U.S. patent application number 11/200499 was filed with the patent office on 2005-12-08 for nucleoside derivatives as inhibitors of rna-dependent rna viral polymerase.
Invention is credited to Bhat, Balkrishen, Cook, Phillip Dan, Eldrup, Anne B., LaFemina, Robert L., Prakash, Thazha P., Rutkowski, Carrie A., Simcoe, Amy L., Valenciano, Mario A..
Application Number | 20050272676 11/200499 |
Document ID | / |
Family ID | 27500770 |
Filed Date | 2005-12-08 |
United States Patent
Application |
20050272676 |
Kind Code |
A1 |
Bhat, Balkrishen ; et
al. |
December 8, 2005 |
Nucleoside derivatives as inhibitors of RNA-dependent RNA viral
polymerase
Abstract
The present invention provides nucleoside compounds and certain
derivatives thereof which are inhibitors of RNA-dependent RNA viral
polymerase. These compounds are inhibitors of RNA-dependent RNA
viral replication and are useful for the treatment of RNA-dependent
RNA viral infection. They are particularly useful as inhibitors of
hepatitis C virus (HCV) NS5B polymerase, as inhibitors of HCV
replication, and/or for the treatment of hepatitis C infection. The
invention also describes pharmaceutical compositions containing
such nucleoside compounds alone or in combination with other agents
active against RNA-dependent RNA viral infection, in particular HCV
infection. Also disclosed are methods of inhibiting RNA-dependent
RNA polymerase, inhibiting RNA-dependent RNA viral replication,
and/or treating RNA-dependent RNA viral infection with the
nucleoside compounds of the present invention.
Inventors: |
Bhat, Balkrishen; (Carlsbad,
CA) ; Eldrup, Anne B.; (Encinitas, CA) ;
Prakash, Thazha P.; (Carlsbad, CA) ; Cook, Phillip
Dan; (Fallbrook, CA) ; LaFemina, Robert L.;
(Schwenksville, PA) ; Simcoe, Amy L.;
(Collegeville, PA) ; Rutkowski, Carrie A.;
(Lansdale, PA) ; Valenciano, Mario A.; (Scotch
Plains, NJ) |
Correspondence
Address: |
MERCK AND CO., INC
P O BOX 2000
RAHWAY
NJ
07065-0907
US
|
Family ID: |
27500770 |
Appl. No.: |
11/200499 |
Filed: |
August 9, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11200499 |
Aug 9, 2005 |
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10431657 |
May 7, 2003 |
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10431657 |
May 7, 2003 |
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10052318 |
Jan 18, 2002 |
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6777395 |
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60263313 |
Jan 22, 2001 |
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60282069 |
Apr 6, 2001 |
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60299320 |
Jun 19, 2001 |
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60344528 |
Oct 25, 2001 |
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Current U.S.
Class: |
514/43 ;
514/265.1; 536/27.2; 544/280 |
Current CPC
Class: |
A61K 9/4858 20130101;
C07D 487/04 20130101; C07H 19/20 20130101; A61K 38/21 20130101;
A61K 45/06 20130101; C07H 19/00 20130101; C07H 19/14 20130101; A61K
31/7064 20130101; C07H 19/12 20130101; C07H 19/06 20130101; C07D
473/00 20130101; Y02P 20/582 20151101; C07H 19/10 20130101; A61K
31/7076 20130101; A61P 1/16 20180101; A61P 31/00 20180101; A61K
31/7056 20130101; A61P 43/00 20180101; C07H 19/16 20130101; A61P
31/14 20180101; A61K 31/7064 20130101; A61K 2300/00 20130101; A61K
31/7056 20130101; A61K 2300/00 20130101; A61K 38/21 20130101; A61K
2300/00 20130101 |
Class at
Publication: |
514/043 ;
536/027.2; 514/265.1; 544/280 |
International
Class: |
A61K 031/7076; A61K
031/519; C07H 019/00; C07D 487/02 |
Claims
What is claimed is:
1. A compound of the structural formula: 183or a pharmaceutically
aceptable salt thereof; wherein R.sup.a and R.sup.h are each
independently selected from the group consisting of hydrogen,
cyano, azido, halogen, hydroxy, mercapto, amino, C.sub.1-4 alkoxy,
C.sub.2-4 alkenyl, C.sub.2-4 alkynyl, and C.sub.1-4 alkyl, wherein
alkyl is unsubstituted or substituted with hydroxy, amino,
C.sub.1-4 alkoxy, C.sub.1-4 alkylthio, or one to three fluorine
atoms; R.sup.b is C.sub.2-4 alkenyl, C.sub.2-4 alkynyl, or
C.sub.1-4 alkyl, wherein alkyl is unsubstituted or substituted with
hydroxy, amino, C.sub.1-4 alkoxy, C.sub.1-4 alkylthio, or one to
three fluorine atoms; R.sup.c is hydrogen, fluorine, hydroxy,
marcapto, C.sub.1-4 alkoxy, or C.sub.1-4 alkyl; or R.sup.b and
R.sup.c together with the carbon atom to which they are attached
form a 3- to 6-membered saturated monocyclic ring system optionally
containing a heteroatom selected from O, S, and NC.sub.0-4 alkyl;
R.sup.d is hydrogen, cyano, nitro, C.sub.1-3 alkyl, NHCONH.sub.2,
CONR.sup.jR.sup.j, CSNR.sup.jR.sup.j, COOR.sup.j,
C(.dbd.NH)NH.sub.2, hydroxy, C.sub.1-3 alkoxy, amino, C.sub.1-4
alkylamino, di(C.sub.1-4 alkyl)amino, halogen, (1,3-oxazol-2-yl),
(1,3-thiazol-2-yl), or (imidazol-2-yl); wherein alkyl is
unsubstituted or substituted with one to three groups independently
selected from halogen, amino, hydroxy, carboxy, and C.sub.1-3
alkoxy; R.sup.e and R.sup.f are each independently hydrogen,
hydroxy, halogen, C.sub.1-4 alkoxy, amino, C.sub.1-4 alkylamino,
di(C.sub.1-4 alkyl)amino, C.sub.3-6 cycloalkylamino, di(C.sub.3-6
cycloalkyl)amino, or C.sub.4-6 cycloheteroalkyl, unsubstituted or
substituted with one to two groups independently selected from
halogen, hydroxy, amino, C.sub.1-4 alkyl, and C.sub.1-4 alkoxy;
R.sup.g is hydrogen, C.sub.1-4 alkyl, C.sub.2-4 alkynyl, halogen,
cyano, carboxy, C.sub.1-4 alkyloxycarbonyl, azido, amino, C.sub.1-4
alkylamino, di(C.sub.1-4 alkyl)amino, hydroxy, C.sub.1-6 alkoxy,
C.sub.1-6 alkylthio, C.sub.1-6 alkylsulfonyl, (C.sub.1-4
alkyl).sub.0-2 aminomethyl, or C.sub.4-6 cycloheteroalkyl,
unsubstituted or substituted with one to two groups independently
selected from halogen, hydroxy, amino, C.sub.1-4 alkyl, and
C.sub.1-4 alkoxy; R.sup.i is hydrogen, C.sub.1-10 alkylcarbonyl,
P.sub.3O.sub.9H.sub.4, P.sub.2O.sub.6H.sub.3, or
P(O)R.sup.mR.sup.n; each R.sup.j is independently hydrogen or
C.sub.1-6 alkyl; R.sup.k and R.sup.l are each independently
hydrogen, methyl, hydroxymethyl, or fluoromethyl; and R.sup.m and
R.sup.n are each independently hydroxy,
OCH.sub.2CH.sub.2SC(.dbd.O)C.sub.1-4 alkyl,
OCH.sub.2O(C.dbd.O)OC.sub.1-4 alkyl, NRCHMeCO.sub.2Me,
OCH(C.sub.1-4 alkyl)O(C.dbd.O)C.sub.1-4 alkyl, 184with the proviso
that when R.sup.a and R.sup.c are .alpha.-hydroxy, R.sup.e is
amino, R.sup.b is .beta.-methyl and R.sup.h is hydrogen or R.sup.h
is .beta.-methyl and R.sup.b is hydrogen, and R.sup.f, R.sup.g,
R.sup.i, R.sup.k, and R.sup.l are hydrogen, then R.sup.d is not
cyano or CONH.sub.2.
2. The compound of claim 1 of the structural formula: 185wherein
R.sup.a is hydrogen, halogen, hydroxy, amino, or C.sub.1-4 alkoxy;
R.sup.b is C.sub.1-3 alkyl, wherein alkyl is optionally substituted
with hydroxy, amino, C.sub.1-3 alkoxy, C.sub.1-3 alkylthio, or one
to three fluorine atoms; R.sup.c is hydroxy, fluoro, or C.sub.1-3
alkoxy; Rd is hydrogen, cyano, methyl, halogen, or CONH.sub.2;
R.sup.g is hydrogen, amino, or C.sub.1-4 alkylamino; R.sup.i is
hydrogen, P.sub.3O.sub.9H.sub.4, P.sub.2O.sub.6H.sub.3, or
PO.sub.3H.sub.2; and R.sup.e and R.sup.f are each independently
hydrogen, halogen, hydroxy, amino, C.sub.1-4 alkylamino,
di(C.sub.1-4 alkyl)amino, or C.sub.3-6 cycloalkylamino; with the
proviso that when R.sup.a and R.sup.c are .alpha.-hydroxy, R.sup.e
is amino, R.sup.b is .beta.-methyl, and R.sup.f, R.sup.g, and
R.sup.i are hydrogen, then R.sup.d is not cyano or CONH.sub.2.
3. The compound of claim 2 wherein R.sup.b is methyl, fluoromethyl,
hydroxymethyl, difluoromethyl, trifluoromethyl, or aminomethyl;
R.sup.c is hydroxy, fluoro, or methoxy; R.sup.a is hydrogen,
fluoro, hydroxy, amino, or methoxy; R.sup.i is hydrogen or
P.sub.3O.sub.9H.sub.4; R.sup.g is hydrogen or amino; R.sup.d is
hydrogen, cyano, methyl, halogen, or CONH.sub.2; and R.sup.e and
R.sup.f are each independently hydrogen, fluoro, hydroxy, or amino;
with the proviso that when R.sup.b is .beta.-methyl, R.sup.a and
R.sup.c are .alpha.-hydroxy, R.sup.e is amino, and R.sup.f,
R.sup.g, and R.sup.i are hydrogen, then R.sup.d is not cyano or
CONH.sub.2.
4. The compound of claim 1 selected from the group consisting of:
4-amino-7-(2-C-methyl-.beta.-D-arabinofuranosyl)-7H-pyrrolo[2,3-d]pyrimid-
ine,
4-amino-7-(2-C-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimi-
dine,
4-methylamino-7-(2-C-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d-
]pyrimidine,
4-dimethylamino-7-(2-C-methyl-.beta.-D-ribofuranosyl)-7H-pyrr-
olo[2,3-d]pyrimidine,
4-cyclopropylamino-7-(2-C-methyl-.beta.-D-ribofurano-
syl)-7H-pyrrolo[2,3-d]pyrimidine,
4-amino-7-(2-C-vinyl-.beta.-D-ribofurano-
syl)-7H-pyrrolo[2,3-d]pyrimidine,
4-amino-7-(2-C-hydroxymethyl-.beta.-D-ri-
bofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine,
4-amino-7-(2-C-fluoromethyl-.bet-
a.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine,
4-amino-5-methyl-7-(2-C-m-
ethyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine,
4-amino-7-(2-C-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine-
-5-carboxylic acid,
4-amino-5-bromo-7-(2-C-methyl-.beta.-D-ribofuranosyl)--
7H-pyrrolo[2,3-d]pyrimidine,
4-amino-5-chloro-7-(2-C-methyl-.beta.-D-ribof-
uranosyl)-7H-pyrrolo[2,3-d]pyrimidine,
4-amino-5-fluoro-7-(2-C-methyl-.bet-
a.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine,
2,4-diamino-7-(2-C-methyl-
-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine,
2-amino-7-(2-C-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine-
,
2-amino-4-cyclopropylamino-7-(2-C-methyl-.beta.-D-ribofuranosyl)-7H-pyrr-
olo[2,3-d]pyrimidine,
2-amino-7-(2-C-methyl-.beta.-D-ribofuranosyl)-7H-pyr-
rolo[2,3-d]pyrimidin-4(3H)-one,
4-amino-7-(2-C-methyl-.beta.-D-ribofuranos-
yl)-7H-pyrrolo[2,3-d]pyrimidine,
4-amino-7-(2-C,2-O-dimethyl-.beta.-D-ribo-
furanosyl)-7H-pyrrolo[2,3-d]pyrimidine,
7-(2-C-methyl-.beta.-D-ribofuranos-
yl)-7H-pyrrolo[2,3-d]pyrimidin-4(3H)-one,
2-amino-5-methyl-7-(2-C,2-O-dime-
thyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidin-4(3H)-one,
4-amino-7-(3-deoxy-2-C-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]py-
rimidine,
4-amino-7-(3-deoxy-2-C-methyl-.beta.-D-arabinofuranosyl)-7H-pyrr-
olo[2,3-d]-pyrimidine,
4-amino-2-fluoro-7-(2-C-methyl-.beta.-D-ribofuranos-
yl)-7H-pyrrolo[2,3-d]pyrimidine,
4-amino-7-(3-C-methyl-.beta.-D-ribofurano-
syl)-7H-pyrrolo[2,3-d]pyrimidine,
4-amino-7-(3-C-methyl-.beta.-D-xylofuran-
osyl)-7H-pyrrolo[2,3-d]pyrimidine,
4-amino-7-(2,4-di-C-methyl-.beta.-D-rib-
ofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine, and
4-amino-7-(3-deoxy-3-fluoro-2-
-C-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine; and
the corresponding 5'-triphosphates; or a pharmaceutically
acceptable salt thereof.
5. The compound of claim 4 selected from the group consisting of:
4-amino-7-(2-C-methyl-.beta.-D-arabinofuranosyl)-7H-pyrrolo[2,3-d]pyrimid-
ine,
4-amino-7-(2-C-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimi-
dine,
4-amino-7-(2-C-fluoromethyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d-
]pyrimidine,
4-amino-5-methyl-7-(2-C-methyl-.beta.-D-ribofuranosyl)-7H-pyr-
rolo[2,3-d]pyrimidine,
4-amino-5-bromo-7-(2-C-methyl-.beta.-D-ribofuranosy-
l)-7H-pyrrolo[2,3-d]pyrimidine,
4-amino-5-chloro-7-(2-C-methyl-.beta.-D-ri-
bofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine,
4-amino-5-fluoro-7-(2-C-methyl-.-
beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine, and
4-amino-7-(2-C,2-O-dimethyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyri-
midine, and the corresponding 5'-triphosphates; or a
pharmaceutically acceptable salt thereof.
6. The compound of claim 5 which is
4-amino-7-(2-C-methyl-.beta.-D-arabino-
furanosyl)-7H-pyrrolo[2,3-d]pyrimidine; or a pharmaceutically
acceptable salt thereof.
7. The compound of claim 5 which is
4-amino-7-(2-C-methyl-.beta.-D-ribofur-
anosyl)-7H-pyrrolo[2,3-d]pyrimidine; or a pharmaceutically
acceptable salt thereof.
8. The compound of claim 5 which is
4-amino-7-(2-C-fluoromethyl-.beta.-D-r-
ibofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine; or a pharmaceutically
acceptable salt thereof.
9. The compound of claim 5 which is
4-amino-5-chloro-7-(2-C-methyl-.beta.--
D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine; or a pharmaceutically
acceptable salt thereof.
10. The compound of claim 5 which is
4-amino-5-bromo-7-(2-C-methyl-.beta.--
D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine; or a pharmaceutically
acceptable salt thereof.
11. The compound of claim 5 which is
4-amino-5-fluoro-7-(2-C-methyl-.beta.-
-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine; or a
pharmaceutically acceptable salt thereof.
12. A pharmaceutical composition comprising a compound of claim 1
and a pharmaceutically acceptable carrier.
13. The pharmaceutical composition of claim 12 useful for
inhibiting RNA-dependent RNA viral polymerase, inhibiting
RNA-dependent RNA replication, and/or treating RNA-dependent RNA
viral infection.
14. The pharmaceutical composition of claim 13 wherein said
RNA-dependent RNA viral polymerase is HCV NS5B polymerase, said
RNA-dependent RNA viral replication is HCV replication, and said
RNA-dependent RNA viral infection is HCV infection.
15. A method of inhibiting RNA-dependent RNA viral polymerase
and/or inhibiting RNA-dependent RNA viral replication comprising
administering to a mammal in need of such inhibition an effective
amount of a compound according to claim 1.
16. The method of claim 15 wherein said RNA-dependent RNA viral
polymerase is HCV NS5B polymerase and said RNA-dependent RNA viral
replication is HCV viral replication.
17. A method of treating RNA-dependent RNA viral infection
comprising administering to a mammal in need of such treatment an
effective amount of a compound according to claim 1.
18. The method of claim 17 wherein said RNA-dependent RNA viral
infection is HCV infection.
19. The method of claim 18 in combination with a therapeutically
effective amount of another agent active against HCV.
20. The method of claim 19 wherein said agent active against HCV is
ribavirin; levovirin; thymosin alpha-1; an inhibitor of NS3 serine
protease; an inhibitor of inosine monophosphate dehydrogenase;
interferon-.alpha. or pegylated interferon-.alpha., alone or in
combination with ribavirin or levovirin.
21. The method of claim 20 wherein said agent active against HCV is
interferon-.alpha. or pegylated interferon-.alpha., alone or in
combination with ribavirin or levovirin.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present invention is related to U.S. provisional
application Ser. No. 60/263,313, filed Jan. 22, 2001; 60/282,069,
filed Apr. 6, 2001; 60/299,320, filed Jun. 19, 2001; and 60/344,528
filed Oct. 25, 2001; the contents of each of which are hereby
incorporated by reference.
FIELD OF THE INVENTION
[0002] The present invention provides nucleoside compounds and
certain derivatives thereof which are inhibitors of RNA-dependent
RNA viral polymerase. These compounds are inhibitors of
RNA-dependent RNA viral replication and are useful for the
treatment of RNA-dependent RNA viral infection. They are
particularly useful as inhibitors of hepatitis C virus (HCV) NS5B
polymerase, as inhibitors of HCV replication, and for the treatment
of hepatitis C infection.
BACKGROUND OF THE INVENTION
[0003] Hepatitis C virus (HCV) infection is a major health problem
that leads to chronic liver disease, such as cirrhosis and
hepatocellular carcinoma, in a substantial number of infected
individuals, estimated to be 2-15% of the world's population. There
are an estimated 4.5 million infected people in the United States
alone, according to the U.S. Center for Disease Control. According
to the World Health Organization, there are more than 200 million
infected individuals worldwide, with at least 3 to 4 million people
being infected each year. Once infected, about 20% of people clear
the virus, but the rest harbor HCV the rest of their lives. Ten to
twenty percent of chronically infected individuals eventually
develop liver-destroying cirrhosis or cancer. The viral disease is
transmitted parenterally by contaminated blood and blood products,
contaminated needles, or sexually and vertically from infected
mothers or carrier mothers to their off-spring. Current treatments
for HCV infection, which are restricted to immunotherapy with
recombinant interferon-.alpha. alone or in combination with the
nucleoside analog ribavirin, are of limited clinical benefit.
Moreover, there is no established vaccine for HCV. Consequently,
there is an urgent need for improved therapeutic agents that
effectively combat chronic HCV infection. The state of the art in
the treatment of HCV infection has been reviewed, and reference is
made to the following publications: B. Dymock, et al., "Novel
approaches to the treatment of hepatitis C virus infection,"
Antiviral Chemistry & Chemotherapy, 11: 79-96 (2000); H. Rosen,
et al., "Hepatitis C virus: current understanding and prospects for
future therapies," Molecular Medicine Today, 5: 393-399 (1999); D.
Moradpour, et al., "Current and evolving therapies for hepatitis
C," European J. Gastroenterol. Hepatol., 11: 1189-1202 (1999); R.
Bartenschlager, "Candidate Targets for Hepatitis C Virus-Specific
Antiviral Therapy," Intervirology, 40: 378-393 (1997); G. M. Lauer
and B. D. Walker, "Hepatitis C Virus Infection," N. Engl. J. Med.,
345: 41-52 (2001); B. W. Dymock, "Emerging therapies for hepatitis
C virus infection," Emerging Drugs, 6: 13-42 (2001); and C. Crabb,
"Hard-Won Advances Spark Excitement about Hepatitis C," Science:
506-507 (2001); the contents of all of which are incorporated by
reference herein in their entirety.
[0004] Different approaches to HCV therapy have been taken, which
include the inhibition of viral serine proteinase (NS3 protease),
helicase, and RNA-dependent RNA polymerase (NS5B), and the
development of a vaccine.
[0005] The HCV virion is an enveloped positive-strand RNA virus
with a single oligoribonucleotide genomic sequence of about 9600
bases which encodes a polyprotein of about 3,010 amino acids. The
protein products of the HCV gene consist of the structural proteins
C, E1, and E2, and the non-structural proteins NS2, NS3, NS4A and
NS4B, and NS5A and NS5B. The nonstructural (NS) proteins are
believed to provide the catalytic machinery for viral replication.
The NS3 protease releases NS5B, the RNA-dependent RNA polymerase
from the polyprotein chain. HCV NS5B polymerase is required for the
synthesis of a double-stranded RNA from a single-stranded viral RNA
that serves as a template in the replication cycle of HCV. NS5B
polymerase is therefore considered to be an essential component in
the HCV replication complex [see K. Ishi, et al., "Expression of
Hepatitis C Virus NS5B Protein: Characterization of Its RNA
Polymerase Activity and RNA Binding," Hepatology, 29: 1227-1235
(1999) and V. Lohmann, et al., "Biochemical and Kinetic Analyses of
NS5B RNA-Dependent RNA Polymerase of the Hepatitis C Virus,"
Virology, 249: 108-118 (1998)]. Inhibition of HCV NS5B polymerase
prevents formation of the double-stranded HCV RNA and therefore
constitutes an attractive approach to the development of
HCV-specific antiviral therapies.
[0006] It has now been found that nucleoside compounds of the
present invention and certain derivatives thereof are potent
inhibitors of RNA-dependent RNA viral replication and in particular
HCV replication. The 5'-triphosphate derivatives of the nucleoside
compounds are inhibitors of RNA-dependent RNA viral polymerase and
in particular HCV NS5B polymerase. The instant nucleoside compounds
and derivatives thereof are useful to treat RNA-dependent RNA viral
infection and in particular HCV infection.
[0007] It is therefore an object of the present invention to
provide nucleoside compounds and certain derivatives thereof which
are useful as inhibitors of RNA-dependent RNA viral polymerase and
in particular as inhibitors of HCV NS5B polymerase.
[0008] It is another object of the present invention to provide
nucleoside derivatives which are useful as inhibitors of the
replication of an RNA-dependent RNA virus and in particular as
inhibitors of the replication of hepatitis C virus.
[0009] It is another object of the present invention to provide
nucleoside compounds and certain derivatives which are useful in
the treatment of RNA-dependent RNA viral infection and in
particular in the treatment of HCV infection.
[0010] It is another object of the present invention to provide
pharmaceutical compositions comprising the novel compounds of the
present invention in association with a pharmaceutically acceptable
carrier.
[0011] It is another object of the present invention to provide
pharmaceutical compositions comprising the nucleoside compounds and
derivatives thereof for use as inhibitors of RNA-dependent RNA
viral polymerase and in particular as inhibitors of HCV NS5B
polymerase.
[0012] It is another object of the present invention to provide
pharmaceutical compositions comprising the nucleoside compounds and
derivatives thereof for use as inhibitors of RNA-dependent RNA
viral replication and in particular as inhibitors of HCV
replication.
[0013] It is another object of the present invention to provide
pharmaceutical compositions comprising the nucleoside compounds and
derivatives thereof for use in the treatment of RNA-dependent RNA
viral infection and in particular in the treatment of HCV
infection.
[0014] It is another object of the present invention to provide
pharmaceutical compositions comprising the nucleoside compounds and
derivatives thereof in combination with other agents active against
an RNA-dependent RNA virus and in particular against HCV.
[0015] It is another object of the present invention to provide
methods for the inhibition of RNA-dependent RNA viral polymerase
and in particular for the inhibition of HCV NS5B polymerase.
[0016] It is another object of the present invention to provide
methods for the inhibition of RNA-dependent RNA viral replication
and in particular for the inhibition of HCV replication.
[0017] It is another object of the present invention to provide
methods for the treatment of RNA-dependent RNA viral infection and
in particular for the treatment of HCV infection.
[0018] It is another object of the present invention to provide
methods for the treatment of RNA-dependent RNA viral infection in
combination with other agents active against RNA-dependent RNA
virus and in particular for the treatment of HCV infection in
combination with other agents active against HCV.
[0019] It is another object of the present invention to provide
nucleoside compounds and certain derivatives thereof and their
pharmaceutical compositions for use as a medicament for the
inhibition of RNA-dependent RNA viral replication and/or the
treatment of RNA-dependent RNA viral infection and in particular
for the inhibition of HCV replication and/or the treatment of HCV
infection.
[0020] It is another object of the present invention to provide for
the use of the nucleoside compounds and certain derivatives thereof
of the present invention and their pharmaceutical compositions for
the manufacture of a medicament for the inhibition of RNA-dependent
RNA viral replication and/or the treatment of RNA-dependent RNA
viral infection and in particular for the inhibition of HCV
replication and/or the treatment of HCV infection.
[0021] These and other objects will become readily apparent from
the detailed description which follows.
SUMMARY OF THE INVENTION
[0022] The present invention provides a method for inhibiting
RNA-dependent RNA viral polymerase, a method for inhibiting
RNA-dependent RNA viral replication, and/or a method for treating
RNA-dependent viral infection in a mammal in need thereof,
comprising administering to the mammal a therapeutically effective
amount of a compound of structural formula I which is of the
stereochemical configuration: 1
[0023] or a pharmaceutically acceptable salt thereof;
[0024] wherein B is selected from the group consisting of 2
[0025] A, G, and L are each independently CH or N;
[0026] D is N, CH, C--CN, C--NO.sub.2, C--C.sub.1-3 alkyl,
C--NHCONH.sub.2, C--CONR.sup.11R.sup.11, C--CSNR.sup.11R.sup.11,
C--COOR.sup.11, C--C(.dbd.NH)NH.sub.2, C-hydroxy, C--C.sub.1-3
alkoxy, C-amino, C--C.sub.1-4 alkylamino, C-di(C.sub.1-4
alkyl)amino, C-halogen, C-(1,3-oxazol-2-yl), C-(1,3-thiazol-2-yl),
or C-(imidazol-2-yl); wherein alkyl is unsubstituted or substituted
with one to three groups independently selected from halogen,
amino, hydroxy, carboxy, and C.sub.1-3 alkoxy;
[0027] E is N or CR.sup.5;
[0028] W is O or S;
[0029] Y is H, C.sub.1-10 alkylcarbonyl, P.sub.3O.sub.9H.sub.4,
P.sub.2O.sub.6H.sub.3, or P(O)R.sup.9R.sup.10;
[0030] R.sup.1 is hydrogen, C.sub.2-4 alkenyl, C.sub.2-4 alkynyl,
or C.sub.1-4 alkyl optionally substituted with amino, hydroxy, or 1
to 3 fluorine atoms and one of R.sup.2 and R.sup.3 is hydroxy or
C.sub.1-4 alkoxy and the other of R.sup.2 and R.sup.3 is selected
from the group consisting of
[0031] hydrogen,
[0032] hydroxy,
[0033] halogen,
[0034] C.sub.1-4 alkyl, optionally substituted with 1 to 3 fluorine
atoms,
[0035] C.sub.1-10 alkoxy, optionally substituted with C.sub.1-3
alkoxy or 1 to 3 fluorine atoms,
[0036] C.sub.2-6 alkenyloxy,
[0037] C.sub.1-4 alkylthio,
[0038] C.sub.1-8 alkylcarbonyloxy,
[0039] aryloxycarbonyl,
[0040] azido,
[0041] amino,
[0042] C.sub.1-4 alkylamino, and
[0043] di(C.sub.1-14 alkyl)amino; or
[0044] R.sup.2 is hydrogen, C.sub.2-4 alkenyl, C.sub.2-4 alkynyl,
or C.sub.1-4alkyl optionally substituted with amino, hydroxy, or 1
to 3 fluorine atoms and one of R.sup.1 and R.sup.3 is hydroxy or
C.sub.1-4 alkoxy and the other of R.sup.1 and R.sup.3 is selected
from the group consisting of
[0045] hydrogen,
[0046] hydroxy,
[0047] halogen,
[0048] C.sub.1-4 alkyl, optionally substituted with 1 to 3 fluorine
atoms,
[0049] C.sub.1-10 alkoxy, optionally substituted with hydroxy,
C.sub.1-3 alkoxy, carboxy, or 1 to 3 fluorine atoms,
[0050] C.sub.2-6 alkenyloxy,
[0051] C.sub.1-4 alkylthio,
[0052] C.sub.1-8 alkylcarbonyloxy,
[0053] aryloxycarbonyl,
[0054] azido,
[0055] amino,
[0056] C.sub.1-4 alkylamino, and
[0057] di(C.sub.1-4 alkyl)amino; or
[0058] R.sup.1 and R.sup.2 together with the carbon atom to which
they are attached form a 3- to 6-membered saturated monocyclic ring
system optionally containing a heteroatom selected from O, S, and
NC.sub.0-4 alkyl;
[0059] R.sup.4 and R.sup.6 are each independently H, OH, SH,
NH.sub.2, C.sub.1-4 alkylamino, di(C.sub.1-4 alkyl)amino, C.sub.3-6
cycloalkylamino, halogen, C.sub.1-4 alkyl, C.sub.1-4 alkoxy, or
CF.sub.3;
[0060] R.sup.5 is H, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6
alkynyl, C.sub.1-4 alkylamino, CF.sub.3, or halogen;
[0061] R.sup.14 is H, CF.sub.3, C.sub.1-4 alkyl, amino, C.sub.1-4
alkylamino, C.sub.3-6 cycloalkylamino, or di(C.sub.1-4
alkyl)amino;
[0062] R.sup.7 is hydrogen, amino, C.sub.1-4 alkylamino, C.sub.3-6
cycloalkylamino, or di(C.sub.1-4 alkyl)amino;
[0063] each R.sup.11 is independently H or C.sub.1-6 alkyl;
[0064] R.sup.8 is H, halogen, CN, carboxy, C.sub.1-4
alkyloxycarbonyl, N.sub.3, amino, C.sub.1-4 alkylamino,
di(C.sub.1-4 alkyl)amino, hydroxy, C.sub.1-6 alkoxy, C.sub.1-6
alkylthio, C.sub.1-6 alkylsulfonyl, or (C.sub.1-4 alkyl).sub.0-2
aminomethyl;
[0065] R.sup.12 and R.sup.13 are each independently hydrogen,
methyl, hydroxymethyl, or fluoromethyl; and
[0066] R.sup.9 and R.sup.10 are each independently hydroxy,
OCH.sub.2CH.sub.2SC(.dbd.O)C.sub.1-4 alkyl,
OCH.sub.2O(C.dbd.O)OC.sub.1-4 alkyl, NHCHMeCO.sub.2Me,
OCH(C.sub.1-4 alkyl)O(C.dbd.O)C.sub.1-4 alkyl, 3
[0067] with the provisos that (a) when R.sup.1 is hydrogen, one of
R.sup.3 and R.sup.4 is hydrogen, and R.sup.2 is fluoro, then the
other of R.sup.3 and R.sup.4 is not hydrogen, halogen, azido,
trifluoromethyl, C.sub.1-4 alkyl, amino, C.sub.1-4 alkylamino,
di(C.sub.1-4 alkyl)amino, or C.sub.1-10 alkoxy; (b) when R.sup.1 is
hydrogen, one of R.sup.3 and R.sup.4 is hydrogen, and R.sup.2 is
halogen, hydroxy, C.sub.1-6 alkoxy, or C.sub.2-6 alkenyloxy, then
the other of R.sup.3 and R.sup.4 is not hydrogen, fluoro, or azido;
and (c) when R.sup.1 and R.sup.3 are hydrogen and R.sup.2 is
hydroxy, then R.sup.4 is not hydroxy.
[0068] The present invention also provides novel compounds of
structural formula IV of the indicated stereochemical configuration
which are useful as inhibitors of RNA-dependent RNA viral
polymerase. The compounds of formula IV are also inhibitors of
RNA-dependent RNA viral replication and are useful for the
treatment of RNA-dependent RNA viral infection: 4
[0069] wherein B is selected from the group consisting of 5
[0070] A, G, and L are each independently CH or N;
[0071] D is N, CH, C--CN, C--NO.sub.2, C--C.sub.1-3 alkyl,
C--NHCONH.sub.2, C--CONR.sup.11R.sup.11, C--CSNR.sup.11R.sup.11,
C--COOR.sup.11, C--C(.dbd.NH)NH.sub.2, C-hydroxy, C--C.sub.1-3
alkoxy, C-amino, C--C.sub.1-4 alkylamino, C-di(C.sub.1-4
alkyl)amino, C-halogen, C-(1,3-oxazol-2-yl), C-(1,3-thiazol-2-yl),
or C-(imidazol-2-yl); wherein alkyl is unsubstituted or substituted
with one to three groups independently selected from halogen,
amino, hydroxy, carboxy, and C.sub.1-3 alkoxy;
[0072] E is N or CR.sup.5;
[0073] W is O or S;
[0074] R.sup.1 is hydrogen, C.sub.2-4 alkenyl, C.sub.2-4 alkynyl,
or C.sub.1-4 alkyl optionally substituted with amino, hydroxy, or 1
to 3 fluorine atoms and one of R.sup.2 and R.sup.3 is hydroxy or
C.sub.1-4 alkoxy and the other of R.sup.2 and R.sup.3 is selected
from the group consisting of hydrogen,
[0075] hydroxy,
[0076] halogen,
[0077] C.sub.1-4 alkyl, optionally substituted with 1 to 3 fluorine
atoms,
[0078] C.sub.1-10 alkoxy, optionally substituted with C.sub.1-3
alkoxy or 1 to 3 fluorine atoms,
[0079] C.sub.2-6 alkenyloxy,
[0080] C.sub.1-4 alkylthio,
[0081] C.sub.1-8 alkylcarbonyloxy,
[0082] aryloxycarbonyl,
[0083] azido,
[0084] amino,
[0085] C.sub.1-4 alkylamino, and
[0086] di(C.sub.1-4 alkyl)amino; or
[0087] R.sup.2 is hydrogen, C.sub.2-4 alkenyl, C.sub.2-4 alkynyl,
or C.sub.1-4 alkyl optionally substituted with amino, hydroxy, or 1
to 3 fluorine atoms and one of R.sup.1 and R.sup.3 is hydroxy or
C.sub.1-4 alkoxy and the other of R.sup.1 and R.sup.3 is selected
from the group consisting of
[0088] hydrogen,
[0089] hydroxy,
[0090] halogen,
[0091] C.sub.1-4 alkyl, optionally substituted with 1 to 3 fluorine
atoms,
[0092] C.sub.1-10 alkoxy, optionally substituted with hydroxy,
C.sub.1-3 alkoxy, carboxy, or 1 to 3 fluorine atoms,
[0093] C.sub.2-6 alkenyloxy,
[0094] C.sub.1-4 alkylthio,
[0095] C.sub.1-8 alkylcarbonyloxy,
[0096] aryloxycarbonyl,
[0097] azido,
[0098] amino,
[0099] C.sub.1-4 alkylamino, and
[0100] di(C.sub.1-4 alkyl)amino; or
[0101] R.sup.1 and R.sup.2 together with the carbon atom to which
they are attached form a 3- to 6-membered saturated monocyclic ring
system optionally containing a heteroatom selected from O, S, and
NC.sub.0-4 alkyl;
[0102] each R.sup.4 is independently H, OH, SH, NH.sub.2, C.sub.1-4
alkylamino, di(C.sub.1-4 alkyl)amino, C.sub.3-6 cycloalkylamino,
halogen, C.sub.1-4 alkyl, C.sub.1-4 alkoxy, or CF.sub.3;
[0103] R.sup.4 and R.sup.6 are each independently H, OH, SH,
NH.sub.2, C.sub.1-4 alkylamino, di(C.sub.1-4 alkyl)amino, C.sub.3-6
cycloalkylamino, halogen, C.sub.1-4 alkyl, C.sub.1-4 alkoxy, or
CF.sub.3;
[0104] R.sup.5 is H, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6
alkynyl, C.sub.1-4 alkylamino, CF.sub.3, or halogen;
[0105] R.sup.14 is H, CF.sub.3, C.sub.1-4 alkyl, amino, C.sub.1-4
alkylamino, C.sub.3-6 cycloalkylamino, or di(C.sub.1-4
alkyl)amino;
[0106] R.sup.7 is hydrogen, amino, C.sub.1-4 alkylamino, C.sub.3-6
cycloalkylamino, or di(C.sub.1-4 alkyl)amino;
[0107] each R.sup.11 is independently H or C.sub.1-6 alkyl;
[0108] R.sup.8 is H, halogen, CN, carboxy, C.sub.1-4
alkyloxycarbonyl, N.sub.3, amino, C.sub.1-4 alkylamino,
di(C.sub.1-4 alkyl)amino, hydroxy, C.sub.1-6 alkoxy, C.sub.1-6
alkylthio, C.sub.1-6 alkylsulfonyl, or (C.sub.1-4 alkyl).sub.0-2
aminomethyl;
[0109] R.sup.12 and R.sup.13 are each independently hydrogen,
methyl, hydroxymethyl, or fluoromethyl; and
[0110] R.sup.9 and R.sup.10 are each independently hydroxy,
OCH.sub.2CH.sub.2SC(.dbd.O)C.sub.1-4 alkyl,
OCH.sub.2O(C.dbd.O)OC.sub.1-4 alkyl, NHCHMeCO.sub.2Me,
OCH(C.sub.1-4 alkyl)O(C.dbd.O)C.sub.1-4 alkyl, 6
[0111] provided that at least one of R.sup.9 and R.sup.10 is not
hydroxy.
