8-Aza-1-deazapurine Nucleosides as Antiviral Agents

'ANKAR

N U C L E O S I D E S & N U C L E O T I D E S , 13(8), 1739-1755 (1994)

8-AZA-l-DEAZAPURINE

NUCLEOSIDES AS A N T I V I R A L AGENTS

P. F r a n c h e t t i , * L . Messini, * L . Cappellacci, " G . A b u Sheikha ", M . Grifantini, * P. Guarracino, A . De M o n t i s , A . G . Loi,° M . E . M a r o n g i u , and P . L a C o l l a +

4

0

Dipartimento °Dipartimento Italy.

di Scienze di Biologia

+

4

4

0

4

0

0

Chimiche, Università di Camerino, 62032 Camerino, Sperimentale, Università di Cagliari, 09124 Cagliari,

Abstract2',3'-Dideoxy-8-aza-l-deazaadenosine ( 2 1 ) and its oc-anomer ( 2 0 ) were synthesized v i a g l y c o s y l a t i o n of 7 - c h l o r o - 3 # - 1 , 2 , 3 - t r i a z o l o [ 4 , 5 - & ] p y r i d i ne with 2,3-dideoxy-5-0-[(l ,l)-dimethylethyl)diphenylsilyl]-D-^/ycer6>-pentofuranosyl c h l o r i d e . The reaction gave a mixture o f a - and P-anomers o f N - , N ^ - and - g l y c o s y l a t e d regioisomers ( 1 2 - 1 5 ) . The a - and p-anomers o f the N^-glycosylated i s o m e r 2 6 and 2 7 were also synthesized through the g l y cosylation o f 8-aza-l-deazaadenine with l - a c e t o x y - 2 , 3 - d i d e o x y - 5 - 0 - [ ( l , l - d i methyiethyl)dimethylsilyl]-D-,g/ycer6>-pentofuranose. These dideoxynucleosides and a series o f previously synthesized 8-aza-l-deazapurine nucleosides were tested for activity against several D N A and R N A v i n i s e s , H I V - 1 inchided. The a - and p - a n o m e r s of 7-chloro-3-(2-deoxy-D-eryf/zr

the glycosylation site should be at N . 4

The structure chemical

was confirmed by the

shift

of

C ( 5 ) was

upfield

1 3

shifted

signal o f the N - g l y c o s y l a t e d isomers desired

spectra

as

which

compared

to

showed the

that

the

corresponding

20 and 2 1 . So it was impossible to obtain

3

the

C-NMR

2 \ 3 ' - d i d e o x y - 8 - a z a - 1-deazaadenosine

by

this

route,

BIOLOGICAL EVALUATION Cytotoxicity the

and

antiviral

8-aza-l-deazapurine

of

the

parent compounds

ferente

drugs

8-aza-

acyclovir

8-Azaadenosine

activity.

nucleosides

and

are

and

Cytotoxicity shown

and

i n Table

antiviral

1-deazaadenine nucleosides

(8-aza-A)

and

cytotoxic to

Vero

1-deazaadenosine

med

as an inhibitor o f v a c c i n i a ( V V ) and vesicular stomatitis

inhibitor nor

o f both

1-deaza-dA

osides,

some

1-deaza-A

V V and showed

resulted

african

any

cells at

(1-deaza-A)

compounds

and

of

those

and o f the

re-

(1) were

the

guanidine.

sole

tiplication,! ?

activity

2, together w i t h

a

swine

l o w concentrations.

potent, fever

although

virus

antiviral activity. A m o n g

7-chloro-derivatives

( 3 a , 4 and 6)

8-Aza-A

virus ( V S V ) m u l not

(ASFV). the

resulted

confir-

very

selective

Neither

8-aza-dA

8-aza- 1-deaza active

nucle-

against

polio

(Sb-1) and coxsackie B l (Coxs). The most potent was the a anomer of the

2'-de-

oxy

n u c l e o s i d e 3a, w h i c h showed selectivity indices o f 41 and

and

Coxs,

respectively.

