U.S. patent application number 10/887292 was filed with the patent office on 2004-12-09 for method for the treatment or prevention of flavivirus infections using nucleoside analogues.
Invention is credited to Cheng, Yun-Xing, Ismaili, Hicham Moulay Alaoui, Lavallee, Jean-Francois, Siddiqui, Arshad, Storer, Richard.
Application Number | 20040248844 10/887292 |
Document ID | / |
Family ID | 22672442 |
Filed Date | 2004-12-09 |
United States Patent
Application |
20040248844 |
Kind Code |
A1 |
Ismaili, Hicham Moulay Alaoui ;
et al. |
December 9, 2004 |
Method for the treatment or prevention of Flavivirus infections
using nucleoside analogues
Abstract
The present invention relates to a method for the treatment or
prevention of Flavivirus infections using nucleoside analogues in a
host comprising administering a therapeutically effective amount of
a compound having the formula I or a pharmaceutically acceptable
salt thereof.
Inventors: |
Ismaili, Hicham Moulay Alaoui;
(Montreal, CA) ; Cheng, Yun-Xing;
(Dollard-des-Ormeaux, CA) ; Lavallee, Jean-Francois;
(Bellefeuille, CA) ; Siddiqui, Arshad;
(Dollard-des-Ormeaux, CA) ; Storer, Richard; (Baie
d'Urfe, CA) |
Correspondence
Address: |
MILLEN, WHITE, ZELANO & BRANIGAN, P.C.
2200 CLARENDON BLVD.
SUITE 1400
ARLINGTON
VA
22201
US
|
Family ID: |
22672442 |
Appl. No.: |
10/887292 |
Filed: |
July 9, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10887292 |
Jul 9, 2004 |
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09785235 |
Feb 20, 2001 |
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6784161 |
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60183349 |
Feb 18, 2000 |
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Current U.S.
Class: |
514/47 ;
514/50 |
Current CPC
Class: |
A61K 45/06 20130101;
A61K 31/7072 20130101; A61P 31/14 20180101; A61K 31/708 20130101;
A61K 31/7056 20130101; A61K 31/7076 20130101; A61P 43/00 20180101;
A61K 31/7068 20130101; A61P 31/12 20180101; A61K 31/7064
20130101 |
Class at
Publication: |
514/047 ;
514/050 |
International
Class: |
A61K 031/7076; A61K
031/7072 |
Claims
1. A method for the treatment or prevention of an hepatitis C
infection in a host comprising administering to said host a
therapeutically effective amount of a compound having the formula
Ib or a pharmaceutically acceptable salt thereof: 42wherein B is a
nucleotide purine radical, a nucleotide pyrimidine radical or an
analogue of a nucleotide purine radical or a nucleotide pyrimidine
radical, wherein said analogue is derived by replacement of a CH
moiety by a nitrogen atom in a nucleotide purine or pyrimidine
radical, replacement of a nitrogen atom by a CH moiety in a
nucleotide purine or pyrimidine radical or both; or derived by
removal of ring substituents of said nucleotide purine radical or
pyrimidine radical; or combinations thereof, and said analogue is
optionally substituted by halogen hydroxyl, amino or C.sub.1-6
alkyl; Ra is H, monophosphate, diphosphate, triphosphate, carbonyl
which is substituted by a straight, branched or cyclic alkyl having
up to 6 C atoms wherein the alkyl is unsubstituted or substituted
by halogen, nitro CONH, COOH O-CQ-6 alkyl O--C.sub.6 alkenyl
O--C.sub.6 alkynyl hydroxyl, amino or COOQ, C.sub.2-6 alkenyl which
is unsubstituted or substituted by halogen nitro, CONH.sub.2, COOH,
O--C_alkyl O--C_alkenyl, O--C, alkynyl, hydroxyl, amino, or COOQ,
C.sub.2-6 alkynyl which is unsubstituted or substituted by halogen,
nitro, CONH, COOH. O--C.sub.1-6 alkyl O--C_alkenyl O--C.sub.2-6
alkynyl, hydroxyl, amino, or COOQ, C.sub.6-10 aryl which is
unsubstituted or mono- or di-substituted with OH SH, amino halogen
or C.sub.1-6 alkyl, or an 43Rc is in each case independently, H,
straight chain, branched chain or cyclic C.sub.1-6 alkyl which is
unsubstituted or substituted by or substituted by halogen, nitro,
CONH. COOH, O--C.sub.1-6 alkyl O--C.sub.--.sub.6 alkenyl.
O--C_).sub.6 alkynyl, hydroxyl, amino, or COOQ, C.sub.2-6 alkenyl
which is unsubstituted or substituted by or substituted by halogen
nitro, CONH.sub.2, COOH, O--C, alkyl, --C_alkenyl, --C_alkynyl
hydroxyl, amino, or COOQ, C.sub.2-6 alkynyl which is unsubstituted
or substituted by or substituted by halogen, nitro, CONH, COOH
O--C, alkyl O--C, alkenyl O--C_alkynyl, hydroxyl amino, or COOQ,
C.sub.6-10 aryl which is unsubstituted or mono- or di-substituted
with OH, SH, amino, halogen or C.sub.1-6 alkyl, or a hydroxy
protecting group; and Z is ORb, Rb is H, straight chain, branched
chain or cyclic C.sub.1-6 alkyl which is unsubstituted or
substituted by or substituted by halogen, nitro, CONH.sub.2, COOH,
O--C_alkyl, O--C_alkenyl, O--C.sub.1-6 alkynyl, hydroxyl, amino, or
COOQ, C.sub.2-6 alkenyl which is unsubstituted or substituted by or
substituted by halogen, nitro, CONH, COOH, --C.sub.1-6alkyl,
O--C.sub.2-6alkenyl, O--C.sub.2-6 alkynyl hydroxyl, amino, or COOQ,
C.sub.2-6 alkynyl which is unsubstituted or substituted by or
substituted by halogen, nitro CONH.sub.2, COOH, O--C_alkyl
O--C_alkenyl O--C_alkynyl hydroxyl amino, or COOQ, C.sub.1-6 acyl,
or a hydroxy protecting group; D.sub.1 and D.sub.2 are each
independently N.sub.3, F, or H, D.sub.1 and D.sub.2 can also be
joined to be .dbd.CH.sub.2, .dbd.CF.sub.2, or C.sub.3-cycloalkyl
which is unsubstituted or substituted by or substituted by halogen,
nitro, CONH.sub.2, COOH, O--C.sub.1-6 alkyl, O--C.sub.2-6 alkenyl,
O--C.sub.7-6 alkynyl, hydroxyl, amino, or COOQ; with the proviso
that when B is adenine, Z is ORb, D.sub.1 is H, D.sub.2 is H and Rb
is H, Ra is not triphosphate or H, wherein said compound is not
administered in conjunction with an interferon.
2. A method according to claim 1 wherein Z is OH.
3. A method according to claim 2 wherein D.sub.1 is H and D.sub.2
is F.
4. A method according to claim 2 wherein Ra is H, monophosphate,
diphosphate, or triphosphate.
5. A method according to claim 2 wherein Ra is triphosphate.
6. A method according to claim 2 wherein Ra is H.
7. A method according to claim 3 wherein Ra is H, monophosphate,
diphosphate, or triphosphate.
8. A method according to claim 3 wherein Ra is triphosphate.
9. A method according to claim 3 wherein Ra is H.
10. A method according to claim 2 wherein B is adenin-9-yl,
guanin-9-yl, inosin-9-yl, 2-amino-purin-9-yl,
2-amino-6-chloro-purin-9-yl, 2-6-diamino-purin-9-yl, thymin-1-yl,
cytosin-1-yl, uracil-1-yl, 3-carboxamido-1,2,4-triazol-1-yl,
3-deaza-adenin-9-yl, 3-deaza-guanin-9-yl, 3-deaza-inosin-9-yl,
3-deaza-2-amino-purin-9-yl, 3-deaza-2-amino-6-chloro-purin-9-yl
3-deaza-2-6-diamino-purin-9-yl, 7-deaza-adenin-9-yl,
7-deaza-guanin-9-yl, 7-deaza-inosin-9-yl,
7-deaza-2-amino-purin-9-yl, 7-deaza-2-amino-6-chloro-purin-9-yl,
7-deaza-2-6-diamino-purin-9-yl, 7-deaza-8-aza-adenin-9-yl,
7-deaza-8-aza-guanin-9-yl, 7-deaza-8-aza-inosin-9-yl,
7-deaza-8-aza-2-amino-purin-9-yl,
7-deaza-8-aza-2-amino-6-chloro-purin-9-- yl,
7-deaza-8-aza-2-6-diamino-purin-9-yl, 8-aza-adenin-9-yl,
8-aza-guanin-9-yl, 8-aza-inosin-9-yl, 8-aza-2-amino-purin-9-yl,
8-aza-2-amino-6-chloro-purin-9-yl, 8-aza-2-6-diamino-purin-9-yl,
5-aza-thymin-1-yl, 5-aza-cytosin-1-yl, 5-aza-uracil-1-yl,
6-aza-thymin-1-yl, 6-aza-cytosin-1-yl, or 6-aza-uracil-1-yl; which
in each case is unsubstituted or substituted by at least one of
NHR.sub.3, C.sub.1-6alkyl, --OC.sub.1-6alkyl, Br, Cl, F, I or OH,
wherein R.sub.3 is H, C.sub.1-6alkyl or C.sub.1-6acyl.
11. A method according to claim 3 wherein B is chosen from
adenin-9-yl, guanin-9-yl, inosin-9-yl, 2-amino-purin-9-yl,
2-amino-6-chloro-purin-9-yl- , 2-6-diamino-purin-9-yl, thymin-1-yl,
cytosin-1-yl, uracil-1-yl, 3-carboxamido-1,2,4-triazol-1-yl,
3-deaza-adenin-9-yl, 3-deaza-guanin-9-yl, 3-deaza-inosin-9-yl,
3-deaza-2-amino-purin-9-yl, 3-deaza-2-amino-6-chloro-purin-9-yl
3-deaza-2-6-diamino-purin-9-yl, 7-deaza-adenin-9-yl,
7-deaza-guanin-9-yl, 7-deaza-inosin-9-yl,
7-deaza-2-amino-purin-9-yl, 7-deaza-2-amino-6-chloro-purin-9-yl,
7-deaza-2-6-diamino-purin-9-yl, 7-deaza-8-aza-adenin-9-yl,
7-deaza-8-aza-guanin-9-yl, 7-deaza-8-aza-inosin-9-yl,
7-deaza-8-aza-2-amino-purin-9-yl,
7-deaza-8-aza-2-amino-6-chloro-purin-9-- yl,
7-deaza-8-aza-2-6-diamino-purin-9-yl, 8-aza-adenin-9-yl,
8-aza-guanin-9-yl, 8-aza-inosin-9-yl, 8-aza-2-amino-purin-9-yl,
8-aza-2-amino-6-chloro-purin-9-yl, 8-aza-2-6-diamino-purin-9-yl,
5-aza-thymin-1-yl, 5-aza-cytosin-1-yl, 5-aza-uracil-1-yl,
6-aza-thymin-1-yl, 6-aza-cytosin-1-yl, or 6-aza-uracil-1-yl; which
in each case is unsubstituted or substituted by at least one of
NHR.sub.3, C.sub.1-6alkyl, --OC.sub.1-6alkyl, Br, Cl, F, I or OH,
wherein R.sub.3 is H, C.sub.1-6alkyl or C.sub.1-6acyl.
