i t r o evaluation a g a i n s t herpes s i m p l e x v i r u s t y p e 1 (HSV-1) and i n f l u e n z a v i r u s t y p e A£, no e v i d e n c e o f i n h i b i t i o n o f v i r a l c y t o p a t h o g e n i c i t y was d e t e c t e d a t doses e x c e e d i n g 320 Mg/ml ( 3 9 ) .

Ô

π

X

a

—I LU Ο LU —I CÛ < > u_ Ο ce

LU CO

Έ D Ζ

0

2

10 20 100 200

CONCENTRATION (μΜ)

Figure 3.

Figure 4.

I n h i b i t i o n o f t h e c y t o p a t h i c e f f e c t o f HIV by ddF-ara-I.

Chemical s t r u c t u r e o f t h e o l i g o a d e n y l a t e ate t r i m e r .

phosphon­

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

152

NUCLE(mDE

HOCH πυυπ2

Q

BzOÇH Τ *

2

1 — r ? CH

"

CH

ÇH

BzOCH

N-OH

\

/

HO

ÇH

^ NH4OH, EtOH

(EtO) P=0

BzOCH

2

2

\ ^ 0 H /

\

ÇH

«

^ "

,)-OH

C H OH 2

(EtO) P=0

2

23

Κ

2

(EtO) P=0

2

f

2

20

OHCO

2

o

2

(EtO) P=0

19

0

Κ

-20°C

2

18

2

0-Γ

2

Ί — r ?

R

(EtO) P=0

2

BzOCH

2

ι — r ?

Of"

2

(EtO) P = 0

ANALOGUES

2

22

21

A c 0 , Py 2

CI

NH

N ^ V ^ HMDS, Δ

l ^* .N U

Ci

V

BzOCH „ 2

AcO

CH I 2

N^V\V

M

2

(EtO) P=0 2

24 Scheme 5.

J L /

Tf

L ^?

BzOCH

A c 0

1)NH /MeOH

J

2

(EtO) P=0 2

\

I

CH

•

2) 1N NaOH

C H

\

A

I 2

MeCN

\

3

2

HOÇH

2

HO

0=P(OH)

25

26

Synthesis of 9-(2-deoxy-2-dihydroxyphosphinylmethyl ) - 0 - D - r i b o f u r a n o s y l ) a d e n i n e

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

2

2

MARQUEZ

153

Nucleoside and Nucleotide Analogues

UV SPECTRUM OF 9-(2'-DEOXY 2'-DIHYDR0XYPH0SPHINYLMETHYLβ -D-RIBOFURANOSYL)ADENINE (26)

-1000* 200

" 220

« 240

1 260

" 280

WAVELENGTH (nm) Figure

5.

Spectral

data

i n support of

structure

26.

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

' 300

NUCLEimDE ANALOGUES

154 Literature Cited 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.

Robins, R.K.; Revankar, G.; McKernan, P.Α.; Murray, B.K.; Kirsi, J.J.; North, J.A. Adv. Enzyme Res. 1985, 24, 29. Lin, J-C.; Pagano, J.S. Pharmac. Ther. 1985, 28, 135. Prisbe, E.J.; Martin, J.C.; McGee, D.P.C.; Barker, M.F.; Smee, D.F.; Duke, A.E.; Matthews, T.R.; Verheyden, J.P.H. J. Med. Chem. 1986, 29, 671. De Clercq, E. Antimicrob. Agents Chemother. 1985, 28, 84. Lim, M-I.; Moyer, J.D.; Cysyk, R.L.; Marquez, V.E. J. Med. Chem. 1984, 27, 1536. Marquez, V.E.; Lim, M-I.; Treanor, S.P.; Plowman, J.; Priest, M.A.; Markovac, Α.; Khan, M.S.; Kaskar, B.; Driscoll, J.S. J. Med. Chem. 1988, 31, 1687. Arita, M.; Adachi, K.; Ohno, M. Nucleic Acid Res., Symp. Ser. 1983, 12, 25. Arita, M.; Okumoto Shuto, S.; Tsujino Res. 1987, 171, 233. Kim, S.K.; Marquez, V.E.; Driscoll, J.S. Laboratory of Medicinal Chemistry, NCI, NIH. Unpublished results. Kim, S.K.; Marquez, V.E. 194th National Meeting of the American Chemical Society, New Orleans, Louisiana, August 30September 4, 1987, CARB 6. De Clercq, E.; Beres, J.; Benrude, W.G. Mol. Pharmacol. 1987, 32, 286. Marquez, V.E.; Driscoll, J.S., Laboratory of Medicinal Chemistry, NCI, NIH. Unpublished results. Kang, G.J.; Cooney, D.A.; Moyer, J.D.; Kelley, J.Α.; Kim, H­ -Y.; Marquez, V.E.; Johns, D.G. J. Biol. Chem. 1988, in press. Tseng, C.K.H.; Marquez, V.E. Tetrahedron Lett. 1985, 26, 3669. X-ray crystallographic analysis for 3-deazaneplanocin A was performed by Dr. Barry M. Goldstein, Department of Biophysics, Medical Center, University of Rochester, Rochester, N.Y. De Clercq, E. Antimicrob. Agents Chemother. 1985, 28, 84. De Clercq, E.; Cools, M. Biochem. Biophys. Res. Commun. 1985, 129, 306. Montgomery, J.Α.; Clayton, S.J.; Thomas, H.J.; Shannon, W.M.; Arnett, G.; Bodner, A.J.; Kion, I.; Cantoni, G.L.; Chiang, P.K. J. Med. Chem. 1982, 25, 626. De Clercq, E.; Bergstrom, D.E.; Holy, Α.; Montgomery, J.A. Antiviral Res. 1984, 4, 119. Marquez, V.E.; Lim, M-I. Med. Res. Rev. 1986, 6, 1. Keller, B.T.; Borchardt, R.T. Biochem. Biophys. Res. Commun. 1984, 120, 131. Glazer, R.I.; Knode, M.C. J. Biol. Chem. 1984, 259, 12964. Glazer, R.I.; Hartman, K.D.; Knode, M.C.; Richard, M.M.; Chiang, P.K.; Tseng, C.K.H.; Marquez, V.E. Biochem. Biophys. Res. Commun. 1986, 135, 688. Glazer, R.I.; Knode, M.C.; Tseng, C.K.H.; Haines, D.R.; Marquez, V.E. Biochem. Pharmacol. 1986, 35, 4523. Marquez, V.E.; Tseng, C.K.H.; Kelley, J.Α.; Mitsuya, H.; Broder, S.; Roth, J.S.; Driscoll, J.S. Biochem. Pharmacol. 1987, 36, 2719.

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

10.

M

A

R

Q

U

E

Z

Nucleoside and Nucleotide Analogues

155

26. York, J.L. J. Org. Chem. 1981, 46, 2171. 27. 3'-deoxy-ara-A was synthesized by Dr. Terrence C. Owen (University of South Florida) by the method of Hansske, F. and Robins, M.J. (J. Am. Chem. Soc. 1983, 105, 6736) under contract N01-CM-437639 to the DS&CB, NCI. 28. Herdewijn, P.; Pauwels, R.; Baba, M.; Balzarini, J.; De Clercq, E. J. Med. Chem. 1987, 30, 2131. 29. Wright, J.Α.; Taylor, N.F.; Fox, J.J. J. Org. Chem. 1969, 34, 2632. 30. Montgomery, J.Α.; Shortnacy, A.T.; Carson, D.A.; Secrist III, J.A. J. Med. Chem. 1986, 29, 2389. 31. Reichman, U.; Watanabe, K.A.; Fox, J.J. Carbohydr. Res. 1975, 42, 233. 32. Cooney, D.A., Laboratory of Pharmacology, NCI, NIH. Unpublished results. 33. Tomaszewski, J.E. Grieshaber C.K. Toxicolog Branch NCI NIH. Unpublished results 34. Ahluwalia, G.; Cooney K.P.; Hao, Z.; Dalai, M.; Broder, S.; Johns, D.G. Biochem. Pharmacol. 1987, 36, 3797. 35. Mitsuya, H.; Broder, S. Proc. Natl. Acad. Sci. USA 1986, 83, 1911. 36. a. Baglioni, C.; D'Alessandro, S.B.; Nilsen, T.S.; den Hartog, J.A.J.; Crea, R.; Van Boom, J.H. J. Biol. Chem. 1981, 156, 3253. b. Chapekar, M.S.; Glazer, R.I. Biochem. Biophys. Res. Commun. 1983, 115, 137. c. Doetsch, P.; Wu, J.M.; Sawada, Y.; Suhadolnik, R.J. Nature. 1981, 291, 355. d. Eppstein, D.A.; Marsh, Y.V.; Schryver, B.B.; Larsen, M.A.; Barnett, J.W.; Verheyden, J.P.H.; Prisbe, E.J. J. Biol. Chem. 1982, 257, 13390. e. Eppstein, D.A.; Marsh, Y.V.; Schryver, B.B. Virology, 1983, 131, 341. f. Eppstein, D.A.; Van der Pas, M.A.; Schryver, B.B.; Sawai, H.; Lesiak, K.; Imai, J.; Torrence, P.F. J. Biol. Chem. 1985, 260, 3666. g. Imai, J.; Johnston, M.I.; Torrence, P.F. J. Biol. Chem. 1982, 257, 12739. h. Lee, C.; Suhadolnik, R.J. FEBS Lett. 1983, 157, 205. i. Sawai, H.; Imai, J.; Lesiak, K.; Johnston, M.I.; Torrence, P.F. J. Biol. Chem. 1983, 258, 1671. j . Suhadolnik, R.J.; Devash, Y.; Reichenbach, N.L.; Flock, M.B.; Wu, J.N. Biochem. Biophys. Res. Commun. 1983, 111 205. 37. Eppstein, D.A.; Schryver, B.B.; Marsh, Y.V. J. Biol. Chem. 1986, 261, 5999. 38. Albrecht, H.P.; Jones, G.H.; Moffatt, J.G. Tetrahedron, 1984, 40, 79. 39. Marquez, V.E.; Tseng, C.K.H., Laboratory of Medicinal Chemistry, NCI, NIH. Unpublished results. RECEIVED January 25, 1989

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Chapter 11

Nucleotide Dimers as Anti Human Immunodeficiency Virus Agents Elliot F. Hahn , Mariano Busso , Abdul M. Mian , and Lionel Resnick 1

2

3

2

IVAX Corporation, 8800 NW 36th Street, Miami, F L 33178 Departments of Dermatology and Pathology, Mount Sinai Medical Center, 4300 Alto Department of Oncology F L 33131 1

2

3

A series of nucleotide homo and heterdimers were synthesized from nucleosides which include azidothymidine, 2', 3'-dideoxyadenosine and 2'3'-dideoxyinosine. The compounds were evaluated with respect to anti-HIV a c t i v i t y , cytotoxicity, i n v i t r o s t a b i l i t y and i n vivo distribution. On an equimolar basis, greater anti-HIV potency and enhanced cytotherapeutic indices were obtained with heterodimers relative to the corresponding monomers. In v i t r o s t a b i l i t y of the dimer phosphate linkage was found to be species dependent. After intravenous administration to rats, the distribution of 3'-azido-3'3'-deoxythymidilyl-(5'5')-2',3'-dideoxy;-5'-adenylic acid, 2-cyanoethyl ester (AZT-P(CyE)ddA) and AZT i n plasma and brain was similar. In 1981, the acquired immunodeficiency syndrome (AIDS) was reported as a new c l i n i c a l entity (1-3). An intensive research effort led to the i d e n t i f i c a t i o n of the e t i o l o g i c agent responsible for the disease. (4-7). The pathogen, human immunodeficiency virus (HIV), i s a non-oncogenic retrovirus closely associated with the l e n t i v i r u s family (8,9). The discovery of HIV led to the development of therapeutic strategies directed against the virus. The different stages i n the replicative cycle of HIV provide various targets at which a n t i v i r a l agents may intervene. 0097-6156/89/0401 -0156$06.00/0 ο 1989 A m e r i c a n C h e m i c a l Society

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

11.