The

other

8-aza-l-deazapurine

13 against

Sb-1

nucleosides

were

inactive. The results shown

i n Table

rence In dA

of cytotoxicity and anti-HIV-1 activity o f the test compounds 3; i n this case 2',3'-dideoxyadenosine

(ddA) was

used

as

are refe-

compound. M T - 4 cells the most cytotoxic compounds were 8-aza-A, followed by

and

1-deaza-A,

aza-1-deaza

which

showed I C 5 0 s

m

8-aza-

the range 0.3-6.4 J I M . A m o n g the

8-

derivatives, 3 a was the most toxic compound (IC50 = 23 u.M). W h e n

T a b l e 2. Cytotoxicity

and antiviral

a

Compd

ic

o f 8-aza- 1-deaza-purine nucleosides

50

Vero

8-aza-A

activity

5.5

b

HSV-1

>5.5

VV

1.0

>5.5

5

cells.

0

Sb-1

>5.5

Coxs

>5.5

VSV

1.2

>396

>396

>396

>396

3

>6

>6

>6

>800

>800

>800

>800

>800

>740

>740

>740

>740

>740

>740

>250

>250

>250

>250

>250

>250

>250

>370

>370

>370

>370

9

27

>370

4

>740

>740

>740

>740

140

50

>740

5

>250

>250

>250

>250

>250

>250

>250

6

>350

>350

>350

>350

140

50

>350

7

>370

>370

>370

>370

>370

>370

>370

8

>790

>790

>790

>790

>790

>790

>790

9

>700

>700

>700

>700

>700

>700

>700

acyclovir

>100

>10

>10

>10

>10

>10

guanidine

>500

>500

>500

125

150

8-aza-dA

>396

>396

6

>6

1-deaza-dA

>800

>800

2

>740

3 3a

1-deaza-A

(1)

0.04 >500

>396

ASFV

E C

in Vero

1.5

>500

Compound dose required to reduce by 50% the number o f viable mock-infected cells after three celi cycles. C o m p o u n d dose required to reduce the number of v i r a i plaques by 5 0 % . Plaque numbers i n untreated cultures were: 125 ( H S V - 1 ) , 110 ( V V ) , 105 ( A S F V ) , 120 (Sb-1), 115 (Coxs), 130 ( V S V ) . a

b

FRANCHETTI ET A L .

1746

T a b l e 3. C y t o t o x i c i t y des i n M T - 4 cells.

anti-HIV-1

and

a

ic

5

effect

8-aza- 1-deaza-purine

b

0

MT4

Compd

of

EC

5

0.3

>0.3

8-aza-dA

1.2

>1.2

6.4

>6.4

(1)

1-deaza-dA

>250

>250

2

64

>64

3

>250

>250

3a

23

>23

4

60

>60

5

174

>174

6

44

>44

7

67

>67

8

>250

>250

9

>250

>250

21

>425

>425

>425

>425

>500

10

26

*

ddA

0

fflV-1

8-aza-A

1-deaza-A

nucleosi

C o m p o u n d dose required to reduce the viability o f mock-infected cells by 50%. b C o m p o u n d dose required to achieve 50% protection o f M T - 4 cells against the HIV-1-induced cytopathogenicity. a

evaluated test

for

inhibition

compounds,

of

the

HIV-1-induced

2',3'-dideoxy

derivatives

cytopathogenicity,

included,

resulted

none

of

effective.

the

Under

the same conditions, d d A showed an E C 5 0 of 10 j a M . Overall,

these

group

at

group

with

toxic group

suggest

that

position 1 i n 8-azaadenosine

and with

determines

results

nitrogen

at

position

8 in

antiviral activities. O n the a chlorine the

atom

appearance

of

at

the

substitution

(or vice versa

of the

1-deazaadenosine) other

hand,

the

nitrogen

with

substitution ablates both

substitution

selective

antiviral

activity

of a C H the

cyto-

o f the

NH2

position 6 i n 8-aza-1-deazaadenine a

a CH

against

nucleosides polio

and

8-AZA-1 - D E A Z A P U R I N E N U C L E O S I D E S

coxsackie viruses. F i n a l l y , position

1,

and

the

C H with

1747

substitution

nitrogen

at

in d d A o f both nitrogen

position

8,

is

with

detrimental

for

C H at

anti-HIV

activity.

