U.S. patent application number 11/005472 was filed with the patent office on 2007-03-15 for 2' and 3'-nucleoside prodrugs for treating flaviviridae infections.
Invention is credited to Gilles Gosselin, Paola LaColla, Jean-Pierre Sommadossi, Richard Storer.
Application Number | 20070060505 11/005472 |
Document ID | / |
Family ID | 30003834 |
Filed Date | 2007-03-15 |
United States Patent
Application |
20070060505 |
Kind Code |
A1 |
Gosselin; Gilles ; et
al. |
March 15, 2007 |
2' and 3'-nucleoside prodrugs for treating Flaviviridae
infections
Abstract
2' and 3'-Prodrugs of 1', 2', 3' or 4'-branched .beta.-D or
.beta.-L nucleosides, or their pharmaceutically acceptable salts
and derivatives are described, which are useful in the prevention
and treatment of Flaviviridae infections and other related
conditions. These modified nucleosides provide superior results
against flaviviruses and pestiviruses, including hepatitis C virus
and viruses generally that replicate through an RNA dependent RNA
reverse transcriptase. Compounds, compositions, methods and uses
are provided for the treatment of Flaviviridae infection, including
HCV infection, that include the administration of an effective
amount of the prodrugs of the present invention, or their
pharmaceutically acceptable salts or derivatives. These drugs may
optionally be administered in combination or alteration with
further anti-viral agents to prevent or treat Flaviviridae
infections and other related conditions.
Inventors: |
Gosselin; Gilles;
(Monpellier, FR) ; Storer; Richard; (Folkstone,
GB) ; LaColla; Paola; (Cagliari, IT) ;
Sommadossi; Jean-Pierre; (Cambridge, MA) |
Correspondence
Address: |
KING & SPALDING LLP
1180 PEACHTREE STREET
ATLANTA
GA
30309
US
|
Family ID: |
30003834 |
Appl. No.: |
11/005472 |
Filed: |
December 6, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10609298 |
Jun 27, 2003 |
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11005472 |
Dec 6, 2004 |
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60392351 |
Jun 28, 2002 |
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60466194 |
Apr 28, 2003 |
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60470949 |
May 14, 2003 |
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Current U.S.
Class: |
424/85.4 ;
514/20.3; 514/26; 514/3.7; 514/4.3; 514/47; 530/322; 536/27.3;
536/6.1 |
Current CPC
Class: |
C07H 19/06 20130101;
A61K 2300/00 20130101; A61P 1/16 20180101; C07H 19/067 20130101;
C07K 9/003 20130101; A61K 45/06 20130101; A61K 31/7072 20130101;
A61K 31/7068 20130101; A61P 31/12 20180101; A61K 31/7068 20130101;
A61K 31/7072 20130101; A61P 43/00 20180101; A61K 38/00 20130101;
A61P 31/14 20180101; A61K 2300/00 20130101 |
Class at
Publication: |
514/008 ;
514/047; 514/026; 536/027.3; 536/006.1; 530/322 |
International
Class: |
A61K 31/7076 20060101
A61K031/7076; C07H 19/16 20060101 C07H019/16; A61K 38/14 20060101
A61K038/14 |
Claims
1. (canceled)
2. A method for the treatment of a host infected with a
Flaviviridae virus, comprising administering an effective treatment
amount of a compound of formula ##STR76## or a pharmaceutically
acceptable salt thereof wherein: Base is a pyrrolopyrimidine:
R.sup.1 is hydrogen, mono, di or triphosphate or a stabilized
phosphate; acyl; an amino acid ester; a carbohydrate; a peptide; or
a pharmaceutically acceptable leaving group which when administered
in vivo is capable of providing a compound wherein R.sup.1 is
independently H or phosphate; and R.sup.2 is acyl; an amino acid
ester; a carbohydrate; a peptide; or a pharmaceutically acceptable
leaving group which when administered in vivo is capable of
providing a compound wherein R.sup.2 is hydrogen; R.sup.6 is alkyl,
alkenyl or alkynyl.
3. The method of claim 2, wherein the virus is hepatitis C.
4. The method of claim 2, wherein the compound or pharmaceutically
acceptable salt, thereof, is administered in combination or
alternation with a second anti-viral agent.
5. The method of claim 2 wherein the second anti-viral agent is
selected from the group consisting of an interferon, a ribavirin,
an interleukin, a NS3 protease inhibitor, a HCV helicase inhibitor,
a polymerase inhibitor a nucleoside, and an inhibitor of
IRES-dependent translation, and a ribozyme.
6. The method of claim 2, wherein the second anti-viral agent is an
interferon.
7. The method of claim 6, wherein the second agent is selected from
the group consisting of pegylated interferon alpha 2a, interferon
alphacon-1, natural interferon, albuferon, interferon beta-1a,
omega interferon, interferon alpha, and interferon gamma-1b.
8. The method of claim 2, wherein the compound or pharmaceutically
acceptable salt thereof is in the form of a dosage unit.
9. The method of claim 8, wherein the dosage unit is a tablet or
capsule.
10. The method of claim 2, wherein the host is a human.
11. The method of claim 2, wherein the compound is in substantially
pure form.
12. The method of claim 2, wherein the compound is at least 90% by
weight of the .beta.-D-isomer.
13. The method of claim 2, wherein the compound or a
pharmaceutically acceptable salt thereof is administered in
combination with a pharmaceutically acceptable carrier to form a
composition.
14. The of claim 13, comprising administering the composition in a
form that is suitable for oral delivery.
15. The method of claim 13, wherein the composition is in the form
of a dosage unit.
16. The method of claim 15, wherein the dosage unit contains 50 to
1000 mg of the compound.
17. The method of claim 16, wherein said dosage unit is a tablet or
capsule.
18. (canceled)
19. The method of claim 13, wherein the compound or
pharmaceutically acceptable salt thereof is in substantially pure
form.
20. The method of claim 13, wherein the pharmaceutically acceptable
carrier is suitable for systemic, topical, parenteral, inhalant or
intravenous delivery.
21. The method of claim 2, wherein R.sup.1 is a mono, di or
triphosphate.
22. The method of claim 2, wherein R.sup.2 is acyl.
23. The method of claim 2, wherein R.sup.2 is an amino acid
ester.
24. The method of claim 2, wherein R.sup.2 is a peptide.
25. The method of claim 2, wherein R.sup.2 is a carbohydrate.
26. The method of claim 2, wherein R.sup.1 is hydrogen.
27. The method of claim 2, wherein R.sup.1 is H and R.sup.6 is
alkyl.
28. The method of claim 21, wherein R.sup.6 is alkyl.
29. The method of claim 23, wherein R.sup.6 is alkenyl.
30. The method of claim 2, wherein R.sup.6 is alkynyl.
31. The method of claim 2, wherein R.sup.6 is
--CH.dbd.CH.sub.2.
32. The method of claim 30, wherein R.sup.6 is ethynyl.
33. The method of claim 28, wherein R.sup.6 is methyl, ethyl, or
propyl.
34. The method of claim 22, wherein acyl is of the formula C(O)R',
wherein R' is a straight, branched, or cyclic alkyl.
35. The method of claim 22, wherein acyl is of the formula C(O)R',
wherein R' is aryl, alkaryl, aralkyl alkoxyalkyl or
aryloxyalkyl.
36. The method of claim 22, wherein acyl is of the formula C(O)R'
wherein R' is aryl.
37. The method of claim 22, wherein R.sup.2 is acetyl.
38. The method of claim 22, wherein R.sup.2 is propionyl, butyryl,
hexanoyl, or 2-propenyl.
39. The method of claim 2, wherein R.sup.2 is an ester of an amino
acid selected from the group consisting of glycine, alanine,
valine, leucine, isoleucine, methionine, phenylalanine, tryptophan,
proline, serine, threonine, cysteine, tyrosine, asparagine,
glutamine, aspartate, glutamate, lysine, arginine and
histidine.
40. The method of claim 2, wherein R.sup.2 is an ester of a
naturally occurring or synthetic .alpha., .beta., .gamma., or
.delta. amino acid.
41. The method of claim 2, wherein R.sup.2 is an ester of an amino
acid in the L configuration.
42. The method of claim 2, wherein R.sup.2 is an ester of
valine.
43. The method of claim 2, wherein host is human.
44. The method of claim 2, wherein: R.sup.1 is H, mono, di or
triphosphate or a stabilized phosphate; R.sup.2 is acyl or an amino
acid ester; and R.sup.6 is methyl, ethyl or propyl.
45. The method of claim 44, wherein R.sup.2 is an ester of an amino
acid selected from the group consisting of glycine, alanine,
valine, leucine, isoleucine, methionine, phenylalanine, tryptophan,
proline, serine, threonine, cysteine, tyrosine, asparagine,
glutamine, aspartate, glutamate, lysine, arginine and
histidine.
46. The method of claim 44, wherein R.sup.2 is an ester of a
naturally occurring or synthetic .alpha., .beta., .gamma., or
.delta. amino acid.
47. The method of claim 44, wherein R.sup.2 is an ester of an amino
acid in the L configuration.
48. The method of claim 44, wherein R.sup.2 is an ester of
valine.
49. The method of claim 44, wherein the compound or
pharmaceutically acceptable salt, thereof, is administered in
combination or alternation with a second anti-viral agent.
50. The method of claim 49 wherein the second anti-viral agent is
selected from the group consisting of an interferon, ribavirin, an
interleukin, a NS3 protease inhibitor, a HCV helicase inhibitor, a
polymerase inhibitor, a nucleoside and an inhibitor of
IRES-dependent translation.
51. A method for the treatment of a host infected with a
Flaviviridae virus, comprising administering an effective treatment
amount of a compound of formula ##STR77## or a pharmaceutically
acceptable salt thereof wherein: Base is a pyrrolopyrimidine base;
R.sup.1 is hydrogen, mono, di or triphosphate or a stabilized
phosphate; acyl; an amino acid ester; a carbohydrate; a peptide; or
a pharmaceutically acceptable leaving group which when administered
in vivo is capable of providing a compound wherein R.sup.1 is
independently H or phosphate; and R.sup.2 is acyl; an amino acid
ester; a carbohydrate; a peptide; or a pharmaceutically acceptable
leaving group which when administered in vivo is capable of
providing a compound wherein R.sup.2 is hydrogen; in combination or
alternation with a second antiviral agent.
52. The method of claim 51 wherein the virus is hepatitis C.
53. The method of claim 51, wherein the host is a human.
54. The method of claim 51, wherein the compound is in
substantially pure form.
55. The method of claim 51, wherein the compound is at least 90% by
weight of the .beta.-D-isomer.
56. The method of claim 51, wherein the compound or a
pharmaceutically acceptable salt thereof is in combination with a
pharmaceutically acceptable carrier.
57. The method of claim 56 wherein the pharmaceutically acceptable
carrier is suitable for oral delivery.
58. The method of claim 56, wherein the compound or a
pharmaceutically acceptable salt thereof, is in the form of a
dosage unit.
59. The method of claim 58, wherein the dosage unit contains 50 to
1000 mg of the compound.
60. The method of claim 56, wherein the pharmaceutically acceptable
carrier is suitable for systemic, topical, parenteral, inhalant or
intravenous delivery.
61. The method of claim 51, wherein R.sup.1 is hydrogen, mono, di
or triphosphate or a stabilized phosphate.
62. The method of claim 51, wherein R.sup.2 is acyl.
63. The method of claim 51, wherein R.sup.2 is an amino acid
ester.
64. The method of claim 51, wherein R.sup.1 is hydrogen.
65. The method of claim 62, wherein R.sup.2 is acetyl.
66. The method of claim 51, wherein R.sup.2 is an ester of a
naturally occurring or synthetic .alpha., .beta., .gamma., or
.delta. amino acid.
67. The method of claim 51, wherein R.sup.2 is an ester of an amino
acid in the L configuration.
68. The method of claim 51, wherein R.sup.2 is an ester of
valine.
69. The method of claim 51, wherein: R.sup.1 is H; and R.sup.2 is
acyl or an amino acid ester.
70. The method of claim 69, wherein R.sup.2 is an amino acid
ester.
71. A method for the treatment of a host infected with a
Flaviviridae virus, comprising administering an effective treatment
amount of a compound of formula: ##STR78## or a pharmaceutically
acceptable salt thereof wherein: Base is a pyrrolopyrimidine base;
R.sup.1 is H or phosphate; R.sup.2 and R.sup.3 are independently H,
phosphate, acyl or an amino acid ester, wherein at least one of
R.sup.2 and R.sup.3 is acyl or an amino acid ester; in combination
or alternation with a second anti-viral agent.
72. The method of claim 71, wherein the second anti-viral agent is
selected from the group consisting of ribavirin, an interleukin, a
helicase inhibitor, and an inhibitor of IRES-dependent
translation.
73. The method of claim 71, wherein the second anti-viral agent is
an interferon.
74. The method of claim 71, wherein the virus is hepatitis C.
75. The method of claim 74, wherein the host is human.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 10/609,298, filed Jun. 27, 2003, which claims the benefit of
priority to U.S. Provisional application No. 60/392,351, filed Jun.
28, 2002; U.S. Provisional Application No. 60/466,194, filed Apr.
28, 2003; and U.S. Provisional application 60/470,949, filed May
14, 2003, the disclosures of each of which are incorporated herein
by reference.
FIELD OF THE INVENTION
[0002] This invention is in the area of pharmaceutical chemistry,
and is in particular, a 2' and/or 3' prodrug of a 1', 2', 3' or
4'-branched nucleosides for the treatment of a Flaviviridae
infection, such as a hepatitis C virus infection.
BACKGROUND OF THE INVENTION
Flaviviridae Viruses
[0003] The Flaviviridae family of viruses comprises at least three
distinct genera: pestiviruses, which cause disease in cattle and
pigs; flaviviruses, which are the primary cause of diseases such as
dengue fever and yellow fever; and hepaciviruses, whose sole member
is HCV. The flavivirus genus includes more than 68 members
separated into groups on the basis of serological relatedness
(Calisher et al., J. Gen. Virol, 1993, 70, 37-43). Clinical
symptoms vary and include fever, encephalitis and hemorrhagic fever
(Fields Virology, Editors: Fields, B. N., Knipe, D. M., and Howley,
P. M., Lippincott-Raven Publishers, Philadelphia, Pa., 1996,
Chapter 31, 931-959). Flaviviruses of global concern that are
associated with human disease include the dengue hemorrhagic fever
viruses (DHF), yellow fever virus, shock syndrome and Japanese
encephalitis virus (Halstead, S. B., Rev. Infect. Dis., 1984, 6,
251-264; Halstead, S. B., Science, 239:476-481, 1988; Monath, T.
P., New Eng. J. Med., 1988, 319, 641-643).
[0004] The pestivirus genus includes bovine viral diarrhea virus
(BVDV), classical swine fever virus (CSFV, also called hog cholera
virus) and border disease virus (BDV) of sheep (Moennig, V. et al.
Adv. Vir. Res. 1992, 41, 53-98). Pestivirus infections of
domesticated livestock (cattle, pigs and sheep) cause significant
economic losses worldwide. BVDV causes mucosal disease in cattle
and is of significant economic importance to the livestock industry
(Meyers, G. and Thiel, H.-J., Advances in Virus Research, 1996, 47,
53-118; Moennig V., et al, Adv. Vir. Res. 1992, 41, 53-98). Human
pestiviruses have not been as extensively characterized as the
animal pestiviruses. However, serological surveys indicate
considerable pestivirus exposure in humans.
[0005] Pestiviruses and hepaciviruses are closely related virus
groups within the Flaviviridae family. Other closely related
viruses in this family include the GB virus A, GB virus A-like
agents, GB virus-B and GB virus-C (also called hepatitis G virus,
HGV). The hepacivirus group (hepatitis C virus; HCV) consists of a
number of closely related but genotypically distinguishable viruses
that infect humans. There are approximately 6 HCV genotypes and
more than 50 subtypes. Due to the similarities between pestiviruses
and hepaciviruses, combined with the poor ability of hepaciviruses
to grow efficiently in cell culture, bovine viral diarrhea virus
(BVDV) is often used as a surrogate to study the HCV virus.
[0006] The genetic organization of pestiviruses and hepaciviruses
is very similar. These positive stranded RNA viruses possess a
single large open reading frame (ORF) encoding all the viral
proteins necessary for virus replication. These proteins are
expressed as a polyprotein that is co- and post-translationally
processed by both cellular and virus-encoded proteinases to yield
the mature viral proteins. The viral proteins responsible for the
replication of the viral genome RNA are located within
approximately the carboxy-terminal. Two-thirds of the ORF are
termed nonstructural (NS) proteins. The genetic organization and
polyprotein processing of the nonstructural protein portion of the
ORF for pestiviruses and hepaciviruses is very similar. For both
the pestiviruses and hepaciviruses, the mature nonstructural (NS)
proteins, in sequential order from the amino-terminus of the
nonstructural protein coding region to the carboxy-terminus of the
ORF, consist of p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B.
[0007] The NS proteins of pestiviruses and hepaciviruses share
sequence domains that are characteristic of specific protein
functions. For example, the NS3 proteins of viruses in both groups
possess amino acid sequence motifs characteristic of serine
proteinases and of helicases (Gorbalenya et al. (1988) Nature
333:22; Bazan and Fletterick (1989) Virology 171:637-639;
Gorbalenya et al. (1989) Nucleic Acid Res. 17.3889-3897).
Similarly, the NS5B proteins of pestiviruses and hepaciviruses have
the motifs characteristic of RNA-directed RNA polymerases (Koonin,
E. V. and Dolja, V. V. (1993) Crit. Rev. Biochem. Molec. Biol.
28:375-430).
[0008] The actual roles and functions of the NS proteins of
pestiviruses and hepaciviruses in the lifecycle of the viruses are
directly analogous. In both cases, the NS3 serine proteinase is
responsible for all proteolytic processing of polyprotein
precursors downstream of its position in the ORF (Wiskerchen and
Collett (1991) Virology 184:341-350; Bartenschlager et al. (1993)
J. Virol. 67:3835-3844; Eckart et al. (1993) Biochem. Biophys. Res.
Comm. 192:399-406; Grakoui et al. (1993) J. Virol. 67:2832-2843;
Grakoui et al. (1993) Proc. Natl. Acad. Sci. USA 90:10583-10587;
Hijikata et al. (1993) J. Virol. 67:4665-4675; Tome et al. (1993)
J. Virol. 67:4017-4026). The NS4A protein, in both cases, acts as a
cofactor with the NS3 serine protease (Bartenschlager et al. (1994)
J. Virol. 68:5045-5055; Failla et al. (1994) J. Virol. 68:
3753-3760; Lin et al. (1994) 68:8147-8157; Xu et al. (1997) J.
Virol. 71:5312-5322). The NS3 protein of both viruses also
functions as a helicase (Kim et al. (1995) Biochem. Biophys. Res.
Comm. 215: 160-166; Jin and Peterson (1995) Arch. Biochem.
Biophys., 323:47-53; Warrener and Collett (1995) J. Virol.
69:1720-1726). Finally, the NS5B proteins of pestiviruses and
hepaciviruses have the predicted RNA-directed RNA polymerases
activity (Behrens et al. (1996) EMBO J. 15:12-22; Lchmannet al.
(1997) J. Virol. 71:8416-8428; Yuan et al. (1997) Biochem. Biophys.
Res. Comm. 232:231-235; Hagedorn, PCT WO 97/12033; Zhong et al.
(1998) J. Virol. 72.9365-9369).
Hepatitis C Virus
[0009] The hepatitis C virus (HCV) is the leading cause of chronic
liver disease worldwide. (Boyer, N. et al. J. Hepatol. 32:98-112,
2000). HCV causes a slow growing viral infection and is the major
cause of cirrhosis and hepatocellular carcinoma (Di Besceglie, A.
M. and Bacon, B. R., Scientific American, October: 80-85, (1999);
Boyer, N. et al. J. Hepatol. 32:98-112, 2000). An estimated 170
million persons are infected with HCV worldwide. (Boyer, N. et al.
J. Hepatol. 32:98-112, 2000). Cirrhosis caused by chronic hepatitis
C infection accounts for 8,000-12,000 deaths per year in the United
States, and HCV infection is the leading indication for liver
transplantation.
[0010] HCV is known to cause at least 80% of posttransfusion
hepatitis and a substantial proportion of sporadic acute hepatitis.
Preliminary evidence also implicates HCV in many cases of
"idiopathic" chronic hepatitis, "cryptogenic" cirrhosis, and
probably hepatocellular carcinoma unrelated to other hepatitis
viruses, such as Hepatitis B Virus (HBV). A small proportion of
healthy persons appear to be chronic HCV carriers, varying with
geography and other epidemiological factors. The numbers may
substantially exceed those for HBV, though information is still
preliminary; how many of these persons have subclinical chronic
liver disease is unclear. (The Merck Manual, ch. 69, p. 901, 16th
ed., (1992)).
[0011] HCV is an enveloped virus containing a positive-sense
single-stranded RNA genome of approximately 9.4 kb. The viral
genome consists of a 5' untranslated region (UTR), a long open
reading frame encoding a polyprotein precursor of approximately
3011 amino acids, and a short 3' UTR. The 5' UTR is the most highly
conserved part of the HCV genome and is important for the
initiation and control of polyprotein translation. Translation of
the HCV genome is initiated by a cap-independent mechanism known as
internal ribosome entry. This mechanism involves the binding of
ribosomes to an RNA sequence known as the internal ribosome entry
site (IRES). An RNA pseudoknot structure has recently been
determined to be an essential structural element of the HCV IRES.
Viral structural proteins include a nucleocapsid core protein (C)
and two envelope glycoproteins, E1 and E2. HCV also encodes two
proteinases, a zinc-dependent metalloproteinase encoded by the
NS2-NS3 region and a serine proteinase encoded in the NS3 region.
These proteinases are required for cleavage of specific regions of
the precursor polyprotein into mature peptides. The carboxyl half
of nonstructural protein 5, NS5B, contains the RNA-dependent RNA
polymerase. The function of the remaining nonstructural proteins,
NS4A and NS4B, and that of NS5A (the amino-terminal half of
nonstructural protein 5) remain unknown.
[0012] A significant focus of current antiviral research is
directed to the development of improved methods of treatment of
chronic HCV infections in humans (Di Besceglie, A. M. and Bacon, B.
R., Scientific American, October: 80-85, (1999)).
Treatment of HCV Infection with Interferon
[0013] Interferons (IFNs) have been commercially available for the
treatment of chronic hepatitis for nearly a decade. IFNs are
glycoproteins produced by immune cells in response to viral
infection. IFNs inhibit replication of a number of viruses,
including HCV, and when used as the sole treatment for hepatitis C
infection, IFN can in certain cases suppress serum HCV-RNA to
undetectable levels. Additionally, IFN can normalize serum amino
transferase levels. Unfortunately, the effect of IFN is temporary
and a sustained response occurs in only 8%-9% of patients
chronically infected with HCV (Gary L. Davis. Gastroenterology
118:S104-S114, 2000). Most patients, however, have difficulty
tolerating interferon treatment, which causes severe flu-like
symptoms, weight loss, and lack of energy and stamina.
[0014] A number of patents disclose Flaviviridae, including HCV,
treatments, using interferon-based therapies. For example, U.S.
Pat. No. 5,980,884 to Blatt et al. discloses methods for
retreatment of patients afflicted with HCV using consensus
interferon. U.S. Pat. No. 5,942,223 to Bazer et al. discloses an
anti-HCV therapy using ovine or bovine interferon-tau. U.S. Pat.
No. 5,928,636 to Alber et al. discloses the combination therapy of
interleukin-12 and interferon alpha for the treatment of infectious
diseases including HCV. U.S. Pat. No. 5,849,696 to Chretien et al.
discloses the use of thymosins, alone or in combination with
interferon, for treating HCV. U.S. Pat. No. 5,830,455 to Valtuena
et al. discloses a combination HCV therapy employing interferon and
a free radical scavenger. U.S. Pat. No. 5,738,845 to Imakawa
discloses the use of human interferon tau proteins for treating
HCV. Other interferon-based treatments for HCV are disclosed in
U.S. Pat. No. 5,676,942 to Testa et al., U.S. Pat. No. 5,372,808 to
Blatt et al., and U.S. Pat. No. 5,849,696. A number of patents also
disclose pegylated forms of interferon, such as U.S. Pat. Nos.
5,747,646, 5,792,834 and 5,834,594 to Hoffmann-La Roche Inc; PCT
Publication No. WO 99/32139 and WO 99/32140 to Enzon; WO 95/13090
and U.S. Pat. Nos. 5,738,846 and 5,711,944 to Schering; and U.S.
Pat. No. 5,908,621 to Glue et al.
[0015] Interferon alpha-2a and interferon alpha-2b are currently
approved as monotherapy for the treatment of HCV. ROFERON.RTM.-A
(Roche) is the recombinant form of interferon alpha-2a.
PEGASYS.RTM. (Roche) is the pegylated (i.e. polyethylene glycol
modified) form of interferon alpha-2a. INTRON.RTM.A (Schering
Corporation) is the recombinant form of Interferon alpha-2b, and
PEG-INTRON.RTM. (Schering Corporation) is the pegylated form of
interferon alpha-2b.
[0016] Other forms of interferon alpha, as well as interferon beta,
gamma, tau and omega are currently in clinical development for the
treatment of HCV. For example, INFERGEN (interferon alphacon-1) by
InterMune, OMNIFERON (natural interferon) by Viragen, ALBUFERON by
Human Genome Sciences, REBIF (interferon beta-1a) by Ares-Serono,
Omega Interferon by BioMedicine, Oral Interferon Alpha by Amarillo
Biosciences, and interferon gamma, interferon tau, and interferon
gamma-1b by InterMune are in development.
Ribivarin
[0017] Ribavirin
(1-.beta.-D-ribofuranosyl-1-1,2,4-triazole-3-carboxamide) is a
synthetic, non-interferon-inducing, broad spectrum antiviral
nucleoside analog sold under the trade name, Virazole (The Merck
Index, 11th edition, Editor: Budavari, S., Merck & Co., Inc.,
Rahway, N.J., p 1304, 1989). U.S. Pat. No. 3,798,209 and RE29,835
disclose and claim ribavirin. Ribavirin is structurally similar to
guanosine, and has in vitro activity against several DNA and RNA
viruses including Flaviviridae (Gary L. Davis. Gastroenterology
118:S104-S114, 2000).
[0018] Ribavirin reduces serum amino transferase levels to normal
in 40% of patients, but it does not lower serum levels of HCV-RNA
(Gary L. Davis. Gastroenterology 118:S104-S114, 2000). Thus,
ribavirin alone is not effective in reducing viral RNA levels.
Additionally, ribavirin has significant toxicity and is known to
induce anemia.
[0019] Ribavirin is not approved for monotherapy against HCV. It
has been approved in combination with interferon alpha-2a or
interferon alpha-2b for the treatment of HCV.
Combination of Interferon and Ribavirin
[0020] The current standard of care for chronic hepatitis C is
combination therapy with an alpha interferon and ribavirin. The
combination of interferon and ribavirin for the treatment of HCV
infection has been reported to be effective in the treatment of
interferon naive patients (Battaglia, A. M. et al., Ann.
Pharmacother. 34:487-494, 2000), as well as for treatment of
patients when histological disease is present (Berenguer, M. et al.
Antivir. Ther. 3(Suppl. 3):125-136, 1998). Studies have show that
more patients with hepatitis C respond to pegylated
interferon-alpha/ribavirin combination therapy than to combination
therapy with unpegylated interferon alpha. However, as with
monotherapy, significant side effects develop during combination
therapy, including hemolysis, flu-like symptoms, anemia, and
fatigue. (Gary L. Davis. Gastroenterology 118:S104-S114, 2000).
[0021] Combination therapy with PEG-INTRON.RTM. (peginterferon
alpha-2b) and REBETOL.RTM. (Ribavirin, USP) Capsules is available
from Schering Corporation. REBETOL.RTM. (Schering Corporation) has
also been approved in combination with INTRON.RTM. A (Interferon
alpha-2b, recombinant, Schering Corporation). Roche's PEGASYS.RTM.
(pegylated interferon alpha-2a) and COPEGUS.RTM. (ribavirin) are
also approved for the treatment of HCV.
[0022] PCT Publication Nos. WO 99/59621, WO 00/37110, WO 01/81359,
WO 02/32414 and WO 03/024461 by Schering Corporation disclose the
use of pegylated interferon alpha and ribavirin combination therapy
for the treatment of HCV. PCT Publication Nos. WO 99/15194, WO
99/64016, and WO 00/24355 by Hoffmann-La Roche Inc also disclose
the use of pegylated interferon alpha and ribavirin combination
therapy for the treatment of HCV.
Additional Methods to Treat Flaviviridae Infections
[0023] The development of new antiviral agents for flaviviridae
infections, especially hepatitis C, is currently underway. Specific
inhibitors of HCV-derived enzymes such as protease, helicase, and
polymerase inhibitors are being developed. Drugs that inhibit other
steps in HCV replication are also in development, for example,
drugs that block production of HCV antigens from the RNA (IRES
inhibitors), drugs that prevent the normal processing of HCV
proteins (inhibitors of glycosylation), drugs that block entry of
HCV into cells (by blocking its receptor) and nonspecific
cytoprotective agents that block cell injury caused by the virus
infection. Further, molecular approaches are also being developed
to treat hepatitis C, for example, ribozymes, which are enzymes
that break down specific viral RNA molecules, and antisense
oligonucleotides, which are small complementary segments of DNA
that bind to viral RNA and inhibit viral replication, are under
investigation. A number of HCV treatments are reviewed by Bymock et
al. in Antiviral Chemistry & Chemotherapy, 11:2; 79-95 (2000)
and De Francesco et al. in Antiviral Research, 58: 1-16 (2003).
[0024] Examples of classes of drugs that are being developed to
treat Flaviviridae infections include: [0025] (1) Protease
inhibitors
[0026] Substrate-based NS3 protease inhibitors (Attwood et al.,
Antiviral peptide derivatives, PCT WO 98/22496, 1998; Attwood et
al., Antiviral Chemistry and Chemotherapy 1999, 10, 259-273;
Attwood et al., Preparation and use of amino acid derivatives as
anti-viral agents, German Patent Pub. DE 19914474; Tung et al.
Inhibitors of serine proteases, particularly hepatitis C virus NS3
protease, PCT WO 98/17679), including alphaketoamides and
hydrazinoureas, and inhibitors that terminate in an electrophile
such as a boronic acid or phosphonate (Llinas-Brunet et al,
Hepatitis C inhibitor peptide analogues, PCT WO 99/07734) are being
investigated.
[0027] Non-substrate-based NS3 protease inhibitors such as
2,4,6-trihydroxy-3-nitro-benzamide derivatives (Sudo K. et al.,
Biochemical and Biophysical Research Communications, 1997, 238,
643-647; Sudo K. et al. Antiviral Chemistry and Chemotherapy, 1998,
9, 186), including RD34082 and RD3-4078, the former substituted on
the amide with a 14 carbon chain and the latter processing a
para-phenoxyphenyl group are also being investigated.
[0028] Sch 68631, a phenanthrenequinone, is an HCV protease
inhibitor (Chu M. et al., Tetrahedron Letters 37:7229-7232, 1996).
In another example by the same authors, Sch 351633, isolated from
the fungus Penicillium griseofulvum, was identified as a protease
inhibitor (Chu M. et al., Bioorganic and Medicinal Chemistry
Letters 9:1949-1952). Nanomolar potency against the HCV NS3
protease enzyme has been achieved by the design of selective
inhibitors based on the macromolecule eglin c. Eglin c, isolated
from leech, is a potent inhibitor of several serine proteases such
as S. griseus proteases A and B, .alpha.-chymotrypsin, chymase and
subtilisin. Qasim M. A. et al., Biochemistry 36:1598-1607,
1997.
[0029] Several U.S. patents disclose protease inhibitors for the
treatment of HCV. For example, U.S. Pat. No. 6,004,933 to Spruce et
al. discloses a class of cysteine protease inhibitors for
inhibiting HCV endopeptidase 2. U.S. Pat. No. 5,990,276 to Zhang et
al. discloses synthetic inhibitors of hepatitis C virus NS3
protease. The inhibitor is a subsequence of a substrate of the NS3
protease or a substrate of the NS4A cofactor. The use of
restriction enzymes to treat HCV is disclosed in U.S. Pat. No.
5,538,865 to Reyes et al. Peptides as NS3 serine protease
inhibitors of HCV are disclosed in WO 02/008251 to Corvas
International, Inc, and WO 02/08187 and WO 02/008256 to Schering
Corporation. HCV inhibitor tripeptides are disclosed in U.S. Pat.
Nos. 6,534,523, 6,410,531, and 6,420,380 to Boehringer Ingelheim
and WO 02/060926 to Bristol Myers Squibb. Diaryl peptides as NS3
serine protease inhibitors of HCV are disclosed in WO 02/48172 to
Schering Corporation. Imidazoleidinones as NS3 serine protease
inhibitors of HCV are disclosed in WO 02/08198 to Schering
Corporation and WO 02/48157 to Bristol Myers Squibb. WO 98/17679 to
Vertex Pharmaceuticals and WO 02/48116 to Bristol Myers Squibb also
disclose HCV protease inhibitors. [0030] (2) Thiazolidine
derivatives which show relevant inhibition in a reverse-phase HPLC
assay with an NS3/4A fusion protein and NS5A/5B substrate (Sudo K.
et al., Antiviral Research, 1996, 32, 9-18), especially compound
RD-1-6250, possessing a fused cinnamoyl moiety substituted with a
long alkyl chain, RD4 6205 and RD4 6193; [0031] (3) Thiazolidines
and benzanilides identified in Kakiuchi N. et al. J. EBS Letters
421, 217-220; Takeshita N. et al. Analytical Biochemistry, 1997,
247, 242-246; [0032] (4) A phenan-threnequinone possessing activity
against protease in a SDS-PAGE and autoradiography assay isolated
from the fermentation culture broth of Streptomyces sp., Sch 68631
(Chu M. et al., Tetrahedron Letters, 1996, 37, 7229-7232), and Sch
351633, isolated from the fungus Penicillium griseofulvum, which
demonstrates activity in a scintillation proximity assay (Chu M. et
al., Bioorganic and Medicinal Chemistry Letters 9, 1949-1952);
[0033] (5) Helicase inhibitors (Diana G. D. et al., Compounds,
compositions and methods for treatment of hepatitis C, U.S. Pat.
No. 5,633,358; Diana G. D. et al., Piperidine derivatives,
pharmaceutical compositions thereof and their use in the treatment
of hepatitis C, PCT WO 97/36554); [0034] (6) Nucleotide polymerase
inhibitors and gliotoxin (Ferrari R. et al. Journal of Virology,
1999, 73, 1649-1654), and the natural product cerulenin (Lohmann V.
et al., Virology, 1998, 249, 108-118); [0035] (7) Antisense
phosphorothioate oligodeoxynucleotides (S-ODN) complementary to
sequence stretches in the 5' non-coding region (NCR) of the virus
(Alt M. et al., Hepatology, 1995, 22, 707-717), or nucleotides
326-348 comprising the 3' end of the NCR and nucleotides 371-388
located in the core coding region of the HCV RNA (Alt M. et al.,
Archives of Virology, 1997, 142, 589-599; Galderisi U. et al.,
Journal of Cellular Physiology, 1999, 181, 251-257); [0036] (8)
Inhibitors of IRES-dependent translation (Ikeda N et al., Agent for
the prevention and treatment of hepatitis C, Japanese Patent Pub.
JP-08268890; Kai Y. et al. Prevention and treatment of viral
diseases, Japanese Patent Pub. JP-10101591); [0037] (9) Ribozymes,
such as nuclease-resistant ribozymes (Maccjak, D. J. et al.,
Hepatology 1999, 30, abstract 995) and those disclosed in U.S. Pat.
No. 6,043,077 to Barber et al., and U.S. Pat. Nos. 5,869,253 and
5,610,054 to Draper et al.; and [0038] (10) Nucleoside analogs have
also been developed for the treatment of Flaviviridae
infections.
[0039] Idenix Pharmaceuticals the use of branched in the treatment
of flaviviruses (including HCV) and pestiviruses in International
Publication Nos. WO 01/90121 and WO 01/92282. Specifically, a
method for the treatment of hepatitis C infection (and flaviviruses
and pestiviruses) in humans and other host animals is disclosed in
the Idenix publications that includes administering an effective
amount of a biologically active 1', 2', 3' or 4'-branched .beta.-D
or .beta.-L nucleosides or a pharmaceutically acceptable salt or
derivative thereof, administered either alone or in combination
with another antiviral agent, optionally in a pharmaceutically
acceptable carrier.
[0040] Other patent applications disclosing the use of certain
nucleoside analogs to treat hepatitis C virus include:
PCT/CA00/01316 (WO 01/32153; filed Nov. 3, 2000) and PCT/CA01/00197
(WO 01/60315; filed Feb. 19, 2001) filed by BioChem Pharma, Inc.
(now Shire Biochem, Inc.); PCT/US02/01531 (WO 02/057425; filed Jan.
18, 2002) and PCT/US02/03086 (WO 02/057287; filed Jan. 18, 2002)
filed by Merck & Co., Inc., PCT/EP01/09633 (WO 02/18404;
published Aug. 21, 2001) filed by Roche, and PCT Publication Nos.
WO 01/79246 (filed Apr. 13, 2001), WO 02/32920 (filed Oct. 18,
2001) and WO 02/48165 by Pharmasset, Ltd.
[0041] PCT Publication No. WO 99/43691 to Emory University,
entitled "2'-Fluoronucleosides" discloses the use of certain
2'-fluoronucleosides to treat HCV.
[0042] Eldrup et al. (Oral Session V, Hepatitis C Virus,
Flaviviridae; 16.sup.th International Conference on Antiviral
Research (Apr. 27, 2003, Savannah, Ga.)) described the structure
activity relationship of 2'-modified nucleosides for inhibition of
HCV.
[0043] Bhat et al. (Oral Session V, Hepatitis C Virus,
Flaviviridae, 2003 (Oral Session V, Hepatitis C Virus,
Flaviviridae; 16.sup.th International Conference on Antiviral
Research (Apr. 27, 2003, Savannah, Ga.); p A75) describe the
synthesis and pharmacokinetic properties of nucleoside analogues as
possible inhibitors of HCV RNA replication. The authors report that
2'-modified nucleosides demonstrate potent inhibitory activity in
cell-based replicon assays.
[0044] Olsen et al. (Oral Session V, Hepatitis C Virus,
Flaviviridae; 16.sup.th International Conference on Antiviral
Research (Apr. 27, 2003, Savannah, Ga.) p A76) also described the
effects of the 2'-modified nucleosides on HCV RNA replication.
[0045] (11) Other miscellaneous compounds including
1-amino-alkylcyclohexanes (U.S. Pat. No. 6,034,134 to Gold et al.),
alkyl lipids (U.S. Pat. No. 5,922,757 to Chojkier et al.), vitamin
E and other antioxidants (U.S. Pat. No. 5,922,757 to Chojkier et
al.), squalene, amantadine, bile acids (U.S. Pat. No. 5,846,964 to
Ozeki et al.), N-(phosphonoacetyl)-L-aspartic acid, (U.S. Pat. No.
5,830,905 to Diana et al.), benzenedicarboxamides (U.S. Pat. No.
5,633,388 to Diana et al.), polyadenylic acid derivatives (U.S.
Pat. No. 5,496,546 to Wang et al.), 2',3'-dideoxyinosine (U.S. Pat.
No. 5,026,687 to Yarchoan et al.), benzimidazoles (U.S. Pat. No.
5,891,874 to Colacino et al.), plant extracts (U.S. Pat. No.
5,837,257 to Tsai et al., U.S. Pat. No. 5,725,859 to Omer et al.,
and U.S. Pat. No. 6,056,961), and piperidenes (U.S. Pat. No.
5,830,905 to Diana et al.). [0046] (12) Other compounds currently
in preclinical or clinical development for treatment of hepatitis C
virus include: Interleukin-10 by Schering-Plough, IP-501 by
Interneuron, Merimebodib (VX-497) by Vertex, AMANTADINE.RTM.
(Symmetrel) by Endo Labs Solvay, HEPTAZYME.RTM. by RPI, IDN-6556 by
Idun Pharma., XTL-002 by XTL., HCV/MF59 by Chiron, CIVACIR.RTM.
(Hepatitis C Immune Globulin) by NABI, LEVOVIRIN.RTM. by
ICN/Ribapharm, VIRAMIDINE.RTM. by ICN/Ribapharm, ZADAXIN.RTM.
(thymosin alpha-1) by Sci Clone, thymosin plus pegylated interferon
by Sci Clone, CEPLENE.RTM. (histamine dihydrochloride) by Maxim, VX
950/LY 570310 by Vertex/Eli Lilly, ISIS 14803 by Isis
Pharmaceutical/Elan, IDN-6556 by Idun Pharmaceuticals, Inc., JTK
003 by AKROS Pharma, BILN-2061 by Boehringer Ingelheim, CellCept
(mycophenolate mofetil) by Roche, T67, a .beta.-tubulin inhibitor,
by Tularik, a therapeutic vaccine directed to E2 by Innogenetics,
FK788 by Fujisawa Healthcare, Inc., IdB 1016 (Siliphos, oral
silybin-phosphatdylcholine phytosome), RNA replication inhibitors
(VP50406) by ViroPharma/Wyeth, therapeutic vaccine by Intercell,
therapeutic vaccine by Epimmune/Genencor, IRES inhibitor by Anadys,
ANA 245 and ANA 246 by Anadys, immunotherapy (Therapore) by Avant,
protease inhibitor by Corvas/SChering, helicase inhibitor by
Vertex, fusion inhibitor by Trimeris, T cell therapy by CellExSys,
polymerase inhibitor by Biocryst, targeted RNA chemistry by PTC
Therapeutics, Dication by Immtech, Int., protease inhibitor by
Agouron, protease inhibitor by Chiron/Medivir, antisense therapy by
AVI BioPharma, antisense therapy by Hybridon, hemopurifier by
Aethlon Medical, therapeutic vaccine by Merix, protease inhibitor
by Bristol-Myers Squibb/Axys, Chron-VacC, a therapeutic vaccine, by
Tripep, Utah 231B by United Therapeutics, protease, helicase and
polymerase inhibitors by Genelabs Technologies, IRES inhibitors by
Immusol, R803 by Rigel Pharmaceuticals, INFERGEN.RTM. (interferon
alphacon-1) by InterMune, OMNIFERON.RTM. (natural interferon) by
Viragen, ALBUFERON.RTM. by Human Genome Sciences, REBIF.RTM.
(interferon beta-1a) by Ares-Serono, Omega Interferon by
BioMedicine, Oral Interferon Alpha by Amarillo Biosciences,
interferon gamma, interferon tau, and Interferon gamma-1b by
InterMune.
[0047] Nucleoside prodrugs have been previously described for the
treatment of other forms of hepatitis. WO 00/09531 (filed Aug. 10,
1999) and WO 01/96353 (filed Jun. 15, 2001) to Idenix
Pharmaceuticals, discloses 2'-deoxy-.beta.-L-nucleosides and their
3'-prodrugs for the treatment of HBV. U.S. Pat. No. 4,957,924 to
Beauchamp discloses various therapeutic esters of acyclovir.
[0048] In light of the fact that HCV infection has reached epidemic
levels worldwide, and has tragic effects on the infected patient,
there remains a strong need to provide new effective pharmaceutical
agents to treat hepatitis C that have low toxicity to the host.
[0049] Further, given the rising threat of other flaviviridae
infections, there remains a strong need to provide new effective
pharmaceutical agents that have low toxicity to the host.
[0050] Therefore, it is an object of the present invention to
provide a compound, method and composition for the treatment of a
host infected with flaviviridae, including hepatitis C virus.
[0051] It is another object of the present invention to provide a
compound, method and composition generally for the treatment of
patients infected with pestiviruses, flaviviruses, or
hepaciviruses.
SUMMARY OF THE INVENTION
[0052] 2'- and 3'-prodrugs of 1', 2', 3' or 4'-branched .beta.-D or
.beta.-L nucleosides, or their pharmaceutically acceptable salts,
or pharmaceutically acceptable formulations containing these
compounds are useful in the prevention and treatment of
Flaviviridae infections and other related conditions such as
anti-Flaviviridae antibody positive and Flaviviridae-positive
conditions, chronic liver inflammation caused by HCV, cirrhosis,
acute hepatitis, fulminant hepatitis, chronic persistent hepatitis,
and fatigue. These compounds or formulations can also be used
prophylactically to prevent or retard the progression of clinical
illness in individuals who are anti-Flaviviridae antibody or
Flaviviridae-antigen positive or who have been exposed to a
Flaviviridae. In one specific embodiment, the Flaviviridae is
hepatitis C. In an alternative embodiment, the compound is used to
treat any virus that replicates through an RNA-dependent RNA
polymerase.
[0053] A method for the treatment of a Flaviviridae infection in a
host, including a human, is also disclosed that includes
administering an effective amount of a 2'- or 3'-prodrug of a
biologically active 1', 2', 3' or 4'-branched .beta.-D or .beta.-L
nucleosides or a pharmaceutically acceptable salt thereof,
administered either alone or in combination or alternation with
another anti-Flaviviridae agent, optionally in a pharmaceutically
acceptable carrier. The term 1', 2', 3' or 4'-branched, as used in
this specification, refers to a nucleoside that has an additional
non-natural substituent in the 1', 2', 3' or 4'-position (i.e.,
carbon is bound to four nonhydrogen substituents). The term
2'-prodrug, as used herein, refers to a 1', 2', 3' or 4'-branched
.beta.-D or .beta.-L nucleoside that has a biologically cleavable
moiety at the 2'-position, including, but not limited to acyl, and
in one embodiment, a natural or synthetic L- or D-amino acid,
preferably an L-amino acid. The term 3'-prodrug, as used herein,
refers to a 1', 2', 3' or 4'-branched .beta.-D or .beta.-L
nucleoside that has a biologically cleavable moiety at the
3'-position, including, but not limited to acyl, and in one
embodiment, a natural or synthetic L- or D-amino acid, preferably
an L-amino acid. Certain other alternative embodiments are also
included.
[0054] In one embodiment, the prodrug of 1', 2', 3' or 4'-branched
.beta.-D or .beta.-L nucleoside includes biologically cleavable
moieties at the 2' and/or 5' positions. Preferred moieties are
natural or synthetic D or L amino acid esters, including D or
L-valyl, though preferably L-amino acid esters, such as L-valyl,
and alkyl esters including acetyl. Therefore, this invention
specifically includes 2'-L or D-amino acid ester and 2',5'-L or
D-diamino acid ester of 1', 2', 3' or 4'-branched .beta.-D or
.beta.-L nucleosides, preferably L-amino acid, with any desired
purine or pyrimidine base, wherein the parent drug optionally has
an EC.sub.50 of less than 15 micromolar, and even more preferably
less than 10 micromolar; 2'-(alkyl or aryl) ester or
2',5'-L-di(alkyl or aryl) ester of 1', 2', 3' or 4'-branched
.beta.-D or .beta.-L nucleosides with any desired purine or
pyrimidine base, wherein the parent drug optionally has an
EC.sub.50 of less than 10 or 15 micromolar; and prodrugs of
2',5'-diesters of 1', 2', 3' or 4'-branched .beta.-D or .beta.-L
nucleosides wherein (i) the 2' ester is an amino acid ester and the
5'-ester is an alkyl or aryl ester; (ii) both esters are amino acid
esters; (iii) both esters are independently alkyl or aryl esters;
and (iv) the 2' ester is independently an alkyl or aryl ester and
the 5'-ester is an amino acid ester, wherein the parent drug
optionally has an EC.sub.50 of less than 10 or 15 micromolar.
[0055] Examples of prodrugs falling within the invention are
2'-L-valine ester of .beta.-D-2'-methyl-cytidine; 2'-L-valine ester
of .beta.-D-2'-methyl-thymidine; 2'-L-valine ester of
.beta.-D-2'-methyl-adenosine; 2'-L-valine ester of
.beta.-D-2'-methyl-guanosine; 2'-L-valine ester of
.beta.-D-2'-methyl-5-fluorocytidine; 2'-L-valine ester of
.beta.-D-2'-methyl-uridine; 2'-acetyl ester of
.beta.-D-2'-methyl-cytidine; 2'-acetyl ester of
.beta.-D-2'-methyl-thymidine; 2'-acetyl ester of
.beta.-D-2'-methyl-adenosine; 2'-acetyl ester of
.beta.-D-2'-methyl-guanosine; 2'-acetyl ester of
.beta.-D-2'-methyl-5-fluoro-cytidine; and 2'-esters of
.beta.-D-2'-methyl-(cytidine, 5-fluorocytidine, guanosine, uridine,
adenosine, or thymidine) wherein (i) the 2' ester is an amino acid
ester; or (ii) the 2' ester is an alkyl or aryl ester.
[0056] Additional examples of prodrugs falling within the invention
are 2',5'-L-divaline ester of .beta.-D-2'-methyl-cytidine
(dival-2'-Me-L-dC); 2',5'-L-divaline ester of
.beta.-D-2'-methyl-thymidine; 2',5'-L-divaline ester of
.beta.-D-2'-methyl-adenosine; 2',5'-L-divaline ester of
.beta.-D-2'-methyl-guanosine; 2',5'-L-divaline ester of
.beta.-D-2'-methyl-5-fluoro-cytidine; 2',5'-L-divaline ester of
.beta.-D-2'-methyl-uridine; 2',5'-diacetyl ester of
.beta.-D-2'-methyl-cytidine; 2',5'-diacetyl ester of
.beta.-D-2'-methyl-thymidine; 2',5'-diacetyl ester of
.beta.-D-2'-methyl-adenosine; 2',5'-diacetyl ester of
.beta.-D-2'-methyl-guanosine; 2',5'-diacetyl ester of
.beta.-D-2'-methyl-5-fluoro-cytidine; and 2',5'-diesters of
.beta.-D-2'-methyl-(cytidine, 5-fluorocytidine, guanosine, uridine,
adenosine, or thymidine) wherein (i) the 2' ester is an amino acid
ester and the 5'-ester is an alkyl or aryl ester; (ii) both esters
are amino acid esters; (iii) both esters are independently alkyl or
aryl esters; or (iv) the 2' ester is an alkyl or aryl ester and the
5'-ester is an amino acid ester.
[0057] In another embodiment, the prodrug of 1', 2', 3' or
4'-branched .beta.-D or .beta.-L nucleoside includes biologically
cleavable moieties at the 3' and/or 5' positions. Preferred
moieties are natural or synthetic D or L amino acid esters,
including D or L-valyl, though preferably L-amino acid esters, such
as L-valyl, and alkyl esters including acetyl. Therefore, this
invention specifically includes 3'-L or D-amino acid ester and
3',5'-L or D-diamino acid ester of 1', 2', 3' or 4'-branched
.beta.-D or .beta.-L nucleosides, preferably L-amino acid, with any
desired purine or pyrimidine base, wherein the parent drug
optionally has an EC.sub.50 of less than 15 micromolar, and even
more preferably less than 10 micromolar; 3'-(alkyl or aryl) ester
or 3',5'-L-di(alkyl or aryl) ester of 1', 2', 3' or 4'-branched
.beta.-D or .beta.-L nucleosides with any desired purine or
pyrimidine base, wherein the parent drug optionally has an
EC.sub.50 of less than 10 or 15 micromolar; and prodrugs of
3',5'-diesters of 1', 2', 3' or 4'-branched .beta.-D or .beta.-L
nucleosides wherein (i) the 3' ester is an amino acid ester and the
5'-ester is an alkyl or aryl ester; (ii) both esters are amino acid
esters; (iii) both esters are independently alkyl or aryl esters;
and (iv) the 3' ester is independently an alkyl or aryl ester and
the 5'-ester is an amino acid ester, wherein the parent drug
optionally has an EC.sub.50 of less than 10 or 15 micromolar.
[0058] Examples of prodrugs falling within the invention are
3'-L-valine ester of .beta.-D-2'-methyl-cytidine; 3'-L-valine ester
of .beta.-D-2'-methyl-thymidine; 3'-L-valine ester of
.beta.-D-2'-methyl-adenosine; 3'-L-valine ester of
.beta.-D-2'-methyl-guanosine; 3'-L-valine ester of
.beta.-D-2'-methyl-5-fluorocytidine; 3'-L-valine ester of
.beta.-D-2'-methyl-uridine; 3'-acetyl ester of
.beta.-D-2'-methyl-cytidine; 3'-acetyl ester of
.beta.-D-2'-methyl-thymidine; 3'-acetyl ester of
.beta.-D-2'-methyl-adenosine; 3'-acetyl ester of
.beta.-D-2'-methyl-guanosine; 3'-acetyl ester of
.beta.-D-2'-methyl-5-fluoro-cytidine; and 3'-esters of
.beta.-D-2'-methyl-(cytidine, 5-fluorocytidine, guanosine, uridine,
adenosine, or thymidine) wherein (i) the 3' ester is an amino acid
ester; or (ii) the 3' ester is an alkyl or aryl ester.
[0059] Additional examples of prodrugs falling within the invention
are 3',5'-L-divaline ester of .beta.-D-2'-methyl-cytidine
(dival-2'-Me-L-dC); 3',5'-L-divaline ester of
.beta.-D-2'-methyl-thymidine; 3',5'-L-divaline ester of
.beta.-D-2'-methyl-adenosine; 3',5'-L-divaline ester of
.beta.-D-2'-methyl-guanosine; 3',5'-L-divaline ester of
.beta.-D-2'-methyl-5-fluoro-cytidine; 3',5'-L-divaline ester of
.beta.-D-2'-methyl-uridine; 3',5'-diacetyl ester of
.beta.-D-2'-methyl-cytidine; 3',5'-diacetyl ester of
.beta.-D-2'-methyl-thymidine; 3',5'-diacetyl ester of
.beta.-D-2'-methyl-adenosine; 3',5'-diacetyl ester of
.beta.-D-2'-methyl-guanosine; 3',5'-diacetyl ester of
.beta.-D-2'-methyl-5-fluoro-cytidine; and 3',5'-diesters of
.beta.-D-2'-methyl-(cytidine, 5-fluorocytidine, guanosine, uridine,
adenosine, or thymidine) wherein (i) the 3' ester is an amino acid
ester and the 5'-ester is an alkyl or aryl ester; (ii) both esters
are amino acid esters; (iii) both esters are independently alkyl or
aryl esters; or (iv) the 3' ester is an alkyl or aryl ester and the
5'-ester is an amino acid ester.
[0060] In another embodiment, the prodrug of 1', 2', 3' or
4'-branched .beta.-D or .beta.-L nucleoside includes biologically
cleavable moieties at the 2', 3' and/or 5' positions. Preferred
moieties are D or L amino acid esters, including D or L-valyl,
though preferably L-amino acid esters, such as L-valyl, and alkyl
esters including acetyl. Therefore, this invention specifically
includes 2',3'-L or D-diamino acid ester and 2',3',5'-L or
D-triamino acid ester of 1', 2', 3' or 4'-branched .beta.-D or
.beta.-L nucleosides, preferably L-amino acid, with any desired
purine or pyrimidine base, wherein the parent drug optionally has
an EC.sub.50 of less than 15 micromolar, and even more preferably
less than 10 micromolar; 2',3'-di(alkyl or aryl) ester or
2',3',5'-L-tri(alkyl or aryl) ester of 1', 2', 3' or 4'-branched
.beta.-D or .beta.-L nucleosides with any desired purine or
pyrimidine base, wherein the parent drug optionally has an
EC.sub.50 of less than 10 or 15 micromolar; and prodrugs of
2',3'-diesters of 1', 2', 3' or 4'-branched .beta.-D or .beta.-L
nucleosides wherein (i) the 2' ester is an amino acid ester and the
3'-ester is an alkyl or aryl ester; (ii) both esters are amino acid
esters; (iii) both esters are independently alkyl or aryl esters;
and (iv) the 2' ester is independently an alkyl or aryl ester and
the 3'-ester is an amino acid ester, wherein the parent drug
optionally has an EC.sub.50 of less than 10 or 15 micromolar.
Further, 2',3',5'-triesters of 1', 2', 3' or 4'-branched .beta.-D
or .beta.-L nucleosides wherein (i) all three esters are amino acid
esters; (ii) all three esters are independently alkyl or aryl
esters; (iii) the 2' ester is an amino acid ester, the 3' ester is
an amino acid ester and the 5'-ester is an alkyl or aryl ester;
(iv) the 2' ester is an amino acid ester, the 3' ester is an alkyl
or aryl ester and the 5'-ester is an alkyl or aryl ester; (v) the
2' ester is an alkyl or aryl ester, the 3' ester is an alkyl or
aryl ester and the 5'-ester is an amino acid ester; (vi) the 2'
ester is an alkyl or aryl ester, the 3' ester is an amino acid
ester and the 5'-ester is an amino acid ester; (vii) the 2' ester
is an alkyl or aryl ester, the 3' ester is an amino acid ester and
the 5'-ester is an alkyl or aryl ester; and (viii) the 2' ester is
an amino acid ester, the 3' ester is an alkyl or aryl ester and the
5'-ester is an amino acid ester; wherein the parent drug optionally
has an EC.sub.50 of less than 10 or 15 micromolar.
[0061] Examples of prodrugs falling within the invention include
2',3'-L-divaline ester of .beta.-D-2'-methyl-cytidine
(dival-2'-Me-L-dC); 2',3'-L-divaline ester of
.beta.-D-2'-methyl-thymidine; 2',3'-L-divaline ester of
.beta.-D-2'-methyl-adenosine; 2',3'-L-divaline ester of
.beta.-D-2'-methyl-guanosine; 2',3'-L-divaline ester of
.beta.-D-2'-methyl-5-fluoro-cytidine; 2',3'-L-divaline ester of
.beta.-D-2'-methyl-uridine; 2',3'-diacetyl ester of
.beta.-D-2'-methyl-cytidine; 2',3'-diacetyl ester of
.beta.-D-2'-methyl-thymidine; 2',3'-diacetyl ester of
.beta.-D-2'-methyl-adenosine; 2',3'-diacetyl ester of
.beta.-D-2'-methyl-guanosine; 2',3'-diacetyl ester of
.beta.-D-2'-methyl-5-fluoro-cytidine; and 2',3'-diesters of
.beta.-D-2'-methyl-(cytidine, 5-fluorocytidine, guanosine, uridine,
adenosine, or thymidine) wherein (i) the 2' ester is an amino acid
ester and the 3'-ester is an alkyl or aryl ester; (ii) both esters
are amino acid esters; (iii) both esters are independently alkyl or
aryl esters; or (iv) the 2' ester is an alkyl or aryl ester and the
3'-ester is an amino acid ester.
[0062] Additional examples of prodrugs falling within the invention
include 2',3',5'-L-trivaline ester of .beta.-D-2'-methyl-cytidine
(trival-2'-Me-L-dC); 2',3',5'-L-trivaline ester of
.beta.-D-2'-methyl-thymidine; 2',3',5'-L-trivaline ester of
.beta.-D-2'-methyl-adenosine; 2',3',5'-L-trivaline ester of
.beta.-D-2'-methyl-guanosine; 2',3',5'-L-trivaline ester of
.beta.-D-2'-methyl-5-fluoro-cytidine; 2',3',5'-L-trivaline ester of
.beta.-D-2'-methyl-uridine; 2',3',5'-triacetyl ester of
.beta.-D-2'-methyl-cytidine; 2',3',5'-triacetyl ester of
.beta.-D-2'-methyl-thymidine; 2',3',5'-triacetyl ester of
.beta.-D-2'-methyl-adenosine; 2',3',5'-triacetyl ester of
.beta.-D-2'-methyl-guanosine; 2',3',5'-triacetyl ester of
.beta.-D-2'-methyl-5-fluoro-cytidine; and 2',3',5'-triesters of
.beta.-D-2'-methyl-(cytidine, 5-fluorocytidine, guanosine, uridine,
adenosine, or thymidine) wherein (i) all three esters are amino
acid esters; (ii) all three esters are independently alkyl or aryl
esters; (iii) the 2' ester is an amino acid ester, the 3' ester is
an amino acid ester and the 5'-ester is an alkyl or aryl ester;
(iv) the 2' ester is an amino acid ester, the 3' ester is an alkyl
or aryl ester and the 5'-ester is an alkyl or aryl ester; (v) the
2' ester is an alkyl or aryl ester, the 3' ester is an alkyl or
aryl ester and the 5'-ester is an amino acid ester; (vi) the 2'
ester is an alkyl or aryl ester, the 3' ester is an amino acid
ester and the 5'-ester is an amino acid ester; (vii) the 2' ester
is an alkyl or aryl ester, the 3' ester is an amino acid ester and
the 5'-ester is an alkyl or aryl ester; and (viii) the 2' ester is
an amino acid ester, the 3' ester is an alkyl or aryl ester and the
5'-ester is an amino acid ester.
[0063] Pharmaceutically acceptable salts of tosylate,
methanesulfonate, acetate, citrate, malonate, tartarate, succinate,
benzoate, ascorbate, .alpha.-ketoglutarate, and
.alpha.-glycerophosphate, formate, fumarate, propionate, glycolate,
lactate, pyruvate, oxalate, maleate, salicyate, sulfate, sulfonate,
nitrate, bicarbonate, hydrobromate, hydrobromide, hydroiodide,
carbonate, and phosphoric acid salts are provided. A particularly
preferred embodiment is the mono or dihydrochloride
pharmaceutically acceptable salts.