12. A method according to claim 2 wherein B is adenin-9-yl,
guanin-9-yl, inosin-9-yl, 2-amino-purin-9-yl,
2-amino-6-chloro-purin-9-yl, 2-6-diamino-purin-9-yl, thymin-1-yl,
cytosin-1-yl, 5-fluoro-cytosin-1-yl, uracil-1-yl, 5-fluorouracil or
1,2,4-triazole-3-carboxamide base.
13. A method according to claim 3 wherein B is adenin-9-yl,
guanin-9-yl, inosin-9-yl, 2-amino-purin-9-yl,
2-amino-6-chloro-purin-9-yl, 2-6-diamino-purin-9-yl, thymin-1-yl,
cytosin-1-yl, 5-fluoro-cytosin-1-yl, uracil-1-yl, 5-fluorouracil or
1,2,4-triazole-3-carboxamide base.
14. A method according to claim 1 wherein the compound of formula I
is: 3'-deoxycytidine; 3'-deoxycytidine-5'triphosphate;
5-Fluoro-3'-deoxycytidine;
5-Fluoro-3'-deoxycytidine-5'triphosphate; 3'-deoxyuridine;
3'-deoxyuridine-5'triphosphate; 5-Fluoro-3'-deoxyuridine- ;
5-Fluoro-3'-deoxyuridine-5'triphosphate; 3'-deoxythymidine;
3'-deoxythymidine-5'triphosphate; 3'-deoxyguanosine;
3'-deoxyguanosine-5'triphosphate; 2-N-acetyl-3'-deoxyguanosine;
2-N-acetyl-3'-deoxyguanosine-5'triphosphate;
5-Methyl-3'-deoxycytidine;
5-Methyl-3'-deoxycytidine-5'triphosphate; 5-Iodo-3'-deoxycytidine;
5-Iodo-3'-deoxycytidine-5'triphosphate; 5-Chloro-3'-deoxycytidine;
5-Chloro-3'-deoxycytidine-5'triphosphate;
3'-fluoro-3'-deoxyguanosine;
3'-fluoro-3'-deoxyguanosine-5'triphosphate; 3'-fluoro
3'-deoxycytidine; 3'-fluoro 3'-deoxycytidine-5'triphosphate;
5-Iodo-3'-deoxycytidine; 5-Iodo-3'-deoxycytidine-5'triphosphate;
5-Chloro-3'-deoxyuridine; 5-Chloro-3'-deoxyuridine-5'triphosphate;
5-Bromo-3'-deoxyuridine; 5-Bromo-3'-deoxyuridine-5'triphosphate;
6-Chloro-3'-deoxyguanosine;
6-Chloro-3'-deoxyguanosine-5'triphosphate;
3'-spirocyclopropyl-3'-deoxygu- anosine;
3'-spirocyclopropyl-3'-deoxyguanosine-5'triphosphate;
3'-difluoro-spirocyclopropyl-3'-deoxyguanosine;
3'-difluoro-spirocyclopro- pyl-3'-deoxyguanosine-5'triphosphate;
3'-methylene-3'-deoxyguanosine;
3'-methylene-3'-deoxyguanosine-5'triphosphate; 3'-difluromethylene
3'-deoxyguanosine; 3'-difluromethylene
3'-deoxyguanosine-5'triphosphate;
3'-spirocyclopropyl-3'-deoxycytidine;
3'-spirocyclopropyl-3'-deoxycytidin- e-5'triphosphate;
3'-difluoro-spirocyclopropyl-3'-deoxycytidine;
3'-difluoro-spirocyclopropyl-3'-deoxycytidine-5'triphosphate;
3'-methylene-3'-deoxycytidine;
3'-methylene-3'-deoxycytidine-5'triphospha- te; 3'-difluromethylene
3'-deoxycytidine; 3'-difluromethylene
3'-deoxycytidine-5'triphosphate;
9-.beta.-D-xylofuranosyl-guanosine;
9-.beta.-D-xylofuranosyl-guanosine-5'triphosphate;
9-.beta.-D-xylofuranosyl-cytidine;
9-.beta.-D-xylofuranosyl-cytidine-5'tr- iphosphate;
3'-azido-3'-deoxycytidine; 3'-azido-3'-deoxycytidine
5'triphosphate; and or a pharmaceutically acceptable salts
thereof.
15. A method according to claim 1I wherein said compound is used in
combination with at least one further therapeutic agent chosen from
ribavirin, amantadine, rimantadine, interleukine-12,
ursodeoxycholic acid (UDCA), glycyrrhizin and silybum marianum.
16. A The method according to claim 2, wherein said compound is
used in combination with at least one further therapeutic agent
chosen from ribavirin, amantadine, rimantadine, interleukine-12,
ursodeoxycholic acid (UDCA), glycyrrhizin and silybum marianum.
17. A The method according to claim 3, wherein said compound is
used in combination with at least one further therapeutic agent
chosen from ribavirin, amantadine, rimantadine, interleukine-12,
ursodeoxycholic acid (UDCA), glycyrrhizin and silybum marianum.
18. A The method according to claim 14, wherein said compound is
used in combination with at least one further therapeutic agent
chosen from ribavirin, amantadine, rimantadine, interleukine-12,
ursodeoxycholic acid (UDCA), glycyrrhizin and silybum marianum.
19. A method according to claim 1, wherein said method is a method
of treatment.
20. A method according to claim 19, wherein Ra is H, monophosphate,
diphosphate, triphosphate, carbonyl substituted by C.sub.1-6 alkyl,
C.sub.2-6, C.sub.2-6 alkynyl, or C.sub.6-10 aryl or 44Rc is, in
each case independently, H, C.sub.1-6 alkyl, C.sub.2-6 alkenyl,
C.sub.2-6 alkynyl, C.sub.6-10 aryl or a hydroxy protecting group
selected from acetyl-2-thioethyl ester, pivaloyloxymethyl ester and
isopropyloxycarbonyloxymethyl ester; and Rb is H, C.sub.1-6 alkyl,
C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-6 acyl, or a hydroxy
protecting group selected from acetyl-2-thioethyl ester,
pivaloyloxymethyl ester and isopropyloxycarbonyloxymethyl
ester.
21. A method according to claim 19, wherein B is adenin-9-yl,
guanin-9-yl, inosin-9-yl, 2-amino-purin-9-yl,
2-amino-6-chloro-purin-9-yl, 2-6-diamino-purin-9-yl, thymin-1-yl,
cytosin-1-yl, uracil-1-yl, or 3-carboxamido-1,2,4-triazol-1-yl,
which in each case is unsubstituted or substituted by at least one
of NHR.sub.3, C.sub.1-6alkyl, --OC.sub.1-6alkyl, Br, Cl, F, I or
OH, wherein R.sub.3 is H, C.sub.1-6alkyl or C.sub.1-6acyl.
22. A method according to claim 19, wherein B is adenin-9-yl,
guanin-9-yl, 2-amino-purin-9-yl, 2-amino-6-chloro-purin-9-yl,
2-6-diamino-purin-9-yl, thymin-1-yl, cytosin-1-yl, uracil-1-yl,
which in each case is unsubstituted or substituted by at least one
of NHR.sub.3, C.sub.1-6alkyl, --OC.sub.1-6alkyl, Br, Cl, F, I or
OH, wherein R.sub.3 is H, C.sub.1-6alkyl or C.sub.1-6acyl.
23. A method according to claim 19, wherein B is guanin-9-yl,
cytosin-1-yl, uracil-1-yl, which in each case is unsubstituted or
substituted by at least one of NHR.sub.3, C.sub.1-6alkyl,
--OC.sub.1-6alkyl, Br, Cl, F, I or OH, wherein R.sub.3 is H,
C.sub.1-6alkyl or C.sub.1-6acyl.
24. A method according to claim 19, wherein B is guanin-9-yl,
cytosin-1-yl, 5'-fluoro-cytosin-1-yl, 5'-fluorouracil-1-yl or
uracil-1-yl.
25. A method according to claim 19, wherein B is 45wherein X is H,
halogen or NHR.sub.10; R.sub.10 is H, C.sub.1-6acyl, C.sub.1-6
alkyl, C.sub.2-6 alkenyl, or C.sub.2-6 alkynyl; Y is H, halogen or
NHR.sub.11; R.sub.11 is H, C.sub.1-6acyl, C.sub.1-6 alkyl,
C.sub.2-6 alkenyl, or C.sub.2-6 alkynyl; Y.sub.2 is H, halogen or
NHR.sub.12; R.sub.12 is H, C.sub.1-6acyl, C.sub.1-6 alkyl,
C.sub.2-6 alkenyl, or C.sub.2-6 alkynyl; R.sub.9 is H, hydroxy
protecting group, C.sub.1-6acyl, C.sub.1-6 alkyl, C.sub.2-6
alkenyl, or C.sub.2-6 alkynyl; Y.sub.3 is H, halogen or NHR.sub.13;
R.sub.13 is H, C.sub.1-6acyl, C.sub.1-6 alkyl, C.sub.2-6 alkenyl,
or C.sub.2-6 alkynyl; R.sub.7 is H, halogen, C.sub.1-6acyl,
C.sub.1-6 alkyl, C.sub.2-6 alkenyl, or C.sub.2-6 alkynyl; and
R.sub.8 is H, halogen, C.sub.1-6acyl, C.sub.1-6 alkyl, C.sub.2-6
alkenyl, or C.sub.2-6 alkynyl.
26. A method according to claim 25, wherein X is H, F, or
NHR.sub.10, R.sub.10 is H, Y is H, F, or NHR.sub.1, R.sub.11 is H,
Y.sub.2 is H, F, or NHR.sub.12, R.sub.12 is H, R.sub.9 is H,
Y.sub.3 is H, F, or NHR.sub.13R.sub.13 is H, R.sub.7 is H, F, or
C.sub.1-6 alkyl, and R.sub.8 is H, F, or C.sub.1-6 alkyl.
27. A method according to claim 19, wherein Z is F or ORb, and ORb
is H or methyl.
28. A method according to claim 19, wherein D.sub.1 and D.sub.2 are
N.sub.3, F, or H in which D.sub.1 and D.sub.2 are not both H, or
D.sub.1 and D.sub.2 together form cyclopropyl,
difluorocyclopropyl-.dbd.CH.sub.2, or .dbd.CF.sub.2.
29. A method according to claim 19, wherein said compound is
administered in an amount of 0.01 to about 750 mg/kg of body weight
per day.
30. A method according to claim 19, wherein said compound is
administered in unit dosages containing 10 to 1500 mg of said
compound per unit dosage.
31. A method according to claim 15, wherein said compound and said
further therapeutic agent are each administered as a formulation
which further contains a pharmaceutically acceptable carrier.
32. A method according to claim 31, wherein said compound and said
further therapeutic agent are sequentially administered, in
separate or combined pharmaceutical formulations.