Adenosine cleosides

deaminase

20 and 21

inhibitory

activity.

tory with K i values o f 1.1 x I O " M and 5.6 x I O 5

- 5

the a-anomer was 5 times more potent than the that 2'-deoxy-8-aza- 1-deaza-A it can

be

role i n the

M respectively. p-anomer.

inhibi-

Interestingly,

Since

we

reported 7

substitution

of the

2 ' - d e o x y - p - D - r i b o s y l moiety

i n h i b i t o r y potency,

important

2',3'-dideoxy-nu-

is a potent inhibitor of A D A ( K i = 1.9 x I O " M ) , 8

concluded that the

at 3'-position i n the the

When

were tested for inhibition of A D A , both were found

pointing interaction

out

that the

hydroxy group decreases

of

h y d r o x y l at

with

about

hydrogen 290

3'-position

with the catalytic site o f the

times

plays

an

enzyme.

E X P E R I M E N T A L SECTION M e l t i n g points were determined on a B u c h i apparatus and are uncorrected. E l e m e n t a l analyses were determined on a C a r l o E r b a M o d e l 1106 analyzer. U l t r a v i o l e t spectra were recorded with an H P 8452 A diode array spectrophotometer driven by an Olivetti M 24. T h i n layer chromatography ( T L C ) was run on s i l i c a gel 60 F-254 plates (Merck); silica gel 60 (Merck) for c o l u m n chromatography was used. N u c l e a r magnetic resonance * H and c spectra were determined, respectively, at 300 ^and 75 M H z with a Varian V X R - 3 0 0 spectrometer. The chemical shift values are expressed in 8 values (parts per m i l l i o n ) relative to tetramethylsilane as an internai standard. A l i exchangeable protons were confirmed by addition of D 0 . Stationary N O E experiments were measured i n ( D ^ D M S O and were run on degassed solutions at 25°C. A presaturation delay o f 1 sec was used, during w h i c h the decoupler low-power was set at 20 d B attenuation. 13

2

Glycosylation with

of

7-chloro-3# -1,2,3-triazolo[4,5-b]pyridine

(10)

2,3-dideoxy-5-0-[(l,l-dimethyIethyl)diphenylsilyl]-D-g/yc*-

ra-pentofuranosyl

chloride

(11).

To a stirred

solution o f I O

9

(2 g,

12.8

mmol) i n dry M e C N (120 m L ) were added powdered K O H (1.9 g, 35.15 mmol) and T D A - 1 (84 jxl, 0.25 mmol). After 10 min the solution of 1 1

(25.6

1 0

mmol

corre-

sponding to lactol) i n T H F was added portionwise (within 20 min). The

mixture

reaction

the

trate

was

stirred

poured

extracted with

cold

silica

gel

into

with

5%

EtOAc

H 0, 2

at r.t.

dried

aq.

for 5 h, the NaHC03

insoluble material

soln. (100

(3x100 m L ) and the and

evaporated.

filtered and

m L ) . The

aqueous

combined organic

F r o m the

c o l u m n ( c y c l o h e x a n e - E t O A c 80:20)

residue

three fractions

fil-

layer

layer was

was

washed

chromatographed were

on

separated.

7-Chloro-3-[2,3-dideoxy-5-0-[(dimethylethyl)diphenylsilyl]-a-D £/vcer0-pentofuranosyl]-3# -l,2,3-triazolo[4,5-&]pyridine

(12).