HAHNETAK

157

Nucleotide Dinners

T h e s e have been t h e s u b j e c t o f a number o f r e v i e w s (10.11) and i n c l u d e : ( 1 ) t h e b i n d i n g s t a g e a t w h i c h CD4 r e c e p t o r d e c o y s a n d d e x t r a n s u l f a t e s a c t , (2) e n t r y i n t o t h e t a r g e t cell where drugs that block fusion or uncoating might intervene, ( 3 ) t r a n s c r i p t i o n o f RNA t o D N A , t h e l e v e l at which reverse t r a n s c r i p t a s e i n h i b i t o r s would f u n c t i o n , (4) i n t e g r a t i o n o f DNA a n d e x p r e s s i o n o f v i r a l genes, where "integrase" inhibitors or "anti-sense" construct would act, (5) viral p r o t e i n p r o d u c t i o n and assembly c o u l d be m o d i f i e d by p r o t e a s e i n h i b i t o r s and agents which affect, myristylation or glycosylation and f i n a l l y , (6) budding o f t h e v i r u s w h i c h may b e p r e v e n t e d b y interferons. The most e f f e c t i v e approach has i n v o l v e d t h e s y n t h e s i s o f i n h i b i t o r s o f r e v e r s e t r a n s c r i p t a s e , t h e u n i q u e enzyme a s s o c i a t e d w i t h t h e r e p l i c a t i o n o f HIV. The r a t i o n a l e for the use of a n t i v i r a l agent on t h e assumption t h a development of progressive d i s e a s e . I n h i b i t i o n o f H I V may permit the regeneration or prevent additional deterioration o f t h e immune s y s t e m . These drugs would p r o v i d e the g r e a t e s t p o s s i b i l i t y of o b t a i n i n g an immediate c l i n i c a l i m p a c t on t h e c o u r s e o f t h e d i s e a s e . Since the discovery of reverse t r a n s c r i p t a s e occurred i n 1970 (12.13) . compounds t h a t inhibit t h e enzyme had been d e v e l o p e d and were a v a i l a b l e f o r e v a l u a t i o n a t the a d v e n t o f t h e AIDS c r i s i s . To d a t e , t h e most p o t e n t and selective anti-HIV compounds a r e a series of 2', 3 dideoxynucleoside analogs. These compounds a r e thought t o be s u c c e s s i v e l y p h o s p h o r y l a t e d by h o s t c e l l enzymes t o y i e l d 2 , 3 - d i d e o x y n u c l e o s i d e - 5 - t r i p h o s p h a t e s , which are analogs of 2 -deoxynucleoside-5 -triphosphates, the natural substrates for cellular DNA polymerases and reverse transcriptase. The 2 ,3 -dideoxynucleoside-5 triphosphates function as substrates for HIV reverse t r a n s c r i p t a s e a n d t e r m i n a t e v i r a l DNA c h a i n e l o n g a t i o n b y i n c o r p o r a t i o n i n t o t h e v i r a l genome ( 1 4 ) . Only 3'-azido2 ,3 -dideoxythymidine (AZT) h a s b e e n a p p r o v e d b y t h e FDA f o r t h e t r e a t m e n t o f HIV i n f e c t i o n . AZT was c h o s e n for c l i n i c a l e v a l u a t i o n on t h e b a s i s o f i t s s e l e c t i v e i n v i t r o antiviral e f f e c t a g a i n s t HIV. The c l i n i c a l t r i a l s with AZT have f o c u s e d on p a t i e n t s w i t h AIDS and AIDS-related complex (ARC). In t h e s e p a t i e n t s , AZT i n d u c e d clinical and l a b o r a t o r y improvements. AZT t h e r a p y a l s o exhibits a degree of success in reversing HIV induced dementia (15.16.17). C u r r e n t s t u d i e s aim t o d e t e r m i n e i f AZT is effective in preventing the development of AIDS in a s y m p t o m a t i c HIV s e r o p o s i t i v e individuals. AZT therapy was a s s o c i a t e d w i t h t o x i c i t i e s t h a t may l i m i t its use, which p r i m a r i l y i n v o l v e d bone marrow s u p p r e s s i o n i n the form of anemia and n e u t r o p e n i a ( 1 5 . 1 6 ) . Therefore, other s t r a t e g i e s w h i c h i n v o l v e c o m b i n a t i o n s o f a n t i HIV agents are also being pursued. The most p r o m i s i n g o f t h e 2 ' , 3 · 1

1

1

1

1

1

1

1

1

1

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

1

158

NUCLEOTIDE

ANALOGUES

dideoxynucleosides to be used individually or in c o m b i n a t i o n w i t h AZT a r e 2 · , 3 · - d i d e o x y c y t i d i n e (ddC), and 2·,3 -dideoxyadenosine (ddA). The u l t i m a t e success of t h e s e a g e n t s a g a i n s t HIV is determined in the clinical arena. Combinations of drugs which permit a reduction in i n d i v i d u a l d o s e s w o u l d be a means o f d e c r e a s i n g t o x i c i t y . 1

Another approach to combination therapy involves the use of either homo or heterodimers of specific dideoxynucleosides. We hypothesized that when the dideoxynucleosides are linked v i a a phosphate bridge the d i m e r i z e d compounds c o u l d p r o v i d e s u p e r i o r p h a r m a c o l o g i c a l effects for the following reasons 1) two different nucleosides are delivered to the c e l l simultaneously, 2) a masked phosphate i s p r e s e n t w i t h masking u n i t a l s o b e i n g active, 3) t h e a g e n t c o u l d f u n c t i o n a s a p r o d r u g , a n d 4) a c t i v i t y as the i n t a c the i n i t i a l phosphorylatio mononucleotides is a l i m i t i n g step for the formation of these a c t i v e metabolites (18). However, AZT i s u n i q u e and the i n i t i a l phosphorylation i s not rate l i m i t i n g . If the nucleotide dimers cross the cell membrane (passive diffusion) and are hydrolysed intracellularly (data indicate that at least part of the dimer crosses the c e l l membrane and i s h y d r o l y s e d intracellularly manuscript i n preparation) , they would y i e l d 1 mole of mononucleotide and nucleoside. It is possible that the nucleotide p r o d u c e d w i l l be f u r t h e r anabolized to its triphosphate l e v e l r a t h e r than be a s u b s t r a t e f o r t h e p h o s p h a t a s e s and produce the nucleoside. Therefore, this process will eliminate the need for the initial obligatory phosphorylation step and may result in a superior therapeutic index. If the nucleotide dimers are hydrolysed before their uptake, the nucleotide phosphatases will convert the mononucleotide into the nucleoside. I n t h i s f a s h i o n , t h e d i m e r s may a c t a s " d e p o t forms" for t h e i r r e s p e c t i v e n u c l e o s i d e s a n d may y i e l d a favorable therapeutic index. We h a v e s y n t h e s i z e d a s e r i e s o f c o m p o u n d s w h i c h are homodimers or heterodimers of s p e c i f i c dideoxynucleosides and h a v e shown t h a t t h e s e a g e n t s p o s s e s s a c t i v i t y against HIV. The phosphate linked dimers of the d i d e o x y n u c l e o s i d e s have t h e g e n e r a l f o r m u l a shown i n F i g . 1 where R and R may b e A Z T , ddA o r d d l and R may b e hydrogen, cyanoethyl or e i t h e r a metal anion or organic anion salt. It should be noted t h a t R and R may b e d e r i v e d f r o m t h e same o r d i f f e r e n t d i d e o x y n u c l e o s i d e s to g e n e r a t e p h o s p h a t e - l i n k e d c o m p o u n d s t h a t a r e e i t h e r homo or hetero dimers. T h e d i m e r s may b e p r e p a r e d b y c o u p l i n g the nucleoside to be e s t e r i f i e d with a nucleoside 5 phosphate in the presence of a a r y l s u l f o n y l condensing agent and a base such as i m i d a z o l e (Fig. 2) . A general procedure for the synthesis of AZT-P-ddA, which is 2

3

1

2

1

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

11.

HAHNETAL.

159

Nucleotide Dimers

applicable for the synthesis of a l l nucleotide dimers is as follows: T h e n u c l e o s i d e - 5 - c y a n o e t h y l p h o s p h a t e was synthesized using a modification of the procedure described by Tener (19). Thus, the barium salt of cyanoethyl phosphate (5.0g) was converted to the pyridinium salt under anhydrous conditions and reacted with AZT (2.0g) in the presence of dicyclohexylcarbodiimide (6.18g) a t room t e m p e r a t u r e f o r 48 h o u r s . T h e r e a c t i o n was s t o p p e d b y a d d i t i o n o f w a t e r (10ml) and the product was purified by column chromatography to o b t a i n 2 . 0 7 g (69%) o f t h e d e s i r e d p r o d u c t . A solution of AZT 5 - c y a n o e t h y l p h o s p h a t e (600mg, 1.5mmol) and 2 , 3 dideoxyadenosine (352mg, 1.5mmol) was p r e p a r e d i n 60 m l of d i s t i l l e d pyridine. T o t h i s s o l u t i o n , 1 . 2 1 g (4mmol) of 2,4,6-triisopropylbenzenesulfonyl c h l o r i d e was a d d e d a n d s t i r r e d f o r 30 m i n , f o l l o w e d b y t h e a d d i t i o n o f 0 . 9 8 4 ml (12 mmol) o f N - m e t h y l - i m i d a z o l e room t e m p e r a t u r e f o under vacuum and the residue was purified by column chromatography using s i l i c a g e l . The c y a n o e t h y l phosphate e s t e r w a s h y d r o l y z e d a t r o o m t e m p e r a t u r e w i t h 6 0 m l o f 15% ammonium h y d r o x i d e s o l u t i o n a n d t h e p r o d u c t was purified by f l a s h chromatography u s i n g s i l i c a g e l t o o b t a i n A Z T - P ddA i n 73% y i e l d . 1

1

1

1

To o b t a i n the c y a n o e t h y l phosphate e s t e r , the crude product prior to hydrolysis w i t h ammonium h y d r o x i d e was p u r i f i e d by chromatography. In initial studies, we determined the anti-HIV activity of the dimers using assays which measured i n h i b i t i o n of syncytium formation, reverse transcriptase p r o d u c t i o n a n d HIV a n t i g e n e x p r e s s i o n . Anti-HIV

Evaluation:

A syncytium i n h i b i t i o n assay that i s a safe, simple, rapid, quantitative and s e n s i t i v e screening system has been developed to detect p o t e n t i a l a n t i - H I V drugs. The assay has been standardized and validated for high capacity anti-HIV screening. Compounds c a n be i d e n t i f i e d and p r i o r i t i z e d b a s e d upon a n t i - H I V e f f e c t s . Potency is expressed as effective dose 50%(ED50), toxicity as i n h i b i t o r y d o s e 50% ( I D 5 0 ) a n d t h e c y t o t h e r a p e u t i c index as ID50/ED50. 1) I n h i b i t i o n o f s y n c y t i u m f o r m a t i o n . MT-2 c e l l s a r e used as targets because of their sensitivity to HIV i n f e c t i o n and the f o r m a t i o n of g i a n t syncytia that are quantifiable. The number and t h e t i m e n e c e s s a r y f o r the production of syncytia i s a function of the input virus inoculum. Target MT-2 c e l l s a r e exposed t o DEAE-Dextran ( 2 5 u g / m l , S i g m a ) f o r 20 m i n u t e s , w a s h e d , a n d i n f e c t e d w i t h

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

160

N U C L E O T I D E ANALOGUES

HIV-l(III-6) (MOI = 0 . 0 0 1 ) a t 37°C in humidified a i r c o n t a i n i n g 5% C 0 . A f t e r 9 6 h o u r s s y n c y t i a a r e c o u n t e d i n a microtiter configuration and compared t o controls. Uninfected and infected MT-2 c e l l s without exposure to drug and uninfected c e l l s exposed t o drugs a r e used as controls. A l l c u l t u r e s a r e performed i n t r i p l i c a t e on two sets of experiments. 2

To i n v e s t i g a t e t h e i n h i b i t o r y e f f e c t s o f t h e d r u g s on H I V - i n d u c e d s y n c y t i a f o r m a t i o n (20) , t h e n u c l e o t i d e d i m e r s were c o m p a r e d t o t h e i r monomers a n d t h e i r c o m b i n a t i o n s a t multiple concentrations ( F i g . 3) . AZT-P-ddA and AZTP(CyE)-ddA exerted the strongest protective e f f e c t against the development of HIV-induced syncytia. AZT-P-ddA and i t s cyanoethyl phosphate d e r i v a t i v e a t a concentration of 0.5um c o m p l e t e l y p r o t e c t e d M T - 2 c e l l s from t h e f o r m a t i o n of syncytia. AZT r e q u i r e lOuM, and t h e c o m b i n a t i o achieve f u l l protection. No a n t i - H I V i n h i b i t o r y effects were seen a t c o n c e n t r a t i o n s below O.OluM.