33. A method according to claim 31, wherein said compound and said
further therapeutic agent are simultaneously administered, in
separate or combined pharmaceutical formulations.
34. A method according to claim 1, wherein said host is a
human.
35. A method according to claim 19, wherein said host is a
human.
36. A method according to claim 2, wherein said host is a
human.
37. A method according to claim 3, wherein said host is a
human.
38. A method according to claim 14, wherein said host is a human.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a method for the treatment
or prevention of Flavivirus infections using nucleoside
analogues.
BACKGROUND OF THE INVENTION
[0002] Hepatitis is a disease occurring throughout the world. It is
generally of viral nature, although there are other causes known.
Viral hepatitis is by far the most common form of hepatitis. Nearly
750,000 Americans are affected by hepatitis each year, and out of
those, more than 150,000 are infected with the hepatitis C virus
(HCV).
[0003] HCV is a positive-stranded RNA virus belonging to the
Flaviviridae family and has closest relationship to the
pestiviruses that include hog cholera virus and bovine viral
diarrhea virus (BVDV). HCV is believed to replicate through the
production of a complementary negative-strand RNA template. Due to
the lack of an efficient culture replication system for the virus,
HCV particles were isolated from pooled human plasma and shown, by
electron microscopy, to have a diameter of about 50-60 nm. The HCV
genome is a single-stranded, positive-sense RNA of about 9,600 bp
coding for a polyprotein of 3009-3030 amino-acids, which is cleaved
co- and pqst-translationally by cellular and two viral proteinases
into mature viral proteins (core, E1, E2, p7, NS2, NS3, NS4A, NS4B,
NS5A, NS5B). It isbelieved that the structural proteins, E1 and E2,
the major glycoproteins are embedded into a viral lipid envelop and
form stable heterodimers. It is also believed that the structural
core protein interacts with the viral RNA genome to form the
nucleocapsid. The nonstructural proteins designated NS2 to NS5
include proteins with enzymatic functions involved in virus
replication and protein processing including a polymerase, protease
and helicase.
[0004] The main source of contamination with HCV is blood. The
magnitude of the HCV infection as a health problem is illustrated
by the prevalence among high-risk groups. For example, 60% to 90%
of hemophiliacs and more than 80% of intravenous drug abusers in
western countries are chronically infected with HCV. For
intravenous drug abusers, the prevalence varies from about 28% to
70% depending on the population studied. The proportion of new HCV
infections associated with post-transfusion has been markedly
reduced lately due to advances in diagnostic tools used to screen
blood donors.
[0005] The only treatment currently available for HCV infection is
interferon-.alpha. (IFN-.alpha.). However, according to different
clinical studies, only 70% of treated patients normalize alanine
aminotransferase (ALT) levels in the serum and after
discontinuation of IFN, 35% to 45% of these responders relapse. In
general, only 20% to 25% of patients have long-term responses to
IFN. Clinical studies have shown that combination treatment with
IFN and ribavirin (RIBA) results in a superior clinical response
than IFN alone. Different genotypes of HCV respond differently to
IFN therapy, genotype 1b is more resistant to IFN therapy than type
2 and 3.
[0006] There is therefore a great need for the further development
of anti-viral agents.
SUMMARY OF THE INVENTION
[0007] The present invention relates to a method for the treatment
or prevention of Flavivirus infections in a host comprising
administering a therapeutically effective amount of a compound
having the formula I or a pharmaceutically acceptable salt thereof:
1
[0008] wherein
[0009] B is chosen from a purine, a pyrimidine or an analogue
thereof;
[0010] Ra is chosen from H, monophosphate, diphosphate,
triphosphate, carbonyl substituted with a C.sub.1-6 alkyl,
C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.6-10 aryl, and 2
[0011] wherein each Rc are independently chosen from H, C.sub.1-6
alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.6-10 aryl and an
hydroxy protecting group; and
[0012] Z is halogen or. ORbi wherein Rb is choseh from of H,
C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-6
acyl, or an hydroxy protecting group
[0013] D.sub.1 and D.sub.2 are independently selected from.
N.sub.3, F, or H, and D.sub.2 can also be joined to be chosen from
C.sub.3-cycloalkyl, -.dbd.CH.sub.2, or -.dbd.CF.sub.2, and
[0014] wherein said compound is in the form of a single enantiomer
or a mixture thereof including racemic mixtures;
[0015] with the proviso that when B is adenine, Z is ORb, D.sub.1
is H, D.sub.2 is H and Rb is H, Ra is not triphosphate or H.
[0016] In another aspect, there is provided a pharmaceutical
formulation comprising the compounds of the invention in
combination with a pharmaceutically acceptable carrier or
excipient.
[0017] Still another aspect, there is provided a method for
treating or preventing a viral infection in a host comprising
administering a combination comprising at least one compound
according to formula I and at least one further therapeutic
agent.
[0018] In another aspect of the invention is the use of a compound
according to formula I, for the preparation of a medicament for
treating or preventing a viral infections in a host.
DETAILED DESCRIPTION OF THE INVENTION
[0019] In one embodiment, the viral infection is chosen from
Flavivirus infections.
[0020] In one embodiment, the Flavivirus infection is chosen from
Hepatitis C virus (HCV), bovine viral diarrhea virus (BVDV), hog
cholera virus and yellow fever virus.
[0021] In an other embodiment, the Flavivirus infection is
Hepatitis C virus.
[0022] In one embodiment, there is also provided a method for
inhibiting or reducing the activity of viral polymerase in a host
comprising administering a therapeutically effective amount of a
compound having the formula I.
[0023] In another embodiment, the viral polymerase is HCV
polymerase.
[0024] The present invention relates to a method for the treatment
or prevention of Flavivirus infections using nucleoside analogues
in a host comprising administering a therapeutically effective
amount of a compound having the formula Ia or a pharmaceutically
acceptable salt thereof: 3
[0025] wherein
[0026] B is chosen from a purine, a pyrimidine or an analogue
thereof;
[0027] Ra is chosen from H, monophosphate, diphosphate,
triphosphate, carbonyl substituted with a C.sub.1-6 alkyl,
C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.6-10 aryl, and -- 4
[0028] wherein each Rc are independently chosen from H, C.sub.1-6
alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.6-10 aryl and an
hydroxy protecting group; and
[0029] Z is halogen or ORb, wherein Rb is chosen from of H,
C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-6
acyl, or an hydroxy protecting group; and
[0030] wherein said compound is in the formof a single enantiomer
or a mixture thereof including racemic mixtures;
[0031] with the proviso that when B is adenine, Z is ORb and Rb is
10H, Ra is not triphosphate or H.
[0032] In one embodiment, the compounds and methods of the present
invention comprise those wherein the following embodiments are
present, either independently or in combination.
[0033] In one embodiment, B is chosen from adenin-9-yl,
guanin-9-yl, inosin-9-yl, 2-amino-purin-9-yl,
2-amino-6-chloro-purin-9-yl, 2-6-diamino-purin-9-yl, thymin-1-yl,
cytosin-1-yl, uracil-1-yl, 3-carboxamido-1,2,4-triazol-1-yl,
1-deaza-adenin-9-yl, 1-deaza-guanin-9-yl, 1-deaza-inosin-9-yl,
1-deaza-2-amino-purin-9-yl, 1-deaza-2-amino-6-chloro-purin-9-yl,
1-deaza-2-6-diamino-purin-9-yl, 3-deaza-adenin-9-yl,
3-deaza-guanin-9-yl, 3-deaza-inosin-9-yl,
3-deaza-2-amino-purin-9-yl, 3-deaza-2-amino-6-chloro-purin-9-yl
3-deaza-2-6-diamino-purin-9-yl, 7-deaza-adenin-9-yl,
7-deaza-guanin-9-yl, 7-deaza-inosin-9-yl,
7-deaza-2-amino-purin-9-yl, 7-deaza-2-amino-6-chloro- -purin-9-yl,
7-deaza-2-6-diamino-purin-9-yl, 7-deaza-8-aza-adenin-9-yl,
7-deaza-8-aza-guanin-9-yl, 7-deaza-8-aza-inosin-9-yl,
7-deaza-8-aza-2-amino-purin-9-yl,
7-deaza-8-aza-2-amino-6-chloro-purin-9-- yl,
7-deaza-8-aza-2-6-diamino-purin-9-yl, 8-aza-adenin-9-yl,
8-aza-guanin-9-yl, 8-aza-inosin-9-yl, 8-aza-2-amino-purin-9-yl,
8-aza-2-amino-6-chloro-purin-9-yl, 8-aza-2-6-diamino-purin-9-yl,
2-aza-adenin-9-yl, 2-aza-guanin-9-yl, 2-aza-inosin-9-yl,
2-aza-2-amino-purin-9-yl, 2-aza-2-amino-6-chloro-purin-9-yl,
2-aza-2-6-diamino-purin-9-yl, 3-deaza-thymin-1-yl,
3-deaza-cytosin-1-yl, 3-deaza-uracil-1-yl, 5-aza-thymin-1-yl,
5-aza-cytqosin-1-yl, 5-aza-uracil-1-yl, 6-aza-thymin-1-yl,
6-aza-cytosin-1-yl, 6-aza-uracil-1-yl each of which is
unsubstituted or substituted by at least one of NHR.sub.3,
C.sub.1-6alkyl, --OC.sub.1-6alkyl, Br, Cl, F, I or OH, wherein
R.sub.3 is H, C.sub.1-6alkyl or C.sub.1-6acyl.
[0034] In one embodiment, B is chosen from adenin-9-yl,
guanin-9-yl, inosin-9-yl, 2-amino-purin-9-yl,
2-amino-6-chloro-purin-9-yl, 2-6-diamino-purin-9-yl, thymin-1-yl,
cytosin-1-yl, uracil-1-yl, 3-carboxamido-1,2,4-triazol-1-yl,
3-deaza-adenin-9-yl, 3-deaza-guanin-9-yl, 3-deaza-inosin-9-yl,
3-deaza-2-amino-purin-9-yl, 3-deaza-2-amino-6-chloro-purin-9-yl
3-deaza-2-6-diamino-purin-9-yl, 7-deaza-adenin-9-yl,
7-deaza-guanin-9-yl, 7-deaza-inosin-9-yl,
7-deaza-2-amino-purin-9-yl, 7-deaza-2-amino-6-chloro-purin-9-yl,
7-deaza-2-6-diamino-purin-9-yl, 7-deaza-8-aza-adenin-9-yl,
7-deaza-8-aza-guanin-9-yl, 7-deaza-8-aza-inosin-9-yl,
7-deaza-8-aza-2-amino-purin-9-yl,
7-deaza-8-aza-2-amino-6-chloro-purin-9-- yl,
7-deaza-8-aza-2-6-diamino-purin-9-yl, 8-aza-adenin-9-yl,
8-aza-guanin-9-yl, 8-aza-inosin-9-yl, 8-aza-2-amino-purin-9-yl,
8-aza-2-amino-6-chloro-purin-9-yl, 8-aza-2-6-diamino-purin-9-yl,
2-aza-adenin-9-yl, 2-aza-guanin-9-yl, 2-aza-inosin-9-yl,
2-aza-2-amino-purin-9-yl, 2-aza-2-amino-6-chloro-purin-9-yl,
2-aza-2-6-diamino-purin-9-yl, 3-deaza-thymin-1-yl,
3-deaza-cytosin-1-yl, 3-deaza-uracil-1-yl, 5-aza-thymin-1-yl,
5-aza-cytosin-1-yl, 5-aza-uracil-1-yl, 6-aza-thymin-1-yl,
6-aza-cytosin-1-yl, 6-aza-uracil-1-yl each of which is
unsubstituted or substituted by at least one of NHR.sub.3,
C.sub.1-6alkyl, --OC.sub.1-6alkyl, Br, Cl, F, I or OH, wherein
R.sub.3 is H, C.sub.1-6alkyl or C.sub.1-6acyl.