-

The

1748

FRANCHETTI ET A L .

material

isolateci

from

with cyclohexane-EtOAc (cyclohexane-EtOAc

third

fraction

was

rechromatographed

by

flash

column

(96:4) as eluent to give 12 as o i l (660 mg, 11.5 %). T L C

96:4): R f 0.26. * H N M R ( M e S O - d ) : 5 1.0 (s, t-Bu); 2.05, 2.52 2

(2m, 2 H , H-3*); 2.63, 2.78

6

(2m, 2 H , H-2'); 3.71 (m, 2 H , H-5'); 4.48 (m, I H , H-4'); 6.85

(dd, J = 2.9, 6.9 H z , I H , H - l ' ) ; 7.40, 7.65 (2m, arom. H); 7.72 (d, J = 5.0 H z , I H , H-6); 8.70 (d, J = 5.0 H z , I H , H-5). Anal. Calcd. for C 2 6 H 2 9 C I N 4 O 2 S Ì : C 63.33; H 5.93; N 11,36. Found; C 63.21; H 6.18; N 11.03. 7-Chloro-3-[2,3-dideoxy-5-0-[(dimethylethyl)diphenylsilyi]-p-D g/yc*r0-pentofuranosyl]-3/f -l,2,3-triazolo[4,5-6]pyridine chromatography

o f fraction

II by flash

column

(13).

Re-

with C 5 H 6 gave 13, as o i l (710

mg, 12 %). T L C ( C H ) : R f 0.13. H N M R ( M e S O - d ) : 5 1.02 (s, t-Bu); 2.08 (m, 2 H , 6

6

l

2

6

H-3'); 2.45, 2.60 (2m, 2 H , H-2'); 3.75 (m, 2 H , H-5*); 4.60 (m, I H , H-4'); 6.72 (dd, J = 2.5, 5.7 H z , I H , H - l ' ) ; 7.20-7.63 (m, arom. H ); 7.73 (d, J = 4.6 H z , I H , H-6); 8.78 (d, J = 4.6 H z , I H , H-5). Anal. Calcd. for C 2 6 H 2 9 C I N 4 O 2 S Ì : C 63.33; H 5.93; N 11.36. Found: C 63.41; H 5.85; N 11.08. 7-Chloro-2-[2,3-dideoxy-5-0-[(dimethylethyl)diphenylsilyl]-p - D £ryc£r0-pentofuranosyl]-2tf -1,2,3-triazolo[4,5-6]pyridine From

the

same

flash

chromatography

o f fraction

II, 14

(14).

was obtained

as o i l

(240 m g , 4 % ) . T L C ( C H ) : R f 0.07. * H N M R ( M e S O - d ) : 5 0.95 (s, t-Bu); 2.20 (m, 6

6

2

6

2 H , H-3'); 2.58, 2.70 (2m, 2 H , H-2'); 3.78 (m, 2 H , H-5'); 4.65 (m, I H , H-4'); 6.65 (d, J = 5.8 H z , I H , H - l ' ) ; 7.15-7.45 (3m, arom. H ); 7.68 (d, J = 4.7 H z , I H , H-6); 8.77 (d, J = 4.7 H z , I H , H - 5 ) . A n a l . Calcd. for C 2 6 H 2 9 C I N 4 O 2 S Ì : C 63.33; H 5.93; N 11.36. Found: C 63.49; H . 6.05; N 11.55, 7-Chloro-l-(2,3-dideoxy-5-0-[(dimethylethyl)diphenylsilyl]-aD-glycero-pentofuranosyl]-IH B y flash

chromatography of fraction

-l,2,3-triazolo[4,5-6]pyridine III with C s H ^ - E t O A c

(99.5:0.5),

(15).

compound

15 was separated as o i l (94 mg, 1.6 %). T L C ( C H - E t O A c 99:1): R f 0.07. U N M R 6

(Me SO-d ): 2

6

6

l

5 0.90 (s, t-Bu); 2.12, 2.60 (2m, 2 H , H-3'); 3.12, 3.38 (2m, 2 H , H-2');

3.65 (m, 2 H , H-5*); 4.40 (m, I H , H-4'); 6.95 (d, J = 6.7 H z , I H , H - l ' ) ; 7.20-7.63 (m, arom. H ) ; 7.83 (d, J = 4.9 H z , I H , H-6); 8.65 (d, J = 4.9 H z , I H , H-5). A n a l . Calcd. for C 2 6 H 2 9 C I N 4 O 2 S Ì : C 63.33; H 5.93; N 11.36. Found: C 63.21; H 6.18; N 11.10. 7-ChIoro-3-(2,3-dideoxy-a-D-g/vctfr0-pentofuranosyl)-3/f -1,2,3triazolo[4,5-£]pyridine

(16).