2)

I n h i b i t o r y e f f e c t on r e v e r s e t r a n s c r i p t a s e p r o d u c t i o n .

Reverse transcriptase assays were performed as p r e v i o u s l y d e s c r i b e d ( 2 0 ) . When M T - 2 c e l l s w e r e i n f e c t e d by HIV, peak r e v e r s e t r a n s c r i p t a s e l e v e l s i n t h e c o n t r o l cultures without d r u g were g r e a t e r than 50,000 CPM/ml (uninfected control cultures gave background counts of 300cpm/ml) ( F i g . 4) . A significant inhibition o f HIV r e v e r s e t r a n s c r i p t a s e p r o d u c t i o n was o b s e r v e d i n a d o s e dependent manner when HIV-infected MT-2 c e l l s were cultured i n the presence of nucleosides and nucleotide dimers. AZT-P-ddA, AZT-P(CyE)-ddA and the combination of AZT + d d A , c o m p l e t e l y i n h i b i t e d t h e p r o d u c t s o f r e v e r s e t r a n s c r i p t a s e a t c o n c e n t r a t i o n s >luM. In comparison t o c o n t r o l s , r e v e r s e t r a n s c r i p t a s e p r o d u c t i o n was p a r t i a l l y inhibited when these compounds were tested at levels >0.1uM. The detection of HIV from culture supernatants (infectious v i r a l yield) correlated with detectable levels of reverse transcriptase and HIV-induced syncytia formation. The n u c l e o t i d e dimers exhibited a higher degree of i n h i b i t i o n f o r the detection of i n f e c t i o u s v i r a l y i e l d when c o m p a r e d t o t h e m o n o m e r s .

3) Inhibition effect.

o f HIV a n t i g e n

expression

and c v t o p a t h i c

A f t e r fourteen days o f i n f e c t i o n , 84% o f M T - 2 c e l l s expressed HIV p24 a n t i g e n as detected by indirect immunofluorescence. At a f i n a l concentration of luM of AZT-P-ddA, AZT-P(CyE)-ddA, o r t h e combination o f AZT + ddA, a >70% i n h i b i t i o n of viral a n t i g e n e x p r e s s i o n was

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

161

Nucleotide Dimers

H A H N ET AL.

ο

R0 J

II

Ρ

OR

2

OR

3

Figure 1.

Structure of Dimers.

0 HO-

II

IB]

IB]

AISOJX

-p—

I

°

OR Figure 2.

v

-

r

°

OR

Coupling Scheme for Dimer Synthesis.

120

ddA ·•- AZT -o AZT • ddA AZT-P(CyE)ddA • AZT-P-ddA

log c o n c e n t r a t i o n

(uM)

Figure 3. Syncytium Inhibition. The number of syncytia was determined 96 hours after exposure of HIV infected MT-2 cells to drug concentrations (lOOuM, lOuM, luM, 0.5uM, O.luM, 0.05uM, O.OluM and 0.005uM). Each value represents the arithmetic mean of triplicate cultures from two sets of experiments. The mean syncytium number in infected cultures without drug was 421 ± 27

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

162

N U C L E O T I D E ANALOGUES

achieved. AZT (luM) or ddA (luM) alone exhibited no inhibition. S i m i l a r r e s u l t s were o b t a i n e d when a s s e s s i n g the i n h i b i t i o n of cytopathic e f f e c t by t h e s e compounds (Fig. 5).

4)

Cvtotherapeutic

evaluation.

The c o m p a r a t i v e HIV i n h i b i t o r y e f f e c t s o f n u c l e o s i d e s and n u c l e o t i d e d i m e r s a r e shown on T a b l e 1 . Using a 14day a s s a y , m o n i t o r i n g c e l l v i a b i l i t y and the expression of HIV a n t i g e n by c e l l u l a r fluorescence, studies were performed t o determine the potency and t o x i c i t y of the compounds. L i n e a r r e g r e s s i o n a n a l y s i s was p e r f o r m e d to d e t e r m i n e t h e ID50 a n d ED50 f o r e a c h c o m p o u n d . The growth i n h i b i t o r 2 cells which were not exposed to the virus, were compared. According to their ID50, t h e compounds c o u l d be c l a s s i f i e d i n t o t h r e e m a j o r g r o u p s . The compounds w i t h the highest toxicity were AZT-P-AZT, AZT + ddA, AZTP(CyE), and AZT. Compounds w i t h m o d e r a t e t o x i c i t y were A Z T - P - d d A , A Z T - P - d d l , and A Z T - P ( C y E ) - d d A . The compounds w i t h t h e l e a s t t o x i c i t y were d d l , ddA, and ddA-P(CyE) . When t h e c y t o t o x i c e f f e c t s o f t h e compounds were tested a g a i n s t t h e h u m a n c e l l l i n e s , H9 a n d U 9 3 7 , s i m i l a r t o x i c p r o f i l e s were s e e n . The a n t i - H I V a c t i v i t y o f t h e compounds a c c o r d i n g to t h e i r ED50 r e v e a l e d two m a j o r p r o f i l e s . The most p o t e n t compounds were AZT + ddA, A Z T - P - d d A , A Z T - P ( C y E ) - d d A , AZTP - d d l , and A Z T - P - A Z T . AZT, ddA, d d l , ddA-P(CyE) , and AZTP(CyE) e x h i b i t e d weaker activities. However, the cytotherapeutic indices of AZT-P-ddA, A Z T - P - d d l , and A Z T - P ( C y E ) - d d A were t h e highest. Based on t h e data obtained from these studies we f o c u s e d on f u r t h e r e v a l u a t i o n o f A Z T - P - d d A and A Z T - P ( C y E ) ddA. F i g u r e 6 shows t h e r e s u l t s o f i n c u b a t i o n o f A Z T - P ddA i n human p l a s m a a t v a r i o u s t e m p e r a t u r e s . The compound is m e t a b o l i z e d a t a r a t e o f a p p r o x i m a t e l y 10% p e r hour. T h i s v a l u e appears t o be s p e c i e s dependent as i s seen i n Fig. 7. A n a l y s i s o f t h e m e t a b o l i t e s i n human p l a s m a ( F i g . 8) s h o w s t h a t t h e p r i n c i p a l c o m p o u n d s f o r m e d a r e A Z T a n d ddl. A s i m i l a r study of the s t a b i l i t y of AZT-P(CyE)-ddA (Fig. 9) at 37° shows that it is metabolized more e x t e n s i v e l y o v e r 3 h o u r a s s a y p e r i o d when c o m p a r e d t o A Z T P-ddA. F i g u r e 10 s h o w s t h a t t h e n a t u r e o f t h e products formed i s also species related. In human p l a s m a , the major metabolite is AZT-P-ddA which is a result of hydrolysis of the triester.

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

11. H A H N E T A L

163

Nucleotide Dimers

80000

Mi no drug B ddA • AZT 0 AZT • ddA • AZT-P(CyE)ddA 1 AZT-P-ddA

0. concentration

(uM)

F i g u r e 4 . I n h i b i t i o n o f Reverse T r a n s c r i p t a s e A c t i v i t y . I n h i b i t i o n o f r e v e r s e t r a n s c r i p t a s e a c t i v i t y from c u l t u r e s u p e r n a t a n t s was determined on day 8 . Each v a l u e r e p r e s e n t s the a r i t h m e t i c mean o f t r i p l i c a t e c u l t u r e s from two s e t s o f experiments. Detectable l e v e l s i n reverse t r a n s c r i p t a s e a c t i v i t y o c c u r when CPM/ml a r e g r e a t e r than 5,000 ( L i n e s , S D ) .

c

EN?

a

b

c

d

e

F i g u r e 5. Assessment o f HIV I n h i b i t i o n by F l u o r e s c e n t and V i a b l e C e l l Count. The i n h i b i t o r y e f f e c t o f luM o f drug on HIV e x p r e s s i o n was a s s e s s e d on day 14 by i n d i r e c t immunofluorescence ( s o l i d columns) and v i a b l e c e l l numbers (open c o l u m n s ) : (a) ddA, (b) AZT, (c) AZT + ddA, (d) AZT-P(CyE)-ddA, and (e) AZT-P-ddA. The r e s u l t s a r e the a r i t h m e t i c mean o f t r i p l i c a t e c u l t u r e s from two s e t s o f experiments ( L i n e s , S D ) .

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NUCLE(XnDE ANALOGUES

Table 1. Comparative Inhibitory Effects o f C o m p o u n d s

25 57 60

4.0 7.0 7.5

100 400 450

AZT ddA ddl AZT + ddA AZT-P-AZT AZT-P(CyE)-ddA AZT-P-ddA AZT-P(CyE) AZT-P-ddl ddA-P(CyE)

CTI

ED50

ID50

Drug

133 40 300 250 30

0.6 1.5 0.7 0.8 3

80 60 210 200 90

ID50: of

uninfected

MT-2

cells

by

50% o n

day

14.

ED50:

D r u g c o n c e n t r a t i o n a c h i e v i n g 50% i n h i b i t i o n of HIV e x p r e s s i o n a s s e s s e d by i m m u n o f l u o r e s c e n c e o n day 14.

CTI:

Cytotherapeutic

index:

ID50/ED50.

The results are expressed as the arithmetic mean of t r i p l i c a t e c u l t u r e s f r o m two s e t s o f e x p e r i m e n t s . Linear r e g r e s s i o n a n a l y s i s was u s e d t o d e t e r m i n e t h e ID50 and ED50.

100 60 Percent

60

40 20

2

3 Hours

F i g u r e 6. S t a b i l i t y o f AZT-P-ddA i n Human Plasma ( i n v i t r o ) a t V a r i o u s Temperatures (4 u g / m l ) .

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

H A H N ET AL.

Nucleotide Dimers

Time (hours)

Figure 7. Stability of AZT-P-ddA in Plasma of Various Species at 37 C (4 ug/ml).

l

3

2

4

Hours

Figure 8 . Disposition of AZT-P-ddA in Human Plasma at 37° (4 ug/ml).

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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NUCLEOTIDE

ANALOGUES

Time (hours)

Figure 9 . Stability of AZT-P(CyE)ddA at 37°C.

2

3

Time (hours)

1

2

3

Figure 10. Stability of AZT-P(CyE)ddA in Human and Rat Plasma.

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

11.

HAHNETAL.

167

Nucleotide Dimers

T a b l e 2. Radioactivity i n Rats Following I.V. Administration o f A Z T (5 mg/kg) A Z T - P ( C y E ) d d A (11.5 mg/kg)

Total

Radioactivity (% o f d o s e )

(H ) 3

a

Time

AZT-P

AZT

(CyE)ddA

Plasma

10

min

7.1

0.069

6.6

0.073

20

min

4.1

0.056

4.6

0.063

40

min

2.9

0.047

2.5

0.032

1 hr 0.028

1. 4

2

1. 1

hr

0 . 030

1.4

0 . 028

0.7

0 . 024

0.6

0.022 3 hr 0.026

a.