[0112] The present invention further provides novel compounds of
structural formula XII of the indicated stereochemical
configuration which are useful as inhibitors of RNA-dependent RNA
viral polymerase and in particular of HCV NS5B polymerase: 7
[0113] wherein R.sup.a and R.sup.h are each independently selected
from the group consisting of hydrogen, cyano, azido, halogen,
hydroxy, mercapto, amino, C.sub.1-4 alkoxy, C.sub.2-4 alkenyl,
C.sub.2-4 alkynyl, and C.sub.1-4 alkyl, wherein alkyl is
unsubstituted or substituted with hydroxy, amino, C.sub.1-4 alkoxy,
C.sub.1-4 alkylthio, or one to three fluorine atoms;
[0114] R.sup.b is C.sub.2-4 alkenyl, C.sub.2-4 alkynyl, or
C.sub.1-4 alkyl, wherein alkyl is unsubstituted or substituted with
hydroxy, amino, C.sub.1-4 alkoxy, C.sub.1-4 alkylthio, or one to
three fluorine atoms;
[0115] R.sup.c is hydrogen, fluorine, hydroxy, mercapto, C.sub.1-4
alkoxy, or C.sub.1-4 alkyl; or R.sup.b and R.sup.c together with
the carbon atom to which they are attached form a 3- to 6-membered
saturated monocyclic ring system optionally containing a heteroatom
selected from O, S, and NC.sub.0-4 alkyl;
[0116] R.sup.d is hydrogen, cyano, nitro, C.sub.1-3 alkyl,
NHCONH.sub.2, CONR.sup.jR.sup.j, CSNR.sup.jR.sup.j, COOR.sup.j,
C(.dbd.NH)NH.sub.2, hydroxy, C.sub.1-3 alkoxy, amino, C.sub.1-4
alkylamino, di(C.sub.1-4 alkyl)amino, halogen, (1,3-oxazol-2-yl),
(1,3-thiazol-2-yl), or (imidazol-2-yl); wherein alkyl is
unsubstituted or substituted with one to three groups independently
selected from halogen, amino, hydroxy, carboxy, and C.sub.1-3
alkoxy;
[0117] R.sup.e and R.sup.f are each independently hydrogen,
hydroxy, halogen, C.sub.1-4 alkoxy, amino, C.sub.1-4 alkylamino,
di(C.sub.1-4 alkyl)amino, C.sub.3-6 cycloalkylamino, di(C.sub.3-6
cycloalkyl)amino, or C.sub.4-6 cycloheteroalkyl, unsubstituted or
substituted with one to two groups independently selected from
halogen, hydroxy, amino, C.sub.1-4 alkyl, and C.sub.1-4 alkoxy;
[0118] R.sup.g is hydrogen, C.sub.1-4 alkyl, C.sub.2-4 alkynyl,
halogen, cyano, carboxy, C.sub.1-4 alkyloxycarbonyl, azido, amino,
C.sub.1-4 alkylamino, di(C.sub.1-4 alkyl)amino, hydroxy, C.sub.1-6
alkoxy, C.sub.1-6 alkylthio, C.sub.1-6 alkylsulfonyl, (C.sub.1-4
alkyl).sub.0-2 aminomethyl, or C.sub.4-6 cycloheteroalkyl,
unsubstituted or substituted with one to two groups independently
selected from halogen, hydroxy, amino, C.sub.1-4 alkyl, and
C.sub.1-4 alkoxy;
[0119] R.sup.i is hydrogen, C.sub.1-10 alkylcarbonyl,
P.sub.3O.sub.9H.sub.4, P.sub.2O.sub.6H.sub.3, or
P(O)R.sup.mR.sup.n;
[0120] each R.sup.j is independently hydrogen or C.sub.1-6
alkyl;
[0121] R.sup.k and R.sup.l are each independently hydrogen, methyl,
hydroxymethyl, or fluoromethyl; and
[0122] R.sup.m and R.sup.n are each independently hydroxy,
OCH.sub.2CH.sub.2SC(.dbd.O)C.sub.1-4 alkyl,
OCH.sub.2O(C.dbd.O)OC.sub.1-4 alkyl, NHCHMeCO.sub.2Me,
OCH(C.sub.1-4 alkyl)O(C.dbd.O)C.sub.1-4 alkyl, 8
[0123] with the proviso that when R.sup.a and R.sup.c are
.alpha.-hydroxy, R.sup.e is amino, R.sup.b is .beta.-methyl and
R.sup.h is hydrogen or R.sup.h is .beta.-methyl and R.sup.b is
hydrogen, and R.sup.f, R.sup.g, R.sup.i, R.sup.k, and R.sup.l are
hydrogen, then R.sup.d is not cyano or CONH.sub.2.
[0124] The compounds of formula XII are also inhibitors of
RNA-dependent RNA viral replication and in particular of HCV
replication and are useful for the treatment of RNA-dependent RNA
viral infection and in particular for the treatment of HCV
infection.
[0125] Also encompassed within the present invention are
pharmaceutical compositions containing the compounds alone or in
combination with other agents active against RNA-dependent RNA
virus and in particular against HCV.
DETAILED DESCRIPTION OF THE INVENTION
[0126] The present invention provides a method for inhibiting
RNA-dependent RNA viral polymerase, a method for inhibiting
RNA-dependent RNA viral replication, and/or a method for treating
RNA-dependent RNA viral infection in a mammal in need thereof
comprising administering to the mammal a therapeutically effective
amount of a compound of structural formula I which is of the
stereochemical configuration: 9
[0127] or a pharmaceutically acceptable salt thereof;
[0128] wherein B is selected from the group consisting of 10
[0129] A, G, and L are each independently CH or N;
[0130] D is N, CH, C--CN, C--NO.sub.2, C--C.sub.1-3 alkyl,
C--NHCONH.sub.2, C--CONR.sup.11R.sup.11, C--CSNR.sup.11R.sup.11,
C--COOR.sup.11, C--C(.dbd.NH)NH.sub.2, C-hydroxy, C--C.sub.1-3
alkoxy, C-amino, C--C.sub.1-4 alkylamino, C-di(C.sub.1-4
alkyl)amino, C-halogen, C-(1,3-oxazol-2-yl), C-(1,3-thiazol-2-yl),
or C-(imidazol-2-yl); wherein alkyl is unsubstituted or substituted
with one to three groups independently selected from halogen,
amino, hydroxy, carboxy, and C.sub.1-3 alkoxy;
[0131] E is N or CR.sup.5;
[0132] W is O or S;
[0133] Y is H, C.sub.1-10 alkylcarbonyl, P.sub.3O.sub.9H.sub.4,
P.sub.2O.sub.6H.sub.3, or P(O)R.sup.9R.sup.10;
[0134] R.sup.1 is hydrogen, C.sub.2-4 alkenyl, C.sub.2-4 alkynyl,
or C.sub.1-4 alkyl optionally substituted with amino, hydroxy, or 1
to 3 fluorine atoms and one of R.sup.2 and R.sup.3 is hydroxy or
C.sub.1-4 alkoxy and the other of R.sup.2 and R.sup.3 is selected
from the group consisting of
[0135] hydrogen,
[0136] hydroxy,
[0137] halogen,
[0138] C.sub.1-4 alkyl, optionally substituted with 1 to 3 fluorine
atoms,
[0139] C.sub.1-10 alkoxy, optionally substituted with C.sub.1-3
alkoxy or 1 to 3 fluorine atoms,
[0140] C.sub.2-6 alkenyloxy,
[0141] C.sub.1-4 alkylthio,
[0142] C.sub.1-8 alkylcarbonyloxy,
[0143] aryloxycarbonyl,
[0144] azido,
[0145] amino,
[0146] C.sub.1-4 alkylamino, and
[0147] di(C.sub.1-4 alkyl)amino; or
[0148] R.sup.2 is hydrogen, C.sub.2-4 alkenyl, C.sub.2-4 alkynyl,
or C.sub.1-4 alkyl optionally substituted with amino, hydroxy, or 1
to 3 fluorine atoms and one of R.sup.1 and R.sup.3 is hydroxy or
C.sub.1-4 alkoxy and the other of R.sup.1 and R.sup.3 is selected
from the group consisting of
[0149] hydrogen,
[0150] hydroxy,
[0151] halogen,
[0152] C.sub.1-4 alkyl, optionally substituted with 1 to 3 fluorine
atoms,
[0153] C.sub.1-10 alkoxy, optionally substituted with hydroxy,
C.sub.1-3 alkoxy, carboxy, or 1 to 3 fluorine atoms,
[0154] C.sub.2-6 alkenyloxy,
[0155] C.sub.1-4 alkylthio,
[0156] C.sub.1-8 alkylcarbonyloxy,
[0157] aryloxycarbonyl,
[0158] azido,
[0159] amino,
[0160] C.sub.1-4 alkylamino, and
[0161] di(C.sub.1-4 alkyl)amino; or
[0162] R.sup.1 and R.sup.2 together with the carbon atom to which
they are attached form a 3- to 6-membered saturated monocyclic ring
system optionally containing a heteroatom selected from O, S, and
NC.sub.0-4 alkyl;
[0163] R.sup.4 and R.sup.6 are each independently H, OH, SH,
NH.sub.2, C.sub.1-4 alkylamino, di(C.sub.1-4 alkyl)amino, C.sub.3-6
cycloalkylamino, halogen, C.sub.1-4 alkyl, C.sub.1-4 alkoxy, or
CF.sub.3;
[0164] R.sup.5 is H, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6
alkynyl, C.sub.1-4 alkylamino, CF.sub.3, or halogen;
[0165] R.sup.14 is H, CF.sub.3, C.sub.1-4 alkyl, amino, C.sub.1-4
alkylamino, C.sub.3-6 cycloalkylamino, or di(C.sub.1-4
alkyl)amino;
[0166] R.sup.7 is hydrogen, amino, C.sub.1-4 alkylamino, C.sub.3-6
cycloalkylamino, or di(C.sub.1-4 alkyl)amino;
[0167] each R.sup.11 is independently H or C.sub.1-6 alkyl;
[0168] R.sup.8 is H, halogen, CN, carboxy, C.sub.1-4
alkyloxycarbonyl, N.sub.3, amino, C.sub.1-4 alkylamino,
di(C.sub.1-4 alkyl)amino, hydroxy, C.sub.1-6 alkoxy, C.sub.1-6
alkylthio, C.sub.1-6 alkylsulfonyl, or (C.sub.1-4 alkyl).sub.0-2
aminomethyl;
[0169] R.sup.12 and R.sup.13 are each independently hydrogen,
methyl, hydroxymethyl, or fluoromethyl; and
[0170] R.sup.9 and R.sup.10 are each independently hydroxy,
OCH.sub.2CH.sub.2SC(.dbd.O)C.sub.1-4 alkyl,
OCH.sub.2O(C.dbd.O)OC.sub.1-4 alkyl, NHCHMeCO.sub.2Me,
OCH(C.sub.1-4 alkyl)O(C.dbd.O)C.sub.1-4 alkyl, 11
[0171] with the provisos that (a) when R.sup.1 is hydrogen, one of
R.sup.3 and R.sup.4 is hydrogen, and R.sup.2 is fluoro, then the
other of R.sup.3 and R.sup.4 is not hydrogen, halogen, azido,
trifluoromethyl, C.sub.1-4 alkyl, amino, C.sub.1-4 alkylamino,
di(C.sub.1-4 alkyl)amino, or C.sub.1-10 alkoxy; (b) when R.sup.1 is
hydrogen, one of R.sup.3 and R.sup.4 is hydrogen, and R.sup.2 is
halogen, hydroxy, C.sub.1-6 alkoxy, or C.sub.2-6 alkenyloxy, then
the other of R.sup.3 and R.sup.4 is not hydrogen, fluoro, or azido;
and (c) when R.sup.1 and R.sup.3 are hydrogen and R.sup.2 is
hydroxy, then R.sup.4 is not hydroxy.
[0172] In one embodiment of the present invention is the method of
inhibiting RNA-dependent RNA viral polymerase, inhibiting
RNA-dependent viral replication, and/or treating RNA-dependent RNA
viral infection with a compound of structural formula II which is
of the stereochemical configuration: 12
[0173] wherein B is 13
[0174] D is N, CH, C--CN, C--NO.sub.2, C--C.sub.1-3 alkyl,
C--NHCONH.sub.2, C--CONR.sup.11R.sup.11, C--CSNR.sup.11R.sup.11,
C--COOR.sup.11, C-hydroxy, C--C.sub.1-3 alkoxy, C-amino,
C--C.sub.1-4 alkylamino, C-di(C.sub.1-4 alkyl)amino, C-halogen,
C-(1,3-oxazol-2-yl), C-(1,3-thiazol-2-yl), or C-(imidazol-2-yl);
wherein alkyl is unsubstituted or substituted with one to three
groups independently selected from halogen, amino, hydroxy,
carboxy, and C.sub.1-3 alkoxy;
[0175] E is N or C--R.sup.5;
[0176] W is O or S;
[0177] Y is H, C.sub.10 alkylcarbonyl, P.sub.3O.sub.9H.sub.4, or
P(O)R.sup.9R.sup.10;
[0178] R.sup.1 is hydrogen, CF.sub.3, or C.sub.1-4 alkyl and one of
R.sup.2 and R.sup.3 is OH or C.sub.1-4 alkoxy and the other of
R.sup.2 and R.sup.3 is selected from the group consisting of
[0179] hydrogen,
[0180] hydroxy,
[0181] halogen,
[0182] C.sub.1-3 alkyl,
[0183] trifluoromethyl,
[0184] C.sub.1-4 alkoxy,
[0185] C.sub.1-4 alkylthio,
[0186] C.sub.1-8 alkylcarbonyloxy,
[0187] aryloxycarbonyl,
[0188] azido,
[0189] amino,
[0190] C.sub.1-4 alkylamino, and
[0191] di(C.sub.1-4 alkyl)amino; or
[0192] R.sup.2 is hydrogen, CF.sub.3, or C.sub.1-4 alkyl and one of
R.sup.1 and R.sup.3 is OH or C.sub.1-4 alkoxy and the other of
R.sup.1 and R.sup.3 is selected from the group consisting of
[0193] hydrogen,
[0194] hydroxy,
[0195] fluoro,
[0196] C.sub.1-4 alkyl,
[0197] trifluoromethyl,
[0198] C.sub.1-4 alkoxy,
[0199] C.sub.1-4 alkylthio,
[0200] C.sub.1-8 alkylcarbonyloxy,
[0201] azido,
[0202] amino,
[0203] C.sub.1-4 alkylamino, and
[0204] di(C.sub.1-4 alkyl)amino; or
[0205] R.sup.1 and R.sup.2 together with the carbon atom to which
they are attached form a 3- to 6-membered saturated monocyclic ring
system optionally containing a heteroatom selected from O, S, and
NC.sub.0-4 alkyl;
[0206] R.sup.4 and R.sup.6 are each independently H, OH, SH,
NH.sub.2, C.sub.1-4 alkylamino, di(C.sub.1-4 alkyl)amino, C.sub.3-6
cycloalkylamino, halogen, C.sub.1-4 alkyl, C.sub.1-4 alkoxy, or
CF.sub.3;
[0207] R.sup.5 is H, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6
alkynyl, C.sub.1-4 alkylamino, CF.sub.3, or halogen;
[0208] R.sup.7 is hydrogen, amino, C.sub.1-4 alkylamino, C.sub.3-6
cycloalkylamino, or di(C.sub.1-4 alkyl)amino;
[0209] each R.sup.11 is independently H or C.sub.1-6 alkyl;
[0210] R.sup.8 is H, halogen, CN, carboxy, C.sub.1-4
alkyloxycarbonyl, N.sub.3, amino, C.sub.1-4 alkylamino,
di(C.sub.1-4 alkyl)amino, hydroxy, C.sub.1-6 alkoxy, C.sub.1-6
alkylthio, C.sub.1-6 alkylsulfonyl, or (C.sub.1-4 alkyl).sub.0-2
aminomethyl; and
[0211] R.sup.9 and R.sup.10 are each independently hydroxy,
OCH.sub.2CH.sub.2SC(.dbd.O)C.sub.1-4 alkyl, or
OCH.sub.2O(C.dbd.O)C.sub.1- -4 alkyl;
[0212] with the provisos that (a) when R.sup.1 is hydrogen, one of
R.sup.3 and R.sup.4 is hydrogen, and R.sup.2 is fluoro, then the
other of R.sup.3 and R.sup.4 is not hydrogen, halogen,
trifluoromethyl, C.sub.1-4 alkyl, amino, C.sub.1-4 alkylamino,
di(C.sub.1-4 alkyl)amino, or C.sub.1-4 alkoxy; (b) when R.sup.1 is
hydrogen, one of R.sup.3 and R.sup.4 is hydrogen, and R.sup.2 is
halogen, hydroxy, or C.sub.1-4 alkoxy, then the other of R.sup.3
and R.sup.4 is not hydrogen, fluoro, or azido; and (c) when R.sup.1
and R.sup.3 are hydrogen and R.sup.2 is hydroxy, then R.sup.4 is
not hydroxy.
[0213] In a second embodiment of the present invention is the
method of inhibiting RNA-dependent RNA viral polymerase, inhibiting
RNA-dependent RNA viral replication, and/or treating RNA-dependent
RNA viral infection with a compound of structural formula III which
is of the stereochemical configuration: 14
[0214] wherein B is 15
[0215] D is N, CH, C--CN, C--NO.sub.2, C--C.sub.1-3 alkyl,
C--NHCONH.sub.2, C--CONR.sup.11R.sup.11, C--CSNR.sup.11R.sup.11,
C--COOR.sup.11, C-hydroxy, C--C.sub.1-3 alkoxy, C-amino,
C--C.sub.1-4 alkylamino, C-di(C.sub.1-4 alkyl)amino, C-halogen,
C-(1,3-oxazol-2-yl), C-(1,3-thiazol-2-yl), or C-(imidazol-2-yl);
wherein alkyl is unsubstituted or substituted with one to three
groups independently selected from halogen, amino, hydroxy,
carboxy, and C.sub.1-3 alkoxy;
[0216] W is O or S;
[0217] Y is H, C.sub.1-10 alkylcarbonyl, P.sub.3O.sub.9H.sub.4,
P.sub.2O.sub.6H.sub.3, or P(O)R.sup.9R.sup.10;
[0218] R.sup.1 is hydrogen, CF.sub.3, or C.sub.1-4 alkyl and one of
R.sup.2 and R.sup.3 is OH or C.sub.1-4 alkoxy and the other of
R.sup.2 and R.sup.3 is selected from the group consisting of
[0219] hydrogen,
[0220] hydroxy,
[0221] fluoro,
[0222] C.sub.1-3 alkyl,
[0223] trifluoromethyl,
[0224] C.sub.1-8 alkylcarbonyloxy,
[0225] C.sub.1-3 alkoxy, and
[0226] amino; or
[0227] R.sup.2 is hydrogen, CF.sub.3, or C.sub.1-4 alkyl and one of
R.sup.1 and R.sup.3 is OH or C.sub.1-4 alkoxy and the other of
R.sup.1 and R.sup.3 is selected from the group consisting of
[0228] hydrogen,
[0229] hydroxy,
[0230] fluoro,
[0231] C.sub.1-3 alkyl,
[0232] trifluoromethyl,
[0233] C.sub.1-8 alkylcarbonyloxy,
[0234] C.sub.1-3 alkoxy, and
[0235] amino; or
[0236] R.sup.1 and R.sup.2 together with the carbon atom to which
they are attached form a 3- to 6-membered saturated monocyclic ring
system optionally containing a heteroatom selected from O, S, and
NC.sub.0-4 alkyl;
[0237] R.sup.6 is H, OH, SH, NH.sub.2, C.sub.1-4 alkylamino,
di(C.sub.1-4 alkyl)amino, C.sub.3-6 cycloalkylamino, halogen,
C.sub.1-4 alkyl, C.sub.1-4 alkoxy, or CF.sub.3;
[0238] R.sup.5 is H, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6
alkynyl, C.sub.1-4 alkylamino, CF.sub.3, or halogen;
[0239] R.sup.7 is hydrogen, amino, C.sub.1-4 alkylamino, C.sub.3-6
cycloalkylamino, or di(C.sub.1-4 alkyl)amino;
[0240] each R.sup.11 is independently H or C.sub.1-6 alkyl;
[0241] R.sup.8 is H, halogen, CN, carboxy, C.sub.1-4
alkyloxycarbonyl, N.sub.3, amino, C.sub.1-4 alkylamino,
di(C.sub.1-4 alkyl)amino, hydroxy, C.sub.1-6 alkoxy, C.sub.1-6
alkylthio, C.sub.1-6 alkylsulfonyl, or (C.sub.1-4 alkyl).sub.0-2
aminomethyl; and
[0242] R.sup.9 and R.sup.10 are each independently hydroxy,
OCH.sub.2CH.sub.2SC(.dbd.O).sub.t-butyl, or
OCH.sub.2O(C.dbd.O)iPr;
[0243] with the provisos that (a) when R.sup.1 is hydrogen and
R.sup.2 is fluoro, then R.sup.3 is not hydrogen, trifluoromethyl,
fluoro, C.sub.1-3 alkyl, amino, or C.sub.1-3 alkoxy; (b) when
R.sup.1 is hydrogen and R.sup.2 is fluoro, hydroxy, or C.sub.1-3
alkoxy, then R.sup.3 is not hydrogen or fluoro; and (c) when
R.sup.1 is hydrogen and R.sup.2 is hydroxy, then R.sup.3 is not
.beta.-hydroxy.
[0244] In a class of this embodiment is the method of inhibiting
RNA-dependent RNA viral polymerase, inhibiting RNA-dependent RNA
viral replication, and/or treating RNA-dependent RNA viral
infection with a compound of structural formula III wherein B is
16
[0245] and W, Y, and the R substituents are as defined under this
second embodiment.
[0246] In a second class of this embodiment is the method of
inhibiting RNA-dependent RNA viral polymerase, inhibiting
RNA-dependent RNA viral replication, and/or treating RNA-dependent
RNA viral infection with a compound of structural formula I wherein
B is 17
[0247] and Y, D, and the R substituents are as defined under this
second embodiment.
[0248] In a third embodiment of the present invention, the
RNA-dependent RNA viral polymerase is a positive-sense
single-stranded RNA-dependent RNA viral polymerase. In a class of
this embodiment, the positive-sense single-stranded RNA-dependent
RNA viral polymerase is a Flaviviridae viral polymerase or a
Picornaviridae viral polymerase. In a subclass of this class, the
Picornaviridae viral polymerase is rhinovirus polymerase,
poliovirus polymerase, or hepatitis A virus polymerase. In a second
subclass of this class, the Flaviviridae viral polymerase is
selected from the group consisting of hepatitis C virus polymerase,
yellow fever virus polymerase, dengue virus polymerase, West Nile
virus polymerase, Japanese encephalitis virus polymerase, Banzi
virus polymerase, and bovine viral diarrhea virus (BVDV)
polymerase. In a subclass of this subclass, the Flaviviridae viral
polymerase is hepatitis C virus polymerase.
[0249] In a fourth embodiment of the present invention, the
RNA-dependent RNA viral replication is a positive-sense
single-stranded RNA-dependent RNA viral replication. In a class of
this embodiment, the positive-sense single-stranded RNA-dependent
RNA viral replication is Flaviviridae viral replication or
Picornaviridae viral replication. In a subclass of this class, the
Picornaviridae viral replication is rhinovirus replication,
poliovirus replication, or hepatitis A virus replication. In a
second subclass of this class, the Flaviviridae viral replication
is selected from the group consisting of hepatitis C virus
replication, yellow fever virus replication, dengue virus
replication, West Nile virus replication, Japanese encephalitis
virus replication, Banzi virus replication, and bovine viral
diarrhea virus replication. In a subclass of this subclass, the
Flaviviridae viral replication is hepatitis C virus
replication.
[0250] In a fifth embodiment of the present invention, the
RNA-dependent RNA viral infection is a positive-sense
single-stranded RNA-dependent viral infection. In a class of this
embodiment, the positive-sense single-stranded RNA-dependent RNA
viral infection is Flaviviridae viral infection or Picornaviridae
viral infection. In a subclass of this class, the Picornaviridae
viral infection is rhinovirus infection, poliovirus infection, or
hepatitis A virus infection. In a second subclass of this class,
the Flaviviridae viral infection is selected from the group
consisting of hepatitis C virus infection, yellow fever virus
infection, dengue virus infection, West Nile virus infection,
Japanese encephalitis virus infection, Banzi virus infection, and
bovine viral diarrhea virus infection. In a subclass of this
subclass, the Flaviviridae viral infection is hepatitis C virus
infection.
[0251] Illustrative of the invention is a method for inhibiting
RNA-dependent RNA viral polymerase, inhibiting RNA-dependent RNA
viral replication, and/or treating RNA-dependent RNA viral
infection wherein the compound is selected from:
[0252] 2'-O-methyl-cytidine,
[0253] 2'-C-methyl-cytidine,
[0254] 3',5'-di-O-octanoyl-2'-O-methyl-cytidine,
[0255] 3'-O-octanoyl-2'-O-methyl-cytidine,
[0256] 2'-C-methyl-adenosine,
[0257] 8-amino-2'-C-methyladenosine,
[0258]
4-amino-7-(2-C-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyri-
midine-5-carbonitrile,
[0259]
4-amino-7-(2-C-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyri-
midine-5-carboxamide,
[0260] 3'-deoxy-3'-methyl-cytidine,
[0261]
4-amino-7-(3-deoxy-3-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3--
d]pyrimidin-5-carboxamide,
[0262] 3'-deoxy-adenosine,
[0263]
4-amino-7-(3-deoxy-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimid-
ine,
[0264]
4-amino-7-(3-deoxy-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimid-
in-5-carboxamide,
[0265] 3'-amino-3'-deoxyadenosine,
[0266]
2-amino-3,4-dihydro-4-oxo-7-(.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-
-d]pyrimidin-5-carboxamide,
[0267]
4-amino-7-(.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidin-5-car-
boxamide,
[0268]
2-amino-3,4-dihydro-4-oxo-7-(.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-
-d]pyrimidin-5-carbonitrile,
[0269]
2-amino-5-ethyl-7-(.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimid-
in-4(3H)-one,
[0270]
6-amino-1-(.beta.-D-ribofuranosyl)-1H-imidazo[4,5-c]pyridin-4(5H)-o-
ne,
[0271] 3'-deoxyguanosine,
[0272]
2-amino-7-(3-deoxy-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimid-
in-4(3H)-one,
[0273] 2'-O-methylguanosine,
[0274]
2-amino-7-(2-O-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyri-
midin-4(3H)-one,
[0275]
2-amino-7-(2-O-methyl-.beta.-D-ribofuranosyl)-5H-pyrrolo[3,2-d]pyri-
midin-4(3H)-one,
[0276]
7-(2-O-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine,
[0277] 3'-deoxycytidine,
[0278]
2-amino-5-methyl-7-(.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimi-
din-4(3H)-one,
[0279]
2-amino-3,4-dihydro-4-oxo-7-(2-O-methyl-.beta.-D-ribofuranosyl)-7H--
pyrrolo-[2,3-d]pyrimidin-5-carbonitrile,
[0280]
2-amino-5-methyl-7-(2-O-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2-
,3-d]pyrimidin-4(3H)-one,
[0281] 8-azidoguanosine,
[0282] 8-aminoguanosine,
[0283] 8-bromoadenosine,
[0284] 8-aminoadenosine,
[0285] 8-bromoguanosine,
[0286] 3'-deoxy-3'-fluorocytidine,
[0287] 3'-deoxy-3'-fluoroguanosine,
[0288]
4-amino-7-(3-deoxy-3-fluoro-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3--
d]pyrimidin-5-carboxamide,
[0289]
2-amino-4-chloro-7-(2-O-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2-
,3-d]pyrimidin-5-carbonitrile,
[0290]
2-amino-4-chloro-5-ethyl-7-(2-O-methyl-.beta.-D-ribofuranosyl)-7H-p-
yrrolo[2,3-d]pyrimidine,
[0291]
2-amino-4-chloro-5-methyl-7-(2-O-methyl-.beta.-D-ribofuranosyl)-7H--
pyrrolo-[2,3-d]pyrimidine,
[0292]
2-amino-7-(2-O-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyri-
midin-4(3H)-thione,
[0293]
2-amino-4-chloro-7-(2-O-methyl-(.beta.-D-ribofuranosyl)-7H-pyrrolo[-
2,3-d]pyrimidine,
[0294]
2-amino-7-(.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine,
[0295]
2-amino-4-chloro-7-(.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimi-
dine,
[0296]
2-amino-4-chloro-5-methyl-7-(.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-
-d]pyrimidine,
[0297]
1-(.beta.-D-ribofuranosyl)-1H-pyrazolo[3,4-d]pyrimidin-4(3H)-one,
[0298]
4-amino-1-(.beta.-D-ribofuranosyl)-1H-pyrazolo[3,4-d]pyrimidine,
[0299] 2-amino-6-chloro-9-(.beta.-D-ribofuranosyl)-9H-purine,
[0300]
2-amino-4-chloro-7-(.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimi-
din-5-carbonitrile,
[0301] 6-methyl-9-(.beta.-D-ribofuranosyl)-9H-purine,
[0302]
2-amino-7-(2-deoxy-2-fluoro-.beta.-D-arabinofuranosyl)-7H-pyrrolo[2-
,3-d]pyrimidin-4(3H)-one,
[0303]
2-amino-4-chloro-7-(2-deoxy-2-fluoro-.beta.-D-arabinofuranosyl)-7H--
pyrrolo[2,3-d]pyrimidine,
[0304]
2-amino-7-(.beta.-D-arabinofuranosyl)-7H-pyrrolo[2,3-d]pyrimidin-4(-
3H)-one,
[0305]
2-amino-7-(.beta.-D-arabinofuranosyl)-3,4-dihydro-4-oxo-7H-pyrrolo[-
2,3-d]pyrimidin-5-carbonitrile,
[0306]
2-amino-5-methyl-7-(.beta.-D-arabinofuranosyl)-7H-pyrrolo[2,3-d]pyr-
imidin-4(3H)-one,
[0307] 9-(.beta.-D-arabinofuranosyl)-9H-purin-6 (1H)-one,
[0308] 1-(.beta.-D-arabinofuranosyl)-1H-cytosine,
[0309]
2-amino-4-chloro-5-methyl-7-(.beta.-D-arabinofuranosyl)-7H-pyrrolo[-
2,3-d]pyrimidine,
[0310] 3'-deoxy-3'-(fluoromethyl)-guanosine,
[0311] 2'-amino-2'-deoxycytidine,
[0312]
4-amino-7-(3-deoxy-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimid-
in-5-carbonitrile,
[0313] 2'-O-methyladenosine,
[0314]
4-amino-7-(2-O-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyri-
midine,
[0315] 3'-amino-3'-deoxy-2'-O-methyl-adenosine,
[0316] 3'-deoxy-3'-methyl-uridine,
[0317]
6-amino-1-(3-deoxy-.beta.-D-ribofuranosyl)-1H-imidazo[4,5-c]pyridin-
-4(5H)-one,
[0318]
6-amino-1-(3-deoxy-3-fluoro-.beta.-D-ribofuranosyl)-1H-imidazo[4,5--
c]pyridin-4(3H)-one,
[0319] 3'-deoxy-3'-fluorouridine,
[0320] 3'-deoxy-3'-fluoroadenosine,
[0321]
2-amino-7-(2-deoxy-.beta.-D-ribofuranosyl)-3,4-dihydro-4-oxo-7H-pyr-
rolo[2,3-d]pyrimidin-5-carbonitrile,
[0322] 3'-deoxy-5-methyl-uridine,
[0323] 3'-deoxy-2'-(2-methoxyethyl)-3'-methyl-5-methyluridine,
[0324] 2'-amino-2'-deoxy-uridine,
[0325] 2-amino-9-(.beta.-D-arabinofuranosyl)-9H-purin-6
(1H)-one,
[0326] 3'-deoxy-3'-methylguanosine,
[0327] 2'-O-[4-(imidazolyl-1)butyl]guanosine,
[0328] 2'-deoxy-2'-fluoroguanosine,
[0329] 2'-deoxyguanosine,
[0330]
2-amino-7-(2-deoxy-2-fluoro-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3--
d]pyrimidin-4(3H)-one,
[0331]
2-amino-7-(3-deoxy-.beta.-D-ribofuranosyl)-3,4-dihydro-4-oxo-7H-pyr-
rolo[2,3-d]pyrimidin-5-carbonitrile,
[0332]
2-amino-5-iodo-7-(.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidi-
n-4(3H)-one,
[0333]
2-amino-7-(3-deoxy-3-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3--
d]pyrimidin-5-carbonitrile,
[0334]
2-amino-7-(.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidin-4(3H)-
-one,
[0335]
2-amino-7-(2-deoxy-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimid-
in-4(3H)-one,
[0336]
2-amino-7-(3-deoxy-3-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3--
d]pyrimidin-4(3H)-one,
[0337]
2-amino-7-(3-deoxy-3-fluoro-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3--
d]pyrimidin-4(3H)-one,
[0338]
6-amino-1-(2-O-methyl-.beta.-D-ribofuranosyl)-1H-imidazo[4,5-c]pyri-
din-4(5H)-one,
[0339]
6-amino-1-(2-deoxy-.beta.-D-ribofuranosyl)-1H-imidazo[4,5-c]pyridin-
-4(5H)-one,
[0340]
6-amino-1-(3-deoxy-3-methyl-.beta.-D-ribofuranosyl)-1H-imidazo[4,5--
c]pyridin-4(5H)-one,
[0341]
6-amino-1-(2-deoxy-2-fluoro-.beta.-D-ribofuranosyl)-1H-imidazo[4,5--
c]pyridin-4(5H)-one,
[0342]
6-amino-1-(.beta.-D-arabinofuranosyl)-1H-imdazo[4,5-c]pyridin-4(5H)-
-one,
[0343] 2'-O-[2-(N,N-diethylaminooxy)ethyl]-5-methyluridine,
[0344] 5-ethynyl-2'-O-(2-methoxyethyl)-cytidine,
[0345] 1-(2-C-methyl-.beta.-D-arabinofuranosyl)uracil,
[0346] 5-methyl-3'-deoxycytidine,
[0347] 2-amino-2'-O-methyladenosine,
[0348] 2'-deoxy-2'-fluoroadenosine,
[0349] 3'-deoxy-3'-fluoroadenosine,
[0350] 3'-deoxy-3'-methyladenosine,
[0351]
2-amino-7-(2-deoxy-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimid-
ine,
[0352]
4-amino-7-(3-deoxy-3-fluoro-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3--
d]pyrimidin-5-carboxamide,
[0353]
4-amino-7-(3-deoxy-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimid-
in-5-carboxamide,
[0354]
4-amino-7-(2-O-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyri-
midine,
[0355]
4-amino-7-(.beta.-D-arabinofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine,
[0356]
4-amino-1-(3-deoxy-3-fluoro-.beta.-D-ribofuranosyl)-1H-imidazo[4,5--
c]pyridine,
[0357]
4-amino-7-(.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine
(tubercidin),
[0358]
4,6-diamino-7-(.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine,
[0359]
2-amino-7-(3-deoxy-3-fluoro-.beta.-D-ribofuranosyl)-7H-pyrrolo-[2,3-
-d]pyrimidin-5-carboxamide,
[0360]
4-amino-1-(3-deoxy-.beta.-D-ribofuranosyl)-1H-imidazo[4,5-c]pyridin-
e,
[0361]
4-amino-1-(3-deoxy-3-methyl-.beta.-D-ribofuranosyl)-1H-imidazo[4,5--
c]pyridine,
[0362]
4-amino-1-(.beta.-D-ribofuranosyl)-1H-imidazo[4,5-c]pyridine,
[0363]
4-amino-1-(2-C-methyl-.beta.-D-ribofuranosyl)-1H-pyrazolo[3,4-d]pyr-
imidine,
[0364]
4-amino-7-(3-deoxy-3-fluoro-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3--
d]pyrimidine; and
[0365] the corresponding 5'-triphosphates,
5'-[bis(isopropyloxycarbonyloxy- methyl)]monophosphates,
5'-mono-(S--C.sub.1-4 alkanoyl-2-thioethyl)monopho- sphates, and
5'-bis-(S--C.sub.1-4 alkanoyl-2-thioethyl)monophosphates
thereof;
[0366] or a pharmaceutically acceptable salt thereof.
