Nucleotide Analogues as Antiviral Agents - American Chemical Society

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

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1

1

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

Downloaded by UNIV LAVAL on July 13, 2016 | http://pubs.acs.org Publication Date: August 22, 1989 | doi: 10.1021/bk-1989-0401.ch002

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Isosteric phosphonate analogs of acyclovir 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

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

NUCLEOTCDE ANALOGUES


18

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.


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 against i n f l u e n z a A.(JJ,22) In a d d i t i o n to i t s a n t i v i r a l a c t i v i t y , amantadine has shown an e f f e c t i n 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


20


N U C L E C m D E ANALOGUES

Scheme 1


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. Since CMV has no v i r a l thymidine kinase the mechanism by which i t i n h i b i t s the v i r u s i s not 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


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 Ganciclovir


Acyclovir

2

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


2. REIST E T AL.


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 approaches have been used (Scheme 4) t o couple the chloromethyl sugar to a guanine analog. The most 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.


24


(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


2

2

2. R E I S T E T AL.


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 t prodrug f o r a c y c l o v i r and has s i g n i 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 y than acyclovir.(23) I t i s 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


26


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 15

(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


2

2



2. REIST E T AL.


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

±5

LA

4_fi

Ο

Ο

Il

II

(BuO) PCH CH CH OCH CI 2

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


2

2


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

Ο

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


30



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 showed adequate a c t i v i t y , i t would be an i n t e r e s t i n g monofunctional analog of g a n c i c l o v i r that cannot 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



2.

REIST E T AL.

(EtO) PCH CH CH — C H 2

2

12

2

31


2

-

(EtO) PCH CH CHCH 2

2

2

3

(EtO) PCH CH CHCH 2

12

12

12 Scheme

2

10


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

(TO

I

«-

ED ue/ml T . I . 50

Cpd

X

11

Η

R

R"

R* H

H

Η

η

V.R. »

2

0.6

100

MCMV

HCMV

HSV-1

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

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


5

5

2.

REIST ET AL.

33


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.


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. Viral Diseases: A Review of Chemotherapy Systems; In "Chemotherapy of Infectious Diseases" (H. 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


Nucleotide Analogues as Antiviral Agents - American Chemical Society

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