0. 5

Percent of radioactivity radioactivity

dose was calculated by present in plasma or brain administered

dividing by total

To assess in vivo disposition of AZT-P-ddA we synthesized radiolabelled substrate containing H in a b i o l o g i c a l l y s t a b l e p o s i t i o n i n t h e AZT m o l e c u l e . This compound was d i l u t e d w i t h u n l a b e l l e d d r u g a n d a d m i n i s t e r e d intravenously (6mg/kg) into rats. B l o o d samples were o b t a i n e d a t 1, 2 and 3 h o u r s a f t e r i n j e c t i o n and a n a l y z e d for radioactive content. The r e s u l t s showed a b o u t 5.5% o f t h e d o s e was p r e s e n t i n s e r u m a f t e r 1 h o u r . This value remained constant over the 3 hour sampling p e r i o d . A s i m i l a r s t u d y was c a r r i e d o u t u s i n g r a d i o l a b e l l e d AZTP ( C y E ) - d d A a n d t h e r e s u l t s w e r e c o m p a r e d t o A Z T ( T a b l e 2) . The d a t a shows t h a t at each time period examined the concentration of both drugs present i n p l a s m a was not significantly different. 3

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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NUCLEOTIDE ANALOGUES

Additional studies are ongoing to determine whether i n v i v o a d v a n t a g e s e x c e e d i n g t h o s e o b s e r v e d i n v i t r o may be a s s o c i a t e d w i t h a d m i n i s t r a t i o n of the dimers. This possibility arises because first they do not have to contend with the fate of the individual components in terms of in vivo metabolism, and second, as intact nucleosides both components will reach the cell simultaneously. Acknowledgment: The a u t h o r s would l i k e thank Dr. C.C. L i n for h i s collaboration. LITERATURE

to

CITED

1.

M. S. G o t t l i e b , R. S c h r o f f , H. M. Schanker, J . D. Weisman New E n g l . J

2.

H. Masur, M. A. Michelis, J . B. Greene, I . Onorato, R. A. Vande Stouwe, R. S. Holzman, G. Wormser, L. Brettman, M. Lange, H. W. Murray and S. Cunningham-Rundles, New Engl. J . Med. 305, 1431 (1981).

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F. P. S i e g a l , C. Lopez, G. S. Brown, S. J. K o r n f i e l d , J . Gold, Z. Hirschman, Parham, M. S i e g a l , Rundles and D. Armstrong , New 305, 1439 (1981).

4.

F. B a r r e - S i n o u s s i , J . C. Chermann, R. Rey, M. T. Nugeyre, S. Chamaret, J. Gruest, C. Dauguet, C. A x l e r - B l i n , F. Brun-Vezinet, C. Rouzioux, W. Rozenbaum and L. Montagnier, Science 220, 868 (1983).

5.

R. C. G a l l o , S. Z. Salahuddin, M. Popovie, G. M. Shearer, M. Kaplan, B. F. Haynes, T. J. P a l k e r , R. R e d f i e l d , J . Oleske, B. S a f a i , G. White, P. F o s t e r and P. D. Markham, Science 224-500 (1984).

6.

S. Broder and R. C. G a l l o , New E n g l . J. Med. 311. 1292 (1984).

7.

M. Popovie, M. G. Sarngadharan, E. Read and R. C. G a l l o , Science 224, 497 (1984).

8.

F. Brun-Vezinet, C. Katlama, D. Roulot, L. Lenoble, M. A l i z o n , J . J. Madjar, M.A. Rey, P. M. Ginard, P. Yeni, F. C l a v e l , S. G a d e l l e , and M. H a r z i c . Lancet i:128 (1987).

Hammer, A. E. J. H a s s e l l , S. S. CunninghamEngl. J . Med.

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HAIIN ET AL.

169

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9.

F. C l a v e l , D. Guetard, F. Brun-Vezinet, S. Chamaret, M. A. Rey, M. O. S a n t o s - F e r r i e r a , A. G. Laurent, C. Dauget, C. Katlama, C. Rouzioux, D. Klatzman, J. L. Champalimaud, and L. Montagnier, Science 233:34 (1986).

10.

E. DeClercq TIPS 8,39

11.

H. Mitsuga and S. Broder Nature 325, 773

12.

D. Baltimore Nature; 226:1209 (1970).

13.

H.M

14.

H. Mitsuya, R. J a r r e t , M. Matsukura, N a t l . Acad S c i , USA 84, 2033 (1987)

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S. Broder, Lane, P. D. Markham, R e d f i e l d , H. Mitsuya, D. E. Groopman, L. Resnick, and A. S. F a u c i ; Lancet

(1987). (1987).

Temin, S. M i z u t a n i , Nature; 226:1211 (1970). e t al Proe

R. W. K l e c k e r , R. R. F. Hoth, E. Gelmann, J . R. C. G a l l o , C. E. Myers ii, 627 (1985).

16.

R. Yarchoan, R. W. Klecker, K. J . Weinhold, P. D. Markham, H. K. L y e r l y , D. T. Durack, E. Gelmann, S. N u s i n o f f Lehrman, R. M. Blum, D. W. Barry, G. M. Shearer, M. A. F i s c h l , H. Mitsuya, R. C. G a l l o , J . M. C o l l i n s , D. P. B o l o g n e s i , C. E. Myers and S. Broder; Lancet i, 575 (1986).

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R. Yarchoan, G. Berg, P. Brouwers, M. A. F i s c h l , A. R. S p i t z e r , A. Wichman, J . Grafman, R. V. Thomas, B. S a f a i , A. B r u n e t t i , C. F. Perno, P. J . Schmidt, S. M. Larson, C. E. Myers and S. Broder; Lancet i, 132 (1987).

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RECEIVED April 17, 1989

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

1879

C h a p t e r 12

Oligonucleotide Analogues as Potential Chemotherapeutic Agents G. Zon Applied Biosystems, Inc., 850 Lincoln Centre Drive, Foster City, C A 94404

In p r i n c i p l e , r e l a t i v e l t i d e s (ca. 15-2 s p e c i f i c a l l y h y b r i d i z e with DNA or RNA and thus be used for novel drug design s t r a t e g i e s i n v o l v i n g targeted i n t e r f e r e n c e of genetic expression at the l e v e l of t r a n s c r i p t i o n or t r a n s l a t i o n . Conceivable chemotherapeutic a p p l i c a t i o n s predicated on sequence-specific h y b r i d i z a t i o n ("antisense" i n h i b i t i o n ) require o l i g o n u c l e o t i d e analogues that are r e s i s t a n t to i n vivo degradation by enzymes such as nucleases. Nuclease-resistant analogues having modified i n t e r n u c l e o s i d e linkages (e.g., methylphosphonates or phosphorothioates) or modified nucleosides (e.g., 2'-O-methylribose or 1'-alpha-anomers) are now r e a d i l y a v a i l a b l e by means o f automated synthesis. Through c o l l a b o r a t i v e i n v e s t i g a t i o n s of various d i f f e r e n t backbone-modified o l i g o n u c l e o t i d e s , we have comparatively studied h y b r i d i z a t i o n (Tm), i n v i t r o i n h i b i t i o n of gene expression (CAT and ras genes), and i n v i t r o i n h i b i t i o n of HIV. Phosphorothioate oligomers at ca. 1 uM e x h i b i t e d potent anti-HIV a c t i v i t y . The mechanism(s) for t h i s a n t i - r e t r o v i r a l a c t i v i t y i s (are) under f u r t h e r i n v e s t i g a tion. Normal c e l l g r o w t h and r e p l i c a t i o n r e q u i r e t r a n s c r i p t i o n o f DNA a n d s u b s e q u e n t t r a n s l a t i o n o f mRNA t o a f f o r d n e c e s s a r y enzymes, s t r u c t u r a l components, r e g u l a t o r y f a c t o r s , and o t h e r t y p e s o f p r o t e i n s . These b i o c h e m i c a l p r o c e s s e s and p r o t e i n p r o d u c t s i n a b e r r a n t o r f o r e i g n form may l e a d t o d i s e a s e , as t h e e n d r e s u l t o f e i t h e r an 0097-6156/89/0401-0170$06.00/0 o 1989 A m e r i c a n Chemical Society

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

12.

ZON

Chemotherapeutic Oligonucleotide Analogues

171

i n h e r i t e d genetic defect, mutagenesis, o r v i r a l infections. A p o t e n t i a l l y general therapeutic strategy i n such cases involves i n h i b i t i o n o f aberrant o r f o r e i g n ("target") gene e x p r e s s i o n by use o f s y n t h e t i c o l i g o n u c l e o t i d e analogues t h a t have sequences c o m p l e m e n t a r y t o s p e c i f i c s e q u e n c e s known t o b e p r e s e n t i n t h e t a r g e t DNA o r RNA. Substrategies include t a r g e t i n g these s i n g l e - s t r a n d e d (ss) analogues a t e i t h e r d o u b l e - s t r a n d e d ( d s ) DNA, t h u s f o r m i n g t r i p l e x e s , o r s s DNA o r RNA, t h u s f o r m i n g d u p l e x e s . A l t e r n a t i v e l y , ss or ds o l i g o n u c l e o t i d e a n a l o g u e s c o u l d b e t a r g e t e d a t regulatory proteins that bind nucleic acids. Early s t u d i e s (1,2) i n t h i s g e n e r a l a r e a b e g a n m o r e t h a n t w o decades ago; however, o n l y w i t h i n t h e l a s t few y e a r s has t h e r e been s i g n i f i c a n t l y i n c r e a s e d a t t e n t i o n and e f f o r t , p e r h a p s due i n p a r t t i biology, sequencing techniques of o l i g o n u c l e o t i d e s . Moreover e n c o u r a g i n g r e s u l t s i n v a r i o u s a n t i v i r a l model s t u d i e s : R o u s s a r c o m a v i r u s Q ) , h e r p e s s i m p l e x v i r u s (HSV) t y p e 1 (4.), v e s i c u l a r s t o m a t i t i s v i r u s ( 5 ) , human i m m u n o d e f i c i e n c y v i r u s (HIV) (£-£), a n d t y p e A i n f l u e n z a v i r u s (2.) . A n u m b e r o f r e v i e w a r t i c l e s ( 1 0 - 1 4 ) o n s u c h " a n t i s e n s e o l i g o n u c l e o t i d e s " have appeared r e c e n t l y and c a n b e c o n s u l t e d f o r much more i n f o r m a t i o n t h a n c a n b e given here. The p r e s e n t r e p o r t i s l i m i t e d t o a n i n t r o d u c t i o n t o concepts i n the design o f anti-RNA o l i g o m e r s , b r i e f comments on t h e i r p r e p a r a t i o n a n d p r o p e r t i e s , and p r e s e n t a t i o n o f p r e l i m i n a r y r e s u l t s obtained w i t h o l i g o n u c l e o t i d e analogues c o n t a i n i n g phosphorothioate l i n k a g e s , which a r e under development by the author through various c o l l a b o r a t i v e i n v e s t i g a t i o n s . This account w i l l h o p e f u l l y provide medicinal and c a r b o h y d r a t e c h e m i s t s w i t h an i n d i c a t i o n o f t h e c u r r e n t p o t e n t i a l , and problems t o address, i n t h e development o f a n t i s e n s e o l i g o n u c l e o t i d e s a s a new c l a s s o f a n t i v i r a l agents. Design of Anti-RNA Oligonucleotide

Analogues

Important Design Factors. Therapeutic applications of o l i g o n u c l e o t i d e s b y means o f s e q u e n c e - s p e c i f i c inhibition o f t h e f u n c t i o n o f RNA r e q u i r e c o n s i d e r a t i o n o f t h e following factors. P r a c t i c a l methods f o r s y n t h e s i s and p u r i f i c a t i o n o f r e l a t i v e l y l a r g e amounts o f s t r u c t u r a l l y m o d i f i e d o l i g o n u c l e o t i d e s (analogues) t h a t have adequate r e s i s t a n c e t o d e p o l y m e r i z a t i o n by nucleases, adequate b i o a v a i l a b i l i t y , and a r e t a k e n up by c e l l s . The c o m p l e m e n t a r y t a r g e t sequence must be a c c e s s i b l e t o t h e a n a l o g u e a n d t h e r e must be an a d e q u a t e r a t e a n d e x t e n t o f a s s o c i a t i o n t o form t h e r e s u l t a n t , p e r f e c t l y base-paired duplex, which adequately i n h i b i t s the intended RNAfunction. There s h o u l d be m i n i m a l s i d e - e f f e c t s (e.g.,