[0367] Further illustrative of the invention is a method for
inhibiting RNA-dependent RNA viral polymerase, inhibiting
RNA-dependent RNA viral replication, and/or treating RNA-dependent
RNA viral infection wherein the compound is selected from:
[0368] 2'-O-methyl-cytidine,
[0369] 2'-C-methyl-cytidine,
[0370] 3',5'-di-O-octanoyl-2'-O-methyl-cytidine,
[0371] 3'-O-octanoyl-2'-O-methyl-cytidine,
[0372]
4-amino-1-(.theta.-D-ribofuranosyl)-1H-pyrazolo[3,4-d]pyrimidine,
[0373]
4-amino-7-(2-C-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyri-
midine-5-carbonitrile,
[0374]
4-amino-7-(2-C-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyri-
midine-5-carboxamide,
[0375] 2'-C-methyladenosine,
[0376] 8-amino-2'-C-methyladenosine,
[0377] 3'-deoxy-3'-methyl-cytidine,
[0378]
4-amino-7-(3-deoxy-3-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3--
d]pyrimidin-5-carboxamide,
[0379] 3'-deoxyadenosine,
[0380]
4-amino-7-(3-deoxy-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimid-
ine,
[0381]
4-amino-7-(3-deoxy-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimid-
in-5-carboxamide,
[0382] 3'-amino-3'-deoxyadenosine,
[0383]
2-amino-3,4-dihydro-4-oxo-7-(.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-
-d]pyrimidin-5-carboxamide,
[0384]
4-amino-7-(.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidin-5-car-
boxamide,
[0385]
2-amino-3,4-dihydro-4-oxo-7-(.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-
-d]pyrimidin-5-carbonitrile,
[0386]
2-amino-5-ethyl-7-(.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimid-
in-4(3H)-one,
[0387]
6-amino-1-(.beta.-D-ribofuranosyl)-1H-imidazo[4,5-c]pyridin-4(5H)-o-
ne,
[0388] 3'-deoxyguanosine,
[0389]
2-amino-7-(3-deoxy-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimid-
in-4(3H)-one,
[0390] 2'-O-methylguanosine,
[0391]
2-amino-7-(2-O-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyri-
midin-4(3H)-one,
[0392]
2-amino-7-(2-O-methyl-.beta.-D-ribofuranosyl)-5H-pyrrolo[3,2-d]pyri-
midin-4-(3H)-one,
[0393]
7-(2-O-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine,
[0394] 3'-deoxy-cytidine,
[0395]
2-amino-5-methyl-7-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimid-
in-4(3H)-one,
[0396]
2-amino-3,4-dihydro-4-oxo-7-(2-O-methyl-.beta.-D-ribofuranosyl)-7H--
pyrrolo-[2,3-d]pyrimidine-5-carbonitrile,
[0397]
2-amino-5-methyl-7-(2-O-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2-
,3-d]pyrimidin-4(3H)-one,
[0398] 8-azidoguanosine,
[0399] 8-aminoguanosine,
[0400] 8-bromoadenosine,
[0401] 8-aminoadenosine,
[0402] 8-bromoguanosine,
[0403] 3'-deoxy-3'-fluorocytidine,
[0404] 3'-deoxy-3'-fluoroguanosine,
[0405]
4-amino-7-(3-deoxy-3-fluoro-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3--
d]-pyrimidin-5-carboxamide,
[0406]
2-amino-4-chloro-7-(2-O-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2-
,3-d]pyrimidin-5-carbonitrile,
[0407]
2-amino-4-chloro-5-ethyl-7-(2-O-methyl-.beta.-D-ribofuranosyl)-7H-p-
yrrolo[2,3-d]pyrimidine,
[0408]
2-amino-4-chloro-5-methyl-7-(2-O-methyl-.beta.-D-ribofuranosyl)-7H--
pyrrolo-[2,3-d]pyrimidine,
[0409]
2-amino-7-(.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine,
[0410]
2-amino-4-chloro-7-(.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimi-
dine,
[0411]
2-amino-4-chloro-5-methyl-7-(.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-
-d]pyrimidine,
[0412]
2-amino-7-(2-deoxy-2-fluoro-.beta.-D-arabinofuranosyl)-7H-pyrrolo[2-
,3-d]pyrimidin-4(3H)-one,
[0413]
4-amino-1-(2-C-methyl-.beta.-D-ribofuranosyl)-1H-pyrazolo[3,4-d]pyr-
imidine,
[0414]
2-amino-7-(.beta.-D-arabinofuranosyl)-7H-pyrrolo[2,3-d]pyrimidin-4(-
3H)-one, and
[0415]
2-amino-7-(.beta.-D-arabinofuranosyl)-3,4-dihydro-4-oxo-7H-pyrrolo[-
2,3-d]pyrimidin-5-carbonitrile; and
[0416] the corresponding 5'-triphosphates,
5'-[bis(isopropyloxycarbonyloxy- methyl)]monophosphates,
5'-mono-(S-pivaloyl-2-thioethyl)monophosphates, and
5'-bis-(S-pivaloyl-2-thioethyl)monophosphates thereof;
[0417] or a pharmaceutically acceptable salt thereof.
[0418] Even further illustrative of the present invention is a
method for inhibiting RNA-dependent RNA viral polymerase,
inhibiting RNA-dependent RNA viral replication, and/or treating
RNA-dependent RNA viral infection wherein the compound is selected
from
[0419] 2'-O-methyl-cytidine,
[0420] 2'-C-methyl-cytidine,
[0421] 3',5'-di-O-octanoyl-2'-O-methyl-cytidine,
[0422] 3'-O-octanoyl-2'-O-methyl-cytidine,
[0423]
4-amino-1-(.theta.-D-ribofuranosyl)-1H-pyrazolo[3,4-d]pyrimidine,
[0424]
4-amino-7-(2-C-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyri-
midine-5-carbonitrile,
[0425]
4-amino-7-(2-C-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyri-
midine-5-carboxamide,
[0426] 2'-C-methyladenosine,
[0427] 8-amino-2'-C-methyladenosine,
[0428] 8-bromoguanosine,
[0429] 8-aminoguanosine,
[0430] 8-aminoadenosine,
[0431]
4-amino-7-(3-deoxy-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimid-
ine,
[0432]
2-amino-4-chloro-5-ethyl-7-(2-O-methyl-.beta.-D-ribofuranosyl)-7H-p-
yrrolo[2,3-d]pyrimidine,
[0433]
2-amino-3,4-dihydro-4-oxo-7-(.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-
-d]pyrimidin-5-carboxamide,
[0434]
4-amino-1-(2-C-methyl-.beta.-D-ribofuranosyl)-1H-pyrazolo[3,4-d]pyr-
imidine,
[0435]
2-amino-4-chloro-7-(2-O-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2-
,3-d]pyrimidin-5-carbonitrile;
[0436] and the corresponding 5'-triphosphates thereof;
[0437]
2'-O-methylcytidine-5'-[bis-(S-pivaloyl-2-thioethyl)phosphate),
[0438]
2-amino-7-(3-deoxy-.beta.-D-ribofuranosyl)-3,4-dihydro-4-oxo-7H-pyr-
rolo[2,3-d]pyrimidine-5'-[bis-(S-pivaloyl-2-thioethyl)phosphate],
[0439]
3'-deoxyguanosine-5'-[bis-(S-pivaloyl-2-thioethyl)phosphate],
and
[0440]
3'-deoxycytidine-5'-[bis-(S-pivaloyl-2-thioethyl)phosphate];
[0441] or a pharmaceutically acceptable salt thereof.
[0442] Yet further illustrative of the invention is a method for
inhibiting RNA-dependent RNA viral polymerase, inhibiting
RNA-dependent RNA viral replication, and/or treating RNA-dependent
RNA viral infection wherein the compound is selected from:
[0443] 2'-O-methylcytidine,
[0444] 2'-C-methylcytidine,
[0445] 3',5'-di-O-octanoyl-2'-O-methyl-cytidine,
[0446] 3'-O-octanoyl-2'-O-methyl-cytidine,
[0447]
4-amino-7-(2-C-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyri-
midine-5-carbonitrile,
[0448]
4-amino-7-(2-C-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyri-
midine-5-carboxamide,
[0449] 2'-C-methyladenosine,
[0450] 8-amino-2'-C-methyladenosine,
[0451]
2'-O-methylcytidine-5'-[bis-(S-pivaloyl-2-thioethyl)phosphate],
[0452]
2-amino-7-(3-deoxy-.beta.-D-ribofuranosyl)-3,4-dihydro-4-oxo-7H-pyr-
rolo[2,3-d]pyrimidine-5'-[bis-(S-pivaloyl-2-thioethyl)phosphate],
and
[0453]
3'-deoxycytidine-5'-[bis-(S-pivaloyl-2-thioethyl)phosphate];
[0454] or a pharmaceutically acceptable salt thereof.
[0455] The present invention also provides novel compounds of
structural formula IV of the indicated stereochemical configuration
which are useful as inhibitors of RNA-dependent RNA viral
polymerase: 18
[0456] wherein B is selected from the group consisting of 19
[0457] A, G, and L are each independently CH or N;
[0458] D is N, CH, C--CN, C--NO.sub.2, C--C.sub.1-3 alkyl,
C--NHCONH.sub.2, C--CONR.sup.11R.sup.11, C--CSNR.sup.11R.sup.11,
C--COOR.sup.11, C-hydroxy, C--C.sub.1-3 alkoxy, C-amino,
C--C.sub.1-4 alkylamino, C-di(C.sub.1-4 alkyl)amino, C-halogen,
C-(1,3-oxazol-2-yl), C-(1,3-thiazol-2-yl), or C-(imidazol-2-yl);
wherein alkyl is unsubstituted or substituted with one to three
groups independently selected from halogen, amino, hydroxy,
carboxy, and C.sub.1-3 alkoxy;
[0459] E is N or CR.sup.5;
[0460] W is O or S;
[0461] R.sup.1 is hydrogen, C.sub.2-4 alkenyl, C.sub.2-4 alkynyl,
or C.sub.1-4 alkyl optionally substituted with amino, hydroxy, or 1
to 3 fluorine atoms and one of R.sup.2 and R.sup.3 is hydroxy or
C.sub.1-4 alkoxy and the other of R.sup.2 and R.sup.3 is selected
from the group consisting of
[0462] hydrogen,
[0463] hydroxy,
[0464] halogen,
[0465] C.sub.1-4 alkyl, optionally substituted with 1 to 3 fluorine
atoms,
[0466] C.sub.1-10 alkoxy, optionally substituted with C.sub.1-3
alkoxy or 1 to 3 fluorine atoms,
[0467] C.sub.2-6 alkenyloxy,
[0468] C.sub.1-4 alkylthio,
[0469] C.sub.1-8 alkylcarbonyloxy, aryloxycarbonyl,
[0470] azido,
[0471] amino,
[0472] C.sub.1-4 alkylamino, and
[0473] di(C.sub.1-4 alkyl)amino; or
[0474] R.sup.2 is hydrogen, C.sub.2-4 alkenyl, C.sub.2-4 alkynyl,
or C.sub.1-4 alkyl optionally substituted with amino, hydroxy, or 1
to 3 fluorine atoms and one of R.sup.1 and R.sup.3 is hydroxy or
C.sub.1-4 alkoxy and the other of R.sup.1 and R.sup.3 is selected
from the group consisting of
[0475] hydrogen,
[0476] hydroxy,
[0477] halogen,
[0478] C.sub.1-4 alkyl, optionally substituted with 1 to 3 fluorine
atoms,
[0479] C.sub.1-10 alkoxy, optionally substituted with hydroxy,
C.sub.1-3 alkoxy, carboxy, or 1 to 3 fluorine atoms,
[0480] C.sub.2-6 alkenyloxy,
[0481] C.sub.1-4 alkylthio,
[0482] C.sub.1-8 alkylcarbonyloxy,
[0483] aryloxycarbonyl,
[0484] azido,
[0485] amino,
[0486] C.sub.1-4 alkylamino, and
[0487] di(C.sub.1-4 alkyl)amino; or
[0488] R.sup.1 and R.sup.2 together with the carbon atom to which
they are attached form a 3- to 6-membered saturated monocyclic ring
system optionally containing a heteroatom selected from O, S, and
NC.sub.0-4 alkyl;
[0489] R.sup.4 and R.sup.6 are each independently H, OH, SH,
NH.sub.2, C.sub.1-4 alkylamino, di(C.sub.1-14 alkyl)amino,
C.sub.3-6 cycloalkylamino, halogen, C.sub.1-4 alkyl, C.sub.1-4
alkoxy, or CF.sub.3;
[0490] R.sup.5 is H, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6
alkynyl, C.sub.1-4 alkylamino, CF.sub.3, or halogen;
[0491] R.sup.14 is H, CF.sub.3, C.sub.1-4 alkyl, amino, C.sub.1-4
alkylamino, C.sub.3-6 cycloalkylamino, or di(C.sub.1-4
alkyl)amino;
[0492] R.sup.7 is hydrogen, amino, C.sub.1-4 alkylamino, C.sub.3-6
cycloalkylamino, or di(C.sub.1-4 alkyl)amino;
[0493] each R.sup.11 is independently H or C.sub.1-6 alkyl;
[0494] R.sup.8 is H, halogen, CN, carboxy, C.sub.1-4
alkyloxycarbonyl, N.sub.3, amino, C.sub.1-4 alkylamino,
di(C.sub.1-4 alkyl)amino, hydroxy, C.sub.1-6 alkoxy, C.sub.1-6
alkylthio, C.sub.1-6 alkylsulfonyl, or (C.sub.1-4 alkyl).sub.0-2
aminomethyl;
[0495] R.sup.12 and R.sup.13 are each independently hydrogen or
methyl; and
[0496] R.sup.9 and R.sup.10 are each independently hydroxy,
OCH.sub.2CH.sub.2SC(.dbd.O)C.sub.1-4 alkyl,
OCH.sub.2O(C.dbd.O)OC.sub.1-4 alkyl, NHCHMeCO.sub.2Me,
OCH(C.sub.1-4 alkyl)O(C.dbd.O)C.sub.1-4 alkyl, 20
[0497] provided that at least one of R.sup.9 and R.sup.10 is not
hydroxy.
[0498] The compounds of formula IV are also inhibitors of
RNA-dependent RNA viral replication and are useful for the
treatment of RNA-dependent RNA viral infection.
[0499] In one embodiment, there are provided novel compounds of
structural formula V which are of the stereochemical configuration:
21
[0500] wherein B is 22
[0501] D is N, CH, C--CN, C--NO.sub.2, C--C.sub.1-3 alkyl,
C--NHCONH.sub.2, C--CONR.sup.11R.sup.11, C--CSNR.sup.11R.sup.11,
C--COOR.sup.11, C-hydroxy, C--C.sub.1-3 alkoxy, C-amino,
C--C.sub.1-4 alkylamino, C-di(C.sub.1-4 alkyl)amino, C-halogen,
C-(1,3-oxazol-2-yl), C-(1,3-thiazol-2-yl), or C-(imidazol-2-yl);
wherein alkyl is unsubstituted or substituted with one to three
groups independently selected from halogen, amino, hydroxy,
carboxy, and C.sub.1-3 alkoxy;
[0502] W is O or S;
[0503] E is N or C--R.sup.5;
[0504] R.sup.1 is hydrogen, C.sub.2-4 alkenyl, C.sub.2-4 alkynyl,
or C.sub.1-4 alkyl optionally substituted with amino, hydroxy, or 1
to 3 fluorine atoms and one of R.sup.2 and R.sup.3 is hydroxy or
C.sub.1-4 alkoxy and the other of R.sup.2 and R.sup.3 is selected
from the group consisting of
[0505] hydrogen,
[0506] hydroxy,
[0507] halogen,
[0508] C.sub.1-3 alkyl,
[0509] trifluoromethyl,
[0510] C.sub.1-4 alkoxy,
[0511] C.sub.1-4 alkylthio,
[0512] C.sub.1-8 alkylcarbonyloxy,
[0513] aryloxycarbonyl,
[0514] azido,
[0515] amino,
[0516] C.sub.1-4 alkylamino, and
[0517] di(C.sub.1-4 alkyl)amino; or
[0518] R.sup.2 is hydrogen, C.sub.2-4 alkenyl, C.sub.2-4 alkynyl,
or C.sub.1-4 alkyl optionally substituted with amino, hydroxy, or 1
to 3 fluorine atoms and one of R.sup.1 and R.sup.3 is hydroxy or
C.sub.1-4 alkoxy and the other of R.sup.1 and R.sup.3 is selected
from the group consisting of
[0519] hydrogen,
[0520] hydroxy,
[0521] fluoro,
[0522] C.sub.1-4 alkyl,
[0523] trifluoromethyl,
[0524] C.sub.1-4 alkoxy,
[0525] C.sub.1-4 alkylthio,
[0526] C.sub.1-8 alkylcarbonyloxy,
[0527] azido,
[0528] amino,
[0529] C.sub.1-4 alkylamino, and
[0530] di(C.sub.1-4 alkyl)amino; or
[0531] R.sup.1 and R.sup.2 together with the carbon atom to which
they are attached form a 3- to 6-membered saturated monocyclic ring
system optionally containing a heteroatom selected from O, S, and
NC.sub.0-4 alkyl;
[0532] R.sup.4 and R.sup.6 are each independently H, OH, SH,
NH.sub.2, C.sub.1-4 alkylamino, di(C.sub.1-4 alkyl)amino, C.sub.3-6
cycloalkylamino, halogen, C.sub.1-4 alkyl, C.sub.1-4 alkoxy, or
CF.sub.3;
[0533] R.sup.5 is H, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6
alkynyl, C.sub.1-4 alkylamino, CF.sub.3, or halogen;
[0534] R.sup.7 is hydrogen, amino, C.sub.1-4 alkylamino, C.sub.3-6
cycloalkylamino, or di(C.sub.1-4 alkyl)amino;
[0535] each R.sup.11 is independently H or C.sub.1-6 alkyl;
[0536] R.sup.8 is H, halogen, CN, carboxy, C.sub.1-4
alkyloxycarbonyl, N.sub.3, amino, C.sub.1-4 alkylamino,
di(C.sub.1-4 alkyl)amino, hydroxy, C.sub.1-6 alkoxy, C.sub.1-6
alkylthio, C.sub.1-6 alkylsulfonyl, or (C.sub.1-4 alkyl).sub.0-2
aminomethyl; and
[0537] R.sup.9 and R.sup.10 are each independently hydroxy,
OCH.sub.2CH.sub.2SC(.dbd.O)C.sub.1-4 alkyl, or
OCH.sub.2O(C.dbd.O)C.sub.1- -4 alkyl, provided that at least one of
R.sup.9 and R.sup.10 is not hydroxy.
[0538] In a second embodiment, there are provided novel compounds
of structural formula VI: 23
[0539] wherein B is 24
[0540] D is N, CH, C--CN, C--NO.sub.2, C--C.sub.1-3 alkyl,
C--NHCONH.sub.2, C--CONR.sup.11R.sup.11, C--CSNR.sup.11R.sup.11,
C--COOR.sup.11, C-hydroxy, C--C.sub.1-3 alkoxy, C-amino,
C--C.sub.1-4 alkylamino, C-di(C.sub.1-4 alkyl)amino, C-halogen,
C-(1,3-oxazol-2-yl), C-(1,3-thiazol-2-yl), or C-(imidazol-2-yl);
wherein alkyl is unsubstituted or substituted with one to three
groups independently selected from halogen, amino, hydroxy,
carboxy, and C.sub.1-3 alkoxy;
[0541] W is O or S;
[0542] R.sup.1 is hydrogen, C.sub.2-4 alkenyl, C.sub.2-4 alkynyl,
or C.sub.1-4 alkyl optionally substituted with amino, hydroxy, or 1
to 3 fluorine atoms and one of R.sup.2 and R.sup.3 is hydroxy or
C.sub.1-4 alkoxy and the other of R.sup.2 and R.sup.3 is selected
from the group consisting of
[0543] hydrogen,
[0544] hydroxy,
[0545] fluoro,
[0546] C.sub.1-3 alkyl,
[0547] trifluoromethyl,
[0548] C.sub.1-3 alkoxy,
[0549] C.sub.1-8 alkylcarbonyloxy, and
[0550] amino; or
[0551] R.sup.2 is hydrogen, C.sub.2-4 alkenyl, C.sub.2-4 alkynyl,
or C.sub.1-4 alkyl optionally substituted with amino, hydroxy, or 1
to 3 fluorine atoms and one of R.sup.1 and R.sup.3 is hydroxy or
C.sub.1-4 alkoxy and the other of R.sup.1 and R.sup.3 is selected
from the group consisting of
[0552] hydrogen,
[0553] hydroxy,
[0554] fluoro,
[0555] C.sub.1-3 alkyl,
[0556] trifluoromethyl,
[0557] C.sub.1-3 alkoxy,
[0558] C.sub.1-8 alkylcarbonyloxy, and
[0559] amino; or
[0560] R.sup.1 and R.sup.2 together with the carbon atom to which
they are attached form a 3- to 6-membered saturated monocyclic ring
system optionally containing a heteroatom selected from O, S, and
NC.sub.0-4 alkyl;
[0561] R.sup.6 is H, OH, SH, NH.sub.2, C.sub.1-4 alkylamino,
di(C.sub.1-4 alkyl)amino, C.sub.3-6 cycloalkylamino, halogen,
C.sub.1-4 alkyl, C.sub.1-4 alkoxy, or CF.sub.3;
[0562] R.sup.5 is H, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6
alkynyl, C.sub.1-4 alkylamino, CF.sub.3, or halogen;
[0563] R.sup.7 is hydrogen, amino, C.sub.1-4 alkylamino, C.sub.3-6
cycloalkylamino, or di(C.sub.1-4 alkyl)amino;
[0564] each R.sup.11 is independently H or C.sub.1-6 alkyl;
[0565] R.sup.8 is H, halogen, CN, carboxy, C.sub.1-4
alkyloxycarbonyl, N.sub.3, amino, C.sub.1-4 alkylamino,
di(C.sub.1-4 alkyl)amino, hydroxy, C.sub.1-6 alkoxy, C.sub.1-6
alkylthio, C.sub.1-6 alkylsulfonyl, or (C.sub.1-4 alkyl).sub.0-2
aminomethyl; and
[0566] R.sup.9 and R.sup.10 are each independently hydroxy,
OCH.sub.2CH.sub.2SC(.dbd.O).sub.t-butyl, or OCH.sub.2O(C.dbd.O)iPr,
provided that at least one of R.sup.9 and R.sup.10 is not
hydroxy.
[0567] Illustrative of the novel compounds of structural formula VI
of the present invention are the following:
[0568]
2'-O-methylcytidine-5'-[bis-(S-pivaloyl-2-thioethyl)phosphate],
[0569]
2-amino-7-(3-deoxy-.beta.-D-ribofuranosyl)-3,4-dihydro-4-oxo-7H-pyr-
rolo[2,3-d]pyrimidine-5'-[bis-(S-pivaloyl-2-thioethyl)phosphate],
[0570]
3'-deoxyguanosine-5'-[bis-(S-pivaloyl-2-thioethyl)phosphate],
[0571]
2'-O-methylguanosine-5'-[bis-(S-acetyl-2-thioethyl)phosphate],
[0572]
2'-O-methylguanosine-5'-[bis-(S-pivaloyl-2-thioethyl)phosphate],
[0573]
8-bromo-2'-O-methylguanosine-5'-[bis-(S-pivaloyl-2-thioethyl)phosph-
ate],
[0574]
2-amino-3,4-dihydro-7-(2-O-methyl-.beta.-D-ribofuranosyl)-4-oxo-7H--
pyrrolo[
[0575]
2,3-d]pyrimidine-5'-[bis-(S-pivaloyl-2-thioethyl)phosphate],
[0576]
2-amino-5-bromo-7-(3-deoxy-.beta.-D-ribofuranosyl)-3,4-dihydro-4-ox-
o-7H-pyrrolo[
[0577]
2,3-d]pyrimidine-5'-[bis-(S-pivaloyl-2-thioethyl)phosphate],
[0578]
5-bromo-2'-O-methylcytidine-5'-[bis-(S-pivaloyl-2-thioethyl)phospha-
te],
[0579] 3'-deoxycytidine-5'-[bis-(S-pivaloyl-2-thioethyl)phosphate],
and
[0580]
2'-O-methylcytidine-5'-[bis(isopropyloxycarbonyloxymethyl)]phosphat-
e.
[0581] The present invention further provides novel compounds of
structural formula XII of the indicated stereochemical
configuration or a pharmaceutically acceptable salt thereof which
are useful as inhibitors of RNA-dependent RNA viral polymerase:
25
[0582] wherein R.sup.a and R.sup.h are each independently selected
from the group consisting of hydrogen, cyano, azido, halogen,
hydroxy, mercapto, amino, C.sub.1-4 alkoxy, C.sub.2-4 alkenyl,
C.sub.2-4 alkynyl, and C.sub.1-4 alkyl, wherein alkyl is
unsubstituted or substituted with hydroxy, amino, C.sub.1-4 alkoxy,
C.sub.1-4 alkylthio, or one to three fluorine atoms;
[0583] R.sup.b is C.sub.2-4 alkenyl, C.sub.2-4 alkynyl, or
C.sub.1-4 alkyl, wherein alkyl is unsubstituted or substituted with
hydroxy, amino, C.sub.1-4 alkoxy, C.sub.1-4 alkylthio, or one to
three fluorine atoms;
[0584] R.sup.c is hydrogen, fluorine, hydroxy, mercapto, C.sub.1-4
alkoxy, or C.sub.1-4 alkyl; or R.sup.b and
[0585] R.sup.c together with the carbon atom to which they are
attached form a 3- to 6-membered saturated monocyclic ring system
optionally containing a heteroatom selected from O, S, and
NC.sub.0-4 alkyl;
[0586] R.sup.d is hydrogen, cyano, nitro, C.sub.1-3 alkyl,
NHCONH.sub.2, CONR.sup.jR.sup.j, CSNR.sup.jR.sup.j, COOR.sup.j,
C(.dbd.NH)NH.sub.2, hydroxy, C.sub.1-3 alkoxy, amino, C.sub.1-4
alkylamino, di(C.sub.1-4 alkyl)amino, halogen, (1,3-oxazol-2-yl),
(1,3-thiazol-2-yl), or (imidazol-2-yl); wherein alkyl is
unsubstituted or substituted with one to three groups independently
selected from halogen, amino, hydroxy, carboxy, and C.sub.1-3
alkoxy;
[0587] R.sup.e and R.sup.f are each independently hydrogen,
hydroxy, halogen, C.sub.1-4 alkoxy, amino, C.sub.1-4 alkylamino,
di(C.sub.1-4 alkyl)amino, C.sub.3-6 cycloalkylamino, di(C.sub.3-6
cycloalkyl)amino, or C.sub.4-6 cycloheteroalkyl, unsubstituted or
substituted with one to two groups independently selected from
halogen, hydroxy, amino, C.sub.1-4 alkyl, and C.sub.1-4 alkoxy;
[0588] R.sup.g is hydrogen, C.sub.1-4 alkyl, C.sub.2-4 alkynyl,
halogen, cyano, carboxy, C.sub.1-4 alkyloxycarbonyl, azido, amino,
C.sub.1-4 alkylamino, di(C.sub.1-4 alkyl)amino, hydroxy, C.sub.1-6
alkoxy, C.sub.1-6 alkylthio, C.sub.1-6 alkylsulfonyl, (C.sub.1-4
alkyl).sub.0-2 aminomethyl, or C.sub.4-6 cycloheteroalkyl,
unsubstituted or substituted with one to two groups independently
selected from halogen, hydroxy, amino, C.sub.1-4 alkyl, and
C.sub.1-4 alkoxy;
[0589] R.sup.i is hydrogen, C.sub.1-10 alkylcarbonyl,
P.sub.3O.sub.9H.sub.4, P.sub.2O.sub.6H.sub.3, or
P(O)R.sup.mR.sup.n;
[0590] each R.sup.j is independently hydrogen or C.sub.1-6
alkyl;
[0591] R.sup.k and R.sup.l are each independently hydrogen, methyl,
hydroxymethyl, or fluoromethyl; and
[0592] R.sup.m and R.sup.n are each independently hydroxy,
OCH.sub.2CH.sub.2SC(.dbd.O)C.sub.1-4 alkyl,
OCH.sub.2O(C.dbd.O)OC.sub.1-4 alkyl, NHCHMeCO.sub.2Me,
OCH(C.sub.1-4 alkyl)O(C.dbd.O)C.sub.1-4 alkyl, 26
[0593] with the proviso that when R.sup.a and R.sup.c are
.alpha.-hydroxy, R.sup.e is amino, R.sup.b is .beta.-methyl and
R.sup.h is hydrogen or R.sup.h is .beta.-methyl and R.sup.b is
hydrogen, and R.sup.f, R.sup.g, R.sup.i, R.sup.k, and R.sup.l are
hydrogen, then R.sup.d is not cyano or CONH.sub.2.
[0594] The compounds of formula XII are also inhibitors of
RNA-dependent RNA viral replication and are useful for the
treatment of RNA-dependent RNA viral infection.
[0595] In one embodiment of the novel compounds of structural
formula XII are the compounds of structural formula XIII: 27
[0596] wherein R.sup.a is hydrogen, halogen, hydroxy, amino, or
C.sub.1-3 alkoxy;
[0597] R.sup.b is C.sub.1-3 alkyl, wherein alkyl is unsubstituted
or substituted with hydroxy, amino, C.sub.1-3 alkoxy, C.sub.1-3
alkylthio, or one to three fluorine atoms;
[0598] R.sup.c is hydroxy, fluoro, or C.sub.1-4 alkoxy;
[0599] R.sup.d is hydrogen, cyano, methyl, halogen, or
CONH.sub.2;
[0600] R.sup.g is hydrogen, amino, or C.sub.1-4 alkylamino;
[0601] R.sup.i is hydrogen, P.sub.3O.sub.9H.sub.4,
P.sub.2O.sub.6H.sub.3, or PO.sub.3H.sub.2; and
[0602] R.sup.e and R.sup.f are each independently hydrogen,
hydroxy, halogen, amino, C.sub.1-4 alkylamino, di(C.sub.1-4
alkyl)amino, or C.sub.3-6 cycloalkylamino;
[0603] with the proviso that when R.sup.a and R.sup.c are
.alpha.-hydroxy, R.sup.e is amino, R.sup.b is methyl, and R.sup.f,
R.sup.g, and R.sup.i are hydrogen, then R.sup.d is not cyano or
CONH.sub.2.
[0604] In a second embodiment of the compounds of structural
formula XII are the compounds of structural formula XIII
wherein:
[0605] R.sup.b is methyl, fluoromethyl, hydroxymethyl,
difluoromethyl, trifluoromethyl, or aminomethyl;
[0606] R.sup.c is hydroxy, fluoro, or methoxy;
[0607] R.sup.a is hydrogen, fluoro, hydroxy, amino, or methoxy;
[0608] R.sup.i is hydrogen or P.sub.3O.sub.9H.sub.4;
[0609] R.sup.g is hydrogen or amino;
[0610] R.sup.d is hydrogen, cyano, methyl, halogen, or CONH.sub.2;
and
[0611] R.sup.e and R.sup.f are each independently hydrogen, fluoro,
hydroxy, or amino;
[0612] with the proviso that when R.sup.b is .beta.-methyl, R.sup.a
and R.sup.c are .alpha.-hydroxy, R.sup.e is amino, and R.sup.f,
R.sup.g, and R.sup.i are hydrogen, then R.sup.d is not cyano or
CONH.sub.2.