[0064] In a first principal embodiment, a compound of Formula (I),
or a pharmaceutically acceptable salt or prodrug thereof, is
provided: ##STR1## wherein: R.sup.1, R.sup.2 and R.sup.3 are
independently H, phosphate (including mono-, di- or triphosphate
and a stabilized phosphate); straight chained, branched or cyclic
alkyl (including lower alkyl); acyl (including lower acyl);
CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted
aryl, sulfonate ester including alkyl or arylalkyl sulfonyl
including methanesulfonyl and benzyl, wherein the phenyl group is
optionally substituted with one or more substituents as described
in the definition of aryl given herein; alkylsulfonyl,
arylsulfonyl, aralkylsulfonyl, a lipid, including a phospholipid;
an amino acid; and amino acid residue, a carbohydrate; a peptide;
cholesterol; or other pharmaceutically acceptable leaving group
which is capable of providing a compound wherein R.sup.1, R.sup.2
and/or R.sup.3 is independently H or phosphate, for example when
administered in vivo; wherein at least one of R.sup.2 and R.sup.3
is not hydrogen; Y is hydrogen, bromo, chloro, fluoro, iodo, OH,
OR.sup.4, NH, NHR.sup.5, NR.sup.4R.sup.5, SH and SR.sup.4; X.sup.1
and X.sup.2 are independently selected from the group consisting of
H, straight chained, branched or cyclic alkyl, CO-alkyl, CO-aryl,
CO-alkoxyalkyl, chloro, bromo, fluoro, iodo, OH, OR.sup.4, NH,
NHR.sup.5, NR.sup.4R.sup.5, SH and SR.sup.4; and R.sup.4 and
R.sup.5 are independently hydrogen, acyl (including lower acyl), or
alkyl (including but not limited to methyl, ethyl, propyl and
cyclopropyl).
[0065] In the embodiments described herein, R.sup.1, R.sup.2 and/or
R.sup.3 can independently be a pharmaceutically acceptable leaving
group which is capable of providing a compound wherein R.sup.1,
R.sup.2 and/or R.sup.3 is independently H or phosphate (including
mono-, di- or triphosphate), for example when administered in
vivo.
[0066] In a second principal embodiment, a compound of Formula II,
or a pharmaceutically acceptable salt or prodrug thereof, is
provided: ##STR2## wherein: R.sup.1, R.sup.2 and R.sup.3 are
independently H, phosphate (including mono-, di- or triphosphate
and a stabilized phosphate); straight chained, branched or cyclic
alkyl (including lower alkyl); acyl (including lower acyl);
CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted
aryl, sulfonate ester including alkyl or arylalkyl sulfonyl
including methanesulfonyl and benzyl, wherein the phenyl group is
optionally substituted with one or more substituents as described
in the definition of aryl given herein; alkylsulfonyl,
arylsulfonyl, aralkylsulfonyl, a lipid, including a phospholipid;
an amino acid; and amino acid residue, a carbohydrate; a peptide;
cholesterol; or other pharmaceutically acceptable leaving group
which when administered in vivo is capable of providing a compound
wherein R.sup.1, R.sup.2 and/or R.sup.3 is independently H or
phosphate; wherein at least one of R.sup.2 and R.sup.3 is not
hydrogen; Y is hydrogen, bromo, chloro, fluoro, iodo, OR.sup.4,
NR.sup.4R.sup.5 or SR.sup.4; X.sup.1 and X.sup.2 are independently
selected from the group consisting of H, straight chained, branched
or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, chloro, bromo,
fluoro, iodo, OR.sup.4, NR.sup.4NR.sup.5 or SR.sup.5; and R.sup.4
and R.sup.5 are independently hydrogen, acyl (including lower
acyl), or alkyl (including but not limited to methyl, ethyl, propyl
and cyclopropyl).
[0067] In a third principal embodiment, a compound of Formula III,
or a pharmaceutically acceptable salt or prodrug thereof, is
provided: ##STR3## wherein: R.sup.1, R.sup.2 and R.sup.3 are
independently H, phosphate (including mono-, di- or triphosphate
and a stabilized phosphate); straight chained, branched or cyclic
alkyl (including lower alkyl); acyl (including lower acyl);
CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted
aryl, sulfonate ester including alkyl or arylalkyl sulfonyl
including methanesulfonyl and benzyl, wherein the phenyl group is
optionally substituted with one or more substituents as described
in the definition of aryl given herein; alkylsulfonyl,
arylsulfonyl, aralkylsulfonyl, a lipid, including a phospholipid;
an amino acid; and amino acid residue, a carbohydrate; a peptide;
cholesterol; or other pharmaceutically acceptable leaving group
which when administered in vivo is capable of providing a compound
wherein R.sup.1, R.sup.2 and/or R.sup.3 is independently H or
phosphate; wherein at least one of R.sup.2 and R.sup.3 is not
hydrogen; Y is hydrogen, bromo, chloro, fluoro, iodo, OR.sup.4,
NR.sup.4R.sup.5 or SR.sup.4; X.sup.1 and X.sup.2 are independently
selected from the group consisting of H, straight chained, branched
or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, chloro, bromo,
fluoro, iodo, OR.sup.4, NR.sup.4NR.sup.5 or SR.sup.5; and R.sup.4
and R.sup.5 are independently hydrogen, acyl (including lower
acyl), or alkyl (including but not limited to methyl, ethyl, propyl
and cyclopropyl).
[0068] In a fourth principal embodiment, a compound of Formula IV,
or a pharmaceutically acceptable salt or prodrug thereof, is
provided: ##STR4## wherein: R.sup.1, R.sup.2 and R.sup.3 are
independently H, phosphate (including mono-, di- or triphosphate
and a stabilized phosphate); straight chained, branched or cyclic
alkyl (including lower alkyl); acyl (including lower acyl);
CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted
aryl, sulfonate ester including alkyl or arylalkyl sulfonyl
including methanesulfonyl and benzyl, wherein the phenyl group is
optionally substituted with one or more substituents as described
in the definition of aryl given herein; alkylsulfonyl,
arylsulfonyl, aralkylsulfonyl, a lipid, including a phospholipid;
an amino acid; and amino acid residue, a carbohydrate; a peptide;
cholesterol; or other pharmaceutically acceptable leaving group
which when administered in vivo is capable of providing a compound
wherein R.sup.1, R.sup.2 and/or R.sup.3 is independently H or
phosphate; wherein at least one of R.sup.2 and R.sup.3 is not
hydrogen; Y is hydrogen, bromo, chloro, fluoro, iodo, OR.sup.4,
NR.sup.4R.sup.5 or SR.sup.4; X.sup.1 is selected from the group
consisting of H, straight chained, branched or cyclic alkyl,
CO-alkyl, CO-aryl, CO-alkoxyalkyl, chloro, bromo, fluoro, iodo,
OR.sup.4, NR.sup.4NR.sup.5 or SR.sup.5; and R.sup.4 and R.sup.5 are
independently hydrogen, acyl (including lower acyl), or alkyl
(including but not limited to methyl, ethyl, propyl and
cyclopropyl).
[0069] In a fifth principal embodiment, a compound of Formula V, or
a pharmaceutically acceptable salt or prodrug thereof, is provided:
##STR5## wherein: R.sup.1, R.sup.2 and R.sup.3 are independently H,
phosphate (including mono-, di- or triphosphate and a stabilized
phosphate); straight chained, branched or cyclic alkyl (including
lower alkyl); acyl (including lower acyl); CO-alkyl, CO-aryl,
CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, sulfonate
ester including alkyl or arylalkyl sulfonyl including
methanesulfonyl and benzyl, wherein the phenyl group is optionally
substituted with one or more substituents as described in the
definition of aryl given herein; alkylsulfonyl, arylsulfonyl,
aralkylsulfonyl, a lipid, including a phospholipid; an amino acid;
and amino acid residue, a carbohydrate; a peptide; cholesterol; or
other pharmaceutically acceptable leaving group which when
administered in vivo is capable of providing a compound wherein
R.sup.1, R.sup.2 and/or R.sup.3 is independently H or phosphate;
wherein at least one of R.sup.2 and R.sup.3 is not hydrogen; Y is
hydrogen, bromo, chloro, fluoro, iodo, OR.sup.4, NR.sup.4R.sup.5 or
SR.sup.4; X.sup.1 is selected from the group consisting of H,
straight chained, branched or cyclic alkyl, CO-alkyl, CO-aryl,
CO-alkoxyalkyl, chloro, bromo, fluoro, iodo, OR.sup.4,
NR.sup.4NR.sup.5 or SR.sup.5; and R.sup.4 and R.sup.5 are
independently hydrogen, acyl (including lower acyl), or alkyl
(including but not limited to methyl, ethyl, propyl and
cyclopropyl).
[0070] In a sixth principal embodiment, a compound of Formula VI,
or a pharmaceutically acceptable salt or prodrug thereof, is
provided: ##STR6## wherein: R.sup.1, R.sup.2 and R.sup.3 are
independently H, phosphate (including mono-, di- or triphosphate
and a stabilized phosphate); straight chained, branched or cyclic
alkyl (including lower alkyl); acyl (including lower acyl);
CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted
aryl, sulfonate ester including alkyl or arylalkyl sulfonyl
including methanesulfonyl and benzyl, wherein the phenyl group is
optionally substituted with one or more substituents as described
in the definition of aryl given herein; alkylsulfonyl,
arylsulfonyl, aralkylsulfonyl, a lipid, including a phospholipid;
an amino acid; and amino acid residue, a carbohydrate; a peptide;
cholesterol; or other pharmaceutically acceptable leaving group
which when administered in vivo is capable of providing a compound
wherein R.sup.1, R.sup.2 and/or R.sup.3 is independently H or
phosphate; wherein at least one of R.sup.2 and R.sup.3 is not
hydrogen; Y is hydrogen, bromo, chloro, fluoro, iodo, OR.sup.4,
NR.sup.4R.sup.5 or SR.sup.4; X.sup.1 is selected from the group
consisting of H, straight chained, branched or cyclic alkyl,
CO-alkyl, CO-aryl, CO-alkoxyalkyl, chloro, bromo, fluoro, iodo,
OR.sup.4, NR.sup.4NR.sup.5 or SR.sup.5; and R.sup.4 and R.sup.5 are
independently hydrogen, acyl (including lower acyl), or alkyl
(including but not limited to methyl, ethyl, propyl and
cyclopropyl).
[0071] In a seventh principal embodiment, a compound selected from
Formulas VII and VIII, or a pharmaceutically acceptable salt or
prodrug thereof, is provided: ##STR7## wherein: Base is a purine or
pyrimidine base as defined herein; R.sup.1, R.sup.2 and R.sup.3 are
independently H, phosphate (including mono-, di- or triphosphate
and a stabilized phosphate); straight chained, branched or cyclic
alkyl (including lower alkyl); acyl (including lower acyl);
CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted
aryl, sulfonate ester including alkyl or arylalkyl sulfonyl
including methanesulfonyl and benzyl, wherein the phenyl group is
optionally substituted with one or more substituents as described
in the definition of aryl given herein; alkylsulfonyl,
arylsulfonyl, aralkylsulfonyl, a lipid, including a phospholipid;
an amino acid; and amino acid residue, a carbohydrate; a peptide;
cholesterol; or other pharmaceutically acceptable leaving group
which when administered in vivo is capable of providing a compound
wherein R.sup.1, R.sup.2 and/or R.sup.3 is independently H or
phosphate; wherein R.sup.2 is not hydrogen; R.sup.6 is alkyl
(including lower alkyl and halogenated alkyl), CH.sub.3, CF.sub.3,
azido, cyano, alkenyl, alkynyl, Br-vinyl, 2-Br-ethyl,
--C(O)O(alkyl), --C(O)O(lower alkyl), --O(acyl), --O(lower acyl),
--O(alkyl), --O(lower alkyl), --O(alkenyl), CF.sub.3, chloro,
bromo, fluoro, iodo, NO.sub.2, NH.sub.2, --NH(lower alkyl),
--NH(acyl), --N(lower alkyl).sub.2, --N(acyl).sub.2; and X is O, S,
SO.sub.2 or CH.sub.2.
[0072] In a eighth principal embodiment, a compound of Formulas IX
and X, or a pharmaceutically acceptable salt or prodrug thereof, is
provided: ##STR8## wherein: Base is a purine or pyrimidine base as
defined herein; R.sup.1, R.sup.2 and R.sup.3 are independently H,
phosphate (including mono-, di- or triphosphate and a stabilized
phosphate); straight chained, branched or cyclic alkyl (including
lower alkyl); acyl (including lower acyl); CO-alkyl, CO-aryl,
CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, sulfonate
ester including alkyl or arylalkyl sulfonyl including
methanesulfonyl and benzyl, wherein the phenyl group is optionally
substituted with one or more substituents as described in the
definition of aryl given herein; alkylsulfonyl, arylsulfonyl,
aralkylsulfonyl, a lipid, including a phospholipid; an amino acid;
and amino acid residue, a carbohydrate; a peptide; cholesterol; or
other pharmaceutically acceptable leaving group which when
administered in vivo is capable of providing a compound wherein
R.sup.1, R.sup.2 and/or R.sup.3 is independently H or phosphate;
wherein R.sup.2 is not hydrogen; R.sup.6 is alkyl (including lower
alkyl and halogenated alkyl), CH.sub.3, CF.sub.3, azido, cyano,
alkenyl, alkynyl, Br-vinyl, 2-Br-ethyl, --C(O)O(alkyl),
--C(O)O(lower alkyl), --O(acyl), --O(lower acyl), --O(alkyl),
--O(lower alkyl), --O(alkenyl), CF.sub.3, chloro, bromo, fluoro,
iodo, NO.sub.2, NH.sub.2, --NH(lower alkyl), --NH(acyl), --N(lower
alkyl).sub.2, --N(acyl).sub.2; and R.sup.7 is hydrogen, OR.sup.3,
hydroxy, alkyl (including lower alkyl), azido, cyano, alkenyl,
alkynyl, Br-vinyl, --C(O)O(alkyl), --C(O)O(lower alkyl), --O(acyl),
--O(lower acyl), --O(alkyl), --O(lower alkyl), --O(alkenyl),
chlorine, bromine, iodine, NO.sub.2, NH.sub.2, --NH(lower alkyl),
--NH(acyl), --N(lower alkyl).sub.2, --N(acyl).sub.2; and X is O, S,
SO.sub.2 or CH.sub.2.
[0073] In a ninth principal embodiment a compound selected from
Formulas XI and XII, or a pharmaceutically acceptable salt or
prodrug thereof, is provided: ##STR9## wherein: Base is a purine or
pyrimidine base as defined herein; R.sup.1, R.sup.2 and R.sup.3 are
independently H, phosphate (including mono-, di- or triphosphate
and a stabilized phosphate); straight chained, branched or cyclic
alkyl (including lower alkyl); acyl (including lower acyl);
CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted
aryl, sulfonate ester including alkyl or arylalkyl sulfonyl
including methanesulfonyl and benzyl, wherein the phenyl group is
optionally substituted with one or more substituents as described
in the definition of aryl given herein; alkylsulfonyl,
arylsulfonyl, aralkylsulfonyl, a lipid, including a phospholipid;
an amino acid; and amino acid residue, a carbohydrate; a peptide;
cholesterol; or other pharmaceutically acceptable leaving group
which when administered in vivo is capable of providing a compound
wherein R.sup.1, R.sup.2 and/or R.sup.3 is independently H or
phosphate; wherein R.sup.2 is not hydrogen; R.sup.6 is alkyl
(including lower alkyl and halogenated alkyl), CH.sub.3, CF.sub.3,
azido, cyano, alkenyl, alkynyl, Br-vinyl, 2-Br-ethyl,
--C(O)O(alkyl), --C(O)O(lower alkyl), --O(acyl), --O(lower acyl),
--O(alkyl), --O(lower alkyl), --O(alkenyl), CF.sub.3, chloro,
bromo, fluoro, iodo, NO.sub.2, NH.sub.2, --NH(lower alkyl),
--NH(acyl), --N(lower alkyl).sub.2, --N(acyl).sub.2; and X is O, S,
SO.sub.2 or CH.sub.2.
[0074] In a tenth principal embodiment the invention provides a
compound of Formula XIII, or a pharmaceutically acceptable salt or
prodrug thereof: ##STR10## wherein: Base is a purine or pyrimidine
base as defined herein; R.sup.1 is H, phosphate (including mono-,
di- or triphosphate and a stabilized phosphate); straight chained,
branched or cyclic alkyl (including lower alkyl); acyl (including
lower acyl); CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl,
CO-substituted aryl, sulfonate ester including alkyl or arylalkyl
sulfonyl including methanesulfonyl and benzyl, wherein the phenyl
group is optionally substituted with one or more substituents as
described in the definition of aryl given herein; alkylsulfonyl,
arylsulfonyl, aralkylsulfonyl, a lipid, including a phospholipid;
an amino acid; and amino acid residue, a carbohydrate; a peptide;
cholesterol; or other pharmaceutically acceptable leaving group
which when administered in vivo is capable of providing a compound
wherein R.sup.1 is H or phosphate; R.sup.6 is alkyl (including
lower alkyl and halogenated alkyl), CH.sub.3, CF.sub.3, azido,
cyano, alkenyl, alkynyl, Br-vinyl, 2-Br-ethyl, --C(O)O(alkyl),
--C(O)O(lower alkyl), --O(acyl), --O(lower acyl), --O(alkyl),
--O(lower alkyl), --O(alkenyl), CF.sub.3, chloro, bromo, fluoro,
iodo, NO.sub.2, NH.sub.2, --NH(lower alkyl), --NH(acyl), --N(lower
alkyl).sub.2, --N(acyl).sub.2; R.sup.7 and R.sup.9 are
independently hydrogen, OR.sup.2, hydroxy, alkyl (including lower
alkyl), azido, cyano, alkenyl, alkynyl, Br-vinyl, --C(O)O(alkyl),
--C(O)O(lower alkyl), --O(acyl), --O(lower acyl), --O(alkyl),
--O(lower alkyl), --O(alkenyl), chlorine, bromine, iodine,
NO.sub.2, NH.sub.2, --NH(lower alkyl), --NH(acyl), --N(lower
alkyl).sub.2, --N(acyl).sub.2; wherein at least one of R.sup.7 and
R.sup.9 is OR.sup.2, wherein the R.sup.2 is independently phosphate
(including mono-, di- or triphosphate and a stabilized phosphate);
straight chained, branched or cyclic alkyl (including lower alkyl);
acyl (including lower acyl); CO-alkyl, CO-aryl, CO-alkoxyalkyl,
CO-aryloxyalkyl, CO-substituted aryl, sulfonate ester including
alkyl or arylalkyl sulfonyl including methanesulfonyl and benzyl,
wherein the phenyl group is optionally substituted with one or more
substituents as described in the definition of aryl given herein;
alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, a lipid, including a
phospholipid; an amino acid; and amino acid residue, a
carbohydrate; a peptide; cholesterol; or other pharmaceutically
acceptable leaving group which when administered in vivo is capable
of providing a compound wherein R.sup.2 is H or phosphate; R.sup.8
and R.sup.10 are independently H, alkyl (including lower alkyl),
chlorine, bromine or iodine; alternatively, R.sup.7 and R.sup.10,
R.sup.8 and R.sup.9, or R.sup.8 and R.sup.10 can come together to
form a pi bond; and X is O, S, SO.sub.2 or CH.sub.2.
[0075] In a eleventh principal embodiment the invention provides a
compound of Formula XIV, or a pharmaceutically acceptable salt or
prodrug thereof: ##STR11## wherein: Base is a purine or pyrimidine
base as defined herein; R.sup.1 is H, phosphate (including mono-,
di- or triphosphate and a stabilized phosphate); straight chained,
branched or cyclic alkyl (including lower alkyl); acyl (including
lower acyl); CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl,
CO-substituted aryl, sulfonate ester including alkyl or arylalkyl
sulfonyl including methanesulfonyl and benzyl, wherein the phenyl
group is optionally substituted with one or more substituents as
described in the definition of aryl given herein; alkylsulfonyl,
arylsulfonyl, aralkylsulfonyl, a lipid, including a phospholipid;
an amino acid; and amino acid residue, a carbohydrate; a peptide;
cholesterol; or other pharmaceutically acceptable leaving group
which when administered in vivo is capable of providing a compound
wherein R.sup.1 is H or phosphate; R.sup.6 is alkyl (including
lower alkyl and halogenated alkyl), CH.sub.3, CF.sub.3, azido,
cyano, alkenyl, alkynyl, Br-vinyl, 2-Br-ethyl, --C(O)O(alkyl),
--C(O)O(lower alkyl), --O(acyl), --O(lower acyl), --O(alkyl),
--O(lower alkyl), --O(alkenyl), CF.sub.3, chloro, bromo, fluoro,
iodo, NO.sub.2, NH.sub.2, --NH(lower alkyl), --NH(acyl), --N(lower
alkyl).sub.2, --N(acyl).sub.2; R.sup.7 and R.sup.9 are
independently hydrogen, OR.sup.2, hydroxy, alkyl (including lower
alkyl), azido, cyano, alkenyl, alkynyl, Br-vinyl, --C(O)O(alkyl),
--C(O)O(lower alkyl), --O(acyl), --O(lower acyl), --O(alkyl),
--O(lower alkyl), --O(alkenyl), chlorine, bromine, iodine,
NO.sub.2, NH.sub.2, --NH(lower alkyl), --NH(acyl), --N(lower
alkyl).sub.2, --N(acyl).sub.2; wherein at least one of R.sup.7 and
R.sup.9 is OR.sup.2, wherein the R.sup.2 is independently phosphate
(including mono-, di- or triphosphate and a stabilized phosphate);
straight chained, branched or cyclic alkyl (including lower alkyl);
acyl (including lower acyl); CO-alkyl, CO-aryl, CO-alkoxyalkyl,
CO-aryloxyalkyl, CO-substituted aryl, sulfonate ester including
alkyl or arylalkyl sulfonyl including methanesulfonyl and benzyl,
wherein the phenyl group is optionally substituted with one or more
substituents as described in the definition of aryl given herein;
alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, a lipid, including a
phospholipid; an amino acid; and amino acid residue, a
carbohydrate; a peptide; cholesterol; or other pharmaceutically
acceptable leaving group which when administered in vivo is capable
of providing a compound wherein R.sup.2 is H or phosphate; R.sup.10
is H, alkyl (including lower alkyl), chlorine, bromine or iodine;
alternatively, or R.sup.7 and R.sup.10 can come together to form a
pi bond; and X is O, S, SO.sub.2 or CH.sub.2.
[0076] In a twelfth principal embodiment, the invention provides a
compound of Formula XV, or a pharmaceutically acceptable salt or
prodrug thereof: ##STR12## wherein: Base is a purine or pyrimidine
base as defined herein; R.sup.1 is H, phosphate (including mono-,
di- or triphosphate and a stabilized phosphate); straight chained,
branched or cyclic alkyl (including lower alkyl); acyl (including
lower acyl); CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl,
CO-substituted aryl, sulfonate ester including alkyl or arylalkyl
sulfonyl including methanesulfonyl and benzyl, wherein the phenyl
group is optionally substituted with one or more substituents as
described in the definition of aryl given herein; alkylsulfonyl,
arylsulfonyl, aralkylsulfonyl, a lipid, including a phospholipid;
an amino acid; and amino acid residue, a carbohydrate; a peptide;
cholesterol; or other pharmaceutically acceptable leaving group
which when administered in vivo is capable of providing a compound
wherein R.sup.1 is H or phosphate; R.sup.6 is alkyl (including
lower alkyl and halogenated alkyl), CH.sub.3, CF.sub.3, azido,
cyano, alkenyl, alkynyl, Br-vinyl, 2-Br-ethyl, --C(O)O(alkyl),
--C(O)O(lower alkyl), --O(acyl), --O(lower acyl), --O(alkyl),
--O(lower alkyl), --O(alkenyl), CF.sub.3, chloro, bromo, fluoro,
iodo, NO.sub.2, NH.sub.2, --NH(lower alkyl), --NH(acyl), --N(lower
alkyl).sub.2, --N(acyl).sub.2; R.sup.7 and R.sup.9 are
independently hydrogen, OR.sup.2, hydroxy, alkyl (including lower
alkyl), azido, cyano, alkenyl, alkynyl, Br-vinyl, --C(O)O(alkyl),
--C(O)O(lower alkyl), --O(acyl), --O(lower acyl), --O(alkyl),
--O(lower alkyl), --O(alkenyl), chlorine, bromine, iodine,
NO.sub.2, NH.sub.2, --NH(lower alkyl), --NH(acyl), --N(lower
alkyl).sub.2, --N(acyl).sub.2; wherein at least one of R.sup.7 and
R.sup.9 is OR.sup.2, wherein each R.sup.2 is independently
phosphate (including mono-, di- or triphosphate and a stabilized
phosphate); straight chained, branched or cyclic alkyl (including
lower alkyl); acyl (including lower acyl); CO-alkyl, CO-aryl,
CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, sulfonate
ester including alkyl or arylalkyl sulfonyl including
methanesulfonyl and benzyl, wherein the phenyl group is optionally
substituted with one or more substituents as described in the
definition of aryl given herein; alkylsulfonyl, arylsulfonyl,
aralkylsulfonyl, a lipid, including a phospholipid; an amino acid;
and amino acid residue, a carbohydrate; a peptide; cholesterol; or
other pharmaceutically acceptable leaving group which when
administered in vivo is capable of providing a compound wherein
R.sup.2 is H or phosphate; R.sup.8 is H, alkyl (including lower
alkyl), chlorine, bromine or iodine; alternatively, R.sup.8 and
R.sup.9 can come together to form a pi bond; X is O, S, SO.sub.2 or
CH.sub.2.
[0077] In a thirteenth principal embodiment, a compound of Formula
XVI, or a pharmaceutically acceptable salt or prodrug thereof, is
provided: ##STR13## wherein: R.sup.1, R.sup.2 and R.sup.3 are
independently H, phosphate (including mono-, di- or triphosphate
and a stabilized phosphate); straight chained, branched or cyclic
alkyl (including lower alkyl); acyl (including lower acyl);
CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted
aryl, sulfonate ester including alkyl or arylalkyl sulfonyl
including methanesulfonyl and benzyl, wherein the phenyl group is
optionally substituted with one or more substituents as described
in the definition of aryl given herein; alkylsulfonyl,
arylsulfonyl, aralkylsulfonyl, a lipid, including a phospholipid;
an amino acid; and amino acid residue, a carbohydrate; a peptide;
cholesterol; or other pharmaceutically acceptable leaving group
which when administered in vivo is capable of providing a compound
wherein R.sup.1, R.sup.2 and/or R.sup.3 is independently H or
phosphate; wherein at least one of R.sup.2 and R.sup.3 is not
hydrogen; Y is hydrogen, bromo, chloro, fluoro, iodo, OH, OR.sup.4,
NH, NHR.sup.5, NR.sup.4R.sup.5, SH and SR.sup.4; X.sup.1 and
X.sup.2 are independently selected from the group consisting of H,
straight chained, branched or cyclic alkyl, CO-alkyl, CO-aryl,
CO-alkoxyalkyl, chloro, bromo, fluoro, iodo, OH, OR.sup.4, NH,
NHR.sup.5, NR.sup.4R.sup.5, SH and SR.sup.4; and R.sup.4 and
R.sup.5 are independently hydrogen, acyl (including lower acyl), or
alkyl (including but not limited to methyl, ethyl, propyl and
cyclopropyl).
[0078] In a fourteenth principal embodiment, a compound of Formula
XVII, or a pharmaceutically acceptable salt or prodrug thereof, is
provided: ##STR14## wherein: R.sup.1, R.sup.2 and R.sup.3 are
independently H, phosphate (including mono-, di- or triphosphate
and a stabilized phosphate); straight chained, branched or cyclic
alkyl (including lower alkyl); acyl (including lower acyl);
CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted
aryl, sulfonate ester including alkyl or arylalkyl sulfonyl
including methanesulfonyl and benzyl, wherein the phenyl group is
optionally substituted with one or more substituents as described
in the definition of aryl given herein; alkylsulfonyl,
arylsulfonyl, aralkylsulfonyl, a lipid, including a phospholipid;
an amino acid; and amino acid residue, a carbohydrate; a peptide;
cholesterol; or other pharmaceutically acceptable leaving group
which when administered in vivo is capable of providing a compound
wherein R.sup.1, R.sup.2 and/or R.sup.3 is independently H or
phosphate; wherein at least one of R.sup.2 and R.sup.3 is not
hydrogen; Y is hydrogen, bromo, chloro, fluoro, iodo, OR.sup.4,
NR.sup.4R.sup.5 or SR.sup.4; X.sup.1 is selected from the group
consisting of H, straight chained, branched or cyclic alkyl,
CO-alkyl, CO-aryl, CO-alkoxyalkyl, chloro, bromo, fluoro, iodo,
OR.sup.4, NR.sup.4NR.sup.5 or SR.sup.5; and R.sup.4 and R.sup.5 are
independently hydrogen, acyl (including lower acyl), or alkyl
(including but not limited to methyl, ethyl, propyl and
cyclopropyl).
[0079] In a fifteenth principal embodiment, a compound selected
from Formulas XVIII and XIX, or a pharmaceutically acceptable salt
or prodrug thereof, is provided: ##STR15## wherein: Base is a
purine or pyrimidine base as defined herein; R.sup.1, R.sup.2 and
R.sup.3 are independently H, phosphate (including mono-, di- or
triphosphate and a stabilized phosphate); straight chained,
branched or cyclic alkyl (including lower alkyl); acyl (including
lower acyl); CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl,
CO-substituted aryl, sulfonate ester including alkyl or arylalkyl
sulfonyl including methanesulfonyl and benzyl, wherein the phenyl
group is optionally substituted with one or more substituents as
described in the definition of aryl given herein; alkylsulfonyl,
arylsulfonyl, aralkylsulfonyl, a lipid, including a phospholipid;
an amino acid; and amino acid residue, a carbohydrate; a peptide;
cholesterol; or other pharmaceutically acceptable leaving group
which when administered in vivo is capable of providing a compound
wherein R.sup.1, R.sup.2 and/or R.sup.3 is independently H or
phosphate; wherein R.sup.2 is not hydrogen; R.sup.6 is alkyl
(including lower alkyl and halogenated alkyl), CH.sub.3, CF.sub.3,
azido, cyano, alkenyl, alkynyl, Br-vinyl, 2-Br-ethyl,
--C(O)O(alkyl), --C(O)O(lower alkyl), --O(acyl), --O(lower acyl),
--O(alkyl), --O(lower alkyl), --O(alkenyl), CF.sub.3, chloro,
bromo, fluoro, iodo, NO.sub.2, NH.sub.2, --NH(lower alkyl),
--NH(acyl), --N(lower alkyl).sub.2, --N(acyl).sub.2; and X is O, S,
SO.sub.2 or CH.sub.2.
[0080] In a sixteenth principal embodiment the invention provides a
compound of Formula XX, or a pharmaceutically acceptable salt or
prodrug thereof: ##STR16## wherein: Base is a purine or pyrimidine
base as defined herein; R.sup.1 is H, phosphate (including mono-,
di- or triphosphate and a stabilized phosphate); straight chained,
branched or cyclic alkyl (including lower alkyl); acyl (including
lower acyl); CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl,
CO-substituted aryl, sulfonate ester including alkyl or arylalkyl
sulfonyl including methanesulfonyl and benzyl, wherein the phenyl
group is optionally substituted with one or more substituents as
described in the definition of aryl given herein; alkylsulfonyl,
arylsulfonyl, aralkylsulfonyl, a lipid, including a phospholipid;
an amino acid; and amino acid residue, a carbohydrate; a peptide;
cholesterol; or other pharmaceutically acceptable leaving group
which when administered in vivo is capable of providing a compound
wherein R.sup.1 is H or phosphate; R.sup.6 is alkyl (including
lower alkyl and halogenated alkyl), CH.sub.3, CF.sub.3, azido,
cyano, alkenyl, alkynyl, Br-vinyl, 2-Br-ethyl, --C(O)O(alkyl),
--C(O)O(lower alkyl), --O(acyl), --O(lower acyl), --O(alkyl),
--O(lower alkyl), --O(alkenyl), CF.sub.3, chloro, bromo, fluoro,
iodo, NO.sub.2, NH.sub.2, --NH(lower alkyl), --NH(acyl), --N(lower
alkyl).sub.2, --N(acyl).sub.2; R.sup.7 and R.sup.9 are
independently hydrogen, OR.sup.2, hydroxy, alkyl (including lower
alkyl), azido, cyano, alkenyl, alkynyl, Br-vinyl, --C(O)O(alkyl),
--C(O)O(lower alkyl), --O(acyl), --O(lower acyl), --O(alkyl),
--O(lower alkyl), --O(alkenyl), chlorine, bromine, iodine,
NO.sub.2, NH.sub.2, --NH(lower alkyl), --NH(acyl), --N(lower
alkyl).sub.2, --N(acyl).sub.2; wherein at least one of R.sup.7 and
R.sup.9 is OR.sup.2, wherein each R.sup.2 is independently
phosphate (including mono-, di- or triphosphate and a stabilized
phosphate); straight chained, branched or cyclic alkyl (including
lower alkyl); acyl (including lower acyl); CO-alkyl, CO-aryl,
CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, sulfonate
ester including alkyl or arylalkyl sulfonyl including
methanesulfonyl and benzyl, wherein the phenyl group is optionally
substituted with one or more substituents as described in the
definition of aryl given herein; alkylsulfonyl, arylsulfonyl,
aralkylsulfonyl, a lipid, including a phospholipid; an amino acid;
and amino acid residue, a carbohydrate; a peptide; cholesterol; or
other pharmaceutically acceptable leaving group which when
administered in vivo is capable of providing a compound wherein
R.sup.2 is H or phosphate; R.sup.8 and R.sup.10 are independently
H, alkyl (including lower alkyl), chlorine, bromine or iodine;
alternatively, R.sup.7 and R.sup.10, R.sup.8 and R.sup.9, or
R.sup.8 and R.sup.10 can come together to form a pi bond; and X is
O, S, SO.sub.2 or CH.sub.2.
[0081] In one embodiment, the amino acid residue is of the formula
C(O)C(R.sup.11)(R.sup.12)(NR.sup.13R.sup.14), wherein
R.sup.11 is the side chain of an amino acid and wherein, as in
proline, R.sup.11 can optionally be attached to R.sup.13 to form a
ring structure; or alternatively, R.sup.11 is an alkyl, aryl,
heteroaryl or heterocyclic moiety;
R.sup.12 is hydrogen, alkyl (including lower alkyl) or aryl;
and
R.sup.13 and R.sup.14 are independently hydrogen, acyl (including
an acyl derivative attached to R.sup.11) or alkyl (including but
not limited to methyl, ethyl, propyl, and cyclopropyl).
[0082] In another preferred embodiment, at least one of R.sup.2 and
R.sup.3 is an amino acid residue, and is preferably L-valinyl.
[0083] The .beta.-D- and .beta.-L-nucleosides of this invention may
inhibit HCV polymerase activity. Nucleosides can be screened for
their ability to inhibit HCV polymerase activity in vitro according
to screening methods set forth more particularly herein. One can
readily determine the spectrum of activity by evaluating the
compound in the assays described herein or with another
confirmatory assay.
[0084] In one embodiment the efficacy of the anti-HCV compound is
measured according to the concentration of compound necessary to
reduce the plaque number of the virus in vitro, according to
methods set forth more particularly herein, by 50% (i.e. the
compound's EC.sub.50). In preferred embodiments the parent of the
prodrug compound exhibits an EC.sub.50 of less than 25, 15, 10, 5,
or 1 micromolar. In one embodiment the efficacy of the
anti-Flaviviridae compound is measured according to the
concentration of compound necessary to reduce the plaque number of
the virus in vitro, according to methods set forth more
particularly herein, by 50% (i.e. the compound's EC.sub.50). In
preferred embodiments the compound exhibits an EC.sub.50 of less
than 15 or 10 micromolar, when measured according to the polymerase
assay described in Ferrari et al., J. Virol., 73:1649-1654, 1999;
Ishii et al., Hepatology, 29:1227-1235, 1999; Lohmann et al., J.
Biol. Chem., 274:10807-10815, 1999; or Yamashita et al, J. Biol.
Chem., 273:15479-15486, 1998.
[0085] In another embodiment, combination and/or alternation
therapy are provided. In combination therapy, an effective dosage
of two or more agents are administered together, whereas during
alternation therapy an effective dosage of each agent is
administered serially. The dosages will depend on absorption,
inactivation, and excretion rates of the drug as well as other
factors known to those of skill in the art. It is to be noted that
dosage values will also vary with the severity of the condition to
be alleviated. It is to be further understood that for any
particular subject, specific dosage regimens and schedules should
be adjusted over time according to the individual need and the
professional judgment of the person administering or supervising
the administration of the compositions.
[0086] The invention also provides combinations of at least two of
the herein described prodrugs. The invention further provides at
least one of the described 2' and 3'-prodrugs in combination or
alternation with a second nucleoside that exhibits activity against
a Flaviviridae virus, including but not limited to a parent drug of
any of the prodrugs defined herein, i.e.
.beta.-D-2'-methyl-cytidine, .beta.-D-2'-methyl-thymidine,
.beta.-D-2'-methyl-adenosine, .beta.-D-2'-methyl-guanosine,
.beta.-D-2'-methyl-5-fluorocytidine and/or
.beta.-D-2'-methyl-uridine. Alternatively, the 2' or 3'-prodrugs
can be administered in combination or alternation with other
anti-Flaviviridae agent exhibits an EC.sub.50 of less than 10 or 15
micromolar, or their prodrugs or pharmaceutically acceptable
salts.
[0087] Nonlimiting examples of antiviral agents that can be used in
combination with the compounds disclosed herein include:
[0088] (1) an interferon and/or ribavirin; (2) Substrate-based NS3
protease inhibitors; (3) Non-substrate-based inhibitors; (4)
Thiazolidine derivatives; (5) Thiazolidines and benzanilides; (6) A
phenan-threnequinone; (7) NS3 inhibitors; (8) HCV helicase
inhibitors; (9) polymerase inhibitors, including RNA-dependent
RNA-polymerase inhibitors; (10) Antisense oligodeoxynucleotides
(11) Inhibitors of IRES-dependent translation; (12)
Nuclease-resistant ribozymes; and (13) other compounds that exhibit
activity against a flaviviridae. The invention further includes
administering the prodrug in combination or alternation with an
immune modulator or other pharmaceutically active modifer of viral
replication, including a biological material such as a protein,
peptide, oligonucleotide, or gamma globulin, including but not
limited to interfereon, interleukin, or an antisense
oligonucleotides to genes which express or regulate Flaviviridae
replication.
[0089] The compounds described herein have a number of enantiomeric
configurations, any of which can be used as desired. The parent
nucleoside framework can exist as a .beta.-D or .beta.-L
nucleoside. In a preferred embodiment, the compound is administered
in a form that is at least 90% of the .beta.-D enantiomer. In
another embodiment, the compound is at least 95% of the .beta.-D
enantiomer. Certain prodrug acyl esters, specifically including
amino acid esters, also have enantiomeric forms. In alternative
embodiments, the compounds are used as racemic mixtures or as any
combination of .beta.-D or .beta.-L parent nucleoside and L or D
amino acid.
[0090] In an alternative embodiment, the parent nucleoside
compounds of any of the 2' or 3'-prodrugs (i.e., the nucleosides
without the 2' or 3' cleavable moieties) provided for the treatment
of a Flaviviridae, and in particular, an HCV infection.
BRIEF DESCRIPTION OF THE FIGURES
[0091] FIG. 1 provides the structure of various non-limiting
examples of nucleosides of the present invention, as well as other
known nucleosides, in particular FIAU and ribavirin.
[0092] FIG. 2 provides a non-limiting example of the steps involved
in esterification of the 1', 2', 3' or 4'-branched .beta.-D or
.beta.-L nucleoside to obtain a 2'-prodrug. The same general
procedure can be used to obtain the 3'-prodrug by selectively
protecting the 2' and 5'-hydroxyl groups or protecting the 2', 3'
and 5'-hydroxyl groups and selectively deprotecting the
3'-hydroxyl.
[0093] FIG. 3 provides a non-limiting example of the steps involved
in esterification of the 1', 2', 3' or 4'-branched .beta.-D or
.beta.-L nucleoside to obtain a 3'-prodrug.
[0094] FIG. 4 provides a non-limiting example of the esterification
of the 1', 2', 3' or 4'-branched .beta.-D or .beta.-L nucleoside to
obtain a 2',3'-prodrug.
[0095] FIG. 5 is an illustration of a process of synthesizing a
.beta.-D-2'-C-methyl-ribofuransyl-cytidine or a 3'-O-L-valine ester
thereof.
[0096] FIG. 6 is an illustration of another process of synthesizing
a .beta.-D-2'-C-methyl-ribofuransyl-cytidine or a 3'-O-L-valine
ester thereof.
[0097] FIG. 7 is a diagram of a process of synthesizing a
.beta.-D-2'-C-methyl-2'-acetyl-ribofuransyl-cytidine-3'-O-L-valine
ester.
[0098] FIG. 8 is a diagram of a process of synthesizing a
.beta.-D-2'-C-methyl-2'-acetyl-ribofuransyl-cytidine-3'-O-L-proline
ester.
[0099] FIG. 9 is a diagram of a process of synthesizing a
.beta.-D-2'-C-methyl-2'-acetyl-ribofuransyl-cytidine-3'-O-L-alanine
ester.
[0100] FIG. 10 is a diagram of a process of synthesizing a
.beta.-D-2'-C-methyl-2'-(cyclohexane
carboxylate)-ribofuransyl-cytidine-3'-O-L-valine ester.
[0101] FIG. 11 is a graph showing the concentration of BVDB
(Log.sub.10 units/ml) over a concentration range of four test
compounds and ribavirin as a control in a cell based assay using de
novo BVDV infected MDBK cells. This graph shows the antiviral
potency of these compounds.
[0102] FIG. 12 is a photocopy of a gel illustrating the
site-specific chain termination of in vitro RNA synthesis by
.beta.-D-2'-C-methyl-ribofuranosyl cytidine triphosphate at
specified guanine residues in RNA templates, as described in
Example 32.
[0103] FIG. 13 is a graph of the titer of bovine viral diarrhea
virus (BVDV) over number of passages of BVDV infected MDBK cells,
indicating eradication of a persistent BVDV infection by prolonged
treatment with .beta.-D-2'-C-methyl-ribofuranosyl cytidine (16 uM)
as described in Example 33. Arrows indicate points at which a
portion of cells were withdrawn from drug treatment.
[0104] FIGS. 14a and 14b are graphs of the concentration of bovine
viral diarrhea virus (BVDV) in MDBK cells persistently infected
with the virus, as described in Example 34. These graphs indicate
the synergy between .beta.-D-2'-C-methyl-ribofuranosyl cytidine and
interferon alpha 2b (IntronA) in reducing the viral titer. FIG. 14a
is a graph of the effect of .beta.-D-2'-C-methyl-ribofuranosyl
cytidine and IntronA on BVDV (strain NY-1) titers in persistently
infected MDBK cells over time. FIG. 14b is a graph of the effect of
.beta.-D-2'-C-methyl-ribofuranosyl cytidine in combination with
IntronA on BVDV (strain I-N-dIns) titers in persistently-infected
MDBK cells.
[0105] FIG. 15a-d illustrate the results of experiments studying
the development of resistance to .beta.-D-2'-C-methyl-ribofuranosyl
cytidine treated MDBK cells, infected with bovine viral diarrhea
virus (BVDV), as described in Example 35. FIG. 15a is a graph of a
representative experiment showing the effect over twenty eight days
of .beta.-D-2'-C-methyl-ribofuranosyl cytidine or IntronA treatment
on BVDV (strain I-N-dIns) titers in persistently infected MDBK
cells. FIG. 15b is a photocopy of a dish plated with infected MDBK
cells that illustrates the size of the foci formed by phenotypes of
the wild-type BVDV (strain I-N-dIns), versus the
.beta.-D-2'-C-methyl-ribofuranosyl cytidine-resistant BVDV
(1-N-dIns 107R), indicating that the resistant virus formed much
smaller foci than the wild-type, I-N-dIns strain. FIG. 15c is a
graph of the titer of BVDV strains I-N-dIns or I-N-dIns-107R over
hours post-infection in infected MDBK cells. FIG. 15d is a graph of
the effect of Intron A on the BVDV viral titer yield in de
novo-infected MDBK cells treated with IntronA.
[0106] FIG. 16 is a graph of the concentration of hepatitis C virus
(Log.sub.10) in individual chimpanzees over days of treatment with
.beta.-D-2'-C-methyl-ribofuranosyl cytidine-3'-O-L-valine ester as
described in Example 36.
[0107] FIG. 17 is a graph of the concentration of hepatitis C virus
in individual chimpanzees over days of treatment with
.beta.-D-2'-C-methyl-ribofuranosyl cytidine-3'-O-L-valine ester as
compared to baseline, as described in Example 36.
[0108] FIG. 18 is a graph of percent of total
.beta.-D-2'-C-methyl-ribofuranosyl cytidine-3'-O-L- valine ester
remaining in samples over time after incubation of the drug in
human plasma at 4.degree. C., 21.degree. C., and 37.degree. C., as
described in Example 37.
[0109] FIG. 19a is a graph showing the relative levels of the di-
and tri-phosphate derivatives of .beta.-D-2'-C-methyl-ribofuranosyl
cytidine and .beta.-D-2'-C-methyl-ribofuranosyl uridine (mUrd)
after incubation of HepG2 cells with 10 .mu.M
.beta.-D-2'-C-methyl-ribofuranosyl cytidine over time, as described
in Example 37. FIG. 19b is a graph of the decay of the
tri-phosphate derivative of .beta.-D-2'-C-methyl-ribofuranosyl
cytidine after incubation of HepG2 cells with 10 .mu.M
.beta.-D-2'-C-methyl-ribofuranosyl cytidine over time. FIG. 19c is
a graph of the concentration of the di- and tri-phosphate
derivatives of .beta.-D-2'-C-methyl-ribofuranosyl cytidine and
.beta.-D-2'-C-methyl-ribofuranosyl uridine (mUrd) after incubation
of HepG2 cells with 10 .mu.M .beta.-D-2'-C-methyl-ribofuranosyl
cytidine at increasing concentrations of the drug .mu.M).
[0110] FIG. 20 is a graph of the concentration (ng/ml) of
.beta.-D-2'-C-methyl-ribofuranosyl cytidine in human serum after
administration of .beta.-D-2'-C-methyl-ribofuranosyl
cytidine-3'-O-L- valine ester to patients, as described in Example
40.
[0111] FIG. 21 is a graph of the median change of the titer of
hepatitis C virus in human patients after administration of
.beta.-D-2'-C-methyl-ribofuranosyl cytidine-3'-O-L- valine ester,
as described in Example 40. The graph indicates change from
baseline in Log.sub.10 HCV RNA by patient visit.
[0112] FIG. 22 is a table of the EC.sub.50 and CC.sub.50 of
representative compounds in a BVDV cell protection assay.
DETAILED DESCRIPTION OF THE INVENTION
[0113] The invention as disclosed herein is a compound, a method
and composition for the treatment of a Flaviviridae infection in
humans and other host animals. The method includes the
administration of an effective HCV or Flaviviridae treatment amount
of a 2'- or 3'-prodrug of a 1', 2', 3' or 4'-branched .beta.-D or
.beta.-L nucleoside as described herein or a pharmaceutically
acceptable salt, derivative or prodrug thereof, optionally in a
pharmaceutically acceptable carrier. The compound of this invention
either possesses antiviral (i.e., anti-HCV) activity, or is
metabolized to a compound that exhibits such activity.
[0114] The 2'- or 3'-prodrug of a 1', 2', 3' or 4'-branched
.beta.-D or .beta.-L nucleoside are acyl derivates of a secondary
or tertiary alcohol alpha to a secondary or tertiary carbon. Due to
the steric hindrance of these prodrugs over the 5'-prodrugs, an
acyl derivative of a primary alcohol, these prodrugs differently
modulate the biological properties of the molecule in vivo. It has
been discovered that the 2'- and 3'-prodrugs of a 1', 2', 3' or
4'-branched .beta.-D or .beta.-L nucleoside can provide a drug with
increased half-life and improved pharmacokinetic profile.
[0115] The 2'- and 3'-prodrugs in a preferred embodiment is a
cleavable acyl group, and most particularly, an amino acid moiety,
prepared from any naturally occurring or synthetic .alpha., .beta.
.gamma. or .delta. amino acid, including but is not limited to,
glycine, alanine, valine, leucine, isoleucine, methionine,
phenylalanine, tryptophan, proline, serine, threonine, cysteine,
tyrosine, asparagine, glutamine, aspartate, glutamate, lysine,
arginine and histidine. In a preferred embodiment, the amino acid
is in the L-configuration. Alternatively, the amino acid can be a
derivative of alanyl, valinyl, leucinyl, isoleuccinyl, prolinyl,
phenylalaninyl, tryptophanyl, methioninyl, glycinyl, serinyl,
threoninyl, cysteinyl, tyrosinyl, asparaginyl, glutaminyl,
aspartoyl, glutaroyl, lysinyl, argininyl, histidinyl,
.beta.-alanyl, .beta.-valinyl, .beta.-leucinyl,
.beta.-isoleuccinyl, .beta.-prolinyl, .beta.-phenylalaninyl,
.beta.-tryptophanyl, .beta.-methioninyl, .beta.-glycinyl,
.beta.-serinyl, .beta.-threoninyl, .beta.-cysteinyl,
.beta.-tyrosinyl, .beta.-asparaginyl, .beta.-glutaminyl,
.beta.-aspartoyl, .beta.-glutaroyl, .beta.-lysinyl,
.beta.-argininyl or .beta.-histidinyl. In one particular,
embodiment, the moiety is a valine ester. One particularly
preferred compound is the 3'-valine ester of
2'-methyl-ribo-cytidine.
[0116] The oral bio-availability of 1', 2', 3' or 4'-branched
.beta.-D or .beta.-L nucleoside as the neutral base and the HCl
salt is low in rodents and non-human primates. It has been
discovered that there is significant competition of 1', 2', 3' or
4'-branched .beta.-D or .beta.-L nucleoside with other nucleosides
or nucleoside analogs for absorption, or transport, from the
gastrointestinal tract and competition of other nucleosides or
nucleoside analogs for the absorption with 1', 2', 3' or
4'-branched .beta.-D or .beta.-L nucleoside. In order to improve
oral bioavailability and reduce the potential for drug-drug
interaction, 2' and 3'-prodrugs of 1', 2', 3' or 4'-branched
.beta.-D or .beta.-L nucleoside were obtained with higher oral
bioavailability than the parent molecule and a reduced effect on
the bioavailability of other nucleosides or nucleoside analogs used
in combination.
[0117] The 2', 3', and/or 5'-mono, di or trivaline ester of a 1',
2', 3' or 4'-branched .beta.-D or .beta.-L nucleoside have higher
oral bioavailability than the parent 1', 2', 3' or 4'-branched
.beta.-D or .beta.-L nucleoside and reduced interaction with other
nucleosides or nucleoside analogs when used in combination as
compared to 1', 2', 3' or 4'-branched .beta.-D or .beta.-L
nucleoside.
[0118] The 2', 3', and/or 5'-mono, di or trivaline ester of a 1',
2', 3' or 4'-branched .beta.-D or .beta.-L nucleoside can be
converted to the parent 1', 2', 3' or 4'-branched .beta.-D or
.beta.-L nucleoside through de-esterification in the
gastrointestinal mucosa, blood or liver. The 2', 3', and/or
5'-mono, di or trivaline ester of a 1', 2', 3' or 4'-branched
.beta.-D or .beta.-L nucleoside can be actively transported from
the gastrointestinal lumen after oral delivery into the bloodstream
by an amino acid transporter function in the mucosa of the
gastrointestinal tract. This accounts for the increase in oral
bioavailability compared to the parent 1', 2', 3' or 4'-branched
.beta.-D or .beta.-L nucleoside that is transported primarily by a
nucleoside transporter function. There is reduced competition for
uptake of the 2', 3', and/or 5'-mono, di or trivaline ester of 1',
2', 3' or 4'-branched .beta.-D or .beta.-L nucleoside with other
nucleosides or nucleoside analogs that are transported by the
nucleoside transporter function and not the amino acid transporter
function. As partial de-esterification of the di or trivaline ester
of 1', 2', 3' or 4'-branched .beta.-D or .beta.-L nucleoside occurs
prior to complete absorption, the mono or divaline ester continues
to be absorbed using the amino acid transporter function.
Therefore, the desired outcome of better absorption, or
bioavailability, and reduced competition with other nucleosides or
nucleoside analogs for uptake into the bloodstream can be
maintained.
[0119] In summary, the present invention includes the following
features: [0120] (a) a 2' and/or 3'-prodrug of a 1', 2', 3' or
4'-branched .beta.-D or .beta.-L nucleoside, as described herein,
and pharmaceutically acceptable salts and compositions thereof,
[0121] (b) a 2' and/or 3'-prodrug of a 1', 2', 3' or 4'-branched
.beta.-D or .beta.-L nucleoside as described herein, and
pharmaceutically acceptable salts and compositions thereof for use
in the treatment and/or prophylaxis of a Flaviviridae infection,
especially in individuals diagnosed as having a Flaviviridae
infection or being at risk of becoming infected by hepatitis C;
[0122] (c) a 2' and/or 3'-prodrug of a 1', 2', 3' or 4'-branched
.beta.-D or .beta.-L nucleoside, or their pharmaceutically
acceptable salts and compositions as described herein substantially
in the absence of the opposite enantiomers of the described
nucleoside, or substantially isolated from other chemical entities;
[0123] (d) processes for the preparation of a 2' and/or 3'-prodrug
of a 1', 2', 3' or 4'-branched .beta.-D or .beta.-L nucleoside, as
described in more detail below; [0124] (e) pharmaceutical
formulations comprising a 2' and/or 3'-prodrug of a 1', 2', 3' or
4'-branched .beta.-D or .beta.-L nucleoside or a pharmaceutically
acceptable salt thereof together with a pharmaceutically acceptable
carrier or diluent; [0125] (f) pharmaceutical formulations
comprising a 2' and/or 3'-prodrug of a 1', 2', 3' or 4'-branched
.beta.-D or .beta.-L nucleoside or a pharmaceutically acceptable
salt thereof together with one or more other effective anti-HCV
agents, optionally in a pharmaceutically acceptable carrier or
diluent; [0126] (g) pharmaceutical formulations comprising a 2'
and/or 3'-prodrug of a 1', 2', 3' or 4'-branched .beta.-D or
.beta.-L nucleoside or a pharmaceutically acceptable salt thereof
together with the parent of a different a 1', 2', 3' or 4'-branched
.beta.-D or .beta.-L nucleoside, optionally in a pharmaceutically
acceptable carrier or diluent; [0127] (h) a method for the
treatment and/or prophylaxis of a host infected with Flaviviridae
that includes the administration of an effective amount of a 2'
and/or 3'-prodrug of a 1', 2', 3' or 4'-branched .beta.-D or
.beta.-L nucleoside, its pharmaceutically acceptable salt or
composition; [0128] (i) a method for the treatment and/or
prophylaxis of a host infected with Flaviviridae that includes the
administration of an effective amount of a 2' and/or 3'-prodrug of
a 1', 2', 3' or 4'-branched .beta.-D or .beta.-L nucleoside, its
pharmaceutically acceptable salt or composition in combination
and/or alternation with one or more effective anti-HCV agent;
[0129] (j) a method for the treatment and/or prophylaxis of a host
infected with Flaviviridae that includes the administration of an
effective amount of a 2' and/or 3'-prodrug of a 1', 2', 3' or
4'-branched .beta.-D or .beta.-L nucleoside, or its
pharmaceutically acceptable salt or composition with the parent of
a different a 1', 2', 3' or 4'-branched .beta.-D or .beta.-L
nucleoside; [0130] (k) a method for the treatment and/or
prophylaxis of a host infected with Flaviviridae that includes the
administration of an effective amount of a 2' and/or 3'-prodrug of
a .beta.-D-2'-methyl-cytidine, or its pharmaceutically acceptable
salt or composition thereof; [0131] (l) a method for the treatment
and/or prophylaxis of a host infected with Flaviviridae that
includes the administration of an effective amount of the
3',5'-divalyl or diacetyl ester of .beta.-D-2'-methyl-cytidine, or
its pharmaceutically acceptable salt or composition thereof; [0132]
(m) use of a 2' and/or 3'-prodrug of a 1', 2', 3' or 4'-branched
.beta.-D or .beta.-L nucleoside, and pharmaceutically acceptable
salts and compositions thereof for the treatment and/or prophylaxis
of a Flaviviridae infection in a host; [0133] (n) use of a 2'
and/or 3'-prodrug of a 1', 2', 3' or 4'-branched .beta.-D or
.beta.-L nucleoside, its pharmaceutically acceptable salt or
composition in combination and/or alternation with one or more
effective anti-HCV agent for the treatment and/or prophylaxis of a
Flaviviridae infection in a host; [0134] (o) use of a 2' and/or
3'-prodrug of a 1', 2', 3' or 4'-branched .beta.-D or .beta.-L
nucleoside, or its pharmaceutically acceptable salt or composition
with the parent of a different a 1', 2', 3' or 4'-branched .beta.-D
or .beta.-L nucleoside for the treatment and/or prophylaxis of a
Flaviviridae infection in a host; [0135] (p) use of a 2' and/or
3'-prodrug of a .beta.-D-2'-methyl-cytidine, or its
pharmaceutically acceptable salt or composition thereof for the
treatment and/or prophylaxis of a Flaviviridae infection in a host;
[0136] (q) use of the 3',5'-divalyl or diacetyl ester of
.beta.-D-2'-methyl-cytidine, or its pharmaceutically acceptable
salt or composition thereof for the treatment and/or prophylaxis of
a Flaviviridae infection in a host; [0137] (r) use of a 2' and/or
3'-prodrug of a 1', 2', 3' or 4'-branched .beta.-D or .beta.-L
nucleoside, and pharmaceutically acceptable salts and compositions
thereof in the manufacture of a medicament for treatment and/or
prophylaxis of a Flaviviridae infection; [0138] (s) use of a 2'
and/or 3'-prodrug of a 1', 2', 3' or 4'-branched .beta.-D or
.beta.-L nucleoside, its pharmaceutically acceptable salt or
composition in combination and/or alternation with one or more
effective anti-HCV agent in the manufacture of a medicament for the
treatment and/or prophylaxis of a Flaviviridae infection in a host;
[0139] (t) use of a 2' and/or 3'-prodrug of a 1', 2', 3' or
4'-branched .beta.-D or .beta.-L nucleoside, or its
pharmaceutically acceptable salt or composition with the parent of
a different a 1', 2', 3' or 4'-branched .beta.-D or .beta.-L
nucleoside in the manufacture of a medicament for the treatment
and/or prophylaxis of a Flaviviridae infection in a host; [0140]
(u) use of a 2' and/or 3'-prodrug of a .beta.-D-2'-methyl-cytidine,
or its pharmaceutically acceptable salt or composition thereof in
the manufacture of a medicament for the treatment and/or
prophylaxis of a Flaviviridae infection in a host; and [0141] (v)
use of the 3',5'-divalyl or diacetyl ester of
.beta.-D-2'-methyl-cytidine, or its pharmaceutically acceptable
salt or composition thereof in the manufacture of a medicament for
the treatment and/or prophylaxis of a Flaviviridae infection in a
host.
[0142] Flaviviridae included within the scope of this invention are
discussed generally in Fields Virology, Editors: Fields, B. N.,
Knipe, D. M., and Howley, P. M., Lippincott-Raven Publishers,
Philadelphia, Pa., Chapter 31, 1996. In a particular embodiment of
the invention, the Flaviviridae is HCV. In an alternate embodiment
of the invention, the Flaviviridae is a flavivirus or pestivirus.
Specific flaviviruses include, without limitation: Absettarov,
Alfuy, Apoi, Aroa, Bagaza, Banzi, Bouboui, Bussuquara, Cacipacore,
Carey Island, Dakar bat, Dengue 1, Dengue 2, Dengue 3, Dengue 4,
Edge Hill, Entebbe bat, Gadgets Gully, Hanzalova, Hypr, Ilheus,
Israel turkey meningoencephalitis, Japanese encephalitis, Jugra,
Jutiapa, Kadam, Karshi, Kedougou, Kokobera, Koutango, Kumlinge,
Kunjin, Kyasanur Forest disease, Langat, Louping ill, Meaban,
Modoc, Montana myotis leukoencephalitis, Murray valley
encephalitis, Naranjal, Negishi, Ntaya, Omsk hemorrhagic fever,
Phnom-Penh bat, Powassan, Rio Bravo, Rocio, Royal Farm, Russian
spring-summer encephalitis, Saboya, St. Louis encephalitis, Sal
Vieja, San Perlita, Saumarez Reef, Sepik, Sokuluk, Spondweni,
Stratford, Tembusu, Tyuleniy, Uganda S, Usutu, Wesselsbron, West
Nile, Yaounde, Yellow fever, and Zika.