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

NUCLEOTIDE ANALOGUES

172

t h a t r e s u l t f r o m a f i n i t e amount o f n o n - s p e c i f i c b i n d i n g t o p o l y n u c l e o t i d e sequences h a v i n g p a r t i a l homology, r e l a t i v e t o the analogue-target duplex. T h e same i s true f o rnon-specific binding t o other c e l l u l a r c o n s t i t u e n t s o r enzymes. Formally analogous design c r i t e r i a obtain f o r conventional (small molecule) a n t i v i r a l agents; nevertheless, because o f t h e comparatively unique nature o f antisense oligonucleotides, i ti s worthwhile t o consider these c r i t e r i a i n a b i t more detail. Structural Modifications. O l i g o n u c l e o t i d e s undergo nuclease-mediated depolymerization a t r a t e s which can be q u i t e f a s t ( h a l f - l i f e < l h ) i n serum (lu). S u b s t i t u t i o n o f one o f t h e n o n - b r i d g i n g oxygens i n t h e n a t u r a l l y o c c u r r i n g l i n k a g e (I) w i t h e i t h e r s u l f u r ( I I ) (1£) , m e t h y l ( I I I (V) (IS.) affords chemicall o l i g o n u c l e o t i d e analogues. ο

ο

ο

II 3Ό-Ρ-05"

I

3Ό-Ρ-05

I

3O-P-05'

I CH

I

II

3

III

0

II 3O-P-05'

3O-P-05'

I

I

NR2

OR

IV

V

3Ό-Ρ-05*

I S"

VI

Rather than modify every linkage, only several o f these m o d i f i c a t i o n s a t t h e 5 ' a n d 3 ' e n d s o f a n o l i g o m e r may provide adequate s t a b i l i t y toward exonucleases (12), although endonucleolytic cleavage s t i l l occurs. This p r o v i d e s a means f o r c o n t r o l l i n g b o t h t h e b i o l o g i c a l h a l f - l i f e o f an o l i g o n u c l e o t i d e analogue and i t s partition coefficient. A d d i t i o n a l l y , i n view of the c h i r a l i t y at phosphorus i n II-V, which r e s u l t s i n 2 d i a s t e r e o m e r s (n=number o f c h i r a l l i n k a g e s ) , u s e o f o n l y terminal modifications reduces t h e stereoisomeric h e t e r o g e n e i t y o f an o l i g o n u c l e o t i d e a n a l o g u e . Another approach along t h i s l i n e i s t o use a c h i r a l linkages, such as V I ( 2 Û ) . A c h i r a l l i n k a g e s w i t h o u t p h o s p h o r u s have n

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

12. Z O N

173

Chemotherapeutic Oligonucleotide Analogues

also been studied, namely, carboxymethyl (2), 3 0-CH C(O)0 5 , carbamate (21,22), 3 0 - C ( 0 ) - N H 5 ' , and s u l f i d e (22), ,

1

2

1

3'C-CH CH2S-C5 , 2

F

moieties.

Among the modified nucleosides that might provide adequate r e s i s t a n c e to nucleases, oligomers c o n t a i n i n g alpha-2'-deoxynucleoside anomers have a t t r a c t e d considerably more a t t e n t i o n (21,25) than those with other types of stereoisomerism (H) or with 2 - O - m e t h y l r i b o nucleosides (2£). Oligonucleotides with base modifications that impart r e s i s t a n c e to nucleases apparently have not been reported. 1

B i o a v a i l a b i l i t y and Uptake by C e l l s . M i l l e r and T s ' o have reported (H) that methylphosphonate analogues (III) prevent expression of h e r p e t i c l e s i o n s when a p p l i e d i n the form of a cream to the HSV-infected ear of a mouse. A t r i t i u m - l a b e l e d methylphosphonat (11) to be d i s t r i b u t e d to a l l organs and t i s s u e s , with the exception of the b r a i n , when i n j e c t e d i n t o the t a i l v e i n of a mouse. By c o n t r a s t , i n a s i m i l a r study with carbon-labeled methylphosphonate analogues, Maguire and Han (22) reported t h a t , on a wet weight b a s i s , peak r a d i o a c t i v i t i e s were i n the order: brain > l i v e r > kidney > lung > heart > muscle. The occurrence times for these peaks were i n the order: muscle > b r a i n = heart = lung > l i v e r > kidney. Indirect evidence for adequate b i o a v a i l a b i l i t y of o l i g o n u c l e o t i d e s with unmodified linkages, and t h e i r uptake by c e l l s , i s provided by reports of i n v i v o a c t i v i t y i n mice against HSV-1 (28.), t i c k - b o r n e e n c e p h a l i t i s v i r u s (22), and c-myc QQ.) . Uptake by c e l l s can be studied with r a d i o l a b e l e d o l i g o n u c l e o t i d e s , by e i t h e r counts alone or autoradiography to v i s u a l i z e the d i s t r i b u t i o n . A l t e r n a t i v e l y , one can use f l u o r e s c e n t l y l a b e l e d oligomers, with e i t h e r f l u o r e s c e n c e - a c t i v a t e d c e l l s o r t i n g (FACS) or fluorescence microscopy. Methylphosphonate analogues are thought to be taken up by passive d i f f u s i o n (H) whereas uptake of n e g a t i v e l y charged, unmodified o l i g o n u c l e o t i d e s and phosphorothioate (II) analogues may involve a more complex process. Our preliminary FACS measurements (Egan, W., Food and Drug A d m i n i s t r a t i o n , personal communication, 1988) with the l a t t e r analogues i n d i c a t e a very r a p i d (<10 min) increase i n c e l l - a s s o c i a t e d fluorescence followed by further increase very slowly or an apparent p l a t e a u . At t h i s time i t i s unknown whether t h i s represents uptake alone or involves an i n i t i a l fast a s s o c i a t i o n with p o s i t i v e l y charged groups on the c e l l surface. A p o t e n t i a l l y major discovery i n t h i s area involves a p u t a t i v e o l i g o n u c l e o t i d e receptor ( S t e i n , C , National I n s t i t u t e s of Health, personal communication, 1988). Also noteworthy are several promising schemes for o b t a i n i n g

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NUCLECmDE ANALOGUES

f a c i l i t a t e d uptake: conjugation of oligonucleotides t o p o l y ( L - l y s i n e ) (21) o r s t e r o i d s (22) a n d p a c k a g i n g o f o l i g o n u c l e o t i d e s i n l i p o s o m e s (22/22) , S e n d a i v i r u s e n v e l o p e (22) , o r e r y t h r o c y t e " g h o s t s " {23.) . S p e c i f i c Binding t o Target and I n h i b i t i o n o f Function. I n 1 9 7 8 Z a m e c n i k a n d c o w o r k e r s (2) c o i n e d t h e w o r d "hybridon" t o refer t o competitive hybridization o f s y n t h e t i c o l i g o n u c l e o t i d e s t o t a r g e t sequences i n order t o b l o c k e i t h e r (1.) R N A D N A polymerization (reverse t r a n s c r i p t i o n ) , ( 2 . ) DNA i n t e g r a t i o n , ( 3 . ) D N A R N A t r a n s c r i p t i o n , (4.) t r a n s l a t i o n , o r (5.) r i b o s o m a l association. A s p l i c e - j u n c t i o n c a n a l s o b e t a r g e t e d (4.) . C a l c u l a t i o n s o f RNA t e r t i a r y s t r u c t u r e t o d e t e r m i n e a c c e s s i b l e s s t a r g e t sequences have been c a r r i e d o u t (24); h o w e v e r , t h e s e a r f questionabl value d t r e p o r t s t o date have t e s t e complementary t o region t r a n s l a t i o n , w h i c h a r e assumed t o be g e n e r a l l y accessible. An i m p o r t a n t development has been recognition that formation o f oligodeoxyribonucleotideRNA h e t e r o d u p l e x e s may l e a d t o s c i s s i o n o f t h e r i b o s t r a n d b y R N a s e H (25), w h i c h a p p a r e n t l y i spresent i n a l l (or most) c e l l s . This allows f o r c a t a l y t i c turnover o f RNA a s o p p o s e d t o 1:1 s t o i c h i o m e t r i c b l o c k a g e . S i g n i f i c a n t l y , phosphorothioate but not alpha-anomeric o l i g o n u c l e o t i d e analogues can function e f f i c i e n t l y i n t h i s m a n n e r (2£). S t a t i s t i c a l l y , a s e q u e n c e o f c a . 17 n u c l e o t i d e s s h o u l d b e f o u n d o n l y o n c e i n t h e human g e n o m e , a n d s h o r t e r s e q u e n c e s (ca. 12-mers) c a n , i n p r i n c i p l e , b e o f u s e a t t h e l e v e l o f mRNA. F r o m DNA p r o b e t e c h n o l o g y , i t i s r e a s o n a b l e t o i n f e r t h a t one base p a i r mismatch i n an RNA t a r g e t - o l i g o n u c l e o t i d e a n a l o g u e d u p l e x c a u s e s s u f f i c i e n t d e s t a b i l i z a t i o n so as t o favor a high degree of b i n d i n g s p e c i f i c i t y , i n t h i s range o f lengths. But t h i s i s t r u e only under thermodynamic c o n t r o l ( a t equilibrium) and s o - c a l l e d stringent conditions ( r e l a t i v e l y high temperature, low s a l t concentration, and l o w c o n c e n t r a t i o n o f o l i g o m e r ) . In contrast, uptake of an o l i g o n u c l e o t i d e analogue by a l i v e c e l l i n v i v o involves non-equilibrium conditions, with regard t o f o r m a t i o n o f o l i g o m e r - R N A ( o r DNA) d u p l e x e s , a n d n o n adjustable non-stringent conditions. This indicates that t h e d e s i r e d d e g r e e o f s p e c i f i c i t y o f d u p l e x f o r m a t i o n may be o f f s e t b y k i n e t i c a l l y c o n t r o l l e d phenomena a n d t h e r a t e a t which t h e "system" approaches a s t a t e o f pseudoequilibrium. I t may t h e r e f o r e b e p o s s i b l e t o m a x i m i z e s p e c i f i c i t y , a t l e a s t i n p a r t , by a d o p t i n g a s t r a t e g y i n which "shorter i sbetter". Y e t t o be determined i s whether t h e synergy o f contiguous antisense oligomers e v i d e n c e d i n c e l l - f r e e s t u d i e s (22) e x t e n d s t o applications i n vivo.

