Nucleotide Analogues as Antiviral Agents - American Chemical Society

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 Pharmacology and Medicine, School of Medicine, University of North Carolina-Chapel Hill, Chapel Hill, NC 27514

Downloaded by 80.82.78.170 on January 17, 2017 | http://pubs.acs.org Publication Date: August 22, 1989 | doi: 10.1021/bk-1989-0401.ch001

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

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5 6

Martin; Nucleotide Analogues as Antiviral Agents 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, are also p o t e n t i a l drug targets. V i r u s - s p e c i f i c DNA polymerases c a t a l y z e DNA- or RNA-specified 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-



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 ACV mono- and triphosphate, r e s p e c t i v e l y , HSVDP represents a Herpes simplex DNA polymerase, and DNA-ACV-MP i s 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



4


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 n or t r a n s l a t i o n . These sequences p e c i f i c n u c l e i c a c i d segments are t y p i c a l l y intended to h y b r i d i z e 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.


1.

McKENNAETAL.

0 HO.


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.

ÔH

HO

HO

0

0

I II

II

=> — C —

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


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 . Fluorine Chem., i n press) (Scheme 4 ) . The d i c h l o r o and dibromo esters (6e, 6g) were made by hypohalogenation (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

a n d

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.



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

7


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



8

0 N

Eto. H

0

N

TMSOÎI

H

0

2

il ll

^;P—c—c—OEt —

H

^P—C—C—OEt —

0

0

0 0 0

TMSOÎI

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 a of 4 and 5. U n l i k e the t r i e s t e r 7, 5 has an e q u i l i b r i u m with i t s hydrate which i s pH-dependent (21; McKenna, 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


1.


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. Compounds 2b-2j and 21-2n were evaluated as 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


10


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 >100 >100 >100 >100

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

a

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

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

>300 >300 >300 >300 >300 >300 >300 >300 >300 >300 >300 >300 >300 >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).


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

11


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

Influenza A/Japan

HSV-1 (F) 0.6/190 μΜ 0.6/52 μΜ

b

a

c

HSV-2 (G) 0.5/190 μΜ 0.8/165 μΜ

HSV-2 (Lovelace) 0.8/190 μΜ 0.8/165 μΜ

PAA, 2a PFA, 1 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.



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

IC

(μΜ)

5 0

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

ICô (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.



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

13


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 Table VI r e v e a l a s t r i k i n g dependence o f IC50 on pH, with a l l three i n h i b i t o r s e x h i b i t i n g lower values at higher pH. 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



14

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


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


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.

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RECEIVED June 15, 1989


Nucleotide Analogues as Antiviral Agents - American Chemical Society

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