[0613] Illustrative of the novel compounds of the present invention
of structural formula XIII which are useful as inhibitors of
RNA-dependent RNA viral polymerase are the following:
[0614]
4-amino-7-(2-C-methyl-.beta.-D-arabinofuranosyl)-7H-pyrrolo[2,3-d]p-
yrimidine,
[0615]
4-amino-7-(2-C-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyri-
midine,
[0616]
4-methylamino-7-(2-C-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3--
d]pyrimidine,
[0617]
4-dimethylamino-7-(2-C-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,-
3-d]pyrimidine,
[0618] 4-cyclopropyl
amino-7-(2-C-methyl-.beta.-D-ribofuranosyl)-7H-pyrrol-
o[2,3-d]pyrimidine,
[0619]
4-amino-7-(2-C-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyri-
midine,
[0620]
4-amino-7-(2-C-hydroxymethyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-
-d]pyrimidine,
[0621]
4-amino-7-(2-C-fluoromethyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3--
d]pyrimidine,
[0622]
4-amino-5-methyl-7-(2-C-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2-
,3-d]pyrimidine,
[0623]
4-amino-7-(2-C-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyri-
midine-5-carboxylic acid,
[0624]
4-amino-5-bromo-7-(2-C-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,-
3-d]pyrimidine,
[0625]
4-amino-5-chloro-7-(2-C-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2-
,3-d]pyrimidine,
[0626]
4-amino-5-fluoro-7-(2-C-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2-
,3-d]pyrimidine,
[0627]
2,4-diamino-7-(2-C-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]-
pyrimidine,
[0628]
2-amino-7-(2-C-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d)pyri-
midine,
[0629]
2-amino-4-cyclopropylamino-7-(2-C-methyl-.beta.-D-ribofuranosyl)-7H-
-pyrrolo[2,3-d]pyrimidine,
[0630]
2-amino-7-(2-C-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyri-
midin-4(3H)-one,
[0631]
4-amino-7-(2-C-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyri-
midine,
[0632]
4-amino-7-(2-C,2-O-dimethyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3--
d]pyrimidine,
[0633]
7-(2-C-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidin-4(-
3H)-one,
[0634] 2-amino-5-methyl-7-(2-C,
2-O-dimethyl-.beta.-D-ribofuranosyl)-7H-py-
rrolo[2,3-d]pyrimidin-4(3H)-one,
[0635]
4-amino-7-(3-deoxy-2-C-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,-
3-d]pyrimidine,
[0636]
4-amino-7-(3-deoxy-2-C-methyl-.beta.-D-arabinofuranosyl)-7H-pyrrolo-
[2,3-d]-pyrimidine,
[0637]
4-amino-2-fluoro-7-(2-C-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2-
,3-d]pyrimidine,
[0638]
4-amino-7-(3-C-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyri-
midine,
[0639]
4-amino-7-(3-C-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyri-
midine,
[0640]
4-amino-7-(2,4-di-C-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d-
]pyrimidine, and
[0641]
4-amino-7-(3-deoxy-3-fluoro-2-C-methyl-.beta.-D-ribofuranosyl)-7H-p-
yrrolo[2,3-d]pyrimidine;
[0642] and the corresponding 5'-triphosphates;
[0643] or a pharmaceutically acceptable salt thereof.
[0644] Further illustrative of the novel compounds of the present
invention of structural formula XIII which are useful as inhibitors
of RNA-dependent RNA viral polymerase are the following:
[0645]
4-amino-7-(2-C-methyl-.beta.-D-arabinofuranosyl)-7H-pyrrolo[2,3-d]p-
yrimidine,
[0646]
4-amino-7-(2-C-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyri-
midine,
[0647]
4-amino-7-(2-C-fluoromethyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3--
d]pyrimidine,
[0648]
4-amino-5-methyl-7-(2-C-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2-
,3-d]pyrimidine,
[0649]
4-amino-5-bromo-7-(2-C-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,-
3-d]pyrimidine,
[0650]
4-amino-5-chloro-7-(2-C-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2-
,3-d]pyrimidine,
[0651]
4-amino-5-fluoro-7-(2-C-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2-
,3-d]pyrimidine, and
[0652]
4-amino-7-(2-C,2-O-dimethyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3--
d]pyrimidine,
[0653] and the corresponding 5'-triphosphates;
[0654] or a pharmaceutically acceptable salt thereof.
[0655] Further structurally novel nucleoside derivatives of the
present invention which are useful as inhibitors of RNA-dependent
RNA viral polymerase are the following:
[0656] 3'-deoxy-3'-methyl-cytidine,
[0657] 3',5'-di-O-octanoyl-2'-O-methyl-cytidine,
[0658] 3'-O-octanoyl-2'-O-methyl-cytidine,
[0659]
4-amino-7-(3-deoxy-3-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3--
d]pyrimidin-5-carboxamide,
[0660]
2-amino-5-ethyl-7-(.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimid-
in-4(3H)-one,
[0661]
2-amino-7-(3-deoxy-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-pyrimidin-
-4(3H)-one,
[0662]
2-amino-7-(2-O-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyri-
midin-4(3H)-one,
[0663]
2-amino-7-(2-O-methyl-.beta.-D-ribofuranosyl)-5H-pyrrolo[3,2-d]pyri-
midin-4(3H)-one,
[0664]
7-(2-O-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine,
[0665]
2-amino-3,4-dihydro-4-oxo-7-(2-O-methyl-.beta.-D-ribofuranosyl)-7H--
pyrrolo-[2,3-d]pyrimidin-5-carbonitrile,
[0666]
2-amino-5-methyl-7-(2-O-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2-
,3-d]pyrimidin-4(3H)-one,
[0667]
2-amino-4-chloro-7-(2-O-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2-
,3-d]pyrimidin-5-carbonitrile,
[0668]
2-amino-4-chloro-5-ethyl-7-(2-O-methyl-.beta.-D-ribofuranosyl)-7H-p-
yrrolo[2,3-d]pyrimidine,
[0669]
2-amino-4-chloro-5-methyl-7-(2-O-methyl-.beta.-D-ribofuranosyl)-7H--
pyrrolo[2,3-d]pyrimidine,
[0670]
2-amino-7-(2-O-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyri-
midin-4(3H)-thione,
[0671]
2-amino-4-chloro-7-(2-O-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2-
,3-d]pyrimidine,
[0672]
2-amino-4-chloro-5-methyl-7-(.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-
-d]pyrimidine,
[0673]
2-amino-7-(2-deoxy-2-fluoro-.beta.-D-arabinofuranosyl)-7H-pyrrolo[2-
,3-d]pyrimidin-4(3H)-one,
[0674]
2-amino-4-chloro-7-(2-deoxy-2-fluoro-.beta.-D-arabinofuranosyl)-7H--
pyrrolo[2,3-d]pyrimidine,
[0675]
2-amino-7-(.beta.-D-arabinofuranosyl)-3,4-dihydro-4-oxo-7H-pyrrolo[-
2,3-d]pyrimidin-5-carbonitrile,
[0676] 9-(.beta.-D-arabinofuranosyl)-9H-purin-6 (1H)-one,
[0677] 3'-amino-3'-deoxy-2'-O-methyl-adenosine,
[0678] 8-amino-2'-C-methyladenosine,
[0679]
6-amino-1-(3-deoxy-.beta.-D-ribofuranosyl)-1H-imidazo[4,5-c]pyridin-
-4(5H)-one,
[0680]
6-amino-1-(3-deoxy-3-fluoro-.beta.-D-ribofuranosyl)-1H-imidazo[4,5--
c]pyridin-4(3H)-one,
[0681]
3'-deoxy-2'-O-(2-methoxyethyl)-3'-methyl-5-methyluridine,
[0682]
2-amino-7-(3-deoxy-.beta.-D-ribofuranosyl)-3,4-dihydro-4-oxo-7H-pyr-
rolo[2,3-d]pyrimidin-5-carbonitrile,
[0683]
2-amino-7-(3-deoxy-3-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3--
d]pyrimidin-5-carbonitrile,
[0684]
2-amino-7-(3-deoxy-3-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3--
d]pyrimidin-4(3H)-one,
[0685]
2-amino-7-(3-deoxy-3-fluoro-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3--
d]pyrimidin-4(3H)-one,
[0686]
6-amino-1-(2-O-methyl-.beta.-D-ribofuranosyl)-1H-imidazo[4,5-c]pyri-
din-4(5H)-one,
[0687]
6-amino-1-(3-deoxy-3-methyl-.beta.-D-ribofuranosyl)-1H-imidazo[4,5--
c]pyridin-4(5H)-one,
[0688]
6-amino-1-(2-deoxy-2-fluoro-.beta.-D-ribofuranosyl)-1H-imidazo[4,5--
c]pyridin-4(5H)-one,
[0689] 1-(2-C-methyl-.beta.-D-arabinofuranosyl)uracil,
[0690]
4-amino-1-(3-deoxy-3-fluoro-.beta.-D-ribofuranosyl)-1H-imidazo[4,5--
c]pyridine,
[0691]
2-amino-7-(-3-deoxy-3-fluoro-.beta.-D-ribofuranosyl)-7H-pyrrolo-[
[0692] 2,3-d]pyrimidin-5-carboxamide,
[0693]
4-amino-1-(2-C-methyl-.beta.-D-ribofuranosyl)-1H-pyrazolo[3,4-d]pyr-
imidine,
[0694]
4-amino-1-(3-deoxy-.beta.-D-ribofuranosyl)-1H-imidazo[4,5-c]pyridin-
e, and
[0695]
4-amino-1-(3-deoxy-3-methyl-.beta.-D-ribofuranosyl)-1H-imidazo[4,5--
c]pyridine;
[0696] and the corresponding 5'-triphosphates;
[0697] or a pharmaceutically acceptable salt thereof.
[0698] In a further embodiment the novel compounds of the present
invention are useful as inhibitors of positive-sense
single-stranded RNA-dependent RNA viral polymerase, inhibitors of
positive-sense single-stranded RNA-dependent RNA viral replication,
and/or for the treatment of positive-sense single-stranded
RNA-dependent RNA viral infection. In a class of this embodiment,
the positive-sense single-stranded RNA-dependent RNA virus is a
Flaviviridae virus or a Picornaviridae virus. In a subclass of this
class, the Picornaviridae virus is a rhinovirus, a poliovirus, or a
hepatitis A virus. In a second subclass of this class, the
Flaviviridae virus is selected from the group consisting of
hepatitis C virus, yellow fever virus, dengue virus, West Nile
virus, Japanese encephalitis virus, Banzi virus, and bovine viral
diarrhea virus (BVDV). In a subclass of this subclass, the
Flaviviridae virus is hepatitis C virus.
[0699] Throughout the instant application, the following terms have
the indicated meanings:
[0700] The alkyl groups specified above are intended to include
those alkyl groups of the designated length in either a straight or
branched configuration. Exemplary of such alkyl groups are methyl,
ethyl, propyl, isopropyl, butyl, sec-butyl, tertiary butyl, pentyl,
isopentyl, hexyl, isohexyl, and the like.
[0701] The term "alkenyl" shall mean straight or branched chain
alkenes of two to six total carbon atoms, or any number within this
range (e.g., ethenyl, propenyl, butenyl, pentenyl, etc.).
[0702] The term "alkynyl" shall mean straight or branched chain
alkynes of two to six total carbon atoms, or any number within this
range (e.g., ethynyl, propynyl, butynyl, pentynyl, etc.).
[0703] The term "cycloalkyl" shall mean cyclic rings of alkanes of
three to eight total carbon atoms, or any number within this range
(i.e., cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,
cycloheptyl, or cyclooctyl).
[0704] The term "cycloheteroalkyl" is intended to include
non-aromatic heterocycles containing one or two heteroatoms
selected from nitrogen, oxygen and sulfur. Examples of 4-6-membered
cycloheteroalkyl include azetidinyl, pyrrolidinyl, piperidinyl,
morpholinyl, thiamorpholinyl, imidazolidinyl, tetrahydrofuranyl,
tetrahydropyranyl, tetrahydrothiophenyl, piperazinyl, and the
like.
[0705] The term "alkoxy" refers to straight or branched chain
alkoxides of the number of carbon atoms specified (e.g., C.sub.1-4
alkoxy), or any number within this range [i.e., methoxy (MeO--),
ethoxy, isopropoxy, etc.].
[0706] The term "alkylthio" refers to straight or branched chain
alkylsulfides of the number of carbon atoms specified (e.g.,
C.sub.1-4 alkylthio), or any number within this range [i.e.,
methylthio (MeS--), ethylthio, isopropylthio, etc.].
[0707] The term "alkylamino" refers to straight or branched
alkylamines of the number of carbon atoms specified (e.g.,
C.sub.1-4 alkylamino), or any number within this range [i.e.,
methylamino, ethylamino, isopropylamino, t-butylamino, etc.].
[0708] The term "alkylsulfonyl" refers to straight or branched
chain alkylsulfones of the number of carbon atoms specified (e.g.,
C.sub.1-6 alkylsulfonyl), or any number within this range [i.e.,
methylsulfonyl (MeSO.sub.2--), ethylsulfonyl, isopropylsulfonyl,
etc.].
[0709] The term "alkyloxycarbonyl" refers to straight or branched
chain esters of a carboxylic acid derivative of the present
invention of the number of carbon atoms specified (e.g., C.sub.1-4
alkyloxycarbonyl), or any number within this range [i.e.,
methyloxycarbonyl (MeOCO--), ethyloxycarbonyl, or
butyloxycarbonyl).
[0710] The term "aryl" includes both phenyl, naphthyl, and pyridyl.
The aryl group is optionally substituted with one to three groups
independently selected from C.sub.1-4 alkyl, halogen, cyano, nitro,
trifluoromethyl, C.sub.1-4 alkoxy, and C.sub.1-4 alkylthio.
[0711] The term "halogen" is intended to include the halogen atoms
fluorine, chlorine, bromine and iodine.
[0712] The term "substituted" shall be deemed to include multiple
degrees of substitution by a named substituent. Where multiple
substituent moieties are disclosed or claimed, the substituted
compound can be independently substituted by one or more of the
disclosed or claimed substituent moieties, singly or plurality.
[0713] The term "5'-triphosphate" refers to a triphosphoric acid
ester derivative of the 5'-hydroxyl group of a nucleoside compound
of the present invention having the following general structural
formula VII: 28
[0714] wherein B, Z, R.sup.1-R.sup.4, R.sup.12 and R.sup.13 are as
defined above. The compounds of the present invention are also
intended to include pharmaceutically acceptable salts of the
triphosphate ester as well as pharmaceutically acceptable salts of
5'-monophosphate and 5'-diphosphate ester derivatives of the
structural formulae VIII and IX, respectively, 29
[0715] The term "5'-(S-acyl-2-thioethyl)phosphate" or "SATE" refers
to a mono- or di-ester derivative of a 5'-monophosphate nucleoside
of the present invention of structural formulae X and XI,
respectively, as well as pharmaceutically acceptable salts of the
mono-ester, 30
[0716] The term "composition", as in "pharmaceutical composition,"
is intended to encompass a product comprising the active
ingredient(s) and the inert ingredient(s) that make up the carrier,
as well as any product which results, directly or indirectly, from
combination, complexation or aggregation of any two or more of the
ingredients, or from dissociation of one or more of the
ingredients, or from other types of reactions or interactions of
one or more of the ingredients. Accordingly, the pharmaceutical
compositions of the present invention encompass any composition
made by admixing a compound of the present invention and a
pharmaceutically acceptable carrier.
[0717] The terms "administration of" and "administering a" compound
should be understood to mean providing a compound of the invention
or a prodrug of a compound of the invention to the individual in
need.
[0718] Another aspect of the present invention is concerned with a
method of inhibiting HCV NS5B polymerase, inhibiting HCV
replication, or treating HCV infection with a compound of the
present invention in combination with one or more agents useful for
treating HCV infection. Such agents active against HCV include, but
are not limited to, ribavirin, levovirin, viramidine, thymosin
alpha-1, interferon-.alpha., pegylated interferon-.alpha.
(peginterferon-.alpha.), a combination of interferon-.alpha. and
ribavirin, a combination of peginterferon-.alpha. and ribavirin, a
combination of interferon-.alpha. and levovirin, and a combination
of peginterferon-.alpha. and levovirin. Interferon-.alpha.
includes, but is not limited to, recombinant interferon-.alpha.2a
(such as Roferon interferon available from Hoffmann-LaRoche,
Nutley, N.J.), pegylated interferon-.alpha.2a (Pegasys.TM.),
interferon-.alpha.2b (such as Intron-A interferon available from
Schering Corp., Kenilworth, N.J.), pegylated interferon-.alpha.2b
(PegIntron.TM.), a recombinant consensus interferon (such as
interferon alphacon-1), and a purified interferon-.alpha. product.
Amgen's recombinant consensus interferon has the brand name
Infergen.RTM.. Levovirin is the L-enantiomer of ribavirin which has
shown immunomodulatory activity similar to ribavirin. Viramidine is
an amidino analog of ribavirin disclosed in WO 01/60379 (assigned
to ICN Pharmaceuticals). In accordance with this method of the
present invention, the individual components of the combination can
be administered separately at different times during the course of
therapy or concurrently in divided or single combination forms. The
instant invention is therefore to be understood as embracing all
such regimes of simultaneous or alternating treatment, and the term
"administering" is to be interpreted accordingly. It will be
understood that the scope of combinations of the compounds of this
invention with other agents useful for treating HCV infection
includes in principle any combination with any pharmaceutical
composition for treating HCV infection. When a compound of the
present invention or a pharmaceutically acceptable salt thereof is
used in combination with a second therapeutic agent active against
HCV, the dose of each compound may be either the same as or
different from the dose when the compound is used alone.
[0719] For the treatment of HCV infection, the compounds of the
present invention may also be administered in combination with an
agent that is an inhibitor of HCV NS3 serine protease, such as
LY570310 (VX-950). HCV NS3 serine protease is an essential viral
enzyme and has been described to be an excellent target for
inhibition of HCV replication. Both substrate and non-substrate
based inhibitors of HCV NS3 protease inhibitors are disclosed in WO
98/17679, WO 98/22496, WO 98/46630, WO 99/07733, WO 99/07734, WO
99/38888, WO 99/50230, WO 99/64442, WO 00/09543, WO 00/59929, WO
01/74768, WO 01/81325, and GB-2337262. HCV NS3 protease as a target
for the development of inhibitors of HCV replication and for the
treatment of HCV infection is discussed in B. W. Dymock, "Emerging
therapies for hepatitis C virus infection," Emerging Drugs, 6:
13-42 (2001).
[0720] Ribavirin, levovirin, and viramidine may exert their
anti-HCV effects by modulating intracellular pools of guanine
nucleotides via inhibition of the intracellular enzyme inosine
monophosphate dehydrogenase (IMPDH). IMPDH is the rate-limiting
enzyme on the biosynthetic route in de novo guanine nucleotide
biosynthesis. Ribavirin is readily phosphorylated intracellularly
and the monophosphate derivative is an inhibitor of IMPDH. Thus,
inhibition of IMPDH represents another useful target for the
discovery of inhibitors of HCV replication. Therefore, the
compounds of the present invention may also be administered in
combination with an inhibitor of IMPDH, such as VX-497, which is
disclosed in WO 97/41211 and WO 01/00622, (assigned to Vertex);
another IMPDH inhibitor, such as that disclosed in WO 00/25780
(assigned to Bristol-Myers Squibb); or mycophenolate mofetil [see
A. C. Allison and E. M. Eugui, Agents Action, 44 (Suppl.): 165
(1993)].
[0721] For the treatment of HCV infection, the compounds of the
present invention may also be administered in combination with the
antiviral agent amantadine (1-aminoadamantane) [for a comprehensive
description of this agent, see J. Kirschbaum, Anal. Profiles Drug
Subs. 12: 1-36 (1983)].
[0722] The compounds of the present invention may also be combined
for the treatment of HCV infection with antiviral 2'-C-branched
ribonucleosides disclosed in R. E. Harry-O'kuru, et al., J. Org.
Chem., 62: 1754-1759 (1997); M. S. Wolfe, et al., Tetrahedron
Lett., 36: 7611-7614 (1995); and U.S. Pat. No. 3,480,613 (Nov. 25,
1969), the contents of which are incorporated by reference in their
entirety. Such 2'-C-branched ribonucleosides include, but are not
limited to, 2'-C-methyl-cytidine, 2'-C-methyl-adenosine,
2'-C-methyl-guanosine, and 9-(2-C-methyl-.beta.-D--
ribofuranosyl)-2,6-diaminopurine.
[0723] By "pharmaceutically acceptable" is meant that the carrier,
diluent, or excipient must be compatible with the other ingredients
of the formulation and not deleterious to the recipient
thereof.
[0724] Also included within the present invention are
pharmaceutical compositions comprising the novel nucleoside
compounds and derivatives thereof of the present invention in
association with a pharmaceutically acceptable carrier. Another
example of the invention is a pharmaceutical composition made by
combining any of the compounds described above and a
pharmaceutically acceptable carrier. Another illustration of the
invention is a process for making a pharmaceutical composition
comprising combining any of the compounds described above and a
pharmaceutically acceptable carrier.
[0725] Also included within the present invention are
pharmaceutical compositions useful for inhibiting RNA-dependent RNA
viral polymerase in particular HCV NS5B polymerase comprising an
effective amount of a compound of this invention and a
pharmaceutically acceptable carrier. Pharmaceutical compositions
useful for treating RNA-dependent RNA viral infection in particular
HCV infection are also encompassed by the present invention as well
as a method of inhibiting RNA-dependent RNA viral polymerase in
particular HCV NS5B polymerase and a method of treating
RNA-dependent viral replication and in particular HCV replication.
Additionally, the present invention is directed to a pharmaceutical
composition comprising a therapeutically effective amount of a
compound of the present invention in combination with a
therapeutically effective amount of another agent active against
RNA-dependent RNA virus and in particular against HCV. Agents
active against HCV include, but are not limited to, ribavirin,
levovirin, viramidine, thymosin alpha-1, an inhibitor of HCV NS3
serine protease, interferon-.alpha., pegylated interferon-.alpha.
(peginterferon-.alpha.), a combination of interferon-.alpha. and
ribavirin, a combination of peginterferon-.alpha. and ribavirin, a
combination of interferon-.alpha. and levovirin, and a combination
of peginterferon-.alpha. and levovirin. Interferon-.alpha.
includes, but is not limited to, recombinant interferon-.alpha.2a
(such as Roferon interferon available from Hoffmann-LaRoche,
Nutley, N.J.), interferon-.alpha.2b (such as Intron-A interferon
available from Schering Corp., Kenilworth, N.J.), a consensus
interferon, and a purified interferon-.alpha. product. For a
discussion of ribavirin and its activity against HCV, see J. O.
Saunders and S. A. Raybuck, "Inosine Monophosphate Dehydrogenase:
Consideration of Structure, Kinetics, and Therapeutic Potential,"
Ann. Rep. Med. Chem., 35: 201-210 (2000).
[0726] Another aspect of the present invention provides for the use
of nucleoside compounds and derivatives thereof and their
pharmaceutical compositions for the manufacture of a medicament for
the inhibition of RNA-dependent RNA viral replication, in
particular HCV replication, and/or the treatment of RNA-dependent
RNA viral infection, in particular HCV infection. Yet a further
aspect of the present invention provides for nucleoside compounds
and derivatives thereof and their pharmaceutical compositions for
use as a medicament for the inhibition of RNA-dependent RNA viral
replication, in particular HCV replication, and/or for the
treatment of RNA-dependent RNA viral infection, in particular HCV
infection.
[0727] The pharmaceutical compositions of the present invention
comprise a compound of structural formula I, IV, or XII as an
active ingredient or a pharmaceutically acceptable salt thereof,
and may also contain a pharmaceutically acceptable carrier and
optionally other therapeutic ingredients.
[0728] The compositions include compositions suitable for oral,
rectal, topical, parenteral (including subcutaneous, intramuscular,
and intravenous), ocular (ophthalmic), pulmonary (nasal or buccal
inhalation), or nasal administration, although the most suitable
route in any given case will depend on the nature and severity of
the conditions being treated and on the nature of the active
ingredient. They may be conveniently presented in unit dosage form
and prepared by any of the methods well-known in the art of
pharmacy.
[0729] In practical use, the compounds of structural formulae I,
IV, and XII can be combined as the active ingredient in intimate
admixture with a pharmaceutical carrier according to conventional
pharmaceutical compounding techniques. The carrier may take a wide
variety of forms depending on the form of preparation desired for
administration, e.g., oral or parenteral (including intravenous).
In preparing the compositions for oral dosage form, any of the
usual pharmaceutical media may be employed, such as, for example,
water, glycols, oils, alcohols, flavoring agents, preservatives,
coloring agents and the like in the case of oral liquid
preparations, such as, for example, suspensions, elixirs and
solutions; or carriers such as starches, sugars, microcrystalline
cellulose, diluents, granulating agents, lubricants, binders,
disintegrating agents and the like in the case of oral solid
preparations such as, for example, powders, hard and soft capsules
and tablets, with the solid oral preparations being preferred over
the liquid preparations.
[0730] Because of their ease of administration, tablets and
capsules represent the most advantageous oral dosage unit form in
which case solid pharmaceutical carriers are obviously employed. If
desired, tablets may be coated by standard aqueous or nonaqueous
techniques. Such compositions and preparations should contain at
least 0.1 percent of active compound. The percentage of active
compound in these compositions may, of course, be varied and may
conveniently be between about 2 percent to about 60 percent of the
weight of the unit. The amount of active compound in such
therapeutically useful compositions is such that an effective
dosage will be obtained. The active compounds can also be
administered intranasally as, for example, liquid drops or
spray.
[0731] The tablets, pills, capsules, and the like may also contain
a binder such as gum tragacanth, acacia, corn starch or gelatin;
excipients such as dicalcium phosphate; a disintegrating agent such
as corn starch, potato starch, alginic acid; a lubricant such as
magnesium stearate; and a sweetening agent such as sucrose, lactose
or saccharin. When a dosage unit form is a capsule, it may contain,
in addition to materials of the above type, a liquid carrier such
as a fatty oil.
[0732] Various other materials may be present as coatings or to
modify the physical form of the dosage unit. For instance, tablets
may be coated with shellac, sugar or both. A syrup or elixir may
contain, in addition to the active ingredient, sucrose as a
sweetening agent, methyl and propylparabens as preservatives, a dye
and a flavoring such as cherry or orange flavor.
[0733] Compounds of structural formulae I, IV, and XII may also be
administered parenterally. Solutions or suspensions of these active
compounds can be prepared in water suitably mixed with a surfactant
such as hydroxy-propylcellulose. Dispersions can also be prepared
in glycerol, liquid polyethylene glycols and mixtures thereof in
oils. Under ordinary conditions of storage and use, these
preparations contain a preservative to prevent the growth of
microorganisms.
[0734] The pharmaceutical forms suitable for injectable use include
sterile aqueous solutions or dispersions and sterile powders for
the extemporaneous preparation of sterile injectable solutions or
dispersions. In all cases, the form must be sterile and must be
fluid to the extent that easy syringability exists. It must be
stable under the conditions of manufacture and storage and must be
preserved against the contaminating action of microorganisms such
as bacteria and fungi. The carrier can be a solvent or dispersion
medium containing, for example, water, ethanol, polyol (e.g.
glycerol, propylene glycol and liquid polyethylene glycol),
suitable mixtures thereof, and vegetable oils.
[0735] Any suitable route of administration may be employed for
providing a mammal, especially a human with an effective dosage of
a compound of the present invention. For example, oral, rectal,
topical, parenteral, ocular, pulmonary, nasal, and the like may be
employed. Dosage forms include tablets, troches, dispersions,
suspensions, solutions, capsules, creams, ointments, aerosols, and
the like. Preferably compounds of structural formulae I, IV, and
XII are administered orally.
[0736] For oral administration to humans, the dosage range is 0.01
to 1000 mg/kg body weight in divided doses. In one embodiment the
dosage range is 0.1 to 100 mg/kg body weight in divided doses. In
another embodiment the dosage range is 0.5 to 20 mg/kg body weight
in divided doses. For oral administration, the compositions are
preferably provided in the form of tablets or capsules containing
1.0 to 1000 milligrams of the active ingredient, particularly, 1,
5, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 400, 500, 600,
750, 800, 900, and 1000 milligrams of the active ingredient for the
symptomatic adjustment of the dosage to the patient to be
treated.
[0737] The effective dosage of active ingredient employed may vary
depending on the particular compound employed, the mode of
administration, the condition being treated and the severity of the
condition being treated. Such dosage may be ascertained readily by
a person skilled in the art. This dosage regimen may be adjusted to
provide the optimal therapeutic response.
[0738] The compounds of the present invention contain one or more
asymmetric centers and can thus occur as racemates and racemic
mixtures, single enantiomers, diastereomeric mixtures and
individual diastereomers. The present invention is meant to
comprehend nucleoside derivatives having the .beta.-D
stereochemical configuration for the five-membered furanose ring as
depicted in the structural formula below, that is, nucleoside
compounds in which the substituents at C-1 and C-4 of the
five-membered furanose ring have the .beta.-stereochemical
configuration ("up" orientation as denoted by a bold line). 31
[0739] The stereochemistry of the substituents at the C-2 and C-3
positions of the furanose ring of the compounds of the present
invention is denoted either by a dashed line which signifies that
the substituent, for example R.sup.2 in structural formula VI, has
the .alpha. (substituent "down") configuration or a squiggly line
which signifies that the substituent, for example R.sup.3 in
structural formula VI, can have either the a (substituent "down")
or .beta. (substituent "up") configuration.
[0740] Some of the compounds described herein contain olefinic
double bonds, and unless specified otherwise, are meant to include
both E and Z geometric isomers.
[0741] Some of the compounds described herein may exist as
tautomers such as keto-enol tautomers. The individual tautomers as
well as mixtures thereof are encompassed with compounds of
structural formulae I, IV, and XII. An example of keto-enol
tautomers which are intended to be encompassed within the compounds
of the present invention is illustrated below: 32
[0742] Compounds of structural formulae I, IV, and XII may be
separated into their individual diastereoisomers by, for example,
fractional crystallization from a suitable solvent, for example
methanol or ethyl acetate or a mixture thereof, or via chiral
chromatography using an optically active stationary phase.
[0743] Alternatively, any stereoisomer of a compound of the
structural formulae I, IV, and XII may be obtained by
stereospecific synthesis using optically pure starting materials or
reagents of known configuration.
[0744] The stereochemistry of the substituents at the C-2 and C-3
positions of the furanose ring of the novel compounds of the
present invention of structural formula XII is denoted by squiggly
lines which signifies that substituents R.sup.a, R.sup.b, R.sup.c
and R.sup.h can have either the I (substituent "down") or .theta.
(substituent "up") configuration independently of one another.
33
[0745] The compounds of the present invention may be administered
in the form of a pharmaceutically acceptable salt. The term
"pharmaceutically acceptable salt" refers to salts prepared from
pharmaceutically acceptable non-toxic bases or acids including
inorganic or organic bases and inorganic or organic acids. Salts of
basic compounds encompassed within the term "pharmaceutically
acceptable salt" refer to non-toxic salts of the compounds of this
invention which are generally prepared by reacting the free base
with a suitable organic or inorganic acid. Representative salts of
basic compounds of the present invention include, but are not
limited to, the following: acetate, benzenesulfonate, benzoate,
bicarbonate, bisulfate, bitartrate, borate, bromide, camsylate,
carbonate, chloride, clavulanate, citrate, dihydrochloride,
edetate, edisylate, estolate, esylate, fumarate, gluceptate,
gluconate, glutamate, glycollylarsanilate, hexylresorcinate,
hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate,
iodide, isothionate, lactate, lactobionate, laurate, malate,
maleate, mandelate, mesylate, methylbromide, methylnitrate,
methylsulfate, mucate, napsylate, nitrate, N-methylglucamine
ammonium salt, oleate, oxalate, pamoate (embonate), palmitate,
pantothenate, phosphate/diphosphate, polygalacturonate, salicylate,
stearate, sulfate, subacetate, succinate, tannate, tartrate,
teoclate, tosylate, triethiodide and valerate. Furthermore, where
the compounds of the invention carry an acidic moiety, suitable
pharmaceutically acceptable salts thereof include, but are not
limited to, salts derived from inorganic bases including aluminum,
ammonium, calcium, copper, ferric, ferrous, lithium, magnesium,
manganic, mangamous, potassium, sodium, zinc, and the like.
Particularly preferred are the ammonium, calcium, magnesium,
potassium, and sodium salts. Salts derived from pharmaceutically
acceptable organic non-toxic bases include salts of primary,
secondary, and tertiary amines, cyclic amines, and basic
ion-exchange resins, such as arginine, betaine, caffeine, choline,
N,N-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol,
2-dimethylaminoethanol, ethanolamine, ethylenediamine,
N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine,
histidine, hydrabamine, isopropylamine, lysine, methylglucamine,
morpholine, piperazine, piperidine, polyamine resins, procaine,
purines, theobromine, triethylamine, trimethylamine,
tripropylamine, tromethamine, and the like.
[0746] Also, in the case of a carboxylic acid (--COOH) or alcohol
group being present in the compounds of the present invention,
pharmaceutically acceptable esters of carboxylic acid derivatives,
such as methyl, ethyl, or pivaloyloxymethyl, or acyl derivatives of
alcohols, such as acetate or maleate, can be employed. Included are
those esters and acyl groups known in the art for modifying the
solubility or hydrolysis characteristics for use as
sustained-release or prodrug formulations.
[0747] Preparation of the Nucleoside Compounds and Derivatives of
the Invention
[0748] The nucleoside compounds and derivatives thereof of the
present invention can be prepared following synthetic methodologies
well-established in the practice of nucleoside and nucleotide
chemistry. Reference is made to the following text for a
description of synthetic methods used in the preparation of the
compounds of the present invention: "Chemistry of Nucleosides and
Nucleotides," L. B. Townsend, ed., Vols. 1-3, Plenum Press, 1988,
which is incorporated by reference herein in its entirety.
[0749] A representative general method for the preparation of
compounds of the present invention is outlined in Scheme 1 below.