[0143] Pestiviruses included within the scope of this invention are
discussed generally in Fields Virology, Editors: Fields, B. N.,
Knipe, D. M., and Howley, P. M., Lippincott-Raven Publishers,
Philadelphia, Pa., Chapter 33, 1996. Specific pestiviruses include,
without limitation: bovine viral diarrhea virus ("BVDV"), classical
swine fever virus ("CSFV," also called hog cholera virus), and
border disease virus ("BDV").
I. Active Compounds
[0144] In a first principal embodiment, a compound of Formula (I),
or a pharmaceutically acceptable salt or prodrug thereof, is
provided: ##STR17## wherein: R.sup.1, R.sup.2 and R.sup.3 are
independently H, phosphate (including mono-, di- or triphosphate
and a stabilized phosphate); straight chained, branched or cyclic
alkyl (including lower alkyl); acyl (including lower acyl);
CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted
aryl, sulfonate ester including alkyl or arylalkyl sulfonyl
including methanesulfonyl and benzyl, wherein the phenyl group is
optionally substituted with one or more substituents as described
in the definition of aryl given herein; alkylsulfonyl,
arylsulfonyl, aralkylsulfonyl, a lipid, including a phospholipid;
an amino acid; and amino acid residue, a carbohydrate; a peptide;
cholesterol; or other pharmaceutically acceptable leaving group
which when administered in vivo is capable of providing a compound
wherein R.sup.1, R.sup.2 and/or R.sup.3 is independently H or
phosphate; wherein at least one of R.sup.2 and R.sup.3 is not
hydrogen; Y is hydrogen, bromo, chloro, fluoro, iodo, OH, OR.sup.4,
NH, NHR.sup.5, NR.sup.4R.sup.5, SH and SR.sup.4; X.sup.1 and
X.sup.2 are independently selected from the group consisting of H,
straight chained, branched or cyclic alkyl, CO-alkyl, CO-aryl,
CO-alkoxyalkyl, chloro, bromo, fluoro, iodo, OH, OR.sup.4, NH,
NHR.sup.5, NR.sup.4R.sup.5, SH and SR.sup.4; and R.sup.4 and
R.sup.5 are independently hydrogen, acyl (including lower acyl), or
alkyl (including but not limited to methyl, ethyl, propyl and
cyclopropyl).
[0145] In a preferred subembodiment, a compound of Formula I, or a
pharmaceutically acceptable salt or prodrug thereof, is provided
wherein:
R.sup.1 is H or phosphate (preferably H);
R.sup.2 and R.sup.3 are independently H, phosphate, acyl or an
amino acid residue, wherein at least one of R.sup.2 and R.sup.3 is
acyl or an amino acid residue;
X.sup.1 is H;
X.sup.2 is H or NH.sub.2; and
Y is hydrogen, bromo, chloro, fluoro, iodo, NH.sub.2 or OH.
[0146] In a second principal embodiment, a compound of Formula II,
or a pharmaceutically acceptable salt or prodrug thereof, is
provided: ##STR18## wherein: R.sup.1, R.sup.2 and R.sup.3 are
independently H, phosphate (including mono-, di- or triphosphate
and a stabilized phosphate); straight chained, branched or cyclic
alkyl (including lower alkyl); acyl (including lower acyl);
CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted
aryl, sulfonate ester including alkyl or arylalkyl sulfonyl
including methanesulfonyl and benzyl, wherein the phenyl group is
optionally substituted with one or more substituents as described
in the definition of aryl given herein; alkylsulfonyl,
arylsulfonyl, aralkylsulfonyl, a lipid, including a phospholipid;
an amino acid; and amino acid residue, a carbohydrate; a peptide;
cholesterol; or other pharmaceutically acceptable leaving group
which when administered in vivo is capable of providing a compound
wherein R.sup.1, R.sup.2 and/or R.sup.3 is independently H or
phosphate; wherein at least one of R.sup.2 and R.sup.3 is not
hydrogen; Y is hydrogen, bromo, chloro, fluoro, iodo, OR.sup.4,
NR.sup.4R.sup.5 or SR.sup.4; X.sup.1 and X.sup.2 are independently
selected from the group consisting of H, straight chained, branched
or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, chloro, bromo,
fluoro, iodo, OR.sup.4, NR.sup.4NR.sup.5 or SR.sup.5; and R.sup.4
and R.sup.5 are independently hydrogen, acyl (including lower
acyl), or alkyl (including but not limited to methyl, ethyl, propyl
and cyclopropyl).
[0147] In a preferred subembodiment, a compound of Formula II, or a
pharmaceutically acceptable salt or prodrug thereof, is provided
wherein:
R.sup.1 is H or phosphate (preferably H);
R.sup.2 and R.sup.3 are independently H, phosphate, acyl or an
amino acid residue, wherein at least one of R.sup.2 and R.sup.3 is
acyl or an amino acid residue;
X.sup.1 is H;
X.sup.2 is H or NH.sub.2; and
Y is hydrogen, bromo, chloro, fluoro, iodo, NH.sub.2 or OH.
[0148] In a third principal embodiment, a compound of Formula III,
or a pharmaceutically acceptable salt or prodrug thereof, is
provided: ##STR19## wherein: R.sup.1, R.sup.2 and R.sup.3 are
independently H, phosphate (including mono-, di- or triphosphate
and a stabilized phosphate); straight chained, branched or cyclic
alkyl (including lower alkyl); acyl (including lower acyl);
CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted
aryl, sulfonate ester including alkyl or arylalkyl sulfonyl
including methanesulfonyl and benzyl, wherein the phenyl group is
optionally substituted with one or more substituents as described
in the definition of aryl given herein; alkylsulfonyl,
arylsulfonyl, aralkylsulfonyl, a lipid, including a phospholipid;
an amino acid; and amino acid residue, a carbohydrate; a peptide;
cholesterol; or other pharmaceutically acceptable leaving group
which when administered in vivo is capable of providing a compound
wherein R.sup.1, R.sup.2 and/or R.sup.3 is independently H or
phosphate; wherein at least one of R.sup.2 and R.sup.3 is not
hydrogen; Y is hydrogen, bromo, chloro, fluoro, iodo, OR.sup.4,
NR.sup.4R.sup.5 or SR.sup.4; X.sup.1 and X.sup.2 are independently
selected from the group consisting of H, straight chained, branched
or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, chloro, bromo,
fluoro, iodo, OR.sup.4, NR.sup.4NR.sup.5 or SR.sup.5; and R.sup.4
and R.sup.5 are independently hydrogen, acyl (including lower
acyl), or alkyl (including but not limited to methyl, ethyl, propyl
and cyclopropyl).
[0149] In a preferred subembodiment, a compound of Formula III, or
a pharmaceutically acceptable salt or prodrug thereof, is provided
wherein:
R.sup.1 is H or phosphate (preferably H);
R.sup.2 and R.sup.3 are independently H, phosphate, acyl or an
amino acid residue, wherein at least one of R.sup.2 and R.sup.3 is
acyl or an amino acid residue;
X.sup.1 is H;
X.sup.2 is H or NH.sub.2; and
Y is hydrogen, bromo, chloro, fluoro, iodo, NH.sub.2 or OH.
[0150] In a fourth principal embodiment, a compound of Formula IV,
or a pharmaceutically acceptable salt or prodrug thereof, is
provided: ##STR20## wherein: R.sup.1, R.sup.2 and R.sup.3 are
independently H, phosphate (including mono-, di- or triphosphate
and a stabilized phosphate); straight chained, branched or cyclic
alkyl (including lower alkyl); acyl (including lower acyl);
CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted
aryl, sulfonate ester including alkyl or arylalkyl sulfonyl
including methanesulfonyl and benzyl, wherein the phenyl group is
optionally substituted with one or more substituents as described
in the definition of aryl given herein; alkylsulfonyl,
arylsulfonyl, aralkylsulfonyl, a lipid, including a phospholipid;
an amino acid; and amino acid residue, a carbohydrate; a peptide;
cholesterol; or other pharmaceutically acceptable leaving group
which when administered in vivo is capable of providing a compound
wherein R.sup.1, R.sup.2 and/or R.sup.3 is independently H or
phosphate; wherein at least one of R.sup.2 and R.sup.3 is not
hydrogen; Y is hydrogen, bromo, chloro, fluoro, iodo, OR.sup.4,
NR.sup.4R.sup.5 or SR.sup.4; X.sup.1 is selected from the group
consisting of H, straight chained, branched or cyclic alkyl,
CO-alkyl, CO-aryl, CO-alkoxyalkyl, chloro, bromo, fluoro, iodo,
OR.sup.4, NR.sup.4R.sup.5 or SR.sup.5; and R.sup.4 and R.sup.5 are
independently hydrogen, acyl (including lower acyl), or alkyl
(including but not limited to methyl, ethyl, propyl and
cyclopropyl).
[0151] In a preferred subembodiment, a compound of Formula IV, or a
pharmaceutically acceptable salt or prodrug thereof, is provided
wherein:
R.sup.1 is H or phosphate (preferably H);
R.sup.2 and R.sup.3 are independently H, phosphate, acyl or an
amino acid residue, wherein at least one of R.sup.2 and R.sup.3 is
acyl or an amino acid residue;
X.sup.1 is H or CH.sub.3; and
Y is hydrogen, bromo, chloro, fluoro, iodo, NH.sub.2 or OH.
[0152] In a fifth principal embodiment, a compound of Formula V, or
a pharmaceutically acceptable salt or prodrug thereof, is provided:
##STR21## wherein: R.sup.1, R.sup.2 and R.sup.3 are independently
H, phosphate (including mono-, di- or triphosphate and a stabilized
phosphate); straight chained, branched or cyclic alkyl (including
lower alkyl); acyl (including lower acyl); CO-alkyl, CO-aryl,
CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, sulfonate
ester including alkyl or arylalkyl sulfonyl including
methanesulfonyl and benzyl, wherein the phenyl group is optionally
substituted with one or more substituents as described in the
definition of aryl given herein; alkylsulfonyl, arylsulfonyl,
aralkylsulfonyl, a lipid, including a phospholipid; an amino acid;
and amino acid residue, a carbohydrate; a peptide; cholesterol; or
other pharmaceutically acceptable leaving group which when
administered in vivo is capable of providing a compound wherein
R.sup.1, R.sup.2 and/or R.sup.3 is independently H or phosphate;
wherein at least one of R.sup.2 and R.sup.3 is not hydrogen; Y is
hydrogen, bromo, chloro, fluoro, iodo, OR.sup.4, NR.sup.4R.sup.5 or
SR.sup.4; X.sup.1 is selected from the group consisting of H,
straight chained, branched or cyclic alkyl, CO-alkyl, CO-aryl,
CO-alkoxyalkyl, chloro, bromo, fluoro, iodo, OR.sup.4,
NR.sup.4NR.sup.5 or SR.sup.5; and R.sup.4 and R.sup.5 are
independently hydrogen, acyl (including lower acyl), or alkyl
(including but not limited to methyl, ethyl, propyl and
cyclopropyl).
[0153] In a preferred subembodiment, a compound of Formula V, or a
pharmaceutically acceptable salt or prodrug thereof, is provided
wherein:
R.sup.1 is H or phosphate (preferably H);
R.sup.2 and R.sup.3 are independently H, phosphate, acyl or an
amino acid residue, wherein at least one of R.sup.2 and R.sup.3 is
acyl or an amino acid residue;
X.sup.1 is H or CH.sub.3; and
Y is hydrogen, bromo, chloro, fluoro, iodo, NH.sub.2 or OH.
[0154] In a sixth principal embodiment, a compound of Formula VI,
or a pharmaceutically acceptable salt or prodrug thereof, is
provided: ##STR22## wherein: R.sup.1, R.sup.2 and R.sup.3 are
independently H, phosphate (including mono-, di- or triphosphate
and a stabilized phosphate); straight chained, branched or cyclic
alkyl (including lower alkyl); acyl (including lower acyl);
CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted
aryl, sulfonate ester including alkyl or arylalkyl sulfonyl
including methanesulfonyl and benzyl, wherein the phenyl group is
optionally substituted with one or more substituents as described
in the definition of aryl given herein; alkylsulfonyl,
arylsulfonyl, aralkylsulfonyl, a lipid, including a phospholipid;
an amino acid; and amino acid residue, a carbohydrate; a peptide;
cholesterol; or other pharmaceutically acceptable leaving group
which when administered in vivo is capable of providing a compound
wherein R.sup.1, R.sup.2 and/or R.sup.3 is independently H or
phosphate; wherein at least one of R.sup.2 and R.sup.3 is not
hydrogen; Y is hydrogen, bromo, chloro, fluoro, iodo, OR.sup.4,
NR.sup.4R.sup.5 or SR.sup.4; X.sup.1 is selected from the group
consisting of H, straight chained, branched or cyclic alkyl,
CO-alkyl, CO-aryl, CO-alkoxyalkyl, chloro, bromo, fluoro, iodo,
OR.sup.4, NR.sup.4NR.sup.5 or SR.sup.5; and R.sup.4 and R.sup.5 are
independently hydrogen, acyl (including lower acyl), or alkyl
(including but not limited to methyl, ethyl, propyl and
cyclopropyl).
[0155] In a preferred subembodiment, a compound of Formula VI, or a
pharmaceutically acceptable salt or prodrug thereof, is provided
wherein:
R.sup.1 is H or phosphate (preferably H);
R.sup.2 and R.sup.3 are independently H, phosphate, acyl or an
amino acid residue, wherein at least one of R.sup.2 and R.sup.3 is
acyl or an amino acid residue;
X.sup.1 is H or CH.sub.3; and
Y is hydrogen, bromo, chloro, fluoro, iodo, NH.sub.2 or OH.
[0156] In a seventh principal embodiment, a compound selected from
Formulas VII and VIII, or a pharmaceutically acceptable salt or
prodrug thereof, is provided: ##STR23## wherein: Base is a purine
or pyrimidine base as defined herein; R.sup.1, R.sup.2 and R.sup.3
are independently H, phosphate (including mono-, di- or
triphosphate and a stabilized phosphate); straight chained,
branched or cyclic alkyl (including lower alkyl); acyl (including
lower acyl); CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl,
CO-substituted aryl, sulfonate ester including alkyl or arylalkyl
sulfonyl including methanesulfonyl and benzyl, wherein the phenyl
group is optionally substituted with one or more substituents as
described in the definition of aryl given herein; alkylsulfonyl,
arylsulfonyl, aralkylsulfonyl, a lipid, including a phospholipid;
an amino acid; and amino acid residue, a carbohydrate; a peptide;
cholesterol; or other pharmaceutically acceptable leaving group
which when administered in vivo is capable of providing a compound
wherein R.sup.1, R.sup.2 and/or R.sup.3 is independently H or
phosphate; wherein R.sup.2 is not hydrogen; R.sup.6 is alkyl
(including lower alkyl and halogenated alkyl), CH.sub.3, CF.sub.3,
azido, cyano, alkenyl, alkynyl, Br-vinyl, 2-Br-ethyl,
--C(O)O(alkyl), --C(O)O(lower alkyl), --O(acyl), --O(lower acyl),
--O(alkyl), --O(lower alkyl), --O(alkenyl), CF.sub.3, chloro,
bromo, fluoro, iodo, NO.sub.2, NH.sub.2, --NH(lower alkyl),
--NH(acyl), --N(lower alkyl).sub.2, --N(acyl).sub.2; and X is O, S,
SO.sub.2 or CH.sub.2.
[0157] In a first subembodiment, a compound of Formula VII or VIII,
or a pharmaceutically acceptable salt or prodrug thereof, is
provided wherein:
Base is a purine or pyrimidine base as defined herein;
R.sup.1 is H or phosphate (preferably H);
R.sup.2 and R.sup.3 are independently H, phosphate, acyl or an
amino acid residue, wherein at least one of R.sup.2 and R.sup.3 is
acyl or an amino acid residue;
R.sup.6 is alkyl; and
X is O, S, SO.sub.2 or CH.sub.2.
[0158] In a second subembodiment, a compound of Formula VII or
VIII, or a pharmaceutically acceptable salt or prodrug thereof, is
provided wherein:
Base is a purine or pyrimidine base as defined herein;
R.sup.1 is H or phosphate (preferably H);
R.sup.2 and R.sup.3 are independently H, phosphate, acyl or an
amino acid residue, wherein at least one of R.sup.2 and R.sup.3 is
an amino acid residue;
R.sup.6 is alkyl; and
X is O, S, SO.sub.2 or CH.sub.2.
[0159] In a third subembodiment, a compound of Formula VII or VIII,
or a pharmaceutically acceptable salt or prodrug thereof, is
provided wherein:
Base is a purine or pyrimidine base as defined herein;
R.sup.1 is H or phosphate (preferably H);
R.sup.2 and R.sup.3 are independently H, phosphate, acyl or an
amino acid residue, wherein at least one of R.sup.2 and R.sup.3 is
acyl or an amino acid residue;
R.sup.6 is alkyl; and
[0160] X is O.
[0161] In a eighth principal embodiment, a compound of Formulas IX
and X, or a pharmaceutically acceptable salt or prodrug thereof, is
provided: ##STR24## wherein: Base is a purine or pyrimidine base as
defined herein; R.sup.1, R.sup.2 and R.sup.3 are independently H,
phosphate (including mono-, di- or triphosphate and a stabilized
phosphate); straight chained, branched or cyclic alkyl (including
lower alkyl); acyl (including lower acyl); CO-alkyl, CO-aryl,
CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, sulfonate
ester including alkyl or arylalkyl sulfonyl including
methanesulfonyl and benzyl, wherein the phenyl group is optionally
substituted with one or more substituents as described in the
definition of aryl given herein; alkylsulfonyl, arylsulfonyl,
aralkylsulfonyl, a lipid, including a phospholipid; an amino acid;
and amino acid residue, a carbohydrate; a peptide; cholesterol; or
other pharmaceutically acceptable leaving group which when
administered in vivo is capable of providing a compound wherein
R.sup.1, R.sup.2 and/or R.sup.3 is independently H or phosphate;
wherein R.sup.2 is not hydrogen; R.sup.6 is alkyl (including lower
alkyl and halogenated alkyl), CH.sub.3, CF.sub.3, azido, cyano,
alkenyl, alkynyl, Br-vinyl, 2-Br-ethyl, --C(O)O(alkyl),
--C(O)O(lower alkyl), --O(acyl), --O(lower acyl), --O(alkyl),
--O(lower alkyl), --O(alkenyl), CF.sub.3, chloro, bromo, fluoro,
iodo, NO.sub.2, NH.sub.2, --NH(lower alkyl), --NH(acyl), --N(lower
alkyl).sub.2, --N(acyl).sub.2; and R.sup.7 is hydrogen, OR.sup.3,
hydroxy, alkyl (including lower alkyl), azido, cyano, alkenyl,
alkynyl, Br-vinyl, --C(O)O(alkyl), --C(O)O(lower alkyl), --O(acyl),
--O(lower acyl), --O(alkyl), --O(lower alkyl), --O(alkenyl),
chlorine, bromine, iodine, NO.sub.2, NH.sub.2, --NH(lower alkyl),
--NH(acyl), --N(lower alkyl).sub.2, --N(acyl).sub.2; and X is O, S,
SO.sub.2 or CH.sub.2.
[0162] In a first subembodiment, a compound of Formula IX or X, or
a pharmaceutically acceptable salt or prodrug thereof, is provided
wherein:
Base is a purine or pyrimidine base as defined herein;
R.sup.1 is H or phosphate (preferably H);
R.sup.2 and R.sup.3 are independently H, phosphate, acyl or an
amino acid residue, wherein at least one of R.sup.2 and R.sup.3 is
acyl or an amino acid residue;
R.sup.6 is alkyl; and
[0163] X is O, S, SO.sub.2 or CH.sub.2.
[0164] In a second subembodiment, a compound of Formula IX or X, or
a pharmaceutically acceptable salt or prodrug thereof, is provided
wherein:
Base is a purine or pyrimidine base as defined herein;
R.sup.1 is H or phosphate (preferably H);
R.sup.2 and R.sup.3 are independently H, phosphate, acyl or an
amino acid residue, wherein at least one of R.sup.2 and R.sup.3 is
an amino acid residue;
R.sup.6 is alkyl; and
X is O, S, SO.sub.2 or CH.sub.2.
[0165] In a third subembodiment, a compound of Formula IX or X, or
a pharmaceutically acceptable salt or prodrug thereof, is provided
wherein:
Base is a purine or pyrimidine base as defined herein;
R.sup.1 is H or phosphate (preferably H);
R.sup.2 and R.sup.3 are independently H, phosphate, acyl or an
amino acid residue, wherein at least one of R.sup.2 and R.sup.3 is
acyl or an amino acid residue;
R.sup.6 is alkyl; and
[0166] X is O.
[0167] In another subembodiments, a compound of Formula X(a), or
its pharmaceutically acceptable salt or prodrug, is provided:
##STR25## wherein: Base is a purine or pyrimidine base as defined
herein; optionally substituted with an amine or cyclopropyl (e.g.,
2-amino, 2,6-diamino or cyclopropyl guanosine); and R.sup.1 and
R.sup.2 are independently H, phosphate (including mono-, di- or
triphosphate and a stabilized phosphate); straight chained,
branched or cyclic alkyl (including lower alkyl); acyl (including
lower acyl); CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl,
CO-substituted aryl, sulfonate ester including alkyl or arylalkyl
sulfonyl including methanesulfonyl and benzyl, wherein the phenyl
group is optionally substituted with one or more substituents as
described in the definition of aryl given herein; alkylsulfonyl,
arylsulfonyl, aralkylsulfonyl, a lipid, including a phospholipid;
an amino acid; and amino acid residue, a carbohydrate; a peptide;
cholesterol; or other pharmaceutically acceptable leaving group
which when administered in vivo is capable of providing a compound
wherein R.sup.1, R.sup.2 and/or R.sup.3 is independently H or
phosphate; wherein R.sup.2 is not hydrogen.
[0168] In a ninth principal embodiment a compound selected from
Formulas XI and XII, or a pharmaceutically acceptable salt or
prodrug thereof, is provided: ##STR26## wherein: Base is a purine
or pyrimidine base as defined herein; R.sup.1, R.sup.2 and R.sup.3
are independently H, phosphate (including mono-, di- or
triphosphate and a stabilized phosphate); straight chained,
branched or cyclic alkyl (including lower alkyl); acyl (including
lower acyl); CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl,
CO-substituted aryl, sulfonate ester including alkyl or arylalkyl
sulfonyl including methanesulfonyl and benzyl, wherein the phenyl
group is optionally substituted with one or more substituents as
described in the definition of aryl given herein; alkylsulfonyl,
arylsulfonyl, aralkylsulfonyl, a lipid, including a phospholipid;
an amino acid; and amino acid residue, a carbohydrate; a peptide;
cholesterol; or other pharmaceutically acceptable leaving group
which when administered in vivo is capable of providing a compound
wherein R.sup.1, R.sup.2 and/or R.sup.3 is independently H or
phosphate; wherein R.sup.2 is not hydrogen; R.sup.6 is alkyl
(including lower alkyl and halogenated alkyl), CH.sub.3, CF.sub.3,
azido, cyano, alkenyl, alkynyl, Br-vinyl, 2-Br-ethyl,
--C(O)O(alkyl), --C(O)O(lower alkyl), --O(acyl), --O(lower acyl),
--O(alkyl), --O(lower alkyl), --O(alkenyl), CF.sub.3, chloro,
bromo, fluoro, iodo, NO.sub.2, NH.sub.2, --NH(lower alkyl),
--NH(acyl), --N(lower alkyl).sub.2, --N(acyl).sub.2; and X is O, S,
SO.sub.2 or CH.sub.2.
[0169] In a first subembodiment, a compound of Formula XI or XII,
or a pharmaceutically acceptable salt or prodrug thereof, is
provided wherein:
Base is a purine or pyrimidine base as defined herein;
R.sup.1 is H or phosphate (preferably H);
R.sup.2 and R.sup.3 are independently H, phosphate, acyl or an
amino acid residue, wherein at least one of R.sup.2 and R.sup.3 is
acyl or an amino acid residue;
R.sup.6 is alkyl; and
X is O, S, SO.sub.2 or CH.sub.2.
[0170] In a second subembodiment, a compound of Formula XI or XII,
or a pharmaceutically acceptable salt or prodrug thereof, is
provided wherein:
Base is a purine or pyrimidine base as defined herein;
R.sup.1 is H or phosphate (preferably H);
R.sup.2 and R.sup.3 are independently H, phosphate, acyl or an
amino acid residue, wherein at least one of R.sup.2 and R.sup.3 is
an amino acid residue;
R.sup.6 is alkyl; and
X is O, S, SO.sub.2 or CH.sub.2.
[0171] In a third subembodiment, a compound of Formula XI or XII,
or a pharmaceutically acceptable salt or prodrug thereof, is
provided wherein:
Base is a purine or pyrimidine base as defined herein;
R.sup.1 is H or phosphate (preferably H);
R.sup.2 and R.sup.3 are independently H, phosphate, acyl or an
amino acid residue, wherein at least one of R.sup.2 and R.sup.3 is
acyl or an amino acid residue;
R.sup.6 is alkyl; and
X is O.
[0172] In a tenth principal embodiment the invention provides a
compound of Formula XIII, or a pharmaceutically acceptable salt or
prodrug thereof: ##STR27## wherein: Base is a purine or pyrimidine
base as defined herein; R.sup.1 is H, phosphate (including mono-,
di- or triphosphate and a stabilized phosphate); straight chained,
branched or cyclic alkyl (including lower alkyl); acyl (including
lower acyl); CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl,
CO-substituted aryl, sulfonate ester including alkyl or arylalkyl
sulfonyl including methanesulfonyl and benzyl, wherein the phenyl
group is optionally substituted with one or more substituents as
described in the definition of aryl given herein; alkylsulfonyl,
arylsulfonyl, aralkylsulfonyl, a lipid, including a phospholipid;
an amino acid; and amino acid residue, a carbohydrate; a peptide;
cholesterol; or other pharmaceutically acceptable leaving group
which when administered in vivo is capable of providing a compound
wherein R.sup.1 is H or phosphate; R.sup.6 is alkyl (including
lower alkyl and halogenated alkyl), CH.sub.3, CF.sub.3, azido,
cyano, alkenyl, alkynyl, Br-vinyl, 2-Br-ethyl, --C(O)O(alkyl),
--C(O)O(lower alkyl), --O(acyl), --O(lower acyl), --O(alkyl),
--O(lower alkyl), --O(alkenyl), CF.sub.3, chloro, bromo, fluoro,
iodo, NO.sub.2, NH.sub.2, --NH(lower alkyl), --NH(acyl), --N(lower
alkyl).sub.2, --N(acyl).sub.2; R.sup.7 and R.sup.9 are
independently hydrogen, OR.sup.2, hydroxy, alkyl (including lower
alkyl), azido, cyano, alkenyl, alkynyl, Br-vinyl, --C(O)O(alkyl),
--C(O)O(lower alkyl), --O(acyl), --O(lower acyl), --O(alkyl),
--O(lower alkyl), --O(alkenyl), chlorine, bromine, iodine,
NO.sub.2, NH.sub.2, --NH(lower alkyl), --NH(acyl), --N(lower
alkyl).sub.2, --N(acyl).sub.2; wherein at least one of R.sup.7 and
R.sup.9 is OR.sup.2, wherein each R.sup.2 is independently
phosphate (including mono-, di- or triphosphate and a stabilized
phosphate); straight chained, branched or cyclic alkyl (including
lower alkyl); acyl (including lower acyl); CO-alkyl, CO-aryl,
CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, sulfonate
ester including alkyl or arylalkyl sulfonyl including
methanesulfonyl and benzyl, wherein the phenyl group is optionally
substituted with one or more substituents as described in the
definition of aryl given herein; alkylsulfonyl, arylsulfonyl,
aralkylsulfonyl, a lipid, including a phospholipid; an amino acid;
and amino acid residue, a carbohydrate; a peptide; cholesterol; or
other pharmaceutically acceptable leaving group which when
administered in vivo is capable of providing a compound wherein
R.sup.2 is H or phosphate; R.sup.8 and R.sup.10 are independently
H, alkyl (including lower alkyl), chlorine, bromine or iodine;
alternatively, R.sup.7 and R.sup.10, R.sup.8 and R.sup.9, or
R.sup.8 and R.sup.10 can come together to form a pi bond; and X is
O, S, SO.sub.2 or CH.sub.2.
[0173] In a first subembodiment, a compound of Formula XIII, or its
pharmaceutically acceptable salt or prodrug, is provided in which:
(1) Base is a purine or pyrimidine base as defined herein; (2)
R.sup.1 is independently H or phosphate (including monophosphate,
diphosphate, triphosphate, or a stabilized phosphate prodrug); acyl
(including lower acyl); alkyl (including lower alkyl); sulfonate
ester including alkyl or arylalkyl sulfonyl including
methanesulfonyl and benzyl, wherein the phenyl group is optionally
substituted with one or more substituents as described in the
definition of aryl given herein; a lipid, including a phospholipid;
an amino acid; a carbohydrate; a peptide; cholesterol; or other
pharmaceutically acceptable leaving group which when administered
in vivo is capable of providing a compound wherein R.sup.1 is
independently H or phosphate; (3) R.sup.6 is alkyl; (4) R.sup.7 and
R.sup.9 are independently OR.sup.2, alkyl, alkenyl, alkynyl,
Br-vinyl, O-alkenyl, chlorine, bromine, iodine, NO.sub.2, amino,
lower alkylamino or di(loweralkyl)amino; wherein at least one of
R.sup.7 and R.sup.9 is OR.sup.2 (and R.sup.2 is not hydrogen); (5)
R.sup.8 and R.sup.10 are independently H, alkyl (including lower
alkyl), chlorine, bromine, or iodine; and (6) X is O, S, SO.sub.2
or CH.sub.2.
[0174] In a second subembodiment, a compound of Formula XIII, or
its pharmaceutically acceptable salt or prodrug, is provided in
which: (1) Base is a purine or pyrimidine base as defined herein;
(2) R.sup.1 is independently H or phosphate (including
monophosphate, diphosphate, triphosphate, or a stabilized phosphate
prodrug); acyl (including lower acyl); alkyl (including lower
alkyl); sulfonate ester including alkyl or arylalkyl sulfonyl
including methanesulfonyl and benzyl, wherein the phenyl group is
optionally substituted with one or more substituents as described
in the definition of aryl given herein; a lipid, including a
phospholipid; an amino acid; a carbohydrate; a peptide;
cholesterol; or other pharmaceutically acceptable leaving group
which when administered in vivo is capable of providing a compound
wherein R.sup.1 is independently H or phosphate; (3) R.sup.6 is
alkyl, alkenyl, alkynyl, Br-vinyl, hydroxy, O-alkyl, O-alkenyl,
chloro, bromo, fluoro, iodo, NO.sub.2, amino, lower alkylamino, or
di(loweralkyl)amino; (4) R.sup.7 and R.sup.9 are independently OH
or OR.sup.2, wherein at least one of R.sup.7 and R.sup.9 is
OR.sup.2 (and R.sup.2 is not hydrogen); (5) R.sup.8 and R.sup.10
are independently H, alkyl (including lower alkyl), chlorine,
bromine, or iodine; and (6) X is O, S, SO.sub.2 or CH.sub.2.
[0175] In a third subembodiment, a compound of Formula XIII, or its
pharmaceutically acceptable salt or prodrug, is provided in which:
(1) Base is a purine or pyrimidine base as defined herein; (2)
R.sup.1 is independently H or phosphate (including monophosphate,
diphosphate, triphosphate, or a stabilized phosphate prodrug); acyl
(including lower acyl); alkyl (including lower alkyl); sulfonate
ester including alkyl or arylalkyl sulfonyl including
methanesulfonyl and benzyl, wherein the phenyl group is optionally
substituted with one or more substituents as described in the
definition of aryl given herein; a lipid, including a phospholipid;
an amino acid; a carbohydrate; a peptide; cholesterol; or other
pharmaceutically acceptable leaving group which when administered
in vivo is capable of providing a compound wherein R.sup.1 is
independently H or phosphate; (3) R.sup.6 is alkyl, alkenyl,
alkynyl, Br-vinyl, hydroxy, O-alkyl, O-alkenyl, chloro, bromo,
fluoro, iodo, NO.sub.2, amino, lower alkylamino or
di(loweralkyl)amino; (4) R.sup.7 and R.sup.9 are independently
OR.sup.2, alkyl, alkenyl, alkynyl, Br-vinyl, O-alkenyl, chlorine,
bromine, iodine, NO.sub.2, amino, lower alkylamino or
di(loweralkyl)amino; wherein at least one of R.sup.7 and R.sup.9 is
OR.sup.2 (and R.sup.2 is not hydrogen); (5) R.sup.8 and R.sup.10
are H; and (6) X is O, S, SO.sub.2 or CH.sub.2.
[0176] In a fourth subembodiment, a compound of Formula XIII, or
its pharmaceutically acceptable salt or prodrug, is provided in
which: (1) Base is a purine or pyrimidine base as defined herein;
(2) R.sup.1 is independently H or phosphate (including
monophosphate, diphosphate, triphosphate, or a stabilized phosphate
prodrug); acyl (including lower acyl); alkyl (including lower
alkyl); sulfonate ester including alkyl or arylalkyl sulfonyl
including methanesulfonyl and benzyl, wherein the phenyl group is
optionally substituted with one or more substituents as described
in the definition of aryl given herein; a lipid, including a
phospholipid; an amino acid; a carbohydrate; a peptide;
cholesterol; or other pharmaceutically acceptable leaving group
which when administered in vivo is capable of providing a compound
wherein R.sup.1 is independently H or phosphate; (3) R.sup.6 is
alkyl, alkenyl, alkynyl, Br-vinyl, hydroxy, O-alkyl, O-alkenyl,
chloro, bromo, fluoro, iodo, NO.sub.2, amino, lower alkylamino, or
di(loweralkyl)amino; (4) R.sup.7 and R.sup.9 are independently
OR.sup.2, alkyl, alkenyl, alkynyl, Br-vinyl, O-alkenyl, chlorine,
bromine, iodine, NO.sub.2, amino, lower alkylamino, or
di(loweralkyl)amino; wherein at least one of R.sup.7 and R.sup.9 is
OR.sup.2 (and R.sup.2 is not hydrogen); (5) R.sup.8 and R.sup.10
are independently H, alkyl (including lower alkyl), chlorine,
bromine, or iodine; and (6) X is O.
[0177] In a fifth subembodiment, a compound of Formula XIII, or its
pharmaceutically acceptable salt or prodrug, is provided in which:
(1) Base is a purine or pyrimidine base as defined herein; (2)
R.sup.1 is independently H or phosphate (including monophosphate,
diphosphate, triphosphate, or a stabilized phosphate prodrug); acyl
(including lower acyl); alkyl (including lower alkyl); sulfonate
ester including alkyl or arylalkyl sulfonyl including
methanesulfonyl and benzyl, wherein the phenyl group is optionally
substituted with one or more substituents as described in the
definition of aryl given herein; a lipid, including a phospholipid;
an amino acid; a carbohydrate; a peptide; cholesterol; or other
pharmaceutically acceptable leaving group which when administered
in vivo is capable of providing a compound wherein R.sup.1 is
independently H or phosphate; (3) R.sup.6 is alkyl; (4) R.sup.7 and
R.sup.9 are independently OH or OR.sup.2, wherein at least one of
R.sup.7 and R.sup.9 is OR.sup.2 (and R.sup.2 is not hydrogen); (5)
R.sup.8 and R.sup.10 are independently H, alkyl (including lower
alkyl), chlorine, bromine or iodine; and (6) X is O, S, SO.sub.2 or
CH.sub.2.
[0178] In a sixth subembodiment, a compound of Formula XIII, or its
pharmaceutically acceptable salt or prodrug, is provided in which:
(1) Base is a purine or pyrimidine base as defined herein; (2)
R.sup.1 is independently H or phosphate (including monophosphate,
diphosphate, triphosphate, or a stabilized phosphate prodrug); acyl
(including lower acyl); alkyl (including lower alkyl); sulfonate
ester including alkyl or arylalkyl sulfonyl including
methanesulfonyl and benzyl, wherein the phenyl group is optionally
substituted with one or more substituents as described in the
definition of aryl given herein; a lipid, including a phospholipid;
an amino acid; a carbohydrate; a peptide; cholesterol; or other
pharmaceutically acceptable leaving group which when administered
in vivo is capable of providing a compound wherein R.sup.1 is
independently H or phosphate; (3) R.sup.6 is alkyl; (4) R.sup.7 and
R.sup.9 are independently OR.sup.2, alkyl (including lower alkyl),
alkenyl, alkynyl, Br-vinyl, O-alkenyl, chlorine, bromine, iodine,
NO.sub.2, amino, lower alkylamino, or di(loweralkyl)amino; wherein
at least one of R.sup.7 and R.sup.9 is OR.sup.2 (and R.sup.2 is not
hydrogen); (5) R.sup.8 and R.sup.10 are H; and (6) X is O, S,
SO.sub.2, or CH.sub.2.
[0179] In a seventh subembodiment, a compound of Formula XIII, or
its pharmaceutically acceptable salt or prodrug, is provided in
which: (1) Base is a purine or pyrimidine base as defined herein;
(2) R.sup.1 is independently H or phosphate (including
monophosphate, diphosphate, triphosphate, or a stabilized phosphate
prodrug); acyl (including lower acyl); alkyl (including lower
alkyl); sulfonate ester including alkyl or arylalkyl sulfonyl
including methanesulfonyl and benzyl, wherein the phenyl group is
optionally substituted with one or more substituents as described
in the definition of aryl given herein; a lipid, including a
phospholipid; an amino acid; a carbohydrate; a peptide;
cholesterol; or other pharmaceutically acceptable leaving group
which when administered in vivo is capable of providing a compound
wherein R.sup.1 is independently H or phosphate; (3) R.sup.6 is
alkyl; (4) R.sup.7 and R.sup.9 are independently OR.sup.2, alkyl
(including lower alkyl), alkenyl, alkynyl, Br-vinyl, O-alkenyl,
chlorine, bromine, iodine, NO.sub.2, amino, lower alkylamino or
di(loweralkyl)amino; wherein at least one of R.sup.7 and R.sup.9 is
OR.sup.2 (and R.sup.2 is not hydrogen); (5) R.sup.8 and R.sup.10
are independently H, alkyl (including lower alkyl), chlorine,
bromine or iodine; and (6) X is O.
[0180] In a eighth subembodiment, a compound of Formula XIII, or
its pharmaceutically acceptable salt or prodrug, is provided in
which: (1) Base is a purine or pyrimidine base as defined herein;
(2) R.sup.1 is independently H or phosphate (including
monophosphate, diphosphate, triphosphate, or a stabilized phosphate
prodrug); acyl (including lower acyl); alkyl (including lower
alkyl); sulfonate ester including alkyl or arylalkyl sulfonyl
including methanesulfonyl and benzyl, wherein the phenyl group is
optionally substituted with one or more substituents as described
in the definition of aryl given herein; a lipid, including a
phospholipid; an amino acid; a carbohydrate; a peptide;
cholesterol; or other pharmaceutically acceptable leaving group
which when administered in vivo is capable of providing a compound
wherein R.sup.1 is independently H or phosphate; (3) R.sup.6 is
alkyl (including lower alkyl), alkenyl, alkynyl, Br-vinyl, hydroxy,
O-alkyl, O-alkenyl, chloro, bromo, fluoro, iodo, NO.sub.2, amino,
lower alkylamino or di(loweralkyl)amino; (4) R.sup.7 and R.sup.9
are independently OH or OR.sup.2, wherein at least one of R.sup.7
and R.sup.9 is OR.sup.2 (and R.sup.2 is not hydrogen); (5) R.sup.8
and R.sup.10 are hydrogen; and (6) X is O, S, SO.sub.2 or
CH.sub.2.
[0181] In a ninth subembodiment, a compound of Formula XIII, or its
pharmaceutically acceptable salt or prodrug, is provided in which:
(1) Base is a purine or pyrimidine base as defined herein; (2)
R.sup.1 is independently H or phosphate (including monophosphate,
diphosphate, triphosphate, or a stabilized phosphate prodrug); acyl
(including lower acyl); alkyl (including lower alkyl); sulfonate
ester including alkyl or arylalkyl sulfonyl including
methanesulfonyl and benzyl, wherein the phenyl group is optionally
substituted with one or more substituents as described in the
definition of aryl given herein; a lipid, including a phospholipid;
an amino acid; a carbohydrate; a peptide; cholesterol; or other
pharmaceutically acceptable leaving group which when administered
in vivo is capable of providing a compound wherein R.sup.1 is
independently H or phosphate; (3) R.sup.6 is alkyl (including lower
alkyl), alkenyl, alkynyl, Br-vinyl, hydroxy, O-alkyl, O-alkenyl,
chloro, bromo, fluoro, iodo, NO.sub.2, amino, lower alkylamino or
di(loweralkyl)amino; (4) R.sup.7 and R.sup.9 are independently OH
or OR.sup.2, wherein at least one of R.sup.7 and R.sup.9 is
OR.sup.2 (and R.sup.2 is not hydrogen); (5) R.sup.8 and R.sup.10
are independently H, alkyl (including lower alkyl), chlorine,
bromine or iodine; and (6) X is O.
[0182] In a tenth preferred subembodiment, a compound of Formula
XIII, or its pharmaceutically acceptable salt or prodrug, is
provided in which: (1) Base is a purine or pyrimidine base as
defined herein; (2) R.sup.1 is independently H or phosphate
(including monophosphate, diphosphate, triphosphate, or a
stabilized phosphate prodrug); acyl (including lower acyl); alkyl
(including lower alkyl); sulfonate ester including alkyl or
arylalkyl sulfonyl including methanesulfonyl and benzyl, wherein
the phenyl group is optionally substituted with one or more
substituents as described in the definition of aryl given herein; a
lipid, including a phospholipid; an amino acid; a carbohydrate; a
peptide; cholesterol; or other pharmaceutically acceptable leaving
group which when administered in vivo is capable of providing a
compound wherein R.sup.1 is independently H or phosphate; (3)
R.sup.6 is alkyl (including lower alkyl), alkenyl, alkynyl,
Br-vinyl, hydroxy, O-alkyl, O-alkenyl, chloro, bromo, fluoro, iodo,
NO.sub.2, amino, lower alkylamino or di(loweralkyl)amino; (4)
R.sup.7 and R.sup.9 are independently OR.sup.2, alkyl (including
lower alkyl), alkenyl, alkynyl, Br-vinyl, O-alkenyl, chlorine,
bromine, iodine, NO.sub.2, amino, lower alkylamino, or
di(loweralkyl)amino; wherein at least one of R.sup.7 and R.sup.9 is
OR.sup.2 (and R.sup.2 is not hydrogen); (5) R.sup.8 and R.sup.10
are hydrogen; and (6) X is O.
[0183] In an eleventh subembodiment, a compound of Formula XIII, or
its pharmaceutically acceptable salt or prodrug, is provided in
which: (1) Base is a purine or pyrimidine base as defined herein;
(2) R.sup.1 is independently H or phosphate; (3) R.sup.6 is alkyl
(including lower alkyl), alkenyl, alkynyl, Br-vinyl, hydroxy,
O-alkyl, O-alkenyl, chloro, bromo, fluoro, iodo, NO.sub.2, amino,
lower alkylamino or di(loweralkyl)amino; (4) R.sup.7 and R.sup.9
are independently OH or OR.sup.2, wherein at least one of R.sup.7
and R.sup.9 is OR.sup.2 (and R.sup.2 is not hydrogen); (5) R.sup.8
and R.sup.10 are hydrogen; and (6) X is O, S, SO.sub.2 or
CH.sub.2.
[0184] In a twelfth subembodiment, a compound of Formula XIII, or
its pharmaceutically acceptable salt or prodrug, is provided in
which: (1) Base is a purine or pyrimidine base as defined herein;
(2) R.sup.1 is independently H or phosphate; (3) R.sup.6 is alkyl;
(4) R.sup.7 and R.sup.9 are independently OH or OR.sup.2, wherein
at least one of R.sup.7 and R.sup.9 is OR.sup.2 (and R.sup.2 is not
hydrogen); (5) R.sup.8 and R.sup.10 are hydrogen; and (6) X is O,
S, SO.sub.2, or CH.sub.2.
[0185] In a thirteenth subembodiment, a compound of Formula XIII,
or its pharmaceutically acceptable salt or prodrug, is provided in
which: (1) Base is a purine or pyrimidine base as defined herein;
(2) R.sup.1 is independently H or phosphate; (3) R.sup.6 is alkyl;
(4) R.sup.7 and R.sup.9 are independently OH or OR.sup.2, wherein
at least one of R.sup.7 and R.sup.9 is OR.sup.2 (and R.sup.2 is not
hydrogen); (5) R.sup.8 and R.sup.10 are independently H, alkyl
(including lower alkyl), chlorine, bromine, or iodine; and (6) X is
O.
[0186] In a fourteenth subembodiment, a compound of Formula XIII,
or its pharmaceutically acceptable salt or prodrug, is provided in
which: (1) Base is a purine or pyrimidine base as defined herein;
(2) R.sup.1 is independently H or phosphate; (3) R.sup.6 is alkyl;
(4) R.sup.7 and R.sup.9 are independently OR.sup.2, alkyl
(including lower alkyl), alkenyl, alkynyl, Br-vinyl, O-alkenyl,
chlorine, bromine, iodine, NO.sub.2, amino, lower alkylamino or
di(loweralkyl)amino; wherein at least one of R.sup.7 and R.sup.9 is
OR.sup.2 (and R.sup.2 is not hydrogen); (5) R.sup.8 and R.sup.10
are hydrogen; and (6) X is O.
[0187] In other subembodiments, a compound of Formula XIII, or its
pharmaceutically acceptable salt or prodrug, is provided in
which:
[0188] (1) Base is adenine; (2) R.sup.1 is hydrogen; (3) R.sup.6 is
methyl; (4) R.sup.7 is hydroxyl (5) R.sup.9 is L-valinyl; (6)
R.sup.8 and R.sup.10 are hydrogen; and (7) X is O;
[0189] (1) Base is guanine; (2) R.sup.1 is hydrogen; (3) R.sup.6 is
methyl; (4) R.sup.7 is hydroxyl (5) R.sup.9 is L-valinyl; (6)
R.sup.8 and R.sup.10 are hydrogen; and (7) X is O;
[0190] (1) Base is cytosine; (2) R.sup.1 is hydrogen; (3) R.sup.6
is methyl; (4) R.sup.7 is hydroxyl (5) R.sup.9 is L-valinyl; (6)
R.sup.8 and R.sup.10 are hydrogen; and (7) X is O;
[0191] (1) Base is thymidine; (2) R.sup.1 is hydrogen; (3) R.sup.6
is methyl; (4) R.sup.7 is hydroxyl (5) R.sup.9 is L-valinyl; (6)
R.sup.8 and R.sup.10 are hydrogen; and (7) X is O;
[0192] (1) Base is uracil; (2) R.sup.1 is hydrogen; (3) R.sup.6 is
methyl; (4) R.sup.7 is hydroxyl (5) R.sup.9 is L-valinyl; (6)
R.sup.8 and R.sup.10 are hydrogen; and (7) X is O;
[0193] (1) Base is adenine; (2) R.sup.1 is phosphate; (3) R.sup.6
is methyl; (4) R.sup.7 is hydroxyl (5) R.sup.9 is L-valinyl; (6)
R.sup.8 and R.sup.10 are hydrogen; and (7) X is O;
[0194] (1) Base is adenine; (2) R.sup.1 is hydrogen; (3) R.sup.6 is
ethyl; (4) R.sup.7 is hydroxyl (5) R.sup.9 is L-valinyl; (6)
R.sup.8 and R.sup.10 are hydrogen; and (7) X is O;
[0195] (1) Base is adenine; (2) R.sup.1 is hydrogen; (3) R.sup.6 is
propyl; (4) R.sup.7 is hydroxyl (5) R.sup.9 is L-valinyl; (6)
R.sup.8 and R.sup.10 are hydrogen; and (7) X is O;
[0196] (1) Base is adenine; (2) R.sup.1 is hydrogen; (3) R.sup.6 is
butyl; (4) R.sup.7 is hydroxyl (5) R.sup.9 is L-valinyl; (6)
R.sup.8 and R.sup.10 are hydrogen; and (7) X is O;
[0197] (1) Base is adenine; (2) R.sup.1 is hydrogen; (3) R.sup.6 is
methyl; (4) R.sup.7 is hydrogen (5) R.sup.9 is L-valinyl; (6)
R.sup.8 and R.sup.10 are hydrogen; and (7) X is O;
[0198] (1) Base is adenine; (2) R.sup.1 is hydrogen; (3) R.sup.6 is
methyl; (4) R.sup.7 is hydroxyl (5) R.sup.9 is L-valinyl; (6)
R.sup.8 and R.sup.10 are hydrogen; and (7) X is S;
[0199] (1) Base is adenine; (2) R.sup.1 is hydrogen; (3) R.sup.6 is
methyl; (4) R.sup.7 is hydroxyl (5) R.sup.9 is L-valinyl; (6)
R.sup.8 and R.sup.10 are hydrogen; and (7) X is SO.sub.2;
[0200] (1) Base is adenine; (2) R.sup.1 is hydrogen; (3) R.sup.6 is
methyl; (4) R.sup.7 is hydroxyl (5) R.sup.9 is L-valinyl; (6)
R.sup.8 and R.sup.10 are hydrogen; and (7) X is CH.sub.2.
[0201] In a eleventh principal embodiment the invention provides a
compound of Formula XIV, or a pharmaceutically acceptable salt or
prodrug thereof: ##STR28## wherein: Base is a purine or pyrimidine
base as defined herein; R.sup.1 is H, phosphate (including mono-,
di- or triphosphate and a stabilized phosphate); straight chained,
branched or cyclic alkyl (including lower alkyl); acyl (including
lower acyl); CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl,
CO-substituted aryl, sulfonate ester including alkyl or arylalkyl
sulfonyl including methanesulfonyl and benzyl, wherein the phenyl
group is optionally substituted with one or more substituents as
described in the definition of aryl given herein; alkylsulfonyl,
arylsulfonyl, aralkylsulfonyl, a lipid, including a phospholipid;
an amino acid; and amino acid residue, a carbohydrate; a peptide;
cholesterol; or other pharmaceutically acceptable leaving group
which when administered in vivo is capable of providing a compound
wherein R.sup.1 is H or phosphate; R.sup.6 is alkyl (including
lower alkyl and halogenated alkyl), CH.sub.3, CF.sub.3, azido,
cyano, alkenyl, alkynyl, Br-vinyl, 2-Br-ethyl, --C(O)O(alkyl),
--C(O)O(lower alkyl), --O(acyl), --O(lower acyl), --O(alkyl),
--O(lower alkyl), --O(alkenyl), CF.sub.3, chloro, bromo, fluoro,
iodo, NO.sub.2, NH.sub.2, --NH(lower alkyl), --NH(acyl), --N(lower
alkyl).sub.2, --N(acyl).sub.2; R.sup.7 and R.sup.9 are
independently hydrogen, OR.sup.2, hydroxy, alkyl (including lower
alkyl), azido, cyano, alkenyl, alkynyl, Br-vinyl, --C(O)O(alkyl),
--C(O)O(lower alkyl), --O(acyl), --O(lower acyl), --O(alkyl),
--O(lower alkyl), --O(alkenyl), chlorine, bromine, iodine,
NO.sub.2, NH.sub.2, --NH(lower alkyl), --NH(acyl), --N(lower
alkyl).sub.2, --N(acyl).sub.2; wherein at least one of R.sup.7 and
R.sup.9 is OR.sup.2, wherein each R.sup.2 is independently
phosphate (including mono-, di- or triphosphate and a stabilized
phosphate); straight chained, branched or cyclic alkyl (including
lower alkyl); acyl (including lower acyl); CO-alkyl, CO-aryl,
CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, sulfonate
ester including alkyl or arylalkyl sulfonyl including
methanesulfonyl and benzyl, wherein the phenyl group is optionally
substituted with one or more substituents as described in the
definition of aryl given herein; alkylsulfonyl, arylsulfonyl,
aralkylsulfonyl, a lipid, including a phospholipid; an amino acid;
and amino acid residue, a carbohydrate; a peptide; cholesterol; or
other pharmaceutically acceptable leaving group which when
administered in vivo is capable of providing a compound wherein
R.sup.2 is H or phosphate; R.sup.10 is H, alkyl (including lower
alkyl), chlorine, bromine or iodine; alternatively, or R.sup.7 and
R.sup.10 can come together to form a pi bond; and X is O, SSO.sub.2
or CH.sub.2.
[0202] In a first subembodiment, a compound of Formula XIV, or its
pharmaceutically acceptable salt or prodrug, is provided in which:
(1) Base is a purine or pyrimidine base as defined herein; (2)
R.sup.1 is independently H; phosphate (including monophosphate,
diphosphate, triphosphate, or a stabilized phosphate prodrug); acyl
(including lower acyl); alkyl (including lower alkyl); sulfonate
ester including alkyl or arylalkyl sulfonyl including
methanesulfonyl and benzyl, wherein the phenyl group is optionally
substituted with one or more substituents as described in the
definition of aryl given herein; a lipid, including a phospholipid;
an amino acid; a carbohydrate; a peptide; cholesterol; or other
pharmaceutically acceptable leaving group which when administered
in vivo is capable of providing a compound wherein R.sup.1 is
independently H or phosphate; (3) R.sup.6 is alkyl (including lower
alkyl), alkenyl, alkynyl, Br-vinyl, hydroxy, O-alkyl, O-alkenyl,
chloro, bromo, fluoro, iodo, NO.sub.2, amino, lower alkylamino, or
di(loweralkyl)amino; (4) R.sup.7 and R.sup.9 are independently
hydrogen, OR.sup.2, alkyl (including lower alkyl), alkenyl,
alkynyl, Br-vinyl, O-alkenyl, chlorine, bromine, iodine, NO.sub.2,
amino, lower alkylamino or di(loweralkyl)-amino; wherein at least
one of R.sup.7 and R.sup.9 is OR.sup.2 (and R.sup.2 is not
hydrogen); (5) R.sup.10 is H; and (6) X is O, S, SO.sub.2, or
CH.sub.2.
[0203] In a second subembodiment, a compound of Fomnula XIV, or its
pharmaceutically acceptable salt or prodrug, is provided in which:
(1) Base is a purine or pyrimidine base as defined herein; (2)
R.sup.1 is independently H; phosphate (including monophosphate,
diphosphate, triphosphate, or a stabilized phosphate prodrug); acyl
(including lower acyl); alkyl (including lower alkyl); sulfonate
ester including alkyl or arylalkyl sulfonyl including
methanesulfonyl and benzyl, wherein the phenyl group is optionally
substituted with one or more substituents as described in the
definition of aryl given herein; a lipid, including a phospholipid;
an amino acid; a carbohydrate; a peptide; cholesterol; or other
pharmaceutically acceptable leaving group which when administered
in vivo is capable of providing a compound wherein R.sup.1 is
independently H or phosphate; (3) R.sup.6 is alkyl (including lower
alkyl), alkenyl, alkynyl, Br-vinyl, hydroxy, O-alkyl, O-alkenyl,
chloro, bromo, fluoro, iodo, NO.sub.2, amino, lower alkylamino or
di(loweralkyl)amino; (4) R.sup.7 and R.sup.9 are independently OH
or OR.sup.2, wherein at least one of R.sup.7 and R.sup.9 is
OR.sup.2 (and R.sup.2 is not hydrogen); (5) R.sup.10 is H, alkyl
(including lower alkyl), chlorine, bromine, or iodine; and (6) X is
O, S, SO.sub.2 or CH.sub.2.
[0204] In a third subembodiment, a compound of Formula XIV, or its
pharmaceutically acceptable salt or prodrug, is provided in which:
(1) Base is a purine or pyrimidine base as defined herein; (2)
R.sup.1 is independently H; phosphate (including monophosphate,
diphosphate, triphosphate, or a stabilized phosphate prodrug); acyl
(including lower acyl); alkyl (including lower alkyl); sulfonate
ester including alkyl or arylalkyl sulfonyl including
methanesulfonyl and benzyl, wherein the phenyl group is optionally
substituted with one or more substituents as described in the
definition of aryl given herein; a lipid, including a phospholipid;
an amino acid; a carbohydrate; a peptide; cholesterol; or other
pharmaceutically acceptable leaving group which when administered
in vivo is capable of providing a compound wherein R.sup.1 is
independently H or phosphate; (3) R.sup.6 is alkyl (including lower
alkyl), alkenyl, alkynyl, Br-vinyl, hydroxy, O-alkyl, O-alkenyl,
chloro, bromo, fluoro, iodo, NO.sub.2, amino, lower alkylamino, or
di(loweralkyl)amino; (4) R.sup.7 and R.sup.9 are independently
hydrogen, OR.sup.2, alkyl (including lower alkyl), alkenyl,
alkynyl, Br-vinyl, O-alkenyl, chlorine, bromine, iodine, NO.sub.2,
amino, lower alkylamino or di(loweralkyl)-amino; wherein at least
one of R.sup.7 and R.sup.9 is OR.sup.2 (and R.sup.2 is not
hydrogen); (5) R.sup.10 is H, alkyl (including lower alkyl),
chlorine, bromine or iodine; and (6) X is O.
[0205] In a fourth subembodiment, a compound of Formula XIV, or its
pharmaceutically acceptable salt or prodrug, is provided in which:
(1) Base is a purine or pyrimidine base as defined herein; (2)
R.sup.1 is independently H; phosphate (including monophosphate,
diphosphate, triphosphate, or a stabilized phosphate prodrug); acyl
(including lower acyl); alkyl (including lower alkyl); sulfonate
ester including alkyl or arylalkyl sulfonyl including
methanesulfonyl and benzyl, wherein the phenyl group is optionally
substituted with one or more substituents as described in the
definition of aryl given herein; a lipid, including a phospholipid;
an amino acid; a carbohydrate; a peptide; cholesterol; or other
pharmaceutically acceptable leaving group which when administered
in vivo is capable of providing a compound wherein R.sup.1 is
independently H or phosphate; (3) R.sup.6 is alkyl (including lower
alkyl), alkenyl, alkynyl, Br-vinyl, hydroxy, O-alkyl, O-alkenyl,
chloro, bromo, fluoro, iodo, NO.sub.2, amino, lower alkylamino or
di(loweralkyl)amino; (4) R.sup.7 and R.sup.9 are independently OH
or OR.sup.2, wherein at least one of R.sup.7 and R.sup.9 is
OR.sup.2 (and R.sup.2 is not hydrogen); (5) R.sup.10 is H; and (6)
X is O, S, SO.sub.2 or CH.sub.2.
[0206] In a fifth subembodiment, a compound of Formula XIV, or its
pharmaceutically acceptable salt or prodrug, is provided in which:
(1) Base is a purine or pyrimidine base as defined herein; (2)
R.sup.1 is independently H; phosphate (including monophosphate,
diphosphate, triphosphate, or a stabilized phosphate prodrug); acyl
(including lower acyl); alkyl (including lower alkyl); sulfonate
ester including alkyl or arylalkyl sulfonyl including
methanesulfonyl and benzyl, wherein the phenyl group is optionally
substituted with one or more substituents as described in the
definition of aryl given herein; a lipid, including a phospholipid;
an amino acid; a carbohydrate; a peptide; cholesterol; or other
pharmaceutically acceptable leaving group which when administered
in vivo is capable of providing a compound wherein R.sup.1 is
independently H or phosphate; (3) R.sup.6 is alkyl (including lower
alkyl), alkenyl, alkynyl, Br-vinyl, hydroxy, O-alkyl, O-alkenyl,
chloro, bromo, fluoro, iodo, NO.sub.2, amino, lower alkylamino or
di(loweralkyl)amino; (4) R.sup.7 and R.sup.9 are independently OH
or OR.sup.2, wherein at least one of R.sup.7 and R.sup.9 is
OR.sup.2 (and R.sup.2 is not hydrogen); (5) R.sup.10 is H, alkyl
(including lower alkyl), chlorine, bromine or iodine; and (6) X is
O.
[0207] In a sixth subembodiment, a compound of Formula XIV, or its
pharmaceutically acceptable salt or prodrug, is provided in which:
(1) Base is a purine or pyrimidine base as defined herein; (2)
R.sup.1 is independently H; phosphate (including monophosphate,
diphosphate, triphosphate, or a stabilized phosphate prodrug); acyl
(including lower acyl); alkyl (including lower alkyl); sulfonate
ester including alkyl or arylalkyl sulfonyl including
methanesulfonyl and benzyl, wherein the phenyl group is optionally
substituted with one or more substituents as described in the
definition of aryl given herein; a lipid, including a phospholipid;
an amino acid; a carbohydrate; a peptide; cholesterol; or other
pharmaceutically acceptable leaving group which when administered
in vivo is capable of providing a compound wherein R.sup.1 is
independently H or phosphate; (3) R.sup.6 is alkyl (including lower
alkyl), alkenyl, alkynyl, Br-vinyl, hydroxy, O-alkyl, O-alkenyl,
chloro, bromo, fluoro, iodo, NO.sub.2, amino, lower alkylamino, or
di(loweralkyl)amino; (4) R.sup.7 and R.sup.9 are independently
hydrogen, OR.sup.2, alkyl (including lower alkyl), alkenyl,
alkynyl, Br-vinyl, O-alkenyl, chlorine, bromine, iodine, NO.sub.2,
amino, lower alkylamino, or di(loweralkyl)amino; wherein at least
one of R.sup.7 and R.sup.9 is OR.sup.2 (and R.sup.2 is not
hydrogen); (5) R.sup.10 is H; and (6) X is O.