[0035] In one embodiment, B is chosen from adenin-9-yl,
guanin-9-yl, inosin-9-yl, 2-amino-purin-9-yl,
2-amino-6-chloro-purin-9-yl, 2-6-diamino-purin-9-yl, thymin-1-yl,
cytosin-1-yl, uracil-1-yl, 3-carboxamido-1,2,4-triazol-1-yl,
3-deaza-adenin-9-yl, 3-deaza-guanin-9-yl, 3-deaza-inosin-9-yl,
3-deaza-2-amino-purin-9-yl, 3-deaza-2-amino-6-chloro-purin-9-yl
3-deaza-2-6-diamino-purin-9-yl, 7-deaza-adenin-9-yl,
7-deaza-guanin-9-yl, 7-deaza-inosin-9-yl,
7-deaza-2-amino-purin-9-yl, 7-deaza-2-amino-6-chloro-purin-9-yl,
7-deaza-2-6-diamino-purin-9-yl, 7-deaza-8-aza-adenin-9-yl,
7-deaza-8-aza-guanin-9-yl, 7-deaza-8-aza-inosin-9-yl,
7-deaza-8-aza-2-amino-purin-9-yl,
7-deaza-8-aza-2-amino-6-chloro-purin-9-- yl,
7-deaza-8-aza-2-6-diamino-purin-9-yl, 8-aza-adenin-9-yl,
8-aza-guanin-9-yl, 8-aza-inosin-9-yl, 8-aza-2-amino-purin-9-yl,
8-aza-2-amino-6-chloro-purin-9-yl, 8-aza-2-6-diamino-purin-9-yl,
5-aza-thymin-1-yl, 5-aza-cytosin-1-yl, 5-aza-uracil-1-yl,
6-aza-thymin-1-yl, 6-aza-cytosin-1-yl, 6-aza-uracil-1-yl
[0036] each of which is unsubstituted or substituted by at least
one of NHR.sub.3, C.sub.1-6alkyl, --OC.sub.1-6alkyl, Br, Cl, F, I
or OH, wherein --R.sub.3 is H, C.sub.1-6alkyl or C.sub.1-6acyl.
[0037] In one embodiment, B is chosen from adenin-9-yl,
guanin-9-yl, inosin-9-yl, 2-amino-purin-9-yl,
2-amino-6-chloro-purin-9-yl, 0.2-6-diamino-purin-9-yl, thymin-1-yl,
cytosin-1-yl, uracil-1-yl, 3-carboxamido-1,2,4-triazol-1-yl each of
which is unsubstituted or substituted by at least one of NHR.sub.3,
C.sub.1-6alkyl, --OC.sub.1-6alkyl, Br, Cl, F, I or OH, wherein
R.sub.3 is H, C.sub.1-6alkyl or C.sub.1-6acyl.
[0038] In a further embodiment, B is chosen from adenin-9-yl,
guanin-9-yl, 2-amino-purin-9-yl, 2-amino-6-chloro-purin-9-yl,
2-6-diamino-purin-9-yl, thymin-1-yl, cytosin-1-yl, uracil-1-yl,
each of which is unsubstituted or substituted by at least one of
NHR.sub.3, C.sub.1-6alkyl, --OC.sub.1-6alkyl, Br, Cl, F, I or OH,
wherein R.sub.3 is H, C.sub.1-6alkyl or C.sub.1-6acyl.
[0039] In a further embodiment, B is chosen from guanin-9-yl,
cytosin-1-yl, uracil-1-yl, each of which is unsubstituted or
substituted by at least one of NHR.sub.3, --C.sub.1-6alkyl,
--OC.sub.1-6alkyl, Br, Cl, F, I or OH, wherein R.sub.3 is H,
C.sub.1-6alkyl or C.sub.1-6acyl.
[0040] In a further embodiment, B is cytosin-1-yl, which is
unsubstituted or substituted by at least one of NHR.sub.3,
C.sub.1-6alkyl, Br, Cl, F, I or OH, wherein R.sub.3 is H,
C.sub.1-6alkyl or C.sub.1-6acyl.
[0041] In a further embodiment, B is guanin-9-yl, which is
unsubstituted or substituted by at least one of NHR.sub.3,
C.sub.1-6alkyl, Br, Cl, F, I or OH, wherein R.sub.3 is H,
C.sub.1-6alkyl or C.sub.1-6acyl.
[0042] In a further embodiment, B is uracil-1-yl, which is
unsubstituted or substituted by at least one of NHR.sub.3,
C.sub.1-6alkyl, Br, Cl, F, I or OH, wherein R.sub.3 is H,
C.sub.1-6alkyl or C.sub.1-6acyl.
[0043] In one embodiment, B is chosen from adenin-9-yl,
guanin-9-yl, inosin-9-yl, 2-amino-purin-9-yl,
2-amino-6-chloro-purin-9-yl, 2-6-diamino-purin-9-yl, thymin-1-yl,
cytosin-1-yl, 5-fluoro-cytosin-1-yl, uracil-1-yl, 5-fluorouracil or
1,2,4-triazole-3-carboxamide base (ribarivin base).
[0044] In one embodiment, B is chosen from adenin-9-yl,
guanin-9-yl, inosin-9-yl, 2-amino-purin-9-yl,
2-amino-6-chloro-purin-9-yl, 2-6-diamino-purin-9-yl, thymin-1-yl,
cytosin-1-yl, 5-fluoro-cytosin-1-yl, uracil-1-yl, or
1,2,4-triazole-3-carboxamide base (ribarivin base).
[0045] In one embodiment, B is chosen from guanin-9-yl,
cytosin-1-yl, 5'-fluoro-cytosin-1-yl, 5'-fluorouracil-1-yl or
uracil-1-yl.
[0046] In one embodiment, B is chosen from guanin-9-yl,
cytosin-1-yl, 5'-fluoro-cytosin-1-yl, 5'-fluorouracil-1-yl or
uracil-1-yl.
[0047] In one embodiment, B is cytosin-1-yl.
[0048] In one embodiment, B is 5-fluoro-cytosin-1-yl.
[0049] In one embodiment, B is 5-fluorouracil.
[0050] In one embodiment, B is guanin-9-yl.
[0051] In oneembodiment, B is uracil-1-yl.
[0052] In a further embodiment, B is 5
[0053] Wherein;
[0054] X is H, halogen or NHR.sub.10, wherein R.sub.10 is H,
C.sub.1-6acyl, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, or C.sub.2-6
alkynyl;
[0055] Y is H, halogen or NHR.sub.11, wherein R.sub.11 is H,
C.sub.1-6acyl, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, or C.sub.2-6
alkynyl;
[0056] Y.sub.2 is H, halogen or NHR.sub.12, wherein R.sub.12 is H,
C.sub.1-6acyl, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, or C.sub.2-6
alkynyl;
[0057] R.sub.9 is H, hydroxy protecting group, C.sub.1-6acyl,
C.sub.1-6 alkyl, C.sub.2-6 alkenyl, or C.sub.2-6 alkynyl;
[0058] Y.sub.3 is H, halogen or NHR.sub.13, wherein R.sub.13 is H,
C.sub.1-6acyl, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, or C.sub.2-6
alkynyl;
[0059] R.sub.7 is H, halogen, C.sub.1-6acyl, C.sub.1-6 alkyl,
C.sub.2-6 alkenyl, or C.sub.2-6 alkynyl;
[0060] R.sub.8 is H, halogen, C.sub.1-6acyl, C.sub.1-6 alkyl,
C.sub.2-6 alkenyl, or C.sub.2-6 alkynyl.
[0061] In one embodiment,
[0062] X is H, halogen or NHR.sub.10, wherein R.sub.10 is H.
[0063] Y is H, halogen or NHR.sub.11, wherein R.sub.1, is H.
[0064] Y.sub.2 is H, halogen or NHR.sub.12, wherein R.sub.12 is
H.
[0065] R.sub.9 is H, hydroxy protecting group, C.sub.1-6 alkyl.
[0066] Y.sub.3 is H, halogen or NHR.sub.13, wherein R.sub.13 is
H.
[0067] R.sub.7 is H, halogen, or C.sub.1-6 alkyl.
[0068] Re is H, halogen or C.sub.1-6 alkyl.
[0069] In a further embodiment,
[0070] X is H, F, or NHR.sub.10 wherein R.sub.10 is H.
[0071] Y is H, F, or NHR.sub.11, wherein R.sub.11 is H.
[0072] Y.sub.2 is H, F, or NHR.sub.12, wherein R.sub.12 is H.
[0073] R.sub.9 is H.
[0074] Y.sub.3 is H, F, or NHR.sub.1., wherein R.sub.13 is H.
[0075] R.sub.7 is H, F, or C.sub.1-6 alkyl.
[0076] R.sub.8 is H, F, or C.sub.1-6 alkyl.
[0077] In one embodiment of the invention, Ra is chosen from H,
monophosphate, diphosphate, and triphosphate.
[0078] In another embodiment of the invention, Ra is H.
[0079] In one embodiment, Z is F or ORb, wherein Rb is chosen from
of H, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl,
C.sub.1-6 acyl, or an hydroxy protecting group.
[0080] In one embodiment, Z is F.
[0081] In one embodiment, Z is ORb, wherein Rb is chosen from of H,
C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-6
acyl, or an hydroxy protecting group.
[0082] In one embodiment, Z is ORb, wherein Rb is chosen from of H,
C.sub.1-6 alkyl, or an hydroxy protecting group.
[0083] In one embodiment, Z is ORb, wherein Rb ischosen from of H,
or methyl.
[0084] In one embodiment, Z is ORb, wherein Rb is H.
[0085] D.sub.1 and D.sub.2 are independently selected from N.sub.3,
F, or H, D and D.sub.2 can also be joined to be chosen from
cyclopropyl, difluorocyclopropyl-.dbd.CH.sub.2, or
-.dbd.CF.sub.2.
[0086] D.sub.1 and D.sub.2 are independently selected from F, or H,
D.sub.1 and D.sub.2 can also be joined to be chosen from
cyclopropyl, difluorocyclopropyl-.dbd.CH.sub.2, or
-.dbd.CF.sub.2.
[0087] D.sub.1 and D.sub.2 are joined and are cyclopropyl.
[0088] D.sub.1 and D.sub.2 are joined and are
difluorocyclopropyl.
[0089] D.sub.1 and D.sub.2 are joined and are -.dbd.CH.sub.2.