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

12. Z O N

ChemotherapeutwOligomwleotia^Ana^

175

Schemes f o r e i t h e r d e c r e a s i n g o r p r e v e n t i n g d i s a s s o c i a t i o n o f an a n t i s e n s e o l i g o m e r from i t s t a r g e t , or displacement by p o l y n u c l e o t i d e / p r o t e i n , i n c l u d e t h e use o f pendant a l k y l a t i n g groups, i n t e r c a l a t o r s , and p h o t o a c t i v a t a b l e groups (10-14). P r e p a r a t i o n and P h y s i c a l C h a r a c t e r i s t i c s o f O l i g o n u c l e o t i d e Analogues S y n t h e s i s and P u r i f i c a t i o n . Oligodeoxyribonucleotides and o l i g o r i b o n u c l e o t i d e s a r e r e a d i l y p r e p a r e d by automated v e r s i o n s o f t h e solid-phase phosphoramidite m e t h o d d e v e l o p e d b y C a r u t h e r s a n d c o w o r k e r s Q2.) (Figure 1). In each c y c l e , coupling a f f o r d s a phosphite linkage w h i c h c a n be e i t h e r o x i d i z e d w i t h I /H20/base t o a phosphotriester precurso sulfurized with S /CS t o a p h o s p h o r a m i d a t e ( I V ) , o r s t a b l e p h o s p h o t r i e s t e r (V) (23.) . O b v i o u s e x t e n s i o n t o m e t h y l p h o s p h o n a m i d i t e s (ΑΏ.) a f f o r d s methylphosphonate l i n k a g e s . I t i s thus p o s s i b l e t o i n t r o d u c e i n t o an o l i g o n u c l e o t i d e a n a l o g u e one o r more o f t h e same o r d i f f e r e n t m o d i f i c a t i o n s i n a n y o r d e r . The a b i l i t y t o assemble oligomers w i t h combinations o f c h a r g e d and n e u t r a l l i n k a g e s p r o v e d t o be u s e f u l , as o u r i n i t i a l work w i t h methylphosphonate and i s o p r o p y l p h o s p h o t r i e s t e r analogues i n d i c a t e d low s o l u b i l i t y i n water, compared t o c h a r g e d o l i g o m e r s , l e a d i n g t o problems w i t h handling and b i o l o g i c a l t e s t i n g , w h i c h w e r e a d d r e s s e d b y s y n t h e s i s o f η-mer a n a l o g u e s having a l t e r n a t i n g linkages: ( I - I I I ) n / 2 and (I-V)n/2. Another approach t o a d j u s t i n g s o l u b i l i t y i s t h e use o f aminoalkyl s u b s t i t u e n t s attached t o e i t h e r phosphorus (41) o r base moieties. N i e l s e n and Caruthers (42.) have described r e g i o s e l e c t i v e Arbuzov-type reactions of internucleoside 2-cyano-l,1-dimethylethylphosphites as a n o v e l and v e r s a t i l e route t o mixed-linkage analogues. Such m o l e c u l e s c a n a l s o be c o n s t r u c t e d by a p p r o p r i a t e a l t e r n a t i o n o f phosphoramidite and H-phosphonate c h e m i s t r y c y c l e s ( Z o n , G., u n p u b l i s h e d d a t a ) , t h e l a t t e r o f w h i c h f e a t u r e s c o u p l i n g b y a c t i v a t i o n o f t h e Hp h o s p h o n a t e monomer w i t h a d a m a n t a n e c a r b o n y l c h l o r i d e t o g i v e a mixed anhydride, and capping by s i m i l a r r e a c t i o n with isopropyl phosphite (42) ( F i g u r e 2 ) . The r e s u l t a n t i n t e r n u c l e o s i d e H-phosphonate l i n k a g e ( s ) c a n be e i t h e r o x i d i z e d t o an u n m o d i f i e d l i n k a g e o r c o n v e r t e d into m o d i f i e d l i n k a g e s such as I I , IV, V (17-44). Stec and c o w o r k e r s (Αϋ) h a v e r e p o r t e d t h e u s e o f a b i s - a m i d i t e r e a g e n t as an a l t e r n a t i v e e n t r y t o e i t h e r phosphoramiditeor H-phosphonate-like chemistry during each c y c l e o f chain extension. 2

8

P o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s has been t r a d i t i o n a l l y used t o p u r i f y s y n t h e t i c o l i g o n u c l e o t i d e s

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

176

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PHOSPHORAMIDITE REAGENT

F i g u r e 1. 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 h e p h o s p h o r a m i d i t e method f o r o l i g o n u c l e o t i d e s y n t h e s i s on a s o l i d s u p p o r t . F o rd e f i n i t i o n s and d e t a i l s , see r e f . 38.

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

ZON

Chemotherapeutic Oligonucleotide Analogues

Β

0=P-H

Β

Ο

L.

F i g u r e 2. Schematic representation of the hydrogen p h o s p h o n a t e method f o r o l i g o n u c l e o t i d e s y n t h e s i s on s o l i d support. F o r d e f i n i t i o n s and d e t a i l s , see r e f

43.

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

178

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i n s m a l l a m o u n t s (< 1 mg), w h i l e i o n - e x c h a n g e H P L C a n d r e v e r s e d - p h a s e ( p a i r e d - i o n ) HPLC h a v e become more p r e v a l e n t methods o f p u r i f i c a t i o n b e c a u s e t h e y c a n be s c a l e d - u p and automated. R e c e n t r e v i e w s (1£) of o l i g o n u c l e o t i d e p u r i f i c a t i o n can be c o n s u l t e d f o r details. An i n c r e a s i n g g r a d i e n t o f a c e t o n i t r i l e i n triethylammonium acetate i s frequently used f o r reversedp h a s e HPLC o f o l i g o n u c l e o t i d e s b e c a u s e t h e e l u e n t s a r e r e l a t i v e l y e a s y t o remove f r o m t h e f i n a l p r o d u c t . Highr e s o l u t i o n p r o t o n NMR c a n b e e m p l o y e d t o c h e c k f o r r e s i d u a l e l u e n t s , w h i l e p h o s p h o r u s NMR i s useful for a n a l y s i s of the chemical homogeneity of the oligomer b a c k b o n e when a p p r o p r i a t e . S t e r e o c h e m i s t r y and S t r e n g t h o f B i n d i n g . A p l o t o f UV a b s o r b a n c e a t 260 nm (A 6o) v s temperature g e n e r a l l y g i v e s an " S " - s h a p e d c u r v inflection point tha which a duplex i s h a l f d i s s o c i a t e d , at the s p e c i f i e d c o n c e n t r a t i o n o f o l i g o m e r s , a d d e d s a l t ( c a t i o n ) a n d pH. A p l o t o f Tm v s . l o g [ c a t i o n a c t i v i t y ] provides thermodynamic c o n s t a n t s f o r duplex f o r m a t i o n , which can be i n t e r p r e t e d i n t e r m s o f t h e s t r e n g t h o f b i n d i n g . The i n f l u e n c e o n Tm o f c o n v e r t i n g a n unmodified p h o s p h o d i e s t e r l i n k a g e (I) i n t o a c h i r a l l i n k a g e , 3ΌP ( 0 ) ( Χ ) - 0 5 ' , s h o u l d d e p e n d o n t h e e l e c t r o n i c n a t u r e o f X, i t s s t e r i c " b u l k " , and t h e a b s o l u t e s t e r e o c h e m i s t r y a t phosphorus. An e l e c t r o n i c a l l y n e u t r a l ( e . g . , X = m e t h y l ) or p o s i t i v e l y charged s u b s t i t u e n t (e.g., X=aminoalkyl) s h o u l d d e c r e a s e t h e d e p e n d e n c e o f Tm o n t h e c a t i o n c o n c e n t r a t i o n , b u t a p p a r e n t l y i t i s n o t known w h e t h e r t h i s i s b e n e f i c i a l under p h y s i o l o g i c a l s a l t c o n d i t i o n s . W i t h r e g a r d t o a b s o l u t e s t e r e o c h e m i s t r y a t p h o s p h o r u s (R or 2 c o n f i g u r a t i o n ) , i n s p e c t i o n of molecular models of d u p l e x e s s u g g e s t s two d i f f e r e n t o r i e n t a t i o n s o f s u b s t i t u e n t X, r e l a t i v e t o t h e h e l i c a l a x i s , e a c h h a v i n g d i f f e r e n t l o c a l i n t e r a c t i o n s with the flanking residues and a s s o c i a t e d w a t e r m o l e c u l e s . We h a v e u t i l i z e d a s e r i e s of backbone m o d i f i e d , self-complementary o c t a d e o x y r i b o n u c l e o t i d e s ( e . g . , R o r ΐ GGAA TTCC) t o e s t a b l i s h the a b s o l u t e c o n f i g u r a t i o n s at phosphorus, by c h e m i c a l a n d NMR m e t h o d s , a n d m e a s u r e t h e c h a n g e i n Tm (ATm) a s s o c i a t e d w i t h the "outward" and "inward" o r i e n t a t i o n s o f X, r e l a t i v e t o t h e u n m o d i f i e d duplex. The l a t t e r o r i e n t a t i o n f o r X = s u l f u r (12), m e t h y l (!&) / o r a l k o x y (12) d e p r e s s e s t h e Tm, w h i c h h a s b e e n a s c r i b e d t o r e p u l s i v e s t e r i c i n t e r a c t i o n s b e t w e e n X a n d t h e 3* proton. The i n f l u e n c e o n Tm o f m u l t i p l e m e t h y l p h o s p h o n a t e m o d i f i c a t i o n s , a n d t h e i n f l u e n c e o n Tm o f t h e n a t u r e o f t h e f l a n k i n g b a s e s , h a s b e e n i n v e s t i g a t e d ( W i l s o n , W. D., Georgia State U n i v e r s i t y , p e r s o n a l communication,1988) i n the self-complementary tetradecadeoxyribonucleotide X

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

12.

179

Chemotherapeutic Oligonucleotide Analogues

ZON

T ( A A T T ) A , w h i c h w h e n u n m o d i f i e d h a s Tm = 35.8°C i n 0.16 M N a C l , as n o t e d i n T a b l e I . Separated m o d i f i c a t i o n s i n t h i s s e r i e s , r e l a t i v e t o c l u s t e r e d ones, h a v e a somewhat l e s s d i s r u p t i v e e f f e c t on h e l i c a l s t a b i l i t y . Incorpor­ a t i o n o f t w e l v e c o n t i g u o u s m o d i f i c a t i o n s l o w e r e d t h e Tm by o n l y 12.6 C. 3

e

Table

Tm

35.8 31.7 30.5

I.

e

( C)

L o c a t i o n of Methylphosphonate Linkages C o r r e s p o n d i n g Tm V a l u e s a t 0.16 M N a C l

Τ

A

A

τ

Τ

A

unmodified X X

τ

Τ

A

Τ

Α

Α

(X),

Τ

and

Α

Χ

X

χ

X

30.4

32.2 33.5 20.9

X

X

X

X

X

X X

χ χ

χ χ

Χ

χ

χ

χ

We h a v e o b t a i n e d o t h e r d a t a o f t h i s s o r t f o r a d d i t i o n a l sequences and f o r p h o s p h o r o t h i o a t e l i n k a g e s ( I I ) , w h i c h l i k e w i s e c a u s e a r o u g h l y 10°C d r o p i n Tm w h e n a r r a n g e d c o n t i g u o u s l y i n 10- t o 3 0 - m e r s . Both methylphosphonate a n d p h o s p h o r o t h i o a t e a n a l o g u e s s h o w Tm c u r v e s t h a t a r e d i s p l a c e d but not s i g n i f i c a n t l y broadened, r e l a t i v e t o the unmodified duplex. The l a t t e r f e a t u r e m i g h t h a v e been unexpected, i n view of the presence o f 2 d i a s t e r e o m e r s , d e p e n d i n g on o n e ' s t h e o r e t i c a l a n a l y s i s and assumptions. n

Anti-HIV A c t i v i t y

of Phosphorothioate

Analogues

P r e l i m i n a r y S t u d i e s . Among t h e g o a l s o f o u r i n i t i a l s t u d i e s were comparison o f t h e e f f i c a c y o f v a r i o u s t y p e s of analogues and t h e use o f q u a n t i t a t i v e a s s a y s . At t h a t time the n e u t r a l methylphosphonates and c h a r g e d , u n m o d i f i e d o l i g o m e r s w e r e known Q,4.)to be a n t i s e n s e i n h i b i t o r s o f p r o t e i n s y n t h e s i s , a n d i t was o f i n t e r e s t to compare them w i t h p a r t i a l l y c h a r g e d methylphosphonates and p h o s p h o t r i e s t e r s , as w e l l as c h a r g e d phosphorothioates. We c h o s e a r e l a t i v e l y s i m p l e e u k a r y o t i c t i s s u e c u l t u r e (CV-1 c e l l s ) t r a n s f e c t i o n a s s a y of p l a s m i d c o n t a i n i n g t h e gene f o r e x p r e s s i o n o f t h e b a c t e r i a l enzyme c h l o r a m p h e n i c o l a c e t y l t r a n s f e r a s e (CAT) c o u p l e d t o v i r a l (SV 40) r e g u l a t o r y s e q u e n c e s a s a m o d e l i n v i t r o system f o r gauging the r e l a t i v e e f f e c t i v e n e s s of these analogues i n the r e g u l a t i o n o f e x p r e s s i o n of a s p e c i f i c p r o t e i n (uQ.) . The t a r g e t was a 2 1 - b a s e s e q u e n c e t h a t b e g a n w i t h AUG, w h i c h c o d e s f o r t h e i n i t i a l methionine. T r a n s f e c t i o n i n t h e p r e s e n c e o f 30 μΜ oligomer l e d to the following order of oligomer