[0208] In a seventh subembodiment, a compound of Formula XIV, or
its pharmaceutically acceptable salt or prodrug, is provided in
which: (1) Base is a purine or pyrimidine base as defined herein;
(2) R.sup.1 is independently H; phosphate (including monophosphate,
diphosphate, triphosphate, or a stabilized phosphate prodrug); acyl
(including lower acyl); alkyl (including lower alkyl); sulfonate
ester including alkyl or arylalkyl sulfonyl including
methanesulfonyl and benzyl, wherein the phenyl group is optionally
substituted with one or more substituents as described in the
definition of aryl given herein; a lipid, including a phospholipid;
an amino acid; a carbohydrate; a peptide; cholesterol; or other
pharmaceutically acceptable leaving group which when administered
in vivo is capable of providing a compound wherein R.sup.1 is
independently H or phosphate; (3) R.sup.6 is alkyl (including lower
alkyl), alkenyl, alkynyl, Br-vinyl, hydroxy, O-alkyl, O-alkenyl,
chloro, bromo, fluoro, iodo, NO.sub.2, amino, lower alkylamino, or
di(loweralkyl)amino; (4) R.sup.7 and R.sup.9 are independently OH
or OR.sup.2, wherein at least one of R.sup.7 and R.sup.9 is
OR.sup.2 (and R.sup.2 is not hydrogen); (5) R.sup.10 is H; and (6)
X is O.
[0209] In an eighth subembodiment, a compound of Formula XIV, or
its pharmaceutically acceptable salt or prodrug, is provided in
which: (1) Base is a purine or pyrimidine base as defined herein;
(2) R.sup.1 is independently H or phosphate; (3) R.sup.6 is alkyl;
(4) R.sup.7 and R.sup.9 are independently hydrogen, OR.sup.2, alkyl
(including lower alkyl), alkenyl, alkynyl, Br-vinyl, O-alkenyl,
chlorine, bromine, iodine, NO.sub.2, amino, lower alkylamino or
di(loweralkyl)-amino; wherein at least one of R.sup.7 and R.sup.9
is OR.sup.2 (and R.sup.2 is not hydrogen); (5) R.sup.10 is H, alkyl
(including lower alkyl), chlorine, bromine or iodine; and (6) X is
O, S, SO.sub.2, or CH.sub.2.
[0210] In a ninth subembodiment, a compound of Formula XIV, or its
pharmaceutically acceptable salt or prodrug, is provided in which:
(1) Base is a purine or pyrimidine base as defined herein; (2)
R.sup.1 is independently H or phosphate; (3) R.sup.6 is alkyl
(including lower alkyl), alkenyl, alkynyl, Br-vinyl, hydroxy,
O-alkyl, O-alkenyl, chloro, bromo, fluoro, iodo, NO.sub.2, amino,
lower alkylamino, or di(loweralkyl)amino; (4) R.sup.7 and R.sup.9
are independently OH or OR.sup.2, wherein at least one of R.sup.7
and R.sup.9 is OR.sup.2 (and R.sup.2 is not hydrogen); (5) R.sup.10
is H; and (6) X is O, S, SO.sub.2, or CH.sub.2.
[0211] In a tenth preferred subembodiment, a compound of Formula
XIV, or its pharmaceutically acceptable salt or prodrug, is
provided in which: (1) Base is a purine or pyrimidine base as
defined herein; (2) R.sup.1 is independently H or phosphate; (3)
R.sup.6 is alkyl; (4) R.sup.7 and R.sup.9 are independently OH or
OR.sup.2, wherein at least one of R.sup.7 and R.sup.9 is OR.sup.2
(and R.sup.2 is not hydrogen); (5) R.sup.10 is H; and (6) X is O,
S, SO.sub.2, or CH.sub.2.
[0212] In even more preferred subembodiments, a compound of Formula
XIV, or its pharmaceutically acceptable salt or prodrug, is
provided in which:
[0213] (1) Base is adenine; (2) R.sup.1 is hydrogen; (3) R.sup.6 is
methyl; (4) R.sup.7 is hydroxyl (5) R.sup.9 is L-valinyl; (6)
R.sup.10 is hydrogen; and (7) X is O;
[0214] (1) Base is guanine; (2) R.sup.1 is hydrogen; (3) R.sup.6 is
methyl; (4) R.sup.7 is hydroxyl (5) R.sup.9 is L-valinyl; (6)
R.sup.10 is hydrogen; and (7) X is O;
[0215] (1) Base is cytosine; (2) R.sup.1 is hydrogen; (3) R.sup.6
is methyl; (4) R.sup.7 is hydroxyl (5) R.sup.9 is L-valinyl; (6)
R.sup.10 is hydrogen; and (7) X is O;
[0216] (1) Base is thymidine; (2) R.sup.1 is hydrogen; (3) R.sup.6
is methyl; (4) R.sup.7 is hydroxyl (5) R.sup.9 is L-valinyl; (6)
R.sup.10 is hydrogen; and (7) X is O;
[0217] (1) Base is uracil; (2) R.sup.1 is hydrogen; (3) R.sup.6 is
methyl; (4) R.sup.7 is hydroxyl (5) R.sup.9 is L-valinyl; (6)
R.sup.10 is hydrogen; and (7) X is O;
[0218] (1) Base is adenine; (2) R.sup.1 is phosphate; (3) R.sup.6
is methyl; (4) R.sup.7 is hydroxyl (5) R.sup.9 is L-valinyl; (6)
R.sup.10 is hydrogen; and (7) X is O;
[0219] (1) Base is adenine; (2) R.sup.1 is hydrogen; (3) R.sup.6 is
ethyl; (4) R.sup.7 is hydroxyl (5) R.sup.9 is L-valinyl; (6)
R.sup.10 is hydrogen; and (7) X is O;
[0220] (1) Base is adenine; (2) R.sup.1 is hydrogen; (3) R.sup.6 is
propyl; (4) R.sup.7 is hydroxyl (5) R.sup.9 is L-valinyl; (6)
R.sup.10 is hydrogen; and (7) X is O;
[0221] (1) Base is adenine; (2) R.sup.1 is hydrogen; (3) R.sup.6 is
butyl; (4) R.sup.7 is hydroxyl (5) R.sup.9 is L-valinyl; (6)
R.sup.10 is hydrogen; and (7) X is O;
[0222] (1) Base is adenine; (2) R.sup.1 is hydrogen; (3) R.sup.6 is
methyl; (4) R.sup.7 is hydroxyl (5) R.sup.9 is L-valinyl; (6)
R.sup.10 is hydrogen; and (7) X is S;
[0223] (1) Base is adenine; (2) R.sup.1 is hydrogen; (3) R.sup.6 is
methyl; (4) R.sup.7 is hydroxyl (5) R.sup.9 is L-valinyl; (6)
R.sup.10 is hydrogen; and (7) X is SO.sub.2; or
[0224] (1) Base is adenine; (2) R.sup.1 is hydrogen; (3) R.sup.6 is
methyl; (4) R.sup.7 is hydroxyl (5) R.sup.9 is L-valinyl; (6)
R.sup.10 is hydrogen; and (7) X is CH.sub.2.
[0225] In an twelfth principal embodiment, the invention provides a
compound of Formula XV, or a pharmaceutically acceptable salt or
prodrug thereof: ##STR29## wherein: Base is a purine or pyrimidine
base as defined herein; R.sup.1 is H, phosphate (including mono-,
di- or triphosphate and a stabilized phosphate); straight chained,
branched or cyclic alkyl (including lower alkyl); acyl (including
lower acyl); CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl,
CO-substituted aryl, sulfonate ester including alkyl or arylalkyl
sulfonyl including methanesulfonyl and benzyl, wherein the phenyl
group is optionally substituted with one or more substituents as
described in the definition of aryl given herein; alkylsulfonyl,
arylsulfonyl, aralkylsulfonyl, a lipid, including a phospholipid;
an amino acid; and amino acid residue, a carbohydrate; a peptide;
cholesterol; or other pharmaceutically acceptable leaving group
which when administered in vivo is capable of providing a compound
wherein R.sup.1 is H or phosphate; R.sup.6 is alkyl (including
lower alkyl and halogenated alkyl), CH.sub.3, CF.sub.3, azido,
cyano, alkenyl, alkynyl, Br-vinyl, 2-Br-ethyl, --C(O)O(alkyl),
--C(O)O(lower alkyl), --O(acyl), --O(lower acyl), --O(alkyl),
--O(lower alkyl), --O(alkenyl), CF.sub.3, chloro, bromo, fluoro,
iodo, NO.sub.2, NH.sub.2, --NH(lower alkyl), --NH(acyl), --N(lower
alkyl).sub.2, --N(acyl).sub.2; R.sup.7 and R.sup.9 are
independently hydrogen, OR.sup.2, hydroxy, alkyl (including lower
alkyl), azido, cyano, alkenyl, alkynyl, Br-vinyl, --C(O)O(alkyl),
--C(O)O(lower alkyl), --O(acyl), --O(lower acyl), --O(alkyl),
--O(lower alkyl), --O(alkenyl), chlorine, bromine, iodine,
NO.sub.2, NH.sub.2, --NH(lower alkyl), --NH(acyl), --N(lower
alkyl).sub.2, --N(acyl).sub.2; wherein at least one of R.sup.7 and
R.sup.9 is OR.sup.2, wherein each R.sup.2 is independently
phosphate (including mono-, di- or triphosphate and a stabilized
phosphate); straight chained, branched or cyclic alkyl (including
lower alkyl); acyl (including lower acyl); CO-alkyl, CO-aryl,
CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, sulfonate
ester including alkyl or arylalkyl sulfonyl including
methanesulfonyl and benzyl, wherein the phenyl group is optionally
substituted with one or more substituents as described in the
definition of aryl given herein; alkylsulfonyl, arylsulfonyl,
aralkylsulfonyl, a lipid, including a phospholipid; an amino acid;
and amino acid residue, a carbohydrate; a peptide; cholesterol; or
other pharmaceutically acceptable leaving group which when
administered in vivo is capable of providing a compound wherein
R.sup.2 is H or phosphate; R.sup.8 is H, alkyl (including lower
alkyl), chlorine, bromine or iodine; alternatively, R.sup.8 and
R.sup.9 can come together to form a pi bond; X is O, S, SO.sub.2 or
CH.sub.2.
[0226] In a first subembodiment, a compound of Formula XV, or its
pharmaceutically acceptable salt or prodrug, is provided in which:
(1) Base is a purine or pyrimidine base as defined herein; (2)
R.sup.1 is independently H; phosphate (including monophosphate,
diphosphate, triphosphate, or a stabilized phosphate prodrug); acyl
(including lower acyl); alkyl (including lower alkyl); sulfonate
ester including alkyl or arylalkyl sulfonyl including
methanesulfonyl and benzyl, wherein the phenyl group is optionally
substituted with one or more substituents as described in the
definition of aryl given herein; a lipid, including a phospholipid;
an amino acid; a carbohydrate; a peptide; cholesterol; or other
pharmaceutically acceptable leaving group which when administered
in vivo is capable of providing a compound wherein R.sup.1 is
independently H or phosphate; (3) R.sup.6 is alkyl; (4) R.sup.7 and
R.sup.9 are independently hydrogen, OR.sup.2, alkyl (including
lower alkyl), alkenyl, alkynyl, Br-vinyl, O-alkenyl, chlorine,
bromine, iodine, NO.sub.2, amino, lower alkylamino or
di(loweralkyl)amino; wherein at least one of R.sup.7 and R.sup.9 is
OR.sup.2 (and R.sup.2 is not hydrogen); (5) R.sup.8 is H, alkyl
(including lower alkyl), chlorine, bromine or iodine; and (6) X is
O, S, SO.sub.2 or CH.sub.2.
[0227] In a second subembodiment, a compound of Formula XV, or its
pharmaceutically acceptable salt or prodrug, is provided in which:
(1) Base is a purine or pyrimidine base as defined herein; (2)
R.sup.1 is independently H; phosphate (including monophosphate,
diphosphate, triphosphate, or a stabilized phosphate prodrug); acyl
(including lower acyl); alkyl (including lower alkyl); sulfonate
ester including alkyl or arylalkyl sulfonyl including
methanesulfonyl and benzyl, wherein the phenyl group is optionally
substituted with one or more substituents as described in the
definition of aryl given herein; a lipid, including a phospholipid;
an amino acid; a carbohydrate; a peptide; cholesterol; or other
pharmaceutically acceptable leaving group which when administered
in vivo is capable of providing a compound wherein R.sup.1 is
independently H or phosphate; (3) R.sup.6 is alkyl (including lower
alkyl), alkenyl, alkynyl, Br-vinyl, hydroxy, O-alkyl, O-alkenyl,
chloro, bromo, fluoro, iodo, NO.sub.2, amino, lower alkylamino or
di-(loweralkyl)amino; (4) R.sup.7 and R.sup.9 are independently OH
or OR.sup.2, wherein at least one of R.sup.7 and R.sup.9 is
OR.sup.2 (and R.sup.2 is not hydrogen); (5) R.sup.8 is H, alkyl
(including lower alkyl), chlorine, bromine, or iodine; and (6) X is
O, S, SO.sub.2 or CH.sub.2.
[0228] In a third subembodiment, a compound of Formula XV, or its
pharmaceutically acceptable salt or prodrug, is provided in which:
(1) Base is a purine or pyrimidine base as defined herein; (2)
R.sup.1 is independently H; phosphate (including monophosphate,
diphosphate, triphosphate, or a stabilized phosphate prodrug); acyl
(including lower acyl); alkyl (including lower alkyl); sulfonate
ester including alkyl or arylalkyl sulfonyl including
methanesulfonyl and benzyl, wherein the phenyl group is optionally
substituted with one or more substituents as described in the
definition of aryl given herein; a lipid, including a phospholipid;
an amino acid; a carbohydrate; a peptide; cholesterol; or other
pharmaceutically acceptable leaving group which when administered
in vivo is capable of providing a compound wherein R.sup.1 is
independently H or phosphate; (3) R.sup.6 is alkyl (including lower
alkyl), alkenyl, alkynyl, Br-vinyl, hydroxy, O-alkyl, O-alkenyl,
chloro, bromo, fluoro, iodo, NO.sub.2, amino, lower alkylamino, or
di(lower-alkyl)amino; (4) R.sup.7 and R.sup.9 are independently
hydrogen, OR.sup.2, alkyl (including lower alkyl), alkenyl,
alkynyl, Br-vinyl, O-alkenyl, chlorine, bromine, iodine, NO.sub.2,
amino, lower alkylamino, or di(loweralkyl)amino; wherein at least
one of R.sup.7 and R.sup.9 is OR.sup.2 (and R.sup.2 is not
hydrogen); (5) R.sup.8 is H; and (6) X is O, S, SO.sub.2 or
CH.sub.2.
[0229] In a fourth subembodiment, a compound of Formula XV, or its
pharmaceutically acceptable salt or prodrug, is provided in which:
(1) Base is a purine or pyrimidine base as defined herein; (2)
R.sup.1 is independently H; phosphate (including monophosphate,
diphosphate, triphosphate, or a stabilized phosphate prodrug); acyl
(including lower acyl); alkyl (including lower alkyl); sulfonate
ester including alkyl or arylalkyl sulfonyl including
methanesulfonyl and benzyl, wherein the phenyl group is optionally
substituted with one or more substituents as described in the
definition of aryl given herein; a lipid, including a phospholipid;
an amino acid; a carbohydrate; a peptide; cholesterol; or other
pharmaceutically acceptable leaving group which when administered
in vivo is capable of providing a compound wherein R.sup.1 is
independently H or phosphate; (3) R.sup.6 is alkyl (including lower
alkyl), alkenyl, alkynyl, Br-vinyl, hydroxy, O-alkyl, O-alkenyl,
chloro, bromo, fluoro, iodo, NO.sub.2, amino, lower alkylamino, or
di(loweralkyl)amino; (4) R.sup.7 and R.sup.9 are independently
hydrogen, OR.sup.2, alkyl (including lower alkyl), alkenyl,
alkynyl, Br-vinyl, O-alkenyl, chlorine, bromine, iodine, NO.sub.2,
amino, lower alkylamino, or di(loweralkyl)amino; wherein at least
one of R.sup.7 and R.sup.9 is OR.sup.2 (and R.sup.2 is not
hydrogen); (5) R.sup.8 is H, alkyl (including lower alkyl),
chlorine, bromine, or iodine; and (6) X is O.
[0230] In a fifth subembodiment, a compound of Formula XV, or its
pharmaceutically acceptable salt or prodrug, is provided in which:
(1) Base is a purine or pyrimidine base as defined herein; (2)
R.sup.1 is independently H; phosphate (including monophosphate,
diphosphate, triphosphate, or a stabilized phosphate prodrug); acyl
(including lower acyl); alkyl (including lower alkyl); sulfonate
ester including alkyl or arylalkyl sulfonyl including
methanesulfonyl and benzyl, wherein the phenyl group is optionally
substituted with one or more substituents as described in the
definition of aryl given herein; a lipid, including a phospholipid;
an amino acid; a carbohydrate; a peptide; cholesterol; or other
pharmaceutically acceptable leaving group which when administered
in vivo is capable of providing a compound wherein R.sup.1 is
independently H or phosphate; (3) R.sup.6 is alkyl (including lower
alkyl), alkenyl, alkynyl, Br-vinyl, hydroxy, O-alkyl, O-alkenyl,
chloro, bromo, fluoro, iodo, NO.sub.2, amino, lower alkylamino, or
di(loweralkyl)amino; (4) R.sup.7 and R.sup.9 are independently OH
or OR.sup.2, wherein at least one of R.sup.7 and R.sup.9 is
OR.sup.2 (and R.sup.2 is not hydrogen); (5) R.sup.8 is H; and (6) X
is O, S, SO.sub.2, or CH.sub.2.
[0231] In a sixth subembodiment, a compound of Formula XV, or its
pharmaceutically acceptable salt or prodrug, is provided in which:
(1) Base is a purine or pyrimidine base as defined herein; (2)
R.sup.1 is independently H; phosphate (including monophosphate,
diphosphate, triphosphate, or a stabilized phosphate prodrug); acyl
(including lower acyl); alkyl (including lower alkyl); sulfonate
ester including alkyl or arylalkyl sulfonyl including
methanesulfonyl and benzyl, wherein the phenyl group is optionally
substituted with one or more substituents as described in the
definition of aryl given herein; a lipid, including a phospholipid;
an amino acid; a carbohydrate; a peptide; cholesterol; or other
pharmaceutically acceptable leaving group which when administered
in vivo is capable of providing a compound wherein R.sup.1 is
independently H or phosphate; (3) R.sup.6 is alkyl (including lower
alkyl), alkenyl, alkynyl, Br-vinyl, hydroxy, O-alkyl, O-alkenyl,
chloro, bromo, fluoro, iodo, NO.sub.2, amino, lower alkylamino, or
di(loweralkyl)amino; (4) R.sup.7 and R.sup.9 are independently OH
or OR.sup.2, wherein at least one of R.sup.7 and R.sup.9 is
OR.sup.2 (and R.sup.2 is not hydrogen); (5) R.sup.8 is H, alkyl
(including lower alkyl), chlorine, bromine, or iodine; and (6) X is
O.
[0232] In a seventh subembodiment, a compound of Formula XV, or its
pharmaceutically acceptable salt or prodrug, is provided in which:
(1) Base is a purine or pyrimidine base as defined herein; (2)
R.sup.1 is independently H; phosphate (including monophosphate,
diphosphate, triphosphate, or a stabilized phosphate prodrug); acyl
(including lower acyl); alkyl (including lower alkyl); sulfonate
ester including alkyl or arylalkyl sulfonyl including
methanesulfonyl and benzyl, wherein the phenyl group is optionally
substituted with one or more substituents as described in the
definition of aryl given herein; a lipid, including a phospholipid;
an amino acid; a carbohydrate; a peptide; cholesterol; or other
pharmaceutically acceptable leaving group which when administered
in vivo is capable of providing a compound wherein R.sup.1 is
independently H or phosphate; (3) R.sup.6 is alkyl (including lower
alkyl), alkenyl, alkynyl, Br-vinyl, hydroxy, O-alkyl, O-alkenyl,
chloro, bromo, fluoro, iodo, NO.sub.2, amino, lower alkylamino, or
di(loweralkyl)amino; (4) R.sup.7 and R.sup.9 are independently
hydrogen, OR.sup.2, alkyl (including lower alkyl), alkenyl,
alkynyl, Br-vinyl, O-alkenyl, chlorine, bromine, iodine, NO.sub.2,
amino, lower alkylamino, or di(loweralkyl)amino; wherein at least
one of R.sup.7 and R.sup.9 is OR.sup.2 (and R.sup.2 is not
hydrogen); (5) R.sup.8 is H; and (6) X is O.
[0233] In an eighth subembodiment, a compound of Formula XV, or its
pharmaceutically acceptable salt or prodrug, is provided in which:
(1) Base is a purine or pyrimidine base as defined herein; (2)
R.sup.1 is independently H or phosphate; (3) R.sup.6 is alkyl
(including lower alkyl), alkenyl, alkynyl, Br-vinyl, hydroxy,
O-alkyl, O-alkenyl, chloro, bromo, fluoro, iodo, NO.sub.2, amino,
lower alkylamino or di(loweralkyl)amino; (4) R.sup.7 and R.sup.9
are independently OH or OR.sup.2, wherein at least one of R.sup.7
and R.sup.9 is OR.sup.2 (and R.sup.2 is not hydrogen); (5) R.sup.8
is H; and (6) X is O, S, SO.sub.2 or CH.sub.2.
[0234] In a ninth subembodiment, a compound of Formula XV, or its
pharmaceutically acceptable salt or prodrug, is provided in which:
(1) Base is a purine or pyrimidine base as defined herein; (2)
R.sup.1 is independently H or phosphate; (3) R.sup.6 is alkyl; (4)
R.sup.7 and R.sup.9 are independently OH or OR.sup.2, wherein at
least one of R.sup.7 and R.sup.9 is OR.sup.2 (and R.sup.2 is not
hydrogen); (5) R.sup.8 is H; and (6) X is O, S, SO.sub.2, or
CH.sub.2.
[0235] In a tenth preferred subembodiment, a compound of Formula
XV, or its pharmaceutically acceptable salt or prodrug, is provided
in which: (1) Base is a purine or pyrimidine base as defined
herein; (2) R.sup.1 is independently H or phosphate; (3) R.sup.6 is
alkyl; (4) R.sup.7 and R.sup.9 are independently OH or OR.sup.2,
wherein at least one of R.sup.7 and R.sup.9 is OR.sup.2 (and
R.sup.2 is not hydrogen); (5) R.sup.8 is H; and (6) X is O.
[0236] In even more preferred subembodiments, a compound of Formula
XV, or its pharmaceutically acceptable salt or prodrug, is provided
in which:
[0237] (1) Base is adenine; (2) R.sup.1 is hydrogen; (3) R.sup.6 is
methyl; (4) R.sup.7 is hydroxyl (5) R.sup.9 is L-valinyl; (6)
R.sup.8 is hydrogen; and (7) X is O;
[0238] (1) Base is guanine; (2) R.sup.1 is hydrogen; (3) R.sup.6 is
methyl; (4) R.sup.7 is hydroxyl (5) R.sup.9 is L-valinyl; (6)
R.sup.8 is hydrogen; and (7) X is O;
[0239] (1) Base is cytosine; (2) R.sup.1 is hydrogen; (3) R.sup.6
is methyl; (4) R.sup.7 is hydroxyl (5) R.sup.9 is L-valinyl; (6)
R.sup.8 is hydrogen; and (7) X is O;
[0240] (1) Base is thymidine; (2) R.sup.1 is hydrogen; (3) R.sup.6
is methyl; (4) R.sup.7 is hydroxyl (5) R.sup.9 is L-valinyl; (6)
R.sup.8 is hydrogen; and (7) X is O;
[0241] (1) Base is uracil; (2) R.sup.1 is hydrogen; (3) R.sup.6 is
methyl; (4) R.sup.7 is hydroxyl (5) R.sup.9 is L-valinyl; (6)
R.sup.8 is hydrogen; and (7) X is O;
[0242] (1) Base is adenine; (2) R.sup.1 is phosphate; (3) R.sup.6
is methyl; (4) R.sup.7 is hydroxyl (5) R.sup.9 is L-valinyl; (6)
R.sup.8 is hydrogen; and (7) X is O;
[0243] (1) Base is adenine; (2) R.sup.1 is hydrogen; (3) R.sup.6 is
ethyl; (4) R.sup.7 is hydroxyl (5) R.sup.9 is L-valinyl; (6)
R.sup.8 is hydrogen; and (7) X is O;
[0244] (1) Base is adenine; (2) R.sup.1 is hydrogen; (3) R.sup.6 is
propyl; (4) R.sup.7 is hydroxyl (5) R.sup.9 is L-valinyl; (6)
R.sup.8 is hydrogen; and (7) X is O;
[0245] (1) Base is adenine; (2) R.sup.1 is hydrogen; (3) R.sup.6 is
butyl; (4) R.sup.7 is hydroxyl (5) R.sup.9 is L-valinyl; (6)
R.sup.8 is hydrogen; and (7) X is O;
[0246] (1) Base is adenine; (2) R.sup.1 is hydrogen; (3) R.sup.6 is
methyl; (4) R.sup.7 is hydroxyl (5) R.sup.9 is L-valinyl; (6)
R.sup.8 is hydrogen; and (7) X is S;
[0247] (1) Base is adenine; (2) R.sup.1 is hydrogen; (3) R.sup.6 is
methyl; (4) R.sup.7 is hydroxyl (5) R.sup.9 is L-valinyl; (6)
R.sup.8 is hydrogen; and (7) X is SO.sub.2; or
[0248] (1) Base is adenine; (2) R.sup.1 is hydrogen; (3) R.sup.6 is
methyl; (4) R.sup.7 is hydroxyl (5) R.sup.9 is L-valinyl; (6)
R.sup.8 is hydrogen; and (7) X.sup.1 is CH.sub.2.
[0249] In a thirteenth principal embodiment, a compound of Formula
XVI, or a pharmaceutically acceptable salt or prodrug thereof, is
provided: ##STR30## wherein: R.sup.1, R.sup.2 and R.sup.3 are
independently H, phosphate (including mono-, di- or triphosphate
and a stabilized phosphate); straight chained, branched or cyclic
alkyl (including lower alkyl); acyl (including lower acyl);
CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted
aryl, sulfonate ester including alkyl or arylalkyl sulfonyl
including methanesulfonyl and benzyl, wherein the phenyl group is
optionally substituted with one or more substituents as described
in the definition of aryl given herein; alkylsulfonyl,
arylsulfonyl, aralkylsulfonyl, a lipid, including a phospholipid;
an amino acid; and amino acid residue, a carbohydrate; a peptide;
cholesterol; or other pharmaceutically acceptable leaving group
which when administered in vivo is capable of providing a compound
wherein R.sup.1, R.sup.2 and/or R.sup.3 is independently H or
phosphate; wherein at least one of R.sup.2 and R.sup.3 is not
hydrogen; Y is hydrogen, bromo, chloro, fluoro, iodo, OH, OR.sup.4,
NH, NHR.sup.5, NR.sup.4R.sup.5, SH and SR.sup.4; X.sup.1 and
X.sup.2 are independently selected from the group consisting of H,
straight chained, branched or cyclic alkyl, CO-alkyl, CO-aryl,
CO-alkoxyalkyl, chloro, bromo, fluoro, iodo, OH, OR.sup.4, NH,
NHR.sup.5, NR.sup.4R.sup.5, SH and SR.sup.4; and R.sup.4 and
R.sup.5 are independently hydrogen, acyl (including lower acyl), or
alkyl (including but not limited to methyl, ethyl, propyl and
cyclopropyl).
[0250] In a preferred subembodiment, a compound of Formula XVI, or
a pharmaceutically acceptable salt or prodrug thereof, is provided
wherein:
R.sup.1 is H or phosphate (preferably H);
R.sup.2 and R.sup.3 are independently H, phosphate, acyl or an
amino acid residue, wherein at least one of R.sup.2 and R.sup.3 is
acyl or an amino acid residue;
X.sup.1 is H;
X is H or NH.sub.2; and
Y is hydrogen, bromo, chloro, fluoro, iodo, NH.sub.2 or OH.
[0251] In a fourteenth principal embodiment, a compound of Formula
XVII, or a pharmaceutically acceptable salt or prodrug thereof, is
provided: ##STR31## wherein: R.sup.1, R.sup.2 and R.sup.3 are
independently H, phosphate (including mono-, di- or triphosphate
and a stabilized phosphate); straight chained, branched or cyclic
alkyl (including lower alkyl); acyl (including lower acyl);
CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted
aryl, sulfonate ester including alkyl or arylalkyl sulfonyl
including methanesulfonyl and benzyl, wherein the phenyl group is
optionally substituted with one or more substituents as described
in the definition of aryl given herein; alkylsulfonyl,
arylsulfonyl, aralkylsulfonyl, a lipid, including a phospholipid;
an amino acid; and amino acid residue, a carbohydrate; a peptide;
cholesterol; or other pharmaceutically acceptable leaving group
which when administered in vivo is capable of providing a compound
wherein R.sup.1, R.sup.2 and/or R.sup.3 is independently H or
phosphate; wherein at least one of R.sup.2 and R.sup.3 is not
hydrogen; Y is hydrogen, bromo, chloro, fluoro, iodo, OR.sup.4,
NR.sup.4R.sup.5 or SR.sup.4; X.sup.1 is selected from the group
consisting of H, straight chained, branched or cyclic alkyl,
CO-alkyl, CO-aryl, CO-alkoxyalkyl, chloro, bromo, fluoro, iodo,
OR.sup.4, NR.sup.4NR.sup.5 or SR.sup.5; and R.sup.4 and R.sup.5 are
independently hydrogen, acyl (including lower acyl), or alkyl
(including but not limited to methyl, ethyl, propyl and
cyclopropyl).
[0252] In a preferred subembodiment, a compound of Formula XVII, or
a pharmaceutically acceptable salt or prodrug thereof, is provided
wherein:
R.sup.1 is H or phosphate (preferably H);
R.sup.2 and R.sup.3 are independently H, phosphate, acyl or an
amino acid residue, wherein at least one of R.sup.2 and R.sup.3 is
acyl or an amino acid residue;
X.sup.1 is H or CH.sub.3; and
Y is hydrogen, bromo, chloro, fluoro, iodo, NH.sub.2 or OH.
[0253] In a fifteenth principal embodiment, a compound selected
from Formulas XVIII and XIX, or a pharmaceutically acceptable salt
or prodrug thereof, is provided: ##STR32## wherein: Base is a
purine or pyrimidine base as defined herein; R.sup.1, R.sup.2 and
R.sup.3 are independently H, phosphate (including mono-, di- or
triphosphate and a stabilized phosphate); straight chained,
branched or cyclic alkyl (including lower alkyl); acyl (including
lower acyl); CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl,
CO-substituted aryl, sulfonate ester including alkyl or arylalkyl
sulfonyl including methanesulfonyl and benzyl, wherein the phenyl
group is optionally substituted with one or more substituents as
described in the definition of aryl given herein; alkylsulfonyl,
arylsulfonyl, aralkylsulfonyl, a lipid, including a phospholipid;
an amino acid; and amino acid residue, a carbohydrate; a peptide;
cholesterol; or other pharmaceutically acceptable leaving group
which when administered in vivo is capable of providing a compound
wherein R.sup.1, R.sup.2 and/or R.sup.3 is independently H or
phosphate; wherein R.sup.2 is not hydrogen; R.sup.6 is alkyl
(including lower alkyl and halogenated alkyl), CH.sub.3, CF.sub.3,
azido, cyano, alkenyl, alkynyl, Br-vinyl, 2-Br-ethyl,
--C(O)O(alkyl), --C(O)O(lower alkyl), --O(acyl), --O(lower acyl),
--O(alkyl), --O(lower alkyl), --O(alkenyl), CF.sub.3, chloro,
bromo, fluoro, iodo, NO.sub.2, NH.sub.2, --NH(lower alkyl),
--NH(acyl), --N(lower alkyl).sub.2, --N(acyl).sub.2; and X is O, S,
SO.sub.2 or CH.sub.2.
[0254] In a first subembodiment, a compound of Formula XVIII and
XIX, or a pharmaceutically acceptable salt or prodrug thereof, is
provided wherein:
Base is a purine or pyrimidine base as defined herein;
R.sup.1 is H or phosphate (preferably H);
R.sup.2 and R.sup.3 are independently H, phosphate, acyl or an
amino acid residue, wherein at least one of R.sup.2 and R.sup.3 is
acyl or an amino acid residue;
R.sup.6 is alkyl; and
X is O, S, SO.sub.2 or CH.sub.2.
[0255] In a second subembodiment, a compound of Formula XVIII and
XIX, or a pharmaceutically acceptable salt or prodrug thereof, is
provided wherein:
Base is a purine or pyrimidine base as defined herein;
R.sup.1 is H or phosphate (preferably H);
R.sup.2 and R.sup.3 are independently H, phosphate, acyl or an
amino acid residue, wherein at least one of R.sup.2 and R.sup.3 is
an amino acid residue;
R.sup.6 is alkyl; and
X is O, S, SO.sub.2 or CH.sub.2.
[0256] In a third subembodiment, a compound of Formula XVIII and
XIX, or a pharmaceutically acceptable salt or prodrug thereof, is
provided wherein:
Base is a purine or pyrimidine base as defined herein;
R.sup.1 is H or phosphate (preferably H);
R.sup.2 and R.sup.3 are independently H, phosphate, acyl or an
amino acid residue, wherein at least one of R.sup.2 and R.sup.3 is
acyl or an amino acid residue;
R.sup.6 is alkyl; and
X is O.
[0257] In a sixteenth principal embodiment the invention provides a
compound of Formula XX, or a pharmaceutically acceptable salt or
prodrug thereof: ##STR33## wherein: Base is a purine or pyrimidine
base as defined herein; R.sup.1 is H, phosphate (including mono-,
di- or triphosphate and a stabilized phosphate); straight chained,
branched or cyclic alkyl (including lower alkyl); acyl (including
lower acyl); CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl,
CO-substituted aryl, sulfonate ester including alkyl or arylalkyl
sulfonyl including methanesulfonyl and benzyl, wherein the phenyl
group is optionally substituted with one or more substituents as
described in the definition of aryl given herein; alkylsulfonyl,
arylsulfonyl, aralkylsulfonyl, a lipid, including a phospholipid;
an amino acid; and amino acid residue, a carbohydrate; a peptide;
cholesterol; or other pharmaceutically acceptable leaving group
which when administered in vivo is capable of providing a compound
wherein R.sup.1 is H or phosphate; R.sup.6 is alkyl (including
lower alkyl), azido, cyano, alkenyl, alkynyl, Br-vinyl, 2-Br-ethyl,
--C(O)O(alkyl), --C(O)O(lower alkyl), --O(acyl), --O(lower acyl),
--O(alkyl), --O(lower alkyl), --O(alkenyl), CF.sub.3, chloro,
bromo, fluoro, iodo, NO.sub.2, NH.sub.2, --NH(lower alkyl),
--NH(acyl), --N(lower alkyl).sub.2, --N(acyl).sub.2; R.sup.7 and
R.sup.9 are independently hydrogen, OR.sup.2, hydroxy, alkyl
(including lower alkyl), azido, cyano, alkenyl, alkynyl, Br-vinyl,
--C(O)O(alkyl), --C(O)O(lower alkyl), --O(acyl), --O(lower acyl),
--O(alkyl), --O(lower alkyl), --O(alkenyl), chlorine, bromine,
iodine, NO.sub.2, NH.sub.2, --NH(lower alkyl), --NH(acyl),
--N(lower alkyl).sub.2, --N(acyl).sub.2; wherein at least one of
R.sup.7 and R.sup.9 is OR.sup.2, wherein each R.sup.2 is
independently phosphate (including mono-, di- or triphosphate and a
stabilized phosphate); straight chained, branched or cyclic alkyl
(including lower alkyl); acyl (including lower acyl); CO-alkyl,
CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl,
sulfonate ester including alkyl or arylalkyl sulfonyl including
methanesulfonyl and benzyl, wherein the phenyl group is optionally
substituted with one or more substituents as described in the
definition of aryl given herein; alkylsulfonyl, arylsulfonyl,
aralkylsulfonyl, a lipid, including a phospholipid; an amino acid;
and amino acid residue, a carbohydrate; a peptide; cholesterol; or
other pharmaceutically acceptable leaving group which when
administered in vivo is capable of providing a compound wherein
R.sup.2 is H or phosphate; R.sup.8 and R.sup.10 are independently
H, alkyl (including lower alkyl), chlorine, bromine or iodine;
alternatively, R.sup.7 and R.sup.10, R.sup.8 and R.sup.9, or
R.sup.8 and R.sup.10 can come together to form a pi bond; and X is
O, S, SO.sub.2 or CH.sub.2.
[0258] In a first subembodiment, a compound of Formula XX, or its
pharmaceutically acceptable salt or prodrug, is provided in which:
(1) Base is a purine or pyrimidine base as defined herein; (2)
R.sup.1 is independently H or phosphate (including monophosphate,
diphosphate, triphosphate, or a stabilized phosphate prodrug); acyl
(including lower acyl); alkyl (including lower alkyl); sulfonate
ester including alkyl or arylalkyl sulfonyl including
methanesulfonyl and benzyl, wherein the phenyl group is optionally
substituted with one or more substituents as described in the
definition of aryl given herein; a lipid, including a phospholipid;
an amino acid; a carbohydrate; a peptide; cholesterol; or other
pharmaceutically acceptable leaving group which when administered
in vivo is capable of providing a compound wherein R.sup.1 is
independently H or phosphate; (3) R.sup.6 is alkyl; (4) R.sup.7 and
R.sup.9 are independently OR.sup.2, alkyl, alkenyl, alkynyl,
Br-vinyl, O-alkenyl, chlorine, bromine, iodine, NO.sub.2, amino,
lower alkylamino or di(loweralkyl)amino; wherein at least one of
R.sup.7 and R.sup.9 is OR.sup.2 (and R.sup.2 is not hydrogen); (5)
R.sup.8 and R.sup.10 are independently H, alkyl (including lower
alkyl), chlorine, bromine, or iodine; and (6) X is O, S, SO.sub.2
or CH.sub.2.
[0259] In a second subembodiment, a compound of Formula XX, or its
pharmaceutically acceptable salt or prodrug, is provided in which:
(1) Base is a purine or pyrimidine base as defined herein; (2)
R.sup.1 is independently H or phosphate (including monophosphate,
diphosphate, triphosphate, or a stabilized phosphate prodrug); acyl
(including lower acyl); alkyl (including lower alkyl); sulfonate
ester including alkyl or arylalkyl sulfonyl including
methanesulfonyl and benzyl, wherein the phenyl group is optionally
substituted with one or more substituents as described in the
definition of aryl given herein; a lipid, including a phospholipid;
an amino acid; a carbohydrate; a peptide; cholesterol; or other
pharmaceutically acceptable leaving group which when administered
in vivo is capable of providing a compound wherein R.sup.1 is
independently H or phosphate; (3) R.sup.6 is alkyl, alkenyl,
alkynyl, Br-vinyl, hydroxy, O-alkyl, O-alkenyl, chloro, bromo,
fluoro, iodo, NO.sub.2, amino, lower alkylamino, or
di(loweralkyl)amino; (4) R.sup.7 and R.sup.9 are independently OH
or OR.sup.2, wherein at least one of R.sup.7 and R.sup.9 is
OR.sup.2 (and R.sup.2 is not hydrogen); (5) R.sup.8 and R.sup.10
are independently H, alkyl (including lower alkyl), chlorine,
bromine, or iodine; and (6) X is O, S, SO.sub.2 or CH.sub.2.
[0260] In a third subembodiment, a compound of Formula XX, or its
pharmaceutically acceptable salt or prodrug, is provided in which:
(1) Base is a purine or pyrimidine base as defined herein; (2)
R.sup.1 is independently H or phosphate (including monophosphate,
diphosphate, triphosphate, or a stabilized phosphate prodrug); acyl
(including lower acyl); alkyl (including lower alkyl); sulfonate
ester including alkyl or arylalkyl sulfonyl including
methanesulfonyl and benzyl, wherein the phenyl group is optionally
substituted with one or more substituents as described in the
definition of aryl given herein; a lipid, including a phospholipid;
an amino acid; a carbohydrate; a peptide; cholesterol; or other
pharmaceutically acceptable leaving group which when administered
in vivo is capable of providing a compound wherein R.sup.1 is
independently H or phosphate; (3) R.sup.6 is alkyl, alkenyl,
alkynyl, Br-vinyl, hydroxy, O-alkyl, O-alkenyl, chloro, bromo,
fluoro, iodo, NO.sub.2, amino, lower alkylamino or
di(loweralkyl)amino; (4) R.sup.7 and R.sup.9 are independently
OR.sup.2, alkyl, alkenyl, alkynyl, Br-vinyl, O-alkenyl, chlorine,
bromine, iodine, NO.sub.2, amino, lower alkylamino or
di(loweralkyl)amino; wherein at least one of R.sup.7 and R.sup.9 is
OR.sup.2 (and R.sup.2 is not hydrogen); (5) R.sup.8 and R.sup.10
are H; and (6) X is O, S, SO.sub.2 or CH.sub.2.
[0261] In a fourth subembodiment, a compound of Formula XX, or its
pharmaceutically acceptable salt or prodrug, is provided in which:
(1) Base is a purine or pyrimidine base as defined herein; (2)
R.sup.1 is independently H or phosphate (including monophosphate,
diphosphate, triphosphate, or a stabilized phosphate prodrug); acyl
(including lower acyl); alkyl (including lower alkyl); sulfonate
ester including alkyl or arylalkyl sulfonyl including
methanesulfonyl and benzyl, wherein the phenyl group is optionally
substituted with one or more substituents as described in the
definition of aryl given herein; a lipid, including a phospholipid;
an amino acid; a carbohydrate; a peptide; cholesterol; or other
pharmaceutically acceptable leaving group which when administered
in vivo is capable of providing a compound wherein R.sup.1 is
independently H or phosphate; (3) R.sup.6 is alkyl, alkenyl,
alkynyl, Br-vinyl, hydroxy, O-alkyl, O-alkenyl, chloro, bromo,
fluoro, iodo, NO.sub.2, amino, lower alkylamino, or
di(loweralkyl)amino; (4) R.sup.7 and R.sup.9 are independently
OR.sup.2, alkyl, alkenyl, alkynyl, Br-vinyl, O-alkenyl, chlorine,
bromine, iodine, NO.sub.2, amino, lower alkylamino, or
di(loweralkyl)amino; wherein at least one of R.sup.7 and R.sup.9 is
OR.sup.2 (and R.sup.2 is not hydrogen); (5) R.sup.8 and R.sup.10
are independently H, alkyl (including lower alkyl), chlorine,
bromine, or iodine; and (6) X is O.
[0262] In a fifth subembodiment, a compound of Formula XX, or its
pharmaceutically acceptable salt or prodrug, is provided in which:
(1) Base is a purine or pyrimidine base as defined herein; (2)
R.sup.1 is independently H or phosphate (including monophosphate,
diphosphate, triphosphate, or a stabilized phosphate prodrug); acyl
(including lower acyl); alkyl (including lower alkyl); sulfonate
ester including alkyl or arylalkyl sulfonyl including
methanesulfonyl and benzyl, wherein the phenyl group is optionally
substituted with one or more substituents as described in the
definition of aryl given herein; a lipid, including a phospholipid;
an amino acid; a carbohydrate; a peptide; cholesterol; or other
pharmaceutically acceptable leaving group which when administered
in vivo is capable of providing a compound wherein R.sup.1 is
independently H or phosphate; (3) R.sup.6 is alkyl; (4) R.sup.7 and
R.sup.9 are independently OH or OR.sup.2, wherein at least one of
R.sup.7 and R.sup.9 is OR.sup.2 (and R.sup.2 is not hydrogen); (5)
R.sup.8 and R.sup.10 are independently H, alkyl (including lower
alkyl), chlorine, bromine or iodine; and (6) X is O, S, SO.sub.2 or
CH.sub.2.
[0263] In a sixth subembodiment, a compound of Formula XX, or its
pharmaceutically acceptable salt or prodrug, is provided in which:
(1) Base is a purine or pyrimidine base as defined herein; (2)
R.sup.1 is independently H or phosphate (including monophosphate,
diphosphate, triphosphate, or a stabilized phosphate prodrug); acyl
(including lower acyl); alkyl (including lower alkyl); sulfonate
ester including alkyl or arylalkyl sulfonyl including
methanesulfonyl and benzyl, wherein the phenyl group is optionally
substituted with one or more substituents as described in the
definition of aryl given herein; a lipid, including a phospholipid;
an amino acid; a carbohydrate; a peptide; cholesterol; or other
pharmaceutically acceptable leaving group which when administered
in vivo is capable of providing a compound wherein R.sup.1 is
independently H or phosphate; (3) R.sup.6 is alkyl; (4) R.sup.7 and
R.sup.9 are independently OR.sup.2, alkyl (including lower alkyl),
alkenyl, alkynyl, Br-vinyl, O-alkenyl, chlorine, bromine, iodine,
NO.sub.2, amino, lower alkylamino, or di(loweralkyl)amino; wherein
at least one of R.sup.7 and R.sup.9 is OR.sup.2 (and R.sup.2 is not
hydrogen); (5) R.sup.8 and R.sup.10 are H; and (6) X is O, S,
SO.sub.2, or CH.sub.2.
[0264] In a seventh subembodiment, a compound of Formula XX, or its
pharmaceutically acceptable salt or prodrug, is provided in which:
(1) Base is a purine or pyrimidine base as defined herein; (2)
R.sup.1 is independently H or phosphate (including monophosphate,
diphosphate, triphosphate, or a stabilized phosphate prodrug); acyl
(including lower acyl); alkyl (including lower alkyl); sulfonate
ester including alkyl or arylalkyl sulfonyl including
methanesulfonyl and benzyl, wherein the phenyl group is optionally
substituted with one or more substituents as described in the
definition of aryl given herein; a lipid, including a phospholipid;
an amino acid; a carbohydrate; a peptide; cholesterol; or other
pharmaceutically acceptable leaving group which when administered
in vivo is capable of providing a compound wherein R.sup.1 is
independently H or phosphate; (3) R.sup.6 is alkyl; (4) R.sup.7 and
R.sup.9 are independently OR.sup.2, alkyl (including lower alkyl),
alkenyl, alkynyl, Br-vinyl, O-alkenyl, chlorine, bromine, iodine,
NO.sub.2, amino, lower alkylamino or di(loweralkyl)amino; wherein
at least one of R.sup.7 and R.sup.9 is OR.sup.2 (and R.sup.2 is not
hydrogen); (5) R.sup.8 and R.sup.10 are independently H, alkyl
(including lower alkyl), chlorine, bromine or iodine; and (6) X is
O.
[0265] In a eighth subembodiment, a compound of Formula XX, or its
pharmaceutically acceptable salt or prodrug, is provided in which:
(1) Base is a purine or pyrimidine base as defined herein; (2)
R.sup.1 is independently H or phosphate (including monophosphate,
diphosphate, triphosphate, or a stabilized phosphate prodrug); acyl
(including lower acyl); alkyl (including lower alkyl); sulfonate
ester including alkyl or arylalkyl sulfonyl including
methanesulfonyl and benzyl, wherein the phenyl group is optionally
substituted with one or more substituents as described in the
definition of aryl given herein; a lipid, including a phospholipid;
an amino acid; a carbohydrate; a peptide; cholesterol; or other
pharmaceutically acceptable leaving group which when administered
in vivo is capable of providing a compound wherein R.sup.1 is
independently H or phosphate; (3) R.sup.6 is alkyl (including lower
alkyl), alkenyl, alkynyl, Br-vinyl, hydroxy, O-alkyl, O-alkenyl,
chloro, bromo, fluoro, iodo, NO.sub.2, amino, lower alkylamino or
di(loweralkyl)amino; (4) R.sup.7 and R.sup.9 are independently OH
or OR.sup.2, wherein at least one of R.sup.7 and R.sup.9 is
OR.sup.2 (and R.sup.2 is not hydrogen); (5) R.sup.8 and R.sup.10
are hydrogen; and (6) X is O, S, SO.sub.2 or CH.sub.2.
[0266] In a ninth subembodiment, a compound of Formula XX, or its
pharmaceutically acceptable salt or prodrug, is provided in which:
(1) Base is a purine or pyrimidine base as defined herein; (2)
R.sup.1 is independently H or phosphate (including monophosphate,
diphosphate, triphosphate, or a stabilized phosphate prodrug); acyl
(including lower acyl); alkyl (including lower alkyl); sulfonate
ester including alkyl or arylalkyl sulfonyl including
methanesulfonyl and benzyl, wherein the phenyl group is optionally
substituted with one or more substituents as described in the
definition of aryl given herein; a lipid, including a phospholipid;
an amino acid; a carbohydrate; a peptide; cholesterol; or other
pharmaceutically acceptable leaving group which when administered
in vivo is capable of providing a compound wherein R.sup.1 is
independently H or phosphate; (3) R.sup.6 is alkyl (including lower
alkyl), alkenyl, alkynyl, Br-vinyl, hydroxy, O-alkyl, O-alkenyl,
chloro, bromo, fluoro, iodo, NO.sub.2, amino, lower alkylamino or
di(loweralkyl)amino; (4) R.sup.7 and R.sup.9 are independently OH
or OR.sup.2, wherein at least one of R.sup.7 and R.sup.9 is
OR.sup.2 (and R.sup.2 is not hydrogen); (5) R.sup.8 and R.sup.10
are independently H, alkyl (including lower alkyl), chlorine,
bromine or iodine; and (6) X is O.
[0267] In a tenth preferred subembodiment, a compound of Formula
XX, or its pharmaceutically acceptable salt or prodrug, is provided
in which: (1) Base is a purine or pyrimidine base as defined
herein; (2) R.sup.1 is independently H or phosphate (including
monophosphate, diphosphate, triphosphate, or a stabilized phosphate
prodrug); acyl (including lower acyl); alkyl (including lower
alkyl); sulfonate ester including alkyl or arylalkyl sulfonyl
including methanesulfonyl and benzyl, wherein the phenyl group is
optionally substituted with one or more substituents as described
in the definition of aryl given herein; a lipid, including a
phospholipid; an amino acid; a carbohydrate; a peptide;
cholesterol; or other pharmaceutically acceptable leaving group
which when administered in vivo is capable of providing a compound
wherein R.sup.1 is independently H or phosphate; (3) R.sup.6 is
alkyl (including lower alkyl), alkenyl, alkynyl, Br-vinyl, hydroxy,
O-alkyl, O-alkenyl, chloro, bromo, fluoro, iodo, NO.sub.2, amino,
lower alkylamino or di(loweralkyl)amino; (4) R.sup.7 and R.sup.9
are independently OR.sup.2, alkyl (including lower alkyl), alkenyl,
alkynyl, Br-vinyl, O-alkenyl, chlorine, bromine, iodine, NO.sub.2,
amino, lower alkylamino, or di(loweralkyl)amino; wherein at least
one of R.sup.7 and R.sup.9 is OR.sup.2 (and R.sup.2 is not
hydrogen); (5) R.sup.8 and R.sup.10 are hydrogen; and (6) X is
O.
[0268] In an eleventh subembodiment, a compound of Formula XX, or
its pharmaceutically acceptable salt or prodrug, is provided in
which: (1) Base is a purine or pyrimidine base as defined herein;
(2) R.sup.1 is independently H or phosphate; (3) R.sup.6 is alkyl
(including lower alkyl), alkenyl, alkynyl, Br-vinyl, hydroxy,
O-alkyl, O-alkenyl, chloro, bromo, fluoro, iodo, NO.sub.2, amino,
lower alkylamino or di(loweralkyl)amino; (4) R.sup.7 and R.sup.9
are independently OH or OR.sup.2, wherein at least one of R.sup.7
and R.sup.9 is OR.sup.2 (and R.sup.2 is not hydrogen); (5) R.sup.8
and R.sup.10 are hydrogen; and (6) X is O, S, SO.sub.2 or
CH.sub.2.
[0269] In a twelfth subembodiment, a compound of Formula XX, or its
pharmaceutically acceptable salt or prodrug, is provided in which:
(1) Base is a purine or pyrimidine base as defined herein; (2)
R.sup.1 is independently H or phosphate; (3) R.sup.6 is alkyl; (4)
R.sup.7 and R.sup.9 are independently OH or OR.sup.2, wherein at
least one of R.sup.7 and R.sup.9 is OR.sup.2 (and R.sup.2 is not
hydrogen); (5) R.sup.8 and R.sup.10 are hydrogen; and (6) X is O,
S, SO.sub.2, or CH.sub.2.
[0270] In a thirteenth subembodiment, a compound of Formula XX, or
its pharmaceutically acceptable salt or prodrug, is provided in
which: (1) Base is a purine or pyrimidine base as defined herein;
(2) R.sup.1 is independently H or phosphate; (3) R.sup.6 is alkyl;
(4) R.sup.7 and R.sup.9 are independently OH or OR.sup.2, wherein
at least one of R.sup.7 and R.sup.9 is OR.sup.2 (and R.sup.2 is not
hydrogen); (5) R.sup.8 and R.sup.10 are independently H, alkyl
(including lower alkyl), chlorine, bromine, or iodine; and (6) X is
O.
[0271] In a fourteenth subembodiment, a compound of Formula XX, or
its pharmaceutically acceptable salt or prodrug, is provided in
which: (1) Base is a purine or pyrimidine base as defined herein;
(2) R.sup.1 is independently H or phosphate; (3) R.sup.6 is alkyl;
(4) R.sup.7 and R.sup.9 are independently OR.sup.2, alkyl
(including lower alkyl), alkenyl, alkynyl, Br-vinyl, O-alkenyl,
chlorine, bromine, iodine, NO.sub.2, amino, lower alkylamino or
di(loweralkyl)amino; wherein at least one of R.sup.7 and R.sup.9 is
OR.sup.2 (and R.sup.2 is not hydrogen); (5) R.sup.8 and R.sup.10
are hydrogen; and (6) X is O.
[0272] In even more preferred subembodiments, a compound of Formula
XX, or its pharmaceutically acceptable salt or prodrug, is provided
in which:
[0273] (1) Base is adenine; (2) R.sup.1 is hydrogen; (3) R.sup.6 is
methyl; (4) R.sup.7 is hydroxyl (5) R.sup.9 is L-valinyl; (6)
R.sup.8 and R.sup.10 are hydrogen; and (7) X is O;
[0274] (1) Base is guanine; (2) R.sup.1 is hydrogen; (3) R.sup.6 is
methyl; (4) R.sup.7 is hydroxyl (5) R.sup.9 is L-valinyl; (6)
R.sup.8 and R.sup.10 are hydrogen; and (7) X is O;
[0275] (1) Base is cytosine; (2) R.sup.1 is hydrogen; (3) R.sup.6
is methyl; (4) R.sup.7 is hydroxyl (5) R.sup.9 is L-valinyl; (6)
R.sup.8 and R.sup.10 are hydrogen; and (7) X is O;
[0276] (1) Base is thymidine; (2) R.sup.1 is hydrogen; (3) R.sup.6
is methyl; (4) R.sup.7 is hydroxyl (5) R.sup.9 is L-valinyl; (6)
R.sup.8 and R.sup.10 are hydrogen; and (7) X is O;
[0277] (1) Base is uracil; (2) R.sup.1 is hydrogen; (3) R.sup.6 is
methyl; (4) R.sup.7 is hydroxyl (5) R.sup.9 is L-valinyl; (6)
R.sup.8 and R.sup.10 are hydrogen; and (7) X is O;
[0278] (1) Base is adenine; (2) R.sup.1 is phosphate; (3) R.sup.6
is methyl; (4) R.sup.7 is hydroxyl (5) R.sup.9 is L-valinyl; (6)
R.sup.8 and R.sup.10 are hydrogen; and (7) X is O;
[0279] (1) Base is adenine; (2) R.sup.1 is hydrogen; (3) R.sup.6 is
ethyl; (4) R.sup.7 is hydroxyl (5) R.sup.9 is L-valinyl; (6)
R.sup.8 and R.sup.10 are hydrogen; and (7) X is O;
[0280] (1) Base is adenine; (2) R.sup.1 is hydrogen; (3) R.sup.6 is
propyl; (4) R.sup.7 is hydroxyl (5) R.sup.9 is L-valinyl; (6)
R.sup.8 and R.sup.10 are hydrogen; and (7) X is O;
[0281] (1) Base is adenine; (2) R.sup.1 is hydrogen; (3) R.sup.6 is
butyl; (4) R.sup.7 is hydroxyl (5) R.sup.9 is L-valinyl; (6)
R.sup.8 and R.sup.10 are hydrogen; and (7) X is O;
[0282] (1) Base is adenine; (2) R.sup.1 is hydrogen; (3) R.sup.6 is
methyl; (4) R.sup.7 is hydrogen (5) R.sup.9 is L-valinyl; (6)
R.sup.8 and R.sup.10 are hydrogen; and (7) X is O;
[0283] (1) Base is adenine; (2) R.sup.1 is hydrogen; (3) R.sup.6 is
methyl; (4) R.sup.7 is hydroxyl (5) R.sup.9 is L-valinyl; (6)
R.sup.8 and R.sup.10 are hydrogen; and (7) X is S;
[0284] (1) Base is adenine; (2) R.sup.1 is hydrogen; (3) R.sup.6 is
methyl; (4) R.sup.7 is hydroxyl (5) R.sup.9 is L-valinyl; (6)
R.sup.8 and R.sup.10 are hydrogen; and (7) X is SO.sub.2;
[0285] (1) Base is adenine; (2) R.sup.1 is hydrogen; (3) R.sup.6 is
methyl; (4) R.sup.7 is hydroxyl (5) R.sup.9 is L-valinyl; (6)
R.sup.8 and R.sup.10 are hydrogen; and (7) X is CH.sub.2.
[0286] Stereochemistry
[0287] It is appreciated that nucleosides of the present invention
have several chiral centers and may exist in and be isolated in
optically active and racemic forms. Some compounds may exhibit
polymorphism. It is to be understood that the present invention
encompasses any racemic, optically-active, diastereomeric,
polymorphic, or stereoisomeric form, or mixtures thereof, of a
compound of the invention, which possess the useful properties
described herein. It being well known in the art how to prepare
optically active forms (for example, by resolution of the racemic
form by recrystallization techniques, by synthesis from
optically-active starting materials, by chiral synthesis, or by
chromatographic separation using a chiral stationary phase).
[0288] In particular, since the 1' and 4' carbons of the nucleoside
are chiral, their nonhydrogen substituents (the base and the CHOR
groups, respectively) can be either cis (on the same side) or trans
(on opposite sides) with respect to the sugar ring system. The four
optical isomers therefore are represented by the following
configurations (when orienting the sugar moiety in a horizontal
plane such that the oxygen atom is in the back): cis (with both
groups "up", which corresponds to the configuration of naturally
occurring .beta.-D nucleosides), cis (with both groups "down",
which is a normaturally occurring .beta.-L configuration), trans
(with the C2' substituent "up" and the C4' substituent "down"), and
trans (with the C2' substituent "down" and the C4' substituent
"up"). The "D-nucleosides" are cis nucleosides in a natural
configuration and the "L-nucleosides" are cis nucleosides in the
non-naturally occurring configuration.
[0289] Likewise, most amino acids are chiral (designated as L or D,
wherein the L enantiomer is the naturally occurring configuration)
and can exist as separate enantiomers.