This scheme illustrates the synthesis of compounds of the present
invention of structural formula 1-7 wherein the furanose ring has
the .beta.-D-ribo configuration. The starting material is a
3,5-bis-O-protected alkyl furanoside, such as methyl furanoside, of
structural formula 1-1. The C-2 hydroxyl group is then oxidized
with a suitable oxidizing agent, such as a chromium trioxide or
chromate reagent or Dess-Martin periodinane, or by Swern oxidation,
to afford a C-2 ketone of structural formula 1-2. Addition of a
Grignard reagent, such as an alkyl, alkenyl, or alkynyl magnesium
halide (for example, MeMgBr, EtMgBr, vinylMgBr, allylMgBr, and
ethynylMgBr) or an alkyl, alkenyl, or alkynyl lithium, such as
MeLi, across the carbonyl double bond of 1-2 in a suitable organic
solvent, such as tetrahydrofuran, diethyl ether, and the like,
affords the C-2 tertiary alcohol of structural formula 1-3. A good
leaving group (such as Cl, Br, and I) is next introduced at the C-1
(anomeric) position of the furanose sugar derivative by treatment
of the furanoside of formula 1-3 with a hydrogen halide in a
suitable organic solvent, such as hydrogen bromide in acetic acid,
to afford the intermediate furanosyl halide 1-4. A C-1 sulfonate,
such methanesulfonate (MeSO.sub.2O--), trifluoromethanesulfonate
(CF.sub.3SO.sub.2O--), or p-toluenesulfonate (--OTs), may also
serve as a useful leaving group in the subsequent reaction to
generate the glycosidic (nucleosidic) linkage. The nucleosidic
linkage is constructed by treatment of the intermediate of
structural formula 1-4 with the metal salt (such as lithium,
sodium, or potassium) of an appropriately substituted
1H-pyrrolo[2,3-d]pyrimidine 1-5, such as an appropriately
substituted 4-halo-1H-pyrrolo[2,3-d]pyrimid- ine, which can be
generated in situ by treatment with an alkali hydride (such as
sodium hydride), an alkali hydroxide (such as potassium hydroxide),
an alkali carbonate (such as potassium carbonate), or an alkali
hexamethyldisilazide (such as NaHMDS) in a suitable anhydrous
organic solvent, such as acetonitrile, tetrahydrofuran,
1-methyl-2-pyrrolidinone, or N,N-dimethylformamide (DMF). The
displacement reaction can be catalyzed by using a phase-transfer
catalyst, such as TDA-1 or triethylbenzylammonium chloride, in a
two-phase system (solid-liquid or liquid-liquid). The optional
protecting groups in the protected nucleoside of structural formula
1-6 are then cleaved following established deprotection
methodologies, such as those described in T. W. Greene and P. G. M.
Wuts, "Protective Groups in Organic Synthesis," 3.sup.rd ed., John
Wiley & Sons, 1999. Optional introduction of an amino group at
the 4-position of the pyrrolo[2,3-d]pyrimidine nucleus is effected
by treatment of the 4-halo intermediate 1-6 with the appropriate
amine, such as alcoholic ammonia or liquid ammonia, to generate a
primary amine at the C-4 position (--NH.sub.2), an alkylamine to
generate a secondary amine (--NHR), or a dialkylamine to generate a
tertiary amine (--NRR'). A 7H-pyrrolo[2,3-d]pyrimidin-4(3H)one
compound may be derived by hydrolysis of 1-6 with aqueous base,
such as aqueous sodium hydroxide. Alcoholysis (such as
methanolysis) of 1-6 affords a C-4 alkoxide (--OR), whereas
treatment with an alkyl mercaptide affords a C-4 alkylthio (--SR)
derivative. Subsequent chemical manipulations well-known to
practitioners of ordinary skill in the art of organic/medicinal
chemistry may be required to attain the desired compounds of the
present invention. 34
[0750] The examples below provide citations to literature
publications, which contain details for the preparation of final
compounds or intermediates employed in the preparation of final
compounds of the present invention. The nucleoside compounds of the
present invention were prepared according to procedures detailed in
the following examples. The examples are not intended to be
limitations on the scope of the instant invention in any way, and
they should not be so construed. Those skilled in the art of
nucleoside and nucleotide synthesis will readily appreciate that
known variations of the conditions and processes of the following
preparative procedures can be used to prepare these and other
compounds of the present invention. All temperatures are degrees
Celsius unless otherwise noted.
EXAMPLE 1
3'-Deoxyguanosine
[0751] 35
[0752] This compound was prepared following the procedures
described in Nucleosides Nucleotides, 13: 1049 (1994).
EXAMPLE 2
3'-Deoxy-3'-fluoroguanosine
[0753] 36
[0754] This compound was prepared following the procedures
described in J. Med. Chem. 34: 2195 (1991).
EXAMPLE 3
8-Azidoguanosine
[0755] 37
[0756] This compound was prepared following the procedures
described in Chem. Pharm. Bull. 16: 1616 (1968).
EXAMPLE 4
8-Bromoguanosine
[0757] 38
[0758] This compound was obtained from commercial sources.
EXAMPLE 5
2'-O-Methylguanosine
[0759] 39
[0760] This compound was obtained from commercial sources.
EXAMPLE 6
3'-Deoxy-3'-(fluoromethyl)guanosine
[0761] 40
[0762] To a solution of
1,2-O-diacetyl-5-O-(p-toluoyl)-3-deoxy-3-(fluorome-
thyl)-D-ribofuranose (257 mg, 0.7 mmol) [prepared by a similar
method as that described for the corresponding 5-O-benzyl
derivative in J. Med. Chem. 36: 353 (1993)] and
N.sup.2-acetyl-O.sup.6-(diphenylcarbamoyl)guani- ne (554 mg, 1.43
mmol) in anhydrous acetonitrile (6.3 mL) was added
bis(trimethylsilyl)acetamide (BSA) (1.03 g, 5 mmol). The reaction
mixture was stirred at reflux for 30 minutes, and the bath was
removed. The reaction mixture was cooled in an ice bath and
TMS-triflate (288 mg, 1.3 mmol) was added with stirring. After
addition was complete, the reaction was heated at reflux for 2 hr.,
the reaction mixture was poured onto ice and extracted with
chloroform (5.times.10 mL). The combined organic layers were washed
with aqueous saturated sodium bicarbonate, brine and dried over
anhydrous Na.sub.2SO.sub.4. The solvent was removed under reduced
pressure and the residue chromatographed over silica gel using 5%
acetone/CH.sub.2Cl.sub.2 as the eluant to furnish the fully
protected corresponding nucleoside derivative. This was dissolved
in 1,4-dioxane (1.5 mL) to which was added 40% MeNH.sub.2/H.sub.2O
(1.3 g, 17 mmol). The reaction mixture was stirred for 1 day,
evaporated and the residue crystallized with ether/MeOH to provide
the title compound (58 mg). .sup.1H NMR (DMSO-d.sub.6): .delta.
2.76-2.67 (m, 1H); 3.55-3.50 (m, 1H), 2.76-2.67 (m, 1H); 3.71-3.66
(m, 1H), 4.08-4.04 (m, 1H), 4.77-4.50 (m, 3H), 5.06 (t, 1H, J=5.3
Hz), 5.69 (d, 1H, J=3.4 Hz), 5.86 (d, 1H, J=5.1 Hz), 6.45 (bs, 2H),
7.97 (s, 1H), 10.59 (s, 1H). .sup.19F NMR (DMSO-d.sub.6): .delta.
-221.46 (m, F).
EXAMPLE 7
2-Amino-3,4-dihydro-4-oxo-7-(.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyri-
midin-5-carboxamide
[0763] 41
[0764] This compound was prepared following the procedures
described in Tetrahedron. Lett. 25: 4793 (1983).
EXAMPLE 8
2-Amino-3,4-dihydro-4-oxo-7-(.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyri-
midin-5-carbonitrile
[0765] 42
[0766] This compound was prepared following the procedures
described in J. Am. Chem. Soc. 98: 7870 (1976).
EXAMPLE 9
2-Amino-5-ethyl-7-(.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidin-4(3H-
)-one
[0767] 43
Step A:
2-Amino-7-(5-O-tert-butyldimethylsilyl-2,3-O-isopropylidene-.beta.-
-D-ribofuranosyl)-4-chloro-5-ethyl-7H-pyrrolo[2,3-d]pyrimidine
[0768] To a stirred suspension of
2-amino-4-chloro-5-ethyl-1H-pyrrolo[2,3-- d]pyrimidine [described
in EP 866070 (1998)] (1.57 g, 8 mmol) in dry MeCN (48 mL) was added
NaH (60% in mineral oil; 0.32 g, 8 mmol), and the mixture was
stirred at room temperature for 1 h. A solution of
5-O-tert-butyldimethylsilyl-2,3-O-isopropylidene-.alpha.-D-ribofuranosyl
chloride [generated in situ from the corresponding lactol (1.95 g,
6.4 mmol) according to Wilcox et al., Tetrahedron Lett., 27: 1011
(1986)] in dry THF (9.6 mL) was added at room temperature, and the
mixture was stirred overnight, then evaporated to dryness. The
residue was suspended in water (100 mL) and extracted with EtOAc
(200+150 mL). The combined extracts were washed with brine, dried
(Na.sub.2SO.sub.4) and evaporated. The residue was purified on a
silica gel column using a solvent system of hexanes/EtOAc: 7/1.
Appropriate fractions were collected and evaporated to dryness to
give the title compound (1.4 g) as a colorless foam.
Step B:
2-Amino-4-chloro-5-ethyl-7-(.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-
-d]pyrimidine
[0769] A mixture of the compound from Step A (1.19 g, 2.5 mmol) in
MeOH (100 mL) and water (50 mL) was stirred with DOWEX H.sup.+ (to
adjust pH of the mixture to 5) at room temperature for 2.5 h. The
mixture was filtered and the resin thoroughly washed with MeOH. The
combined filtrate and washings were evaporated and the residue
coevaporated several times with water to yield the title compound
(0.53 g) as a white solid.
Step C:
2-Amino-5-ethyl-7-(.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimi-
din-4(3H)-one
[0770] A mixture of the compound from Step B (104 mg, 0.32 mmol) in
2N aqueous NaOH (10 mL) was stirred at reflux temperature for 15
min. The solution was cooled in ice bath, neutralized with 2 N
aqueous HCl, and evaporated to dryness. The residue was suspended
in MeOH, mixed with silica gel, and evaporated. The solid residue
was placed onto a silica gel column (packed in a solvent mixture of
CH.sub.2Cl.sub.2/MeOH: 10/1) which was eluted with a solvent system
of CH.sub.2Cl.sub.2/MeOH: 10/1 and 5/1. The fractions containing
the product were collected and evaporated to dryness to yield the
title compound (48 mg) as a white solid.
[0771] .sup.1H NMR (CD.sub.3OD): .delta. 1.22 (t, 3H), 2.69 (q,
2H), 3.69, 3.80 (2m, 2H), 4.00 (m, 1H), 4.22 (m, 1H), 4.45 (t, 1H),
5.86 (d, 1H, J=6.0 Hz), 6.60 (d, 1H, J=1.2 Hz).
EXAMPLE 10
2-Amino-7-(3-deoxy-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidin-4(3H-
)-one
[0772] 44
Step A:
2-Amino-7-(2,3-anhydro-.beta.-D-ribofuranosyl)-4-methoxy-7H-pyrrol-
o[2,3-d]pyrimidine
[0773] To a mixture of
2-amino-7-(.beta.-D-ribofuranosyl)-4-chloro-7H-pyrr-
olo[2,3-d]pyrimidine (1.8 g, 6.0 mmol) in acetonitrile (80 mL) were
added a solution of H.sub.2O/CH.sub.3CN (1:9, 1.08 mL) and then
.alpha.-acetoxyisobutyryl bromide (3.5 mL, 24 mmol). After 2 h
stirring at room temperature, saturated aqueous NaHCO.sub.3 (170
mL) was added and the mixture was extracted with EtOAc (300+200
mL). The combined organic phase was washed with brine (100 mL),
dried (Na.sub.2SO.sub.4) and evaporated to a pale yellow foamy
residue. This was suspended in anhydrous MeOH (80 mL) and stirred
overnight with 25 mL of DOWEX OH.sup.- resin (previously washed
with anhydrous MeOH). The resin was filtered, washed thoroughly
with MeOH and the combined filtrate evaporated to give a pale
yellow foam (1.92 g).
Step B:
2-Amino-7-(3-deoxy-.beta.-D-ribofuranosyl)-4-methoxy-7H-pyrrolo[2,-
3-d]pyrimidine
[0774] A solution of LiEt.sub.3BH/THF (1M, 75 mL, 75 mmol) was
added dropwise to a cold (ice bath) deoxygenated (Ar, 15 min)
solution of the compound from Step A (1.92 g) under Ar. Stirring at
0.degree. C. was continued for 4 h. At this point the reaction
mixture was acidified with 5% aqueous acetic acid (110 mL), then
purged with Ar for 1 h and and finally evaporated to a solid
residue. Purification on a silica gel column using
MeOH/CH.sub.2Cl.sub.2 as eluent yielded target compound as a
colourless foam (1.01 g).
Step C:
2-Amino-7-(3-deoxy-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimi-
dine-4(3H)-one
[0775] A mixture of compound from Step B (0.4 g, 1.4 mmol) in 2N
aqueous NaOH (40 mL) was stirred at reflux temperature for 3 h. The
solution was cooled in ice bath, neutralized with 2 N aqueous HCl
and evaporated to dryness. The residue was suspended in MeOH, mixed
with silica and evaporated. The residue was placed onto a silica
gel column which was eluted with CH.sub.2Cl.sub.2/MeOH: 10/1 and
5/1 to give the title compound as white solid (0.3 g).
[0776] .sup.1H NMR (DMSO-d.sub.6): .delta. 1.85, 2.12 (2m, 2H),
3.55, 3.46 (2dd, 2H), 4.18 (m, 1H); 4.29 (m, 1H), 4.85 (7, 1H),
5.42 (d, 1H) 5.82 (d, 1H, J=2.4 Hz), 6.19 (s, 2H), 6.23 (d, 1H,
J=3.6 Hz), 6.87 (d, 1H), 10.31 (s, 1H).
EXAMPLE 11
2-Amino-7-(2-O-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidin-4-
(3H)-one
[0777] 45
Step A:
2-Amino-4-chloro-7-(5-t-butyldimethylsilyl-2,3-O-isopropylidene-.b-
eta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine
[0778] HMPT (10.65 ml, 55 mmol) was added portionwise over 30 min.
to a solution of
5-O-tert-butyldimethylsilyl-2,3-O-isopropylidene-D-ribofurano- se
(13.3 g, 44 mmol), dry THF (135 mL), CCl.sub.4 (5.62 mL, 58 mmol)
under N.sub.2 at -76.degree. C. After 30 min., the temp. was raised
to -20.degree. C. In a separate flask, a suspension of
2-amino-4-chloro-1H-pyrrolo-[2,3-d]-pyrimidine (15 g, 89 mmol) in
CH.sub.3CN (900 mL) was treated at 15.degree. C. with 60% NaH (3.60
g., 90 mmol.). The reaction was stirred 30 min whereupon the
previous reaction mixture was cannulated with vigorous stirring.
The reaction was stirred 16 hrs. and then concentrated in vacuo.
The resulting semisolid was added to ice/water/EtOAc and extracted
with EtOAc (3.times.200 mL), dried NaSO.sub.4, filtered and
evaporated. The resulting oil was chromatographed on silica gel
(EtOAc/Hexane 1/1) to afford the product as an oil (9.0 g).
Step B:
2-Amino-4-chloro-7-(.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrim-
idine
[0779] A solution of the compound from Step A (5.76 g, 13 mmol) in
MeOH/H.sub.2O (1200 mL/600 mL) and Dowex WX8-400 (4.8 g) was
stirred 16 hrs. at room temperature. The resin was filtered off and
the filtrate evaporated to afford the title compound as a white
solid; yield 3.47 g.
[0780] .sup.1H NMR (DMSO-d.sub.6): .delta. 3.56 (m, 2H), 3.86 (m,
1H), 4.07 (m, 1H), 4.32 (m, 1H), 4.99 (t, 1H), 5.10 (d, 1H), 5.30
(d, 1H), 6.00 (d, 1H), 6.38 (d, 1H), 6.71 (s br, 2H), 7.39 (d,
1H).
Step C:
2-Amino-4-chloro-7-(2-O-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[-
2,3-d]pyrimidine
[0781] A solution of the compound from Step B (1.0 g, 3.3 mmol) in
dry DMF (100 mL) at 15.degree. C. was treated with 60% NaH (0.14 g,
3.5 mmol). After 30 min., iodomethane (47 g, 3.3 mmol) was added
portionwise to the stirred solution. The reaction was stirred at
room temperature for 16 hrs. and then evaporated at a temperature
below 40.degree. C. The resulting solid was chromatographed on
silica gel to afford the product as a white solid; yield 0.81
g.
[0782] .sup.1H NMR (DMSO-d.sub.6): .delta. 3.25 (s, 3H), 3.54 (m,
2H), 3.87 (m, 1H), 4.07 (m, 1H), 4.22 (m, 1H), 5.01 (m, 1H), 5.16
(d, 1H), 6.07 (d, 1H), 6.37 (d, 1H), 6.70 (s br, 2H), 7.40 (s, 1H).
Mass spectrum: m/z 316 (M+1).sup.+.
Step D:
2-Amino-7-(2-O-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyr-
imidin-4(3H)-one
[0783] A solution of the compound from Step C (80 mg, 0.25 mmol) in
NaOH/H.sub.2O (1.6 g/20 ml) was heated at reflux for 7 hrs.,
whereupon the solution was adjusted with dilute HCl to a pH of 7
and then evaporated. Chromatography of the resulting solid on
silica gel with EtOAc/MeOH 8/2 afforded the product as a white
solid; yield 64 mg.
[0784] .sup.1H NMR (DMSO-d.sub.6): .delta. 3.25 (s, 3H), 3.52 (m,
2H) 3.81 (m, 1H), 4.00 (m, 1H), 4.19 (m, 1H), 5.10 (s br, 2H), 5.95
(d, 1H), 6.27 (d, 1H), 6.33 (s br, 2H), 6.95 (d, 1H), 10.55 (s br,
1H).
EXAMPLE 12
2-Amino-5-methyl-7-(.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidin-4(3-
H)-one
[0785] 46
[0786] This compound is described in Biochemistry, 33: 2703 (1994)
and was synthesized by the following procedure:
Step A:
2-Amino-7-(5-O-tert-butyldimethylsilyl-2,3-O-isopropylidene-.beta.-
-D-ribofuranosyl)-4-chloro-5-methyl-7H-pyrrolo[2,3-d]pyrimidine
[0787] To a stirred suspension of
2-amino-4-chloro-5-methyl-1H-pyrrolo[2,3- -d]pyrimidine (Liebigs
Ann. Chem. 1984, 4, 708) (0.91 g, 5 mmol) in dry MeCN (30 ml) was
added Nail (60% in mineral oil; 0.2 g, 5 mmol) and the mixture was
stirred at room temperature for 0.5 h. A solution of
5-O-tert-butyldimethylsilyl-2,3-O-isopropylidene-.alpha.-D-ribofuranosyl
chloride [generated in situ from the corresponding lactol (1.22 g,
4 mmol) according to Tetrahedron Lett. 27: 1011 (1986)] in dry THF
(6 mL) was added at room temperature, and the mixture was stirred
overnight, then evaporated to dryness. The residue was suspended in
water (100 mL) and extracted with EtOAc (2.times.100 mL). The
combined organic extracts were washed with brine, dried
(Na.sub.2SO.sub.4) and evaporated. The residue was purified on a
silica gel column using a solvent system of hexanes/EtOAc: 7/1 and
5/1. Appropriate fractions were collected and evaporated to dryness
to give the title compound (0.7 g) as a colorless foam.
Step B:
2-Amino-4-chloro-5-methyl-7-(.beta.-D-ribofuranosyl)-7H-pyrrolo[2,-
3-d]pyrimidine
[0788] A mixture of the intermediate from Step A (0.67 g, 1.4 mmol)
in MeOH (70 ml) and water (35 ml) was stirred with DOWEX H.sup.+
(to adjust pH of the mixture to 5) at room temperature for 4 h. The
mixture was filtered and the resin thoroughly washed with MeOH. The
combined filtrate and washings were evaporated and the residue
coevaporated several times with water to yield the title compound
(0.37 g) as a white solid.
Step C:
2-Amino-5-methyl-7-(.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrim-
idin-4(3H)-one
[0789] A mixture of intermediate from Step B (100 mg, 0.32 mmol) in
2N aqueous NaOH (20 mL) was stirred at reflux temperature for 1.5
h. The solution was cooled in ice bath, neutralized with 2 N
aqueous HCl and evaporated to dryness. The residue was suspended in
MeOH, mixed with silica gel and evaporated. The solid residue was
placed onto a silica gel column (packed in a solvent mixture of
CH.sub.2Cl.sub.2/MeOH: 10/1) which was eluted with a solvent system
of CH.sub.2Cl.sub.2/MeOH: 10/1 and 5/1. The fractions containing
the product were collected and evaporated to dryness to yield the
title compound (90 mg) as a white solid.
[0790] .sup.1H NMR (DMSO-d.sub.6): .delta. 2.15 (d, 3H), 3.47, 3.50
(2m, 2H), 3.75 (m, 1H), 3.97 (m, 1H), 4.17 (m, 1H), 4.89 (t, 1H),
4.96 (d, 1H), 5.14 (d, 1H), 5.80 (d, 1H, J=6.4 Hz), 6.14 (s, 2H),
6.60 (q, 1H, J=1.2 Hz), 10.23 (s, 1H).
EXAMPLE 13
2-Amino-3,4-dihydro-4-oxo-7-(2-O-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo-
[2,3-d]pyrimidine-5-carbonitrile
[0791] 47
Step A:
2-Amino-4-chloro-7-.beta.-D-ribofuranosyl-7H-pyrrolo[2,3-d]pyrimid-
ine-5-carbonitrile
[0792] This intermediate was prepared according to J. Chem. Soc.
Perkin Trans. 1. 2375 (1989).
Step B:
2-Amino-4-chloro-7-[3,5-O-(1,1,3-tetraisopropyldisiloxane-1,3-diyl-
)-.beta.-D-ribofuranosyl]-7H-pyrrolo[2,3-d]pyrimidine-5-carbonitrile
[0793] To a solution of the compound from Step A (1.64 g, 5.00
mmol) in DMF (30 mL) was added imidazole (0.681 g, 10.0 mmol). The
solution was cooled to 0.degree. C. and
1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (1.58 g, 5.00 mmol)
was added dropwise. The bath was removed and the solution stirred
at room temperature for 30 minutes, evaporated in vacuo to an oil,
taken up in ethyl acetate (150 mL) and washed with saturated
aqueous sodium bicarbonate (50 mL) and with water (50 mL). The
organic phase was dried over magnesium sulfate, filtered and
evaporated in vacuo. The residue was purified on silica gel using
ethyl acetate/hexane (1:2) as eluent. Fractions containing the
product were pooled and evaporated in vacuo to give the desired
product (2.05 g) as a colorless foam.
[0794] .sup.1H NMR (DMSO-d.sub.6): .delta. 1.03 (m, 28H), 3.92 (m,
1H), 4.01 (m, 1H), 4.12 (m, 1H), 4.24 (m, 2H), 5.67 (m, 1H), 5.89
(s, 1H), 7.17 (bs, 2H), 8.04 (s, 1H).
Step C:
2-Amino-4-chloro-7-[2-O-methyl-.beta.-D-ribofuranosyl]-7H-pyrrolo[-
2,3-d]pyrimidine-5-carbonitrile
[0795] To a pre-cooled solution (0.degree. C.) of the compound from
Step B (1.70 g, 3.00 mmol) in DMF (30 mL) was added methyl iodide
(426 mg, 3.00 mmol) and then NaH (60% in mineral oil) (120 mg, 3.00
mmol). The mixture was stirred at rt for 30 minutes and then poured
into a stirred mixture of saturated aqueous ammonium chloride (100
mL) and ethyl acetate (100 mL). The organic phase was washed with
water (100 mL), dried over magnesium sulfate, filtered and
evaporated in vacuo. The resulting oily residue was co-evaporated
three times from acetonitrile (10 mL), taken up in THF (50 mL) and
tetrabutylammonium fluoride (1.1 mmol/g on silica) (4.45 g, 6.00
mmol) was added. The mixture was stirred for 30 minutes, filtered
and the filtrate evaporated in vacuo. The crude product was
purified on silica using methanol/dichloromethane (7:93) as eluent.
Fractions containing the product were pooled and evaporated in
vacuo to give the desired product (359 mg) as a colorless
solid.
[0796] .sup.1H NMR (DMSO-d.sub.6): .delta. 3.30 (s, 3H), 3.56 (m,
2H) 3.91 (m, 1H), 4.08 (m, 1H), 4.23 (m, 1H), 5.11 (m, 1H), 5.23
(m, 1H), 7.06 (m, 1H), 7.16 (bs, 2H), 8.38 (s, 1H).
Step D:
2-Amino-3,4-dihydro-4-oxo-7-[2-O-methyl-.beta.-D-ribofuranosyl]-7H-
-pyrrolo[2,3-d]pyrimidine-5-carbonitrile
[0797] To a solution of the compound from Step D in DMF (5.0 mL)
and dioxane (3.5 mL) was added syn-pyridinealdoxime (336 mg, 2.75
mmol) and then tetramethylguanidine (288 mg, 2.50 mmol). The
resulting solution was stirred overnight at rt, evaporated in vacuo
and and co-evaporated three times from acetonitrile (20 mL). The
oily residue was purified on silica gel using
methanol/dichloromethane (7:93) as eluent. Fractions containing the
product were pooled and evaporated in vacuo to give the desired
product (103 mg) as a colorless solid.
[0798] .sup.1H NMR (DMSO-d.sub.6): .delta. 3.30 (s, 3H), 3.57 (m,
2H), 3.86 (m, 1H), 4.00 (m, 1H), 4.21 (m, 1H), 5.07 (m, 1H), 5.17
(m, 1H), 5.94 (m, 1H), 6.56 (bs, 2H), 7.93 (s, 1H), 10.82 (bs,
1H).
EXAMPLE 14
2-Amino-5-methyl-7-(2-O-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[23-d]pyr-
imidin-4(3H)-one
[0799] 48
Step A:
2-Amino-4-chloro-5-methyl-7-(2-O-methyl-.beta.-D-ribofuranosyl)-7H-
-pyrrolo[2,3-d]-pyrimidine
[0800] Into a solution of the compound from Example 12, Step B (188
mg, 0.6 mmol) in anhydrous DMF (6 mL) was added NaH (60% in mineral
oil; 26 mg, 0.66 mmol). The mixture was stirred at room temperature
for 0.5 h and then cooled. MeI (45 .mu.L) was added at 0.degree. C.
and the reaction mixture allowed to warm to 15.degree. C. in 5 h.
Then the mixture was poured into ice-water (20 mL) and extracted
with CH.sub.2Cl.sub.2 (100+50 mL). The combined organic extracts
were washed with water (50 mL), brine (50 mL) and dried
(Na.sub.2SO.sub.4). The evaporated residue was purified on a silica
gel column with a solvent system of CH.sub.2Cl.sub.2/MeOH: 30/1.
Appropriate fractions were pooled and evaporated to yield the title
compound (50 mg) as a colorless glass.
Step B:
2-Amino-7-(2-O-methyl-.beta.-D-ribofuranosyl)-5-methyl-7H-pyrrolo[-
2,3-d]pyrimidin-4(3H)-one
[0801] A solution of the compound from Step A (50 mg, 0.15 mmol) in
0.5M NaOMe/MeOH (4 mL) was stirred at reflux temperature for 1.5 h.
The mixture was cooled, mixed with silica gel and evaporated to
dryness. The silica gel was loaded onto a silica gel column and
eluted with a solvent system of CH.sub.2Cl.sub.2/MeOH: 30/1. The
fractions containing the product were collected and evaporated to
yield 2-amino-7-(2-O-methyl-.bet-
a.-D-ribofuranosyl)-4-methoxy-5-methyl-7H-pyrrolo[2,3-d]pyrimidine
(40 mg). This was mixed with 2 N aqueous NaOH (4 mL) and stirred at
reflux temperature for 10 h. The mixture was cooled in ice bath,
neutralized with 2 N aqueous HCl and evaporated. The solid residue
was suspended in MeOH, mixed with silica gel and evaporated. The
silica gel was loaded onto a silica gel column and eluted with a
solvent system of CH.sub.2Cl.sub.2/MeOH: 5/1. Appropriate fractions
were pooled and evaporated to give the title compound (40 mg) as a
white solid.
[0802] .sup.1H NMR (DMSO-d.sub.6): .delta. 2.18 (s, 3H), 3.26 (s,
3H), 3.45, 3.52 (2m, 2H), 3.82 (m, 1H), 3.97 (dd, 1H), 4.20 (m,
1H), 4.99 ((t, 1H), 5.10 (d, 1H), 5.94 (d, 1H, J=7.0 Hz), 6.19 (bs,
2H), 6.68 (s, 1H), 10.60 (br, 1H).
EXAMPLE 15
2-Amino-7-(2-deoxy-2-fluoro-.beta.-D-arabinofuranosyl)-7H-pyrrolo[2,3-d]py-
rimidin 4(3H)-one
[0803] 49
[0804] This compound was prepared following the procedures
described in J. Med. Chem. 38: 3957 (1995).
EXAMPLE 16
2-Amino-7-(.beta.-D-arabinofuranosyl)-7H-pyrrolo[2,3-d]pyrimidin-4(3H)-one
[0805] 50
[0806] This compound was prepared following the procedures
described in J. Org. Chem. 47: 226 (1982).
EXAMPLE 17
2-Amino-7-(.beta.-D-arabinofuranosyl)-3,4-dihydro-4-oxo-7H-pyrrolo[2,3-d]p-
yrimidine-5-carbonitrile
[0807] 51
Step A:
2-Amino-7-(.beta.-D-arabinofuranosyl)-4-chloro-7H-pyrrolo[2,3-d]py-
rimidine-5-carbonitrile
[0808] This intermediate was prepared according to J. Chem. Soc.
Perkin Trans. 1, 2375 (1989).
Step B:
2-Amino-7-(.beta.-D-arabinofuranosyl)-3,4-dihydro-4-oxo-7H-pyrrolo-
[2,3-d]pyrimidine-5-carbonitrile
[0809] To a solution of the compound from Step A (163 mg, 0.50
mmol) in DMF (5.0 mL) and dioxane (3.5 mL) was added
syn-pyridinealdoxime (336 mg, 2.75 mmol) and then
tetramethylguanidine (288 mg, 2.50 mmol). The resulting solution
was stirred overnight at rt, evaporated in vacuo and and
co-evaporated three times from acetonitrile (20 mL). The oily
residue was purified on silica using methanol/dichloromethane (1:4)
as eluent. Fractions containing the product were pooled and
evaporated in vacuo to give the desired product (72 mg) as a
colorless solid.
[0810] .sup.1H NMR (DMSO-d.sub.6): .delta. 3.60 (m, 2H), 3.73 (m,
1H), 4.01 (m, 2H), 5.06 (m, 1H), 5.48 (m, 2H), 6.12 (m, 1H), 6.52
(bs, 2H), 7.70 (s, 1H), 10.75 (bs, 1H).
EXAMPLE 18
2-Amino-5-methyl-7-(.beta.-D-arabinofuranosyl)-7H-pyrrolo[2,3-d]pr
din-4(3H)-one
[0811] 52
Step A
2-Amino-7-(2,3,5-tri-O-benzyl-.beta.-D-arabinofuranosyl)-4-chloro-5-
-methyl-7H-pyrrolo[2,3-d]pyrimidine
[0812] To a solution of 1-O-p-nitrobenzyl-D-arabinofuranose (3.81
g, 6.70 mmol) in DCM was bubbled HBr until TLC (hexane/ethylacetate
(2:1)) showed complete reaction (about 30 min). The reation mixture
was filtered and evaporated in vacuo. The oily residue was taken up
in acetonitrile (10 mL) and added to a vigorously stirred
suspension of 2-amino-4-chloro-5-methyl-7H-pyrrolo[2,3-d]pyrimidine
(Liebigs Ann. Chem. (1984), 4,708) (1.11 g, 6.00 mmol) KOH (1.12 g,
20.0 mmol) and tris[2-(2-methoxyethoxy)ethyl]amine (0.216 g, 0.67
mmol) in acetonitrile (80 mL). The resulting suspension was stirred
at rt for 30 min, filtered and evaporated in vacuo. The crude
product was purified on silica using hexane/ethylacetate (3:1) as
the eluent. Fractions containing the product were pooled and
evaporated in vacuo to give the desired product (1.13 g) as a
colorless foam.
Step B:
2-Amino-7-.beta.-D-arabinofuranosyl-4-chloro-5-methyl-7H-pyrrolo[2-
,3-d]pyrimidine
[0813] To a precooled (-78.degree. C.) solution of the compound
from Step A (0.99 g, 1.7 mmol) in dichloromethane (30 mL) was added
borontrichloride (1M in dichloromethane) (17 mL, 17.0 mmol) over a
10 min. The resulting solution was stirred at -78.degree. C. for 1
h, allowed to warm to -15.degree. C. and stirred for another 3 h.
The reaction was quenched by addition of methanol/dichloromethane
(1:1) (15 mL), stirred at -15.degree. C. for 30 min, and pH
adjusted to 7.0 by addtion of NH.sub.4OH. The mixture was
evaporated in vacuo and the resulting oil purified on silica using
methanol/dichloromehane (1:9) as eluent. Fractions containing the
product were pooled and evaporated in vacuo to give the desired
product (257 mg) as a colorless foam.
Step C:
2-Amino-7-(.beta.-D-arabinofuranosyl)-5-methyl-7H-pyrrolo[2,3-d]py-
rimidin-4(3H)-one
[0814] To the compound from Step B (157 mg, 0.50 mmol) was added
NaOH (2M, aqueous) (2 mL). The resulting solution was stirred at
relux for 1 h, cooled and neutralized by addition of HCl (2M,
aqueous). The mixture was evaporated in vacuo and the crude product
purified on silica using methanol/dichloromehane (2:8) as eluent.
Fractions containing the product were pooled and evaporated in
vacuo to give the desired product (53 mg) as a colorless
powder.
[0815] .sup.1H NMR (DMSO-d.sub.6): .delta. 2.13 (d, 3H), 3.58 (m,
2H), 3.71 (m, 1H), 4.00 (m, 2H), 5.09 (m, 1H), 6.22 (bs, 2H), 5.50
(m, 2H), 6.12 (m, 1H), 6.64 (s, 1H), 10.75 (bs, 1H).
EXAMPLE 19
2-Amino-7-(3-deoxy-3-fluoro-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrim-
idin-4(3H)-one
[0816] 53
[0817] A solution
1-O-acetyl-2-O-benzyl-5-O-(p-toluoyl)-3-deoxy-3-fluoro-D-
-ribofuranose (410 mg, 1.01 mmol) (prepared by a modified method
described for similar sugar derivatives, Helv. Chim. Acta 82: 2052
(1999) and J. Med. Chem. 1991, 34, 2195) in anhydrous
CH.sub.2Cl.sub.2 (1.5 mL) was cooled to -15.degree. C. in a dry
ice/CH.sub.3CN bath. After cooling the reaction mixture for 10 min.
under the argon atmosphere, 33% HBr/AcOH (370 .mu.L, 1.5 equiv.)
was added slowly over 20 min keeping the bath temperature around
-15.degree. C. After the addition was complete, the reaction
mixture was stirred at -10.degree. C. for 1 hr. The solvent was
removed under reduced pressure and the residue azeotroped with
anhydrous toluene (5.times.10 mL). In a separate flask,
2-amino-4-chloro-7H-pyrrolo- [2,3-d]pyrimidine (210 mg, 1.2 mmol)
was suspended in anhydrous CH.sub.3CN (10 mL) and cooled to
-10.degree. C. To this was added 60% NaH dispersion in oil (57 mg)
in two portions, and the reaction mixture was stirred for 45 min.
during which time the solid dissolved and the bath temperature rose
to 0.degree. C. The bath was removed and stirring was continued for
about 20 additional min. It was cooled back to -10.degree. C. and
the bromo sugar, prepared above, was taken up in anhydrous
CH.sub.3CN (1.5 mL) and added slowly to the anion of nucleobase.