The protected

12

(600 m g , 1.21 m m o l )

dissol-

ved i n T H F , was stirred with B U 4 N F 1.1 M i n T H F (17 m L ) at r. t. for 30 m i n . The mixture

was evaporated,

c o l u mn

eluting

with

and the residue

CHCl3-MeOH

(99:1);

was chromatographed rechromatography

on a s i l i c a g e l

with

EtOAc-CGH^

(8:2) yielded 16 as o i l (145 mg, 47 %). T L C ( C H C l - M e O H 99:1): R f 0.22. U V ( N a O H 3

8-AZA-1 - D E A Z A P U R I N E N U C L E O S I D E S

0.1 N ) : X

1749

256 nm (e 4600), 284 (e 2600). * H N M R ( M e S O - r f ) : 8 1.90, 2.45 (2m,

max

2

6

2 H , H-3'); 2.65, 2.77 (2m, 2 H , H-2'); 3.48 (m, 2 H , H-5'); 4.38 (m, I H , H-4'); 4.82 (t, J = 5.8 H z , I H , OH-5'); 6.85 (dd, J = 3.2, 6.7 H z , I H , H - l ' ) ; 7.77 ( d, J = 4.9 H z , I H , H-6); 8.78 (d, J = 4.9 H z , I H , H-5).

1

3

C N M R ( M e S O - d ) : 151.8 (C 5); 146.2 ( C 3a); 134.8 2

6

(C 7); 134.6 (C 7a); 120.8 ( C 6); 87.4 (C 1'); 82.2 (C 4'); 63.4 (C 5'); 30.6 ( C 2'); 26.8 (C 3').

Anal. Calcd. for C I Q H J C 1 N 0 : C 54.79; H 5.06; N 25.56. Found: C 54.66; H 5.22; 1

4

2

N 25.70. 7-Chloro-3-(2,3-dideoxy-P-D«g/ycer0-pentofuranosyl]-31? -1,2,3triazolo[4,5-6]pyridine mg,

(17).

This

compound

was obtained

from

13

(700

1.42 mmol) as described for 16. The reaction mixture was purified by flash

chromatography

C H C l 3 - M e O H (90:10) and then by silica gel column

with

using

C H C l 3 - M e O H (99:1) as eluent, to give 17 as o i l (220 mg, 61 %). T L C ( C H C l - M e O H 3

99:1): R f 0.34. U V ( N a O H 0.1 N ) : X

m

a

282 nm (e 9500). * H N M R ( M e S O - d ) : 8

x

2

6

1.95, 2.38 (2m, 2 H , H-3'); 2.58 (m, 2 H , H-2'); 3.52 (m, 2 H , H-5'); 4.50 (m, I H , H-4'); 4.88 (t, J = 5.6 H z , I H , OH-5'); 6.70 (dd, J = 3.0, 11.6 H z , I H , H - l ' ) ; 7.73 (d, J = 4.8 H z , I H , H-6); 8.78 (d, J = 4.8 H z , I H , H-5).

1

3

C N M R ( M e S O - d ) : 151.9 ( C 5); 146.6 ( C 2

6

3a); 135.2 (C 7 and C 7a); 121.0 (C 6); 86.8 (C 1'); 83.5 (C 4'); 64.0 ( C 5'); 31.3 ( C 2'); 27.4 ( C 3'). Anal. Calcd. for C Ui ÌO

I C 1 N 0 : C 54.79; H 5.06; N 25.56. Found: C 54.58; 4

2

H 5.13; N 25.48. 7-Chloro-2-(2,3-dideoxy-J3-D-g/ycer0-pentofuranosyI]-2H -1,2,3triazolo[4,5-6]pyridine 17

starting

tography C H 6

1

2

(18).