[0290] Examples of methods to obtain optically active materials are
known in the art, and include at least the following. [0291] i)
physical separation of crystals--a technique whereby macroscopic
crystals of the individual enantiomers are manually separated. This
technique can be used if crystals of the separate enantiomers
exist, i.e., the material is a conglomerate, and the crystals are
visually distinct; [0292] ii) simultaneous crystallization--a
technique whereby the individual enantiomers are separately
crystallized from a solution of the racemate, possible only if the
latter is a conglomerate in the solid state; [0293] iii) enzymatic
resolutions--a technique whereby partial or complete separation of
a racemate by virtue of differing rates of reaction for the
enantiomers with an enzyme; [0294] iv) enzymatic asymmetric
synthesis--a synthetic technique whereby at least one step of the
synthesis uses an enzymatic reaction to obtain an enantiomerically
pure or enriched synthetic precursor of the desired enantiomer;
[0295] v) chemical asymmetric synthesis--a synthetic technique
whereby the desired enantiomer is synthesized from an achiral
precursor under conditions that produce asymmetry (i.e., chirality)
in the product, which may be achieved using chiral catalysts or
chiral auxiliaries; [0296] vi) diastereomer separations--a
technique whereby a racemic compound is reacted with an
enantiomerically pure reagent (the chiral auxiliary) that converts
the individual enantiomers to diastereomers. The resulting
diastereomers are then separated by chromatography or
crystallization by virtue of their now more distinct structural
differences and the chiral auxiliary later removed to obtain the
desired enantiomer; [0297] vii) first- and second-order asymmetric
transformations--a technique whereby diastereomers from the
racemate equilibrate to yield a preponderance in solution of the
diastereomer from the desired enantiomer or where preferential
crystallization of the diastereomer from the desired enantiomer
perturbs the equilibrium such that eventually in principle all the
material is converted to the crystalline diastereomer from the
desired enantiomer. The desired enantiomer is then released from
the diastereomer; [0298] viii) kinetic resolutions--this technique
refers to the achievement of partial or complete resolution of a
racemate (or of a further resolution of a partially resolved
compound) by virtue of unequal reaction rates of the enantiomers
with a chiral, non-racemic reagent or catalyst under kinetic
conditions; [0299] ix) enantiospecific synthesis from non-racemic
precursors--a synthetic technique whereby the desired enantiomer is
obtained from non-chiral starting materials and where the
stereochemical integrity is not or is only minimally compromised
over the course of the synthesis; [0300] x) chiral liquid
chromatogaphy--a technique whereby the enantiomers of a racemate
are separated in a liquid mobile phase by virtue of their differing
interactions with a stationary phase. The stationary phase can be
made of chiral material or the mobile phase can contain an
additional chiral material to provoke the differing interactions;
[0301] xi) chiral gas chromatography--a technique whereby the
racemate is volatilized and enantiomers are separated by virtue of
their differing interactions in the gaseous mobile phase with a
column containing a fixed non-racemic chiral adsorbent phase;
[0302] xii) extraction with chiral solvents--a technique whereby
the enantiomers are separated by virtue of preferential dissolution
of one enantiomer into a particular chiral solvent; [0303] xiii)
transport across chiral membranes--a technique whereby a racemate
is placed in contact with a thin membrane barrier. The barrier
typically separates two miscible fluids, one containing the
racemate, and a driving force such as concentration or pressure
differential causes preferential transport across the membrane
barrier. Separation occurs as a result of the non-racemic chiral
nature of the membrane which allows only one enantiomer of the
racemate to pass through. II. Definitions
[0304] The term "alkyl", as used herein, unless otherwise
specified, refers to a saturated straight, branched, or cyclic,
primary, secondary, or tertiary hydrocarbon of typically C.sub.1 to
C.sub.10, and specifically includes methyl, CF.sub.3, CCl.sub.3,
CFCl.sub.2, CF.sub.2Cl, ethyl, CH.sub.2CF.sub.3, CF.sub.2CF.sub.3,
propyl, isopropyl, cyclopropyl, butyl, isobutyl, secbutyl, t-butyl,
pentyl, cyclopentyl, isopentyl, neopentyl, hexyl, isohexyl,
cyclohexyl, cyclohexylmethyl, 3-methylpentyl, 2,2-dimethylbutyl,
and 2,3-dimethylbutyl. The term includes both substituted and
unsubstituted alkyl groups, and particularly includes halogenated
alkyl groups, and even more particularly fluorinated alkyl groups.
Non-limiting examples of moieties with which the alkyl group can be
substituted are selected from the group consisting of halogen
(fluoro, chloro, bromo or iodo), hydroxyl, amino, alkylamino,
arylamino, alkoxy, aryloxy, nitro, cyano, sulfonic acid, sulfate,
phosphonic acid, phosphate, or phosphonate, either unprotected, or
protected as necessary, as known to those skilled in the art, for
example, as taught in Greene, et al., Protective Groups in Organic
Synthesis, John Wiley and Sons, Second Edition, 1991, hereby
incorporated by reference.
[0305] The term "lower alkyl", as used herein, and unless otherwise
specified, refers to a C.sub.1 to C.sub.4 saturated straight,
branched, or if appropriate, a cyclic (for example, cyclopropyl)
alkyl group, including both substituted and unsubstituted
moieties.
[0306] The term "alkylamino" or "arylamino" refers to an amino
group that has one or two alkyl or aryl substituents, respectively.
Unless otherwise specifically stated in this application, when
alkyl is a suitable moiety, lower alkyl is preferred. Similarly,
when alkyl or lower alkyl is a suitable moiety, unsubstituted alkyl
or lower alkyl is preferred.
[0307] The term "protected" as used herein and unless otherwise
defined refers to a group that is added to an oxygen, nitrogen, or
phosphorus atom to prevent its further reaction or for other
purposes. A wide variety of oxygen and nitrogen protecting groups
are known to those skilled in the art of organic synthesis.
[0308] The term "aryl", as used herein, and unless otherwise
specified, refers to phenyl, biphenyl, or naphthyl, and preferably
phenyl. The term includes both substituted and unsubstituted
moieties. The aryl group can be substituted with any described
moiety, including, but not limited to, one or more moieties
selected from the group consisting of halogen (fluoro, chloro,
bromo or iodo), hydroxyl, amino, alkylamino, arylamino, alkoxy,
aryloxy, nitro, cyano, sulfonic acid, sulfate, phosphonic acid,
phosphate, or phosphonate, either unprotected, or protected as
necessary, as known to those skilled in the art, for example, as
taught in Greene, et al., Protective Groups in Organic Synthesis,
John Wiley and Sons, Second Edition, 1991.
[0309] The term "alkaryl" or "alkylaryl" refers to an alkyl group
with an aryl substituent. The term aralkyl or arylalkyl refers to
an aryl group with an alkyl substituent.
[0310] The term "halo", as used herein, includes chloro, bromo,
iodo, and fluoro.
[0311] The term "purine" or "pyrimidine" base includes, but is not
limited to, adenine, N.sup.6-alkylpurines, N.sup.6-acylpurines
(wherein acyl is C(O)(alkyl, aryl, alkylaryl, or arylalkyl),
N.sup.6-benzylpurine, N.sup.6-halopurine, N.sup.6-vinylpurine,
N.sup.6-acetylenic purine, N.sup.6-acyl purine,
N.sup.6-hydroxyalkyl purine, N.sup.6-alkylaminopurine,
N.sup.6-thioalkyl purine, N.sup.2-alkylpurines,
N.sup.2-alkyl-6-thiopurines, thymine, cytosine, 5-fluorocytosine,
5-methylcytosine, 6-azapyrimidine, including 6-azacytosine, 2-
and/or 4-mercaptopyrmidine, uracil, 5-halouracil, including
5-fluorouracil, C.sup.5-alkylpyrimidines,
C.sup.5-benzylpyrimidines, C.sup.5-halopyrimidines,
C.sup.5-vinylpyrimidine, C.sup.5-acetylenic pyrimidine,
C.sup.5-acyl pyrimidine, C.sup.5-hydroxyalkyl purine,
C.sup.5-amidopyrimidine, C.sup.5-cyanopyrimidine,
C.sup.5-iodopyrimidine, C.sup.6-iodo-pyrimidine, C.sup.5--Br-vinyl
pyrimidine, C.sup.6--Br-vinyl pyrimidine, C.sup.5-nitropyrimidine,
C.sup.5-amino-pyrimidine, N.sup.2-alkylpurines,
N.sup.2-alkyl-6-thiopurines, 5-azacytidinyl, 5-azauracilyl,
triazolopyridinyl, imidazolopyridinyl, pyrrolopyrimidinyl, and
pyrazolopyrimidinyl. Purine bases include, but are not limited to,
guanine, adenine, hypoxanthine, 2,6-diaminopurine, and
6-chloropurine. Functional oxygen and nitrogen groups on the base
can be protected as necessary or desired. Suitable protecting
groups are well known to those skilled in the art, and include
trimethylsilyl, dimethylhexylsilyl, t-butyldimethylsilyl, and
t-butyldiphenylsilyl, trityl, alkyl groups, and acyl groups such as
acetyl and propionyl, methanesulfonyl, and p-toluenesulfonyl.
[0312] The term "acyl" or "O-linked ester" refers to a group of the
formula C(O)R', wherein R' is an straight, branched, or cyclic
alkyl (including lower alkyl), carboxylate reside of amino acid,
aryl including phenyl, alkaryl, aralkyl including benzyl,
alkoxyalkyl including methoxymethyl, aryloxyalkyl such as
phenoxymethyl; or substituted alkyl (including lower alkyl), aryl
including phenyl optionally substituted with chloro, bromo, fluoro,
iodo, C.sub.1 to C.sub.4 alkyl or C.sub.1 to C.sub.4 alkoxy,
sulfonate esters such as alkyl or aralkyl sulphonyl including
methanesulfonyl, the mono, di or triphosphate ester, trityl or
monomethoxy-trityl, substituted benzyl, alkaryl, aralkyl including
benzyl, alkoxyalkyl including methoxymethyl, aryloxyalkyl such as
phenoxymethyl. Aryl groups in the esters optimally comprise a
phenyl group. In particular, acyl groups include acetyl,
trifluoroacetyl, methylacetyl, cyclpropylacetyl, propionyl,
butyryl, hexanoyl, heptanoyl, octanoyl, neo-heptanoyl,
phenylacetyl, 2-acetoxy-2-phenylacetyl, diphenylacetyl,
.alpha.-methoxy-.alpha.-trifluoromethyl-phenylacetyl, bromoacetyl,
2-nitro-benzeneacetyl, 4-chloro-benzeneacetyl,
2-chloro-2,2-diphenylacetyl, 2-chloro-2-phenylacetyl,
trimethylacetyl, chlorodifluoroacetyl, perfluoroacetyl,
fluoroacetyl, bromodifluoroacetyl, methoxyacetyl,
2-thiopheneacetyl, chlorosulfonylacetyl, 3-methoxyphenylacetyl,
phenoxyacetyl, tert-butylacetyl, trichloroacetyl,
monochloro-acetyl, dichloroacetyl, 7H-dodecafluoro-heptanoyl,
perfluoro-heptanoyl, 7H-dodeca-fluoroheptanoyl,
7-chlorododecafluoro-heptanoyl, 7-chloro-dodecafluoro-heptanoyl,
7H-dodecafluoroheptanoyl, 7H-dodeca-fluoroheptanoyl,
nona-fluoro-3,6-dioxa-heptanoyl, nonafluoro-3,6-dioxaheptanoyl,
perfluoroheptanoyl, methoxybenzoyl, methyl
3-amino-5-phenylthiophene-2-carboxyl,
3,6-dichloro-2-methoxy-benzoyl,
4-(1,1,2,2-tetrafluoro-ethoxy)-benzoyl, 2-bromo-propionyl,
omega-aminocapryl, decanoyl, n-pentadecanoyl, stearyl,
3-cyclopentyl-propionyl, 1-benzene-carboxyl, O-acetylmandelyl,
pivaloyl acetyl, 1-adamantane-carboxyl, cyclohexane-carboxyl,
2,6-pyridinedicarboxyl, cyclopropane-carboxyl,
cyclobutane-carboxyl, perfluorocyclohexyl carboxyl,
4-methylbenzoyl, chloromethyl isoxazolyl carbonyl,
perfluorocyclohexyl carboxyl, crotonyl,
1-methyl-1H-indazole-3-carbonyl, 2-propenyl, isovaleryl,
1-pyrrolidinecarbonyl, 4-phenylbenzoyl.
[0313] The term "amino acid" includes naturally occurring and
synthetic .alpha., .beta. .gamma. or .delta. amino acids, and
includes but is not limited to, amino acids found in proteins, i.e.
glycine, alanine, valine, leucine, isoleucine, methionine,
phenylalanine, tryptophan, proline, serine, threonine, cysteine,
tyrosine, asparagine, glutamine, aspartate, glutamate, lysine,
arginine and histidine. In a preferred embodiment, the amino acid
is in the L-configuration. Alternatively, the amino acid can be a
derivative of alanyl, valinyl, leucinyl, isoleuccinyl, prolinyl,
phenylalaninyl, tryptophanyl, methioninyl, glycinyl, serinyl,
threoninyl, cysteinyl, tyrosinyl, asparaginyl, glutaminyl,
aspartoyl, glutaroyl, lysinyl, argininyl, histidinyl,
.beta.-alanyl, .beta.-valinyl, .beta.-leucinyl,
.beta.-isoleuccinyl, .beta.-prolinyl, .beta.-phenylalaninyl,
.beta.-tryptophanyl, .beta.-methioninyl, .beta.-glycinyl,
.beta.-serinyl, .beta.-threoninyl, .beta.-cysteinyl,
.beta.-tyrosinyl, .beta.-asparaginyl, .beta.-glutaminyl,
.beta.-aspartoyl, .beta.-glutaroyl, .beta.-lysinyl,
.beta.-argininyl or .beta.-histidinyl.
[0314] As used herein, the term "substantially free of" or
"substantially in the absence of" refers to a nucleoside
composition that includes at least 85 or 90% by weight, preferably
95%, 98%, 99% or 100% by weight, of the designated enantiomer of
that nucleoside. In a preferred embodiment, in the methods and
compounds of this invention, the compounds are substantially free
of enantiomers.
[0315] Similarly, the term "isolated" refers to a nucleoside
composition that includes at least 85, 90%, 95%, 98%, 99% to 100%
by weight, of the nucleoside, the remainder comprising other
chemical species or enantiomers.
[0316] The term "host", as used herein, refers to an unicellular or
multicellular organism in which the virus can replicate, including
cell lines and animals, and preferably a human. Alternatively, the
host can be carrying a part of the Flaviviridae viral genome, whose
replication or function can be altered by the compounds of the
present invention. The term host specifically refers to infected
cells, cells transfected with all or part of the Flaviviridae
genome and animals, in particular, primates (including chimpanzees)
and humans. In most animal applications of the present invention,
the host is a human patient. Veterinary applications, in certain
indications, however, are clearly anticipated by the present
invention (such as chimpanzees).
[0317] The term "pharmaceutically acceptable salt or prodrug" is
used throughout the specification to describe any pharmaceutically
acceptable form (such as an ester, phosphate ester, salt of an
ester or a related group) of a nucleoside compound which, upon
administration to a patient, provides the nucleoside compound.
Pharmaceutically acceptable salts include those derived from
pharmaceutically acceptable inorganic or organic bases and acids.
Suitable salts include those derived from alkali metals such as
potassium and sodium, alkaline earth metals such as calcium and
magnesium, among numerous other acids well known in the
pharmaceutical art. Pharmaceutically acceptable prodrugs refer to a
compound that is metabolized, for example hydrolyzed or oxidized,
in the host to form the compound of the present invention. Typical
examples of prodrugs include compounds that have biologically
labile protecting groups on a functional moiety of the active
compound. Prodrugs include compounds that can be oxidized, reduced,
aminated, deaminated, hydroxylated, dehydroxylated, hydrolyzed,
dehydrolyzed, alkylated, dealkylated, acylated, deacylated,
phosphorylated, dephosphorylated to produce the active compound.
The compounds of this invention possess antiviral activity against
a Flaviviridae, or are metabolized to a compound that exhibits such
activity.
III. Prodrugs and Derivatives
Pharmaceutically Acceptable Salts
[0318] In cases where compounds are sufficiently basic or acidic to
form stable nontoxic acid or base salts, administration of the
compound as a pharmaceutically acceptable salt may be appropriate.
Examples of pharmaceutically acceptable salts are organic acid
addition salts formed by addition of acids, which form a
physiological acceptable anion, for example, tosylate,
methanesulfonate, acetate, citrate, malonate, tartarate, succinate,
benzoate, ascorate, .alpha.-ketoglutarate,
.alpha.-glycerophosphate, formate, fumarate, propionate, glycolate,
lactate, pyruvate, oxalate, maleate, and salicylate. Suitable
inorganic salts may also be formed, including, sulfate, nitrate,
bicarbonate, carbonate salts, hydrobromate and phosphoric acid. In
a preferred embodiment, the salt is a mono- or di-hydrochloride
salt.
[0319] Pharmaceutically acceptable salts may be obtained using
standard procedures well known in the art, for example by reacting
a sufficiently basic compound such as an amine with a suitable acid
affording a physiologically acceptable anion. Alkali metal (for
example, sodium, potassium or lithium) or alkaline earth metal (for
example calcium) salts of carboxylic acids can also be made. In one
embodiment, the salt is a hydrochloride salt of the compound. In a
further embodiment, the pharmaceutically acceptable salt is a
dihydrochloride salt.
Nucleotide Prodrug Formulations
[0320] The nucleosides described herein can be administered as a
nucleotide prodrug to increase the activity, bioavailability,
stability or otherwise alter the properties of the nucleoside. A
number of nucleotide prodrug ligands are known. In general,
alkylation, acylation or other lipophilic modification of the
mono-, di- or triphosphate of the nucleoside reduces polarity and
allows passage into cells. Examples of substituent groups that can
replace one or more hydrogens on the phosphate moiety are alkyl,
aryl, steroids, carbohydrates, including sugars, 1,2-diacylglycerol
and alcohols.
[0321] In an alternative embodiment, the compound is administered
as a phosphonate, phosphorothioate or SATE derivative.
[0322] Many are described in R. Jones and N. Bischoferger,
Antiviral Research, 1995, 27:1-17. Any of these can be used in
combination with the disclosed nucleosides to achieve a desired
effect. Nonlimiting examples of U.S. patents that disclose suitable
lipophilic substituents that can be covalently incorporated into
the nucleoside, preferably at the 5'-OH position of the nucleoside
or lipophilic preparations, include U.S. Pat. No. 5,149,794 (Sep.
22, 1992, Yatvin et al.); U.S. Pat. No. 5,194,654 (Mar. 16, 1993,
Hostetler et al., U.S. Pat. No. 5,223,263 (Jun. 29, 1993, Hostetler
et al.); U.S. Pat. No. 5,256,641 (Oct. 26, 1993, Yatvin et al.);
U.S. Pat. No. 5,411,947 (May 2, 1995, Hostetler et al.); U.S. Pat.
No. 5,463,092 (Oct. 31, 1995, Hostetler et al.); U.S. Pat. No.
5,543,389 (Aug. 6, 1996, Yatvin et al.); U.S. Pat. No. 5,543,390
(Aug. 6, 1996, Yatvin et al.); U.S. Pat. No. 5,543,391 (Aug. 6,
1996, Yatvin et al.); and U.S. Pat. No. 5,554,728 (Sep. 10, 1996;
Basava et al.), all of which are incorporated herein by reference.
Foreign patent applications that disclose lipophilic substituents
that can be attached to the nucleosides of the present invention,
or lipophilic preparations, include WO 89/02733, WO 90/00555, WO
91/16920, WO 91/18914, WO 93/00910, WO 94/26273, WO 96/15132, EP 0
350 287, EP 93917054.4, and WO 91/19721.
[0323] The active nucleoside can also be provided as a 2', 3'
and/or 5'-phosphoether lipid or a 2', 3' and/or 5'-ether lipid, as
disclosed in the following references, which are incorporated by
reference herein: Kucera, L. S., N. Iyer, et al. 1990 AIDS Res.
Hum. Retro Viruses. 6:491-501; Piantadosi, C., J. Marasco C. J., et
al. 1991 J. Med. Chem. 34:1408.1414; Hosteller, K. Y., D. D.
Richman, et al. 1992 Antimicrob. Agents Chemother. 36:2025.2029;
Hosetler, K. Y., L. M. Stuhmiller, 1990. J. Biol. Chem.
265:61127.
[0324] Nonlimiting examples of U.S. patents that disclose suitable
lipophilic substituents that can be covalently incorporated into
the nucleoside, preferably at the 2', 3' and/or 5'-OH position of
the nucleoside or lipophilic preparations, include U.S. Pat. No.
5,149,794 (Sep. 22, 1992, Yatvin et al.); U.S. Pat. No. 5,194,654
(Mar. 16, 1993, Hostetler et al., U.S. Pat. No. 5,223,263 (Jun. 29,
1993, Hostetler et al.); U.S. Pat. No. 5,256,641 (Oct. 26, 1993,
Yatvin et al.); U.S. Pat. No. 5,411,947 (May 2, 1995, Hostetler et
al.); U.S. Pat. No. 5,463,092 (Oct. 31, 1995, Hostetler et al.);
U.S. Pat. No. 5,543,389 (Aug. 6, 1996, Yatvin et al.); U.S. Pat.
No. 5,543,390 (Aug. 6, 1996, Yatvin et al.); U.S. Pat. No.
5,543,391 (Aug. 6, 1996, Yatvin et al.); and U.S. Pat. No.
5,554,728 (Sep. 10, 1996; Basava et al.), all of which are
incorporated herein by reference. Foreign patent applications that
disclose lipophilic substituents that can be attached to the
nucleosides of the present invention, or lipophilic preparations,
include WO 89/02733, WO 90/00555, WO 91/16920, WO 91/18914, WO
93/00910, WO 94/26273, WO 96/15132, EP 0 350 287, EP 93917054.4,
and WO 91/19721.
[0325] Aryl esters, especially phenyl esters, are also provided.
Nonlimiting examples are disclosed in DeLambert et al., J. Med.
Chem. 37: 498 (1994). Phenyl esters containing a carboxylic ester
ortho to the phosphate are also provided. Khamnei and Torrence, J.
Med. Chem.; 39:4109-4115 (1996). In particular, benzyl esters,
which generate the parent compound, in some cases using
substituents at the ortho- or para-position to accelerate
hydrolysis, are provided. Examples of this class of prodrugs are
described by Mitchell et al., J. Chem. Soc. Perkin Trans. 12345
(1992); Brook, et al. WO 91/19721; and Glazier et al. WO
91/19721.
[0326] Cyclic phosphonate esters are also provided. Nonlimiting
examples are disclosed in Hunston et al., J. Med. Chem. 27: 440-444
(1984) and Starrett et al. J. Med. Chem. 37: 1857-1864 (1994).
Additionally, cyclic 3',5'-phosphate esters are provided.
Nonlimiting examples are disclosed in Meier et al. J. Med. Chem.
22: 811-815 (1979). Cyclic 1',3'-propanyl phosphonate and phosphate
esters, such as ones containing a fused aryl ring, i.e. the
cyclosaligenyl ester, are also provided (Meier et al., Bioorg. Med.
Chem. Lett. 7: 99-104 (1997)). Unsubstituted cyclic 1',3'-propanyl
esters of the monophosphates are also provided (Farquhar et al., J.
Med. Chem. 26: 1153 (1983); Farquhar et al., J. Med. Chem. 28: 1358
(1985)) were prepared. In addition, cyclic 1',3'-propanyl esters
substituted with a pivaloyloxy methyloxy group at C-1' are provided
(Freed et al., Biochem. Pharmac. 38: 3193 (1989); Biller et al.,
U.S. Pat. No. 5,157,027).
[0327] Cyclic phosphoramidates are known to cleave in vivo by an
oxidative mechanism. Therefore, in one embodiment of the present
invention, a variety of substituted 1',3' propanyl cyclic
phosphoramidates are provided. Non-limiting examples are disclosed
by Zon, Progress in Med. Chem. 19, 1205 (1982). Additionally, a
number of 2'- and 3'-substituted proesters are provided.
2'-Substituents include methyl, dimethyl, bromo, trifluoromethyl,
chloro, hydroxy, and methoxy; 3'-substituents including phenyl,
methyl, trifluoromethyl, ethyl, propyl, i-propyl, and cyclohexyl. A
variety of 1'-substituted analogs are also provided.
[0328] Cyclic esters of phosphorus-containing compounds are also
provided. Non-limiting examples are described in the following:
[0329] [1] di and tri esters of phosphoric acids as reported in
Nifantyev et al., Phosphorus, Sulfur Silicon and Related Eelements,
113: 1 (1996); Wijnberg et al., EP-180276 A1; [0330] [2] phosphorus
(III) acid esters. Kryuchkov et al., Izv. Akad. Nauk SSSR, Ser.
Khim. 6: 1244 (1987). Some of the compounds were claimed to be
useful for the asymmetric synthesis of L-Dopa precursors. Sylvain
et al., DE3512781 A1; [0331] [3] phosphoramidates. Shih et al.,
Bull. Inst. Chem. Acad. Sin, 41: 9 (1994); Edmundson et al., J.
Chem. Res. Synop. 5: 122 (1989); and [0332] [4] phosphonates.
Neidlein et al., Heterocycles 35: 1185 (1993).
[0333] Further, nonlimiting examples of U.S. and International
Patent Applications that disclose suitable cyclic phosphoramidate
prodrugs include U.S. Pat. No. 6,312,662; WO 99/45016; WO 00/52015;
WO 01/47935; and WO 01/18013 to Erion, et al. from Metabasis
Therapeutics, Inc. Specifically, prodrugs of Formula A below are
provided: ##STR34## wherein: [0334] together V and Z are connected
via an additional 3-5 atoms to form a cyclic group containing 5-7
atoms, optionally 1 heteroatom, substituted with hydroxy, acyloxy,
alkoxycarbonyloxy, or aryloxycarbonyloxy attached to a carbon atom
that is three atoms from both O groups attached to the phosphorus;
or [0335] together V and Z are connected via an additional 3-5
atoms to form a cyclic group, optionally containing 1 heteroatom,
that is fused to an aryl group at the beta and gamma position to
the O attached to the phosphorus; [0336] together V and W are
connected via an additional 3 carbon atoms to form an optionally
substituted cyclic group containing 6 carbon atoms and substituted
with one substituent selected from the group consisting of hydroxy,
acyloxy, alkoxycarbonyloxy, alkylthiocarbonyloxy, and
aryloxycarbonyloxy, attached to one of said carbon atoms that is
three atoms from an O attached to the phosphorus; [0337] together Z
and W are connected via an additional 3-5 atoms to form a cyclic
group, optionally containing one heteroatom, and V must be aryl,
substituted aryl, heteroaryl, or substituted heteroaryl; [0338]
together W and W' are connected via an additional 2-5 atoms to form
a cyclic group, optionally containing 0-2 heteroatoms, and V must
be aryl, substituted aryl, heteroaryl, or substituted heteroaryl;
[0339] Z is selected from the group consisting of --CHR.sup.2OH,
--CHR.sup.2OC(O)R.sup.3, --CHR.sup.2OC(S)R.sup.3,
--CHR.sup.2OC(S)OR.sup.3, --CHR.sup.2OC(O)SR.sup.3,
--CHR.sup.2OCO.sub.2R.sup.3, --OR.sup.2, --SR.sup.2,
--CHR.sup.2N.sub.3, --CH.sup.2 aryl, --CH(aryl)OH,
--CH(CH.dbd.CR.sup.2.sub.2)OH, --CH(C.ident.CR.sup.2)OH, --R.sup.2,
--NR.sup.2.sub.2, --OCOR.sup.3, --OCO.sub.2R.sup.3, --SCOR.sup.3,
--SCO.sub.2R.sup.3, --NHCOR.sup.2, --NHCO.sub.2R.sup.3,
--CH.sub.2NHaryl, --(CH.sub.2).sub.p--OR.sup.12, and
--(CH.sub.2).sub.p--SR.sup.12; [0340] p is an integer 2 or 3;
[0341] with the provisos that: [0342] a) V, Z, W, W' are not all
--H; and [0343] b) when Z is --R.sup.2, then at least one of V, W,
and W' is not --H, alkyl, aralkyl, or alicyclic; [0344] R.sup.2 is
selected from the group consisting of R.sup.3 and --H; [0345]
R.sup.3 is selected from the group consisting of alkyl, aryl,
alicyclic, and aralkyl; [0346] R.sup.12 is selected from the group
consisting of --H, and lower acyl; [0347] M is the biologically
active agent, and that is attached to the phosphorus in Formula A
via the 2', 3' and/or 5'-hydroxyl. IV. Combination or Alternation
Therapy
[0348] The active compounds of the present invention can be
administered in combination or alternation with another
anti-flavivirus or pestivirus agent, or in particular an anti-HCV
agent. In combination therapy, effective dosages of two or more
agents are administered together, whereas in alternation or
sequential-step therapy, an effective dosage of each agent is
administered serially or sequentially. The dosages given will
depend on absorption, inactivation and excretion rates of the drug
as well as other factors known to those of skill in the art. It is
to be noted that dosage values will also vary with the severity of
the condition to be alleviated. It is to be further understood that
for any particular subject, specific dosage regimens and schedules
should be adjusted over time according to the individual need and
the professional judgment of the person administering or
supervising the administration of the compositions. In preferred
embodiments, an anti-HCV (or anti-pestivirus or anti-flavivirus)
compound that exhibits an EC.sub.50 of 10-15 .mu.M, or preferably
less than 1-5 .mu.M, is desirable.
[0349] It has been recognized that drug-resistant variants of
flaviviruses, pestiviruses or HCV can emerge after prolonged
treatment with an antiviral agent. Drug resistance most typically
occurs by mutation of a gene that encodes for an enzyme used in
viral replication. The efficacy of a drug against the viral
infection can be prolonged, augmented, or restored by administering
the compound in combination or alternation with a second, and
perhaps third, antiviral compound that induces a different mutation
from that caused by the principle drug. Alternatively, the
pharmacokinetics, biodistribution or other parameter of the drug
can be altered by such combination or alternation therapy. In
general, combination therapy is typically preferred over
alternation therapy because it induces multiple simultaneous
stresses on the virus.
[0350] Any of the viral treatments described in the Background of
the Invention can be used in combination or alternation with the
compounds described in this specification. Nonlimiting examples
include: [0351] 1) Protease Inhibitors
[0352] Examples include substrate-based NS3 protease inhibitors
(Attwood et al., Antiviral peptide derivatives, PCT WO 98/22496,
1998; Attwood et al., Antiviral Chemistry and Chemotherapy 1999,
10, 259-273; Attwood et al., Preparation and use of amino acid
derivatives as anti-viral agents, German Patent Pub. DE 19914474;
Tung et al. Inhibitors of serine proteases, particularly hepatitis
C virus NS3 protease, PCT WO 98/17679), including alphaketoamides
and hydrazinoureas, and inhibitors that terminate in an
electrophile such as a boronic acid or phosphonate (Llinas-Brunet
et al, Hepatitis C inhibitor peptide analogues, PCT WO 99/07734);
Non-substrate-based NS3 protease inhibitors such as
2,4,6-trihydroxy-3-nitro-benzamide derivatives (Sudo K. et al.,
Biochemical and Biophysical Research Communications, 1997, 238,
643-647; Sudo K. et al. Antiviral Chemistry and Chemotherapy, 1998,
9, 186), including RD3-4082 and RD3-4078, the former substituted on
the amide with a 14 carbon chain and the latter processing a
para-phenoxyphenyl group; and Sch 68631, a phenanthrenequinone, an
HCV protease inhibitor (Chu M. et al., Tetrahedron Letters
37:7229-7232, 1996).
[0353] Sch 351633, isolated from the fungus Penicillium
griseofulvum, was identified as a protease inhibitor (Chu M. et
al., Bioorganic and Medicinal Chemistry Letters 9:1949-1952). Eglin
c, isolated from leech, is a potent inhibitor of several serine
proteases such as S. griseus proteases A and B,
.alpha.-chymotrypsin, chymase and subtilisin. Qasim M. A. et al.,
Biochemistry 36:1598-1607, 1997.
[0354] U.S. patents disclosing protease inhibitors for the
treatment of HCV include, for example, U.S. Pat. No. 6,004,933 to
Spruce et al. which discloses a class of cysteine protease
inhibitors for inhibiting HCV endopeptidase 2; U.S. Pat. No.
5,990,276 to Zhang et al. which discloses synthetic inhibitors of
hepatitis C virus NS3 protease; U.S. Pat. No. 5,538,865 to Reyes et
a; WO 02/008251 to Corvas International, Inc, and WO 02/08187 and
WO 02/008256 to Schering Corporation. HCV inhibitor tripeptides are
disclosed in U.S. Pat. Nos. 6,534,523, 6,410,531, and 6,420,380 to
Boehringer Ingelheim and WO 02/060926 to Bristol Myers Squibb.
Diaryl peptides as NS3 serine protease inhibitors of HCV are
disclosed in WO 02/48172 to Schering Corporation. Imidazoleidinones
as NS3 serine protease inhibitors of HCV are disclosed in WO
02/08198 to Schering Corporation and WO 02/48157 to Bristol Myers
Squibb. WO 98/17679 to Vertex Pharmaceuticals and WO 02/48116 to
Bristol Myers Squibb also disclose HCV protease inhibitors. [0355]
2) Thiazolidine derivatives which show relevant inhibition in a
reverse-phase HPLC assay with an NS3/4A fusion protein and NS5A/5B
substrate (Sudo K. et al., Antiviral Research, 1996, 32, 9-18),
especially compound RD-1-6250, possessing a fused cinnamoyl moiety
substituted with a long alkyl chain, RD4 6205 and RD4 6193; [0356]
3) Thiazolidines and benzanilides identified in Kakiuchi N. et al.
J. EBS Letters 421, 217-220; Takeshita N. et al. Analytical
Biochemistry, 1997, 247, 242-246; [0357] 4) A phenan-threnequinone
possessing activity against protease in a SDS-PAGE and
autoradiography assay isolated from the fermentation culture broth
of Streptomyces sp., Sch 68631 (Chu M. et al., Tetrahedron Letters,
1996, 37, 7229-7232), and Sch 351633, isolated from the fungus
Penicillium griseofulvum, which demonstrates activity in a
scintillation proximity assay (Chu M. et al., Bioorganic and
Medicinal Chemistry Letters 9, 1949-1952); [0358] 5) Helicase
inhibitors (Diana G. D. et al., Compounds, compositions and methods
for treatment of hepatitis C, U.S. Pat. No. 5,633,358; Diana G. D.
et al., Piperidine derivatives, pharmaceutical compositions thereof
and their use in the treatment of hepatitis C, PCT WO 97/36554);
[0359] 6) Nucleotide polymerase inhibitors and gliotoxin (Ferrari
R. et al. Journal of Virology, 1999, 73, 1649-1654), and the
natural product cerulenin (Lohmann V. et al., Virology, 1998, 249,
108-118); [0360] 7) Antisense phosphorothioate
oligodeoxynucleotides (S-ODN) complementary to sequence stretches
in the 5' non-coding region (NCR) of the virus (Alt M. et al.,
Hepatology, 1995, 22, 707-717), or nucleotides 326-348 comprising
the 3' end of the NCR and nucleotides 371-388 located in the core
coding region of the HCV RNA (Alt M. et al., Archives of Virology,
1997, 142, 589-599; Galderisi U. et al., Journal of Cellular
Physiology, 1999, 181, 251-257); [0361] 8) Inhibitors of
IRES-dependent translation (Ikeda N et al., Agent for the
prevention and treatment of hepatitis C, Japanese Patent Pub.
JP-08268890; Kai Y. et al. Prevention and treatment of viral
diseases, Japanese Patent Pub. JP-10101591); [0362] 9) Ribozymes,
such as nuclease-resistant ribozymes (Maccjak, D. J. et al.,
Hepatology 1999, 30, abstract 995) and those disclosed in U.S. Pat.
No. 6,043,077 to Barber et al., and U.S. Pat. Nos. 5,869,253 and
5,610,054 to Draper et al.; and [0363] 10) Nucleoside analogs have
also been developed for the treatment of Flaviviridae infections.
[0364] 11) Any of the compounds described by Idenix Pharmaceuticals
in International Publication Nos. WO 01/90121 and WO 01/92282;
[0365] 12) Other patent applications disclosing the use of certain
nucleoside analogs to treat hepatitis C virus include:
PCT/CA00/01316 (WO 01/32153; filed Nov. 3, 2000) and PCT/CA01/00197
(WO 01/60315; filed Feb. 19, 2001) filed by BioChem Pharma, Inc.
(now Shire Biochem, Inc.); PCT/US02/01531 (WO 02/057425; filed Jan.
18, 2002) and PCT/US02/03086 (WO 02/057287; filed Jan. 18, 2002)
filed by Merck & Co., Inc., PCT/EP01/09633 (WO 02/18404;
published Aug. 21, 2001) filed by Roche, and PCT Publication Nos.
WO 01/79246 (filed Apr. 13, 2001), WO 02/32920 (filed Oct. 18,
2001) and WO 02/48165 by Pharmasset, Ltd. [0366] 13) PCT
Publication No. WO 99/43691 to Emory University, entitled
"2'-Fluoronucleosides" discloses the use of certain
2'-fluoronucleosides to treat HCV. [0367] 14) Other miscellaneous
compounds including 1-amino-alkylcyclohexanes (U.S. Pat. No.
6,034,134 to Gold et al.), alkyl lipids (U.S. Pat. No. 5,922,757 to
Chojkier et al.), vitamin E and other antioxidants (U.S. Pat. No.
5,922,757 to Chojkier et al.), squalene, amantadine, bile acids
(U.S. Pat. No. 5,846,964 to Ozeki et al.),
N-(phosphonoacetyl)-L-aspartic acid, (U.S. Pat. No. 5,830,905 to
Diana et al.), benzenedicarboxamides (U.S. Pat. No. 5,633,388 to
Diana et al.), polyadenylic acid derivatives (U.S. Pat. No.
5,496,546 to Wang et al.), 2',3'-dideoxyinosine (U.S. Pat. No.
5,026,687 to Yarchoan et al.), benzimidazoles (U.S. Pat. No.
5,891,874 to Colacino et al.), plant extracts (U.S. Pat. No.
5,837,257 to Tsai et al., U.S. Pat. No. 5,725,859 to Omer et al.,
and U.S. Pat. No. 6,056,961), and piperidenes (U.S. Pat. No.
5,830,905 to Diana et al.). [0368] 15) Any other compounds
currently in preclinical or clinical development for treatment of
hepatitis C virus including: Interleukin-10 by Schering-Plough,
IP-501 by Interneuron, Merimebodib (VX-497) by Vertex,
AMANTADINE.RTM. (Symmetrel) by Endo Labs Solvay, HEPTAZYME.RTM. by
RPI, IDN-6556 by Idun Pharma., XTL-002 by XTL., HCV/MF59 by Chiron,
CIVACIR.RTM. (Hepatitis C Immune Globulin) by NABI, LEVOVIRIN.RTM.
by ICN/Ribapharm, VIRAMIDINE.RTM. by ICN/Ribapharm, ZADAXIN.RTM.
(thymosin alpha-1) by Sci Clone, thymosin plus pegylated interferon
by Sci Clone, CEPLENE.RTM. (histamine dihydrochloride) by Maxim, VX
950/LY 570310 by Vertex/Eli Lilly, ISIS 14803 by Isis
Pharmaceutical/Elan, IDN-6556 by Idun Pharmaceuticals, Inc., JTK
003 by AKROS Pharma, BILN-2061 by Boehringer Ingelheim, CellCept
(mycophenolate mofetil) by Roche, T67, a .beta.-tubulin inhibitor,
by Tularik, a therapeutic vaccine directed to E2 by Innogenetics,
FK788 by Fujisawa Healthcare, Inc., IdB 1016 (Siliphos, oral
silybin-phosphatdylcholine phytosome), RNA replication inhibitors
(VP50406) by ViroPharma/Wyeth, therapeutic vaccine by Intercell,
therapeutic vaccine by Epimmune/Genencor, IRES inhibitor by Anadys,
ANA 245 and ANA 246 by Anadys, immunotherapy (Therapore) by Avant,
protease inhibitor by Corvas/SChering, helicase inhibitor by
Vertex, fusion inhibitor by Trimeris, T cell therapy by CellExSys,
polymerase inhibitor by Biocryst, targeted RNA chemistry by PTC
Therapeutics, Dication by Immtech, Int., protease inhibitor by
Agouron, protease inhibitor by Chiron/Medivir, antisense therapy by
AVI BioPharma, antisense therapy by Hybridon, hemopurifier by
Aethlon Medical, therapeutic vaccine by Merix, protease inhibitor
by Bristol-Myers Squibb/Axys, Chron-VacC, a therapeutic vaccine, by
Tripep, Utah 231B by United Therapeutics, protease, helicase and
polymerase inhibitors by Genelabs Technologies, IRES inhibitors by
Immusol, R803 by Rigel Pharmaceuticals, INFERGEN.RTM. (interferon
alphacon-1) by InterMune, OMNIFERON.RTM. (natural interferon) by
Viragen, ALBUFERON.RTM. by Human Genome Sciences, REBIF.RTM.
(interferon beta-1a) by Ares-Serono, Omega Interferon by
BioMedicine, Oral Interferon Alpha by Amarillo Biosciences,
interferon gamma, interferon tau, and Interferon gamma-1b by
InterMune. V. Pharmaceutical Compositions
[0369] Hosts, including humans, infected with pestivirus,
flavivirus, HCV or another organism replicating through a
RNA-dependent RNA viral polymerase, or for treating any other
disorder described herein, can be treated by administering to the
patient an effective amount of the active compound or a
pharmaceutically acceptable prodrug or salt thereof in the presence
of a pharmaceutically acceptable carrier or dilutent. The active
materials can be administered by any appropriate route, for
example, orally, parenterally, intravenously, intradermally,
subcutaneously, or topically, in liquid or solid form.
[0370] A preferred dose of the compound for pestivirus, flavivirus
or HCV infection or any other condition described herein will be in
the range from about 1 to 50 mg/kg, preferably 1 to 20 mg/kg, of
body weight per day, more generally 0.1 to about 100 mg per
kilogram body weight of the recipient per day. Lower doses may be
preferable, for example doses of 0.5-100 mg, 0.5-50 mg, 0.5-10 mg,
or 0.5-5 mg per kilogram body weight per day. Even lower doses may
be useful, and thus ranges can also include from 0.1-0.5 mg per
kilogram body weight per day. The effective dosage range of the
pharmaceutically acceptable salts and prodrugs can be calculated
based on the weight of the parent nucleoside to be delivered. If
the salt or prodrug exhibits activity in itself, the effective
dosage can be estimated as above using the weight of the salt or
prodrug, or by other means known to those skilled in the art.
[0371] The compound is conveniently administered in a unit of any
suitable dosage form, including but not limited to one containing 7
to 3000 mg, preferably 70 to 1400 mg of active ingredient per unit
dosage form. An oral dosage of 50-1000 mg is usually convenient,
including in one or multiple dosages of 50, 100, 200, 250, 300,
400, 500, 600, 700, 800, 900 or 1000 mgs. Lower doses may be
preferable, for example from 10-100 or 1-50 mg. Also contemplated
are doses of 0.1-50 mg, or 0.1-20 mg or 0.1-10.0 mg. Furthermore,
lower doses may be utilized in the case of administration by a
non-oral route, as, for example, by injection or inhalation.
[0372] Ideally the active ingredient should be administered to
achieve peak plasma concentrations of the active compound of from
about 0.2 to 70 .mu.M, preferably about 1.0 to 10 .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
administered as a bolus of the active ingredient.
[0373] The concentration of active compound in the drug composition
will depend on absorption, inactivation and excretion rates of the
drug as well as other factors known to those of skill in the art.
It is to be noted that dosage values will also vary with the
severity of the condition to be alleviated. It is to be further
understood that for any particular subject, specific dosage
regimens should be adjusted over time according to the individual
need and the professional judgment of the person administering or
supervising the administration of the compositions, and that the
concentration ranges set forth herein are exemplary only and are
not intended to limit the scope or practice of the claimed
composition. The active ingredient may be administered at once, or
may be divided into a number of smaller doses to be administered at
varying intervals of time.
[0374] A preferred mode of administration of the active compound is
oral. Oral compositions will generally include an inert diluent or
an edible carrier. They may be enclosed in gelatin capsules or
compressed into tablets. For the purpose of oral therapeutic
administration, the active compound can be incorporated with
excipients and used in the form of tablets, troches, or capsules.
Pharmaceutically compatible binding agents, and/or adjuvant
materials can be included as part of the composition.
[0375] The tablets, pills, capsules, troches and the like can
contain any of the following ingredients, or compounds of a similar
nature: a binder such as microcrystalline cellulose, gum tragacanth
or gelatin; an excipient such as starch or lactose, a
disintegrating agent such as alginic acid, Primogel, or corn
starch; a lubricant such as magnesium stearate or Sterotes; a
glidant such as colloidal silicon dioxide; a sweetening agent such
as sucrose or saccharin; or a flavoring agent such as peppermint,
methyl salicylate, or orange flavoring. When the dosage unit form
is a capsule, it can contain, in addition to material of the above
type, a liquid carrier such as a fatty oil. In addition, dosage
unit forms can contain various other materials which modify the
physical form of the dosage unit, for example, coatings of sugar,
shellac, or other enteric agents.
[0376] The compound can be administered as a component of an
elixir, suspension, syrup, wafer, chewing gum or the like. A syrup
may contain, in addition to the active compounds, sucrose as a
sweetening agent and certain preservatives, dyes and colorings and
flavors.
[0377] The compound or a pharmaceutically acceptable prodrug or
salts thereof can also be mixed with other active materials that do
not impair the desired action, or with materials that supplement
the desired action, such as antibiotics, antifungals,
anti-inflammatories, or other antivirals, including other
nucleoside compounds. Solutions or suspensions used for parenteral,
intradermal, sucutaneous, or topical application can include the
following components: a sterile diluent such as water for
injection, saline solution, fixed oils, polyethylene glycols,
glycerine, propylene glycol or other synthetic solvents;
antibacterial agents such as benzyl alcohol or methyl parabens;
antioxidants such as ascorbic acid or sodium bisulfite; chelating
agents such as ethylenediaminetetraacetic acid; buffers such as
acetates, citrates or phosphates and agents for the adjustment of
tonicity such as sodium chloride or dextrose. The parental
preparation can be enclosed in ampoules, disposable syringes or
multiple dose vials made of glass or plastic.
[0378] If administered intravenously, preferred carriers are
physiological saline or phosphate buffered saline (PBS).
[0379] In a preferred embodiment, the active compounds are prepared
with carriers that will protect the compound against rapid
elimination from the body, such as a controlled release
formulation, including implants and microencapsulated delivery
systems. biodegradable, biocompatible polymers can be used, such as
ethylene vinyl acetate, polyanhydrides, polyglycolic acid,
collagen, polyorthoesters and polylactic acid. Methods for
preparation of such formulations will be apparent to those skilled
in the art. The materials can also be obtained commercially from
Alza Corporation.
[0380] Liposomal suspensions (including liposomes targeted to
infected cells with monoclonal antibodies to viral antigens) are
also preferred as pharmaceutically acceptable carriers. These may
be prepared according to methods known to those skilled in the art,
for example, as described in U.S. Pat. No. 4,522,811 (which is
incorporated herein by reference in its entirety). For example,
liposome formulations may be prepared by dissolving appropriate
lipid(s) (such as stearoyl phosphatidyl ethanolamine, stearoyl
phosphatidyl choline, arachadoyl phosphatidyl choline, and
cholesterol) in an inorganic solvent that is then evaporated,
leaving behind a thin film of dried lipid on the surface of the
container. An aqueous solution of the active compound or its
monophosphate, diphosphate, and/or triphosphate derivatives is then
introduced into the container. The container is then swirled by
hand to free lipid material from the sides of the container and to
disperse lipid aggregates, thereby forming the liposomal
suspension.
VI. Processes for the Preparation of Active Compounds
[0381] The nucleosides of the present invention can be synthesized
by any means known in the art. In particular, the synthesis of the
present nucleosides can be achieved by either alkylating the
appropriately modified sugar, followed by glycosylation or
glycosylation followed by alkylation of the nucleoside. The
following non-limiting embodiments illustrate some general
methodology to obtain the nucleosides of the present invention.
A. General Synthesis of 1'-C-Branched Nucleosides
[0382] 1'-C-Branched ribonucleosides of the following structure:
##STR35## wherein BASE is a purine or pyrimidine base as defined
herein; R.sup.7 and R.sup.9 are independently hydrogen, OR.sup.2,
hydroxy, alkyl (including lower alkyl), azido, cyano, alkenyl,
alkynyl, Br-vinyl, --C(O)O(alkyl), --C(O)O(lower alkyl), --O(acyl),
--O(lower acyl), --O(alkyl), --O(lower alkyl), --O(alkenyl),
chlorine, bromine, iodine, NO.sub.2, NH.sub.2, --NH(lower alkyl),
--NH(acyl), --N(lower alkyl).sub.2, --N(acyl).sub.2; R.sup.8 and
R.sup.10 are independently H, alkyl (including lower alkyl),
chlorine, bromine or iodine; alternatively, R.sup.7 and R.sup.9,
R.sup.7 and R.sup.10, R.sup.8 and R.sup.9, or R.sup.8 and R.sup.10
can come together to form a pi bond; R.sup.1 and R.sup.2 are
independently H; phosphate (including monophosphate, diphosphate,
triphosphate, or a stabilized phosphate prodrug); acyl (including
lower acyl); alkyl (including lower alkyl); sulfonate ester
including alkyl or arylalkyl sulfonyl including methanesulfonyl and
benzyl, wherein the phenyl group is optionally substituted with one
or more substituents as described in the definition of aryl given
herein; a lipid, including a phospholipid; an amino acid; a
carbohydrate; a peptide; cholesterol; or other pharmaceutically
acceptable leaving group which is capable of providing a compound
wherein R.sup.1 or R.sup.2 is independently H or phosphate, for
example when administered in vivo; R.sup.6 is an alkyl, chloro-,
bromo-, fluoro-, or iodo-alkyl (i.e. CF.sub.3), alkenyl, or alkynyl
(i.e. allyl); and X is O, S, SO.sub.2 or CH.sub.2 can be prepared
by one of the following general methods. 1) Modification from the
Lactone
[0383] The key starting material for this process is an
appropriately substituted lactone. The lactone can be purchased or
can be prepared by any known means including standard
epimerization, substitution and cyclization techniques. The lactone
can be optionally protected with a suitable protecting group,
preferably with an acyl or silyl group, by methods well known to
those skilled in the art, as taught by Greene et al. Protective
Groups in Organic Synthesis, John Wiley and Sons, Second Edition,
1991. The protected lactone can then be coupled with a suitable
coupling agent, such as an organometallic carbon nucleophile, such
as a Grignard reagent, an organolithium, lithium dialkylcopper or
R.sup.6--SiMe.sub.3 in TBAF with the appropriate non-protic solvent
at a suitable temperature, to give the 1'-alkylated sugar.
[0384] The optionally activated sugar can then be coupled to the
BASE by methods well known to those skilled in the art, as taught
by Townsend Chemistry of Nucleosides and Nucleotides, Plenum Press,
1994. For example, an acylated sugar can be coupled to a silylated
base with a Lewis acid, such as tin tetrachloride, titanium
tetrachloride or trimethylsilyltriflate in the appropriate solvent
at a suitable temperature.
[0385] Subsequently, the nucleoside can be deprotected by methods
well known to those skilled in the art, as taught by Greene et al.
Protective Groups in Organic Synthesis, John Wiley and Sons, Second
Edition, 1991.
[0386] In a particular embodiment, the 1'-C-branched ribonucleoside
is desired. The synthesis of a ribonucleoside is shown in Scheme 1.
Alternatively, deoxyribo-nucleoside is desired. To obtain these
nucleosides, the formed ribonucleoside can optionally be protected
by methods well known to those skilled in the art, as taught by
Greene et al. Protective Groups in Organic Synthesis, John Wiley
and Sons, Second Edition, 1991, and then the 2'-OH can be reduced
with a suitable reducing agent. Optionally, the 2'-hydroxyl can be
activated to facilitate reduction; i.e. via the Barton reduction.
##STR36## 2. Alternative Method for the Preparation of
1'-C-Branched Nucleosides
[0387] The key starting material for this process is an
appropriately substituted hexose. The hexose can be purchased or
can be prepared by any known means including standard
epimerization, such as alkaline treatment, substitution and
coupling techniques. The hexose can be selectively protected to
give the appropriate hexa-furanose, as taught by Townsend Chemistry
of Nucleosides and Nucleotides, Plenum Press, 1994.
[0388] The 1'-hydroxyl can be optionally activated to a suitable
leaving group such as an acyl group or a chloro, bromo, fluoro,
iodo via acylation or halogenation, respectively. The optionally
activated sugar can then be coupled to the BASE by methods well
known to those skilled in the art, as taught by Townsend Chemistry
of Nucleosides and Nucleotides, Plenum Press, 1994. For example, an
acylated sugar can be coupled to a silylated base with a Lewis
acid, such as tin tetrachloride, titanium tetrachloride or
trimethylsilyltriflate in the appropriate solvent at a suitable
temperature. Alternatively, a halo-sugar can be coupled to a
silylated base with the presence of trimethylsilyltriflate.
[0389] The 1'-CH.sub.2--OH, if protected, can be selectively
deprotected by methods well known in the art. The resultant primary
hydroxyl can be functionalized to yield various C-branched
nucleosides. For example, the primary hydroxyl can be reduced to
give the methyl, using a suitable reducing agent. Alternatively,
the hydroxyl can be activated prior to reduction to facilitate the
reaction; i.e. via the Barton reduction. In an alternate
embodiment, the primary hydroxyl can be oxidized to the aldehyde,
then coupled with a carbon nucleophile, such as a Grignard reagent,
an organolithium, lithium dialkylcopper or R.sup.6--SiMe.sub.3 in
TBAF with the appropriate non-protic solvent at a suitable
temperature.
[0390] In a particular embodiment, the 1'-C-branched ribonucleoside
is desired. The synthesis of a ribonucleoside is shown in Scheme 2.
Alternatively, deoxyribo-nucleoside is desired. To obtain these
nucleosides, the formed ribonucleoside can optionally be protected
by methods well known to those skilled in the art, as taught by
Greene et al. Protective Groups in Organic Synthesis, John Wiley
and Sons, Second Edition, 1991, and then the 2'-OH can be reduced
with a suitable reducing agent. Optionally, the 2'-hydroxyl can be
activated to facilitate reduction; i.e. via the Barton reduction.
##STR37##
[0391] In addition, the L-enantiomers corresponding to the
compounds of the invention can be prepared following the same
general methods (1 or 2), beginning with the corresponding L-sugar
or nucleoside L-enantiomer as starting material.
B. General Synthesis of 2'-C-Branched Nucleosides
[0392] 2'-C-Branched ribonucleosides of the following structure:
##STR38## wherein BASE is a purine or pyrimidine base as defined
herein; R.sup.7 and R.sup.9 are independently hydrogen, OR.sup.2,
hydroxy, alkyl (including lower alkyl), azido, cyano, alkenyl,
alkynyl, Br-vinyl, --C(O)O(alkyl), --C(O)O(lower alkyl), --O(acyl),
--O(lower acyl), --O(alkyl), --O(lower alkyl), --O(alkenyl),
chlorine, bromine, iodine, NO.sub.2, NH.sub.2, --NH(lower alkyl),
--NH(acyl), --N(lower alkyl).sub.2, --N(acyl).sub.2; R.sup.10 is H,
alkyl (including lower alkyl), chlorine, bromine or iodine;
alternatively, R.sup.7 and R.sup.9, or R.sup.7 and R.sup.10 can
come together to form a pi bond; R.sup.1 and R.sup.2 are
independently H; phosphate (including monophosphate, diphosphate,
triphosphate, or a stabilized phosphate prodrug); acyl (including
lower acyl); alkyl (including lower alkyl); sulfonate ester
including alkyl or arylalkyl sulfonyl including methanesulfonyl and
benzyl, wherein the phenyl group is optionally substituted with one
or more substituents as described in the definition of aryl given
herein; a lipid, including a phospholipid; an amino acid; a
carbohydrate; a peptide; cholesterol; or other pharmaceutically
acceptable leaving group which when administered in vivo is capable
of providing a compound wherein R.sup.1 or R.sup.2 is independently
H or phosphate; R.sup.6 is an alkyl, chloro-, bromo-, fluoro-,
iodo-alkyl (i.e. CF.sub.3), alkenyl, or alkynyl (i.e. allyl); and X
is O, S, SO.sub.2 or CH.sub.2 can be prepared by one of the
following general methods. 1. Glycosylation of the Nucleobase with
an Appropriately Modified Sugar
[0393] The key starting material for this process is an
appropriately substituted sugar with a 2'-OH and 2'-H, with the
appropriate leaving group (LG), for example an acyl group or a
chloro, bromo, fluoro or iodo. The sugar can be purchased or can be
prepared by any known means including standard epimerization,
substitution, oxidation and reduction techniques. The substituted
sugar can then be oxidized with the appropriate oxidizing agent in
a compatible solvent at a suitable temperature to yield the
2'-modified sugar. Possible oxidizing agents are Jones reagent (a
mixture of chromic acid and sulfuric acid), Collins's reagent
(dipyridine Cr(VI) oxide, Corey's reagent (pyridinium
chlorochromate), pyridinium dichromate, acid dichromate, potassium
permanganate, MnO.sub.2, ruthenium tetroxide, phase transfer
catalysts such as chromic acid or permanganate supported on a
polymer, Cl.sub.2-pyridine, H.sub.2O.sub.2-ammonium molybdate,
NaBrO.sub.2--CAN, NaOCl in HOAc, copper chromite, copper oxide,
Raney nickel, palladium acetate, Meerwin-Pondorf-Verley reagent
(aluminum t-butoxide with another ketone) and
N-bromosuccinimide.
[0394] Then coupling of an organometallic carbon nucleophile, such
as a Grignard reagent, an organolithium, lithium dialkylcopper or
R.sup.6--SiMe.sub.3 in TBAF with the ketone with the appropriate
non-protic solvent at a suitable temperature, yields the
2'-alkylated sugar. The alkylated sugar can be optionally protected
with a suitable protecting group, preferably with an acyl or silyl
group, by methods well known to those skilled in the art, as taught
by Greene et al. Protective Groups in Organic Synthesis, John Wiley
and Sons, Second Edition, 1991.
[0395] The optionally protected sugar can then be coupled to the
BASE by methods well known to those skilled in the art, as taught
by Townsend Chemistry of Nucleosides and Nucleotides, Plenum Press,
1994. For example, an acylated sugar can be coupled to a silylated
base with a Lewis acid, such as tin tetrachloride, titanium
tetrachloride or trimethylsilyltriflate in the appropriate solvent
at a suitable temperature. Alternatively, a halo-sugar can be
coupled to a silylated base with the presence of
trimethylsilyltriflate.
[0396] Subsequently, the nucleoside can be deprotected by methods
well known to those skilled in the art, as taught by Greene et al.
Protective Groups in Organic Synthesis, John Wiley and Sons, Second
Edition, 1991.
[0397] In a particular embodiment, the 2'-C-branched ribonucleoside
is desired. The synthesis of a ribonucleoside is shown in Scheme 3.
Alternatively, deoxyribo-nucleoside is desired. To obtain these
nucleosides, the formed ribonucleoside can optionally be protected
by methods well known to those skilled in the art, as taught by
Greene et al. Protective Groups in Organic Synthesis, John Wiley
and Sons, Second Edition, 1991, and then the 2'-OH can be reduced
with a suitable reducing agent. Optionally, the 2'-hydroxyl can be
activated to facilitate reduction; i.e. via the Barton reduction.
##STR39## 2. Modification of a Pre-Formed Nucleoside
[0398] The key starting material for this process is an
appropriately substituted nucleoside with a 2'-OH and 2'-H. The
nucleoside can be purchased or can be prepared by any known means
including standard coupling techniques. The nucleoside can be
optionally protected with suitable protecting groups, preferably
with acyl or silyl groups, by methods well known to those skilled
in the art, as taught by Greene et al. Protective Groups in Organic
Synthesis, John Wiley and Sons, Second Edition, 1991.
[0399] The appropriately protected nucleoside can then be oxidized
with the appropriate oxidizing agent in a compatible solvent at a
suitable temperature to yield the 2'-modified sugar. Possible
oxidizing agents are Jones reagent (a mixture of chromic acid and
sulfuric acid), Collins's reagent (dipyridine Cr(VI) oxide, Corey's
reagent (pyridinium chlorochromate), pyridinium dichromate, acid
dichromate, potassium permanganate, MnO.sub.2, ruthenium tetroxide,
phase transfer catalysts such as chromic acid or permanganate
supported on a polymer, Cl.sub.2-pyridine, H.sub.2O.sub.2-ammonium
molybdate, NaBrO.sub.2--CAN, NaOCl in HOAc, copper chromite, copper
oxide, Raney nickel, palladium acetate, Meerwin-Pondorf-Verley
reagent (aluminum t-butoxide with another ketone) and
N-bromosuccinimide.
[0400] Subsequently, the nucleoside can be deprotected by methods
well known to those skilled in the art, as taught by GreeneGreene
et al. Protective Groups in Organic Synthesis, John Wiley and Sons,
Second Edition, 1991.
[0401] In a particular embodiment, the 2'-C-branched ribonucleoside
is desired. The synthesis of a ribonucleoside is shown in Scheme 4.
Alternatively, deoxyribo-nucleoside is desired. To obtain these
nucleosides, the formed ribonucleoside can optionally be protected
by methods well known to those skilled in the art, as taught by
Greene et al..sub.--Protective Groups in Organic Synthesis, John
Wiley and Sons, Second Edition, 1991, and then the 2'-OH can be
reduced with a suitable reducing agent. Optionally, the 2'-hydroxyl
can be activated to facilitate reduction; i.e. via the Barton
reduction. ##STR40## 3. Synthesis of
.beta.-D-2'-C-Methyl-Ribofuranosyl Cytidine-3'-O-L- Valine
Ester
[0402] In one synthesis method, depicted in FIG. 5, the synthesis
comprises reacting cytosine, BSA and SnCl.sub.4/acetonitrile with
1,2,3,5-tetra-O-benzoyl-2-C-methyl-.beta.-D-ribofuranose (FIG. 5,
compound 1) to form
4-amino-1-(3,4-dibenzoyloxy-5-benzoyloxymethyl-3-methyl-tetrahydro-furan--
2-yl)-1H-pyrimidin-2-one (FIG. 5, compound 2); and reacting (FIG.