After the addition was complete, the reaction mixture was stirred
for an additional 45 min allowing the temperature of the reaction
to rise to 0.degree. C. The bath was removed and the reaction
allowed to stir at room temperature for 3 hr. Methanol was added
carefully to the reaction mixture and the separated solid removed
by filtration. The solvent was removed under reduced pressure and
the residual oil dissolved in EtOAc (50 mL) and washed with water
(3.times.20 mL). The organic layer was dried over Na.sub.2SO.sub.4
and concentrated to give an oil. It was purified by column
chromatography to furnish fully protected 2-amino-7-(5-O-(p-toluoy-
l)-2-O-benzyl-3-deoxy-3-fluoro-.beta.-D-ribofuranosyl)-4-chloro-7H-pyrrolo-
[2,3-d]pyrimidine (190 mg) as an .alpha./.beta. mixture (1:1).
After conversion of 4-chloro to 4-oxo by heating the compound with
2N NaOH/dioxane mixture at 105.degree. C. and after the usual
workup the residue was debenzylated using 20 mol % w/w of 10% Pd/C
and ammonium formate in refluxing methanol to give title compound
after purification by HPLC; yield 10%. ESMS: calcd. for C,
H.sub.11H.sub.13FN.sub.4O.sub.4 284.24, found 283.0 (M+1).
EXAMPLE 20
2-Amino-3,4-dihydro-4-oxo-7-(2-deoxy-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,-
3-d]pyrimidine-5-carbonitrile
[0818] 54
[0819] This compound was prepared following the procedures
described in Synthesis 1327 (1998).
EXAMPLE 21
6-Amino-1-(.beta.-D-ribofuranosyl)-1H-imidazo[4,5-c]pyridin-4(5H)-one
[0820] 55
[0821] This compound was prepared following the conditions
described in J. Am. Chem. Soc. 97: 2916 (1975).
EXAMPLE 22
2-Amino-7-(2-O-methyl-.beta.-D-ribofuranosyl)-5H-pyrrolo[3,2-d]pyrimidin-4-
-(3H)-one
[0822] 56
[0823] To a suspension of
2-amino-5H-pyrrolo[3,2-d]pyrimidin-4(3H)-one (9-deazaguanine)
(0.454 g, 3.0 mmol) (prepared according to J. Org. Chem. 1978, 43,
2536) and 2-O-methyl-1,3,5-tri-O-benzoyl-.beta.-D-ribofuranose
(1.54 g, 3.2 mmol) in dry nitromethane (23 mL) at 60.degree. C. was
added stannic chloride (0.54 mL, 4.5 mmol). The reaction mixture
was maintained at this temperature for 0.5 hr., cooled and poured
onto ice-cold saturated sodium bicarbonate solution (70 mL). The
insoluble material was filtered through florisil and washed with
ethyl acetate (3.times.50 mL). The filtrate was extracted with
ethyl acetate (2.times.50 mL), and organic layer was washed with
water (2.times.50 mL), dried over Na.sub.2SO.sub.4 and evaporated
to dryness. Chromatography of the resulting foam on silica gel with
CH.sub.2Cl.sub.2/MeOH(14:1) afforded the benzoylated product (0.419
g, 30% yield). To a suspension of the benzoylated product (0.25 g)
in MeOH (2.4 mL) was added t-butylamine (0.52 m]L) and stirring at
room temperature was continued for 24 hrs. followed by addition of
more t-butylamine (0.2 mL). The reaction mixture was stirred at
ambient temperature overnight, concentrated in vacuum and the
residue was purified by flash chromatography over silica gel using
CH.sub.2Cl.sub.2/MeOH (85:15) as eluent giving the desired compound
as a foam (0.80 g).
[0824] .sup.1H NMR (200 MHz, DMSO-d.sub.6): .delta. Hz3.28 (s, 3H),
3.40-3.52 (in, 3H), 3.87-3.90 (m, 1H), 4.08-4.09 (m, 1H), 4.67 (d,
1H, J=5.2 Hz), 4.74 (d, 1H, J=7.0 Hz), 5.62 and 5.50 (2 bs, 3H),
7.14 (d, 1H, J=2.6 Hz), 10.43 (s, 1H), 11.38 (s, 1H); Mass
spectrum: calcd. for C.sub.12H.sub.16N.sub.4O.sub.5: 296.28; found:
295.11.
EXAMPLE 23
6-Amino-1-(3-deoxy-.beta.-D-ribofuranosyl)-1H-imidazo[4,5-c]pyridine-4(5H)-
-one (3'-deoxy-3-deaza-guanosine)
[0825] 57
Step A: 3-Deoxy-4-O-p-toluoyl-2-O-acetyl-.beta.-D-ribofuranosyl
acetate
[0826] A solution of
3-deoxy-4-O-p-toluoyl-1,2-O-isopropylidene-.beta.-D-r- ibofuranose
(Nucleosides Nucleotides 1994, 13, 1425 and Nucleosides Nucleotides
1992, 11, 787) (5.85 g, 20 mmol) in 64 mL of 80% acetic acid was
stirred at 85.degree. C. overnight. The reaction mixture was
concentrated and co-evaporated with toluene. The residue was
dissolved in 90 mL of pyridine. Acetic anhydride (6 mL) was added
at 0.degree. C., and the reaction mixture was stiffed at rt for 6
h. After condensation, the residue was dissolved in ethyl acetate
and washed with aqueous sodium bicarbonate solution, water and
brine. The organic phase was dried and concentrated.
Chromatographic purification on a silica gel column using 3:1 and
2:1 hexanes-EtOAc as eluent provided 5.51 g of the title compound
as a clear oil.
[0827] .sup.1H NMR (CDCl.sub.3): .delta. 1.98 (s, 3H), 2.09 (s,
3H), 2.15-2.35 (m, 2H), 2.41 (s, 3H), 4.27-4.42 (m, 1H), 4.46-4.58
(m, 1H), 4.65-4.80 (m, 1H), 5.21-5.28 (m, 1H), 6.20 (s, 1H),
7.19-7.31 (m, 2H), 7.90-8.01 (m, 2H).
Step B: Methyl
5-cyanomethyl-1-(3-deoxy-4-O-p-toluoyl-2-O-acetyl-.beta.-D--
ribofuranosyl)-1H-imidazole-4-carboxylate
[0828] A mixture of methyl
5(4)-(cyanomethyl)-1H-imidazole-4(5)-carboxylat- e (J. Am. Chem.
Soc. 1976, 98, 1492 and J. Org. Chem. 1963, 28, 3041) (1.41 g, 8.53
mmol), 1,1,1,3,3,3-hexamethyldisilazane (20.5 mL) and ammonium
sulfate (41 mg) was refluxed at 125.degree. C. under Ar atmosphere
for 18 h. After evaporation, the residue was dissolved in 10 mL of
dichloroethane. A solution of the compound from Step A (2.86 g, 8.5
mmol) in 10 mL of dichloroethane was added followed by addition of
SnCl.sub.4 (1.44 mL, 3.20 g). The resulted reaction mixture was
stirred at rt overnight and diluted with chloroform. The mixture
was washed with aqueous sodium bicarbonate, water and brine. The
organic phase was dried and concentrated. Chromatographic
purification of the residue on a silica gel column using 1:1, 1:2,
and 1:3 hexanes-EtOAc as eluent provided 2.06 g of the title
compound as a white foam.
[0829] .sup.1H NMR (CDCl.sub.3): .delta. 2.15 (s, 3H), 2.28-2.40
(m, 2H), 2.38 (s, 3H), 3,87 (s, 3H), 4.46 (dd, 2H, J=7.6, 2.0 Hz),
4.50-4.57 (m, 1H), 4.68-4.75 (m, 1H), 4.76-4.83 (m, 1H), 5.41 (d,
1H, J=5.6 Hz), 5.91 (s, 1H), 7.24-7.28 (m, 2H), 7.80 (s, 1H),
7.82-7.90 (m, 2H); .sup.13C NMR (CDCl.sub.3) .delta. 13.1, 20.7,
21.6, 31.5, 51.8, 63.5, 77.9, 79.2, 89.8, 115.1, 126.2, 129.3,
129.5, 131.7, 135.1, 144.3, 163.1, 166.1, 170.3.
Step C:
6-Amino-1-(3-deoxy-.beta.-D-ribofuranosyl)-1H-imidazo[4,5-c]pyridi-
ne-4(5H)-one
[0830] A solution of the compound from Step B (2.00 g, 4.53 mmol)
in methanol (30 mL) was saturated with ammonia at 0.degree. C.
Concentrated ammonium hydroxide (30 mL) was added and the sealed
metal reactor was heated at 85.degree. C. for 5 h. After cooling to
rt, the reaction mixture was transferred directly onto a silica gel
column. Elution with 4:1, 3:1 and 2:1 CHCl.sub.3-MeOH provided 0.79
g of the title compound as a white solid.
[0831] .sup.1H NMR (DMSO-d.sub.6): .delta. 2.41-2.46 (m, 1H),
2.52-2.58 (m, 1H), 3.48-3.55 (m, 1H), 3.60-3.70 (m, 1H), 4.27-4.36
(m, 2H), 4.97 (t, 1H, J=5.6 Hz), 5.44 (s, 1H), 5.47 (s, 1H), 5.60
(s, 2H), 5.66, (d, 1H, J=4.4 Hz), 7.90 (s, 1H), 10.33 (s, 1H);
.sup.13C NMR (DMSO d.sub.6) .delta. 34.1, 62.4, 70.4, 74.7, 80.4,
91.6, 123.0, 136.3, 141.9, 147.6, 156.5.
EXAMPLE 24
6-Amino-1-(3-deoxy-3-fluoro-.beta.-D-ribofuranosyl)-1H-imidazo[4,5-c]pyrid-
in-4(3H)-one
[0832] 58
[0833] This compound was prepared in a manner similar to the
preparation of
2-amino-7-(3-deoxy-3-fluoro-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]p-
yrimidin-4(3H)-one (Example 23).
EXAMPLE 25
1-(.beta.-D-Ribofuranosyl)-1H-pyrazolo[3,4-d]pyrimidin-4(3H)-one
(Allopurinol riboside)
[0834] 59
[0835] This compound was obtained from commercial sources.
EXAMPLE 26
9-(.beta.-D-Arabinofuranosyl)-9H-purin-6 (1H)-one
[0836] 60
[0837] This compound was prepared following the conditions
described in J. Med. Chem. 18: 721 (1975).
EXAMPLE 27
2-Amino-7-(2-O-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidin-4-
(3H) thione
[0838] 61
[0839] A solution of the compound from Example 11, Step C (1.5 g, 5
mmol), thiourea (0.4 g, 5.2 mmol.) in abs. EtOH was refluxed for 16
hrs. The solution was evaporated and the resulting oil
chromatographed on silica gel (EtOAc/MeOH: 9/1) to afford the
desired product as a foam.
[0840] .sup.1H NMR (DMSO-d.sub.6): .delta. 3.30 (s, 3H), 5.00-5.06
(t, 1H), 5.19 (d, 1H), 5.95 (d, 1H), 6.43 (d, 1H), (d, 1H).
EXAMPLE 28
2-Amino-7-(.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine
[0841] 62
[0842] This compound was obtained from commercial sources.
EXAMPLE 29
2-Amino-4-chloro-7-(2-O-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]py-
rimidin-5-carbonitrile
[0843] 63
[0844] This compound was prepared as described in Example 13, Steps
A-C.
EXAMPLE 30
2-Amino-4-chloro-5-ethyl-7-(2-O-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[-
23-d]pyrimidine
[0845] 64
Step A:
2-Amino-4-chloro-5-ethyl-7-[3,5-O-(tetraisopropyldisiloxane-1,3-di-
yl)-(.beta.-D-ribofuranosyl]-7H-pyrrolo[2,3-d]pyrimidine
[0846] To a solution of
2-amino-4-chloro-5-ethyl-7-(.beta.-D-ribofuranosyl-
)-7H-pyrrolo[2,3-d]pyrimidine (0.300 g, 0.913 mmol) in pyridine (8
mL) was added 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (0.317
g, 1.003 mmol) dropwise. The solution stirred at rt overnight,
evaporated in vacuo to an oil, and evaporated repeatedly from
acetonitrile. The crude product was purified on silica using 5%
methanol in dichloromethane as eluent. Fractions containing the
product were pooled and evaporated in vacuo to give the desired
product (254 mg) as a colorless solid.
Step B:
2-Amino-4-chloro-5-ethyl-7-(2-O-methyl-.beta.-D-ribofuranosyl)-7H--
pyrrolo[2,3-d]pyrimidine
[0847] To a pre-cooled solution (0.degree. C.) of the compound from
step A (192 mg, 0.337 mmol) in DMF (3 mL) was added methyl iodide
(45.4 mg, 0.320 mmol) and then NaH (60% in mineral oil) (8.10 mg,
0.320 mmol). The mixture was stirred at rt for 45 minutes and then
poured into a stirred mixture of saturated aqueous ammonium
chloride (10 mL) and ethyl acetate (10 mL). The organic phase phase
was washed with brine (10 mL) and dried over MgSO.sub.4 and
evaporated in vacuo. The resulting oily residue was taken up in THF
(5 mL) and tetrabutylammonium fluoride (1.1 mmol/g on silica)
(0.529 g, 0.582 mmol) was added. The mixture was stirred for 30
minutes, filtered and the filtrate evaporated in vacuo. The crude
product was purified on silica using 10% methanol in
dichloromethane as eluent. Fractions containing the product were
pooled and evaporated in vacuo to give the desired product (66 mg)
as a colorless solid.
[0848] .sup.1H NMR (DMSO-d.sub.6): .delta. 1.15 (t, 3H), 2.65 (q,
2H), 3.20 (s, 3H), 3.51 (m, 2H), 3.84 (m, 1H), 4.04 (m, 1H), 4.21
(m, 1H), 4.99 (m, 2H), 5.15 (m, 2H), 6.07 (m, 2H), 6.62 (s br, 2H),
7.06 (s, 2H).
EXAMPLE 31
2-Amino-4-chloro-5-methyl-7-(2-O-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo-
[2,3-d]pyrimidine
[0849] 65
[0850] This compound was prepared as described in Example 14, Step
A.
[0851] .sup.1H NMR (CD.sub.3OD): .delta. 2.33 (s, 3H), 3.39 (s,
1H), 3.72, 3.83 (2dd, 2H), 4.03 (m, 1H), 4.17 (t, 1H), 4.39 (dd,
1H), 5.98 (d, 1H, J=5.9 Hz), 6.7 (bs, 2H), 7.01 (s, 1H).
EXAMPLE 32
2-Amino-4-chloro-7-(2-O-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]py-
rimidine
[0852] 66
[0853] This compound was synthesized as described in Example 11,
Steps A-C.
EXAMPLE 33
2-Amino-4-chloro-7-(.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine
[0854] 67
[0855] This compound was prepared following the procedures
described in Helv. Chim. Acta 73: 1879 (1990).
EXAMPLE 34
2-Amino-4-chloro-5-methyl-7-(.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyri-
midine
[0856] 68
[0857] The compound was prepared as described in Example 12, Steps
A-B.
[0858] .sup.1H NMR (DMSO-d.sub.6): .delta. 2.29 (s, 3H), 3.54 (m,
2H), 3.84 (m, 1H), 4.04 (dd, 1H, J.sub.1=3.0, J.sub.2=4.9 Hz),
4.80-5.50 (bs, 3H), 4.28 (t, 1H), 5.98 (d, 1H, J=6.5 Hz), 6.7 (bs,
2H), 7.13 (s, 1H).
EXAMPLE 35
2-Amino-4-chloro-5-ethyl-7-(.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrim-
idine
[0859] 69
[0860] This compound was prepared as described in Example 9, Steps
A-B.
[0861] .sup.1H NMR (DMSO-d.sub.6): .delta. 2.00 (t, 3H), 2.69 (q,
2H), 3.48 (dd, 1H, J.sub.1=4.2 Hz, J.sub.2=11.8 Hz), 3.56 (dd, 1H,
J.sub.1=4.3 Hz, J.sub.2=11.8 Hz), 3.80 (m, 1H), 4.02 (dd, 1H,
J.sub.1=3.1 Hz, J.sub.2=5.0 Hz), 4.62 (t, 1H), 5.0 (bs, 2H), 5.2
(bs, 1H), 5.60 (d, 1H, J=6.4 Hz), 6.61 (bs, 2H), 7.09 (s, 1H).
EXAMPLE 36
2-Amino-6-chloro-9-(.beta.-D-ribofuranosyl)-9H-purine
[0862] 70
[0863] This compound was obtained from commercial sources.
EXAMPLE 37
2-Amino-4-chloro-7-(.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine-5--
carbonitrile
[0864] 71
[0865] This compound was prepared following the procedures
described in J. Chem. Soc. Perkin Trans. 1, 2375 (1989).
EXAMPLE 38
2-Amino-4-chloro-7-(2-deoxy-2-fluoro-.beta.-D-arabinofuranosyl)-7H-pyrrolo-
[2,3-d]pyrimidine
[0866] 72
[0867] This compound was prepared following the procedures
described in J. Med. Chem. 38: 3957 (1995).
EXAMPLE 39
2-Amino-4-chloro-5-methyl-7-(.beta.-D-arabinofuranosyl)-7H-pyrrolo[2,3-d]p-
yrimidine
[0868] 73
[0869] The compound was prepared as described in Example 18, Steps
A-B.
[0870] .sup.1H NMR (DMSO-d.sub.6): .delta. 2.24 (s, 3H), 3.60 (m,
3H), 3.98 (m, 2H), 4.98 (m, 1H), 5.43 (bs, 2H), 6.25 (s, 1H), 6.57
(bs, 2H), 7.01 (s, 1H).
EXAMPLE 40
2'-O-Methylcytidine
[0871] 74
[0872] This compound was obtained from commercial sources.
EXAMPLE 41
3'-Deoxy-3'-methylcytidine
[0873] 75
[0874] This compound was prepared following the procedures
described in U.S. Pat. No. 3,654,262 (1972), which is incorporated
by reference herein in its entirety.
EXAMPLE 42
3'-Deoxycytidine
[0875] 76
[0876] This compound was obtained from commercial sources.
EXAMPLE 43
3'-Deoxy-3'-fluorocytidine
[0877] 77
[0878] This compound was prepared following the procedures
described in J. Med. Chem. 34: 2195 (1991).
EXAMPLE 44
1-(.beta.-D-Arabinofuranosyl)-1H-cytosine
[0879] 78
[0880] This compound was obtained from commercial sources.
EXAMPLE 45
2'-Amino-2'-deoxycytidine
[0881] 79
[0882] This compound was obtained from commercial sources.
EXAMPLE 46
3'-Deoxy-3'-methyluridine
[0883] 80
[0884] This compound was prepared following procedures described in
U.S. Pat. No. 3,654,262, which is incorporated by reference herein
in its entirety.
EXAMPLE 47
3'-Deoxy-3'-fluorouridine
[0885] 81
[0886] This compound was prepared following procedures described in
J. Med. Chem. 34: 2195 (1991) and FEBS Lett. 250: 139 (1989).
EXAMPLE 48
3'-Deoxy-5-methyluridine
[0887] 82
[0888] This compound was obtained from commercial sources.
EXAMPLE 49
3'-Deoxy-2'-O-(2-methoxyethyl)-3'-methyl-5-methyluridine
[0889] 83
Step A:
5'-O-(tert-butyldiphenylsilyl)-3'-O-(3-tert-butylphenoxythiocarbon-
yl)-2'-O-(2-methoxyethyl)-5-methyluridine
[0890] This compound was synthesized by the reaction of the
corresponding 5'-protected-2'-substituted-5-methyluridine with
3'-t-butylphenoxy chlorothionoformate following the similar
procedure for the preparation of 3'-phenoxythiocarbonyl-2'-deoxy
derivative (Synthesis 1994, 1163).
Step B:
5'-O-(tert-Butyldiphenylsilyl)-3'-deoxy-2'-O-(2-methoxyethyl)-3'-(-
2-phenylethenyl)-5-methyluridine
[0891] To a solution of
5'-O-(tert-butyldiphenylsilyl)-3'-O-(3-tert-butylp-
henoxythiocarbonyl)-2'-O-(2-methoxyethyl)-5-methyluridine (15.0 g,
20.0 mmol) in 150 mL of benzene was added PhCH.dbd.CHSnBu3 (18.7 g,
50 mmol). The resulting solution was degassed three times with
argon at rt and 45.degree. C. After AIBN (1.0 g, 6.1 mmol) was
added, the resulting solution was refluxed for 2 h. Another portion
of AIBN (1.0 g, 6.1 mmol) was added after cooling to about
40.degree. C. and refluxed for 2 h. This procedure was repeated
until the starting material disappeared. The solvent was evaporated
and the residue was purified by flash chromatography on a silica
gel column using 10:1 and 5:1 hexanes-EtOAc as eluent to give 1.74
g of 5'-O-(tert-butyldiphenylsilyl)-3'-deoxy-2'-O-(2--
methoxyethyl)-3'-(2-phenylethenyl).sub.5-methyluridine as a white
foam.
[0892] .sup.1H NMR (CDCl.sub.3): .delta. 1.13, (s, 9H), 1.43 (s,
3H), 3.18-3.30 (m, 1H), 3.37 (s, 3H), 3.58-3.62 (m, 2H), 3.79-3.80
(m, 2H), 4.06-4.37 (m, 4H), 4.95 (s, 1H), 6.25-6.40 (m, 1H), 6.62
(d, 1H, J=16 Hz), 7.27-7.71 (m, 16H), 9.21 (s, 1H); .sup.13C NMR
(CDCl.sub.3) .delta. 11.9, 19.6, 27.2, 45.3, 59.0, 62.1, 70.2,
72.0, 84.6, 87.1, 90.2, 110.4, 122.8, 126.4, 127.8, 128.0, 128.3,
128.6, 130.0, 132.7, 133.5, 134.7, 135.3, 135.4, 136.9, 150.3,
154.1; HRMS (FAB) m/z 641.302 (M+H).sup.+
(C.sub.37H.sub.45N.sub.2O.sub.6Si requires 641.304).
Step C:
5'-O-(tert-Butyldiphenylsilyl)-3'-deoxy-3'-(hydroxymethyl)-2'-O-(2-
-methoxyethyl)-5-methyluridine
[0893] To a solution of
5'-O-(tert-butyldiphenylsilyl)-3'-deoxy-2'-O-(2-me-
thoxyethyl)-3'-(2-phenylethenyl)-5-methyluridine. (5.0 g, 7.8 mmol)
and N-methylmorpholine N-oxide (NMO) (1.47 g, 12.5 mmol) in 150 mL
of dioxane was added a catalytic amount of osmium tetraoxide (4%
aqueous solution, 2.12 mL, 85 mg, 0.33 mmol). The flask was covered
by aluminum foil and the reaction mixture was stirred at rt
overnight. A solution of NaIO.sub.4 (5.35 g, 25 mmol) in 5 mL of
water was added to the above stirred reaction mixture. The
resulting reaction mixture was stirred for 1 h at 0.degree. C. and
2 h at rt, followed by addition of 10 mL of ethyl acetate. The
mixture was filtered through a celite pad and washed with ethyl
acetate. The filtrate was washed 3 times with 10% aqueous
Na.sub.2S.sub.2O.sub.3 solution until the color of aqueous phase
disappeared. The organic phase was further washed with water and
brine, dried (Na.sub.2SO.sub.4) and concentrated. The aldehyde thus
obtained was dissolved in 130 mL of ethanol-water (4:1, v/v).
Sodium borohydride (NaBH.sub.4) (1.58 g, 40 mmol) was added in
portions at 0.degree. C. The resulting reaction mixture was stirred
at rt for 2 h and then treated with 200 g of ice water. The mixture
was extracted with ethyl acetate. The organic phase was washed with
water and brine, dried (Na.sub.2SO.sub.4) and concentrated. The
resulted residue was purified by flash chromatography on a silica
gel column using 2:1, 1:1 and 1:2 hexanes-EtOAc as eluents to give
1.6 g of 5'-O-(tert-butyldiphenylsilyl)--
3'-deoxy-3'-(hydroxymethyl)-2'-O-(2-methoxyethyl)-5-methyluridine
as a white foam.
[0894] .sup.1H NMR (CDCl.sub.3): .delta. 1.09 (s, 9H), 1.50 (s,
3H), 2.25 (bs, 1H), 2.52-2.78 (m, 1H), 3.38 (s, 3H), 3.52-4.25 (m,
10H), 5.86 (s, 1H), 7.38-7.70 (m, 11H), 9.95 (bs, 1H); .sup.13C NMR
(CDCl.sub.3): .delta. 12.1, 19.5, 27.1, 43.1, 58.2, 58.8, 63.1,
69.5, 71.6, 82.3, 86.1, 89.8, 110.5, 128.0, 130.2, 132.5, 133.2,
135.1, 135.3, 136.5, 150.5, 164.4; HRMS (FAB) m/z 569.268
(M+H).sup.+ (C.sub.30H.sub.41N.sub.2O.sub.7- Si requires
569.268).
Step D:
5'-O-(tert-Butyldiphenylsilyl)-3'-deoxy-3'-(iodomethyl)-2'-O-(2-me-
thoxyethyl)-5-methyluridine
[0895] To a solution of
5'-O-(tert-butyldiphenylsilyl)-3'-deoxy-3'-(hydrox-
ymethyl)-2'-O-(2-methoxyethyl)-5-methyluridine (1.34 g, 2.35 mmol)
in 25 mL of anhydrous DMF under stirring was added sequentially at
0.degree. C. 2,6-lutidine (0.55 mL, 0.51 g, 4.7 mmol, 2.0 equiv)
and methyl triphenoxy-phosphonium iodide (1.28 g, 2.83 mmol). The
resulting reaction mixture was stirred at 0.degree. C. for 1 h and
at rt for 2 h. The reaction mixture was diluted with 10 mL of ethyl
acetate and washed twice with 0.1 N Na.sub.2S.sub.2O.sub.3 aqueous
solution to remove iodine. The organic phase was further washed
with aqueous NaHCO.sub.3 solution, water, and brine. The aqueous
phases were back extracted with ethyl acetate. The combined organic
phases were dried (Na.sub.2SO.sub.4) and concentrated. The
resulting residue was purified by flash chromatography on a silica
gel column using 5:1, 3:1 and then 1:1 hexanes-EtOAc to provide
1.24 g of 5'-O-(tert-butyldiphenylsilyl)-3'-deoxy-3'-(iodomethyl)-
-2'-O-(2-methoxyethyl)-5-methyluridine as a white foam.
[0896] .sup.1H NMR (CDCl.sub.3): .delta. 1.13 (s, 9H), 1.62 (s,
3H), 2.64-2.85 (m, 2H), 3.20-3.35 (m, 1H), 3.38 (s, 3H), 3.50-4.25
(m, 8H), 5.91 (s, 1H), 7.32-7.50 (m, 6H), 7.60 (s, 1H), 7.62-7.78
(m, 4H), 10.46 (s, 1H); .sup.13C NMR (CDCl.sub.3): .delta. 12.4,
19.5, 27.2, 45.0, 58.0, 62.5, 70.3, 71.9, 83.3, 85.6, 88.9, 110.5,
128.1, 128.2, 130.1, 130.3, 132.4, 132.9, 135.0, 135.4, 135.6,
150.7, 164.7; HRMS (FAB) m/z 679.172 (M+H).sup.+
(C.sub.30H.sub.40IN.sub.2O.sub.6Si requires 679.170).
Step E:
3'-Deoxy-3'-(iodomethyl)-2'-O-(2-methoxyethyl)-5-methyluridine
[0897] A solution of
5'-O-(tert-butyldiphenylsilyl)-3'-deoxy-3'-(iodomethy-
l)-2'-O-(2-methoxyethyl)-5-methyluridine (1.12 g, 1.65 mmol) and
triethylamine trihydrofluoride (1.11 mL, 1.1 g, 6.7 mmol) in 20 mL
of THF was stirred at rt for 24 h. The reaction mixture was diluted
with 50 mL of ethyl acetate and washed with water and brine. The
organic phase was dried (Na.sub.2SO.sub.4) and concentrated. The
residue was purified by flash chromatography on a silica gel
column. Gradient elution with 2:1, 1:2 and then 1:3 hexanes-EtOAc
provided 504 mg of the title compound as a white foam.
[0898] .sup.1H NMR (CD.sub.3OD): .delta. 1.87 (s, 3H), 2.47-2.75
(m, 1H), 3.18-3.37 (m, 2H), 3.40 (s, 3H), 3.59-3.70 (m, 2H),
3.71-3.90 (m, 2H), 3.92-4.17 (m, 4H), 5.87 (s, 1H), 8.17 (s, 1H);
.sup.13C NMR (CD.sub.3OD): .delta. 12.5, 45.2, 59.2, 60.9, 71.0,
72.9, 85.4, 87.3, 89.7, 110.5, 138.0, 152.1, 166.6; HRMS (FAB) m/z
441.053 (M+H).sup.+ (C.sub.14H.sub.22IN.sub.2O.sub.6 requires
441.052).
Step F:
3'-Deoxy-5'-O-(4-methoxytrityl)-3'-(iodomethyl)-2'-O-(2-methoxyeth-
yl)-5-methyluridine
[0899] A mixture of
3'-deoxy-3'-(iodomethyl)-2'-O-(2-methoxyethyl)-5-methy- luridine
(472 mg, 1.1 mmol), diisopropylethylamine (0.79 mL, 0.586 g, 4.5
mmol), and p-anisyl chlorodiphenyl methane (4'-methoxytrityl
chloride, MMT-Cl) (1.32 g, 4.27 mmol) in 6 mL of ethyl acetate and
4 mL of THF was stirred at rt for 48 h. The reaction mixture was
diluted with ethyl acetate and washed with water, followed by
brine. The organic phase was dried (Na.sub.2SO.sub.4) and
concentrated. The crude product was purified by flash
chromatography on a silica gel column. Gradient elution with 3:1,
2:1, 1:1, and then 1:3 hexanes-EtOAc provided 690 mg of the title
compound as a white foam.
[0900] .sup.1H NMR (CDCl.sub.3): .delta. 1.46 (s, 3H), 2.70-2.89
(m, 2H), 3.19-3.31 (m, 2H), 3.39 (s, 3H), 3.58-3.70 (m, 3H), 3.80
(s, 3H), 3.80-3.94 (m, 1H), 4.05-4.25 (m, 3H), 5.89 (s, 1H), 6.85
(s, 1H), 6.89 (s, 1H), 7.24-7.48 (m, 12H), 7.78 (s, 1H), 9.69 (s,
1H); .sup.13C NMR (CDCl.sub.3): .delta. 12.3, 45.3, 55.3, 58.9,
61.6, 70.2, 71.9, 82.6, 85.6, 87.1, 89.1, 110.5, 113.4, 127.4,
128.2, 128.4, 130.5, 134.7, 135.3, 143.6, 143.7, 150.5, 158.9,
164.6.
[0901] HRMS (FAB) m/z 735.155 (M+Na).sup.+
(C.sub.34H.sub.37IN.sub.2O.sub.- 7Na requires 735.154).
Step G:
3'-Deoxy-5'-O-(4-methoxytrityl)-3'-methyl-2'-(2-methoxyethyl)-5-me-
thyluridine
[0902] A mixture of ammonium phosphinate (410 mg, 5.1 mmol) and
1,1,1,3,3,3-hexamethyldisilazane (1.18 mL, 0.90 g, 5.59 mmol) was
heated at 100-110.degree. C. for 2 h under nitrogen atmosphere with
condenser. The intermediate BTSP(bis[trimethylsilyl]phosphinate)
was cooled to 0.degree. C. and 5 mL of dichloromethane was
injected. To this mixture was injected a solution of
3'-deoxy-5'-O-(4-methoxytrityl)-3'-(iodomethyl-
)-2'-O-(2-methoxyethyl)-5-methyluridine (0.78 g, 1.1 mmol) and
diisopropylethylamine (0.39 mL, 287 mg, 2.23 mmol) in 7 mL of
dichloromethane. After the reaction mixture was stirred at rt
overnight, a mixture of THF--MeOH--NEt.sub.3 (3/6/0.3 mL) was added
and continued to stir for 1 h. The reaction mixture was filtered
through a pad of celite and washed with dichloromethane. The
solvent was evaporated and the residue was purified by flash
chromatography on a silica gel column using 2:1, 1:1, and then 1:2
hexanes-EtOAc as eluent providing 380 mg of the title compound.
[0903] .sup.1H NMR (CDCl.sub.3): .delta. 0.97 (d, 3H, J=6.8 Hz),
1.41 (s, 3H), 2.35-2.55 (m, 1H), 3.27 (dd, 1H, J=11.0, 3.0 Hz),
3.37 (s, 3H), 3.54-3.68 (m, 3H), 3.79 (s, 3H), 3.75-3.87 (m, 1H),
3.94 (d, 1H, J=5.0 Hz), 4.03-4.16 (m, 2H), 5.84 (s, 1H), 6.83 (s,
1H), 6.87 (s, 1H), 7.20-7.37 (m, 8H), 7.39-7.50 (m, 4H), 7.86 (s,
1H), 9.50 (s, 1H); .sup.13C NMR (CDCl.sub.3): .delta. 8.7, 12.1,
35.6, 55.3, 59.0, 61.7, 69.8, 72.1, 85.4, 86.4, 86.7, 89.8, 110.0,
113.3, 127.2, 128.0, 128.4, 130.4, 135.0, 135.7, 143.9, 150.5,
158.8, 164.6.
[0904] HRMS (FAB) m/z 609.256 (M+Na).sup.+
(C.sub.34H.sub.38N.sub.2O.sub.7- Na requires 609.257).
Step H:
3'-Deoxy-3'-methyl-2'-O-(2-methoxyethyl)-5-methyluridine
[0905] Trifluoroacetic acid (1.5 mL) was added dropwise to a
stirred solution of
3'-deoxy-5'-O-(4-methoxytrityl)-3'-methyl-2'-O-(2-methoxyethy-
l)-5-methyluridine (370 mg, 0.63 mmol) in 50 mL of chloroform at
0.degree. C. The mixture was stirred at rt for 30 min,
concentrated, and then dissolved in ethyl acetate. The solution was
washed with dilute sodium bicarbonate and brine. The organic phase
was dried (Na.sub.2SO.sub.4) and concentrated. The resulting
residue was purified by flash chromatography on a silica gel
column. Elution with 1:1, 1:3 and then 0:1 hexanes-EtOAc provided
170 mg of the title compound as a white foam.
[0906] .sup.1H NMR (CDCl.sub.3): .delta. 1.03 (d, 3H, J=6.8 Hz),
1.83 (s, 3H), 2.20-2.40 (m, 1H), 3.10-3.28 (m, 1H), 3.35 (s, 3H),
3.50-4.15 (m, 10H), 5.81 (s, 1H), 7.89 (s, 1H), 9.77 (s, 1H);
.sup.13C NMR (CDCl.sub.3): .delta. 8.9, 12.4, 34.7, 59.0, 60.6,
69.7, 72.0, 86.3, 89.8, 109.7, 136.9, 150.4, 164.7. HRMS (FAB) m/z
315.154 (M+H).sup.+ (C.sub.14H.sub.23N.sub.2O.sub.6 requires
315.155).
EXAMPLE 50
2'-Amino-2'-deoxyuridine
[0907] 84
[0908] This compound was prepared following the procedures
described in J. Org. Chem. 61: 781 (1996).
EXAMPLE 51
3'-Deoxyuridine
[0909] 85
[0910] This compound was obtained from commercial sources.
EXAMPLE 52
2'-C-Methyladenosine
[0911] 86
[0912] This compound was prepared following the conditions
described in J. Med. Chem. 41: 1708 (1998).