[0090] D.sub.1 and D.sub.2 are joined and are -.dbd.CF.sub.2.
[0091] In one embodiment, D.sub.1 is H or F.
[0092] In one embodiment, D.sub.2 is H or F.
[0093] In one embodiment, D.sub.1 is H.
[0094] In one embodiment, D.sub.2 is H.
[0095] In one embodiment, D.sub.1 is F.
[0096] In one embodiment, D.sub.2 is F.
[0097] In one embodiment, D.sub.1 is N.sub.3 and D.sub.2 is H.
[0098] In one embodiment, D.sub.1 is H and D.sub.2 is N.sub.3.
[0099] In one embodiment, D.sub.1 is N.sub.3 and D.sub.2 is F.
[0100] In one embodiment, D.sub.1 is F and D.sub.2 is N.sub.3.
[0101] In one embodiment, D.sub.1 is H and D.sub.2 is F.
[0102] In one embodiment, D.sub.1 is F and D.sub.2 is H.
[0103] In one embodiment, D.sub.1 and D.sub.2 are H.
[0104] In one embodiment, D.sub.1 and D.sub.2 are F.
[0105] In a further embodiment, the present invention relates to a
method for the treatment or prevention of Flavivirus infections
using nucleoside analogues in a host comprising administering a
therapeutically effective amount of a compound having the formula
Ib or a pharmaceutically acceptable salt thereof: 6
[0106] wherein Ra, B, D.sub.1, D.sub.2 and Z are as defined
above.
[0107] In a further embodiment, the present invention relates toa
method for the treatment or prevention of Flavivirus infections
using nucleoside analogues in a host comprising administering a
therapeutically effective amount of a compound having the formula
Ic or a pharmaceutically acceptable salt thereof: 7
[0108] wherein Ra, B, D.sub.1, D.sub.2 and Z are as defined
above.
[0109] In a further embodiment, the present invention relates to a
method for the treatment or prevention of Flavivirus infections
using nucleoside analogues in a host comprising administering a
therapeutically effective amount of a compound having the formula
Id or a pharmaceutically acceptable salt thereof: 8
[0110] wherein Ra, B, D.sub.1, D.sub.2 and Z are as defined
above.
[0111] In a further embodiment, the present invention relates to a
method for the treatment- or prevention of Flavivirus infections
using nucleoside analogues in a host comprising administering a
therapeutically effective amount of a compound having the formula
Ie or a pharmaceutically acceptable salt thereof: 9
[0112] wherein Ra, B, D.sub.1, D.sub.2 and Z are as defined
above.
[0113] In a further embodiment, the present invention relates to a
method for the treatment or prevention of Flavivirus infections
using nucleoside analogues in a host comprising administering a
therapeutically effective amount of a compound having the formula
If or a pharmaceutically acceptable salt thereof: 10
[0114] wherein Ra, B, and Z are as defined above.
[0115] In a further embodiment, the present invention relates to a
method for the treatment or prevention of Flavivirus infections
using nucleoside analogues in a host comprising administering a
therapeutically effective amount of a compound having the formula
Ig or a pharmaceutically acceptable salt thereof: 11
[0116] wherein Ra, B, and Z are as defined above.
[0117] In a further embodiment, the present invention relates to a
method for the treatment or prevention of Flavivirus infections
using nucleoside analogues in a host comprising administering a
therapeutically effective amount of a compound having the formula
Ih or a pharmaceutically acceptable salt thereof: 12
[0118] wherein Ra, B, and Z are as defined above.
[0119] In a further embodiment, the present invention relates to a
method for the treatment or prevention of Flavivirus infections
using nucleoside analogues in a host comprising administering a
therapeutically effective amount of a compound having the formula
Ii or a pharmaceutically acceptable salt thereof: 13
[0120] wherein Ra, B, and Z are as defined above.
[0121] In one embodiment, a compound of formula (I) is chosen
from:
1 3'-deoxycytidine Z = H, Compound #1, 3'-deoxycytidine-
5'triphosphate Z = triphosphate, Compound #2 14 5-Fluoro-
3'-deoxycytidine Z = H, Compound #3 5-Fluoro- 3'-deoxycytidine-
5'triphosphate Z = triphosphate, Compound #4 15 3'-deoxyuridine Z =
H, Compound #5 3'-deoxyuridine- 5'triphosphate Z = triphosphate,
Compound #6 16 5-Fluoro- 3'-deoxyuridine Z = H, Compound #7
5-Fluoro- 3'-deoxyuridine- 5'triphosphate Z = triphosphate,
Compound #8 17 3'-deoxythymidine Z = H, Compound #9
3'-deoxythymidine- 5'triphosphate Z = triphosphate, Compound #10 18
3'-deoxyguanosine Z = H, Compound #11 3'-deoxyguanosine-
5'triphosphate Z = triphosphate, Compound #12 19 2-N-acetyl-
3'-deoxyguanosine Z = H, Compound #13 2-N-acetyl-
3'-deoxyguanosine- 5'triphosphate Z = triphosphate, Compound #14 20
5-Methyl- 3'-deoxycytidine Z = H, Compound #15, 5-Methyl-
3'-deoxycytidine- 5'triphosphate Z = triphosphate, Compound #16 21
5-Iodo- 3'-deoxycytidine Z = H, Compound #17, 5-Iodo-
3'-deoxycytidine- 5'triphosphate Z = triphosphate, Compound #18 22
5-Chloro- 3'-deoxycytidine Z = H, Compound #19, 5- = Chloro-
3'-deoxycytidine- 5'triphosphate Z = triphosphate, Compound #20 23
3'-fluoro- 3'-deoxyguanosine Z = H, Compound #21 3'-fluoro-
3'-deoxyguanosine- 5'triphosphate Z = triphosphate, Compound #22 24
3'-fluoro- 3'-deoxycytidine Z = H, Compound #23, 3'-fluoro-
3'-deoxycytidine- 5'triphosphate Z = triphosphate, Compound #24 25
5-Iodo- 3'-deoxycytidine Z = H, Compound #25, 5- = Iodp-
3'-deoxycytidine- 5'triphosphate Z = triphosphate, Compound #26 26
5-Chloro- 3'-deoxyuridine Z = H, Compound #27 5-Chloro-
3'-deoxyuridine- 5'triphosphate Z = triphosphate, Compound #28 27
5-Bromo- 3'-deoxyuridine Z = H, Compound #29 5-Bromo-
3'-deoxyuridine- 5'triphosphate Z = triphosphate, Compound #30 28
6-Chloro- 3'-deoxyguanosine Z = H, Compound #31 6-Chloro-
3'-deoxyguanosine- 5'triphosphate Z = triphosphate, Compound #32 29
3'-spirocyclopropyl- 3'-deoxyguanosine Z = H, Compound #33
3'-spirocyclopropyl- 3'-deoxyguanosine 5'triphosphate Z =
triphosphate, Compound #34 30 3'-difluoro- spirocyclopropyl-
3'-deoxyguanosine Z = H, Compound #35 3'difluoro- spirocyclopropyl-
3'-deoxyguanosine 5'triphosphate Z = triphosphate, Compound #36 31
3'-methylene- 3'-deoxyguanosine Z = H, Compound #37 3'-methylene-
3'-deoxyguanosine 5'triphosphate Z = triphosphate, Compound #38 32
3'-difluromethylene 3'-deoxyguanosine Z = H, Compound #39
3'-difluromethylene 3'-deoxyguanosine 5'triphosphate Z =
triphosphate, Compound #40 33 3'-spiro- cyclopropyl- 3'-deoxycy-
tidine Z = H, Compound #41 3'-spiro- cyclopropyl- 3'-deoxycy-
tidine-5'triphosphate Z = triphosphate Compound #42 34 3'-difluoro-
spirocyclopropyl- 3'- deoxycytidine Z = H, Compound #43
3'-difluoro- spirocyclopropyl- 3'-deoxycytidine- 5'triphosphate Z =
triphosphate, Compound #44 35 3'-methylene- 3'-deoxycytidine Z = H,
Compound #45 3'-methylene- 3'-deoxycytidine- 5'triphosphate Z =
triphosphate, Compound #46 36 3'-difluromethylene 3'-deoxycytidine
Z = H, Compound #47 3'-difluromethylene 3'-deoxycytidine-
5'triphosphate Z = triphosphate, Compound #48 37
9-.beta.-D-xylofuranosyl- guanosine Z = H, Compound #49
9-.beta.-D-xylofuranosyl- guanosine- 5'triphosphate Z =
triphosphate, Compound #50 38 9-.beta.-D-xylofuranosyl- cytidine Z
= H, Compound #51 9-.beta.-D-xylofuranosyl- cytidine-
5'triphosphate Z = triphosphate, Compound #52 39 3'-azido-
3'-deoxycytidine Z = H, Compound #53 3'-azido- 3'-deoxycytidine
5'triphosphate Z = triphosphate, Compound #54 40
[0122] It will be appreciated by those skilled in the art that the
compounds of formula (I) contain at least three chiral centres and
which are marked by 1, 2 and 3. When D.sub.1 and D.sub.2 are
different, the compounds of formula (I) contain at least four
chiral centres which are marked by 1, 2, 3 and 4. The compounds of
formula (I) thus exist in the form of different optical isomers
(e.g .beta.-L and .beta.-D) and geometric isomers trans or .alpha.
and cis or .beta.. All such enantiomers, geometric isomers and
mixtures thereof including racemic mixtures are included within the
scope of the invention. The single optical isomer or enantiomer can
be obtained by method well known in the art, such as chiral HPLC,
enzymatic resolution and the use of chiral auxiliary.
[0123] According to one embodiment, the atoms marked by 1 and 2 are
in the cis or .beta. configuration.
[0124] According to one embodiment, the atoms marked by 1 and 2 are
in the cis or .beta. configuration while the atom marked by 3 is in
a trans or .alpha. configuration with respect to the atom 1 and
2.
[0125] According to one embodiment, compounds of formula I of the
present invention are provided substantially in the form of the
.beta.-D configuration.
[0126] According to one embodiment, compounds of formula I of the
present invention are provided substantially in the form of the
.beta.-L configuration.
[0127] By "substantially" is meant that there is more one
enantiomer then of the other enantiomer.
[0128] In another embodiment, the compounds of formula I of the
present invention are at least 95% free of the corresponding
.beta.-D enantiomer.
[0129] In another embodiment, the compounds of formula I of the
present invention are at least 97% free of the corresponding
.beta.-D enantiomer.
[0130] Still in another embodiment, the compounds of formula I of
the present invention are at least-99% free of the corresponding
.beta.-D enantiomer.
[0131] In another embodiment, the compounds of formula I of the
present invention are at least 95% free of--the corresponding
.beta.-L enantiomer.
[0132] In another embodiment, the compounds of formula I of the
present invention are at least 97% free of the corresponding
.beta.-L enantiomer.
[0133] Still in another embodiment, the compounds of formula I of
the present invention are at least 99% free of the corresponding
.beta.-L enantiomer.
