Design of Inhibitors of Herpes Simplex Virus Thymidine Kinase - ACS

Chapter 7

Design of Inhibitors of Herpes Simplex Virus Thymidine Kinase

Downloaded by TUFTS UNIV on June 4, 2018 | https://pubs.acs.org Publication Date: August 22, 1989 | doi: 10.1021/bk-1989-0401.ch007

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 herpes simplex virus thymidine kinase in 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

Martin; Nucleotide Analogues as Antiviral Agents 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 the bacterial and mammalian enzymes but s u r p r i s i n g l y t h e s e s t u d i e s (25.) d i d not i n c l u d e t h e v i r a l enzyme.

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


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


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

HSV-1

Rl

R2

R3

3

Η

Η

Η

8

4 5 6

Me n-Bu

Η

Η

50

Η

Η

50

Et

Η

3

7

Br

Η

Η

1

8 9

Η

Cl

Η

1.3

Η

Η

Compound


[μΜ]

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


7. MARTIN E T AJL

107


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

of

1

HO

IC50

Compound

HSV-1

10 11 12 13 14 15 16

[μΜ]

R 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


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 .

t h e amide

derivatives

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


with

1

HO

IC50

Compound

HSV-1

18 19 20 21 22 23 24

[μΜ]

R 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


7.

109


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 l p h o s p h o r y l a t i o n by v i r a l TK f o r e x p r e s s i o n of a c t i v i t y . The e v a l u a t i o n o f 26 i n v i v o has not been r e p o r t e d 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


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.


7. MARTIN ET A K

111


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 o d e c r e a s e d a c t i v i t y . When t h e s e s t u d i e s 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 d d e r i v a t i v e s i t was f o u n d t h a t 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.


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 >200

>200 >200 >200

>60,000 >83,000 >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


7. MARTIN E T A K


113

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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A

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RECEIVED January 4, 1989


Design of Inhibitors of Herpes Simplex Virus Thymidine Kinase - ACS

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