EXAMPLE 53
3'-Deoxyadenosine (Cordycepin)
[0913] 87
[0914] This compound was obtained from commercial sources.
EXAMPLE 54
3'-Amino-3'-deoxyadenosine
[0915] 88
[0916] This compound was prepared following the conditions
described in Tetrahedron Lett. 30: 2329 (1989).
EXAMPLE 55
8-Bromoadenosine
[0917] 89
[0918] This compound was obtained from commercial sources.
EXAMPLE 56
2'-O-Methyladenosine
[0919] 90
[0920] This compound was obtained from commercial sources.
EXAMPLE 57
3'-Deoxy-3'-fluoroadenosine
[0921] 91
[0922] This compound was prepared following the procedures
described in J. Med. Chem. 34: 2195 (1991).
EXAMPLE 58
6-Methyl-9-(.beta.-D-ribofuranosyl)-9H-purine
[0923] 92
[0924] This compound was prepared following the procedures
described in Nucleosides, Nucleotides, Nucleic Acids 19: 1123
(2000).
EXAMPLE 59
2',3',5'-tri-O-acetyl-8-methylsulfonyladenosine
[0925] 93
EXAMPLE 60
1-Methyl-9-[2,3,5-tri-O-(p-toluoyl)-.beta.-D-ribofuranosyl]-9H-purine-6
(1H)-thione
[0926] 94
EXAMPLE 61
4-Amino-7-(2-C-methyl-.beta.-D-arabinofuranosyl)-7H-pyrrolo[2,3-d]pyrimidi-
ne
[0927] 95
[0928] To chromium trioxide (1.57 g, 1.57 mmol) in dichloromethane
(DCM) (10 mL) at 0.degree. C. was added acetic anhydride (145 mg,
1.41 mmol) and then pyridine (245 mg, 3.10 mmol). The mixture was
stirred for 15 min, then a solution of
7-[3,5-O-[1,1,3,3-tetrakis(1-methylethyl)-1,3-dis-
iloxanediyl]-.beta.-D-ribofuranosyl]-7H-pyrrolo[2,3-d]pyrimidin-4-amine
[for preparation, see J. Am. Chem. Soc. 105: 4059 (1983)] (508 mg,
1.00 mmol) in DCM (3 mL) was added. The resulting solution was
stirred for 2 h and then poured into ethyl acetate (10 mL), and
subsequently filtered through silica gel using ethyl acetate as the
eluent. The combined filtrates were evaporated in vacuo, taken up
in diethyl ether/THF (1:1) (20 mL), cooled to -78.degree. C. and
methylmagnesium bromide (3M, in THF) (3.30 mL, 10 mmol) was added
dropwise. The mixture was stirred at -78.degree. C. for 10 min,
then allowed to come to room temperature (rt) and quenched by
addition of saturated aqueous ammonium chloride (10 mL) and
extracted with DCM (20 mL). The organic phase was evaporated in
vacuo and the crude product purified on silica gel using 5%
methanol in dichloromethane as eluent. Fractions containing the
product were pooled and evaporated in vacuo. The resulting oil was
taken up in THF (5 mL) and tetrabutylammonium fluoride (TBAF) on
silica (1.1 mmol/g on silica) (156 mg) was added. The mixture was
stirred at rt for 30 min, filtered, and evaporated in vacuo. The
crude product was purified on silica gel using 10% methanol in
dichloromethane as eluent. Fractions containing the product were
pooled and evaporated in vacuo to give the desired compound (49 mg)
as a colorless solid.
[0929] .sup.1H NMR (DMSO-d.sub.6): .delta. 1.08 (s, 3H), 3.67 (m,
2H), 3.74 (m, 1H), 3.83 (m, 1H), 5.19 (m, 1H), 5.23 (m, 1H), 5.48
(m, 1H), 6.08 (1H, s), 6.50 (m, 1H), 6.93 (bs, 2H), 7.33 (m, 1H),
8.02 (s, 1H).
EXAMPLE 62
4-Amino-7-(2-C-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine
[0930] 96
Step A:
3,5-Bis-O-(2,4-dichlorophenylmethyl)-1-O-methyl-.alpha.-D-ribofura-
nose
[0931] A mixture of
2-O-acetyl-3,5-bis-O-(2,4-dichlorophenylmethyl)-1-O-me-
thyl-.alpha.-D-ribofuranose [for preparation, see: Helv. Chim. Acta
78: 486 (1995)] (52.4 g, 0.10 mol) in methanolic K.sub.2CO.sub.3
(500 mL, saturated at room temperature) was stirred at room
temperature for 45 min. and then concentrated under reduced
pressure. The oily residue was suspended in CH.sub.2Cl.sub.2 (500
mL), washed with water (300 mL+5.times.200 mL) and brine (200 mL),
dried (Na.sub.2SO.sub.4), filtered, and concentrated to give the
title compound (49.0 g) as colorless oil, which was used without
further purification in Step B below.
[0932] .sup.1H NMR (DMSO-d.sub.6): .delta. 3.28 (s, 3H, OCH.sub.3),
3.53 (d, 2H, J.sub.5,4=4.5 Hz, H-5a, H-5b), 3.72 (dd, 1H,
J.sub.3,4=3.6 Hz, J.sub.3,2=6.6 Hz, H-3), 3.99 (ddd, 1H,
J.sub.2,1=4.5 Hz, J.sub.2,OH-2=9.6 Hz, H-2), 4.07 (m, 1H, H-4),
4.50 (s, 2H, CH.sub.2Ph), 4.52, 4.60 (2d, 2H, J.sub.gem=13.6 Hz,
CH.sub.2Ph), 4.54 (d, 1H, OH-2), 4.75 (d, 1H, H-1), 7.32-7.45,
7.52-7.57 (2m, 10H, 2Ph).
[0933] .sup.13C NMR (DMSO-d.sub.6) .delta. 55.40, 69.05, 69.74,
71.29, 72.02, 78.41, 81.45, 103.44, 127.83, 127.95, 129.05, 129.28,
131.27, 131.30, 133.22, 133.26, 133.55, 133.67, 135.45, 135.92.
Step B:
3,5-Bis-O-(2,4-dichlorophenylmethyl)-1-O-methyl-.alpha.-D-erythro--
pentofuranos-2-ulose
[0934] To an ice-cold suspension of Dess-Martin periodinane (50.0
g, 118 mmol) in anhydrous CH.sub.2Cl.sub.2 (350 mL) under argon
(Ar) was added a solution of the compound from Step A (36.2 g, 75
mmol) in anhydrous CH.sub.2Cl.sub.2 (200 mL) dropwise over 0.5 h.
The reaction mixture was stirred at 0.degree. C. for 0.5 h and then
at room temperature for 3 days. The mixture was diluted with
anhydrous Et.sub.2O (600 mL) and poured into an ice-cold mixture of
Na.sub.2S.sub.2O.sub.3.5H.sub.2O (180 g) in saturated aqueous
NaHCO.sub.3 (1400 mL). The layers were separated, and the organic
layer was washed with saturated aqueous NaHCO.sub.3 (600 mL), water
(800 mL) and brine (600 mL), dried (MgSO.sub.4), filtered and
evaporated to give the title compound (34.2 g) as a colorless oil,
which was used without further purification in Step C below.
[0935] .sup.1H NMR (CDCl.sub.3) .delta. 3.50 (s, 3H, OCH.sub.3),
3.79 (dd, 1H, J.sub.5a,5b=11.3 Hz, J.sub.5a,4=3.5 Hz, H-5a), 3.94
(dd, 1H, J.sub.5b,4=2.3 Hz, H-5b), 4.20 (dd, 1H, J.sub.3,1=1.3 Hz,
J.sub.3,4=8.4 Hz, H-3), 4.37 (ddd, 1H, H-4), 4.58, 4.69 (2d, 2H,
J.sub.gem=13.0 Hz, CH.sub.2Ph), 4.87 (d, 1H, H-1), 4.78, 5.03 (2d,
2H, J.sub.gem=12.5 Hz, CH.sub.2Ph), 7.19-7.26, 7.31-7.42 (2m, 10H,
2Ph).
[0936] .sup.13C NMR (DMSO-d.sub.6) .delta. 55.72, 69.41, 69.81,
69.98, 77.49, 78.00, 98.54, 127.99, 128.06, 129.33, 129.38, 131.36,
131.72, 133.61, 133.63, 133.85, 133.97, 134.72, 135.32, 208.21.
Step C:
3,5-Bis-O-(2,4-dichlorophenylmethyl)-2-C-methyl-1-O-methyl-.alpha.-
-D-ribofuranose
[0937] To a solution of MeMgBr in anhydrous Et.sub.2O (0.48 M, 300
mL) at -55.degree. C. was added dropwise a solution of the compound
from Step B (17.40 g, 36.2 mmol) in anhydrous Et.sub.2O (125 mL).
The reaction mixture was allowed to warm to -30.degree. C. and
stirred for 7 h at -30.degree. C. to -15.degree. C., then poured
into ice-cold water (500 mL) and the mixture vigorously stirred at
room temperature for 0.5 h. The mixture was filtered through a
Celite pad (10.times.5 cm) which was thoroughly washed with
Et.sub.2O. The organic layer was dried (MgSO.sub.4), filtered and
concentrated. The residue was dissolved in hexanes (.intg.30
.mu.L), applied onto a silica gel column (10.times.7 cm, prepacked
in hexanes) and eluted with hexanes and hexanes/EtOAc (9/1) to give
the title compound (16.7 g) as a colorless syrup.
[0938] .sup.1H NMR (CDCl.sub.3): .delta. 1.36 (d, 3H,
J.sub.Me,OH=0.9 Hz, 2C-Me), 3.33 (q, 1H, OH), 3.41 (d, 1H,
J.sub.3,4=3.3 Hz), 3.46 (s, 3H, OCH.sub.3), 3.66 (d, 2H,
J.sub.5,4=3.7 Hz, H-5a, H-5b), 4.18 (apparent q, 1H, H-4), 4.52 (s,
1H, H-1), 4.60 (s, 2H, CH.sub.2Ph), 4.63, 4.81 (2d, 2H,
J.sub.gem=13.2 Hz, CH.sub.2Ph), 7.19-7.26, 7.34-7.43 (2m, 10H,
2Ph).
[0939] .sup.13C NMR (CDCl.sub.3): .delta. 24.88, 55.45, 69.95,
70.24, 70.88, 77.06, 82.18, 83.01, 107.63, 127.32, 129.36, 130.01,
130.32, 133.68, 133.78, 134.13, 134.18, 134.45, 134.58.
Step D:
4-Chloro-7-[3,5-bis-O-(2,4-dichlorophenylmethyl)-2-C-methyl-.beta.-
-D-ribofuranosyl]-7H-pyrrolo[2,3-d]pyrimidine
[0940] To a solution of the compound from Step C (9.42 g, 19 mmol)
in anhydrous dichloromethane (285 mL) at 0.degree. C. was added HBr
(5.7 M in acetic acid, 20 mL, 114 mmol) dropwise. The resulting
solution was stirred at 0.degree. C. for 1 h and then at rt for 3
h, evaporated in vacuo and co-evaporated with anhydrous toluene
(3.times.40 mL). The oily residue was dissolved in anhydrous
acetonitrile (50 mL) and added to a solution of sodium salt of
4-chloro-1H-pyrrolo[2,3-d]pyrimidine [for preparation, see J. Chem.
Soc., 131 (1960)] in acetonitrile [generated in situ from
4-chloro-1H-pyrrolo[2,3-d]pyrimidine (8.76 g, 57 mmol) in anhydrous
acetonitrile (1000 m]L), and NaH (60% in mineral oil, 2.28 g, 57
mmol), after 4 h of vigorous stirring at room temperature]. The
combined mixture was stirred at room temperature for 24 h, and then
evaporated to dryness. The residue was suspended in water (250 mL)
and extracted with EtOAc (2.times.500 mL). The combined extracts
were washed with brine (300 mL), dried over Na.sub.2SO.sub.4,
filtered and evaporated. The crude product was purified on a silica
gel column (10 cm.times.10 cm) using ethyl acetate/hexane (1:3 and
1:2) as the eluent. Fractions containing the product were combined
and evaporated in vacuo to give the desired product (5.05 g) as a
colorless foam.
[0941] .sup.1H NMR (CDCl.sub.3): .delta. 0.93 (s, 3H, CH.sub.3),
3.09 (s, 1H, OH), 3.78 (dd, 1H, J.sub.5',5"=10.9 Hz, J.sub.5',4=2.5
Hz, H-5'), 3.99 (dd, 1H, J.sub.5,4=2.2 Hz, H-5"), 4.23-4.34 (m, 2H,
H-3', H-4'), 4.63, 4.70 (2d, 2H, J.sub.gem=12.7 Hz, CH.sub.2Ph),
4.71, 4.80 (2d, 2H, J.sub.gem=12.1 Hz, CH.sub.2Ph), 6.54 (d, 1H,
J.sub.5,6=3.8 Hz, H-5), 7.23-7.44 (m, 10H, 2Ph).
[0942] .sup.13C NMR (CDCl.sub.3): .delta. 21.31, 69.10, 70.41,
70.77, 79.56, 80.41, 81.05, 91.11, 100.57, 118.21, 127.04, 127.46,
127.57, 129.73, 129.77, 130.57, 130.99, 133.51, 133.99, 134.33,
134.38, 134.74, 135.21, 151.07, 151.15 152.47.
Step E:
4-Chloro-7-(2-C-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]py-
rimidine
[0943] To a solution of the compound from Step D (5.42 g, 8.8 mmol)
in dichloromethane (175 mL) at -78.degree. C. was added boron
trichloride (1M in dichloromethane, 88 mL, 88 mmol) dropwise. The
mixture was stirred at -78.degree. C. for 2.5 h, then at
-30.degree. C. to -20.degree. C. for 3 h. The reaction was quenched
by addition of methanol/dichloromethane (1:1) (90 mL) and the
resulting mixture stirred at -15.degree. C. for 30 min., then
neutralized with aqueous ammonia at 0.degree. C. and stirred at
room temperature for 15 min. The solid was filtered and washed with
CH.sub.2Cl.sub.2/MeOH (1/1, 250 mL). The combined filtrate was
evaporated, and the residue was purified by flash chromatography
over silica gel using CH.sub.2Cl.sub.2 and CH.sub.2Cl.sub.2:MeOH
(99:1, 98:2, 95:5 and 90:10) gradient as the eluent to furnish
desired compound (1.73 g) as a colorless foam, which turned into an
amorphous solid after treatment with MeCN.
[0944] .sup.1H NMR (DMSO-d.sub.6) .delta. 0.64 (s, 3H, CH.sub.3),
3.61-3.71 (m, 1H, H-5'), 3.79-3.88 (m, 1H, H-5"), 3.89-4.01 (m, 2H,
H-3', H-4'), 5.15-5.23 (m, 3H, 2'-OH, 3'-OH, 5'-OH), 6.24 (s, 1H,
H-1'), 6.72 (d, 1H, J.sub.5,6=3.8 Hz, H-5), 8.13 (d, 1H, H-6), 8.65
(s, 1H, H-2).
[0945] .sup.13C NMR (DMSO-d.sub.6) .delta. 20.20, 59.95, 72.29,
79.37, 83.16, 91.53, 100.17, 117.63, 128.86, 151.13, 151.19,
151.45.
Step F:
4-Amino-7-(2-C-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyr-
imidine
[0946] To the compound from Step E (1.54 g, 5.1 mmol) was added
methanolic ammonia (saturated at 0.degree. C.; 150 mL). The mixture
was heated in a stainless steel autoclave at 85.degree. C. for 14
h, then cooled and evaporated in vacuo. The crude mixture was
purified on a silica gel column with CH.sub.2Cl.sub.2/MeOH (9/1) as
eluent to give the title compound as a colorless foam (0.8 g),
which separated as an amorphous solid after treatment with MeCN.
The amorphous solid was recrystallized from methanol/acetonitrile;
m.p. 222.degree. C.
[0947] .sup.1H NMR (DMSO-d.sub.6): .delta. 0.62 (s, 3H, CH.sub.3),
3.57-3.67 (m, 1H, H-5'), 3.75-3.97 (m, 3H, H-5", H-4', H-3'), 5.00
(s, 1H, 2'-OH), 5.04 (d, 1H, J.sub.3'OH,3'=6.8 Hz, 3'-OH), 5.06 (t,
1H, J.sub.5'OH,5',5"=5.1 Hz, 5'-OH), 6.11 (s, 1H, H-1'), 6.54 (d,
1H, J.sub.5,6=3.6 Hz, H-5), 6.97 (br s, 2H, NH.sub.2), 7.44 (d, 1H,
H-6), 8.02 (s, 1H, H-2).
[0948] .sup.13C NMR (DMSO-d.sub.6): .delta. 20.26, 60.42, 72.72,
79.30, 82.75, 91.20, 100.13, 103.08, 121.96, 150.37, 152.33,
158.15.
[0949] LC-MS: Found: 279.10 (M-H.sup.+); calc. for
C.sub.12H.sub.16N.sub.4- O.sub.4+H.sup.+: 279.11.
EXAMPLE 63
4-Amino-7-(3-deoxy-3-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrim-
idin carboxamide
[0950] 97
Step A:
4-Amino-6-bromo-7-(2-O-acetyl-5-O-benzoyl-3-deoxy-3-methyl-.beta.--
D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidin-5-carbonitrile
[0951] BSA (0.29 mL, 2.0 mmol) was added into a stirred suspension
of 4-amino-6-bromo-5-cyano-1H-pyrrolo[2,3-d]pyrimidine (0.24 g, 1
mmol; prepared according to Nucleic Acid Chemistry, Part IV,
Townsend, L. B. and Tipson, R. S.; Ed.; Wiley-Interscience: New
York, 1991, pp. 16-17 and Synthetic Commun. 1998, 28, 3835) in dry
acetonitrile (10 mL) at room temperature under argon. After 15 min,
1,2-di-O-acetyl-5-O-benzoyl-3-deox- y-3-methyl-D-ribofuranose (J.
Med. Chem. (1976), 19, 1265) (0.36 g, 1.0 mmol) was added along
with TMSOTf (0.54 g, 3 mmol). The mixture was stirred at room
temperature for 5 min and then at 80.degree. C. for 0.5 h. The
solution was cooled, diluted with ethyl acetate (50 mL) and poured
into ice-cold saturated aqueous NaHCO.sub.3 (15 mL). The layers
were separated. The organic layer was washed with brine (15 mL),
dried (Na.sub.2SO.sub.4) and then evaporated. The residue was
purified on silica gel column using a solvent system of
hexanes/EtOAc: 3/1. Appropriate fractions were collected and
evaporated to provide the title compound as colorless foam (0.21
g).
Step B:
4-Amino-7-(2-O-acetyl-5-O-benzoyl-3-deoxy-3-methyl-.beta.-D-ribofu-
ranosyl)-7H-pyrrolo[2,3-d]pyrimidin-5-carbonitrile
[0952] To a suspension of the title compound from Step A (183 mg,
0.35 mmol) in EtOH (9 mL) were added ammonium formate (0.23 g, 3.6
mmol) and 10% palladium on activated carbon (20 mg) and the mixture
was heated at reflux for 1.5 h. The hot reaction mixture was
filtered through Celite and washed with hot EtOH. The solvent was
removed and the residue treated with MeOH. The pale yellow solid
was filtered thus yielding 105 mg of pure title compound. The
filtrate was evaporated and purified on a silica gel column with a
solvent system of CH.sub.2Cl.sub.2/MeOH: 50/1 to afford an
additional 63 mg of title compound as a white solid.
Step C:
4-Amino-7-(3-deoxy-3-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-
-d]pyrimidin-5-carboxamide
[0953] A mixture of the compound from Step B (51 mg, 0.12 mmol),
ethanolic ammonia (5 mL, saturated at 0.degree. C.), aqueous
ammonia (5 mL, 30%) and aqueous hydrogen peroxide (1 mL, 35%) was
stirred room temperature for 8 h. The solution was evaporated and
the residue purified on silica gel column with a solvent system of
CH.sub.2Cl.sub.2/MeOH: 10/1 to give the title compound as a white
solid (28 mg). .sup.1H-MNR (CD.sub.3OD): .delta. 1.12 (d, 3H, J=6.8
Hz), 2.40 (m, 1H), 3.76 (dd, 1H, J.sub.1=12.8 Hz, J.sub.2=4.0 Hz),
3.94-4.04 (m, 2H), 4.33 (d, 1H, J=5.4 Hz), 6.13 (s, 1H), 8.11 (s,
1H), 8.16 (s, 1H).
EXAMPLE 64
4-Amino-7-(3-deoxy-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine-5-c-
arboxamide
[0954] 98
[0955] This compound was prepared following the procedures
described in J. Med. Chem. 26: 25 (1983).
EXAMPLE 65
4-Amino-7-(.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carboxami-
de (Sangivamycin)
[0956] 99
[0957] This compound was obtained from commercial sources.
EXAMPLE 66
7-(2-O-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine
[0958] 100
[0959] This compound was prepared following the procedures
described in J. Org. Chem. 39: 1891(1974).
EXAMPLE 67
4-Amino-7-(3-deoxy-3-fluoro-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrim-
idine-5-carboxamide
[0960] 101
[0961] This compound was prepared following the procedures
described in Chem. Pharm. Bull. 41: 775 (1993).
EXAMPLE 68
4-Amino-7-(3-deoxy-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine-5-c-
arbonitrile
[0962] 102
[0963] This compound was prepared following the procedures
described in J. Med. Chem. 30: 481(1987).
EXAMPLE 69
4-Amino-7-(2-O-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine
[0964] 103
[0965] This compound was prepared following the procedures
described in J. Org. Chem. 39: 1891(1974).
EXAMPLE 70
3'-Amino-3'-deoxy-2'-O-methyladenosine
[0966] 104
[0967] This compound is obtained by the methylation of
appropriately protected 3'-amino-3'-deoxyadenosine derivative
(Example 54).
EXAMPLE 71
4-Amino-7-(3-deoxy-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine
[0968] 105
[0969] This compound was prepared following the following procedure
described in Can. J. Chem. 55: 1251 (1977).
EXAMPLE 72
[0970] General Process to SATE Prodrug Moiety
[0971] S-Acyl-2-Thioethyl (SATE) pronucleotides are discussed in C.
R. Wagner, V. V. Iyer, and E. J. McIntee, "Pronucleotides: Toward
the In Vivo Delivery of Antiviral and Anticancer Nucleotides," Med.
Res. Rev., 20: 1-35 (2000), which is incorporated by reference
herein in its entirety. SATE derivatives of nucleosides are also
disclosed U.S. Pat. Nos. 5,770,725; 5,849,905; and 6,020,482, the
contents of each of which are incorporated by reference herein in
their entirety.
Bis(S-acetyl-2-thioethyl)-N,N-diisopropylphosphoramidite
[0972] 2-Mercaptoethanol (5 g, 64 mmol) was dissolved in
CH.sub.2Cl.sub.2 (50 mL). To this solution was added triethylamine
(7.67 mL, 57.6 mmol), and the reaction mixture was cooled in an ice
bath to 0.degree. C. Acetic anhydride (4.54 mL, 48 mmol) was added
dropwise in 10 min, and the reaction mixture was stirred for 1 h at
0.degree. C. The reaction mixture was then allowed to come to room
temperature over a period of 2 h. The reaction mixture was diluted
with CH.sub.2Cl.sub.2 (50 mL), washed with water (75 mL), 5%
aqueous NaHCO.sub.3 (75 mL) and brine (75 mL). The organic phase
was dried over anhydrous Na.sub.2SO.sub.4 and concentrated in vacuo
to give an oil. The oil was then dissolved in anhydrous THF (40 mL)
and anhydrous triethylamine (7.76 mL) was added. To this mixture
was added activated molecular sieves (4 .ANG.) and was kept at room
temperature for 10 min. The reaction mixture was cooled in an ice
bath to 0.degree. C. and diisopropylphosphoramidous dichloride
(6.47 g, 32.03 mmol) was added. The reaction mixture was stirred at
0.degree. C. for 2 h under inert atmosphere. Hexane (40 mL) was
added to the reaction mixture and the precipitate formed was
filtered. The filtrate was concentrated to one fourth of the
volume, purified by loaded silica gel column chromatography and
eluted with hexane containing 3% triethylamine and incremental
amount of ethyl acetate (0 to 7%) to give the title compound as an
oil (2.36 g).
[0973] .sup.1H NMR (CDCl.sub.3): .delta. 1.17 (s, 6H), 1.21 (s,
6H), 2.36 (s, 6H), 3.14 (t, J=6.44 Hz), 3.51-3.84 (m, 6H); .sup.13C
NMR (CDCl.sub.3): .delta. 24.47, 24.61, 30.48, 42.85, 43.1, 61.88,
62.23, 195.26; .sup.13P NMR (CDCl.sub.3): .delta. 146.96.
EXAMPLE 73
2'-O-Methylguanosine-5'-[bis-(S-acetyl-2-thioethyl)phosphate]
[0974] 106
Step A:
N.sup.2-(4-monomethoxytrityl)-2'-O-methylguanosine-5'-[bis-(S-acet-
yl-2-thioethyl)phosphate]
[0975] N.sup.2-(4-monomethoxytrityl)-2'-O-methylguanosine (0.74 g,
1.31 mmol) was mixed with 1H-tetrazole (0.061 g, 0.87 mmol) and
dried over P.sub.2O.sub.5 in vacuo overnight. To this mixture was
added anhydrous acetonitrile (8 mL). To the turbid solution,
bis(S-acetyl-2-thioethyl)N,N- -diisopropylphosphoramidite (0.3 g,
0.87 mmol) was added slowly and the reaction mixture was stirred at
ambient temperature under inert atmosphere for 2 h. Solvent was
removed in vacuo. The residue was cooled to -40.degree. C. and a
solution of 3-chloroperbenzoic acid (0.2 g) in CH.sub.2Cl.sub.2 (7
mL) was added. The solution was allowed to warm up to room
temperature over 1 h. Sodium hydrogensulfite (10% aqueous solution,
2 mL) was added to reduce the excess of 3-chloroperbenzoic acid.
The organic phase separated, diluted with CH.sub.2Cl.sub.2 (20 mL),
washed with saturated aqueous Na.sub.2CO.sub.3 (10 mL), water (10
mL), dried over Na.sub.2SO.sub.4 and evaporated to dryness. The
residue was purified by silica gel column chromatography and eluted
with CH.sub.2Cl.sub.2 containing incremental amount of MeOH (5 to
10%) as eluent to yield the title compound (0.36 g) as a foam.
[0976] .sup.1H NMR (DMSO-d.sub.6): .delta. 2.35 (s, 6H), 2.97 (s,
3H), 3.11 (t, 4H, J=6.0 Hz), 3.5 (m, 1H), 3.74 (s, 3H), 3.72-3.83
(m, 2H), 3.97-4.11 (m, 6H), 5.1 (d, 1H, J=6.4 Hz), 5.29 (d, 1H,
J=3.1 Hz), 6.89 (d, 2H, J=8.8 Hz), 7.15-7.37 (m, 12H), 7.68 (s,
1H), 7.73 (s, 1H), 10.72 (s, 1H); .sup.13C NMR (CDCl.sub.3):
.delta. 30.36, 55.38, 57.99, 66.08, 66.19, 67.22, 69.15, 70.49,
81.18, 81.57, 86.64, 113.04, 117.99, 126.66, 127.71, 128.67,
130.04, 136.09, 136.56, 144.51, 144.82, 149.52, 151.29, 158.15,
194.56; .sup.13P NMR (CDCl.sub.3): .delta. -2.04; MS (API-ES)
852.10 [M-H].sup.+.
Step B:
2'-O-methylguanosine-5'-[bis-(S-acetyl-2-thioethyl)phosphate]
[0977]
N.sup.2-(4-monomethoxytrityl)-2'-O-methylguanosine-5'-[bis-(S-acety-
l-2-thioethyl)phosphate] (0.2 g, 0.23 mmol) was dissolved in acetic
acid: MeOH: H.sub.2O, 36:1 and heated at 55.degree. C. for 24 h.
Solvent was removed and the residue was purified by HPLC on reverse
phase column (Hamilton PRP-1, 250.times.22 mm, A=Acetonitrile,
B=H.sub.2O 20 to 100 B in 65 min, flow 10 mL min.sup.-1). Fractions
containing the product were pooled together and evaporated to give
the title compound (40% yield).
[0978] .sup.13P NMR (CDCl.sub.3): .delta. -0.72; MS (API-ES) m/z
582.1 [M+H].sup.+.
EXAMPLE 74
2'-O-Methylguanosine-5'-[bis-(S-pivaloyl-2-thioethyl)phosphate]
[0979] 107
Step A:
Bis(S-pivaloyl-2-thioethyl)-N,N-diisopropylphosphoramidite
[0980] S-pivaloyl-2-thioethanol (6.3 g, 39.6 mmol) was dissolved in
anhydrous THF (100 mL). To this solution was added activated
molecular sieves (4A.degree.) and kept at room temperature for 30
min. Anhydrous triethylamine (7.9 mL, 59.4 mmol) was added and the
reaction mixture was cooled in an ice bath to 0.degree. C. To this
mixture diisopropylphosphoramidous dichloride (4 g, 19.8 mmol) was
added dropwise. The mixture was stirred the reaction mixture at
0.degree. C. for 2 h under inert gas atmosphere. Hexane (100 mL)
was added to the reaction mixture, and the precipitate formed was
filtered. The filtrate was concentrated to one fourth of the
volume. This was purified by flash silica gel column chromatography
using hexane containing 2% triethylamine and incremental amount of
ethyl acetate (0 to 3%) as eluent to give the title compound as an
oil (5.23 g).
[0981] .sup.1H NMR (CDCl.sub.3): .delta. 1.13-1.31 (m, 30H), 1.21
(s, 6H), 3.09 (t, J=6.6 Hz, 4H), 3.51-3.84 (m, 6H); .sup.3C NMR
(CDCl.sub.3): .delta. 24.47, 24.61, 27.32, 30.00, 42.85, 43.1,
46.32, 61.98, 62.33, 206.1; .sup.13P NMR (CDCl.sub.3): .delta.
148.51.
Step B:
2'-O-methylguanosine-5'-[bis-(S-pivaloyl-2-thioethyl)phosphate]
[0982] N.sup.2-(4-monomethoxytrityl)-2'-O-methylguanosine (0.6 g,
1.05 mmol) was mixed with 1H-tetrazole (0.05 g, 0.7 mmol) and dried
over P.sub.2O.sub.5 in vacuo overnight. To this mixture anhydrous
acetonitrile (13.8 mL) was added. The reaction mixture was cooled
to 0.degree. C. in an ice bath and
bis(S-pivaloyl-2-thioethyl)N,N-diisopropylphosphoramidite (0.32 g,
0.7 mmol) was added slowly. The reaction mixture was stirred at
0.degree. C. for 5 minutes. The ice bath was removed and the
reaction mixture was allowed to stir at room temperature under an
inert atmosphere for 2 h. Solvent was removed in vacuo. The residue
was cooled to -40.degree. C. and a solution of 3-chloroperbenzoic
acid (0.24 g, 1.4 mmol, 57-80%) in CH.sub.2Cl.sub.2 (10 mL) was
added. The solution was allowed to warm up to -10.degree. C. over 1
h. Sodium hydrogensulfite (10% aqueous solution, 10 mL) was added
to reduce the excess of 3-chloroperbenzoic acid. The organic phase
separated, diluted with CH.sub.2Cl.sub.2 (50 mL), washed with
saturated aqueous Na.sub.2CO.sub.3 (40 mL), water (40 mL), dried
over Na.sub.2SO.sub.4 and evaporated to dryness. The residue was
chromatographed on a flash silica gel column using a
CH.sub.2Cl.sub.2 containing incremental amount of MeOH (0 to 5%) as
eluent. Fractions containing the product were pooled together and
evaporated. The residue was dissolved in a solution of acetic
acid/water/methanol (10 mL, 3:1:6) and heated at 55.degree. C. for
24 h. Evaporated the solution in vacuum to get an oil. The oil was
dissolved in 20% MeOH in water and purified by HPLC on C-18 column
(Luna C-18, 250.times.2.12 mm, A=water, B=acetonitrile, 20 to 10% B
in 65 min., flow 10 mL min.sup.-1, .lambda. 260 nm) to yield the
title compound (0.082 g).
[0983] .sup.1H NMR (DMSO-d.sub.6): .delta. 1.18 (s, 18H), 3.08 (m,
4H), 3.33 (s, 3H) 3.94-4.10 (m, 6H), 4.14-4.21 (m, 2H), 4.29 (m,
1H), 5.42 (d, 1H, J=5.4 Hz), 5.81 (d, 1H, J=5.8 Hz), 6.49 (bs, 2H),
7.86 (s, 1H), 10.66 (bs, 1H); .sup.13P NMR (DMSO-d.sub.6): .delta.
-0.71; MS (API-ES) m/z 664.2 [M-H].sup.-.
EXAMPLE 75
8-Bromo-2'-O-methyl
guanosine-5'-[bis-(S-pivaloyl-2-thioethyl)phosphate]
[0984] 108
[0985] This compound was synthesized according to the procedure
used for the synthesis of Example 74 starting with
8-bromo-N.sup.2-(4-monomethoxyt- rityl)-2'-O-methylguanosine (0.46
g, 0.63 mmol). Other reagents used were 1H-tetrazole (0.034 g, 0.49
mmol), bis(S-pivaloyl-2-thioethyl)N,N-diisopr- opylphosphoramidite
(0.22 g, 0.49 mmol), acetonitrile (8.3 ml), 3-chloroperbenzoic acid
(0.17 g, 0.98 mmol, 57-80%) in CH.sub.2Cl.sub.2 (4 mL). The title
compound was isolated in 13% yield (0.061 g).
[0986] .sup.1H NMR (DMSO-d.sub.6): .delta. 1.14 and 1.16 (m, 18H),
3.06 (m, 4H), 3.32 (s, 3H) 3.96-4.06 (m, 5H), 4.18-4.3 (m, 2H),
4.46 (d, 1H, J=2.4 Hz), 4.66 (t, 1H, J=2.6 Hz), 5.37 (d, 1H, J=2.6
Hz), 5.78 (d, 1H, J=2.8 Hz), 6.62 (bs, 2H), 10.99 (bs, 1H);
.sup.13P NMR (DMSO-d.sub.6) 6-0.79; MS (API-ES) m/z 742.13 and
744.13 [M-H].sup.-.
EXAMPLE 76
2-Amino-3,4-dihydro-7-(2-O-methyl-.beta.-D-ribofuranosyl)-4-oxo-7H-pyrrolo-
[2,3-d]pyrimidine-5'-Ibis-(S-pivaloyl-2-thioethyl)phosphate]
[0987] 109
[0988] This compound was synthesized according to the procedure
used for the synthesis of Example 74 starting with
7-deaza-N.sup.2-(4-monomethoxyt- rityl)-2'-O-methylguanosine (0.47
g, 0.82 mmol). Other reagents used were 1H-tetrazole (0.044 g, 0.63
mmol), bis(S-pivaloyl-2-thioethyl)N,N-diisopr- opylphosphoramidite
(0.29 g, 0.63 mmol), acetonitrile (11 mL), 3-chloroperbenzoic acid
(0.21 g, 1.26 mmol, 57-80%) in CH.sub.2Cl.sub.2 (5.2 mL). The title
compound was isolated in 29% yield (0.158 g).