[0134] There is also provided pharmaceutically acceptable salts of
the compounds of formula I of the present invention. By the term
pharmaceutically acceptable salts of the compounds of formula (I)
are meant those derived from pharmaceutically acceptable inorganic
and organic acids and bases. Examples of suitable acids include
hydrochloric, hydrobromic, sulphuric, nitric, perchloric, fumaric,
maleic, phosphoric, glycollic, lactic, salicylic, succinic,
toluene-p-sulphonic, tartaric, acetic, citric, methanesulphonic,
formic, benzoic, malonic, naphthalene-2-sulphonic and
benzenesulphonic acids.
[0135] Salts derived from appropriate bases include alkali metal
(e.g. sodium), alkaline earth metal (e.g. magnesium), ammonium and
NR.sub.4+ (where R is C.sub.1-4 alkyl) salts.
[0136] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. All
publications, patent applications, patents, and other references
mentioned herein are incorporated by reference in their entirety.
In case of conflict, the present specification, including
definitions, will control. In addition, the materials, methods, and
examples are illustrative only and not intended to be limiting.
[0137] As used in the present application, "compound(s) of formula
(I)" refers to all compounds identified by formula (I) and formulae
(Ia) to (Ii).
[0138] As used in this application, the term "purine or pyrimidine
or an analogue thereof" is meant a purine or pyrimidine base found
in nucleotide or an analogue thereof which mimics such bases in
that their structures (the kinds of atoms and their arrangement)
are similar to the normal bases but may possess additional or lack
certain of the functional properties of the normal bases. Such
analogues include those derived by replacement of a CH moiety by a
nitrogen atom (for example, 5-azapyrimidines such as 5-azacytosine)
or vice versa (for example 7-deazapurines, such as 7-deazadenosine
or 7-deazaguanosine) or both (e.g. 7-deaza, 8-azapurines).
Analogues of such bases also include those compounds wherein ring
substituents are either incorporated, removed or modified by
conventional substituents known in the art e.g. halogen, hydroxyl,
amino, C1-6 alkyl. Such purine or pyrimidine base, analogues and
derivatives will be well known to those skilled in the art.
[0139] As used in this application, the term "alkyl" represents an
unsubstituted or substituted (by a halogen, nitro, CONH.sub.2,
COOH, O--C.sub.1-6 alkyl, O--C.sub.2-6 alkenyl, O--C.sub.2-6
alkynyl, hydroxyl, amino, or COOQ, wherein Q is C.sub.1-6 alkyl;
C.sub.2-6 alkenyl; C.sub.2-6 alkynyl) straight chain, branched
chain or cyclic hydrocarbon moiety (e.g. isopropyl, ethyl,
fluorohexyl or cyclopropyl). The term alkyl is also meant to
include alkyls in which one or more hydrogen atoms is replaced by
an halogen, more preferably, the halogen is fluoro (e.g. CF.sub.3--
or CF.sub.3CH.sub.2--)
[0140] As used in this application, the term "cycloalkyl"
represents an "alkyl" as defined above which forms a ring.
[0141] The terms "alkenyl" and "alkynyl" represent an alkyl
containing at least one unsaturated group (e.g. allyl).
[0142] The term "hydroxy protecting group" is well known in the
field of organic chemistry. Such protecting groups may be found in
T. Greene, Protective Groups In Organic Synthesis, (John Wiley
& Sons, 1981). Example of hydroxy protecting groups include but
are not limited to acetyl-2-thioethyl ester, pivaloyloxymethyl
ester and isopropyloxycarbonyloxymethyl ester.
[0143] The term "aryl" represents an unsaturated carbocyclic
moiety, optionally mono- or di-substituted with OH, SH, amino,
halogen or C.sub.1-6 alkyl.
[0144] The term "heteroaryl" represents an aryl wherein at least
one carbon ring atom is substituted by an heteroatom (e.g. N, O, or
S).
[0145] The term "aminoalkyl" represents an alkyl which is
covalently bonded to the adjacent atom through a nitrogen atom.
[0146] The term "thioalkyl" represents an alkyl which is covalently
bonded to the adjacent atom through a sulfur atom.
[0147] The term "alkoxy" represents an alkyl which is covalently
bonded to the adjacent atom through an oxygen atom.
[0148] Halogen are chosen from F, Cl, I, and Br.
[0149] The term "host" represents any mammals including humans.
[0150] In one embodiment, the host is human.
[0151] The compounds of the present invention are can be prepared
by methods well known in the art. For example, such methods are
described in the following references J.Med.Chem. 1991, 34,
693-701; Chem. Pharm. Bull. 1995, 43(11) 2005-2009; J.Org.Chem.
1989, 54, 631-635; Can.J.Chem. 1975, 53(19), 2975-2977; Nucleosides
Nucleotides, 1990, 9(8), 1045-60 and Chemistry of Nucleosides and
Nucleotides edited by Leroy B. Towsend, 1988 Plenum Press Volumes 1
and 2; Synthesis of 2'-.beta.-fluoro- and
3'-.beta.-fluoro-substituted guanine nucleosides. Effect of sugar
conformational shifts on nucleophilic displacement of the
2'-hydroxy and 3'-hydroxy group with DAST. J. Org. Chem., 57(26),
(1992) 7315-21. Synthesis and antiviral and cytostatic properties
of 3'-deoxy-3'-fluoro- and
2'-azido-3'-fluoro-2',3'-dideoxy-D-ribofuranosides of natural
heterocyclic bases. J. Med. Chem. 34(7), (1991) 2195-202. Synthesis
of 9-(3-deoxy-3-fluoro-.beta.-D-ribofuranosyl)guanine, a new potent
antiviral agent. J. Chem. Soc., Chem. Commun. (1989) (1989), (14),
955-7. Synthesis and antiviral activity evaluation of
3'-fluoro-3'-deoxyribonucl- eosides: broad-spectrum antiviral
activity of 3'-fluoro-3'-deoxyadenosine. Antiviral Res. (1989),
12(3), 133-50. 3'-Fluoro-3'-deoxyribonucleoside 5'-triphosphates:
synthesis and use as terminators of RNA biosynthesis. FEBS Lett.
(1989), 0.250(2), 139-41. Reaction of 1-(2',3'-epoxy-.beta.-D--
lyxofuranosyl)uracil with hydrogen fluoride. The unexpected
formation of 1-(3'-fluoro-3'-deoxy-.beta.-D-ribofuranosyl)uracil.
J. Heterocycl. Chem. (1989), 21(3), 773-5. Synthesis of
3'-deoxy-3'-fluorouridine. J. Carbohydr., Nucleosides, Nucleotides
(1989), 2(3), 191-5. Synthesis of the 2'-deoxy-21-fluoro and
3'-deoxy-3'-fluoro analogs of 8-bromoadenosine. Nucleic Acids Symp.
Ser. (1989), 37(Symposium on Nucleic Acids Chemistry, 1997), 17-18.
Synthesis of 8-substituted analogs of 3'-deoxy-31-fluoroadenosine.
Nucleosides Nucleotides (1989), 0.17(1-3), 115-122. A new synthesis
of 3'-fluoro-3'-deoxyadenosine. Nucleosides Nucleotides (1989),
10(1-3), 719-21. Synthesis of 3'-fluoro-3'-deoxyadenosine starting
from adenosine. Synthesis (1989), (10), 900-5. Synthesis of
3'-deoxy-3'-fluoroadenosine by chemical transglycosidation. Z.
Chem. (1989), 29(6); 209-10. Stereoselective synthesis of
3'-deoxy-3'-fluoroadenosine. Bull. Chem. Soc. Jpn. (1989), 62(6),
2119-20. Synthesis of nucleosides fluorinated in the sugar moiety.
The application of diethylaminosulfur trifluoride to the synthesis
of fluorinated nucleosides. Nucleosides Nucleotides (1989), 8(1),
65-96. Preparation of difluorouridines as antitumor agents.
Efficient removal of sugar O-tosyl groups and heterocycle halogens
from purine nucleosides with sodium naphthalenide. Tetrahedron
(1989), 53(18), 6295-6302. Synthesis of fluoro and azido
derivatives of purine nucleosides from nucleoside 2',3'-cyclic
sulfates. Bioorg. Khim. (1989), 20(11), 1226-30. Synthesis of
modified oligomeric 2'-5' A analogs: potential antiviral agents.
Helv. Chim. Acta (1989), 74(1), 7-23. Diethylaminosulfur
trifluoride (DAST) as a fluorinating agent of pyrimidine
nucleosides having a 2',3'-vicinal diol system. Chem. Pharm. Bull.
(1989), 38(5), 1136-9. Synthesis of 9-(3-deoxy- and
2,3-dideoxy-3-fluoro-.beta.-D-xylofu- ranosyl)guanines as potential
antiviral agents. Tetrahedron Lett. (1989), 30(24), 3171-4.
Synthesis and anti-HIV activity of various 2'- and 3'-substituted
2',3'-dideoxyadenosines: a structure-activity analysis. J. Med.
Chem. (1989)., 30(11), 2131-7. Adenosine 2',3'-ribo-epoxide.
Synthesis, intramolecular degradation, and transformation into
3'-substituted xylofuranosyl nucleosides and the lyxo-epoxide. J.
Org. Chem. (1989), 39(11), 1564-70. Fluoro sugar analogs of
arabinosyl- and xylosylcytosines. J. Med. Chem. (1989), 13(2),
269-72. 9-(3-Deoxy-3-fluoro-.beta.-D-xylofuranosyl)adenine and
9-(3-deoxy-3-fluoro-.beta.-D-arabinofuranosyl)adenine. Carbohyd.
Res. (1989), 6(3), 347-54. 3',3'-Difluoro-3.sup.1-deoxythymidine:
comparison of anti-HIV activity to 3'-fluoro-3'-deoxythymidine. J.
Med. Chem. (1989), 35(18), 3369-72. Nucleic acid related compounds.
83. Synthesis of 3'deoxyadenosine-3'-spirocyclopropane,
31-deoxyuridine-3'-spirocyclopropa- ne, and
5'-deoxy-4',51-methanoadenosine. Tetrahedron Lett. (1989), 35(21),
3445-8. Synthesis of 2',3'-didehydro-21,31-dideoxy-3'-C-methyl
substituted nucleosides. Nucleosides Nucleotides (1989), 12(8),
865-77. 2',3'-Didehydro-2',3'-dideoxy-2'(and3')-methylnucleosides
via [3,3]-sigmatropic rearrangements of 2'(and
3')-methylene-3.sup.1'(and 2')--O-thiocarbonyl derivatives and
radical reduction of a 2'-chloro-3.sup.1-methylene analog. Can. J.
Chem. (1989), 71(2), 186-91. Synthesis and biological activity of
2' (and 3')-deoxy-2.sup.1 (and 3')-methylenenucleoside analogs that
function as mechanism-based inhibitors of S-adenosyl-L-homocysteine
hydrolase and/or ribonucleotide reductase. J. Med. Chem. (1989),
35(12), 2283-93. Synthesis and anticancer and antiviral activities
of various 2'- and 3'-methylidene-substituted nucleoside analogs
and crystal structure of 2'-deoxy-2'-methylidenecytidine
hydrochloride. J. Med. Chem. (1989), 34(8), 2607-15.