5, compound 2) with NaOMe/MeOH to provide
4-amino-1-(3,4-dihydroxy-5-hydroxymethyl-3-methyl-tetrahydro-furan-2-yl)--
1H-pyrimidin-2-one (FIG. 5, compound 3), also known as
2-C-methyl-.beta.-D-ribofuranose. The use of cytosine as a starting
material rather than benzoyl-cytosine improves the "atom economy"
of the process and simplifies purification at later steps.
[0403] The next steps in this process comprise reacting (FIG. 5,
compound 3) with Me.sub.2NCH(OMe).sub.2 in DMF to form (FIG. 5,
compound 4),
N-[1-(3,4-dihydroxy-5-hydroxymethyl-3-methyl-tetrahydro-furan-2-yl)-2-oxo-
-1,2-dihydro-pyrimidin-4-yl]-N,N-dimethyl-formamidine, which is the
amino-protected form of (FIG. 5, compound 3); reacting (FIG. 5,
compound 4) with TBDPSCl and imidazole in DCM to provide the
5'-silyl-protected form of (FIG. 5, compound 4) as
N'-{1-[5-(tert-butyl-diphenyl-silanyloxymethyl)-3,4-dihydroxy-3-methyl-te-
trahydro-furan-2-yl]-2-oxo-1,2-dihydro-pyrimidin-4-yl}-N,N-dimethyl-formam-
idine (FIG. 5, compound 5), where the use of DCM provides the
advantage of having greater control over disilyl by-product
formation; reacting (FIG. 5, compound 5) with N-Boc-L-valine, EDC
and DMAP in DCM at room temperature to form
2-tert-butoxycarbonylamino-3-methyl-butyric acid
2-(tert-butyl-diphenyl-silanyloxy-methyl)-5-[4-(dimethylamino-methyleneam-
ino)-2-oxo-2H-pyrimidin-1-yl]-4-hydroxy-4-methyl-tetrahydro-furan-3-yl
ester (FIG. 5, compound 6); removing the silyl and amino-protecting
groups by reacting (FIG. 5, compound 6) with NH.sub.4F in MeOH in
the presence of approximately 10 mole equivalents of ethyl acetate
to prevent cleavage of the 3'-O-valinyl ester by liberated ammonia,
and refluxing the mixture to provide
2-tert-butoxycarbonylamino-3-methyl-butyric acid
5-(4-amino-2-oxo-2H-pyrimidin-1-yl)-4-hydroxy-2-hydroxymethyl-4-methyl-te-
trahydro-furan-3-yl ester to provide (FIG. 5, compound 7); and
finally, reacting (FIG. 5, compound 7) with HCl in EtOH to provide
2-amino-3-methyl-butyric acid
5-(4-amino-2-oxo-2H-pyrimidin-1-yl)-4-hydroxy-2-hydroxymethyl-4-methyl-te-
trahydro-furan-3-yl ester, dihydrochloride salt (FIG. 5, compound
8) as a final product.
6. Alternative Synthesis of .beta.-D-2'-C-Methyl-Ribofuranosyl
Cytidine-3'-O-L- Valine Ester
[0404] In another method to synthesize the compounds of the
invention, shown in FIG. 6, benzoylcytosine, BSA and
SnCl.sub.4/acetonitrile are reacted with
1,2,3,5-tetra-O-benzoyl-2-C-methyl-.beta.-D-ribofuranose (FIG. 6,
compound 1a) to form
4-benzoylamino-1-(3,4-dibenzoyloxy-5-benzoyloxymethyl-3-methyl-tetrahydro-
-furan-2-yl)-1H-pyrimidin-2-one (FIG. 6, compound 2a); reacting
(FIG. 6, compound 2a) with NH.sub.3 in methanol and
chromatographically separating the product,
4-amino-1-(3,4-dihydroxy-5-hydroxymethyl-3-methyl-tetrahydro-furan-2-yl)--
1H-pyrimidin-2-one (FIG. 6, compound 3a), also known as
.beta.-D-2'-C-methyl-cytidine; reacting (FIG. 6, compound 3a) with
Me.sub.2NCH(OMe).sub.2 in DMF at room temperature for 1.5 hours to
form
N-[1-(3,4-dihydroxy-5-hydroxymethyl-3-methyl-tetrahydro-furan-2-yl)-2-oxo-
-1,2-dihydro-pyrimidin-4-yl]-N,N-dimethyl-formamidine (FIG. 6,
compound 4a); reacting (FIG. 6, compound 4a) with TBDPSCl and
pyridine at room temperature for 6 hours to provide
N'-{1-[5-(tert-butyl-diphenyl-silanyloxymethyl)-3,4-dihydroxy-3-methyl-te-
trahydro-furan-2-yl]-2-oxo-1,2-dihydro-pyrimidin-4-yl}-N,N-dimethyl-formam-
idine (FIG. 6, compound 5a); reacting (FIG. 6, compound 5a) with
N-Boc-L-valine, DEC and DMAP in THF/DMF at room temperature for 2
days and subjecting the product formed from this reaction to HPLC
in order to provide 2-tert-butoxycarbonylamino-3-methyl-butyric
acid
2-(tert-butyl-diphenyl-silanyloxy-methyl)-5-[4-(dimethylaminomethyleneami-
no)-2-oxo-2H-pyrimidin-1-yl]-4-hydroxy-4-methyl-tetrahydro-furan-3-yl
ester (FIG. 6, compound 6a); refluxing (FIG. 6, compound 6a) with
NH.sub.4F in MeOH for about 3 hours to remove the silyl and
amino-protecting groups, and subjecting the product to
chromatographic purification to provide
2-tert-butoxycarbonylamino-3-methyl-butyric acid
5-(4-amino-2-oxo-2H-pyrimidin-1-yl)-4-hydroxy-2-hydroxymethyl-4-methyl-te-
trahydro-furan-3-yl ester (FIG. 6, compound 7a); and finally
reacting (FIG. 6, compound 7a) with HCl in EtOAc at room
temperature to provide 2-amino-3-methyl-butyric acid
5-(4-amino-2-oxo-2H-pyrimidin-1-yl)-4-hydroxy-2-hydroxymethyl-4-methyl-te-
trahydro-furan-3-yl ester, dihydrochloride salt (FIG. 6, compound
8a) as a final product.
[0405] The synthesis of 2'-C-methyl-cytidine-3'-O-L-valine ester
(val-mcyd) is depicted in scheme 5 and scheme 6, and described
below. ##STR41## Step 1: Synthesis of Scheme 5, Compound 9:
2-C-Methyl-D-ribonic-.gamma.-lactone
[0406] De-ionized water (100 mL) was stirred in a 250 mL 3-necked
round bottom flask, equipped with an overhead stirrer, a stirring
shaft, a digital temperature read-out device and an argon line.
Argon was bubbled into water for thirty minutes and D-fructose
(20.0 g, 0.111 mole) was added and the solution became clear in a
few minutes. Calcium oxide (12.5 g, 0.223 mole) was added in
portions over a period of five minutes and the mixture was
vigorously stirred. An exotherm was observed and reaction
temperature reached 39.6.degree. C. after 10 minutes from the start
of the calcium oxide addition. After about fifteen minutes, the
reaction mixture developed a yellow color that deepened with time.
After three hours, an aliquot was withdrawn for TLC analysis. The
aliquot was acidified to pH 2 using saturated aqueous solution of
oxalic acid. The resulting white suspension was evaporated under
reduced pressure to remove the water. Toluene (2 mL) was added to
the residue and the mixture was evaporated under reduced pressure
(at 45-50.degree. C.) to remove any trace of water. The residual
solid was re-constituted in 2 mL of 1:1 tetrahydrofuran:methanol
mixture. After thorough mixing, the suspension was allowed to
settle and the supernatant clear solution was spotted for TLC
(silica plate was developed in 2% methanol in ethyl acetate and
stained in 1% alkaline potassium permanganate dip. The plate was
then heated, using a heat gun, until the appearance of yellowish
spots on the pink background). The desired lactone typically
appears at an R.sub.f value of 0.33 under the above conditions.
More polar by-products and unreacted material are detected in the
R.sub.f value range of 0.0 to 0.2.
[0407] Although product formation was observed after 3 hours, the
reaction was allowed to continue for 22 hours during which time the
reaction mixture was stirred at 25.degree. C. At the end of this
period, pH of the mixture was 13.06. Carbon dioxide gas was bubbled
into the reaction mixture for about 2.5 hours (pH was 7.25). The
formed calcium carbonate solid was removed by vacuum filtration,
filter cake washed with 50 mL of de-ionized water. The aqueous
layers were combined and treated with oxalic acid (5.0 g, 0.056
mole) and the mixture was vigorously stirred at 25.degree. C. for
30 minutes (The initial dark color largely disappeared and the
mixture turned into a milky white slurry). The pH of the mixture at
this stage is typically 2-3. The slurry mixture was stirred at
45-50.degree. C. overnight. The mixture was then evaporated under
reduced pressure and at 45-50.degree. C. to remove 75 mL of water.
Sodium chloride (30 g) and tetrahydrofuran (100 mL) were added to
the aqueous slurry (about 75 mL) and the mixture was vigorously
stirred at 25.degree. C. for 30 minutes. The layers were separated
and the aqueous layer was stirred for 10 minutes with 75 mL of
fresh tetrahydrofuran. This process was repeated for three times
and the tetrahydrofuran solutions were combined and stirred with 10
g of anhydrous magnesium sulfate for 30 minutes. The mixture was
filtered and the magnesium sulfate filter cake was washed with 60
mL of tetrahydrofuran. The filtrate was evaporated under reduced
pressure and at 40.degree. C. to give 10.86 g of crude product as a
dark orange semisolid. (For scale up runs tetrahydrofuran will be
replaced with acetone instead of evaporation of crude product to
dryness). Crude product was stirred with acetone (20 mL) at
20.degree. C. for 3 hours. Product was collected by vacuum
filtration and the filter cake washed with 12 mL of acetone to give
the desired product 9 as white crystalline solid. Product was dried
in vacuum to give 2.45 g (13.6% yield). Melting point of compound
9: 158-162.degree. C. (literature melting point: 160-161.degree.
C.). .sup.1H NMR (DMSO-d.sub.6) .delta. ppm 5.69 (s, 1H, exch. With
D.sub.2O), 5.41 (d, 1H, exch. With D.sub.2O), 5.00 (t, 1H, exch.
With D.sub.2O), 4.15 (m, 1H), 3.73 (m, 2H), 3.52 (m, 1H), 1.22 (s,
3H). .sup.13C NMR (DMSO-d.sub.6) .delta. ppm 176.44, 82.95, 72.17,
72.02, 59.63, 20.95. (C.sub.6H.sub.10O.sub.5: calcd C, 44.45; H,
6.22. Found: C, 44.34; H, 6.30).
Step 2: Synthesis of Scheme 5, Compound 10:
2,3,5-Tri-O-benzoyl-2-C-methyl-D-ribonic-.gamma.-lactone
[0408] A mixture of lactone 1 (3.0 g, 18.50 mmol.),
4-dimethylaminopyridine (0.45 g, 3.72 mmol.) and triethylamine
(25.27 g, 249.72 mmol.) in 1,2-dimethoxy ethane (50 mL) was stirred
at 25.degree. C. under argon atmosphere for thirty minutes. This
white suspension was cooled to 5.degree. C. and benzoyl chloride
(11.7 g, 83.23 mmol.) was added over a period of fifteen minutes.
The mixture was stirred at 25.degree. C. for two hours. TLC
analysis (silica, 2% methanol in ethyl acetate) indicated complete
consumption of starting material. Ice cold water (100 g) was added
to the reaction mixture and stirring was continued for thirty
minutes. The formed white solids were collected by vacuum
filtration and filter cake washed with cold water (50 mL). This
crude product was stirred with tert-butyl methyl ether (60 mL) at
20.degree. C. for thirty minutes, then filtered, filter cake washed
with tert-butyl methyl ether (25 mL) and dried in vacuum to give
7.33 g (83.4% yield) of compound 10 as a white solid in 97.74%
purity (HPLC/AUC). Melting point of compound 10: 137-140.degree. C.
(literature melting point: 141-142.degree. C.). .sup.1H NMR
(CDCl.sub.3) .delta. ppm 8.04 (d, 2H), 7.92 (d, 2H), 7.73 (d, 2H),
7.59 (t, 1H), 7.45 (m, 4H), 7.32 (t, 2H), 7.17 (t, 2H), 5.51 (d,
1H), 5.17 (m, 1H), 4.82-4.66 (d of an AB quartet, 2H) 1.95, (s,
3H). .sup.13C NMR (CDCl.sub.3) .delta. ppm 172.87, 166.17, 166.08,
165.58, 134.06, 133.91, 133.72, 130.09, 129.85, 129.80, 129.37,
128.78, 128.60, 128.49, 127.96, 127.89, 79.67, 75.49, 72.60, 63.29,
23.80. TOF MS ES+ (M+1: 475).
Step 3: Synthesis of Scheme 5, compound 11:
2,3,5-Tri-O-benzoyl-2-C-methyl-.beta.-D-ribofuranose:
[0409] A solution of Red-Al (65 wt. % in toluene, 2.0 mL, 6.56
mmol.) in anhydrous toluene (2.0 mL) was stirred at 0.degree. C.
under argon atmosphere. A solution of anhydrous ethanol (0.38 mL,
6.56 mmol.) in anhydrous toluene (1.6 mL) was added to the toluene
solution over a period of five minutes. The resulting mixture was
stirred at 0.degree. C. for fifteen minutes and 2 mL (2.18 mmol.)
of this Red-Al/ethanol reagent was added to a cold (-5.degree. C.)
solution of 2,3,5-tri-O-benzoyl-2-C-methyl-D-ribonolactone (475 mg,
1.0 mmol.) in anhydrous toluene (10 mL) over a period of 10
minutes. The reaction mixture was stirred at -5.degree. C. for
forty minutes. TLC analysis (silica plates, 35% ethyl acetate in
heptane) indicated complete consumption of starting material. HPLC
analysis indicated only 0.1% of starting material remaining. The
reaction was quenched with acetone (0.2 mL), water (15 mL) and 1 N
HCl (15 mL) at 0.degree. C. and allowed to warm to room
temperature. 1 N HCl (5 mL) was added to dissolve the inorganic
salts (pH: 2-3). The mixture was extracted with ethyl acetate
(3.times.25 mL) and the organic solution washed with brine (25 mL),
dried (anhydrous sodium sulfate, 10 g) and solvent removed under
reduced pressure and at temperature of 40.degree. C. to give the
desired product 11 in quantitative yield (480 mg). This material
was used as is for the subsequent step.
Step 4: Synthesis of Scheme 5, Compound 12:
1,2,3,5-tetra-O-benzoyl-2-C-methyl-.beta.-D-ribofuranose:
[0410] Benzoyl chloride (283 mg, 2.0 mmol.) was added, over a
period of five minutes, to a cold solution (5.degree. C.) of
compound 11 (480 mg, 1.0 mmol.), 4-dimethylaminopyridine (12.3 mg,
0.1 mmol.) and triethylamine (506 mg, 5.0 mmol.) in anhydrous
tetrahydrofuran (5 mL). The reaction mixture was stirred at room
temperature and under argon atmosphere overnight. HPLC analysis
indicated 0.25% of un-reacted starting material. The reaction was
quenched by adding ice-cold water (10 g) and saturated aqueous
solution of sodium bicarbonate. Tetrahydrofuran was removed under
reduced pressure and the mixture was extracted with ethyl acetate
(50 mL). The organic solution was washed with water (25 mL), brine
(25 mL), dried (anhydrous sodium sulfate, 12 g) and solvent removed
under reduced pressure to give 650 mg of thick oily product. This
crude product was stirred with 5 mL of tert-butyl methyl ether for
5 minutes and heptane (5 mL) and water (0.1 mL) were added and
stirring was continued for an additional period of two hours at
20.degree. C. Solids were collected by vacuum filtration and filter
caked washed with 1:1 heptane:tert-butyl methyl ether solution (6
mL) and tert-butyl methyl ether (2 mL). Drying the solid in vacuum
gave 300 mg (52%) of desired product 12 (98.43% pure by HPLC/AUC)
as a white solid that melted at 154-156.3.degree. C. (literature
melting point: 155-156.degree. C.). .sup.1H NMR (CDCl.sub.3)
.delta. ppm 8.13 (m, 4H), 8.07 (d, 2H), 7.89 (d, 2H), 7.63 (m, 3H),
7.48 (m, 6H), 7.15 (m, 3H), 7.06 (s, 1H), 5.86 (dd, 1H), 4.79 (m,
1H), 4.70-4.52 (d of an AB quartet, 2H), 1.95, (s, 3H). .sup.13C
NMR (CDCl.sub.3) .delta. ppm 166.31, 165.83, 165.01, 164.77,
134.01, 133.86, 133.70, 133.17, 130.44, 130.13, 129.97, 129.81,
129.59, 129.39, 129.07, 128.84, 128.76, 128.37, 98.01, 86.87,
78.77, 76.35, 64.05, 17.07. (C.sub.34H.sub.28O.sub.9: calcd C,
70.34; H, 4.86. Found: C, 70.20; H, 4.95). ##STR42## Step 5:
Synthesis of Scheme 6, Compound 13:
4-Amino-1-(3,4-dibenzoyloxy-5-benzyloxymethyl-3-methyl-tetrahydro-fur-
an-2-yl)-1H-pyrimidine-2-one
[0411] Cytosine (89 g, 0.80 mol) was suspended in acetonitrile (900
ml) in a 12 L round bottomed flask equipped with a reflux
condenser, overhead stirrer and an argon inlet adapter. The
suspension was stirred at 20.degree. C. under argon atmosphere and
N,O-bis(trimethylsilyl)acetamide (537 ml, 2.2 mol) was added in one
portion. The resulting solution was heated to 80.degree. C. and
stirred for an additional hour at the same temperature.
1,2,3,5-tetra-O-benzoyl-2-C-methyl-.quadrature.-D-ribofuranose
(425.0 g, 0.73 mol) was suspended in acetonitrile (4000 ml) and
added to the reaction mixture. The reaction mixture became clear
after a few minutes and the temperature dropped to ca. 50.degree.
C. Tin(IV) chloride (154 ml, 1.31 mol) was added over a period of
15 minutes and stirring was continued at 80.degree. C. After one
hour, an aliquot of reaction mixture was quenched by adding aqueous
sodium bicarbonate solution and extracting the aqueous layer with
ethyl acetate. The ethyl acetate layer was examined by TLC (silica
gel, 20% ethyl acetate in heptane, R.sub.f for sugar derivative:
0.40). TLC analysis indicated the complete consumption of the sugar
derivative. Desired product was detected by TLC using 10% methanol
in dichloromethane (R.sub.f: 0.37). The reaction was also monitored
by HPLC (Method # 2). Reaction mixture was cooled to 20.degree. C.
and quenched by adding saturated aqueous sodium bicarbonate
solution (3000 ml) over a period of 30 minutes (observed an
exotherm when added the first few drops of the sodium bicarbonate
solution). Solid sodium bicarbonate (1350 g) was added in portions
to avoid foaming. The mixture was checked to make sure that its pH
is .gtoreq.7. Agitation was stopped and layers were allowed to
separate for 20 minutes. The aqueous layer was drained and stirred
with ethyl acetate (1500 ml) and the mixture was allowed to
separate (30 minutes). The organic layer was isolated and combined
with the acetonitrile solution. The organic solution was washed
with brine (500 ml) and then solvent stripped to a volume of ca.
750 ml. Product can be used as is in the subsequent reaction. It
may also be further stripped to white foamy solid, in quantitative
yield. Structure of compound 13 was confirmed by .sup.1H NMR
analysis.
Step 6: Synthesis of Scheme 6, Compound mCyd:
4-Amino-1-(3,4-dihydroxy-5-hydroxymethyl-3-methyl-tetrahydro-furan-2-yl)--
1H-pyrimidine-2-one
[0412] Sodium methoxide (13.8 g, 0.26 mol) was added to a solution
of compound 10 (416 g, 0.73 mol) in methanol (2000 ml). The
reaction mixture was stirred at room temperature and monitored by
TLC (silica gel, 10% methanol in dichloromethane, R.sub.f of
compound 9: 0.53) and (silica gel, 30% methanol in dichloromethane,
R.sub.f of compound 11: 0.21). Product started to precipitate after
30 minutes and TLC indicated reaction completion after two hours.
The reaction was also monitored by HPLC (Method # 2). Methanol was
removed under reduced pressure to a volume of ca. 500 ml chased
with ethanol (2.times.500 ml) to a volume of ca. 500 ml. The
residual thick slurry was diluted with 750 ml of ethanol and the
mixture was stirred at 20.degree. C. for one hour. Product was
collected by filtration, filter cake washed with ethanol (100 ml)
and tert-butyl-methyl ether (100 ml) and dried to give 168 g (90%
yield for the two steps) of product 11 in purity of >97%
(HPLC/AUC). Product was also analyzed by .sup.1H and .sup.13C
NMR.
Step 7: Synthesis of Scheme 6, Compound 14:
2-Tert-butoxycarbonylamino-3-methyl-butyric acid
5-(4-amino-2-oxo-2H-pyrimidin-1-yl)-4-hydroxy-2-hydroxymethyl-4-methyl-te-
trahydro-furan-3-yl ester
[0413] A solution of N-(tert-butoxycarbonyl)-L-valine (46.50 g, 214
mmol.), carbonyldiimidazole (34.70 g, 214 mmol.), and anhydrous
tetrahydrofuran (1000 mL) in a 2 L round bottom flask, was stirred
at 25.degree. C. under argon for 1.5 hours and then at
40-50.degree. C. for 20 minutes. In a separate 5 L 5-necked round
bottom flask, equipped with an overhead stirrer, cooling tower,
temperature probe, addition funnel, and an argon line was added
4-amino-1-(3,4-dihydroxy-5-hydroxymethyl-3-methyl-tetrahydro-furan-2-yl)--
1H-pyrimidine-2-one (50.0 g, 195 mmol.) and anhydrous
N,N-dimethylformamide (1000 mL). This mixture was heated at
100.degree. C. for 20 minutes until all of the pyrimidine-2-one
derivative compound went into solution, and then triethyl amine
(500 mL) and 4-dimethylaminopyridine (2.38 g, 19 mmol) were added
to the solution. The mixture was next heated at 97.degree. C. for
20 minutes and the tetrahydrofuran solution was added slowly
through an addition funnel over a period of 2 hours, maintaining
the temperature no lower than 82.degree. C. The reaction mixture
was heated at 82.degree. C. for 1 hour and monitored by HPLC
(product=68%, SM=11%, and impurity at about 12 min=17%, excluding
dimethylaminopyridine). The reaction mixture was cooled to room
temperature and then triethylamine and tetrahydrofuran were removed
under vacuum at 30.degree. C. The solution was then neutralized
with acetic acid to a pH of 7.69. N,N-dimethylformamidine was
removed under vacuum at 35.degree. C. and chased with ethyl acetate
(2.times.200 mL). The crude product was stirred with ethyl acetate
(500 mL) and water (300 mL). The two layers were separated and the
aqueous layer was extracted with ethyl acetate (500 mL). The
combined organic layers were washed with an aqueous saturated brine
solution (500 mL). Next the organic layer was extracted with an
aqueous solution of malonic acid (4.times.400 mL, 10 wt. %). The
organic layer was checked by TLC (silica, 20% methanol in
dichloromethane) to make sure that all the desired product was
removed from the organic layer. The acidic aqueous extracts were
combined and cooled in an ice bath and neutralized with
triethylamine to a pH of 7.40 so that the solids fell out of
solution. Ethyl acetate then was added to the aqueous layer. The
white solids were collected by vacuum filtration. Drying the
obtained solids in vacuum gave 81.08 g of 99.01 pure (HPLC) first
crop.
Step 8: Synthesis of Scheme 6, val-mCyd-2-Amino-3-methyl-butyric
acid 5-(4-amino-2-oxo-2H-pyrimidine-1-yl)-4-hydroxy-2
hydroxy-methyl-4-methyl-tetrahydro-furan-3-yl ester
(dihydrochloride salt)
[0414] A solution of compound 14 (21.0 g, 0.046 mol) in ethanol
(168 ml) was stirred in a round bottomed flask equipped with an
overhead stirrer, temperature probe, argon line and hydrogen
chloride gas bubbler. Hydrogen chloride gas (22 g) was bubbled into
the clear solution over a period of one hour. The reaction
temperature was kept below 30.degree. C. using an ice-water bath.
Solid formation started after a few minutes of introducing the
hydrogen chloride gas. After 4 hours, HPLC (method # 3) showed only
0.8% of starting material. Solids were collected by filtration and
filter cake washed with ethanol (20 ml) and di-ethyl ether (100
ml). After drying product under vacuum for 16 hours, 19.06 g
(96.5%) of val-mCyd was obtained in 97.26% purity (HPLC, method #
3); m.p. 210.degree. C. (brown), 248-250.degree. C. (melted);
.sup.1H NMR (DMSO-d.sub.6) .delta. ppm 10.0 (s, 1H, 1/2NH.sub.2,
D.sub.2O exchangeable), 8.9-8.6 (2 br s, 4H, 1/2NH.sub.2, NH.sub.3,
D.sub.2O exchangeable), 8.42 (d, 1H, H-6, J.sub.5-6=7.9 Hz), 6.24
(d, 1H, H-5, J.sub.5-6=7.9 Hz), 5.84 (s, 1H, H-1'), 5.12 (d, 1H,
H-3', J.sub.3'-4'=8.8 Hz), 4.22 (d, 1H, H-4, J.sub.3'-4'=8.7 Hz),
4.0-3.9 (m, 1H, CH), 3.8-3.5 (m, 2H, H-5', H-5''), 2.3-2.1 (m, 1H,
CH), 1.16 (s, 3H, CH.sub.3), 1.0 (m, 6H, (CH.sub.3).sub.2CH);
FAB>0 (GT) 713 (2M+H).sup.+, 449 (M+G+H).sup.+, 357 (M+H).sup.+,
246 (S).sup.+, 112 (B+2H).sup.+; FAB<0 (GT) 747 (2M+Cl).sup.-,
483 (M+G+Cl).sup.-, 391 (M+Cl).sup.-, 355 (M-H).sup.-, 116
(Val).sup.-, 110 (B).sup.-, 35 (Cl).sup.-.
[0415] Two different HPLC methods were used to analyze the above
compounds. Both methods use the following reverse phase column. In
method 1, the column was run at a flow rate of 1.00 ml/min of an
acetonitrile/water linear gradient for a 20 minute run time.
Five-minute equilibration was allowed between runs. The
measurements were at 254 nm. TABLE-US-00001 TABLE A Retention time
of key intermediates: Scheme 5, Compound Retention Time Compund 10
10.2 min Compund 11 9.4 min Compund 12 12.9 min
[0416] In the second method, identification was determined at 272
nm. A Waters Novapak C18, 3.9.times.150 mm ID, 4 .mu.m particle
size, 60 .ANG. pore size or equivalent can be used. The
chromatographic conditions are as follows: injection volume=10
.mu.l, column temperature=25.degree. C., flow rate=1.00 ml/min,
ultraviolet detector at 272 nm, run time is 35 minutes. The system
suitability requirement for the percent relative standard deviation
for the reference standard is not more than 1.0%. TABLE-US-00002
TABLE B Purity and impurities are determined at 272 nm Solvent A -
20 nM Solvent B - triethylammonium Acetonitrile, Time (minutes)
acetate buffer HPLC grade. 0.00 100.0 0.0 10.00 85.0 15.0 25.00 5.0
95.0 35.0 5.0 95.0
[0417] TABLE-US-00003 TABLE C Retention times of key intermediates
and final drug substance Scheme 6, Compound Retention Time
(minutes) Compound mCyd 2.7-2.8 Compund 14 15.5 val-mCyd 9.1
[0418] A process of synthesizing a
.beta.-D-2'-C-methyl-2'-acetyl-ribofuransyl-cytidine-3'-O-L-valine
ester is detailed in FIG. 7. A process of synthesizing a
.beta.-D-2'-C-methyl-2'-acetyl-ribofuransyl-cytidine-3'-O-L-proline
ester is detailed in FIG. 8. A process for synthesizing a
.beta.-D-2'-C-methyl-2'-acetyl-ribofuransyl-cytidine-3'-O-L-alanine
ester is depicted in FIG. 9. A process of synthesizing a
.beta.-D-2'-C-methyl-2'-(cyclohexane
carboxylate)-ribofuransyl-cytidine-3'-O-L-valine ester is depicted
in FIG. 10. These processes can be accomplished using techniques
similar to those described above.
C. General Synthesis of 3'-C-Branched Nucleosides
[0419] 3'-C-Branched ribonucleosides of the following structure:
##STR43## wherein BASE is a purine or pyrimidine base as defined
herein; R.sup.7 and R.sup.9 are independently hydrogen, OR.sup.2,
hydroxy, alkyl (including lower alkyl), azido, cyano, alkenyl,
alkynyl, Br-vinyl, --C(O)O(alkyl), --C(O)O(lower alkyl), --O(acyl),
--O(lower acyl), --O(alkyl), --O(lower alkyl), --O(alkenyl),
chlorine, bromine, iodine, NO.sub.2, NH.sub.2, --NH(lower alkyl),
--NH(acyl), --N(lower alkyl).sub.2, --N(acyl).sub.2; R.sup.8 is H,
alkyl (including lower alkyl), chlorine, bromine or iodine;
alternatively, R.sup.7 and R.sup.9, or R.sup.8 and R.sup.9 can come
together to form a pi bond; R.sup.1 and R.sup.2 are independently
H; phosphate (including monophosphate, diphosphate, triphosphate,
or a stabilized phosphate prodrug); acyl (including lower acyl);
alkyl (including lower alkyl); sulfonate ester including alkyl or
arylalkyl sulfonyl including methanesulfonyl and benzyl, wherein
the phenyl group is optionally substituted with one or more
substituents as described in the definition of aryl given herein; a
lipid, including a phospholipid; an amino acid; a carbohydrate; a
peptide; cholesterol; or other pharmaceutically acceptable leaving
group which when administered in vivo is capable of providing a
compound wherein R.sup.1 or R.sup.2 is independently H or
phosphate; R.sup.6 is an alkyl, chloro-, fluoro-, bromo-,
iodo-alkyl (i.e. CF.sub.3), alkenyl, or alkynyl (i.e. allyl); and X
is O, S., SO.sub.2 or CH.sub.2 can be prepared by one of the
following general methods. 1. Glycosylation of the Nucleobase with
an Appropriately Modified Sugar
[0420] The key starting material for this process is an
appropriately substituted sugar with a 3'-OH and 3'-H, with the
appropriate leaving group (LG), for example an acyl group or a
chloro, bromo, fluoro, iodo. The sugar can be purchased or can be
prepared by any known means including standard epimerization,
substitution, oxidation and reduction techniques. The substituted
sugar can then be oxidized with the appropriate oxidizing agent in
a compatible solvent at a suitable temperature to yield the
3'-modified sugar. Possible oxidizing agents are Jones reagent (a
mixture of chromic acid and sulfuric acid), Collins's reagent
(dipyridine Cr(VI) oxide, Corey's reagent (pyridinium
chlorochromate), pyridinium dichromate, acid dichromate, potassium
permanganate, MnO.sub.2, ruthenium tetroxide, phase transfer
catalysts such as chromic acid or permanganate supported on a
polymer, Cl.sub.2-pyridine, H.sub.2O.sub.2-ammonium molybdate,
NaBrO.sub.2--CAN, NaOCl in HOAc, copper chromite, copper oxide,
Raney nickel, palladium acetate, Meerwin-Pondorf-Verley reagent
(aluminum t-butoxide with another ketone) and
N-bromosuccinimide.
[0421] Then coupling of an organometallic carbon nucleophile, such
as a Grignard reagent, an organolithium, lithium dialkylcopper or
R.sup.6--SiMe.sub.3 in TBAF with the ketone with the appropriate
non-protic solvent at a suitable temperature, yields the
3'-C-branched sugar. The 3'-C-branched sugar can be optionally
protected with a suitable protecting group, preferably with an acyl
or silyl group, by methods well known to those skilled in the art,
as taught by Greene et al. Protective Groups in Organic Synthesis,
John Wiley and Sons, Second Edition, 1991.
[0422] The optionally protected sugar can then be coupled to the
BASE by methods well known to those skilled in the art, as taught
by Townsend Chemistry of Nucleosides and Nucleotides, Plenum Press,
1994. For example, an acylated sugar can be coupled to a silylated
base with a Lewis acid, such as tin tetrachloride, titanium
tetrachloride or trimethylsilyltriflate in the appropriate solvent
at a suitable temperature. Alternatively, a halo-sugar can be
coupled to a silylated base with the presence of
trimethylsilyltriflate.
[0423] Subsequently, the nucleoside can be deprotected by methods
well known to those skilled in the art, as taught by Greene et al.
Protective Groups in Organic Synthesis, John Wiley and Sons, Second
Edition, 1991.
[0424] In a particular embodiment, the 3'-C-branched ribonucleoside
is desired. The synthesis of a ribonucleoside is shown in Scheme 7.
Alternatively, deoxyribo-nucleoside is desired. To obtain these
nucleosides, the formed ribonucleoside can optionally be protected
by methods well known to those skilled in the art, as taught by
Greene et al. Protective Groups in Organic Synthesis, John Wiley
and Sons, Second Edition, 1991, and then the 2'-OH can be reduced
with a suitable reducing agent. Optionally, the 2'-hydroxyl can be
activated to facilitate reduction; i.e. via the Barton reduction.
##STR44## 2. Modification of a Pre-Formed Nucleoside
[0425] The key starting material for this process is an
appropriately substituted nucleoside with a 3'-OH and 3'-H. The
nucleoside can be purchased or can be prepared by any known means
including standard coupling techniques. The nucleoside can be
optionally protected with suitable protecting groups, preferably
with acyl or silyl groups, by methods well known to those skilled
in the art, as taught by Greene et al. Protective Groups in Organic
Synthesis, John Wiley and Sons, Second Edition, 1991.
[0426] The appropriately protected nucleoside can then be oxidized
with the appropriate oxidizing agent in a compatible solvent at a
suitable temperature to yield the 2'-modified sugar. Possible
oxidizing agents are Jones reagent (a mixture of chromic acid and
sulfuric acid), Collins's reagent (dipyridine Cr(VI) oxide, Corey's
reagent (pyridinium chlorochromate), pyridinium dichromate, acid
dichromate, potassium permanganate, MnO.sub.2, ruthenium tetroxide,
phase transfer catalysts such as chromic acid or permanganate
supported on a polymer, Cl.sub.2-pyridine, H.sub.2O.sub.2-ammonium
molybdate, NaBrO.sub.2--CAN, NaOCl in HOAc, copper chromite, copper
oxide, Raney nickel, palladium acetate, Meerwin-Pondorf-Verley
reagent (aluminum t-butoxide with another ketone) and
N-bromosuccinimide.
[0427] Subsequently, the nucleoside can be deprotected by methods
well known to those skilled in the art, as taught by GreeneGreene
et al. Protective Groups in Organic Synthesis, John Wiley and Sons,
Second Edition, 1991.
[0428] In a particular embodiment, the 3'-C-branched ribonucleoside
is desired. The synthesis of a ribonucleoside is shown in Scheme 8.
Alternatively, deoxyribo-nucleoside is desired. To obtain these
nucleosides, the formed ribonucleoside can optionally be protected
by methods well known to those skilled in the art, as taught by
Greene et al. Protective Groups in Organic Synthesis, John Wiley
and Sons, Second Edition, 1991, and then the 2'-OH can be reduced
with a suitable reducing agent. Optionally, the 2'-hydroxyl can be
activated to facilitate reduction; i.e. via the Barton reduction.
##STR45##
[0429] In another embodiment of the invention, the L-enantiomers
are desired. Therefore, the L-enantiomers can be corresponding to
the compounds of the invention can be prepared following the same
foregoing general methods, beginning with the corresponding L-sugar
or nucleoside L-enantiomer as starting material.
D. General Synthesis of 4'-C-Branched Nucleosides
[0430] 4'-C-Branched ribonucleosides of the following structure:
##STR46## wherein BASE is a purine or pyrimidine base as defined
herein; R.sup.7 and R.sup.9 are independently hydrogen, OR.sup.2,
hydroxy, alkyl (including lower alkyl), azido, cyano, alkenyl,
alkynyl, Br-vinyl, --C(O)O(alkyl), --C(O)O(lower alkyl), --O(acyl),
--O(lower acyl), --O(alkyl), --O(lower alkyl), --O(alkenyl),
chlorine, bromine, iodine, NO.sub.2, NH.sub.2, --NH(lower alkyl),
--NH(acyl), --N(lower alkyl).sub.2, --N(acyl).sub.2; R.sup.8 and
R.sup.10 are independently H, alkyl (including lower alkyl),
chlorine, bromine or iodine; alternatively, R.sup.7 and R.sup.9,
R.sup.7 and R.sup.10, R.sup.8 and R.sup.9, or R.sup.8 and R.sup.10
can come together to form a pi bond; R.sup.1 and R.sup.2 are
independently H; phosphate (including monophosphate, diphosphate,
triphosphate, or a stabilized phosphate prodrug); acyl (including
lower acyl); alkyl (including lower alkyl); sulfonate ester
including alkyl or arylalkyl sulfonyl including methanesulfonyl and
benzyl, wherein the phenyl group is optionally substituted with one
or more substituents as described in the definition of aryl given
herein; a lipid, including a phospholipid; an amino acid; a
carbohydrate; a peptide; cholesterol; or other pharmaceutically
acceptable leaving group which when administered in vivo is capable
of providing a compound wherein R.sup.1 is independently H or
phosphate; R.sup.6 is an alkyl, halogeno-alkyl (i.e. CF.sub.3),
alkenyl, or alkynyl (i.e. allyl); and X is O, S, SO.sub.2 or
CH.sub.2 can be prepared by the following general method.
Modification from the Pentodialdo-Furanose
[0431] The key starting material for this process is an
appropriately substituted pentodialdo-furanose. The
pentodialdo-furanose can be purchased or can be prepared by any
known means including standard epimerization, substitution and
cyclization techniques.
[0432] In a preferred embodiment, the pentodialdo-furanose is
prepared from the appropriately substituted hexose. The hexose can
be purchased or can be prepared by any known means including
standard epimerization (e.g. via alkaline treatment), substitution
and coupling techniques. The hexose can be either in the furanose
form, or cyclized via any means known in the art, such as
methodology taught by Townsend Chemistry of Nucleosides and
Nucleotides, Plenum Press, 1994, preferably by selectively
protecting the hexose, to give the appropriate hexafuranose.
[0433] The 4'-hydroxymethylene of the hexafuranose then can be
oxidized with the appropriate oxidizing agent in a compatible
solvent at a suitable temperature to yield the 4'-aldo-modified
sugar. Possible oxidizing agents are Swern reagents, Jones reagent
(a mixture of chromic acid and sulfuric acid), Collins's reagent
(dipyridine Cr(VI) oxide, Corey's reagent (pyridinium
chlorochromate), pyridinium dichromate, acid dichromate, potassium
permanganate, MnO.sub.2, ruthenium tetroxide, phase transfer
catalysts such as chromic acid or permanganate supported on a
polymer, Cl.sub.2-pyridine, H.sub.2O.sub.2-ammonium molybdate,
NaBrO.sub.2--CAN, NaOCl in HOAc, copper chromite, copper oxide,
Raney nickel, palladium acetate, Meerwin-Pondorf-Verley reagent
(aluminum t-butoxide with another ketone) and N-bromosuccinimide,
though preferably using H.sub.3PO.sub.4, DMSO and DCC in a mixture
of benzene/pyridine at room temperature.
[0434] Then, the pentodialdo-furanose can be optionally protected
with a suitable protecting group, preferably with an acyl or silyl
group, by methods well known to those skilled in the art, as taught
by Greene et al. Protective Groups in Organic Synthesis, John Wiley
and Sons, Second Edition, 1991. In the presence of a base, such as
sodium hydroxide, the protected pentodialdo-furanose can then be
coupled with a suitable electrophilic alkyl, halogeno-alkyl (i.e.
CF.sub.3), alkenyl or alkynyl (i.e. allyl), to obtain the
4'-alkylated sugar. Alternatively, the protected
pentodialdo-furanose can be coupled with the corresponding
carbonyl, such as formaldehyde, in the presence of a base, such as
sodium hydroxide, with the appropriate polar solvent, such as
dioxane, at a suitable temperature, which can then be reduced with
an appropriate reducing agent to give the 4'-alkylated sugar. In
one embodiment, the reduction is carried out using PhOC(S)Cl, DMAP,
preferably in acetonitrile at room temperature, followed by
treatment of ACCN and TMSS refluxed in toluene.
[0435] The optionally activated sugar can then be coupled to the
BASE by methods well known to those skilled in the art, as taught
by Townsend Chemistry of Nucleosides and Nucleotides, Plenum Press,
1994. For example, an acylated sugar can be coupled to a silylated
base with a Lewis acid, such as tin tetrachloride, titanium
tetrachloride or trimethylsilyltriflate in the appropriate solvent
at a suitable temperature.
[0436] Subsequently, the nucleoside can be deprotected by methods
well known to those skilled in the art, as taught by Greene et al.
Protective Groups in Organic Synthesis, John Wiley and Sons, Second
Edition, 1991.
[0437] In a particular embodiment, the 4'-C-branched ribonucleoside
is desired. Alternatively, deoxyribo-nucleoside is desired. To
obtain these deoxyribo-nucleosides, a formed ribo-nucleoside can
optionally be protected by methods well known to those skilled in
the art, as taught by Greene et al. Protective Groups in Organic
Synthesis, John Wiley and Sons, Second Edition, 1991, and then the
2'-OH can be reduced with a suitable reducing agent. Optionally,
the 2'-hydroxyl can be activated to facilitate reduction; i.e. via
the Barton reduction.
[0438] In another embodiment of the invention, the L-enantiomers
are desired. Therefore, the L-enantiomers can be corresponding to
the compounds of the invention can be prepared following the same
foregoing general methods, beginning with the corresponding
L-pentodialdo-furanose as starting material.
E. General Synthesis of 2' and/or 3'-Prodrugs
[0439] The key starting material for this process is an
appropriately substituted 1', 2', 3' or 4'-branched .beta.-D or
.beta.-L nucleosides. The branched nucleoside can be purchased or
can be prepared by any known means including the techniques
disclosed herein. The branched nucleoside can be optionally
protected with a suitable protecting group, preferably with a silyl
group, by methods well known to those skilled in the art, as taught
by Greene et al. Protective Groups in Organic Synthesis, John Wiley
and Sons, Second Edition, 1991. The optionally protected branched
nucleoside can then be coupled with a suitable acyl doner, such as
an acyl chloride and/or an acyl anhydride with the appropriate
protic or aprotic solvent at a suitable temperature, to give the 2'
and/or 3' prodrug of 1', 2', 3' or 4'-branched .beta.-D or .beta.-L
nucleoside. (Synthetic Communications, 1978, 8(5), 327-333; J. Am.
Chem. Soc., 1999, 121(24), 5661-5664.) Alternatively, the
optionally protected branched nucleoside can then be coupled with a
suitable acyl, such as a carboxylic acid, such as alkanoic acid
and/or amino acid residue, optionally with a suitable coupling
agent, with the appropriate aprotic solvent at a suitable
temperature, to give the 2' and/or 3' prodrug of 1', 2', 3' or
4'-branched .beta.-D or .beta.-L nucleoside. Possible coupling
reagents are any reagents that promote coupling, including but are
not limiting to, Mitsunobu reagents (e.g. diisopropyl
azodicarboxylate and diethyl azodicarboxylate) with
triphenylphosphine or various carbodiimides. In one embodiment, for
a 3'-prodrug of a 2'-branched nucleoside, the nucleoside is
preferably not protected and is directly coupled to an alkanoic
acid or amino acid residue with an appropriate coupling reagent,
such as a carbodiimide.
[0440] For example, simple amino-alcohols can be esterified using
acid chlorides in refluxing acetonitrile-benzene mixture (See
Scheme 9 below: Synthetic Communications, 1978, 8(5), 327-333;
hereby incorporated by reference). Alternatively, esterification
can be achieved using an anhydride, as described in J. Am. Chem.
Soc., 1999, 121(24), 5661-5664, which is hereby incorportated by
reference. See FIGS. 2, 3 and 4. ##STR47##
[0441] The present invention is described by way of illustration,
in the following examples. It will be understood by one of ordinary
skill in the art that these examples are in no way limiting and
that variations of detail can be made without departing from the
spirit and scope of the present invention.
EXAMPLE 1
Preparation of 1'-C-Methylriboadenine via
6-amino-9-(1-deoxy-.beta.D-psicofuranosyl)purine
[0442] Melting points were determined on a Mel-temp II apparatus
and are uncorrected. NMR spectra were recorded on a Bruker 400 AMX
spectrometer at 400 MHz for .sup.1H NMR and 100 MHz for .sup.13C
NMR with TMS as internal standard. Chemical shifts (.delta.) are
reported in parts per million (ppm), and signals are reported as s
(singlet), d (doublet), t (triplet), q (quartet), m (multiplet), or
bs (broad singlet). IR spectra were measured on a Nicolet 510P
FT-IR spectrometer. Mass spectra were recorded on a Micromass
Autospec high-resolution mass spectrometer. TLC were performed on
Uniplates (silica gel) purchased from Analtech Co. Column
chromatography was performed using either silica gel-60 (220440
mesh) for flash chromatography or silica gel G (TLC grade, >440
mesh) for vacuum flash column chromatography. UV spectra were
obtained on a Beckman DU 650 spectrophotometer. Elemental analysis
was performed by Atlantic Microlab, Inc., Norcross, Ga., or
Galbraith Laboratories, Inc., Knoxyille, Tenn. HPLC was performed
with a Waters HPLC system (Millipore Corporation, Milford, Mass.)
equipped with a Model 600 controller, a Model 996 photodiode array
detector and a Model 717 plus autosampler. Millennium 2010 software
was used for system control, data acquisition and processing. A
chiralyser polarimetric detector, Perkin-Elmer Model 241MC
polarimeter (Wilton, Conn.), was used for the determination of
optical rotations.
[0443] The title compound can be prepared according to a published
procedure (J. Farkas, and F. Sorm, "Nucleic acid components and
their analogues. XCIV. Synthesis of
6-amino-9-(1-deoxy-.beta.-D-psicofuranosyl)purine", Collect. Czech.
Chem. Commun. 1967, 32, 2663-2667. J. Farkas", Collect. Czech.
Chem. Commun. 1966, 31, 1535) (Scheme 10). ##STR48##
[0444] In a similar manner, but using the appropriate sugar and
pyrimidine or purine bases, the following nucleosides of Formula I
are prepared. ##STR49## wherein R.sup.1, R.sup.2, R.sup.3, X.sup.1,
X.sup.2, and Y are defined in Table 1. Alternatively, the following
nucleosides of Formula IV are prepared, using the appropriate sugar
and pyrimidine or purine bases. ##STR50## wherein R.sup.1, R.sup.2,
R.sup.3, X.sup.1, Y are defined in Table 2. Alternatively, the
following nucleosides of Formula VII are prepared, using the
appropriate sugar and pyrimidine or purine bases. ##STR51## wherein
R.sup.1, R.sup.2, R.sup.3, R.sup.6, X, and Base are defined in
Table 3.
[0445] Alternatively, the following nucleosides of Formula VIII are
prepared, using the appropriate sugar and pyrimidine or purine
bases. ##STR52## wherein R.sup.1, R.sup.2, R.sup.6, X, and Base are
defined in Table 4. Alternatively, the following nucleosides of
Formula XXI are prepared, using the appropriate sugar and
pyrimidine or purine bases. ##STR53## wherein R.sup.1, R.sup.2,
R.sup.6, X and Base are defined in Table 5.
[0446] Alternatively, the following nucleosides of Formula XIII are
prepared, using the appropriate sugar and pyrimidine or purine
bases. ##STR54## wherein R.sup.1, R.sup.6, R.sup.7, R.sup.8, X,
Base, R.sup.10 and R.sup.9 are defined in Table 6.
EXAMPLE 2
Preparation of 2'-C-methylriboadenine
[0447] The title compound was prepared according to a published
procedure (R. E. Harry-O'kuru, J. M. Smith, and M. S. Wolfe, "A
short, flexible route toward 2'-C-branched ribonucleosides", J.
Org. Chem. 1997, 62, 1754-1759) (Scheme 11). ##STR55##
[0448] The 3'-prodrug of the 2'-branched nucleoside was prepared
according to published procedure (Synthetic Communications, 1978,
8(5), 327-333; J. Am. Chem. Soc., 1999, 121(24), 5661-5664).
Alternatively, the 2'-branched nucleoside can be esterified without
protection (Scheme 11b). Carbonyldiimidazole (377 mg, 2.33 mmol)
was added to a solution of N-(tert-butoxycarbonyl)-L-valine (507
mg, 2.33 mmol) in 15 mL of anhydrous tetrahydrofuran. The mixture
was stirred at 20.degree. C. for one hour and at 50.degree. C. for
10 minutes and then added to a solution of
4-Amino-1-(3,4-dihydroxy-5-hydroxymethyl-3-methyl-tetrahydro-furan-2-y-
l)-1H-pyrimidine-2-one (500 mg, 1.95 mmol),
4-(dimethylamino)pyridine (25 mg, 0.195 mmol), triethylamine (5 mL)
in anhydrous N,N-dimethylformamide (10 mL), which is also stirring
at 50.degree. C. The reaction mixture was stirred at 50.degree. C.
for one hour and then examined by HPLC*. HPLC analysis indicated
the formation of 52% of the desired ester, 17% of starting material
in addition to undesired by-products. The 3'-OH of
4-amino-1-(3,4-dihydroxy-5-hydroxymethyl-3-methyl-tetrahydro-furan-2-yl)--
1H-pyrimidine-2-one tends to react selectively when coupled with
BOC-Val. ##STR56##
[0449] The product was analyzed by HPLC using a reverse phase
column: Waters part # WAT086344; Nova-Pak C18, 60 .ANG. pore size,
4 .mu.m particle size, 3.9.times.150 mm. Chromatograms were
generated using a Waters 2695 HPLC and 996 PDA detector. Mobile
Phase: HPLC grade acetonitrile and water were bought from JT Baker
and 1M solution of triethylammonium acetate from Fluka.
Flow rate: 1.00 mL/min. of an acetonitrile/20 mM aqueous
triethylammonium acetate buffer gradient as described below.
System is equilibrated for five minutes between runs.
[0450] Wave length: 272 nm. TABLE-US-00004 TABLE D Column
Specifications Time % Acetonitrile % Buffer 0.00 0.00 100.0 15.00
80.0 20.0 30.00 80.0 20.0
[0451] TABLE-US-00005 TABLE E Description of compounds vs.
retention times: Compound RETENTION TIME (IN MINUTES) Desired ester
8.3 DMAP 3.7 (Broad Peak) Starting material 2.7
[0452] In a similar manner, but using the appropriate sugar and
pyrimidine or purine bases, the following nucleosides of Formula II
are prepared. ##STR57## wherein R.sup.2, R.sup.3, X.sup.1, X.sup.2,
and Y are defined in Table 7.
[0453] Alternatively, the following nucleosides of Formula V are
prepared, using the appropriate sugar and pyrimidine or purine
bases. ##STR58## wherein R.sup.1, R.sup.2, R.sup.3, X.sup.1 and Y
are defined in Table 8.
[0454] Alternatively, the following nucleosides of Formula IX are
prepared, using the appropriate sugar and pyrimidine or purine
bases. ##STR59## wherein R.sup.1, R.sup.2, R.sup.3, R.sup.6, X, and
Base are defined in Table 9.
[0455] Alternatively, the following nucleosides of Formula X are
prepared, using the appropriate sugar and pyrimidine or purine
bases. ##STR60## wherein R.sup.1, R.sup.2, R.sup.7, R.sup.6, X, and
Base are defined in Table 10.
[0456] Alternatively, the following nucleosides of Formula XXII are
prepared, using the appropriate sugar and pyrimidine or purine
bases. ##STR61## wherein R.sup.1, R.sup.2, R.sup.6, X, and Base are
defined in Table 11.
[0457] Alternatively, the following nucleosides of Formula XIII are
prepared, using the appropriate sugar and pyrimidine or purine
bases. ##STR62## wherein R.sup.1, R.sup.6, R.sup.7, X, Base,
R.sup.9 and R.sup.10 are defined in Table 12.
EXAMPLE 3
Preparation of 3'-C-methylriboadenine
[0458] The title compound can be prepared according to a published
procedure (R. F. Nutt, M. J. Dickinson, F. W. Holly, and E. Walton,
"Branched-chain sugar nucleosides. III. 3'-C-methyladenine", J.
Org. Chem. 1968, 33, 1789-1795) (Scheme 12). ##STR63##
[0459] In a similar manner, but using the appropriate sugar and
pyrimidine or purine bases, the following nucleosides of Formula
III are prepared. ##STR64## wherein R.sup.1, R.sup.2, R.sup.3,
X.sup.1, X.sup.2, and Y are defined in Table 13.
[0460] Alternatively, the following nucleosides of Formula VI are
prepared, using the appropriate sugar and pyrimidine or purine
bases. ##STR65## wherein R.sup.1, R.sup.2, R.sup.3, X.sup.1, and Y
are defined in Table 14.
[0461] Alternatively, the following nucleosides of Formula XI are
prepared, using the appropriate sugar and pyrimidine or purine
bases. ##STR66## wherein R.sup.1, R.sup.2, R.sup.3, R.sup.6, X, and
Base are defined in Table 15.
[0462] Alternatively, the following nucleosides of Formula XII are
prepared, using the appropriate sugar and pyrimidine or purine
bases. ##STR67## wherein R.sup.1, R.sup.2, R.sup.6, X and Base are
defined in Table 16.
[0463] Alternatively, the following nucleosides of Formula XXIII
are prepared, using the appropriate sugar and pyrimidine or purine
bases. ##STR68## wherein R.sup.1, R.sup.2, R.sup.6, X and Base are
defined in Table 17.
[0464] Alternatively, the following nucleosides of Formula XV are
prepared, using the appropriate sugar and pyrimidine or purine
bases. ##STR69## wherein R.sup.1, R.sup.6, R.sup.7, X, Base,
R.sup.8 and R.sup.9 are defined in Table 18.
EXAMPLE 4
Preparation of
1-O-Methyl-2,3-O-Isopropylidene-.beta.-D-Ribofuranose (AA)
[0465] The title compound can be prepared according to a published
procedure (Leonard, N. J.; Carraway, K. L. "5-Amino-5-deoxyribose
derivatives. Synthesis and use in the preparation of "reversed"
nucleosides" J. Heterocycl. Chem. 1966, 3, 485-489).
[0466] A solution of 50.0 g (0.34 mole) of dry D-ribose in 1.0 L of
acetone, 100 mL of 2,2-dimethoxypropane, 200 mL of methanol
containing 20 mL of methanol saturated with hydrogen chloride at
0.degree. C. was stirred overnight at room temperature. The
resulting solution was neutralized with pyridine and evaporated
under reduced pressure. The resulting oil was partitioned between
400 mL of water and 400 mL of methylene chloride. The water layer
was extracted twice with methylene chloride (400 mL). The combined
organic extracts were dried over sodium sulfate and evaporated
under reduced pressure. The residue was purified by silica gel
column chromatography [eluent: stepwise gradient of methanol (1-2%)
in methylene chloride] to give pure AA (52.1 g, 75%) as a yellow
syrup. .sup.1H-NMR (CDCl.sub.3): .delta. 5.00 (s, 1H, H-1), 4.86
(d, 1H, H-2, J.sub.2-3=5.9 Hz), 4.61 (d, 1H, H-3, J.sub.3-2=5.9
Hz), 4.46 (t, 1H, H-4, J.sub.4-5=2.7 Hz), 3.77-3.61 (m, 2H, H-5 and
H-5'), 3.46 (s, 1H, OCH.sub.3), 3.0-2.4 (br s, 1H, OH-5), 1.51 (s,
3H C.sub.3), 1.34 (s, 3H C.sub.3); MS (matrix GT): FAB>0 m/z 173
(M-OCH3).sup.+.
EXAMPLE 5
Preparation of
1-O-Methyl-2,3-O-Isopropylidene-.beta.-D-Pentodialdo-Ribofuranose
(BB)
[0467] The title compound can be prepared according to a published
procedure (Jones, G. H.; Moffatt, J. G. Oxidation of carbohydrates
by the sulfoxide-carbodiimide and related methods. Oxidation with
dicyclohexylcarbodiimide-DMSO, diisopropylcarbodiimide-DMSO, acetic
anhydride-DMSO, and phosphorus pentoxide-DMSO: in Methods in
Carbohydrate Chemistry; Whisler, R. L. and Moffatt, J. L. Eds;
Academic Press: New York, 1972; 315-322).
[0468] Compound AA was co-evaporated twice with anhydrous pyridine.
Dicyclohexylcarbodi-imide (DCC, 137.8 g, 0.67 mol) was added to a
solution of AA (68.2 g, 0.33 mole) in anhydrous benzene (670 mL),
DMSO (500 mL) and pyridine (13.4 mL). To the resulting solution,
cooled to 0.degree. C., was added a solution of anhydrous
crystalline orthophosphoric acid (16.4 g, 0.167 mmol) in anhydrous
DMSO (30 mL). The mixture was stirred for 1.5 hours at 0.degree. C.
and 18 hours at room temperature under argon atmosphere, diluted
with ethyl acetate (1000 mL). A solution of oxalic acid dihydrate
(63.1 g, 038 mol) in DMSO (30 mL) was added and the reaction
mixture was stirred at room temperature during 1 hour and then
filtered to eliminate precipitated dicyclohexylurea (DCU). The
filtrate was concentrated to a volume of about 600 mL under reduced
pressure and neutralized with a saturated aqueous sodium hydrogen
carbonate solution (400 mL). Brine (200 mL) was added and the
organic layer was extracted with ethyl acetate (4.times.1000 mL).
The combined organic layers were concentrated to a volume of about
2000 mL, washed with a saturated aqueous sodium hydrogen carbonate
solution (2.times.700 mL), and with brine (2.times.700 mL) before
being dried over sodium sulfate and evaporated under reduced
pressure. A small fraction of the crude residue was purified on
silica gel chromatography [eluent: chloroform/ethyl ether, 8:2] in
order to confirm the structure of BB which was obtained as a pale
yellow solid. .sup.1H-NMR (CDCl.sub.3): .delta. 9.61 (s, 1H, H-5),
5.12 (s, 1H, H-1), 5.08 (d, 1H, H-2, J.sub.2-3=5.9 Hz), 4.53 (d,
1H, H-3, J.sub.3-2=6.0 Hz), 4.51 (s, 1H, H-4), 3.48 (s, 1H,
OCH.sub.3), 1.56 (s, 3H C.sub.3), 1.36 (s, 3H CH.sub.3); MS (matrix
GT): FAB>0 m/z 203 (M+H).sup.+, 171 (M-OCH.sub.3).sup.+.
EXAMPLE 6
Preparation of
4-C-Hydroxymethyl-1-O-methyl-2,3-O-isopropylidene-.beta.-D-Ribofuranose
(CC)
[0469] The title compound can be prepared according to a published
procedure (Leland, D. L.; Kotick, M. P. Carbohydr. Res. 1974, 38,
C9-C11; Jones, G. H.; Taniguchi, M., et al. J. Org. Chem. 1979, 44,
1309-1317; Gunic, E.; Girardet, J.-L.; et al. Bioorg. Med. Chem.
2001, 9, 163-170).
[0470] To a solution of the crude material (BB) obtained above and
37% aqueous formaldehyde (167 mL) in dioxane (830 mL) was added
aqueous sodium hydroxyde (2N, 300 mL). The mixture was stirred at
room temperature for 4 hours and neutralized by addition of Dowex
50 W X 2 (H.sup.+ form). The resin was filtered, washed with
methanol, and the combined filtrates were concentrated to dryness
and coevaporated several times with absolute ethanol. Sodium
formate which was precipitated from absolute ethanol was removed by
filtration, the filtrate was concentrated to dryness and the
residue was purified by silica gel column chromatography [eluent:
stepwise gradient of methanol (04%) in chloroform] to give pure CC
(42.2 g, 54% from AA), which was recrystallized from cyclohexane.
Mp=94-95 (dec.) (lit. 94-96.5; 97-98: Refs: 3,4), .sup.1H-NMR
(DMSO-d.sub.6): .delta. 4.65 (s, 1H, H-1), 4.44-4.37 (m, 3H, H-2,
H-3 and OH-6), 4.27 (t, 1H, OH-5, J=5.6 Hz, J=6.0 Hz), 3.42-3.34
(m, 2H, H-5 and H-6) 3.29 (dd, 1H, H-5', J.sub.5'-OH=5.4 Hz,
J5-5'=11.4 Hz), 3.11 (dd, 1H, H-6', J.sub.6'-OH=5.7 Hz, J6-6'=10.9
Hz), 3.03 (s, 3H, OCH.sub.3), 1.48 (s, 3H C.sub.3), 1.05 (s, 3H
C.sub.3); MS (matrix GT): FAB>0 m/z 469 (2M+H).sup.+, 235
(M+H).sup.+, 203 (M-OCH.sub.3)+ FAB<0 m/z 233 (M-H).sup.-.