Compound

18

from 14 ( 2 3 0 m g , 0.46 mmol)

with C H C 1 - C H 3

6

1 2

was prepared and

purified

as described by

flash

for

chroma-

(85:15) to give an o i l (70 mg, 60 % ) . T L C (CHC13-

85:15): R f 0.15. U V ( N a O H 0.1 N ) : X

m

a

x

282 nm (e 10500). * H N M R ( M e 2 S O -

dt): 8 2.20 (m, 2 H , H-3'); 2.52, 2.65 (2m, 2 H , H-2'); 3.57 (m, 2 H , H-5'); 4.60 (m, I H , H-4*); 4.80 (t, J = 5.7 H z , I H , OH-5'); 6.70 (dd, J = 2.4, 5.6 H z , I H , H - l ' ) ; 7.75 (d, J = 4.7 Hz, I H , H-6); 8.81 (d, J = 4.7 H z , I H , H-5).

1

3

C N M R (Me SO-^6)*- 155.9 (C 3a); 152.9 2

(C 5); 134.8 (C 7a); 133.8 ( C 7); 122.7 (C 6); 95.6 (C 1'); 84.4 ( C 4'); 64.0 ( C 5'); 32.6 ( C 2'); 26.6 (C 3'). A n a l . Calcd. for C\QU\ 0 : 4

C 54.79; H 5.06; N 25.56. Found: C

2

54.68; H 5.00; N 25.67. 7 - C h l o r o - l - ( 2 , 3 - d i d e o x y - a - D - g / y c e r 0 - p e n t o f u r a n o s y l ] - l # -1,2,3triazolo[4,5-6]pyridine ded,

after

chromatographic

(99:1), compound

(19).

Deprotection

separation

of

15 (90 m g , 0.18 m m o l )

on silica gel column w i t h

19 as white solid (40 mg, 87 %); m.p. 114-115

M e O H 99:1): R f 0.19. U V ( N a O H 0.1 N ) : A .

m a x

yiel-

CHCl3-MeOH

° C . T L C (CHCI3-

264 nm (e 12100). * H N M R ( M e 2 S O -

de): 8 2.20 (m, 2 H , H-3'); 2.62, 3.05 (2m, 2 H , H-2'); 3.27 (m, 2 H , H-5'); 4.37 (m, I H , H-4'); 4.65 (t, J = 5.6 H z , I H , OH-5'); 6.98 (dd, J = 2.5, 7.0 H z , I H , H - l ' ) ; 7.82 (d, J = 4.9

1750

FRANCHETTI ET AL.

H z , I H , H-6); 8.70 (d, J = 4.9 H z , I H , H-5).

1

3

C N M R ( M e S O - r f ) : 158.2 ( C 3a); 149.7 2

6

(C 5), 127.6 (C 7); 123.8 (C 6); 123.2 ( C 7a); 88.6 ( C 1*); 83.5 (C 4'); 63.6 (C 5'); 31.1 ( C 2'); 27.1 ( C 3*). A n a l . Calcd. for C ^ H ] Ì C 1 N 0 : C 54.79; H 5.06; N 25.56. Found: C 4

2

54.70; H 5.11; N 25.45. 7 - A mino-3-(2,3-di deoxy-a-D-g/y cero-pentofuranosyl]-3# triazolo[4,5-6]pyridine ammonia

( 2 0 ) . To

(20 m L ) was added,

compound

16

and the mixture

-1,2,3-

(130 m g , 0.4 mmol)

was heated

i n a steel

liquid reaction

vessel at 70 ° C for 27 h. The excess ammonia was removed and the residue o i l was cromatographed

on a silica gel column with C H C l 3 - M e O H (90:10) to give 2 0

as white solid (30 m g , 25 %); m.p. 170-172 °C. T L C ( C H C l 3 - M e O H 90:10): R f 0.35. U V (MeOH): X

m2LX

258 nm (e 4100); 266 (e 3900); 306 (e 7200);

( N a O H 0.1 N ) : 256

(e 8300); 304 (e 6500). H N M R (Me SO-