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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ANALOGUES

inhibitory effect: phosphorothioate > methylphosphonate > a l t e r n a t i n g methylphosphonate and u n m o d i f i e d > a l t e r n a t i n g e t h y l p h o s p h o t r i e s t e r and u n m o d i f i e d > u n m o d i f i e d = a l t e r n a t i n g i s o p r o p y l p h o s p h o t r i e s t e r and u n m o d i f i e d , w i t h t h e f i r s t c a s e b e i n g 84% and t h e l a s t two c a s e s b e i n g 35% and 0%, r e s p e c t i v e l y . A c e l l - f r e e t r a n s l a t i o n assay i n r a b b i t r e t i c u l o c y t e l y s a t e , w h i c h had been d e v e l o p e d f o r e v a l u a t i n g methylphosphonate a n a l o g u e s as i n h i b i t o r s o f r a s - p 2 1 , was made a v a i l a b l e t o us f o r c o m p a r a t i v e s t u d i e s w i t h p h o s p h o r o t h i o a t e s (Chang, Ε . H., Uniformed S e r v i c e s U n i v e r s i t y o f H e a l t h S c i e n c e , p e r s o n a l communication, 1988). An 11-mer m e t h y l p h o s p h o n a t e complementary t o t h e 5 end o f t h e mRNA c a u s e d -45% and -95% i n h i b i t i o n o f p21 t r a n s l a t i o n a t 50 μΜ and 200 μΜ, r e s p e c t i v e l y . Oligomers h a v i n g u n m o d i f i e d l i n k a g e s o r a l t e r n a t i n g u n m o d i f i e d and methylphosphonate linkage the phosphorothioate i n h i b i t i o n a t 12.5 μΜ and 50 μΜ, r e s p e c t i v e l y . The s p e c i f i c i t y o f the p h o s p h o r o t h i o a t e s i n t h i s system i s s t i l l under i n v e s t i g a t i o n . 1

A n t i - H I V A c t i v i t y In v i t r o . Based on t h e presumed i m p o r t a n c e o f t h e a r t / t r s (rev) e n c o d e d p r o t e i n i n t h e r e g u l a t i o n o f HIV, a 14-mer sequence and v a r i o u s presumed i r r e l e v a n t ( c o n t r o l ) sequences were s t u d i e d i n a c y t o p a t h i c e f f e c t i n h i b i t i o n a s s a y ( 2 ) . None o f t h e t e s t e d methylphosphonate and u n m o d i f i e d s e q u e n c e s showed s t a t i s t i c a l l y s i g n i f i c a n t a c t i v i t y , whereas t o o u r s u r p r i s e a l l o f the p h o s p h o r o t h i o a t e s (except v e r y s h o r t ones, 5-mers) e x h i b i t e d some l e v e l o f a c t i v i t y . A 28-mer p o l y m e r o f dC was t h e most p o t e n t p h o s p h o r o t h i o a t e t e s t e d , showing complete i n h i b i t i o n a t 0.5 - 1.0 μΜ. Subsequent e x p e r i m e n t s w i t h t h i s m a t e r i a l a t 1 μΜ employed a S o u t h e r n b l o t a n a l y s i s t o d e m o n s t r a t e complete i n h i b i t i o n o f de novo v i r a l DNA s y n t h e s i s . Recent u n p u b l i s h e d r e s u l t s suggest t h a t p h o s p h o r o t h i o a t e o l i g o m e r s can i n h i b i t R N A — D N A t r a n s c r i p t i o n by b i n d i n g t o v i r a l r e v e r s e t r a n s c r i p t a s e (RT) (Cohen, J . S. National I n s t i t u t e s of Health, Private communication, 1988). In a d d i t i o n t o t h i s p r e s u m p t i v e mechanism f o r s e q u e n c e - n o n s p e c i f i r. R T - d i r e c t e d a n t i v i r a l a c t i v i t y o f phosphorothioate oligomers i n non-infected c e l l s , a s e q u e n c e - s p e c i f i c mechanism has b e e n f o u n d more r e c e n t l y i n an a s s a y f o r i n h i b i t i o n o f v i r a l e x p r e s s i o n o f HIV i n c h r o n i c a l l y i n f e c t e d T - c e l l s (Matsukura, M., N a t i o n a l I n s t i t u t e s o f H e a l t h , p r i v a t e cummunication, 1988). The a c t i v e p h o s p h o r o t h i o a t e sequence i s t h e e x t e n d e d (28-mer) v e r s i o n o f t h e a f o r e m e n t i o n e d a n t i - a r t / t r s (rev) oligomer. The r e s u l t s o f N o r t h e r n b l o t a n a l y s i s s u g g e s t t h a t t h e r e a r e marked e f f e c t s on RNA; however, t h e d e t a i l s have y e t t o be e s t a b l i s h e d . In any e v e n t , a t t h i s time the phosphorothioate analogues are e s p e c i a l l y r

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

12.

ZON

Chemotherapeutic Oligonucleotide Analogues

i n t e r e s t i n g as p o t e n t i a l a n t i - r e t r o v i r a l a g e n t s i n t h a t t h e y t h a t may h a v e t w o , i n d e p e n d e n t m e c h a n i s m s o f a c t i o n which operate at d i f f e r e n t points i n the v i r u s lifecycle.

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RECEIVED January 4, 1989

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Author Index Bapat, Ashok, 1 Bronson, Joanne J., 72,88 Busso, Mariano, 156 Cheng, Yung-Chi, 1 Datema, Roelf, 116 De Clercq, Erik, 51 Duncan, I. B., 103 Ghazzouli, Ismail, 72,88 Hahn, Elliot F., 156 Harutunian, Vahak, 1 Hitchcock, Michael J. M., 72,88 Holy, Antonin, 51 Kern, Earl R., 72,88 Khawli, Leslie Α., 1 Kim, Choung Un, 72 Levy, Jeffrey Ν., 1 Marquez, Victor E., 140 Martin, J. Α., 103

Martin, John C, 72,88 McKenna, Charles Ε., 1 Mian, Abdul M., 156 Olofsson, Sigvard, 116 Pong, R. Y., 17 Reist, E. J., 17 Resnick, Lionel, 156 Sidwell, R. W., 17 Smee, Donald F., 124 Starnes, Milbrey C, 1 Sturm, P. Α., 17 Tanga, M. J., 17 Thomas, G. J., 103 Tolman Votruba Webb, Robert R., II, 88 Ye, Ting-Gao, 1 Zon, G., 170

Affiliation Index Applied Biosystems, Inc., 171 Bristol-Myers Company, 72,88,116 Czechoslovak Academy of Sciences, 51 Gôteborgs Universitet, 116 IVAX Corporation, 156 Katholieke Universiteit Leuven, 51 Merck Sharp & Dohme Research Laboratories 35 Mount Sinai Medical Center, 156

National Cancer Institute, NIH, 140 Nucleic Acid Research Institute, 124 Products Limited, 103 International, 17 University of Alabama, 72,88 University of Miami Medical School, 156 University of North Carolina-Chapel Hill, 1 University of Southern California, 1 University, 17

R

S

U

o

R

t

c

h

e

I

a

h

S t a t e

Subject Index A

Acyclonucleoside phosphonates antiviral activity against herpes viruses, 30,32* syntheses, 23-28 toxicity, 30,33 Acyclovir activation by viral thymidine kinase, 105 antiviral activity, 19 selectivity for herpes simplex virus, 19,21 structure, 18f,22/ Acyclovir phosphate, structure, 21—22 Adenosine 5'-sulfamate, antiviral activity, 125

Acyclic nucleoside analogues antiviral activity, 53-54 structures, 54 Acyclic nucleotide analogues, structure—activity investigation, 55-59 Acyclo sugar structure, relationship with substrate activity, 42,44/,45 Acyclonucleoside(s) relative phosphorylation rates, 45,47/ viral activation, 35-36,37/

185

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Author Index Bapat, Ashok, 1 Bronson, Joanne J., 72,88 Busso, Mariano, 156 Cheng, Yung-Chi, 1 Datema, Roelf, 116 De Clercq, Erik, 51 Duncan, I. B., 103 Ghazzouli, Ismail, 72,88 Hahn, Elliot F., 156 Harutunian, Vahak, 1 Hitchcock, Michael J. M., 72,88 Holy, Antonin, 51 Kern, Earl R., 72,88 Khawli, Leslie Α., 1 Kim, Choung Un, 72 Levy, Jeffrey Ν., 1 Marquez, Victor E., 140 Martin, J. Α., 103

Martin, John C, 72,88 McKenna, Charles Ε., 1 Mian, Abdul M., 156 Olofsson, Sigvard, 116 Pong, R. Y., 17 Reist, E. J., 17 Resnick, Lionel, 156 Sidwell, R. W., 17 Smee, Donald F., 124 Starnes, Milbrey C, 1 Sturm, P. Α., 17 Tanga, M. J., 17 Thomas, G. J., 103 Tolman Votruba Webb, Robert R., II, 88 Ye, Ting-Gao, 1 Zon, G., 170

Affiliation Index Applied Biosystems, Inc., 171 Bristol-Myers Company, 72,88,116 Czechoslovak Academy of Sciences, 51 Gôteborgs Universitet, 116 IVAX Corporation, 156 Katholieke Universiteit Leuven, 51 Merck Sharp & Dohme Research Laboratories 35 Mount Sinai Medical Center, 156

National Cancer Institute, NIH, 140 Nucleic Acid Research Institute, 124 Products Limited, 103 International, 17 University of Alabama, 72,88 University of Miami Medical School, 156 University of North Carolina-Chapel Hill, 1 University of Southern California, 1 University, 17

R

S

U

o

R

t

c

h

e

I

a

h

S t a t e

Subject Index A

Acyclonucleoside phosphonates antiviral activity against herpes viruses, 30,32* syntheses, 23-28 toxicity, 30,33 Acyclovir activation by viral thymidine kinase, 105 antiviral activity, 19 selectivity for herpes simplex virus, 19,21 structure, 18f,22/ Acyclovir phosphate, structure, 21—22 Adenosine 5'-sulfamate, antiviral activity, 125

Acyclic nucleoside analogues antiviral activity, 53-54 structures, 54 Acyclic nucleotide analogues, structure—activity investigation, 55-59 Acyclo sugar structure, relationship with substrate activity, 42,44/,45 Acyclonucleoside(s) relative phosphorylation rates, 45,47/ viral activation, 35-36,37/

185

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

NUCLEOTIDE ANALOGUES

186 Amantadine antiviral activity, 19 structure, 18f A n t i - R N A oligonucleotide analogues bioavailability and uptake by cells, 173-174 design factors, 171-172 inhibition of function, 174-175 specific binding to target, 174-175 structural modifications, 172-173 Antiviral activity, phosphonylmethyl ethers of nucleosides, 59-64 Antiviral activity of phosphonylmethyl ether derivatives, 9-[2-(phosphonylmethoxy)ethyl]adenine, 79,80-81/ Antiviral agents chemical structures, 141,142/" FDA-licensed compounds, 17,18/"

Cyclic 2'-norguanine monophosphate antiviral activity against herpes virus, 47-48 structure, 48 Cyclopentenylcytosine antiviral activity, 141,143/ chemistry, 141-142 mechanism of action, 143 Cytomegalovirus, inhibition by (phosphonylmethoxy)ethyl derivatives of purine and pyrimidine bases, 73

D 3-Deazaneplanocin A antiviral activity, 144,145/ chemistry, 142,144,147

important properties, 2 nucleoside analogues, 3 nucleotide analogues, 3 - 4 oligonucleotide analogues, 4 pyrophosphate analogues, 2—14 Ara A antiviral activity, 19 structure, 17,l$f 3'-Azido-2'3'-dideoxytnymidine in vivo disposition, 167/,168 stability in plasma, 162,164-165/ treatment of human immunodeficiency virus infection, 157-158 viral inhibition mechanism, 3 Azidothymidine antiviral activity, 19 structure, 17,18f

Β

£-D-ribofuranosyl)adenine antiviral activity, 151 chemistry, 150,151/,152,153/ 2'-Deoxy-5-ethyluridine derivatives, inhibition of thymidine kinases, 109-110r,lll,112/ 2'-Deoxy-5-iodouridine derivatives, inhibition of thymidine kinase, 106,107/ 2 '3 '-Dideoxy-2 ' -fluoro-flra -adenosine antiviral activity, 148 chemistry, 146,147/,148-149 mechanism of action, 150 2'3'-Dideoxy-2'-fluoro-flrfl-inosine antiviral activity, 148,151/ chemistry, 146,147/,148-149 mechanism of action, 150 D N A polymerases assays for herpes virus and human reaction mixtures, 8 - 9 preparation, 8