EXAMPLE 7
Preparation of
6-O-Monomethoxytrityl-4-C-hydroxymethyl-1-O-methyl-2,3-O-isopropylidene-.-
beta.-D-ribofuranose (DD)
[0471] The title compound can be prepared according to a published
procedure (Gunic, E.; Girardet, J.-L.; et al. Bioorg. Med. Chem.
2001, 9, 163-170).
[0472] To a solution of CC (41.0 g, 175 mmol) in pyridine (700 ml)
was added by portions dimethoxytrityl chloride (60.5 g, 178 mmol)
at 4.degree. C. The reaction mixture was stirred for 3 hours at
room temperature. After addition of methanol, the reaction mixture
was concentrated (200 ml) and then dissolved with ethyl acetate (2
L). The organic layer was washed with a 5% aqueous sodium hydrogen
carbonate solution, with water and dried over sodium sulfate and
then evaporated to dryness. Purification by silica gel column
chromatography [eluent: ethyl acetate/hexane 15/85] afforded pure
DD (63.0 g, 68%) as a syrup. .sup.1H-NMR (CDCl.sub.3): .delta.
7.5-6.9 (m, 13H, MMTr), 4.89 (s, 1H, H-1), 4.72-4.62 (m, 3H, H-2,
H-3 and OH-5), 3.82 (dd, 1H, H-5, J.sub.5-OH=5.5 Hz, J5-5'=10.5
Hz), 3.79 (s, 6H, OCH3), 3.54 (dd, 1H, H-5', J.sub.5'-OH=4.9 Hz,
J.sub.5'-5=10.5 Hz), 3.31 (s, 3H, OCH.sub.3), 3.24 (d, 1H, H-6,
J.sub.6-6'=9.2 Hz), 3.13 (d, 1H, H-6', J.sub.6'-6=9.2 Hz), 1.24 (s,
3H C.sub.3), 1.15 (s, 3H C.sub.3); MS (matrix GT): FAB>0 m/z 303
(DMTr).sup.+.
EXAMPLE 8
Preparation of
5-O-Benzoyl-4-C-hydroxymethyl-1-O-methyl-2,3-O-isopropylidene-.beta.-D-ri-
bo-furanose (EE)
[0473] The title compound can be prepared according to a published
procedure (Gunic, E.; Girardet, J.-L.; Pietrzkowski, Z.; Esler, C.;
Wang, G. "Synthesis and cytotoxicity of 4'-C- and 5'-C-substituted
Toyocamycins" Bioorg. Med. Chem. 2001, 9, 163-170).
[0474] To a solution of DD (2.51 g, 4.68 mmol) in anhydrous
pyridine (37 mL) was added under argon benzoyl chloride (1.09 mL,
9.36 mmol) and the reaction mixture was stirred for 13 hours at to
room temperature. Then the reaction was cooled to 0.degree. C. and
stopped with ice-cold water (100 mL). The water layer was extracted
with methylene chloride (30.quadrature. 200 mL). The combined
organic layers were washed with a saturated aqueous sodium hydrogen
carbonate solution (2.times.150 mL), with water (1.times.150 mL)
and then dried over sodium sulfate and evaporated under reduced
pressure. The residue was dissolved in 80% acetic acid (70.2 mL)
and the mixture was stirred at room temperature for 3 hr and
concentrated to dryness. Purification by silica gel column
chromatography [eluent: chloroform] afforded pure EE (1.40 g, 88%)
as a syrup. .sup.1H-NMR (CDCl.sub.3): .delta. 8.1-7.4 (m, 5H,
C.sub.6H.sub.5CO), 5.08 (s, 1H, H-1), 4.77 (dd, 2H, H-2 and H-3,
J=6.1 Hz, J=8.2 Hz), 4.51 (q, 2H, H-5 and H-5', J=11.5 Hz,
J.sub.5-5'=23.8 Hz), 3.91 (t, 2H, H-6 and H-6', J=12.3 Hz), 4.38
(s, 1H, OCH.sub.3), 2.2-1.8 (brs, 1H, OH-6), 1.57 (s, 3H C.sub.3),
1.38 (s, 3H C.sub.3); MS (matrix GT): FAB>0 m/z 677
(2M+H).sup.+, 339 (M+H).sup.+, 307 (M-OCH.sub.3).sup.+, 105
(C.sub.6H.sub.5CO).sup.+ FAB<0 m/z 121
(C.sub.6H.sub.5CO.sub.2).sup.-.
EXAMPLE 9
Preparation of
5-O-Benzoyl-4-C-methyl-1-O-methyl-2,3-O-isopropylidene-.beta.-D-ribofuran-
ose (FF)
[0475] The title compound can be prepared according to a published
procedure (Gunic, E.; Girardet, J.-L.; et al. Bioorg. Med. Chem.
2001, 9, 163-170).
[0476] A solution of EE (37.6 g, 0.111 mol),
4-dimethylaminopyridine (DMAP, 40.7 g, 0.333 mol) and
phenoxythiocarbonyle chloride in anhydrous acetonitrile (1000 mL)
was stirred at room temperature for 1 hour and concentrated to
dryness. The residue was dissolved in methylene chloride (500 mL)
and successively washed with 0.2 M hydrochloric acid (2.times.500
mL) and water (500 mL) before being dried over sodium sulfate,
evaporated under reduced pressure and coevaporated several times
with anhydrous toluene. The crude material was dissolved in
anhydrous toluene (880 mL) and tris(trimethylsilyl)silane (TMSS,
42.9 mL, 0.139 mol), and 1,1'-azobis(cyclohexanecarbonitrile)
(ACCN, 6.8 g, 27.8 mmol) were added. The reaction mixture was
stirred under reflux for 45 minutes, cooled to room temperature and
concentrated under reduced pressure. The resulting residue was
purified by silica gel column chromatography [eluent: stepwise
gradient of diethyl ether (5-20%) in petroleum ether] to give pure
FF (26.4 g, 74%) as a pale yellow syrup. .sup.1H-NMR
(DMSO-d.sub.6): .delta. 8.0-7.5 (m, 5H, C.sub.6H.sub.5CO), 4.85 (s,
1H, H-1), 4.63 (dd, 2H, H-2 and H-3, J=6.1 Hz, J=11.6 Hz), 4.24 (d,
1H, H-5, J.sub.5-5'=11.1 Hz), 4.10 (d, 1H, H-5', J.sub.5'-5=11.1
Hz), 3.17 (s, 1H, OCH.sub.3), 1.38 (s, 3H C.sub.3), 1.30 (s, 3H
C.sub.3), 1.25 (s, 3H C.sub.3); MS (matrix GT): FAB>0 m/z 291
(M-OCH.sub.3).sup.+, 105 (C.sub.6H.sub.5CO).sup.+ FAB<0 m/z 121
(C.sub.6H.sub.5CO.sub.2).sup.-.
EXAMPLE 10
Preparation of
5-O-Benzoyl-4-C-Methyl-1,2,3-O-acetyl-.alpha.,.beta.-D-ribofuranose
(GG)
[0477] Compound FF (22.5 g, 70 mmol) was suspended in a 80% aqueous
acetic acid solution (250 mL). The solution was heated at
100.degree. C. for 3 hours. The volume was then reduced by half and
coevaporated with absolute ethanol and pyridine. The oily residue
was dissolved in pyridine (280 mL) and then cooled at 0.degree. C.
Acetic anhydride (80 mL) and 4-dimethylamino-pyridine (500 mg) were
added. The reaction mixture was stirred at room temperature for 3
hours and then concentrated under reduced pressure. The residue was
dissolved with ethyl acetate (1 L) and successively washed with a
saturated aqueous sodium hydrogen carbonate solution, a 1 M
hydrochloric acid and water. The organic layer was dried over
sodium sulfate and evaporated under reduced pressure. The resulting
residue was purified by silica gel column chromatography [eluent:
stepwise gradient of diethyl ether (30-40%) in petroleum ether] to
give pure GG (16.2 g, 60%) as a pale yellow syrup. A small fraction
of the material was re-purified on silica gel chromatography [same
eluent: system] in order separate the .alpha. and the .beta.
anomers.
[0478] .alpha. anomer: .sup.1H-NMR (DMSO-d.sub.6): .delta. 8.1-7.5
(m, 5H, C.sub.6H.sub.5CO), 6.34 (pt, 1H, H-1, J=2.4 Hz, J=2,1 Hz),
5.49 (m, 2H, H-2 and H-3), 4.33 (q, 2H, H-5 and H-5', J=11.6 Hz,
J=18.7 Hz), 2.15 (s, 3H, CH.sub.3CO.sub.2), 2.11 (s, 3H,
CH.sub.3CO.sub.2), 2.07 (s, 3H, CH.sub.3CO.sub.2), 1.37 (s, 3H,
CH.sub.3); MS (matrix GT): FAB>0 m/z 335
(M-CH.sub.3CO.sub.2.sup.-).sup.+, 275
(M-CH.sub.3CO.sub.2.sup.-+H).sup.+, 105 (C.sub.6H.sub.5CO).sup.+,
43 (CH.sub.3CO).sup.+ FAB<0 m/z 121
(C.sub.6H.sub.5CO.sub.2).sup.-, 59 (CH.sub.3CO.sub.2).sup.-.
[0479] .beta. anomer: .sup.1H-NMR (DMSO-d.sub.6): .delta. 8.1-7.5
(m, 5H, C.sub.6H.sub.5CO), 5.99 (s, 1H, H-1), 5.46 (d, 1H, H-2,
J.sub.2-3=5.3 HZ), 5.30 (d, 1H, H-2, J.sub.2-3=5.3 Hz), 4.39 (d,
1H, H-5, J.sub.5-5'=11.7 Hz), 4.19 (d, 1H, H-5', J.sub.5'-5=11.7
Hz), 2.10 (s, 3H, CH.sub.3CO.sub.2), 2.06 (s, 3H,
CH.sub.3CO.sub.2), 2.02 (s, 3H, CH.sub.3CO.sub.2), 1.30 (s, 3H,
CH.sub.3); MS (matrix GT): FAB>0 m/z 335
(M-CH.sub.3CO.sub.2.sup.-).sup.+, 275
(M-CH.sub.3CO.sub.2.sup.-+H).sup.+, 105 (C.sub.6H.sub.5CO).sup.+,
43 (CH.sub.3CO).sup.+ FAB<0 m/z 121
(C.sub.6H.sub.5CO.sub.2).sup.-, 59 (CH.sub.3CO.sub.2).sup.-.
EXAMPLE 11
Preparation of
1-(5-O-Benzoyl-4-C-methyl-2,3-O-acetyl-.beta.-D-ribofuranosyl)uracil
(HH)
[0480] A suspension of uracil (422 mg, 3.76 mmol) was treated with
hexamethyldisilazane (HMDS, 21 mL) and a catalytic amount of
ammonium sulfate during 17 hours under reflux. After cooling to
room temperature, the mixture was evaporated under reduced
pressure, and the residue, obtained as a colorless oil, was diluted
with anhydrous 1,2-dichloroethane (7.5 mL). To the resulting
solution was added GG (0.99 g, 2.51 mmol) in anhydrous
1,2-dichloroethane (14 mL), followed by addition of trimethylsilyl
trifluoromethanesulfonate (TMSTf, 0.97 mL, 5.02 mmol). The solution
was stirred for 2.5 hours at room temperature under argon
atmosphere, then diluted with chloroform (150 mL), washed with the
same volume of a saturated aqueous sodium hydrogen carbonate
solution and finally with water (2.times.100 mL). The organic phase
was dried over sodium sulfate, then evaporated under reduced
pressure. The resulting crude material was purified by silica gel
column chromatography [eluent: stepwise gradient of methanol (0-2%)
in chloroform] to afford pure HH (1.07 g, 95%) as a foam.
.sup.1H-NMR (DMSO-d6): .delta. 11.48 (s, 1H, NH), 8.1-7.5 (m, 6H,
C.sub.6H.sub.5CO and H-6), 5.94 (d, 1H, H-1', J.sub.1'-2'=3.3 Hz),
5.61 (m, 3H, H-5, H-2' and H-3'), 4.47 (d, 1H, H-5',
J.sub.5'-5''=11.7 Hz), 4.35 (d, 1H, H-5'', J.sub.5''-5'=11.7 Hz),
2.12 (s, 3H, CH.sub.3CO.sub.2), 2.09 (s, 3H, CH.sub.3CO.sub.2),
1.38 (s, 3H, CH.sub.3); MS (matrix GT): FAB>0 m/z 893
(2M+H).sup.+, 447 (M+H).sup.+, 335 (S).sup.+, 113 (BH.sub.2).sup.+,
105 (C.sub.6H.sub.5CO).sup.+, 43 (CH.sub.3CO).sup.+ FAB<0 m/z
891 (2M-H).sup.-, 445 (M-H).sup.-, 121 (C.sub.6HsCO.sub.2).sup.-,
111 (B).sup.-, 59 (CH.sub.3CO.sub.2).sup.-.
EXAMPLE 12
Preparation of 1-(4-C-methyl-.beta.-D-ribofuranosyl)uracil (II)
[0481] The title compound can be prepared according to a published
procedure from HH (Waga, T.; Nishizaki, T.; et al. Biosci.
Biotechnol. Biochem. 1993, 57, 1433-1438).
[0482] A solution of HH (610 mg, 1.37 mmol) in methanolic ammonia
(previously saturated at -10.degree. C.) (27 mL) was stirred at
room temperature overnight. The solvent was evaporated under
reduced pressure and the residue was partitioned between methylene
chloride (40 mL) and water (40 mL). The aqueous layer was washed
with methylene chloride (2.times.40 mL), concentrated under reduced
pressure and coevaporated several times with absolute ethanol.
Recrystallization from a mixture absolute ethanol/methanol gave II
(215 mg, 61%) as a colorless and crystalline solid. Mp: 226-227
(dec.) (lit. 227: Ref. 6); UV (H.sub.2O): .lamda..sub.max=259 nm
(.epsilon.=10100), .lamda..sub.min=228 nm (.epsilon.=2200); HPLC
99.56%, .sup.1H-NMR (DMSO-d.sub.6): .delta. 11.28 (s, 1H, NH), 7.89
(d, 1H, H-6, J.sub.6-5=8.1 Hz), 5.80 (d, 1H, H-1', J.sub.1'-2'=7.1
Hz), 5.64 (d, 1H, H-5, J.sub.5-6=8.1 Hz), 5.24 (d, 1H, OH-2',
J.sub.OH-2'=6.5 Hz), 5.18 (t, 1H, OH-5' J.sub.OH-5'=J.sub.OH-5'=5.2
Hz), 5.01 (d, 1H, OH-3', J.sub.OH-3'=5.0 Hz), 4.28 (dd, 1H, H-2',
J=6.5 Hz, J=12.2 Hz), 3.90 (t, 1H, H-3',
J.sub.3'-2'=J.sub.3'-OH'=5.1 Hz), 3.30 (m, 2H, H-5' and H-5''),
1.06 (s, 3H, CH.sub.3); MS (matrix GT): FAB>0 m/z 517
(2M+H).sup.+, 259 (M+H).sup.+, 147 (S).sup.+ FAB<0 m/z 515
(2M-H).sup.-, 257 (M-H).sup.-.
EXAMPLE 13
Preparation of
1-(5-O-Benzoyl-4-C-methyl-2,3-O-acetyl-.beta.-D-ribofuranosyl)4-thio-urac-
il (JJ)
[0483] Lawesson's reagent (926 mg, 2.29 mmol) was added under argon
to a solution of HH (1.46 g, 3.27 mmol) in anhydrous
1,2-dichloroethane (65 mL) and the reaction mixture was stirred
overnight under reflux. The solvent was evaporated under reduced
pressure and the residue was purified by silica gel column
chromatography [eluent: stepwise gradient of methanol (1-2%) in
chloroform] to give pure JJ (1.43 g, 95%) as a yellow foam.
.sup.1H-NMR (DMSO-d.sub.6): .delta. 12.88 (s, 1H, NH), 8.1-7.5 (m,
6H, C.sub.6H.sub.5CO and H-6), 6.27 (d, 1H, H-1', J.sub.1'-2'=7.51
Hz), 5.91 (br s, 1H, H-5) 5.64 (m, 2H, H-2' and H-3'), 4.47 (d, 1H,
H-5', J.sub.5'-5''=11.7 Hz), 4.36 (d, 1H, H-5', J.sub.5'-5''=11.7
Hz), 2.11 (s, 3H, CH.sub.3CO.sub.2), 2.09 (s, 3H,
CH.sub.3CO.sub.2), 1.39 (s, 3H, CH.sub.3); MS (matrix GT): FAB>0
m/z 925 (2M+H).sup.+, 463 (M+H).sup.+, 335 (S).sup.+, 129
(BH.sub.2).sup.+, 105 (C.sub.6H.sub.5CO).sup.+, 43
(CH.sub.3CO).sup.+ FAB<0 m/z 461 (M-H).sup.-, 127 (B).sup.-, 121
(C.sub.6H.sub.5CO.sub.2).sup.-, 59 (CH.sub.3CO.sub.2).sup.-.
EXAMPLE 14
Preparation of 1-(4-C-methyl-.beta.-D-ribofuranosyl)4-thio-uracil
(KK)
[0484] A solution of JJ (500 mg, 1.08 mmol) in methanolic ammonia
(previously saturated at -10.degree. C.) (27 mL) was stirred at
room temperature overnight. The solvent was evaporated under
reduced pressure and the residue was partitioned between methylene
chloride (40 ml) and water (40 mL). The aqueous layer was washed
with methylene chloride (2.times.40 mL), concentrated under reduced
pressure. The crude material was purified by silica gel column
chromatography [eluent: stepwise gradient of methanol (5-7%) in
methylene chloride] to give pure KK (188 mg, 63%), which was
lyophilized. Mp: 65-70 (dec.); UV (methanol): .lamda..sub.max=330
mm (.epsilon.=20000) 246 nm (.epsilon.=4200), .lamda..sub.min=275
nm (.epsilon.=1500); .sup.1H-NMR (DMSO-d.sub.6): .delta. 12.51
(brs, 1H, NH), 7.81 (d, 1H, H-6, J.sub.6-5=7.6 Hz), 6.30 (d, 1H,
H-5, J.sub.5-6=7.5 Hz), 5.77, (d, 1H, H-1', J.sub.1'-2'=6.7 Hz),
5.32 (d, 1H, OH-2', J.sub.OH-2'=6.1 Hz), 5.20 (t, 1H, OH-5'
J.sub.OH-5'=J.sub.OH-5''=5.2 Hz), 5.03 (d, 1H, OH-3',
J.sub.OH-3'=5.2 Hz), 4.17 (dd, 1H, H-2', J=6.2 Hz, J=12.0 Hz), 3.89
(t, 1H, H-3', J.sub.3'-2'=J.sub.3'-OH'=5.1 Hz), 3.35 (m, 2H, H-5'
and H-5''), 1.02 (s, 3H, CH.sub.3); MS (matrix GT): FAB>0 m/z
275 (M+H).sup.+, 147 (S).sup.+, 129(BH.sub.2).sup.+ FAB<0 m/z
547 (2M-H).sup.-, 273 (M-H).sup.-, 127 (B).sup.-.
EXAMPLE 15
Preparation of 1-(4-C-methyl-.beta.-D-ribofuranosyl)cytosine,
hydrochloric form (LL)
[0485] Compound KK (890 mg, 1.93 mmol) was treated with methanolic
ammonia (previously saturated at -10.degree. C.), (12 mL) at
100.degree. C. in a stainless-steel bomb for 3 hours, then cooled
to room temperature. The solvent was evaporated under reduced
pressure and the residue was partitioned between methylene chloride
(40 mL) and water (40 mL). The aqueous layer was washed with
methylene chloride (2.times.40 mL), concentrated under reduced
pressure. The crude material was purified by silica gel column
chromatography [eluent: methylene chloride/methanol/ammonium
hydroxide 65:30:5]. The collected fractions were evaporated under
reduced pressure and in absolute ethanol (6.3 mL). To the solution
was added a 2N hydrochloric acid solution (1.5 mL) and the mixture
was stirred before being concentrated under reduced pressure. The
procedure was repeated twice and LL was precipitated from absolute
ethanol. Mp: 213-214 (dec.); UV (methanol): .lamda..sub.max=280 nm
(.epsilon.=9800), .lamda..sub.min=245 nm (.epsilon.=3600);
.sup.1H-NMR (DMSO-d.sub.6): .delta. 9.82 (s, 1H, NH.sub.2), 8.72
(s, 1H, NH.sub.2), 8.34 (d, 1H, H-6, J.sub.6-5=7.8 Hz), 6.21 (d,
1H, H-5, J.sub.5-6=7.8 Hz), 5.83 (d, 1H, H-1', J.sub.1'-2'=5.8 Hz),
4.22 (d, 1H, OH-2', J.sub.OH-2'=6.5 Hz), 5.6-4.7 (m, 3H, OH-2',
OH-3' and OH-5'), 4.28 (t, 1H, H-2', J=5.6 Hz), 3.99 (d, 1H, H-3',
J=5.3 Hz), 3.43 (m, 2H, H-5' and H-5''), 1.14 (s, 3H, CH.sub.3); MS
(matrix GT): FAB>0 m/z 515 (2M+H).sup.+, 258 (M+H).sup.+, 147
(S).sup.+, 112 (BH.sub.2).sup.+ FAB<0 m/z 256 (M-H).sup.-.
EXAMPLE 16
Preparation of
1-(5-O-Benzoyl-4-C-methyl-2,3-O-acetyl-.beta.-D-ribofuranosyl)thymine
(MM)
[0486] A suspension of thymine (384 mg, 3.04 mmol) was treated with
hexamethyldisilazane (HMDS, 17 mL) and a catalytic amount of
ammonium sulfate overnight under reflux. After cooling to room
temperature, the mixture was evaporated under reduced pressure, and
the residue, obtained as a colorless oil, was diluted with
anhydrous 1,2-dichloroethane (6 mL). To the resulting solution was
added GG (1.0 g, 2.53 mmol) in anhydrous 1,2-dichloroethane (14
mL), followed by addition of trimethylsilyl
trifluoromethanesulfonate (TMSTf, 0.98 mL, 5.06 mmol). The solution
was stirred for 5 hours at room temperature under argon atmosphere,
then diluted with chloroform (150 mL), washed with the same volume
of a saturated aqueous sodium hydrogen carbonate solution and
finally with water (2.times.100 mL). The organic phase was dried
over sodium sulfate, then evaporated under reduced pressure. The
resulting crude material was purified by silica gel column
chromatography [eluent: 2% of methanol in chloroform] to afford
pure MM (1.09 g, 94%) as a foam. .sup.1H-NMR (DMSO-d.sub.6):
.delta. 11.47 (s, 1H, NH), 8.1-7.4 (m, 6H, C.sub.6H.sub.5CO and
H-6), 5.98 (d, 1H, H-1', J=5.0 Hz), 5.5-5.7 (m, 2H, H-2' and H-3'),
4.42 (dd, 2H, H-5' and H-5'', J=11.6 Hz, J=31.6 Hz), 2.12 (s, 3H,
CH.sub.3CO.sub.2), 2.09 (s, 3H, CH.sub.3CO.sub.2), 1.60 (s, 1H,
CH.sub.3), 1.37 (s, 3H, CH.sub.3); MS (matrix GT): FAB>0 m/z 461
(M+H).sup.+, 335 (S).sup.+, 105 (C.sub.6H.sub.5CO).sup.+, 43
(CH.sub.3CO).sup.+ FAB<0 m/z 459 (M-H).sup.-, 125 (B).sup.-, 121
(C.sub.6H.sub.5CO.sub.2).sup.-, 59 (CH.sub.3CO.sub.2).sup.-.
EXAMPLE 17
Preparation of 1-(4-C-methyl-.beta.-D-ribofuranosyl)thymine
(NN)
[0487] The title compound can be prepared according to a published
procedure from MM (Waga, T.; Nishizaki, T.; et al. Biosci.
Biotechnol. Biochem. 1993, 57, 1433-1438).
[0488] A solution of MM (1.09 g, 2.37 mmol) in methanolic ammonia
(previously saturated at -10.degree. C.) (60 mL) was stirred at
room temperature overnight. The solvent was evaporated under
reduced pressure and the residue was partitioned between methylene
chloride (60 mL) and water (60 mL). The aqueous layer was washed
with methylene chloride (2.times.60 mL), concentrated under reduced
pressure and coevaporated several times with absolute ethanol.
Recrystallization from methanol gave NN (450 mg, 70%) as a
colorless and crystalline solid. Mp: 258-260 (dec.) (lit. 264: Ref.
6); UV (H.sub.2O): .lamda..sub.max=264.4 nm (.epsilon.=8800),
.lamda..sub.min=232.0 nm (.epsilon.=2200); .sup.1H-NMR
(DMSO-d.sub.6): .delta. 11.29 (s, 1H, NH), 7.75 (s, 1H, H-6), 5.82
(d, 1H, H-1', J.sub.1'-2'=7.2 Hz), 5.19 (m, 2H, OH-2', OH-5'), 5.02
(d, 1H, OH-3', J.sub.OH-3'=5.0 Hz), 4.21 (dd, 1H, H-2', J=6.4 Hz,
J=12.3 Hz), 3.92 (t, 1H, H-3', J.sub.3'-2'=J.sub.3'-OH'=5.0 Hz),
3.30 (m, 2H, H-5' and H-5''), 1.78 (s, 3H, CH.sub.3), 1.09 (s, 3H,
CH.sub.3); MS (matrix GT): FAB>0 m/z 545 (2M+H).sup.+, 365
(M+G+H).sup.+, 273 (M+H).sup.+, 147 (S).sup.+, 127 (B+2H).sup.+,
FAB<0 m/z 543 (2M-H).sup.-, 271 (M-H).sup.-, 125 (B).sup.-;
[.alpha.].sub.D.sup.20-32.0 (c=0.5 in H.sub.2O, litt. -26.4).
EXAMPLE 18
Preparation of
1-(5,2,3-Tri-O-acetyl-4-C-methyl-.beta.-D-ribofuranosyl)thymine
(OO)
[0489] A solution of NN (200 mg, 0.735 mmol) in anhydrous pyridine
(7.4 ml) was treated with acetic anhydride (1.2 mL) and stirred at
room temperature for 3 hours. The solvent was evaporated under
reduced pressure, and the residue was purified by silica gel column
chromatography [eluent: stepwise gradient of methanol (0-5%) in
methylene chloride] to afford pure OO (0.400 g, quantitative yield)
as a foam. .sup.1H-NMR (DMSO-d.sub.6): .delta. 11.45 (s, 1H, NH),
7.56 (s, 1H, H-6), 5.90 (d, 1H, H-1', J.sub.1'-2'=4.8 Hz), 5.5-5.4
(m, 2H, H-2' and H-3'), 4.3-4.0 (m, 2H, H-5' and H-5''), 2.1-2.0
(m, 9H, 3 CH.sub.3CO.sub.2), 1.78 (s, 1H, CH.sub.3), 1.20 (s, 3H,
CH.sub.3); MS (matrix GT): FAB>0 m/z 797 (2M+H).sup.+, 399
(M+H).sup.+, 339 (M-CH.sub.3CO.sub.2).sup.+, 273 (S).sup.+, 127
(BH.sub.2).sup.+, 43 (CH.sub.3CO).sup.+ FAB<0 m/z 795
(2M-H).sup.-, 397 (M-H).sup.-, 355 (M-CH.sub.3CO).sup.-, 125
(B).sup.-, 59 (CH.sub.3CO.sub.2).sup.-.
EXAMPLE 19
Preparation of
1-(5,2,3-Tri-O-acetyl-4-C-methyl-.beta.-D-ribofuranosyl)-4-thio-thymine
(PP)
[0490] Lawesson's reagent (119 mg, 0.29 mmol) was added under argon
to a solution of OO (0.167 g, 4.19 mmol) in anhydrous
1,2-dichloroethane (11 mL) and the reaction mixture was stirred
overnight under reflux. The solvent was evaporated under reduced
pressure and the residue was purified by silica gel column
chromatography [eluent: stepwise gradient of methanol (1-2%) in
chloroform] to give pure PP (0.165 g, 95%) as a yellow foam.
.sup.1H-NMR (DMSO-d.sub.6): .delta. 12.81 (s, 1H, NH), 7.64 (s, 1H,
H-6), 5.84(d, 1H, H-1', J.sub.1'-2'=4.66 Hz), 5.5-5.4 (m, 2H, H-2'
and H-3'), 4.11 (dd, 2H, H-5' and H-5'', J=11.7 Hz, J=31.3 Hz),
2.0-1.8 (m, 12H, 3 CH.sub.3CO.sub.2 and CH.sub.3), 1.33 (s, 3H,
CH.sub.3); MS (matrix GT): FAB>0 m/z 829 (2M+H).sup.+, 415
(M+H).sup.+, 273 (S).sup.+, 143 (BH.sub.2).sup.+, 43
(CH.sub.3CO).sup.+ FAB<0 m/z 827 (2M-H).sup.-, 413 (M-H).sup.-,
141 (B).sup.-, 59 (CH.sub.3CO.sub.2).sup.-.
[0491] In a similar manner, the following nucleosides of Formula
XVII are prepared, using the appropriate sugar and pyrimidine
bases. ##STR70## wherein R.sup.1, R.sup.2, R.sup.3, X.sup.1 and Y
are defined in Table 19.
EXAMPLE 20
Preparation of
1-(4-C-methyl-.beta.-D-ribofuranosyl)-5-methyl-cytosine (QQ),
hydrochloride form
[0492] Compound PP (0.160 g, 0.386 mmol) was treated with
methanolic ammonia (previously saturated at -10.degree. C.), (10
mL) at 100.degree. C. in a stainless-steel bomb for 3 hours, then
cooled to room temperature. The solvent was evaporated under
reduced pressure and the residue was partitioned between methylene
chloride (30 mL) and water (30 mL). The aqueous layer was washed
with methylene chloride (2.times.30 mL), concentrated under reduced
pressure. The crude material was purified by silica gel column
chromatography [eluent: 20% methanol in methylene chloride] to
afford 1-(4-C-methyl-.beta.-D-ribofuranosyl)-5-methyl-cytosine (60
mg, 57%). This compound was dissolved in EtOH 100 (1.5 mL), treated
with a 2N hydrochloric acid solution (0.3 mL), and the mixture was
stirred before being concentrated under reduced pressure. The
procedure was repeated twice and QQ was precipitated from absolute
ethanol. Mp: 194-200 (dec.); UV (H.sub.2O): .lamda..sub.max=275.6
.lamda..sub.min (.epsilon.=7300), .lamda..sub.min=255 nm
(.epsilon.=4700); HPLC 100%, .sup.1H-NMR (DMSO-d.sub.6): .delta.
9.34 and 9.10 (2s, 2H, NH.sub.2), 8.21 (s, 1H, H-6), 5.80 (d, 1H,
H-1', J.sub.1'-2'=6.0 Hz), 5.3-4.3 (m, 3H, OH-2', OH-3' and OH-5'),
4.21 (t, 1H, H-2', J=5.7 Hz), 3.98 (d, 1H, H-3', J=5.3 Hz), 3.5-3.3
(m, 2H, H-5' and H-5''), 1.97 (s, 3H, CH.sub.3), 1.12 (s, 3H,
CH.sub.3).
EXAMPLE 21
Preparation of
O-6-Diphenylcarbamoyl-N.sup.2-isobutyryl-9-(2,3-di-O-acetyl-5-O-benzoyl-4-
-C-methyl-.beta.-D-ribofuranosyl)guanine (RR)
[0493] To a suspension of
O-6-diphenylcarbamoyl-N.sup.2-isobutyrylguanine (1.80 g, 4.33 mmol)
in anhydrous toluene (20 mL) was added
N,O-bis(trimethylsilyl)acetamide (1.92 mL, 7.9 mmol). The reaction
mixture was allowed to warm under reflux for 1 hour. Compound GG
(1.55 g, 3.93 mmol) was dissolved in toluene (10 mL) and
trimethylsilyltrifluoromethanesulfonate (TMSTf) (915 mL, 4.72 mmol)
was added. The mixture was heated under reflux for 30 minutes. The
solution was then cooled to room temperature and neutralized with a
5% aqueous sodium hydrogen carbonate solution. The reaction mixture
was diluted with ethyl acetate (200 mL). The organic phase was
washed with a 5% aqueous sodium hydrogen carbonate solution (150
mL) and with water (2.times.150 mL). The organic layer was dried
over Na.sub.2SO.sub.4 and evaporated to dryness. The residue was
purified by silica gel column chromatography [eluent: stepwise
gradient of diethyl ether (70-90%) in petroleum ether] to afford
pure RR (1.62 g, 55%) as a foam.
EXAMPLE 22
Preparation of 9-(4-C-methyl-.beta.-D-ribofuranosyl)guanine
(SS)
[0494] The title compound can be prepared according to a published
procedure from RR (Waga, T.; Nishizaki, T.; et al. Biosci.
Biotechnol. Biochem. 1993, 57, 1433-1438).
[0495] A solution of RR (1.50 g, mmol) in methanolic ammonia
(previously saturated at -10.degree. C.) (20 mL) was stirred at
room temperature overnight. The solvent was evaporated under
reduced pressure and the residue was partitioned between methylene
chloride (60 mL) and water (60 mL). The aqueous layer was washed
with methylene chloride (2.times.60 mL), concentrated under reduced
pressure. The residue was purified by an RP18 column chromatography
[eluent water/acetonitrile 95/5] to afford pure SS (380 mg, 60%).
Recrystallization from water gave S as a crystalline solid.
Mp>300 (dec.), UV (H.sub.2O): .lamda..sub.max=252 nm
(.epsilon.=14500), .sup.1H-NMR (DMSO-d.sub.6): .delta. 10.64 (s,
1H, NH), 7.95 (s, 1H, H-8), 6.45 (sl, 2H, NH.sub.2), 5.68 (d, 1H,
H-1', J.sub.1'-2'=7.45 Hz), 5.31 (d, 1H, OH, OH-2', J.sub.OH-2'=6.8
Hz), 5.17 (t, 1H, OH, OH-5', J=5.5 Hz), 5.07 (d, 1H, OH-3',
J.sub.OH-3'=4.5 Hz), 4.65 (dd, 1H, H-2', J=7.1 Hz, J=12.2 Hz), 4.00
(t, 1H, H-3', J.sub.3'-2'=J.sub.3'-OH'=4.8 Hz), 3.41 (m, 2H, H-5'
and H-5''), 1.12 (s, 3H, CH.sub.3); MS (matrix GT): FAB>0 m/z
595 (2M+H).sup.+, 390 (M+G+H).sup.+, 298 (M+H).sup.+, 152
(B+2H).sup.+, FAB<0 m/z 593 (2M-H).sup.-, 296 (M-H).sup.-, 150
(B).sup.-.
EXAMPLE 23
9-(2,3-di-O-acetyl-5-O-benzoyl-4-C-methyl-.beta.D-ribofuranosyl)adenine
(TT)
[0496] A solution of GG (1.10 g, 2.79 mmol) in anhydrous
acetonitrile (50 ml) was treated with adenine (452.4 mg, 3.35 mmol)
and stannic chloride (SnCl.sub.4, 660 .mu.L, 5.58 mmol) and stirred
at room temperature overnight. The solution was concentrated under
reduced pressure, diluted with chloroform (100 mL) and treated with
a cold saturated aqueous solution of NaHCO.sub.3 (100 ml). The
mixture was filtered on celite, and the precipitate was washed with
hot chloroform. The filtrates were combined, washed with water (100
ml) and brine (100 ml), dried (Na.sub.2SO.sub.4), and evaporated
under reduced pressure. The residue was purified by silica gel
column chromatography [eluent: stepwise gradient of methanol (3-5%)
in dichloromethane] to afford pure TT (977 mg, 77%) as a white
foam. .sup.1H-NMR (DMSO-d.sub.6): .delta. 8.31-7.49 (m, 7H,
C.sub.6H.sub.5CO, H-2 and H-8), 7.37 (ls, 2H, NH.sub.2) 6.27 (m,
2H, H-1' and H-3'), 5.90 (m, 1H, H-2'), 4.60 (d, 1H, H-5', J=11.7
Hz), 4.35 (d, 1H, H-5''), 2.17 (s, 3H, CH.sub.3CO.sub.2), 2.06 (s,
3H, CH.sub.3CO.sub.2), 1.42 (s, 3H, CH.sub.3).
EXAMPLE 24
Preparation of 9-(4-C-methyl-.beta.-D-ribofuranosyl)adenine
(UU)
[0497] The title compound can be prepared according to a published
procedure from TT (Waga, T.; Nishizaki, T.; et al. Biosci.
Biotechnol. Biochem. 1993, 57, 1433-1438).
[0498] A solution of TT (970 mg, 2.08 mmol) in methanolic ammonia
(previously saturated at -10.degree. C.) (50 mL) was stirred at
room temperature overnight. The solvent was evaporated under
reduced pressure and the residue was partitioned between methylene
chloride (100 ml) and water (100 ml). The aqueous layer was washed
with methylene chloride (2.times.100 mL), and concentrated under
reduced pressure. The residue was purified by silica gel column
chromatography [eluent: stepwise gradient of methanol (10-30%) in
ethyl acetate] to afford pure U (554 mg, 95%). Crystallization from
methanol/ethyl acetate gave UU as a white solid. Mp: 96-97 (dec.);
.sup.1H-NMR (DMSO-d.sub.6): .delta. 8.33 (s, 1H, H-2), 8.13 (s, 1H,
H-8), 7.36 (brs, 2H, NH.sub.2), 5.84 (d, 1H, H-1', J.sub.1'-2'=7.4
Hz), 5.69 (dd, 1H, OH-5', J=4.2 Hz and J=7.8 Hz), 5.33 (d, 1H,
OH-2', J=6.6 Hz), 5.13 (d, 1H, OH-3', J=4.4 Hz), 4.86 (m, 1H,
H-2'), 4.04 (t, 1H, H-3'), 3.58-3.32 (m, 2H, H-5' and H-5''), 1.15
(s, 3H, CH.sub.3); MS (matrix GT): FAB>0 m/z 563 (2M+H).sup.+,
374 (M+G+H).sup.+, 282 (M+H).sup.+, 136 (B+2H).sup.+, FAB<0 m/z
561 (2M-H).sup.-, 280 (M-H).sup.-, 134 (B).sup.-.
[0499] In a similar manner, the following nucleosides of Formula
XVI are prepared, using the appropriate sugar and purine bases.
##STR71## wherein R.sup.1, R.sup.2, R.sup.3, X.sup.1, X.sup.2, and
Y are defined in Table 20.
[0500] Alternatively, the following nucleosides of Formula XVIII
are prepared, using the appropriate sugar and pyrimidine or purine
bases. ##STR72## wherein R.sup.1, R.sup.2, R.sup.3, R.sup.6, X and
Base are defined in Table 21.
[0501] Alternatively, the following nucleosides of Formula XIX are
prepared, using the appropriate sugar and pyrimidine or purine
bases. ##STR73## wherein R.sup.1, R.sup.2, R.sup.6, X and Base are
defined in Table 22.
[0502] Alternatively, the following nucleosides of Formula XXIV are
prepared, using the appropriate sugar and pyrimidine or purine
bases. ##STR74## wherein R.sup.1, R.sup.2, R.sup.6, X, and Base are
defined in Table 23.
[0503] Alternatively, the following nucleosides of Formula XX are
prepared, using the appropriate sugar and pyrimidine or purine
bases. ##STR75## wherein R.sup.1, R.sup.6, R.sup.7, R.sup.8, X,
Base, R.sup.10 and R.sup.9 are defined in Table 24.
[0504] Tables 1-24 set out examples of species within the present
invention. When the amino acid appears in the table, it is
considered to be a specific and independent disclosure of each of
the esters of .alpha., .beta. .gamma. or .delta. glycine, alanine,
valine, leucine, isoleucine, methionine, phenylalanine, tryptophan,
proline, serine, threonine, cysteine, tyrosine, asparagine,
glutamine, aspartate, glutamate, lysine, arginine and histidine in
the D and L-configurations. When the term acyl is used in the
tables, it is meant to be a specific and independent disclosure of
any of the acyl groups as defined herein, including but not limited
to acetyl, trifluoroacetyl, methylacetyl, cyclopropylacetyl,
cyclopropylcarboxy, propionyl, butyryl, hexanoyl, heptanoyl,
octanoyl, neo-heptanoyl, phenylacetyl, diphenylacetyl,
.alpha.-trifluoromethyl-phenylacetyl, bromoacetyl,
4-chloro-benzeneacetyl, 2-chloro-2,2-diphenylacetyl,
2-chloro-2-phenylacetyl, trimethylacetyl, chlorodifluoroacetyl,
perfluoroacetyl, fluoroacetyl, bromodifluoroacetyl,
2-thiopheneacetyl, tert-butylacetyl, trichloroacetyl,
monochloro-acetyl, dichloroacetyl, methoxybenzoyl,
2-bromo-propionyl, decanoyl, n-pentadecanoyl, stearyl,
3-cyclopentyl-propionyl, 1-benzene-carboxyl, pivaloyl acetyl,
1-adamantane-carboxyl, cyclohexane-carboxyl,
2,6-pyridinedicarboxyl, cyclopropane-carboxyl,
cyclobutane-carboxyl, 4-methylbenzoyl, crotonyl,
1-methyl-1H-indazole-3-carbonyl, 2-propenyl, isovaleryl,
4-phenylbenzoyl.
F. Biological Assays
[0505] Compounds can exhibit anti-flavivirus or pestivirus activity
by inhibiting flavivirus or pestivirus polymerase, by inhibiting
other enzymes needed in the replication cycle, or by other
pathways.
Phosphorylation Assay of Nucleoside to Active Triphosphate
[0506] To determine the cellular metabolism of the compounds, HepG2
cells were obtained from the American Type Culture Collection
(Rockville, Md.), and were grown in 225 cm.sup.2 tissue culture
flasks in minimal essential medium supplemented with non-essential
amino acids, 1% penicillin-streptomycin. The medium was renewed
every three days, and the cells were subcultured once a week. After
detachment of the adherent monolayer with a 10 minute exposure to
30 mL of trypsin-EDTA and three consecutive washes with medium,
confluent HepG2 cells were seeded at a density of
2.5.times.10.sup.6 cells per well in a 6-well plate and exposed to
10 .mu.M of [.sup.3H] labeled active compound (500 dpm/pmol) for
the specified time periods. The cells were maintained at 37.degree.
C. under a 5% CO.sub.2 atmosphere. At the selected time points, the
cells were washed three times with ice-cold phosphate-buffered
saline (PBS). Intracellular active compound and its respective
metabolites were extracted by incubating the cell pellet overnight
at -20.degree. C. with 60% methanol followed by extraction with an
additional 20 .mu.L of cold methanol for one hour in an ice bath.
The extracts were then combined, dried under gentle filtered air
flow and stored at -20.degree. C. until HPLC analysis.
Bioavailability Assay in Cynomolgus Monkeys
[0507] Within 1 week prior to the study initiation, the cynomolgus
monkey was surgically implanted with a chronic venous catheter and
subcutaneous venous access port (VAP) to facilitate blood
collection and underwent a physical examination including
hematology and serum chemistry evaluations and the body weight was
recorded. Each monkey (six total) receives approximately 250 .mu.Ci
of .sup.3H activity with each dose of active compound at a dose
level of 10 mg/kg at a dose concentration of 5 mg/mL, either via an
intravenous bolus (3 monkeys, IV), or via oral gavage (3 monkeys,
PO). Each dosing syringe was weighed before dosing to
gravimetrically determine the quantity of formulation administered.
Urine samples were collected via pan catch at the designated
intervals (approximately 18-0 hours pre-dose, 0-4, 4-8 and 8-12
hours post-dosage) and processed. Blood samples were collected as
well (pre-dose, 0.25, 0.5, 1, 2, 3, 6, 8, 12 and 24 hours
post-dosage) via the chronic venous catheter and VAP or from a
peripheral vessel if the chronic venous catheter procedure should
not be possible. The blood and urine samples were analyzed for the
maximum concentration (C.sub.max), time when the maximum
concentration was achieved (T.sub.max), area under the curve (AUC),
half life of the dosage concentration (T.sup.1/2), clearance (CL),
steady state volume and distribution (V.sub.ss) and bioavailability
(F).
Bone Marrow Toxicity Assay
[0508] Human bone marrow cells were collected from normal healthy
volunteers and the mononuclear population were separated by
Ficoll-Hypaque gradient centrifugation as described previously by
Sommadossi J-P, Carlisle R. Antimicrobial Agents and Chemotherapy
1987; 31:452-454; and Sommadossi J-P, Schinazi R F, et al.
Biochemical Pharmacology 1992; 44:1921-1925. The culture assays for
CFU-GM and BFU-E were performed using a bilayer soft agar or
methylcellulose method. Drugs were diluted in tissue culture medium
and filtered. After 14 to 18 days at 37.degree. C. in a humidified
atmosphere of 5% CO.sub.2 in air, colonies of greater than 50 cells
were counted using an inverted microscope. The results are
presented as the percent inhibition of colony formation in the
presence of drug compared to solvent control cultures.
Mitochondria Toxicity Assay
[0509] HepG2 cells were cultured in 12-well plates as described
above and exposed to various concentrations of drugs as taught by
Pan-Zhou X-, Cui L, et al. Antimicrob. Agents Chemother. 2000;
44:496-503. Lactic acid levels in the culture medium after 4 day
drug exposure were measured using a Boehringer lactic acid assay
kit. Lactic acid levels were normalized by cell number as measured
by hemocytometer count.
Cytotoxicity Assay
[0510] Cells were seeded at a rate of between 5.times.10.sup.3 and
5.times.10.sup.4/well into 96-well plates in growth medium
overnight at 37.degree. C. in a humidified CO.sub.2 (5%)
atmosphere. New growth medium containing serial dilutions of the
drugs was then added. After incubation for 4 days, cultures were
fixed in 50% TCA and stained with sulforhodamine B. The optical
density was read at 550 nm. The cytotoxic concentration was
expressed as the concentration required to reduce the cell number
by 50% (CC.sub.50).
Cell Protection Assay (CPA)
[0511] The assay was performed essentially as described by
Baginski, S. G.; Pevear, D. C.; et al. PNAS USA 2000, 97(14),
7981-7986. MDBK cells (ATCC) were seeded onto 96-well culture
plates (4,000 cells per well) 24 hours before use. After infection
with BVDV (strain NADL, ATCC) at a multiplicity of infection (MOI)
of 0.02 plaque forming units (PFU) per cell, serial dilutions of
test compounds were added to both infected and uninfected cells in
a final concentration of 0.5% DMSO in growth medium. Each dilution
was tested in quadruplicate. Cell densities and virus inocula were
adjusted to ensure continuous cell growth throughout the experiment
and to achieve more than 90% virus-induced cell destruction in the
untreated controls after four days post-infection. After four days,
plates were fixed with 50% TCA and stained with sulforhodamine B.
The optical density of the wells was read in a microplate reader at
550 nm. The 50% effective concentration (EC.sub.50) values were
defined as the compound concentration that achieved 50% reduction
of cytopathic effect of the virus.
Plaque Reduction Assay
[0512] For each compound the effective concentration was determined
in duplicate 24-well plates by plaque reduction assays. Cell
monolayers were infected with 100 PFU/well of virus. Then, serial
dilutions of test compounds in MEM supplemented with 2% inactivated
serum and 0.75% of methyl cellulose were added to the monolayers.
Cultures were further incubated at 37.degree. C. for 3 days, then
fixed with 50% ethanol and 0.8% Crystal Violet, washed and
air-dried. Then plaques were counted to determine the concentration
to obtain 90% virus suppression.
Yield Reduction Assay
[0513] For each compound the concentration to obtain a 6-log
reduction in viral load was determined in duplicate 24-well plates
by yield reduction assays. The assay was performed as described by
Baginski, S. G.; Pevear, D. C.; et al. PNAS USA 2000, 97(14),
7981-7986, with minor modifications. Briefly, MDBK cells were
seeded onto 24-well plates (2.times.105 cells per well) 24 hours
before infection with BVDV (NADL strain) at a multiplicity of
infection (MOI) of 0.1 PFU per cell. Serial dilutions of test
compounds were added to cells in a final concentration of 0.5% DMSO
in growth medium. Each dilution was tested in triplicate. After
three days, cell cultures (cell monolayers and supernatants) were
lysed by three freeze-thaw cycles, and virus yield was quantified
by plaque assay. Briefly, MDBK cells were seeded onto 6-well plates
(5.times.105 cells per well) 24 h before use. Cells were inoculated
with 0.2 mL of test lysates for 1 hour, washed and overlaid with
0.5% agarose in growth medium. After 3 days, cell monolayers were
fixed with 3.5% formaldehyde and stained with 1% crystal violet
(w/v in 50% ethanol) to visualize plaques. The plaques were counted
to determine the concentration to obtain a 6-log reduction in viral
load.
EXAMPLE 25
Antiviral Potency of Test Compounds in a Cell Based Assay
[0514] The titer of BVDB (Log.sub.10 units/ml) were identified
after treatment of infected MDBK cells with increasing
concentrations of four test compounds. Ribavirin was used as a
standard. This data is shown in FIG. 11. The graph shows the
antiviral potency of these compounds.
EXAMPLE 26
Cellular Pharmacology of 2'-C-methyl-cytidine-3'-O-L-valine ester
(Val-mCyd)
Phosphorylation Assay of Nucleoside to Active Triphosphate
[0515] To determine the cellular metabolism of the compounds, HepG2
cells were obtained from the American Type Culture Collection
(Rockville, Md.), and were grown in 225 cm.sup.2 tissue culture
flasks in minimal essential medium supplemented with non-essential
amino acids, 1% penicillin-streptomycin. The medium was renewed
every three days, and the cells were subcultured once a week. After
detachment of the adherent monolayer with a 10 minute exposure to
30 mL of trypsin-EDTA and three consecutive washes with medium,
confluent HepG2 cells were seeded at a density of
2.5.times.10.sup.6 cells per well in a 6-well plate and exposed to
10 .mu.M of [.sup.3H] labeled active compound (500 dpm/pmol) for
the specified time periods. The cells were maintained at 37.degree.
C. under a 5% CO.sub.2 atmosphere. At the selected time points, the
cells were washed three times with ice-cold phosphate-buffered
saline (PBS). Intracellular active compound and its respective
metabolites were extracted by incubating the cell pellet overnight
at -20.degree. C. with 60% methanol followed by extraction with an
additional 20 .mu.L of cold methanol for one hour in an ice bath.
The extracts were then combined, dried under gentle filtered air
flow and stored at -20.degree. C. until HPLC analysis.
[0516] Antiviral nucleosides and nucleoside analogs were generally
converted into the active metabolite, the 5'-triphosphate (TP)
derivatives by intracellular kinases. The nucleoside-TPs then exert
their antiviral effect by inhibiting the viral polymerase during
virus replication. In primary human hepatocyte cultures, in a human
hepatoma cell line (HepG2), and in a bovine kidney cell line
(MDBK), mCyd was converted into a major metabolite,
2'-C-methyl-cytidine-5'-triphosphate (mCyd-TP), along with smaller
amounts of a uridine 5'-triphosphate derivative,
2'-C-methyl-uridine-5'-triphosphate (mUrd-TP). mCyd-TP is
inhibitory when tested in vitro against the BVDV replication
enzyme, the NS5B RNA dependent RNA polymerase, and is thought to be
responsible for the antiviral activity of mCyd.
[0517] The cellular metabolism of mCyd was examined using MDBK
cells, HepG2 cells and human primary hepatocytes exposed to 10
.mu.M [.sup.3H]-mCyd. High-pressure liquid chromatography (HPLC)
analysis demonstrated that mCyd was phosphorylated in all three
cell types, with mCyd-TP being the predominant metabolite after 24
h. The metabolic profile obtained over a 48-hour exposure of human
hepatoma HepG2 cells to 10 .mu.M [.sup.3H]-mCyd was tested. In
HepG2 cells, levels of mCyd-TP peaked at 41.5.+-.13.4 .mu.M after
24 hours (see Table 25) and fell slowly thereafter. In primary
human hepatocytes, the peak mCyd-TP concentration at 24 hours was 4
fold lower at 10.7.+-.6.7 .mu.M. MDBK bovine kidney cells yielded
intermediate levels of mCyd-TP (30.1.+-.6.9 .mu.M at 24 hours).
[0518] Exposure of hepatocytes to mCyd led to production of a
second 5'-triphosphate derivative, mUrd-TP. In HepG2 cells exposed
to 10 .mu.M [.sup.3H]-mCyd, the mUrd-TP level reached 1.9.+-.1.6
.mu.M at 24 hours, compared to 8.1.+-.3.4 .mu.M in MDBK cells and
3.2.+-.2.0 .mu.M in primary human hepatocytes. While MDBK and HepG2
cells produced comparable total amounts of phosphorylated species
(approximately 43 versus 47 .mu.M, respectively) at 24 h, mUrd-TP
comprised 19% of the total product in MDBK cells versus only 4% in
HepG2 cells. mUrd-TP concentration increased steadily over time,
however reached a plateau or declined after 24 hours.
TABLE-US-00006 TABLE 25 Activation of mCyd (10 .mu.M) in
Hepatocytes and MDBK Cells Metabolite (.mu.M) Cells.sup.a n mCyd-MP
mUrd-MP mCyd-DP mUrd-DP mCyd-TP mUrd-TP HepG2 6 ND ND 3.7 .+-. 2.1
ND 41.5 .+-. 13.4 1.9 .+-. 1.6 Human Primary Hepatocytes 5 ND ND
1.15 .+-. 1.1 0.26 .+-. 0.4 C 10.7 .+-. 6.7 3.2 .+-. 2.0 MDBK
Bovine Kidney Cells 7 ND ND 4.2 .+-. 2.7 0.76 .+-. 0.95 30.1 .+-.
6.9 8.1 .+-. 3.4 .sup.aCells were incubated for 24 hours with
[.sup.3H]-mCyd, specific activity: HepG2 assay = 0.5 Ci/mmol; human
and monkey hepatocyte assay = 1.0 Ci/mmol. .sup.bThe concentrations
of metabolites were determined as pmoles per million cells. One
pmole per million cells is roughly equivalent to 1 .mu.M. ND, not
detected.
[0519] The apparent intracellular half-life of the mCyd-TP was
13.9.+-.2.2 hours in HepG2 cells and 7.6.+-.0.6 hours in MDBK
cells: the data were not suitable for calculating the half life of
mUrd-TP. Other than the specific differences noted above, the
phosphorylation pattern detected in primary human hepatocytes was
qualitatively similar to that obtained using HepG2 or MDBK
cells.
EXAMPLE 27
Cell Cytotoxicity
Mitochondria Toxicity Assay
[0520] HepG2 cells were cultured in 12-well plates as described
above and exposed to various concentrations of drugs as taught by
Pan-Zhou X-R, Cui L, et al. Antimicrob. Agents Chemother. 2000;
44:496-503. Lactic acid levels in the culture medium after 4 day
drug exposure were measured using a Boehringer lactic acid assay
kit. Lactic acid levels were normalized by cell number as measured
by hemocytometer count.
Cytotoxicity Assays
[0521] Cells were seeded at a rate of between 5.times.10.sup.3 and
5.times.10.sup.4/well into 96-well plates in growth medium
overnight at 37.degree. C. in a humidified CO.sub.2 (5%)
atmosphere. New growth medium containing serial dilutions of the
drugs was then added. After incubation for 4 days, cultures were
fixed in 50% TCA and stained with sulforhodamine B. The optical
density was read at 550 nm. The cytotoxic concentration was
expressed as the concentration required to reduce the cell number
by 50% (CC.sub.50).
[0522] Conventional cell proliferation assays were used to assess
the cytotoxicity of mCyd and its cellular metabolites in rapidly
dividing cells. The inhibitory effect of mCyd was determined to be
cytostatic in nature since mCyd showed no toxicity in confluent
cells at concentrations far in excess of the corresponding
CC.sub.50 for a specific cell line. mCyd was not cytotoxic to
rapidly growing Huh7 human hepatoma cells or H9c2 rat myocardial
cells at the highest concentration tested (CC.sub.50>250 .mu.M).
The mCyd CC.sub.50 values were 132 and 161 .mu.M in BHK-21 hamster
kidney and HepG2 human hepatoma cell lines, respectively. The
CC.sub.50 for mCyd in HepG2 cells increased to 200 .mu.M when the
cells were grown on collagen-coated plates for 4 or 10 days. For
comparison, CC.sub.50 values of 35-36 .mu.M were derived when
ribavirin was tested in HepG2 and Huh7 cells. In the MDBK bovine
kidney cells used for BVDV antiviral studies, the CC.sub.50 of mCyd
was 36 .mu.M. A similar CC.sub.50 value (34 .mu.M) was determined
for mCyd against MT-4 human T-lymphocyte cells. In addition, mCyd
was mostly either non-cytotoxic or weakly cytotoxic
(CC.sub.50>50 to >200 .mu.M) to numerous other cell lines of
human and other mammalian origin, including several human carcinoma
cell lines, in testing conducted by the National Institutes of
Health (NIH) Antiviral Research and Antimicrobial Chemistry
Program. Exceptions to this were rapidly proliferating HFF human
foreskin fibroblasts and MEF mouse embryo fibroblasts, where mCyd
showed greater cytotoxicity (CC.sub.50s 16.9 and 2.4 .mu.M,
respectively). Again, mCyd was much less toxic to stationary phase
fibroblasts.
[0523] The cytotoxic effect of increasing amounts of mCyd on
cellular DNA or RNA synthesis was examined in HepG2 cells exposed
to [.sup.3H]-thymidine or [.sup.3H]-uridine. In HepG2 cells, the
CC.sub.50s of mCyd required to cause 50% reductions in the
incorporation of radiolabeled thymidine and uridine into cellular
DNA and RNA, were 112 and 186 .mu.M, respectively. The CC.sub.50
values determined for ribavirin (RBV) for DNA and RNA synthesis,
respectively, were 3.16 and 6.85 .mu.M. These values generally
reflect the CC.sub.50s of 161 and 36 .mu.M determined for mCyd and
RBV, respectively, in conventional cell proliferation cytotoxicity
assays. To assess the incorporation of mCyd into cellular RNA and
DNA, HepG2 cells were exposed to 10 .mu.M [.sup.3H]-mCyd or control
nucleosides (specific activity 5.6-8.0 Ci/mmole, labeled in the
base) for 30 hours. Labeled cellular RNA or DNA species were
separately isolated and incorporation was determined by
scintillation counting. Exposure of HepG2 cells to mCyd resulted in
very low levels of incorporation of the ribonucleoside analog into
either cellular DNA or RNA (0.0013-0.0014 pmole/.mu.g of nucleic
acid). These levels were similar to the 0.0009 and 0.0013
pmole/.mu.g values determined for the incorporation of ZDV and ddC,
respectively, into RNA: since these deoxynucleosides were not
expected to incorporate into RNA, these levels likely reflect the
assay background. The incorporation of ZDV and ddC into DNA was
significantly higher (0.103 and 0.0055 pmole/.mu.g, respectively).
Ribavirin (RBV) incorporated into both DNA and RNA at levels
10-fold higher than mCyd. TABLE-US-00007 TABLE 26a Cellular Nucleic
Acid Synthesis and Incorporation Studies in HepG2 Cells (10 .mu.M
Drug and Nucleoside Controls) CC.sub.50 (.mu.M) Incorporated drug
amount DNA RNA pmole/.mu.g pmole/.mu.g Compound
([.sup.3H]Thymidine) ([.sup.3H]Uridine) DNA RNA mCyd 112.3 .+-.
34.5 186.1 .+-. 28.2 0.0013 .+-. 0.0008.sup.a 0.0014 .+-.
0.0008.sup.a ZDV nd nd 0.103 .+-. 0.0123.sup.a 0.0009 .+-.
0.0003.sup.a ddC nd nd 0.0055.sup.b 0.0013.sup.b Ribavirin 3.16
.+-. 0.13 6.85 .+-. 1.83 0.0120.sup.b 0.0132.sup.c .sup.aData
represent mean of three experiments .sup.bData represent one
experiment .sup.cData represent mean of two experiments nd, not
determined
[0524] TABLE-US-00008 TABLE 26b Cytotoxicity of mCyd in Mammalian
Cell Lines Cell Line.sup.a n CC.sub.50 (.mu.M) Huh 7 7 >250 Hep
G2 6 161 .+-. 19 Hep G2.sup.b 2 >200 MDBK 7 36 .+-. 7 BHK-21 2
132 .+-. 6 H9c2 2 >250 .sup.aAll Cytotoxicity testing was done
under conditions of rapid cell division .sup.bCells were grown on
collagen coated plates for 4 or 10 d
Bone Marrow Toxicity Assay
[0525] Human bone marrow cells were collected from normal healthy
volunteers and the mononuclear population were separated by
Ficoll-Hypaque gradient centrifugation as described previously by
Sommadossi J-P, Carlisle R. Antimicrobial Agents and Chemotherapy
1987; 31:452-454; and Sommadossi J-P, Schinazi R F, et al.
Biochemical Pharmacology 1992; 44:1921-1925. The culture assays for
CFU-GM and BFU-E were performed using a bilayer soft agar or
methylcellulose method. Drugs were diluted in tissue culture medium
and filtered. After 14 to 18 days at 37.degree. C. in a humidified
atmosphere of 5% CO.sub.2 in air, colonies of greater than 50 cells
were counted using an inverted microscope. The results are
presented as the percent inhibition of colony formation in the
presence of drug compared to solvent control cultures.