Stereoselective addition of a Wittig reagent to give a single
nucleoside oxaphospetane diastereoisomer. Synthesis of 2' (and
3')-deoxy-2' (and 3')-methyleneuridine (and cytidine) derivatives
from uridine ketonucleosidesSynthesis (1989), (4), 282-8. A novel
example of unsaturated branched chain sugar nucleoside:
3'-deoxy-3'-methylideneadeno- sine. Helv. Chim. Acta (1989), 64(2),
425-9. Synthesis of 2' (and 3')-deoxy-2' (and
3')-methyleneadenosines and bis(methylene)furan
4',5'-didehydro-5'-deoxy-2'(and 3.sup.1)-methyleneadenosines.
Inhibitors of S-adenosyl-L-homocysteine hydrolase and
ribonucleotide reductase. J. Org. Chem. (1989), 56(25), 7108-13.
Radical and palladium-catalyzed deoxygenation of the allylic
alcohol systems in the sugar moiety of pyrimidine nucleosides.
Nucleosides Nucleotides (1989), 11(2-4), 197-226. Synthesis and NMR
spectra of some new carbohydrate modified uridine phosphoramidites.
Nucleosides Nucleotides (1989), 16(7-9), 1529-1532. New method for
the preparation of 3'- and 2'-phosphoramidites of 2'- and
3'-difluoromethyleneuridine. Tetrahedron (1989), 52(23), 7929-7938.
Nucleic acid related compounds. 83. Synthesis of
3'deoxyadenosine-3'-spir- ocyclopropane,
3'-deoxyuridine-3'-spirocyclopropane, and
5'-deoxy-4',5'-methanoadenosine. Tetrahedron Lett. (1989), 35(21),
3445-8. Some compounds of the present invention are commercially
available at Sigma or Aldrich.
[0152] According to one embodiment, it will be appreciated that the
amount of a compound of formula I of the present invention required
for use in treatment will vary not only with the particular
compound selected but also with the route of administration, the
nature of the condition for which treatment is required and the age
and condition of the patient and will be ultimately at the
discretion of the attendant physician or veterinarian. In general
however a suitable dose will be in the range of from about 0.01 to
about 750 mg/kg of body weight per day, preferably in the range of
0.5 to 60 mg/kg/day, most preferably in the range of 1 to 20
mg/kg/day.
[0153] The desired dose according to one embodiment is conveniently
presented in a single dose or as divided dose administered at
appropriate intervals, for example as two, three, four or more
doses per day.
[0154] In another embodiment, the compound is conveniently
administered in unit dosage form; for example containing 10 to 1500
mg, conveniently 20 to 1000 mg, most conveniently 50 to 700 mg of
active ingredient per unit dosage form.
[0155] According to another embodiment of the present invention,
the active ingredient is administered to achieve peak plasma
concentrations of the active compound of from about 1 to about 75
.mu.M, preferably about 2 to 50 .mu.M, most preferably about 3 to
about 30 .mu.M. This may be achieved, for example, by the
intravenous injection of a 0.1 to 5% solution of the active
ingredient, optionally in saline, or orally administered as a bolus
containing about 1 to about 500 mg of the active ingredient.
Desirable blood levels may be maintained by a continuous infusion
to provide about 0.01 to about 5.0 mg/kg/hour or by intermittent
infusions containing about 0.4 to about 15 mg/kg of the active
ingredient.
[0156] While it is possible that, for use in therapy, a compound of
formula I of the present invention may be administered as the raw
chemical, it is preferable according to one embodiment of the
invention, to present the active ingredient as a pharmaceutical
formulation. The embodiment of the invention thus further provides
a pharmaceutical formulation comprising a compound of formula (I)
or a pharmaceutically acceptable salt thereof together with one or
more pharmaceutically acceptable carriers therefor and, optionally,
other therapeutic and/or prophylactic ingredients. The carrier(s)
must be "acceptable" in the sense of being compatible with the
other ingredients of the formulation and not deleterious to the
recipient thereof. According to one embodiment of the present
invention, pharmaceutical formulations include but are not limited
to those suitable for oral, rectal, nasal, topical (including
buccal and sub-lingual), transdermal, vaginal or parenteral
(including intramuscular, sub-cutaneous and intravenous)
administration or in a form suitable for administration by
inhalation or insufflation. The formulations may, where
appropriate, be conveniently presented in discrete dosage units and
may be prepared by any of the methods well known in the art of
pharmacy. All methods according to this embodiment include the step
of bringing into association the active compound with liquid
carriers or finely divided solid carriers or both and then, if
necessary, shaping the product into the desired formulation.
[0157] According to another embodiment, pharmaceutical formulation
suitable for oral administration are conveniently presented as
discrete units such as capsules, cachets or tablets each containing
a predetermined amount of the active ingredient; as a powder or
granules. In another embodiment, the formulation is presented as a
solution, a suspension or as an emulsion. Still in another
embodiment, the active ingredient is presented as a bolus,
electuary or paste.
[0158] Tablets and capsules for oral administration may contain
conventional excipients such as binding agents, fillers,
lubricants, disintegrants, or wetting agents. The tablets may be
coated according to methods well known in the art. Oral liquid
preparations may be in the form of, for example, aqueous or oily
suspensions, solutions, emulsions, syrups or elixirs, or may be
presented as a dry product for constitution with water or other
suitable vehicle before use. Such liquid preparations may contain
conventional additives such as suspending agents, emulsifying
agents, non-aqueous vehicles (which may include edible oils), or
preservatives.
[0159] The compounds of formula I according to an embodiment of the
present invention are formulated for parenteral administration
(e.g. by injection, for example bolus injection or continuous
infusion) and may be presented in unit dose form in ampoules,
pre-filled syringes, small volume infusion or in multi-dose
containers with an added preservative. The compositions may take
such forms as suspensions, solutions, or emulsions in oily or
aqueous vehicles, and may contain formulatory agents such as
suspending, stabilizing an/or dispersing agents. Alternatively, the
active ingredient may be in powder form, obtained by aseptic
isolation of sterile solid or by lyophilisation from solution, for
constitution with a suitable vehicle, e.g. sterile, pyrogen-free
water, before use.
[0160] For topical administration to the epidermis, the compounds
of formula I, according to one embodiment of the present invention,
are formulated as ointments, creams or lotions, or as a transdermal
patch. Such transdermal patches may contain penetration enhancers
such as linalool, carvacrol, thymol, citral, menthol and
t-anethole. Ointments and creams may, for example, be formulated
with an aqueous or oily base with the addition of suitable
thickening and/or gelling agents. Lotions may be formulated with an
aqueous or oily base and will in general also contain one or more
emulsifying agents, stabilizing agents, dispersing agents,
suspending agents, thickening agents, or colouring agents.
[0161] Formulations suitable for topical administration in the
mouth include lozenges comprising active ingredient in a flavoured
base, usually sucrose and acacia or tragacanth; pastilles
comprising the active ingredient in an inert base such as gelatin
and glycerin or sucrose and acacia; and mouthwashes comprising the
active ingredient in a suitable liquid carrier.
[0162] Pharmaceutical formulations suitable for rectal
administration wherein the carrier is a solid. In another
embodiment, they are presented as unit dose suppositories. Suitable
carriers include cocoa butter and other materials commonly used in
the art, and the suppositories may be conveniently formed by
admixture of the active compound with the softened or melted
carrier(s) followed by chilling and shaping in moulds.
[0163] According to one embodiment, the formulations suitable for
vaginal administration are presented as pessaries, tampons, creams,
gels, pastes, foams or sprays containing in addition to the active
ingredient such carriers as are known in the art to be
appropriate.
[0164] For intra-nasal administration the compounds, in one
embodiment of the invention, are used as a liquid spray or
dispersible powder or in the form of drops. Drops may be formulated
with an aqueous or non-aqueous base also comprising one more
dispersing agents, solubilising agents or suspending agents. Liquid
sprays are conveniently delivered from pressurized packs.
[0165] For administration by inhalation the compounds, according to
one embodiment of the invention are conveniently delivered from an
insufflator, nebulizer or a pressurized pack or other convenient
means of delivering an aerosol spray. In another embodiment,
pressurized packs comprise a suitable propellant such as
dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In
another embodiment, the dosage unit in the pressurized aerosol is
determined by providing a valve to deliver a metered amount.
[0166] Alternatively, in another embodiment, for administration by
inhalation or insufflation, the compounds of formula I according to
the present invention are in the form of a dry powder composition,
for example a powder mix of the compound and a suitable powder base
such as lactose or starch. In another embodiment, the powder
composition is presented in unit dosage form in, for example,
capsules or cartridges or e.g. gelatin or blister packs from which
the powder may be administered with the aid of an inhalator or
insufflator.
[0167] In one embodiment, the above described formulations are
adapted to give sustained release of the active ingredient.
[0168] The compounds of the invention may also be used in
combination with other antiviral agents.
[0169] In one embodiment, the compounds of the invention may be
employed together with at least one other antiviral agent chosen
from protease inhibitors, polymerase inhibitors, and helicase
inhibitors.
[0170] As used in this application, the term "interferon" include:
interferon likes molecules such as interferon (IFN), interferon
.alpha.-2a, interferon .alpha.-2b, consensus interferon (CIFN) and
other types of interferons.
[0171] In one embodiment, the compounds of the invention may be
employed together with at least one other antiviral agent chosen
from interferon (IFN), interferon .alpha.-2a, interferon
.alpha.-2b, consensus interferon (CIFN), ribavirin, amantadine,
rimantadine, interleukine-12, ursodeoxycholic acid (UDCA),
glycyrrhizin and silybum marianum.
[0172] In one embodiment, the compounds of the invention may be
employed together with at least one other antiviral agent chosen
from Interferon-.alpha., Ribavirin and Amantadine.
[0173] In one embodiment, the compounds of the invention may be
employed together with at least one other antiviral agent chosen
from Interferon-.alpha. and Ribavirin (REBETRON).
[0174] In one embodiment, the compounds of the invention may be
employed together Interferon-.alpha..
[0175] In one embodiment, the compounds of the invention may be
employed together with Ribavirin.
[0176] The combinations referred to above may conveniently be
presented for use in the form of a pharmaceutical formulation and
thus pharmaceutical formulations comprising a combination as
defined above together with a pharmaceutically acceptable carrier
therefor comprise a further aspect of the invention.
[0177] The individual components of such combinations may be
administered either sequentially or simultaneously in separate or
combined pharmaceutical formulations.
[0178] When the compound (I) or a pharmaceutically acceptable salts
thereof is used in combination with a second therapeutic agent
active against the same virus the dose of each compound may be
either the same as or differ from that when the compound is used
alone.
[0179] Appropriate doses will be readily appreciated by those
skilled in the art.
[0180] The following examples are provided to illustrate various
embodiments of the present invention and shall not be considered as
limiting in scope.