Base-modified nucleotide analogues, antiviral activity, 140-141 Biochemical studies, phosphonylmethyl ethers of nucleosides, 64-68 Biological role of terminal glycosylation of viral glycoproteins processing of N-linked oligo­ saccharides, 117,118f role in Sindbis v i r u s - B H K cell system, 117,119,120r (£)-5-(2-Bromovinyl)-2'-deoxyuridine, inhibition of viral glycoproteins, 121

C Chemotherapy of viral infections, problems, 17 Combined prodrugs, description, 14

G Ganciclovir antiviral activity, 21 energy contour plot, 36,3
In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

187

INDEX H α-Halo phosphonates antiviral activities, 10,11/ inhibitor concentrations for DNA polymerases, 9,10r percent inhibition of DNA polymerases, 9,10ί synthesis, 6-7 α-Halogenated pyrophosphate analogues, antiviral activity, 5 Herpes simplex virus(es) disease symptoms, 103 role of thymidine kinase in infections, 103-104 system treatment by (5)-9-[3-hydroxy2-(phosphonylmethoxy)propyl] derivatives, 96-98 Herpes simplex virus infections, therapeuti regimens, 121-122 Herpes thymidine kinase acyclo sugar structure-substrate activity, 42,44/",45 molecular models, 36-43 substrate requirements, 36 Herpes virus antiviral activity 9- [2-(phosphonylmethoxy)ethyl]adenine, 79,80/ (phosphonylmethoxy)ethyl derivatives, 81,82-83/ 9-[2-(phosphonylmethoxy)ethyl]guanine, 84-85/ Herpes virus DNA polymerase inhibition studies antiviral activities, 10,11/ α-halo phosphonates, 9,10/ inhibitor concentrations, 9,10/ HIV-1 reverse transcriptase inhibition studies effect of template, 11,12/ α-οχο phosphonates, 11,12-13/ HSV—1 DNA polymerase, inhibition by nucleoside triphosphate analogues, 65,66/ Human immunodeficiency virus development of therapeutic strategies, 156 inhibition of reverse transcriptase, 157—168 stages in replicative cycle, 156-157 N -(Hydroxyalkyl)guanines, herpes thymidine kinase substrates, 42/ 9-[(2-Hydroxyethoxy)methyl]guanine antiviral activity, 3 viral inhibition mechanism, 3 AT-[3-Hydroxy-2-(phosphonylmethoxy)propyl] derivatives of heterocyclic bases abbreviations, 60/ activity against retroviruses, 63/,64 antiviral activity, 57,60,61-62/,63 biochemical studies, 64-68 synthesis, 57-59

(S)-9-[3-Hydroxy-2-(phosphonylmethoxy)propyljadenine antiviral activity, 59-60,88 biochemical studies, 64—68 broad-spectrum antiviral activity, 72 effect of structure on antiviral activity, 73 in vitro antiviral activity, 94,95/ inhibition of cytomegalovirus, 73 of HSV-1 DNA polymerase, 65,66/ of reverse transcriptase, 67,68/ ofribonucleotidereductase, 66,67/ mechanism of antiviral activity, 89 pharmacokinetics in mice, 96/ structure, 73,89 structure-antiviral activity relationship, 56,57/

infections in mice, 96,97/ toxicity to mice, 95/,96 (5)-9-[3-Hydroxy-2-(phosphonylmethoxy)propyljcytidine in vitro antiviral activity, 94,95/ structure, 89 synthesis, 90,93-94 systemic efficacy against murine cytomegalovirus in mice, 99,100/ systemic treatment of herpes simplex viral infections in mice, 96-97,98/ topical efficacy against herpes simplex virus, 99/ toxicity to mice, 95/,96 (S)-9-[3-Hydroxy-2-(phosphonylmethoxy)propyljguanine in vitro antiviral activity, 94,95/ structure, 89 synthesis, 90-93 systemic treatment of herpes simplex virus infection, 96,97/ toxicity to mice, 95/,96 (S)-9-[3-Hydroxy-2-(phosphonylmethoxy)propyljthymidine, structure, 89

2

Inhibition of terminal N~ and O-glycosylation specific for herpes virus infected cells by (£)-5-(2-bromovinyl)2'-deoxyuridine, 121 development of inhibitors, 119 of sugar nucleotide translocation, 119,12Qf Inhibition studies herpes virus DNA polymerases, 9,10-11/ HIV-1 reverse transcriptase, 11,12-13/ Inhibitors of viral nucleic acid polymerases, design, 2

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

NUCLEOTIDE ANALOGUES

188

Isopolarity, requirement of analogues for antiviral activity, 52 Isosteric phosphonate, structure, 21-22 IUDR antiviral activity, 19 structure, 17,18/

Nucleotide dimers—Continued structure, 158,161/ treatment of human immunodeficiency virus infection, 158 Nucleotide halo phosphonate analogues inhibition of HIV-1 reverse transcriptase, 13-14 structures, 13-14

Κ α-Keto phosphonates ketone-hydrate equilibria, 8 synthesis, 7-8

M

O-linked oligosaccharides, synthesis, 116-117 Oligonucleotide analogues anti human immunodeficiency virus activity, 179-181 antiviral activity, 4

Molecular modeling for thymidine structure-substrate study binding scenarios, 40,41/,42/,43/ energy contour plots, 36,38-39/ structure-substrate relationship for modified guanines, 36,40/ Murine cytomegalovirus, systemic efficacy of (S)-9-[3-hydroxy-2-(phosphonylmethoxy)propyljcytidine, 99,100/

Ν N-linked oligosaccharides, processing, 116-117,118/* Nucleocidin, activity, 125 Nucleoside antiviral agents, examples, 3 Nucleoside derivatives, dissociation constants, 109/.110 Nucleotide antiviral activity, 88 herpes virus specific inhibitors of protein glycosylation, 116-122 Nucleotide analogues categories, 140—141 formation, 88 Nucleotide antiviral analogues, viral inhibition mechanism, 3—4 Nucleotide dimers comparative inhibitory effects of compounds, 162,164/ coupling scheme for synthesis, 158-159,161/ cytotherapeutic evaluation, 162-168 inhibition of human immunodeficiency antigen expression and cytopathic effect, 160,162,163/ inhibition of reverse transcriptase activity, 160,163/ inhibition of syncytium formation, 159-160,161/

inhibition of aberrant gene expression, 171 phosphoramidite synthetic method, 175,176/ purification, 175,178 stereochemistry, 178 strength of binding, 178,179/ synthesis, 175,176-177/ α-Οχο phosphonates inhibition of HIV-1 reverse transcriptase, 11,12/,13 pH dependence of HIV-1 reverse transcriptase inhibition, 13/ α-Οχο pyrophosphate analogues, antiviral activity, 5

Ρ 2

N -Phenylguanine derivatives, inhibition of thymidine kinase, 105,106/ Phosphate-modified nucleotide analogues, antiviral activity, 140-141 5'-Phosphonate derivatives of nucleotides, inhibition of viral infections, 124-125 Phosphonoacetic acid antiviral activity, 4 effect of substituents on antiviral activity, 5—6 structure, 4—5 Phosphonoformic acid antiviral activity, 2—3 structure, 4-5 9-(Phosphonylalkyl)adenines, side chains, 56-57,58/ 9-[(Phosphonylmethoxy)alkyl]adenines, side chains, 56,57/ (Phosphonylmethoxy)ethyl derivatives, antiviral activity against herpes viruses, 81,82-83/

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

189

INDEX

N-[2-(Phosphonylmethoxy)ethyl] derivatives of heterocyclic bases abbreviations, 60/ activity against retroviruses, 63/,64 antiviral activity, 61-62/,63 biochemical studies, 64-68 inhibition of HSV-1 DNA polymerase, 66/ of ribonucleotide reductase, 66,67/ synthesis, 59 (Phosphonylmethoxy)ethyl purine and pyrimidine derivatives, synthesis, 74-77 9-[2-(Phosphonylmethoxy)ethyl]adenine antiviral activity, 72,79,80-81/ structure, 73 synthesis, 74-75 9-[2-(Phosphonylmethoxy)ethyl]guanine analogues antiviral activity against herpes viruses, 84—85/ synthesis, 77-79 9-[3-(Phosphonylmethoxy)propyl]guanine, synthesis, 78 Phosphonylmethyl ether derivatives antiviral activity, 79-85 synthesis, 74-79 Phosphonylmethyl ethers of acyclic nucleotides antiviral activity, 53-54 structures, 54 Phosphonylmethyl ethers of 9-(2,3-dihydroxypropyl)adenine structure-antiviral activity relationship, 56 synthesis of isomers, 55 Phosphonylmethyl ethers of nucleosides antiviral activity, 59-64 biochemical studies, 64-68 effect of ether linkage on antiviral activity, 52-54 structures, 52 5'-0-Phosphonylmethyl nucleosides antiviral activity, 53 synthesis, 52-53 Phosphorothioate analogues, anti human immunodeficiency virus activity, 179-181 Pyrophosphate analogues antiviral activity, 4 structure, 2,4—5 synthesis, 6—8

Reverse transcriptase function in human immunodeficiency virus, 157 inhibition by acyclic nucleoside triphosphate analogues, 67,68/ inhibitors, 157 production inhibition by nucleotide dimers, 160,163/ Ribavirin antiviral activity, 19 structure, 17,18/ Ribavirin 5'-sulfamate antiviral activity against various viruses, 125,126/ antiviral activity under varying multiplicities of viral infection, 126,127/ effects

on incorporation of uridine and amino acids into virus-infected cells, 128,129/ on lethal viral infection in mice, 127,128/ on viral and cellular polypeptide synthesis, 129,131/ future research, 137 inhibition of DNA synthesis, 132,133/ of methylation, 133 of polyamine synthesis, 134,135/ of protein translation, 130,131/132-133/ of sulfuration, 134-135/,136 mode of antiviral action, 127-131 structure, 125 uptake into cells, 136/ Ribonucleotide reductase, inhibition by acyclic nucleoside triphosphate analogues, 66,67/

Structure-activity relationship, acyclic nucleotide analogues, 55-59 Sugar-modified nucleotide analogues, antiviral activity, 140-141 Sugar nucleotide translocation, schematic diagram, 119,120f Syncytium formation, inhibition by nucleotide dimers, 159-160,161/ Syncytium inhibition assay, description, 159

R

Τ

Rauscher MuLV, antiviral efficacy of 9-[2(phosphonylmethoxy)ethyl]adenine, 79,80/ Retroviruses, antiviral efficacy of 9-[2-(phosphonylmethoxy)ethyl]adenine, 79,80/

Terminal glycosylation description, 116-117 of viral glycoproteins, biological role, 117,118f,l 19,120/

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

NUCLEOTIDE ANALOGUES

190 Thymidine energy contour plot, 36,38/" structure, 35 Thymidine derivatives, inhibition of thymidine kinase, 107,108*,109 Thymidine kinase function, 19-20,35 herpes, See also Herpes thymidine kinase inhibition by acyclovir, 105 by 2'-deoxy-5-ethyluridine derivatives, 109-1 10*,111,112* by 2'-deoxy-5-iodouridine derivatives, 106,107* by N -phenylguanine derivatives, 105,106* by product analogues, 106-112 by substrate analogues, 104-105,106* by thymidine derivatives, 107,108*,10 by trifluorothymidine, 106 mechanism of action, 104 2

Thymidine kinase—Continued role in herpes simplex viral infections, 103-104 substrate specificity, 19 Trifluorothymidine antiviral activity, 19 inhibition of thymidine kinase, 106 structure, 17,18/"

V Viral diseases, FDA-licensed compounds, 17,18/ Viral glycoproteins, biological role of terminal glycosylation, 117,118/*,119,120*

In Nucleotide Analogues as Antiviral Agents; Martin, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.