Cell Protection Assay (CPA)
[0526] The assay was performed essentially as described by
Baginski, S. G.; Pevear, D. C.; et al. PNAS USA 2000, 97(14),
7981-7986. MDBK cells (ATCC) were seeded onto 96-well culture
plates (4,000 cells per well) 24 hours before use. After infection
with BVDV (strain NADL, ATCC) at a multiplicity of infection (MOI)
of 0.02 plaque forming units (PFU) per cell, serial dilutions of
test compounds were added to both infected and uninfected cells in
a final concentration of 0.5% DMSO in growth medium. Each dilution
was tested in quadruplicate. Cell densities and virus inocula were
adjusted to ensure continuous cell growth throughout the experiment
and to achieve more than 90% virus-induced cell destruction in the
untreated controls after four days post-infection. After four days,
plates were fixed with 50% TCA and stained with sulforhodamine B.
The optical density of the wells was read in a microplate reader at
550 nm. The 50% effective concentration (EC.sub.50) values were
defined as the compound concentration that achieved 50% reduction
of cytopathic effect of the virus.
[0527] The myelosuppressive effects of certain nucleoside analogs
have highlighted the need to test for potential effects of
investigational drugs on the growth of human bone marrow progenitor
cells in clonogenic assays. In particular, anemia and neutropenia
are the most common drug-related clinical toxicities associated
with the anti-HIV drug zidovudine (ZDV) or the ribavirin (RBV)
component of the standard of care combination therapy used for HCV
treatment. These toxicities have been modeled in an in vitro assay
that employed bone marrow cells obtained from healthy volunteers
(Sommadossi J-P, Carlisle R. Antimicrob. Agents Chemother. 1987;
31(3): 452-454). ZDV has been previously shown to directly inhibit
human granulocyte-macrophage colony-forming (CFU-GM) and erythroid
burst-forming M in this model (BFU-E) activity at clinically
relevant concentrations of 1-2 (Berman E, et al. Blood 1989;
74(4):1281-1286; Yoshida Y, Yoshida C. AIDS Res. Hum. Retroviruses
1990; 6(7):929-932; Lerza R, et al. Exp. Hematol. 1997;
25(3):252-255; Domsife R E, Averett D R. Antimicrob. Agents
Chemother. 1996; 40(2):514-519; Kurtzberg J, Carter S G. Exp.
Hematol. 1990; 18(10):1094-1096; Weinberg R S, et al. Mt. Sinai J.
Med. 1998; 65(1):5-13). Using human bone marrow clonogenic assays,
the CC.sub.50 values of mCyd in CFU-GM and BFU-E were 14.1.+-.4.5
and 13.9.+-.3.2 .mu.M (see Table 27). mCyd was significantly less
toxic to bone marrow cells than both ZDV and RBV (Table 27).
TABLE-US-00009 TABLE 27 Bone Marrow Toxicity of mCyd in Granulocyte
Macrophage Progenitor and Erythrocyte Precursor Cells CFU-GM.sup.a
BFU-E.sup.a Compound CC.sub.50 (.mu.M) CC.sub.50 (.mu.M) mCyd 14.1
.+-. 4.5 .mu.M 13.9 .+-. 3.2 ZDV 0.89 .+-. 0.47 0.35 .+-. 0.28 RBV
7.49 .+-. 2.20 0.99 .+-. 0.24 .sup.aData from 3 independent
experiments for RBV and 5-8 independent experiments for mCyd and
ZDV. All experiments were done in triplicate.
Effect on Mitochondrial Function
[0528] Antiviral nucleoside analogs approved for HIV therapy such
as ZDV, stavudine (d4T), didanosine (ddI), and zalcitabine (ddC)
have been occasionally associated with clinically limiting delayed
toxicities such as peripheral neuropathy, myopathy, and
pancreatitis (Browne M J, et al. J. Infect. Dis. 1993;
167(1):21-29; Fischl M A, et al. Ann. Intern. Med. 1993;
18(10):762-769; Richman D D, et al. N. Engl. J. Med. 1987; 317(4):
192-197; Yarchoan R, et al. Lancet 1990; 336(8714):526-529). These
clinical adverse events have been attributed by some experts to
inhibition of mitochondrial function due to reduction in
mitochondrial DNA (mtDNA) content and nucleoside analog
incorporation into mtDNA. In addition, one particular nucleoside
analog, fialuridine
(1,-2'-deoxy-2'-fluoro-1-.beta.-D-arabinofuranosyl-5-iodo-uracil;
FIAU), caused hepatic failure, pancreatitis, neuropathy, myopathy
and lactic acidosis due to direct mitochondrial toxicity (McKenzie
R, et al. N. Engl. J. Med. 1995; 333(17):1099-1105).
Drug-associated increases in lactic acid production can be
considered a marker of impaired mitochondrial function or oxidative
phosphorylation. (Colacino, J. M. Antiviral Res. 1996 29(2-3):
125-39).
[0529] To assess the potential of mCyd to produce mitochondrial
toxicity, several in vitro studies were conducted using the human
hepatoma cell lines HepG2 or Huh7. These studies included analysis
of lactic acid production, mtDNA content, and determination of
changes in morphology (e.g., loss of cristae, matrix dissolution
and swelling, and lipid droplet formation) of mitochondrial
ultrastructure.
[0530] The effects of mCyd on mitochondria are presented in Table
28. No differences were observed in lactic acid production in
mCyd-treated cells versus untreated cells at up to 50 .mu.M mCyd in
Huh7 cells or 10 .mu.M mCyd in HepG2 cells. A modest (38%) increase
in lactic acid production was seen in HepG2 cells treated with 50
.mu.M mCyd. The significance of this finding is unclear,
particularly since mCyd is unlikely to attain a plasma
concentration of 50 .mu.M in the clinic. For comparison, lactic
acid production increases by 100% over control cells in cells
treated with 10 .mu.M FIAU (Cui L, Yoon, et al. J. Clin. Invest.
1995; 95:555-563). Exposure of HepG2 cells to mCyd for 6 or 14 days
at concentrations up to 50 .mu.M had no negative effect on
mitochondrial DNA content compared to a 56 or 80% reduction in
ddC-treated cells, respectively.
[0531] Following M mCyd, the ultrastructure of HepG2 cells, and in
.quadrature.14 days of exposure to 10 particular mitochondria, was
examined by transmission electron microscopy. No changes in cell
architecture, or in mitochondrial number or morphology (including
cristae), were observed in the majority of cells. In 17% of the
cells, 1 to 2 mitochondria out of an average of 25 per cell
appeared enlarged. Such minor changes would be unlikely to have any
significant impact on mitochondrial function. ddC-treated cells
showed abnormal mitochondrial morphology with loss of cristae, and
the accumulation of fat droplets. (Medina, D. J., C. H. Tsai, et
al. Antimicrob. Agents Chemother. 1994 38(8): 1824-8; Lewis W, et
al. J. Clin. Invest. 1992; 89(4):1354-1360, Lewis, L. D., F. M.
Hamzeh, et al. Antimicrob. Agents Chemother. 1992 36(9): 2061-5).
TABLE-US-00010 TABLE 28 Effect of mCyd on Hepatocyte Proliferation,
Mitochondrial Function, and Morphology in HepG2 Cells Electron
L-Lactate mtDNA/nuclear DNA Microscopy.sup.c (% of Control.sup.a)
(% of Control.sup.b) Lipid Conc HepG2 Huh7 6 day 14 day Droplet
Agent (.mu.M) Cells Cells Treatment Treatment Form. Mito.Morphol.
Cont. 0 100 100 100 100 Negative Normal mCyd 10 98.6 .+-. 7.3 98.0
.+-. 12.3 117.3 .+-. 17.5 99.7 .+-. 23.9 Negative Normal.sup.d 50
138.0 .+-. 8.9 97.1 .+-. 10.1 158.2 .+-. 17.5 83.0 .+-. 15.5 nd nd
ddC 1 nd nd 44.3 .+-. 9.3 19.6 .+-. 8.2 nd nd 10 nd nd nd nd
Positive Loss of Cristae
Effect on Human DNA Polymerases .alpha., .beta., and .gamma.
[0532] The cellular DNA polymerases are responsible for normal
nuclear and mitochondrial DNA synthesis and repair. Nucleoside
analog triphosphates are potential inhibitors of DNA polymerases
and hence could disrupt critical cell functions. In particular, the
inhibition of human polymerase .gamma., the enzyme responsible for
mitochondrial DNA synthesis, has been linked to defects in
mitochondrial function (Lewis, W., E. S. Levine, et al. Proceedings
of the National Academy of Sciences, USA 1996 93(8): 3592-7).
Experiments were undertaken to determine if mCyd-TP inhibited human
DNA polymerases. As shown in Table 29 mCyd-TP was not a substrate
for human DNA polymerases .alpha., .beta., or .gamma.. Even 1 mM
mCyd-TP failed to inhibit these enzymes by 50% in the majority of
replicate assays and IC.sub.50 values could only be determined to
be in excess of 880-1000 .mu.M. In contrast, ddC was a potent
inhibitor of all three human DNA polymerases and of polymerases
.beta. and .gamma. in particular (IC.sub.50s of 4.8 and 2.7 .mu.M,
respectively). Potent inhibition was also seen for the control
drug, actinomycin D, a known inhibitor of DNA-dependent-DNA
polymerases. TABLE-US-00011 TABLE 29 Inhibition of Human
Polymerases by mCyd-Triphosphate IC.sub.50 (.mu.M) mCyd-TP.sup.a
ddC-TP.sup.b Act. D.sup.a Pol .alpha. >1000 78 .+-. 23.4 5.8
.+-. 3.1 Pol .beta. .gtoreq.883.3 .+-. 165 4.8 .+-. 1 7.9 .+-. 3
Pol .gamma. .gtoreq.929.3 .+-. 100 2.7 .+-. 1 15.5 .+-. 4
.sup.aMean .+-. S.D. from 4 data sets .sup.bMean .+-. S.D. from 2
data sets .sup.aHepG2 or huh7 cells were treated with compounds for
4 days, data represent at least three independent experiments
.sup.bHepG2 cells were treated with compounds for 6 and 14 days,
data represents at least three independent experiments .sup.cHepG2
cells were treated with compounds for 14 days .sup.d17% cells (11
of 64) contained 1 or 2 enlarged mitochondria out of 25 in two
independent experiments nd, not determined
EXAMPLE 28
In Vitro Antiviral Activity Against Bvdv
[0533] Compounds can exhibit anti-flavivirus or pestivirus activity
by inhibiting flavivirus or pestivirus polymerase, by inhibiting
other enzymes needed in the replication cycle, or by other
pathways.
Plaque Reduction Assay
[0534] For each compound the effective concentration was determined
in duplicate 24-well plates by plaque reduction assays. Cell
monolayers were infected with 100 PFU/well of virus. Then, serial
dilutions of test compounds in MEM supplemented with 2% inactivated
serum and 0.75% of methyl cellulose were added to the monolayers.
Cultures were further incubated at 37.degree. C. for 3 days, then
fixed with 50% ethanol and 0.8% Crystal Violet, washed and
air-dried. Then plaques were counted to determine the concentration
to obtain 90% virus suppression.
Yield Reduction Assay
[0535] For each compound the concentration to obtain a 6-log
reduction in viral load was determined in duplicate 24-well plates
by yield reduction assays. The assay was performed as described by
Baginski, S. G.; Pevear, D.C.; Seipel, M.; et al. PNAS USA 2000,
97(14), 7981-7986, with minor modifications. Briefly, MDBK cells
were seeded onto 24-well plates (2.times.105 cells per well) 24
hours before infection with BVDV (NADL strain) at a multiplicity of
infection (MOI) of 0.1 PFU per cell. Serial dilutions of test
compounds were added to cells in a final concentration of 0.5% DMSO
in growth medium. Each dilution was tested in triplicate. After
three days, cell cultures (cell monolayers and supernatants) were
lysed by three freeze-thaw cycles, and virus yield was quantified
by plaque assay. Briefly, MDBK cells were seeded onto 6-well plates
(5.times.105 cells per well) 24 h before use. Cells were inoculated
with 0.2 mL of test lysates for 1 hour, washed and overlaid with
0.5% agarose in growth medium. After 3 days, cell monolayers were
fixed with 3.5% formaldehyde and stained with 1% crystal violet
(w/v in 50% ethanol) to visualize plaques. The plaques were counted
to determine the concentration to obtain a 6-log reduction in viral
load.
[0536] Studies on the antiviral activity of mCyd in cultured cells
were conducted. The primary assay used to determine mCyd antiviral
potency was a BVDV-based cell-protection assay (CPA). This assay
measures the ability of mCyd to protect growing MDBK bovine kidney
cells from destruction by a cytopathic NADL strain of BVDV. The
cytotoxicity of the test drug on uninfected cells was measured in
parallel. The antiviral activities of mCyd and ribavirin in the CPA
are compared in Table 30a. mCyd effectively protected de
novo-infected MDBK cells in a concentration-dependent manner with
an EC.sub.50=0.67.+-.0.22 .mu.M (Table 30a). mCyd afforded complete
cytoprotection at concentrations well below the CC.sub.50 for mCyd
in this assay (38.+-.9 .mu.M). In the CPA, as well as in other
assays described below, ribavirin showed no clear antiviral effect:
significant (50% or more) cell protection was not achieved in most
assays as the cytotoxicity of ribavirin overlaps and masks the
protective effect. Thus, ribavirin gave a CC.sub.50 of 4.3.+-.0.6
.mu.M and an EC.sub.50>4.3 .mu.M in the CPA.
[0537] For Tables 30a-30o below, cell lines utilized include MT-4
for HIV; Vero 76, African green monkey kidney cells for SARS; BHK
for Bovine Viral Diarrhea Virus; Sb-1 for poliovirus Sabin type-1;
CVB-2, CVB-3, CVB-4, and CVA-9 for Coxsackieviruses B-2, B-3, B-4
and A-9; and REO-1 for double-stranded RNA viruses. Note:
BVDV=bovine viral diarrhea virus; YFV=yellow fever virus;
DENV=dengue virus; WNV=West Nile virus; CVB-2=Coxsackie B-2 virus;
Sb-1=Sabin type 1 poliomyelitis virus; and REO=double-stranded RNA
Reovirus. TABLE-US-00012 TABLE 30a In Vitro Activity of mCyd
Against BVDV in the Cell Protection Assay Compound n EC.sub.50,
.mu.M CC.sub.50, .mu.M mCyd 11 0.67 .+-. 0.22 38 .+-. 9 RBV 3
>4.3 4.3 .+-. 0.6
[0538] TABLE-US-00013 TABLE 30b CC.sub.50 Test Results for
.beta.-D-2'-C-methyl-cytidine (Compound G), 3'-O-valinyl ester of
.beta.-D-2'-C-methyl-cytidine dihydrochloride salt (Compound M),
and .beta.-D-2'-C-methyl-uracil (Compound N) Compound CC.sub.50
BVDV YFV DENV 2 WNV CVB-2 Sb-1 REO G 34 2.3 54 95 80 12 11.5 13 M
24 5.8 82 >100 82 12 14 22 N >100 18 100 > or =100 80
>100 55 >100
[0539] TABLE-US-00014 TABLE 30c CC.sub.50 and EC.sub.50 Test
Results for .beta.-D-2'-C-methyl-cytidine (Compound G) CC.sub.50
CC.sub.50 CC.sub.50 EC.sub.50 EC.sub.50 EC.sub.50 EC.sub.50
EC.sub.50 EC.sub.50 Compound MT-4 Vero 76 BHK Sb-1 CVB-2 CVB-3
CVB-4 CVA-9 REO-1 G 34 >100 >100 6 11 9 13 26 13
[0540] TABLE-US-00015 TABLE 30d CC.sub.50 and EC.sub.50 Test
Results for .beta.-D-2'-C-methyl-adenosine (Compound A) and
.beta.-D-2'-C-methyl-2-amino adenosine (Compound B) CC.sub.50
CC.sub.50 CC.sub.50 EC.sub.50 EC.sub.50 EC.sub.50 EC.sub.50
EC.sub.50 EC.sub.50 Compound MT-4 Vero 76 BHK Sb-1 CVB-2 CVB-3
CVB-4 CVA-9 REO-1 A 4 80 70 10 10 14 13 12 >70 B >100 >100
50 90 75 23 32 39 2
[0541] TABLE-US-00016 TABLE 30e CC.sub.50 and EC.sub.50 Test
Results for .beta.-D-2'-C-methyl-guanosine (Compound C) and
.beta.-D-2'-C-methyl-6-chloro-guanosine (Compound D) CC.sub.50
CC.sub.50 CC.sub.50 EC.sub.50 EC.sub.50 EC.sub.50 EC.sub.50
EC.sub.50 EC.sub.50 Compound MT-4 Vero 76 BHK Sb-1 CVB-2 CVB-3
CVB-4 CVA-9 REO-1 C >100 >100 100 22 30 22 12 46 2 D >100
>100 30 50 25 21 25 37 0.4
[0542] TABLE-US-00017 TABLE 30f CC.sub.50 and EC.sub.50 Test
Results for 3',5'-di-O-valinyl ester of
.beta.-D-2'-C-methyl-guanosine dihydrochloride salt (Compound E)
CC.sub.50 CC.sub.50 CC.sub.50 EC.sub.50 EC.sub.50 EC.sub.50
EC.sub.50 EC.sub.50 EC.sub.50 Compound MT-4 Vero 76 BHK Sb-1 CVB-2
CVB-3 CVB-4 CVA-9 REO-1 E >100 >100 100 30 33 30 35 40 2
[0543] TABLE-US-00018 TABLE 30g CC.sub.50 and EC.sub.50 Test
Results for .beta.-D-2'-C-methyl-cytidine (Compound G) CC.sub.50
CC.sub.50 CC.sub.50 EC.sub.50 EC.sub.50 EC.sub.50 EC.sub.50
EC.sub.50 EC.sub.50 Compound MT-4 Vero 76 BHK Sb-1 CVB-2 CVB-3
CVB-4 CVA-9 REO-1 G 34 >100 >100 6 11 9 13 26 13
[0544] TABLE-US-00019 TABLE 30h CC.sub.50 and EC.sub.50 Test
Results for .beta.-D-2'-C-ethynyl-adenosine (Compound H) CC.sub.50
CC.sub.50 CC.sub.50 EC.sub.50 EC.sub.50 EC.sub.50 EC.sub.50
EC.sub.50 EC.sub.50 Compound MT-4 Vero 76 BHK Sb-1 CVB-2 CVB-3
CVB-4 CVA-9 REO-1 H 4.6 60 15 1 1.5 1 2 2.5 6
[0545] TABLE-US-00020 TABLE 30i CC.sub.50 and EC.sub.50 Test
Results for .beta.-D-2'-C-ethynyl-cytidine (Compound I) CC.sub.50
CC.sub.50 CC.sub.50 EC.sub.50 EC.sub.50 EC.sub.50 EC.sub.50
EC.sub.50 EC.sub.50 Compound MT-4 Vero 76 BHK Sb-1 CVB-2 CVB-3
CVB-4 CVA-9 REO-1 I >or = 100 >100 >100 26 33 33 24 59
>100
[0546] TABLE-US-00021 TABLE 30j CC.sub.50 and EC.sub.50 Test
Results for .beta.-D-2-amino-adenosine (Compound J) CC.sub.50
CC.sub.50 CC.sub.50 EC.sub.50 EC.sub.50 EC.sub.50 EC.sub.50
EC.sub.50 EC.sub.50 Compound MT-4 Vero 76 BHK Sb-1 CVB-2 CVB-3
CVB-4 CVA-9 REO-1 J 50 >100 >100 40 53 55 50 53 >100
[0547] TABLE-US-00022 TABLE 30k CC.sub.50 Test Results for
.beta.-D-2'-C-methyl-adenosine (Compound A),
.beta.-D-2'-C-methyl-2-amino adenosine (Compound B), and
.beta.-D-2'-C-methyl-2-amino-6-cyclopropyl adenosine(Compound K)
Com- DENV CVB- Sb- pound CC.sub.50 BVDV YFV 2 WNV 2 1 REO A 4.0 1.2
2.7 2.7 3.6 7 7 >70 B >100 2.1 0.8 0.7 0.3 76 90 2 K >100
18 10 4.9 3.5 >100 >100 9.5
[0548] TABLE-US-00023 TABLE 30l CC.sub.50 Test Results for
.beta.-D-2'-C-methyl-guanosine (Compound C),
.beta.-D-2'-C-methyl-1-(methyl-2-oxo-2-phenyl ethyl)guanosine
(Compound L), and .beta.-D-2'-C-methyl-6-chloro guanosine (Compound
D) Com- DENV CVB- Sb- pound CC.sub.50 BVDV YFV 2 WNV 2 1 REO C
>100 3.5 1.2 1.4 0.6 29 50 2 L >100 12 6 4.4 3 >100
>100 12 D >100 0.7 1.0 0.7 0.3 25 50 0.4
[0549] TABLE-US-00024 TABLE 30m CC.sub.50 Test Results for
3',5'-di-O-valinyl ester of .beta.-D-2'-C-methyl-guanosine
dihydrochloride salt (Compound E) Com- DENV CVB- Sb- pound
CC.sub.50 BVDV YFV 2 WNV 2 1 REO E >100 4.9 1.0 1.4 1 33 55
2.1
[0550] TABLE-US-00025 TABLE 30n CC.sub.50 Test Results for
.beta.-D-2'-C-ethynyl-adenosine (Compound H) Com- DENV CVB- Sb-
pound CC.sub.50 BVDV YFV 2 WNV 2 1 REO H 4.6 0.4 2.0 1.1 1 1.2 0.7
6
[0551] TABLE-US-00026 TABLE 30o CC.sub.50 Test Results for
.beta.-D-2'-C-methyl-cytidine (Compound G), 3'-O-valinyl ester of
.beta.-D-2'-C-methyl-cytidine dihydrochloride salt (Compound M),
and .beta.-D-2'-C-methyl-uracil (Compound N) Com- DENV CVB- Sb-
pound CC.sub.50 BVDV YFV 2 WNV 2 1 REO G 34 2.3 54 95 80 12 11.5 13
M 24 5.8 82 >100 82 12 14 22 N >100 18 100 > or =100 80
>100 55 >100
[0552] The overall antiviral potency of mCyd was determined against
different strains of BVDV and both cytopathic (cp) and
noncytopathic (ncp) biotypes in cell protection assays as well as
in plaque reduction and yield reduction assays. The latter assays
measure the output of infectious virus from cells and hence provide
a stringent test of antiviral efficacy. The different data sets
from all three assays show agreement as summarized in Table 31. The
range of 50% and 90% effective inhibitory concentration (EC.sub.50
and EC.sub.90) values for mCyd was 0.3 to 2.8 .mu.M and 0.87 to 4.8
.mu.M, respectively.
[0553] In the BVDV yield reduction assay, subcytotoxic
concentrations (circa 20 .mu.M) of mCyd suppressed de novo BVDV
production by up to 6 logs, to the point where no infectious virus
was detected. A 4 log.sub.10 effective reduction in BVDV production
(EC.sub.4log10 or EC.sub.99.99) was attained between 6.0 and 13.9
.mu.M mCyd. In contrast, interferon alpha 2b (IFN a2b), although
active against BVDV in this assay (EC.sub.50 2.6 IU per ml), never
gave more than 2 logs of viral reduction, even at 1000 IU per ml.
Thus, the antiviral effect of mCyd against BVDV was much greater
than that of IFN.alpha.2b or RBV.
EXAMPLE 29
In Vitro Antiviral Activity Against Other Positive-Strand RNA
Viruses
[0554] mCyd has been tested for efficacy against positive-strand
RNA viruses other than BVDV. Data obtained are summarized in Table
31 and 32. Against flaviruses, mCyd showed modest activity. The
composite EC.sub.50 ranges (in .mu.M) determined from both sites
were: West Nile virus (46-97); Yellow Fever virus (9-80); and
Dengue Virus (59-95). For mCyd against the alpha virus, Venezuelan
Equine Encephalitis virus, EC.sub.50 values were 1.3-45 .mu.M. mCyd
was broadly active against Picomoviruses, such as Polio virus
(EC.sub.50=6 .mu.M), Coxsackie virus (EC.sub.50=15 .mu.M),
Rhinovirus types 5 and 14 (EC.sub.50s=<0.1 and 0.6 .mu.g/ml) and
Rhinovirus type 2 (EC.sub.50 2-10 .mu.M). mCyd was generally
inactive against all RNA and DNA viruses tested except for the
positive-strand RNA viruses. mCyd was also found to have no
activity against HIV in MT-4 human T lymphocyte cells or HBV in
HepG2.2.15 cells. TABLE-US-00027 TABLE 31 In Vitro Antiviral
Activity of mCyd Against Plus-Strand RNA Viruses Antiviral Efficacy
(.mu.M) Method of Assay Virus Type Cell Type n EC.sub.50 EC.sub.90
EC.sub.4 log Cell Protection Assay BVDV NADL cp MDBK 11 0.67 .+-.
0.22 Yield Reduction Assay BVDV NADL cp MDBK 3 2.77 .+-. 1.16 4.8
.+-. 1.55 13.9 .+-. 3.07 BVDV New York-1 ncp MDBK 6 0.30 .+-. 0.07
0.87 .+-. 0.18 6.03 .+-. 1.41 BVDV I-NADL cp MDBK 1 0.68 1.73 8.22
BVDV I-N-dIns ncp MDBK 1 0.59 1.49 7.14 Plaque Reduction Assay BVDV
NADL cp MDBK 3 2.57 .+-. 0.35 4.63 .+-. 0.72 Cell Protection Assay
West Nile Virus BHK 3 63-97 Cell Protection Assay Yellow Fever
Virus 17D BHK 1 60-80 DENV-2 BHK 2 95 Cell Protection Assay DENV-4
BHK 1 59 Polio Virus Plaque Reduction Sb-1 VERO 1 6 Assay Plaque
Reduction Assay Coxsackie Virus B2 VERO 1 15 cp, cytopathic virus;
ncp noncytopathic virus 1-NADL cp and I-N-dIns ncp represent
recombinant BVDV viruses
[0555] TABLE-US-00028 TABLE 32 In Vitro Antiviral Activity,
Selectivity, and Cytotoxicity of mCvd Virus (Cell line).sup.a
EC.sub.50.sup.b (.mu.M) CC.sub.50.sup.c (.mu.M) WNV (Vero) 46
114-124 YFV (Vero) 9-30 150->200 VEE (Vero) 1.3-45 >200 HSV-1
(HFF).sup.d >100 >100 HSV-2 (HFF).sup.d >100 >100 VZV
(HFF).sup.d >20 67.8 EBV (Daudi).sup.d 25.5 >50 HCMV
(HFF).sup.d 9.9-15.6 67-73 MCMV (MEF) >0.8 2.4 Influenza A/H1N1
(MDCK) >200 >200 Influenza A/H3N2 (MDCK) >20 45-65
Influenza B (MDCK) >200 55-140 Adenovirus type 1 (A549) >200
>200 Parainfluenza type 3 >200 >200 (MA-104) Rhinovirus
type 2 (KB) 2-10 >200 Rhinovirus type 5 (KB).sup.d 0.6 20-30
Rhinovirus type 14 <0.1 20->100 (HeLa-Ohio).sup.d RSV type A
(MA-104) >200 200 Punta Toro A (LLC-MK2) >200 >200
.sup.aHFF, human foreskin fibroblast; Daudi, Burkitt's B-cell
lymphoma; MDCK, canine kidney cells; CV-1, African green monkey
kidney cells; KB, human nasopharyngeal carcinoma; MA-104, Rhesus
monkey kidney cells; LLC-MK2, Rhesus monkey kidney cells; A549,
Human lung carcinoma cells; MEF, mouse embryo fibroblast; Vero,
African green monkey kidney cells; HeLa, human cervical
adenocarcinoma cells. .sup.bEC.sub.50 = 50% effective
concentration. .sup.cCC.sub.50 = 50% cytotoxic concentration.
.sup.dResult presented in .mu.g/mL rather than .mu.M.
EXAMPLE 30
Multiplicity of Infection (MOI) and Antiviral Efficacy
[0556] The cell protection assay format was used to test the effect
of increasing the amount of BVDV virus on the EC.sub.50 of mCyd.
Increasing the MOI of BVDV in this assay from 0.04 to 0.16, caused
the EC.sub.50 of mCyd to increase linearly from 0.5 .mu.M to
approximately 2.2 .mu.M.
EXAMPLE 31
Viral Rebound in mCyd Treated Cells
[0557] The effect of discontinuing treatment with mCyd was tested
in MDBK cells persistently infected with a noncytopathic strain
(strain I-N-dIns) of BVDV. Upon passaging in cell culture, these
cells continuously produce anywhere from 10.sup.6 to >10.sup.7
infectious virus particles per ml of media. This virus can be
measured by adding culture supernatants from treated MDBK (BVDV)
cells to uninfected MDBK cells and counting the number of resultant
viral foci after disclosure by immunostaining with a BVDV-specific
antibody. Treatment of a persistently infected cell line with 4
.mu.M mCyd for one cell passage (3 days) reduced the BVDV titer by
approximately 3 log.sub.10 from pretreatment and control cell
levels of just under 107 infectious units per ml. At this point,
mCyd treatment was discontinued. Within a single passage, BVDV
titers rebounded to untreated control levels of just over 107
infectious units per ml.
EXAMPLE 32
Mechanism of Action
[0558] In standard BVDV CPA assays, mCyd treatment results in a
marked increase in total cellular RNA content as cells grow,
protected from the cytopathic effects of BVDV. This was coupled
with a marked decrease in the production of BVDV RNA due to mCyd.
Conversely, in the absence of mCyd, total cellular RNA actually
decreases as BVDV RNA rises due to the destruction of the cells by
the cytopathic virus. To further test the effect of mCyd on viral
and cellular RNAs, the accumulation of intracellular BVDV RNA was
monitored in MDBK cells 18-hours post infection (after
approximately one cycle of virus replication) using Real Time
RT-PCR. In parallel, a cellular housekeeping ribosomal protein mRNA
(rig S15 mRNA) was also quantitated by RT-PCR using specific
primers. The results showed that mCyd dramatically reduced BVDV RNA
levels in de novo-infected MDBK cells with an EC.sub.50 of 1.7
.mu.M and an EC.sub.90 of 2.3 .mu.M. The maximum viral RNA
reduction was 4 log.sub.10 at the highest inhibitor concentration
tested (125 .mu.M). No effect on the level of the rig S15 cellular
mRNA control was observed. Together, the preceding findings suggest
that mCyd inhibits BVDV by specifically interfering with viral
genome RNA synthesis without impacting cellular RNA content. This
idea is further supported by the observation (Table 26a) that
inhibition of RNA synthesis as measured by [.sup.3H]-uridine uptake
in HepG2 cells requires high concentrations of mCyd (EC.sub.50=186
.mu.M).
[0559] In in vitro studies using purified BVDV NS5B RNA-dependent
RNA polymerase (Kao, C. C., A. M. Del Vecchio, et al. 1999.
Virology 253(1): 1-7) and synthetic RNA templates, mCyd-TP
inhibited RNA synthesis with an IC.sub.50 of 0.74 .mu.M and was a
competitive inhibitor of BVDV NS5B RNA-dependent RNA polymerase
with respect to the natural CTP substrate. The inhibition constant
(K.sub.i) for mCyd-TP was 0.16 .mu.M and the Michaelis-Menten
constant (K.sub.m) for CTP was 0.03 .mu.M. Inhibition of RNA
synthesis by mCyd-TP required the presence of a cognate G residue
in the RNA template. The effect of mCyd-TP on RNA synthesis in the
absence of CTP was investigated in more detail using a series of
short (21 mer) synthetic RNA templates containing a single G
residue, which was moved progressively along the template. Analysis
of the newly synthesized transcripts generated from these templates
in the presence of mCyd-TP revealed that RNA elongation continued
only as far as the G residue, then stopped (FIG. 12). In templates
containing more than one G residue, RNA synthesis stopped at the
first G residue encountered by the polymerase. These data strongly
suggest that m-Cyd-TP is acting as a non-obligate chain terminator.
The mechanism of this apparent chain termination is under further
investigation.
EXAMPLE 33
Eradication of a Persistent Bvdv Infection
[0560] The ability of mCyd to eradicate a viral infection was
tested in MDBK cells persistently infected with a noncytopathic
strain of BVDV (strain I-N-dIns). (Vassilev, V. B. and R. O. Donis
Virus Res. 2000 69(2): 95-107.) Compared to untreated cells,
treatment of persistently infected cells with 16 .mu.M mCyd reduced
virus production from more than 6 logs of virus per ml to
undetectable levels within two cell passages (3 to 4 days per
passage). No further virus production was seen upon continued
treatment with mCyd through passage 12. At passages 8, 9 and 10
(arrows, FIG. 13), a portion of cells was cultured for two further
passages in the absence of drug to give enough time for mCyd-TP to
decay and virus replication to resume. The culture media from the
cells were repeatedly tested for the re-emergence of virus by
adding culture supernatants from treated MDBK (BVDV) cells to
uninfected MDBK cells and counting the resultant viral foci after
disclosure by immunostaining with a BVDV-specific antibody.
Although this assay can detect a single virus particle, no virus
emerged from the cells post drug treatment. Thus, treatment with
mCyd for 8 or more passages was sufficient to eliminate virus from
the persistently infected cells.
EXAMPLE 34
Combination Studies with Interferon Alpha 2B
[0561] The first study, performed in MDBK cells persistently
infected with the New York-1 (NY-1) strain of BVDV, compared the
effect of monotherapy with either mCyd (8 .mu.M) or interferon
alpha 2b (200 IU/ml), or the two drugs in combination (FIG. 14A).
In this experiment, 8 .mu.M mCyd alone reduced viral titers by
approximately 3.5 log.sub.10 after one passage to a level that was
maintained for two more passages. Interferon alpha 2b alone was
essentially inactive against persistent BVDV infection
(approximately 0.1 log.sub.10 reduction in virus titer) despite
being active against de novo BVDV infection. However, the
combination of mCyd plus interferon alpha 2b reduced virus to
undetectable levels by the second passage and clearly showed better
efficacy to either monotherapy.
[0562] In a follow up study (FIG. 14B) of MDBK cells persistently
infected with the I-N-dIns noncytopathic strain of BVDV, mCyd was
supplied at fixed doses of 0, 2, 4 and 8 .mu.M, while interferon
alpha 2b was titrated from 0 to 2,000 IU per ml. Again, interferon
alpha 2b was essentially inactive (0.1 log reduction in viral
titer), while mCyd alone inhibited BVDV (strain I-N-dIns)
propagation in a dose-dependent manner. mCyd at 8 .mu.M reduced
virus production by 6.2 log.sub.10, to almost background
levels.
EXAMPLE 35
Resistance Development
[0563] In early cell culture studies, repeated passaging of a
cytopathic strain of BVDV in MDBK cells in the presence of mCyd
failed to generate resistant mutants, suggesting that the isolation
mCyd-resistant BVDV mutants is difficult. However, studies in cell
lines persistently infected with noncytopathic forms of BVDV led to
the selection of resistant virus upon relatively prolonged
treatment with mCyd at suboptimal therapeutic concentrations of
drug (2 to 8 .mu.M, depending on the experiment). In the
representative experiment shown in FIG. 15A, the virus was no
longer detectable after two passages in the presence of 8 .mu.M
mCyd, but re-emerged by passage 6. The lower titer of the
re-emergent virus is apparent from the data: resistant virus
typically has a 10 fold or more lower titer than the wild-type
virus and was easily suppressed by co-therapy with IntronA (FIG.
15A). The phenotype of the virus that re-emerged was remarkably
different from the initial wild-type virus: as shown in FIG. 15B,
it yielded much smaller foci (typically, 3 to 10 times smaller in
diameter then those of the wild-type virus). This phenotype did not
change after prolonged passaging in culture in the presence of the
inhibitor (at least 72 days), however, it quickly reverted to the
wild-type phenotype (large foci) after the discontinuation of the
treatment.
[0564] RT-PCR sequencing of the resistant mutant was used to
identify the mutation responsible for resistance. Sequencing
efforts were focused on the NS5B RNA-dependent RNA polymerase
region of BVDV, which was assumed to be the likely target for a
nucleoside inhibitor. A specific S405T amino-acid substitution was
identified at the start of the highly conserved B domain motif of
the polymerase. The B domain is part of the polymerase active site
and is thought to be involved in nucleoside binding (Lesburg, C.
A., M. B. Cable, et al. Nature Structural Biology 1999 6(10):
937-43). Resistance to nucleosides has been mapped to this domain
for other viruses such as HBV (Ono et al, J. Clin. Invest. 2001
February; 107(4):449-55). To confirm that this mutation was
responsible for the observed resistance, the mutation was
reintroduced into the backbone of a recombinant molecular clone of
BVDV. The resulting clone was indistinguishable in phenotypic
properties from the isolated mutant virus, confirming that the
S405T mutation is responsible for resistance and that the NS5B
RNA-dependent RNA polymerase is the molecular target for mCyd. The
highly conserved nature of this motif at the nucleotide sequence
(Lai, V. C., C. C. Kao, et al. J. Virol. 1999 73(12): 10129-36) and
structural level among positive-strand RNA viruses (including HCV)
allows a prediction that the equivalent mutation in the HCV NS5B
RNA-dependent RNA polymerase would likely be S282T.
[0565] S405T mutant BVDV was refractory to mCyd up to the highest
concentrations that could be tested (EC.sub.50>32 .mu.M), but
was also significantly impaired in viability compared to wild-type
virus. As noted above, the S405T mutant exhibited a 1-2 log.sub.10
lower titer than wild-type. BVDV and produced much smaller viral
plaques. In addition, the mutant virus showed a marked reduction in
the rate of a single cycle of replication (>1000-fold lower
virus titer at 12 h), and accumulated to about 100 fold lower
levels than the wild-type virus even after 36 h of replication
(FIG. 15C). The virus also quickly reverted to wild-type virus upon
drug withdrawal. Finally, the mutant was also more sensitive
(.about.40 fold) to treatment with IFN alpha 2b than wild-type as
shown in FIG. 15D.
[0566] A second, additional mutation, C446S, was observed upon
further passaging of the S405T mutant virus in the presence of
drug. This mutation occurs immediately prior to the essential GDD
motif in the C domain of BVDV NS5B RNA-dependent RNA polymerase.
Preliminary studies suggest that a virus bearing both mutations
does not replicate significantly better than the S405T mutant,
hence the contribution of this mutation to viral fitness remains
unclear. Further studies to characterize resistance development are
ongoing.
EXAMPLE 36
In Vivo Antiviral Activity of Val-mCyd in an Animal Efficacy
Model
[0567] Chimpanzees chronically infected with HCV are the most
widely accepted animal model of HCV infection in human patients
(Lanford, R. E., C. Bigger, et al. Ilar J. 2001 42(2): 117-26;
Grakoui, A., H. L. Hanson, et al. Hepatology 2001 33(3): 489-95). A
single in vivo study of the oral administration of val-mCyd in the
chimpanzee model of chronic hepatitis C virus infection has been
conducted.
[0568] HCV genotyping on the five chimpanzees was performed by the
Southwest Foundation Primate Center as part of their mandated
internal Health and Maintenance Program, designed to ascertain the
disease status of all animals in the facility to identify potential
safety hazards to employees. The five chimpanzees used in this
study exhibited a high HCV titer in a genotyping RT PCR assay that
distinguishes genotype 1 HCV from all other genotypes, but does not
distinguish genotype 1a from 1b. This indicates that the
chimpanzees used in this study were infected with genotype 1 HCV
(HCV-1). TABLE-US-00029 TABLE 33 Summary of Val-mCyd In Vivo
Activity Study in the Chimpanzee Model of Chronic HCV Infection
Species Val-mCyd Doses Frequency/Route Study Study Description (N)
(mg/kg) (n) of Administration Endpoints One-week antiviral
Chimpanzee 10 and 20 QD .times. 7 days Serum HCV activity of mCyd
(5) (2 each) (PO) RNA, serum in chronically [equivalent
chemistries, hepatitis C virus to 8.3 and 16.6 CBCs, general
(genotype 1)- mpk of free well being, infected chimpanzees base],
and and clinical vehicle control observations (1)
Seven-Day Antiviral Activity Study in the Chimpanzee Model of
Chronic Hepatitis C Virus Infection
[0569] Four chimpanzees (2 animals per dose group at 10 mg/kg/day
or 20 mg/kg/day) received val-mCyd dihydrochloride, freshly
dissolved in a flavorful fruit drink vehicle. These doses were
equivalent to 8.3 and 16.6 mg/kg/day of the val-mCyd free base,
respectively. A fifth animal dosed with vehicle alone provided a
placebo control. The study design included three pretreatment
bleeds to establish the baseline fluctuation of viral load and
three bleeds during the one week of treatment (on days 2, 5 and 7
of therapy) to evaluate antiviral efficacy. The analysis was
completed at the end of the one-week dosing period, with no further
follow up.
HCV RNA Determination
[0570] Serum levels of HCV RNA throughout the study were determined
independently by two clinical hospital laboratories. HCV RNA was
assayed using a quantitative RT-PCR nucleic acid amplification test
(Roche Amplicor HCV Monitor Test, version 2.0). This assay has a
lower limit of detection (LLOD) of 600 IU/mL and a linear range of
600-850,000 IU/mL.
[0571] To aid in interpretation of the viral load declines seen
during therapy, emphasis was placed on determining (i) the extent
of fluctuations in baseline HCV viral load in individual animals,
and (ii) the inherent variability and reproducibility of the HCV
viral load assay. To address these issues, full viral load data
sets obtained from the two laboratories were compared. The results
from both sites were found to be closely comparable and affirmed
both the stability of the pretreatment HCV viral loads as well as
the reliability of the HCV Roche Amplicor assay. To present the
most balanced view of the study, the mean values derived by
combining both data sets were used to generate the results
presented in FIGS. 16 and 17. FIG. 16 presents the averaged data
for dose cohorts, while FIG. 17 presents the individual animal
data. The changes in viral load from baseline seen during therapy
for each animal at each site are also summarized in Table 34.
[0572] The HCV viral load analysis from the two sites revealed that
pretreatment HCV viral loads were (i) very similar among all five
animals and all 3 dose groups, and (ii) very stable over the 3-week
pretreatment period. The mean pretreatment log.sub.10 viral load
and standard deviations among the five individual animals were
5.8.+-.0.1 (site 1) and 5.6.+-.0.1 (site 2). These data indicate
that the c.v. (coefficient of variance) of the assay is only around
2% at both sites. The largest fluctuation in HCV viral load seen in
any animal during pretreatment was approximately 0.3
log.sub.10.
[0573] As seen in FIGS. 16 and 17, once a day oral delivery of
val-mCyd produced a rapid antiviral effect that was not seen for
the placebo animal, nor during the pretreatment period. Viral
titers were substantially reduced from baseline after two days of
therapy for all animals receiving val-mCyd, and tended to fall
further under continued therapy in the two treatment arms. By the
end of treatment (day 7), the mean reductions from baseline HCV
viral load were 0.83 log.sub.10 and 1.05 log.sub.10 for the 8.3 and
16.6 mg/kg/day dose groups, respectively. The titer of the placebo
animal remained essentially unchanged from baseline during the
therapy period.
[0574] An analysis of the data from the two quantification sites on
the changes in baseline HCV viral load in response to therapy is
presented in Table 34. Overall, the two data sets agree well,
confirming the reliability of the assay. With the exception of
animal 501, the difference in viral load between the two sites was
generally 0.3 log.sub.10 or less, similar to the fluctuation
observed during the pretreatment period. For animal 501, the
discrepancy was closer to 0.5 log.sub.10. The viral load drop seen
in response to therapy varied from 0.436 (animal 501, site 1) to
1.514 log.sub.10 (animal 497, site 2). The latter corresponds to a
change in HCV viral load from 535,000 (pretreatment) to 16,500 (day
7) genomes per ml. TABLE-US-00030 TABLE 34 Summary of Changes in
Baseline Log.sub.10 HCV RNA Viral Load During Therapy Dose Animal
(mpk) ID Site Day 2 Day 5 Day 7 0 499 1 -0.00041 -0.11518 0.14085 2
-0.06604 0.10612 -0.16273 8.3 500 1 -1.15634 -0.40385 -0.80507 2
-1.07902 -0.55027 -1.06259 8.3 501 1 -0.25180 -0.36179 -0.43610 2
-0.45201 -0.71254 -0.90034 16.6 497 1 -0.72148 -0.90704 -1.27723 2
-0.85561 -1.01993 -1.51351 16.6 498 1 -0.29472 -0.28139 -0.60304 2
-0.65846 -0.55966 -0.69138
Exposure of Chimpanzees to mCyd
[0575] Limited HPLC analysis were performed to determine the
concentration of mCyd attained in the sera of chimpanzees following
dosing with val-mCyd. In sera drawn 1 to 2 hours post dose on days
2 and 5 of dosing, mCyd levels were typically between 2.9 and 12.1
.mu.M (750 and 3100 ng/mL, respectively) in treated animals. No
mCyd was detected in pretreatment sera or in the placebo control
sera. Within 24 hours of the final dose, serum levels of mCyd had
fallen to 0.2 to 0.4 .mu.M (50 and 100 ng/mL, respectively). No
mUrd was detected in any sera samples although the methodology used
has a lower limit of quantification of 0.4 .mu.M (100 ng/mL) for
mUrd.
Safety of mCyd in the Chimpanzee Model of Chronic HCV Infection
[0576] Chimpanzees were monitored by trained veterinarians
throughout the study for weight loss, temperature, appetite, and
general well being, as well as for blood chemistry profile and
CBCs. No adverse events due to drug were noted. The drug appeared
to be well tolerated by all four treated animals. All five animals
lost some weight during the study and showed some aspartate
aminotransferase (AST) elevations, but these are normal occurrences
related to sedation procedures used, rather than study drug. A
single animal experienced an alanine aminotransferase (ALT) flare
in the pretreatment period prior to the start of dosing, but the
ALT levels diminished during treatment. Thus, this isolated ALT
event was not attributable to drug.
EXAMPLE 37
In Vitro Metabolism
[0577] Studies were conducted to determine the stability of
val-mCyd and mCyd in human plasma. Val-mCyd was incubated in human
plasma at 0, 21 or 37.degree. C. and samples analyzed at various
time points up to 10 hours (FIG. 18). At 37.degree. C., val-mCyd
was effectively converted to mCyd, with only 2% of the input
val-mCyd remaining after 10 hours. The in vitro half-life of
val-mCyd in human plasma at 37.degree. C. was 1.81 hours. In
studies of the in vitro stability of mCyd in human plasma, or upon
treatment with a crude preparation enriched in human
cytidine/deoxycytidine deaminase enzymes, mCyd remained essentially
unchanged and no deamination to the uridine derivative of mCyd
(mUrd) occurred after incubation at 37.degree. C. Only in rhesus
and cynomologus monkey plasma was limited deamination observed.
Incubation of mCyd at 37.degree. C. in cynomologus monkey plasma
yielded 6.7 and 13.0% of mUrd deamination product after 24 and 48
hours, respectively, under conditions where control cytidine
analogs were extensively deaminated.
[0578] In addition to the TP derivatives of mCyd and mUrd, minor
amounts of mCyd-5'-diphosphate, mCyd-DP, roughly 10% the amount of
the corresponding TP, were seen in all three cell types. Lesser
amounts of mUrd-DP were detected only in two cell types (primary
human hepatocytes and MDBK cells). No monophosphate (MP)
metabolites were detected in any cell type. There was no trace of
any intracellular mUrd and no evidence for the formation of
liponucleotide metabolites such as the 5'-diphosphocholine species
seen upon the cellular metabolism of other cytidine analogs.
[0579] FIG. 19 shows the decay profiles of mCyd-TP determined
following exposure of HepG2 cells to 10 .mu.M [.sup.3H]-mCyd for 24
hours. The apparent intracellular half-life of the mCyd-TP was
13.9.+-.2.2 hours in HepG2 cells and 7.6.+-.0.6 hours in MDBK
cells: the data were not suitable for calculating the half life of
mUrd-TP. The long half life of mCyd-TP in human hepatoma cells
supports the notion of once-a-day dosing for val-mCyd in clinical
trials for HCV therapy. Phosphorylation of mCyd occurred in a
dose-dependent manner up to 50 .mu.M drug in all three cell types,
as shown for HepG2 cells in FIG. 19C. Other than the specific
differences noted above, the phosphorylation pattern detected in
primary human hepatocytes was qualitatively similar to that
obtained using HepG2 or MDBK cells.
Contribution of mUrd
[0580] In addition to the intracellular active moiety, mCyd-TP,
cells from different species have been shown to produce variable
and lesser amounts of a second triphosphate, mUrd-TP, via
deamination of intracellular mCyd species. The activity of mUrd-TP
against BVDV NS5B RNA-dependent RNA polymerase has not been tested
to date but is planned. To date, data from exploratory cell culture
studies on the antiviral efficacy and cytotoxicity of mUrd suggest
that mUrd (a) is about 10-fold less potent than mCyd against BVDV;
(b) has essentially no antiviral activity against a wide spectrum
of other viruses; and (c) is negative when tested at high
concentrations in a variety of cytotoxicity tests (including bone
marrow assays, mitochondrial function assays and incorporation into
cellular nucleic acid). Based on these results, it appears that the
contribution of mUrd to the overall antiviral activity or
cytotoxicity profile of mCyd is likely to be minor. Extensive
toxicology coverage for the mUrd metabolite of mCyd exists from
subchronic studies conducted with val-mCyd in the monkey.
EXAMPLE 38
Cellular Pathways for Metabolic Activation
[0581] The nature of the enzyme responsible for the phosphorylation
of mCyd was investigated in substrate competition experiments.
Cytidine (Cyd) is a natural substrate of cytosolic uridine-cytidine
kinase (UCK), the pyrimidine salvage enzyme responsible for
conversion of Cyd to Cyd-5'-monophosphate (CMP). The intracellular
phosphorylation of mCyd to mCyd-TP was reduced in the presence of
cytidine or uridine in a dose-dependent fashion with EC.sub.50
values of 19.17.+-.4.67 .mu.M for cytidine and 20.92.+-.7.10 .mu.M
for uridine. In contrast, deoxycytidine, a substrate for the enzyme
deoxycytidine kinase (dCK), had little effect on the formation of
mCyd-TP with an EC.sub.50>100 .mu.M. The inhibition of mCyd
phosphorylation by both cytidine and uridine, but not
deoxycytidine, suggests that mCyd is phosphorylated by the
pyrimidine salvage enzyme, uridine-cytidine kinase (Van Rompay, A.
R., A. Norda, et al. Mol Pharmacol 2001 59(5): 1181-6). Further
studies are required to confirm the proposed role of this kinase in
the activation of mCyd.
EXAMPLE 39
Pathways for the Cellular Biosynthesis of mUrd-TP
[0582] As outlined above, mUrd-TP is a minor metabolite arising to
varying extents in cells from different species. mUrd does not
originate via extracellular deamination of mCyd since mUrd is not
seen in the cell medium which also lacks any deamination
activities. The cellular metabolism data are consistent with the
idea that mUrd-TP arises via the biotransformation of intracellular
mCyd species. Consideration of the known ribonucleoside metabolic
pathways suggests that the most likely routes involve deamination
of one of two mCyd species by two distinct deamination enzymes:
either mCyd-MP by a cytidylate deaminase (such as deoxycytidylate
deaminase, dCMPD), or of mCyd by cytidine deaminase (CD). Further
phosphorylation steps lead to mUrd-TP. These possibilities are
under further investigation.
EXAMPLE 40
Clinical Evaluation of Val-mCyd
[0583] Patients who met eligibility criteria were randomized into
the study at Baseline (Day 1), the first day of study drug
administration. Each dosing cohort was 12 patients, randomized in a
10:2 ratio to treatment with drug or matching placebo. Patients
visited the study center for protocol evaluations on Days 1, 2, 4,
8, 11, and 15. After Day 15, study drug was stopped. Thereafter,
patients attended follow-up visits on Days 16, 17, 22, and 29.
Pharmacokinetic sampling was performed on the first and last days
of treatment (Day 1 and Day 15) on all patients, under fasting
conditions.
[0584] The antiviral effect of val-mCyd was assessed by (i) the
proportion of patients with a .gtoreq.1.0 log.sub.10 decrease from
baseline in HCV RNA level at Day 15, (ii) the time to a .gtoreq.1.0
log.sub.10 decrease in serum HCV RNA level, (iii) the change in HCV
RNA level from Day 1 to Day 15, (iv) the change in HCV RNA level
from Day 1 to Day 29, (v) the proportion of patients who experience
return to baseline in serum HCV RNA level by Day 29, and (vi) the
relationship of val-mCyd dose to HCV RNA change from Day 1 to Day
15.
Clinical Pharmacokinetics of mCyd after Oral Administration of
Escalating Doses of Val-mCyd
[0585] Pharmacokinetics were evaluated over a period of 8 h after
the first dose on day 1 and after the last dose on day 15, with
24-h trough levels monitored on days 2, 4, 8, 11 and 16, and a 48-h
trough on day 17. Plasma concentrations of mCyd, mUrd and Val-mCyd
were measured by a HPLC/MS/MS methodology with a lower limit of
quantitation (LOQ) at 20 ng/ml.
[0586] The pharmacokinetics of mCyd was analyzed using a
non-compartmental approach. As presented in the tables below, the
principal pharmacokinetic parameters were comparable on day 1 and
day 15, indicative of no plasma drug accumulation after repeated
dosing. The plasma exposure also appears to be a linear function of
dose. As shown in the tables below, principal pharmacokinetic
parameters of drug exposure (Cmax and AUC) doubled as doses
escalated from 50 to 100 mg. TABLE-US-00031 TABLE 35
Pharmacokinetic parameters of mCyd at 50 mg C.sub.max T.sub.max
AUC.sub.0-inf t.sub.1/2 Parameters (ng/ml) (h) (ng/ml .times. h)
(h) Day 1 Mean 428.1 2.5 3118.7 4.1 SD 175.5 1.1 1246.4 0.6 CV %
41.0 43.2 40.0 13.8 Day 15 Mean 362.7 2.2 3168.4 4.6 SD 165.7 1.0
1714.8 1.3 CV % 45.7 46.9 54.1 28.6
[0587] TABLE-US-00032 TABLE 36 Pharmacokinetic parameters of mCyd
at 100 mg C.sub.max T.sub.max AUC.sub.0-inf t.sub.1/2 Parameters
(ng/ml) (h) (ng/ml .times. h) (h) Day 1 Mean 982.1 2.6 6901.7 4.4
SD 453.2 1.0 2445.7 1.1 CV % 46.1 36.2 35.4 25.2 Day 15 Mean 1054.7
2.0 7667.5 4.2 SD 181.0 0.0 1391.5 0.5 CV % 17.2 0.0 18.1 11.7
[0588] The mean day 1 and day 15 plasma kinetic profiles of mCyd at
50 and 100 mg are depicted in the FIG. 20.
[0589] In summary, following oral administration of val-mCyd, the
parent compound mCyd was detectable in the plasma of HCV-infected
subjects. mCyd exhibits linear plasma pharmacokinetics in these
subjects across the two dose levels thus far examined. There was no
apparent accumulation of mCyd in subjects' plasma following 15 days
of daily dosing at the doses thus far examined.
Antiviral Activity of mCyd after Oral Administration of Escalating
Doses of Val-mCyd Starting at 50 mg/day for 15 Days in HCV-Infected
Patients
[0590] Serum HCV RNA level were determined with the use of the
Amplicor HCV Monitor.TM. assay v2.0 (Roche Molecular Systems,
Branchburg, N.J., USA), which utilizes polymerase chain reaction
(PCR) methods. The lower limit of quantification (LLOQ) with this
assay was estimated to be approximately 600 IU/mL and the upper
limit of quantification (ULOQ) with this assay was estimated to be
approximately 500,000 IU/mL.
[0591] Serum samples for HCV RNA were obtained at screening (Day
-42 to -7) to determine eligibility for the study. The Screening
serum HCV RNA values must be .gtoreq.5 log.sub.10 IU/mL by the
Amplicor HBV Monitor.TM. assay at the central study laboratory.
[0592] During the study period, serum samples for HCV RNA were
obtained at Baseline (Day 1), and at every protocol-stipulated
post-Baseline study visit (Days 2, 4, 8, 11, 15, 16, 17, 22, and
29). Serum samples for HCV RNA were also collected during
protocol-stipulated follow-up visits for patients prematurely
discontinued from the study.
[0593] The antiviral activity associated with the first two cohorts
(50 and 100 mg per day) in the ongoing study is summarized in the
following tables and graphs. Although the duration of dosing was
short (15 days) and the initial dose levels low, there were already
apparent effects on the levels of HCV RNA in the plasma of infected
patients. TABLE-US-00033 TABLE 37 Summary Statistics of HCV RNA in
Log.sub.10 Scale Day Treatment -1 1 2 4 8 11 15 16 17 22 29 Placebo
N 6 5 5 4 4 4 4 4 3 4 3 Median 6.45 6.25 6.25 6.52 6.42 6.28 6.58
6.51 6.64 6.35 6.61 Mean 6.45 6.28 6.40 6.48 6.36 6.34 6.54 6.52
6.50 6.40 6.40 StdErr 0.25 0.12 0.15 0.18 0.24 0.16 0.11 0.19 0.31
0.23 0.30 50 mg N 10 10 10 10 10 10 10 10 10 10 10 Median 6.81 6.69
6.58 6.55 6.56 6.46 6.57 6.45 6.54 6.73 6.67 Mean 6.72 6.72 6.60
6.56 6.62 6.47 6.57 6.57 6.54 6.64 6.71 StdErr 0.11 0.11 0.12 0.06
0.10 0.09 0.08 0.11 0.08 0.10 0.09 100 mg N 11 10 10 10 9 10 10 9 9
10 4 Median 6.75 6.93 6.80 6.46 6.59 6.56 6.41 6.40 6.72 6.66 6.71
Mean 6.60 6.68 6.52 6.43 6.42 6.36 6.30 6.23 6.65 6.53 6.67 StdErr
0.16 0.24 0.23 0.21 0.24 0.22 0.22 0.23 0.16 0.18 0.17
[0594] TABLE-US-00034 TABLE 38 Summary Statistics of Change From
Baseline (Day 1) in Log.sub.10 HCV RNA Day Treatment 2 4 8 11 15 16
17 22 29 Placebo N 5 4 4 4 4 4 3 4 3 Median 0.17 0.21 0.15 0.08
0.31 0.21 0.27 0.17 0.09 Mean 0.12 0.22 0.10 0.08 0.28 0.25 0.15
0.14 0.09 StdErr 0.09 0.12 0.16 0.06 0.15 0.10 0.18 0.09 0.16 50 mg
N 10 10 10 10 10 10 10 10 10 Median -0.07 -0.13 -0.06 -0.26 -0.10
-0.13 -0.21 -0.09 -0.04 Mean -0.13 -0.16 -0.11 -0.26 -0.15 -0.15
-0.18 -0.09 -0.01 StdErr 0.05 0.07 0.05 0.06 0.08 0.05 0.07 0.06
0.10 100 mg N 10 10 9 10 10 9 9 10 4 Median -0.12 -0.24 -0.20 -0.28
-0.43 -0.49 -0.24 -0.19 -0.12 Mean -0.16 -0.25 -0.21 -0.32 -0.38
-0.39 -0.18 -0.15 0.13 StdErr 0.07 0.10 0.16 0.13 0.12 0.14 0.15
0.13 0.28
[0595] The clinical evaluation of val-mCyd in the tested patients
is shown in FIG. 21. This figure depicts the median change from
baseline in Log.sub.10 HCV RNA by visit.
EXAMPLE 41
Evaluation of Test Compounds
[0596] Several of the compounds described herein were tested in the
BVDV cell protection assay described above. FIG. 22 is a table of
the EC.sub.50 and CC.sub.50 of representative compounds in a BVDV
cell protection assay, to show the efficacy of the compounds.
[0597] This invention has been described with reference to its
preferred embodiments. Variations and modifications of the
invention, will be obvious to those skilled in the art from the
foregoing detailed description of the invention. It is intended
that all of these variations and modifications be included within
the scope of this invention. TABLE-US-00035 LENGTHY TABLE
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TABLE-US-00059 LENGTHY TABLE The patent application contains a
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An electronic copy of the table will also be available from the
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1.19(b)(3).
Sequence CWU 1
1
6 1 21 RNA artificial Synthetic RNA template (21mer) with a 6G
residue 1 cauaugcucu uaaucuuuuc c 21 2 21 RNA artificial Synthetic
RNA template (21mer) with a 6G and 7G residue 2 cauauggucu
uaaucuuuuc c 21 3 21 RNA artificial Synthetic RNA template (21mer)
with a 6G and 9G residue 3 cauaugcugu uaaucuuuuc c 21 4 21 RNA
artificial Synthetic RNA template (21mer) with a 6C and 7G residue
4 cauaucgucu uaaucuuuuc c 21 5 21 RNA artificial Synthetic RNA
template (21mer) with a 6C and 9G residue 5 cauauccugu uaaucuuuuc c
21 6 21 RNA artificial Synthetic RNA template (21mer) with a 6C and
15G residue 6 cauauccucu uaauguuuuc c 21
* * * * *
References