EXAMPLE 1
Preparation of 3'-DEOXYCYTIDINE 5'-TRIPHOSPHATE TRIAMMONIUM SALT
(Compound #2)
[0181] 41
[0182] Procedure: To a stirring suspension of
3'-deoxy-2'-acetoxycytidine (15.0 mg, 0.056 mmol) in dry DMF (0.60
ml) was added dry pyridine (0.20 ml) followed by a freshly prepared
solution of 2-chloro-4H-1,3,2-benzodio- xaphosphorin-4-one. 0. 5M
in 1,4-dioxane (111 .mu.l, 0.056 mmol). The mixture was stirred 30
minutes at room temperature, then tributylamine (36 .mu.l, 0.152
mmol) and a solution of tributylammonium pyrophosphate 0.5 M in DMF
(101 .mu.l, 0.051 mmol) were added simultaneously. The mixture was
stirred another 30 minutes. A solution of I2 1% in
pyridine/H.sub.2O (98:2) (1.01 ml, 0.081 mmol of I) was added and
the mixture was stirred 30 minutes. The excess of iodine was
destroyed by adding 0.2 ml of aqueous sodium bisulfite 5%. The
mixture was stirred 15 minutes, then it was concentrated under
reduced pressure to remove all solvents. The residue was dissolved
in water, washed two times with methylene chloride and once with
ethyl acetate. The aqueous layer was concentrated and purified by
charcoal column as follow: about 400 mg of charcoal, placed over a
thin layer of Celite in a funnel with fritted disk, was prewashed
by passing methanol, then deionized water (by vaccuum). The crude
residue was diluted in a minimum of water, acidified to pH 1-2 by
adding few drops of HCl 1N, then placed on the top of the charcoal
column. The column was eluted with deionized water (35 ml) in order
to remove inorganic salts, then 0.5 N ammonia (15 ml) to collect
the desired triphosphate. The collected triphophate wasconcentrated
and diluted in deionized water (1 ml) and concentrated NH4OH (2
ml). The mixture was stirred one hour at room temperature to cleave
the acetyl group, then concentrated to dryness. The residue was
purified on a pad of C18 RP silica gel eluting with deionized water
(the desired triphosphate comes out fast). The fractions containing
the desired triphosphate were collected and lyophilized to give,
the 3'-deoxycytidine 5'-triphosphate triammonium salt as a
yellowish solid (18 mg, 69% yield, purity>85% evaluated by 1H
and 31P-NMR). .sup.1H NMR (400 MHz, D.sub.2O) .delta.: 7.90 (d, 1H,
7.5 Hz), 5.99 (d, 1H, 7.5 Hz), 5.73 (s, 1H), 4.55 (s, 1H), 4.35 (d,
1H, 5.0 Hz), 4.26 (m, 1H), 4.04 (m, 1H), 2.05 (m, 1H), 1.94 (m, 1H)
ppm. .sup.31P NMR (162 MHz, D.sub.2O) .delta.: -5.9 (br.s), -10.4
(d, 19 Hz), -21.5 (br.s) ppm. In a similar manner, the compounds of
the invention can be obtained.
EXAMPLE 2
Evaluation of Triphosphate Analogues
[0183] In The HCV RNA-Dependent RNA Polymerase AssayThe following
references which are referenced in the example are all incorporated
by reference:
[0184] 1. Behrens, S., Tomei, L., De Francesco, R. (1989) EMBO 15,
12-22
[0185] 2. Harlow, E, and Lane, D. (1989) Antibodies: A Laboratory
Manual. Cold Spring Harbord Laboratory. Cold Spring Harbord.
NY.
[0186] 3. Lohmann, V., Korner, F., Herian, U., and Bartenschlager,
R. (1989) J. Virol. 71, 8416-8428
[0187] Compounds were evaluated using an in vitro polymerase assay
containing purified recombinant HCV RNA-dependent RNA polymerase
(NS5B protein). HCV NS5B was expressed in insect cells using a
recombinant baculovirus as vector. The experimental procedures used
for the cloning, expression and purification of the HCV NS5B
protein are described below. Following are details of the
RNA-dependent RNA polymerase assays used to test the compounds.
[0188] Expression of the HCV NS5B Protein in Insect Cells:
[0189] The cDNA encoding the entire NS5B protein of HCV-Bk strain,
genotype 1b, was amplified by PCR using a plasmid containing a cDNA
version of the full-length HCV genome as template. The
oligonucleotides used to amplify this HCV region were designed to
introduce a NheI site followed by an ATG at the 5' end of the NS5B
coding region as well as a BamHI site at the 3'end immediately
downstream of the translation stop codon. The amplified sequence,
of 1.8 kb, was digested with NheI and BamHI and ligated to a
predigested pBlueBacII plasmid (Invitrogen). The resulting
recombinant plasmid was designated pBac/NS5B. Sf9 cells were
co-transfected with 3 .mu.g of pBac/NS5B, together with 1 .mu.g of
linearized baculovirus DNA (Invitrogen), as described in the
manufacturer's protocol. Following two rounds of plaque
purification, an NS5B-recombinant baculovirus, BacNS5B, was
isolated. The presence of the recombinant NS5B protein was
determined by western blot analysis (Harlow and Lane, 1988) of
BacNS5B-infected Sf9 cells, using a HCV NS5B specific rabbit
polyclonal antiserum (anti-NS5B). Infections of Sf9 cells with this
plaque purified virus were performed in one-liter spinner flasks at
a cell density of 1.2.times.10.sup.6 cells/ml and a multiplicity of
infection of 5.
[0190] Preparation of a Soluble Recombinant NS5B Protein:
[0191] Sf9 cells were infected as described above. Sixty hours
post-infection, cells were harvested then washed twice with
phosphate buffer saline (PBS). Total proteins were solubilized as
described in Lohmann et al. (1989) with some modifications. In
brief, proteins were extracted in three steps, S1, S2, S3, using
lysis buffers (LB) I, LB II and LB III (Lohmann et al, 1997). The
composition of LBII was modified to contain 0.1% triton X-100 and
150 mM NaCl to reduce the amount of solubilized NS5B protein at
this step. In addition, sonication of cell extracts was avoided
throughout the protocol to preserve the integrity of the protein
structure.
[0192] Purification of Recombinant NS5B Using Fast Protein Liquid
Chromatography (FPLC):
[0193] Soluble NS5B protein in the S3 fraction was diluted to lower
the NaCl concentration to 300 mM, then it incubated batchwise with
DEAE sepharose beads (Amersham-Pharmacia) for 2 hrs at 0.4?C, as
described by Behrens et al. (1989). Unbound material was cleared by
centrifugation for 15 min at 4.degree. C., at 25 000 rpm using a
SW41 rotor (Beckman). The supernatant was further diluted to lower
the NaCl concentration to 200 mM and subsequently loaded, with a
flow rate of 1 ml/min, on a 5 ml HiTrap.RTM. heparin column
(Amersham-Pharmacia) connected to an FPLC.RTM. system
(Amersham-Pharmacia). Bound proteins were eluted in 1 ml fractions,
using a continuous NaCl gradient of 0.2 to 1 M, over a 25 ml
volume. NS5B-containing fractions were identified by sodium dodecyl
sulfate polyacrylamide gel electrophoresis (SDS-PAGE), followed by
western blotting using the anti-NS5B antiserum at a dilution of
1:2000. Positive fractions were pooled and the elution buffer was
exchanged against a 50 mM NaPO.sub.4 pH 7.0, 20% glycerol, 0.5%
triton X-100 and 10 mM DTT, using a PD-10 column
(Amersham-Pharmacia). The sample was then loaded onto a 1 ml
HiTrap.RTM. SP column (Amersham-Pharmacia), with a flow rate of 0.1
ml/min. Bound proteins were eluted using a continuous 0 to 1 M NaCl
gradient over a 15 ml volume. Eluted fractions were analyzed by
SDS-PAGE and western blotting. Alternatively, proteins were
visualized, following SDS-PAGE, by silver staining using the Silver
Stain Plus kit (BiORad) as described by the manufacturer. Positive
fractions were tested for RdRp activity (see below) and the most
active ones were pooled, and stored as a 40% glycerol solution at
-70.degree. C.
[0194] In Vitro RNA-Dependent RNA Polymerase Assays Used to
Evaluate the Triphosphate Form of Nucleoside Analogues:
[0195] RdRp assays were conducted using in vitro transcribed
heteropolymeric RNA templates.
[0196] RdRp reactions were performed in a total volume of 50 .mu.l
of a buffer consisting of 20 mM Tris-HCl pH 7.5, 1 mM DTT, 50 mM
NaCl, 0.5 mM MnCl.sub.2 and 5 mM MgCl.sub.2. Standard HCV RdRp
reactions contained 200 ng of purified NS5B protein. The substrate
mixture included in the, assay depended on the base of the
nucleoside triphosphate to be tested (adenine, guanine, cytosine or
uracil analogue). The NTP substrate with a similar base to that of
the inhibitor, was added at twice the measured Km. This
concentration included 5 uCi (3000 Ci/mmol) of a. [.sup.32p]
version of this nucleotide. The remaining three substrates were
used at 100 .mu.M. The measured Kms for the four substrates were as
follows: 18 .mu.M for ATP, 0.5 .mu.M for CTP and GTP, and 1.2 .mu.M
for UTP. Following a two hour incubation at 22.degree. C.,
reactions were stopped by the addition of 100 .mu.g of sonicated
salmon sperm DNA (Life Technologies) and 1 ml of 10%
trichloroacetic acid (TCA)-0.5% tetrasodium pyrophosphate (PPi).
Nucleic acids were precipitated at 4.degree. C. for 30 min after
which samples were filtered on GF/C glass microfiber filters
(Millipore). Membranes were subsequently washed with 25 ml of a 1%
TCA-0.1% PPi solution, then air dried. Incorporated radioactivity
was quantified using a liquid scintillation counter
(1450-Microbeta, Wallac).
[0197] Heteropolymeric RNA templates were generated by run-off
transcription. As template for these transcription reactions, a
recombinant pcDNA3 plasmid (Invitrogen) containing a cDNA version
of the HCV genome was used and referred to as pcDNA/HCVfl. In vitro
transcriptions were performed using the MEGAscript.TM. kit
(Ambion), as suggested by the manufacturer. In brief, the plasmid
pcDNA/HCVfl was linearized with EcOR1 to generate a truncated HCV
transcript of about 6900 nucleotides. Linearized DNA was extracted
with a one to one volume of phenol/chloroform, precipitated with
ethanol, then-1 .mu.g of this linearized DNA was used as template
in T7 RNA polymerase-driven in vitro transcription reactions.
Transcripts were extracted using the TRIZOL.RTM. reagent (Life
Technologies) and an aliquot (1 .mu.g) was used as template in RdRp
assays.
2 HCV polymerase Compound IC.sub.50 COMPOUND#2 0.036 .mu.M
COMPOUND#4 0.3 .mu.M COMPOUND#6 0.26 .mu.M COMPOUND#8 1.98 .mu.M
COMPOUND#10 6.4 .mu.M COMPOUND#12 0.048 .mu.M COMPOUND#14 3.1 .mu.M
COMPOUND#16 0.36 .mu.M COMPOUND#18 6.88 .mu.M COMPOUND#20 0.18
.mu.M COMPOUND#22 0.12 .mu.M COMPOUND#24 0.055 .mu.M COMPQUND#26
0.91 .mu.M COMPOUND#28 2.1 .mu.M COMPOUND#30 2.9 .mu.M COMPOUND#32
6.8 .mu.M COMPOUND#54 9.0 .mu.M
* * * * *