[0989] .sup.1H NMR (DMSO-d.sub.6): .delta. 1.14 (s, 18H), 3.06 (m,
4H), 3.31 (s, 3H) 3.96-4.26 (m, 9H), 5.35 (d, 1H, J=2.6 Hz), 5.78
(d, 1H, J=5.2 Hz), 5.99 (d, 1H, J=6.6 Hz), 6.27 (m, 3H), 6.86 (d,
1H, J=3.6 Hz), 10.39 (s, 1H); .sup.13P NMR (DMSO-d.sub.6): .delta.
-0.72; MS (API-ES) m/z 663.20 [M-H].sup.-; HRMS Calcd for
C.sub.26H.sub.42N.sub.4O.sub.10PS.- sub.2 665.2074 found
665.2071.
EXAMPLE 77
3'-Deoxyguanosine-5'-[bis-(S-pivaloyl-2-thioethyl)phosphate]
[0990] 110
[0991] N.sup.2-(4-Monomethoxytrityl)-3'-deoxyguanosine (0.20 g,
0.35 mmol) was mixed with 1H-tetrazole (0.019 g, 0.27 mmol) and
dried over P.sub.2O.sub.5 in vacuo overnight. To this mixture
anhydrous acetonitrile (4.7 mL) was added to give a turbid
solution. The reaction mixture was cooled to 0.degree. C. in an ice
bath and bis(S-pivaloyl-2-thioethyl)N,N--
diisopropylphosphoramidite (0.12 g, 0.27 mmol) was added slowly.
The reaction mixture was stirred at 0.degree. C. for 5 minutes. The
ice bath was removed and the reaction mixture was allowed to come
to room temperature. The reaction mixture was stirred at room
temperature under an inert gas atmosphere for 2 h. Solvent was
removed in vacuo. The residue was cooled to -40.degree. C. and a
solution of 3-chloroperbenzoic acid (0.12 g, 0.7 mmol, 57-80%) in
CH.sub.2Cl.sub.2 (2.2 mL) was added. The solution was allowed to
warm up to -10.degree. C. over 1 h. Sodium hydrogensulfite (10%
aqueous solution, 2 mL) was added to reduce the excess of
3-chloroperbenzoic acid. The organic phase was separated, diluted
with CH.sub.2Cl.sub.2 (30 mL), washed with saturated aqueous
Na.sub.2CO.sub.3 (20 mL), water (20 mL), dried over
Na.sub.2SO.sub.4 and evaporated to dryness. The residue was
chromatographed on a flash silica gel column using CH.sub.2Cl.sub.2
containing incremental amount of MeOH (0 to 5%) as eluent.
Fractions containing the product were pooled and evaporated. The
residue was dissolved in a solution of acetic acid/water/methanol
(5 mL, 3:1:6) and heated at 55.degree. C. for 24 h. Evaporated the
solution in vacuum to get an oil. The oil was dissolved in 20% MeOH
in water and purified by HPLC on C-18 column (Luna C-18,
250.times.2.12 mm, A=water, B=acetonitrile, 20 to 10% B in 65 min.,
flow 10 mL min.sup.-1, .lambda. 260 nm) to yield the title compound
(0.027 g).
[0992] .sup.1H NMR (DMSO-d.sub.6): .delta. 1.15 (s, 18H), 1.92-2.01
(m, 1H), 2.17-2.28 (m, 1H), 3.04 (t, 4H, J=6.2 Hz), 3.91-4.23 (m,
6H), 4.37-4.55 (m, 2H), 5.67 (m, 2H), 6.45 (bs, 2H), 7.75 (s, 1H),
10.61 (s, 1H); .sup.13P NMR (DMSO-d.sub.6): .delta. -0.75; MS
(API-ES) m/z 634.2 [M-H].sup.-.
EXAMPLE 78
2-Amino-7-(3-deoxy-.beta.-D-ribofuranosyl)-3,4-dihydro-4-oxo-7H-pyrrolo[2,-
3-d]pyrimidine-5'-[bis-(S-pivaloyl-2-thioethyl)phosphate]
[0993] 111
[0994]
2-(4-Monomethoxytrityl)amino-7-(3-deoxy-.beta.-D-ribofuranosyl)-7H--
pyrrolo[2,3-d]pyrimidin-4(3H)-one (0.30 g, 0.52 mmol) was mixed
with 1H-tetrazole (0.028 g, 0.40 mmol) and dried over
P.sub.2O.sub.5 in vacuo overnight. To this mixture anhydrous
acetonitrile (7 mL) was added, and the solution was cooled to
0.degree. C. in an ice bath.
Bis(S-pivaloyl-2-thioethyl)-N,N-diisopropylphosphoramidite (0.18 g,
0.40 mmol) was added slowly. The reaction mixture was allowed to
come to at room temperature and stirred at room temperature under
an inert atmosphere for 2 h. The solvent was removed in vacuo. The
residue was cooled to -40.degree. C., and a solution of
3-chloroperbenzoic acid (0.14 g, 0.8 mmol, 57-80%) in
CH.sub.2Cl.sub.2 (5 mL) was added. The solution was allowed to warm
up to -10.degree. C. over 2 h. Sodium hydrogensulfite (10% aqueous
solution, 5 mL) was added to reduce the excess of
3-chloroperbenzoic acid. The organic phase was separated, diluted
with CH.sub.2Cl.sub.2 (50 mL), washed with saturated aqueous
Na.sub.2CO.sub.3 (40 mL), water (40 mL), dried over
Na.sub.2SO.sub.4 and evaporated to dryness. The residue was
chromatographed on a flash silica gel column using CH.sub.2Cl.sub.2
containing incremental amount of MeOH (0 to 5%) as eluent.
Fractions containing the product were pooled and evaporated. The
residue was dissolved in a solution of acetic acid/water/methanol
(10 mL, 3:1:6) and heated at 55.degree. C. for 24 h. The solution
was evaporated to give an oil. The oil was dissolved in 20% MeOH in
water and purified by HPLC on C-18 column (Luna C18, 250.times.2.12
mm, A=water, B=acetonitrile 20 to 10% B in 65 mL, flow 10 mL/min,
.lambda. 260 nm) to give the title compound (0.053 g).
[0995] .sup.1H NMR (DMSO-d.sub.6): .delta. 1.16 (s, 18H), 1.91-2.01
(m, 1H), 2.17-2.25 (m, 1H), 3.05 (t, 4H, J=6.2 Hz), 3.92-4.2 (m,
6H), 4.35 (bs, 2H), 5.56 (d, 1H, J=4.2 Hz), 5.86 (d, 1H, J=2.4 Hz),
6.24 (m, 3H), 6.77 (d, 1H, J=3.6 Hz), 10.36 (s, 1H); .sup.13P NMR
(DMSO-d.sub.6): .delta. -0.89; HRMS (MALDI) Calcd for
C.sub.25H.sub.39N.sub.4O.sub.9PS.su- b.2.635.1969 found
635.1964.
EXAMPLE 79
2-Amino-5-bromo-7-(3-deoxy-.beta.-D-ribofuranosyl)-3,4-dihydro-4-oxo-7H-py-
rrolo[2,3-d]pyrimidine-5'-[bis-(S-pivaloyl-2-thioethyl)phosphate]
[0996] 112
[0997]
2-(4-Monomethoxytrityl)amino-5-bromo-7-(3-deoxy-.beta.-D-ribofurano-
syl)-7H-pyrrolo[2,3-d]pyrimidin-4(3H)-one (0.066 g, 0.17 mmol) was
mixed with imidazole triflate (0.017 g, 0.17 mmol) and dried over
P.sub.2O.sub.5 in vacuo overnight. To this mixture anhydrous
acetonitrile (7 mL) and
bis(S-pivaloyl-2-thioethyl)N,N-diisopropylphosphoramidite (0.97 g,
0.24 mmol) were added slowly. The reaction mixture was stirred
under an inert atmosphere for 18 h. Solvent was removed in vacuo.
The residue was cooled to -40.degree. C. and a solution of
3-chloroperbenzoic acid (0.059 g, 0.34 mmol, 57-80%) in
CH.sub.2Cl.sub.2 (2 mL) was added. The solution was allowed to warm
up to -10.degree. C. over 2 h. Sodium hydrogensulfite (10% aqueous
solution, 5 mL) was added to reduce the excess of
3-chloroperbenzoic acid. The organic phase was separated, diluted
with CH.sub.2Cl.sub.2 (30 mL), washed with saturated aqueous
Na.sub.2CO.sub.3 (20 mL), water (20 mL), dried over
Na.sub.2SO.sub.4 and evaporated to dryness. The residue was
chromatographed on flash silica gel column using CH.sub.2Cl.sub.2
containing incremental amount of MeOH (0 to 5%) as eluent.
Fractions containing the product were pooled and evaporated. The
residue was dissolved in a solution of acetic acid/water/methanol
(3 mL, 3:1:6) and heated at 55.degree. C. for 24 h. The solution
was evaporated to give an oil. The oil was dissolved in 20% MeOH in
water and purified by HPLC on C-18 column (Luna C18, 250.times.2.12
mm, A=water, B=acetonitrile 20 to 10% B in 65 mL, flow 10 mL
min.sup.-1, .lambda. 260 nm) to afford the title compound (0.036
g).
[0998] .sup.1H NMR (DMSO-d.sub.6): .delta. 1.17 (s, 18H), 1.87-2.03
(m, 1H), 2.17-2.26 (m, 1H), 3.05 (t, 4H, J=6.4 Hz), 3.92-4.2 (m,
6H), 4.37 (bs, 2H), 5.70 (d, 1H, J=4.4 Hz), 5.85 (d, 1H, J=2.6 Hz),
6.36 (bs, 2H), 6.93 (s, 1H), 10.51 (s, 1H); .sup.13P NMR
(DMSO-d.sub.6): .delta. -0.89; MS (AP-ES) m/z 711.11 and 713.09
[M-H].sup.-; HRMS (MALDI) Calcd for
C.sub.25H.sub.38BrN.sub.4O.sub.9PS.sub.2.713.1074 and 715.1074
found 713.1081 and 715.102.
EXAMPLE 80
2'-O-Methylcytidine-5'-[bis-(S-pivaloyl-2-thioethyl)phosphate]
[0999] 113
[1000] N.sup.4-(4,4'-Dimethoxytrityl)-2'-O-methylcytidine (0.49 g,
0.86 mmol) was mixed with 1H-tetrazole (0.06 g, 0.86 mmol) and
dried over P.sub.2O.sub.5 in vacuo overnight. To this mixture
anhydrous acetonitrile (6 mL) and
bis-(S-pivaloyl-2-thioethyl)-N,N-diisopropylphosphoramidite (0.39
g, 0.86 mmol) were added at 0.degree. C. The reaction mixture was
allowed to come to room temperature and stirred under an inert
atmosphere for 18 h. Solvent was removed in vacuo. The residue was
cooled to -40.degree. C. and a solution of 3-chloroperbenzoic acid
(0.3 g, 1.72 mmol, 57-80%) in CH.sub.2Cl.sub.2 (5.5 mL) was added.
The solution was allowed to warm up to -10.degree. C. over 2 h.
Sodium hydrogensulfite (10% aqueous solution, 5 mL) was added to
reduce the excess of 3-chloroperbenzoic acid. The organic phase was
separated, diluted with CH.sub.2Cl.sub.2 (30 mL), washed with
saturated aqueous Na.sub.2CO.sub.3 (20 m]L), water (20 mL), dried
over Na.sub.2SO.sub.4 and evaporated to dryness. The residue was
chromatographed on a flash silica gel column using CH.sub.2Cl.sub.2
containing incremental amount of MeOH (0 to 10%) as eluent.
Fractions containing the product were pooled and evaporated. The
residue was dissolved in a solution of acetic acid/water/methanol
(10 mL, 3:1:6) and heated at 55.degree. C. for 24 h. The solution
was evaporated to give an oil. The oil was dissolved in 20% MeOH in
water and purified by HPLC on C-18 column (Luna C18, 250.times.2.12
mm, A=water, B=acetonitrile 20 to 10% B in 65 mL, flow 10 mL
min.sup.-1, .lambda. 260 nm) to yield the title compound (0.076
g).
[1001] .sup.1H NMR (DMSO-d.sub.6): .delta. 1.18 (s, 18H), 3.12 (t,
4H, J=6.4 Hz), 3.39 (s, 3H), 3.69 (t, 1H, J=4.2 Hz), 3.93-4.3 (m,
8H), 5.29 (d, 1H, J=6.2 Hz), 5.72 (d, 1H, J=7.4 Hz), 5.86 (d, 1H,
J=4 Hz), 7.21 (bs, 2H), 7.58 (d, 1H, J=7.4 Hz); .sup.13P NMR
(CD.sub.3CN): .delta. -0.64; MS (AP-ES) m/z 625.69 [M+H].sup.+;
HRMS (MALDI) Calcd for C.sub.24H.sub.40N.sub.3O.sub.10PS.sub.2Na
648.1785 found 648.1804.
EXAMPLE 81
5-Bromo-2'-O-methylcytidine-5'-[bis-(S-pivaloyl-2-thioethyl)phosphate]
[1002] 114
Step A: 5-Bromo-3'-O-(t-butyldimethyl)silyl-2'-O-methylcytidine
[1003] 2'-O-Methylcytidine (1.5 g, 5.83 mmol) was mixed with
imidazole (3.97 g, 58.32 mmol) and dried in vacuo. This mixture was
dissolved in anhydrous DMF (4 mL) and t-butyldimethylsilyl chloride
(4.41 g, 29.25 mmol) was added and the reaction mixture was stirred
for 18 h at room temperature under an inert atmosphere. Reaction
mixture was diluted with water (100 mL) and extracted with ethyl
acetate (2.times.60 mL). The organic phase was dried over anhydrous
Na.sub.2SO.sub.4 and evaporated. The residue was purified by silica
gel column chromatography and eluted with ethyl acetate/hexane,
6:4. Fractions containing the product were pooled and evaporated.
The product obtained (2.76 g) was dissolved in acetonitrile (19.43
mL), LiBr (0.623 g, 7.18 mmol) and stirred to get a clear solution.
To this ammonium ceric (IV) nitrate (6.24 g, 11.37 mmol) was added
and the reaction mixture was allowed to stir at room temperature
for 3 h. Solvent was removed in vacuum. The residue obtained was
taken in ethyl acetate (100 mL) and washed with water (80 mL). The
organic phase was separated, dried over anhydrous Na.sub.2SO.sub.4
and evaporated. Residue purified by silca gel column chromatography
and eluted with 5% MeOH in CH.sub.2Cl.sub.2. The product obtained
(2.66 g) was dissolved in 80% acetic acid in water and heated at
50.degree. C. for 6 h. The solvent was removed and the residue
purified on a silica gel column and eluted with 5% MeOH in
CH.sub.2Cl.sub.2 to give the title compound (0.85 g).
[1004] .sup.1H NMR (DMSO-d.sub.6): .delta. 0.78 (s, 6H), 0.85 (s,
9H), 3.31 (s, 3H), 3.44-3.6 (m, 2H), 3.69-3.9 (m, 2H), 4.24 (m,
1H), 5.29 (t, 1H, J=4.4 Hz), 5.76 (d, 1H, J=3.2 Hz), 7.06 (bs, 1H),
7.88 (bs, 1H), 8.39 (s, 1H).
Step B:
5-Bromo-2`-O-methylcytidine-S`-[bis-(S-pivaloyl-2-thioethyl)phosph-
ate]
[1005] 5-Bromo-3'-O-(t-butyldimethyl)silyl-2'-O-methylcytidine
(0.093 g, 0.21 mmol) was mixed with 1H-tetrazole (0.03 g, 0.42
mmol) and dried over P.sub.2O.sub.5 in vacuo overnight. To this
mixture anhydrous acetonitrile (2 mL).
Bis-(S-pivaloyl-2-thioethyl)-N,N-diisopropylphosphoramidite (0.2 g,
0.42 mmol) was added at 0.degree. C. The reaction mixture was
allowed to come to room temperature and stirred under an inert
atmosphere for 4 h. Solvent was removed in vacuo. The residue was
cooled to -40.degree. C. and a solution of 3-chloroperbenzoic acid
(0.072 g, 0.42 mmol, 57-80%) in CH.sub.2Cl.sub.2 (2 mL) was added.
The solution was allowed to warm up to -10.degree. C. over 2 h.
Sodium hydrogensulfite (10% aqueous solution, 2 mL) was added to
reduce the excess of 3-chloroperbenzoic acid. The organic phase
separated, diluted with CH.sub.2Cl.sub.2 (30 mL), washed with
saturated aqueous Na.sub.2CO.sub.3 (20 mL), water (20 mL), dried
over Na.sub.2SO.sub.4 and evaporated to dryness. The residue was
dissolved in THF (2.1 mL) and triethylamine trihydrofluoride (0.17
g, 1.1 mmol). The reaction mixture was stirred at room temperature
for 18 h. The solution was evaporated to give an oil. The oil was
dissolved in ethyl acetate (30 mL) and washed with water (20 mL),
5% aqueous NaHCO.sub.3 and brine (20 mL). The organic phase was
dried over anhydrous Na.sub.2SO.sub.4 and evaporated. The residue
was dissolved in 20% MeOH in water and purified by HPLC on C-18
column (Luna C18, 250.times.2.12 mm, A=water, B=acetonitrile 20 to
10% B in 65 mL, flow 10 mL min.sup.-1, .lambda. 260 nm) to give the
title compound (0.054 g).
[1006] .sup.1H NMR (DMSO-d.sub.6): .delta. 1.17 (s, 18H), 3.11 (t,
4H, J=6.2 Hz), 3.39 (s, 3H), 3.75 (t, 1H, J=4.8 Hz), 3.93-4.3 (m,
8H), 5.23 (d, 1H; J=6.4 Hz), 5.8 (d, 1H, J=3.8 Hz), 7.07 (bs, 1H),
7.89 (s, 1H) 7.94 (bs, 1H); .sup.13P NMR (CD.sub.3CN): 8-0.34; MS
(AP-ES) m/z 702.00 and 704.00 [M-H].sup.-; HRMS (MALDI) Calcd for
C.sub.24H.sub.39BrN.sub.3O- .sub.10PS.sub.2Na 726.0890 and 728.0890
found 726.0893 and 728.086.
EXAMPLE 82
3'-Deoxycytidine-5'-[bis-(S-pivaloyl-2-thioethyl)phosphate]
[1007] 115
Step A: N.sup.4-(4,4'-dimethoxytrityl)-3'-deoxycytidine
[1008] 3'-Deoxycytidine (0.8 g, 3.54 mmol) was mixed with imidazole
(2.41 g, 35.4 mmol) and dried over P.sub.2O.sub.5 in vacuum
overnight at 40.degree. C. The mixture was dissolved in anhydrous
DMF and t-butyldimethylsilyl chloride (2.68 g, 17.78 mmol) was
added and the reaction mixture was stirred under an argon
atmosphere for 18 h at room temperature. The reaction mixture was
diluted with water (100 mL) and extracted with ethyl acetate
(2.times.75 mL). The organic phase was separated, dried over
anhydrous Na.sub.2SO.sub.4 and evaporated. The residue was purified
by silica gel column chromatography and eluted with ethyl
acetate/hexane (6:4) to yield
2',5'-bis(t-butyldimethylsilyl)-3'-de- oxycytidine (1.27 g). This
was then mixed with DMAP (0.34 g, 2.79 mmol) and dried in vacuum.
This mixture was dissolved in anhydrous pyridine (8 mL) and
4,4'-dimethoxytrityl chloride (1.89 g, 5.58 mmol) was added. The
reaction mixture was stirred at room temperature under an argon
atmosphere for 18 h. Solvent was removed in vacuo. The residue
obtained was taken in ethyl acetate (100 mL) and washed with 5%
NaHCO.sub.3 in water (75 mL) and brine (75 ml). The organic phase
was dried over anhydrous Na.sub.2SO.sub.4 and evaporated. The
residue obtained was dissolved in THF (28 mL). To this
triethylamine trihydrofluoride (2.26 mL, 13.74 mmol) and
triethylamine (0.95 mL, 6.87 mmol) were added and stirred at room
temperature for 18 h. Solvent was removed and the residue dissolved
in ethyl acetate (50 mL), washed with water (50 mL) and 5%
NaHCO.sub.3 in water (50 mL). The organic phase was dried over
anhydrous Na.sub.2SO.sub.4 and evaporated. The residue obtained was
purified by silica gel column chromatography and eluted with 5%
MeOH in CH.sub.2Cl.sub.2 to yield the title compound (0.66 g).
[1009] .sup.1H NMR (DMSO-d.sub.6): .delta. 1.66 (m, 1H), 1.85 (m,
1H), 3.47 (m, 1H), 3.63 (m, 1H), 3.71 (s, 6H), 4.00 (bs, 1H), 4.19
(m, 1H), 4.96 (t, 1H, J=5.2 Hz), 5.39 (bs, 1H), 5.53 (s, 1H), 6.17
(bs, 1H), 6.83 (d, 4H, J=8.8 Hz), 7.04-7.22 (m, 9H), 7.77 (d, 1H,
J=7.6 Hz), 8.27 (bs, 1H); MS (AP-ES) m/z 528.1 [M-H].sup.-.
Step B:
3'-Deoxycytidine-5'-[bis-(S-pivaloyl-2-thioethyl)phosphate]
[1010] This compound was synthesized following the similar
synthetic procedure used for the synthesis of Example 80 starting
with N.sup.4-(4,4'-dimethoxytrityl)-3'-deoxycytidine (0.3 g, 0.57
mmol). Other reagents used for the synthesis were 1H-tetrazole
(0.04 g, 0.57 mmol), acetonitrile (4 mL),
bis-(S-pivaloyl-2-thioethyl)-N,N-diisopropylphosphor- amidite (0.52
g, 1.14 mmol) and 3-chloroperbenzoic acid (0.2 g, 1.14 mmol,
57-80%) in CH.sub.2Cl.sub.2 (3.6 mL). The product was isolated in
22% yield (0.073 g) after HPLC purification.
[1011] .sup.1H NMR (200 MHz, DMSO-d.sub.6): .delta. 1.17 (s, 18H),
1.84 (m, 2H), 3.11 (t, 4H, J=6.4 Hz), 3.93-4.31 (m, 8H), 4.39 (m,
1H), 5.55 (d, 1H, J=4.2 Hz), 5.67 (dd, 2H, J=7.4 and 1.8 Hz), 7.1
(bs, 2H), 7.56 (d, 1H, J=7.4 Hz); .sup.13P NMR (CD.sub.3CN):
.delta. -0.71; MS (AP-ES) m/z 596.1 [M+H].sup.+; HRMS (MALD) Calcd
for C.sub.23H.sub.38N.sub.3O.sub- .9PS.sub.2Na 618.1679 found
618.1600.
EXAMPLE 83
2'-O-Methylcytidine-5'-[bis(isopropyloxycarbonyloxymethyl)]phosphate
[1012] 116
[1013] Phosphonomethoxy nucleoside analogs are discussed in C. R.
Wagner, V. V. Iyer, and E. J. McIntee, "Pronucleotides: Toward the
In Vivo Delivery of Antiviral and Anticancer Nucleotides," Med.
Res. Rev., 20: 1-35 (2000), which is incorporated by reference
herein in its entirety. They are also disclosed U.S. Pat. Nos.
5,922,695; 5,977,089; 6,043,230; and 6,069,249, the contents of
each of which are incorporated by reference herein in their
entirety.
Step A: iso-Propyl chloromethyl carbonate
[1014] This was prepared according to Antiviral Chemistry &
Chemotherapy 8: 557 (1997).
Step B: 2'-O-Methylcytidine-5'-phosphate
[1015] This intermediate was prepared as described in Tetrahedron
Lett. 50: 5065 (1967).
Step C: 2'-O-Methylcytidine-5'-[bis(isopropyloxycarbonyloxy
methyl)]phosphate
[1016] 2'-O-Methylcytidine-5'-phosphate (0.4 g, 1.19 mmol) was
dried over P.sub.2O.sub.5 in vacuum overnight at 40.degree. C. It
was then suspended in anhydrous DMF (4 mL). To this mixture was
added diisopropylethylamine (0.86 mL, 4.92 mmol) and iso-propyl
chloromethyl carbonate (1.56 g, 7.34 mmol). The mixture was heated
at 50.degree. C. for 1 h. The reaction mixture was then allowed to
come to room temperature. The reaction mixture was stirred at room
temperature for 48 h and then filtered. The filtrate was diluted
with water (100 mL) and extracted with CH.sub.2Cl.sub.2 (3.times.50
mL). The organic phase was dried over anhydrous Na.sub.2SO.sub.4
and evaporated. The residue was dissolved in 20% MeOH in water and
purified by HPLC on C-18 column (Luna C18, 250.times.2.12 mm,
A=water, B=acetonitrile 20 to 10% B in 65 ML, flow 10 mL
min.sup.-1, .lambda. 260 nm) to give the title compound (2.5
mg).
[1017] .sup.13P NMR (CD.sub.3CN): .delta. -3.09; MS (AP-ES) m/z
570.1 [M+H].sup.+.
EXAMPLE 84
2'-O-Methylcytidine-5'-[(2-decyloxy-3-dodecylthio-1-propyl)phosphate]
[1018] 117
[1019] The procedure is described for similar nucleoside analogs in
German Patent 408366 (1992) and J. Acquired Immune Defic. Syndr.
2000, 23, 227. The reaction of the appropriately protected
2'-O-methylcytidine with
(2-decyloxy-3-dodecylthio-1-propyl)phosphate [prepared by the
reaction of 2-decyloxy-3-dodecylthio-1-propanol with POCl.sub.3 in
ether in presence of triethylamine] under refluxing conditions in a
toluene-ether mixture furnishes the desired compound.
EXAMPLE 85
2'-O-Methylcytidine-5'-[rac-(3-octadecylthio-2-palmitoyloxy-1-propyl)phosp-
hate]
[1020] 118
[1021] This compound is synthesized by the reaction of
2'-O-methylcytidine-5'-monophosphoromorpholidate with
rac-1-S-octadecyl-2-O-palmitoyl-1-thioglycerol in pyridine
following the similar procedure described for AZT and ddC in J.
Med. Chem. 39: 1771 (1996).
EXAMPLE 86
Nucleoside 5'-Triphosphates
[1022] The nucleoside 5'-triphosphates of the present invention
were prepared according to the general procedures described in
Chem. Rev. 100: 2047 (2000).
EXAMPLE 87
Purification and Purity Analysis of Nucleoside 5'-Triphosphates
[1023] Triphosphates were purified by anion exchange (AX)
chromatography using a 30.times.100 mm Mono Q column (Pharmacia)
with a buffer system of 50 mM Tris, pH 8. Elution gradients were
typically from 40 mM NaCl to 0.8 M NaCl in two column volumes at
6.5 mL/min. Appropriate fractions from anion exchange
chromatography were collected and desalted by reverse-phase (RP)
chromatography using a Luna C18 250.times.21 mm column (Phenomenex)
with a flow rate of 10 ml/min. Elution gradients were generally
from 1% to 95% methanol in 14 min at a constant concentration of 5
mM triethylammonium acetate (TEAA).
[1024] Mass spectra of the purified triphosphates were determined
using on-line HPLC mass spectrometry on a Hewlett-Packard (Palo
Alto, Calif.) MSD 1100. A Phenomenex Luna (C18(2)), 150.times.2 mm,
plus 30.times.2 mm guard column, 3-.mu.m particle size was used for
RP HPLC. A 0 to 50% linear gradient (15 min) of acetonitrile in 20
mM TEAA (triethylammonium acetate) pH 7 was performed in series
with mass spectral detection in the negative ionization mode.
Nitrogen gas and a pneumatic nebulizer were used to generate the
electrospray. The mass range of 150-900 was sampled. Molecular
masses were determined using the HP Chemstation analysis
package.
[1025] The purity of the purified triphosphates was determined by
analytical RP and AX HPLC. RP BPLC with a Phenomonex Luna or
Jupiter column (250.times.4.6 mm), 5-.mu.m particle size was
typically run with a 2-70% acetonitrile gradient in 15 min in 100
mM TEAA, pH 7. AX HPLC was performed on a 1.6.times.5 mm Mono Q
column (Pharmacia). Triphosphates were eluted with a gradient of 0
to 0.4 M NaCl at constant concentration of 50 mM Tris, pH 8. Purity
of the triphosphates was generally >80%.
EXAMPLE 88
Nucleoside 5'-Monophosphates
[1026] The nucleoside 5'-monophosphates of the present invention
were prepared according to the general procedure described in
Tetrahedron Lett. 50: 5065 (1967).
EXAMPLE 89
2-Amino-9-(.beta.-D-arabinofuranosyl)-9H-purin-6 (1H)-one
[1027] 119
[1028] This compound was obtained from commercial sources.
EXAMPLE 90
3'-Deoxy-3'-methylguanosine
[1029] 120
[1030] This compound was prepared following procedures described in
U.S. Pat. No. 3,654,262 (1972).
EXAMPLE 91
2'-O-[4-(Imidazolyl-1)butyl]guanosine
[1031] 121
Step A: 2'-O-[4-(Imidazolyl-1)butyl]-2-aminoadenosine
[1032] A solution 2-aminoadenosine (7.36 g, 26 mmol) in dry DMF
(260 mL) was treated portionwise with 60% NaH (3.92 g, 1000 mmol).
After 1 hr., a solution of bromobutylimidazole (9.4 g, 286 mmol) in
DMF (20 ml) was added. After 16 hrs., the solution was conc. in
vacuo, partitioned between H.sub.2O/EtOAc and separated. The
aqueous layer was evaporated, and the residue was chromatographed
on silica gel (CHCl.sub.3/MeOH) to afford the title nucleoside as a
white solid; yield 4.2 g.
[1033] .sup.1H NMR (DMSO-d.sub.6): .delta. 1.39 (t, 2H), 1.67 (t,
2H), 3.3-3.7 (m, 4H), 3.93 (m, 3H), 4.29 (m, 2H), 4.40 (d, 1H),
5.50 (5, 1H), 5.72 (d, 1H), 5.82 (bs, 2H), 6.72 (bs, 2H), 6.86 (s,
1H), 7.08 (s, 1H), 7.57 (s, 1H). 7.91 (s, 1H).
Step B: 2'-O-[4-(Imidazolyl-1)butyl]guanosine
[1034] A mixture of the intermediate from Step A (3.2 g, 8 mmol) in
H.sub.2O (200 mL), DMSO (10 mL), trisodium phosphate (10 g), and
adenosine deaminase (0.3 g) was stirred at room temperature and pH
7. The solution was filtered and and then evaporated. The resulting
solid was crystallized from EtOAc/MeOH to afford the title compound
as a white solid; yield 2.6 g.
[1035] .sup.1H NMR (DMSO-d.sub.6): .delta. 1.39 (t, 2H), 1.67 (t,
2H), 3.3-3.7 (m, 4H), 3.93 (m, 3H), 4.29 (m, 2H), 5.10 (t, 1H),
5.20 (d, 1H), 5.79 (d, 1H), 6.50 (bs, 2H), 6.86 (s, 1H), 7.08 (s,
1H), 7.57 (s, 1H) 7.9 (s, 1H).
EXAMPLE 92
2'-Deoxy-2'-fluoroguanosine
[1036] 122
[1037] This compound was prepared following the conditions
described in Chem. Pharm. Bull. 29: 1034 (1981).
EXAMPLE 93
2'-Deoxyguanosine
[1038] 123
[1039] This compound was obtained from commercial sources.
EXAMPLE 94
2-Amino-7-(2-deoxy-2-fluoro-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrim-
idin-4(3H)-one
[1040] 124
Step A:
2-Amino-4-chloro-7-(2,3,5-tri-O-benzyl-(.beta.-D-arabinofuranosyl)-
-7H-pyrrolo[2,3-d]-pyrimidine
[1041] To a suspension of
2-amino-4-chloro-1H-pyrrolo[2,3-d]pyrimidine [Liebigs Ann. Chem. 1:
137 (1983)] (3.03 g, 18 mmol) in anhydrous MeCN (240 mL), powdered
KOH (85%; 4.2 g, 60 mmol) and tris[2-(2-methoxyethoxy)-
-ethyl]amine (0.66 mL, 2.1 mmol) were added and the mixture was
stirred at room temperature for 10 min. Then a solution of
2,3,5-tri-O-benzyl-D-arab- inofuranosyl bromide [prepared from
corresponding 1-O-p-nitrobenzoate (11.43 g, 20.1 mmol) according to
Seela et al., J. Org. Chem. (1982), 47, 226] in MeCN (10 mL) was
added and stirring continued for another 40 min. Solid was filtered
off, washed with MeCN (2.times.25 mL) and combined filtrate
evaporated. The residue was purified on a silica gel column with a
solvent system of hexanes/EtOAc: 7/1, 6/1 and 5/1. Two main zones
were separated. From the more rapidly migrating zone was isolated
the .alpha. anomer (0.74 g) and from the slower migrating zone the
desired .alpha. anomer (4.01 g).
Step B:
2-Amino-7-(.beta.-D-arabinofuranosyl)-4-chloro-7H-pyrrolo[2,3-d]py-
rimidine
[1042] To a solution of the compound from Step A (4.0 g, 7 mmol) in
CH.sub.2Cl.sub.2 (150 ml) at -78.degree. C. was added a solution of
1.0 M BCl.sub.3 in CH.sub.2Cl.sub.2 (70 mL, 70 mmol) during 45 min.
The mixture was stirred at -78.degree. C. for 3 h and at
-20.degree. C. for 2.5 h. MeOH--CH.sub.2Cl.sub.2 (70 mL, 1:1) was
added to the mixture, which was then stirred at -20.degree. C. for
0.5 h and neutralized with conc. aqueous NH.sub.3 at 0.degree. C.
The mixture was stirred at room temperature for 10 min. and then
filtered. The solid was washed with MeOH--CH.sub.2Cl.sub.2 (70 mL,
1:1) and the combined filtrate evaporated. The residue was purified
on a silica gel column with a solvent system of
CH.sub.2Cl.sub.2/MeOH: 20/1 to give the desired nucleoside (1.18 g)
as a white solid.
Step C:
2-Amino-7-[3,5-O-(1,3,3-tetraisopropyldisiloxane-1,3-diyl)-.beta.--
D-arabinofuranosyl]-4-chloro-7H-pyrrolo[2,3-d]pyrimidine
[1043] The compound from Step B (0.87 g, 2.9 mmol) and imidazole
(0.43 g, 5.8 mmol were dissolved in DMF (3.5 mL).
1,3-Dichloro-1,1,3,3-tetraisopro- pyldisiloxane (1.0 mL) was added
to the solution. The reaction mixture was stirred at room
temperature for 1 h and then evaporated. The residue was
partitioned between CH.sub.2Cl.sub.2 (150 mL) and water (30 mL).
The layers were separated. The organic layer was dried
(Na.sub.2SO.sub.4) and evaporated. The residue was purified on a
silica gel column with a solvent system of hexanes/EtOAc: 7/1 and
5/1 to give the title compound (1.04 g).
Step D:
2-Amino-7-[2-O-acetyl-3,5-O-(1,1,3,3-tetraisopropyldisiloxane-1,3--
diyl)-.beta.-D-arabinofuranosyl]-4-chloro-7H-pyrrolo[2,3-d]pyrimidine
[1044] A mixture of the compound from Step C (0.98 g, 1.8 mmol) in
MeCN (12 mL), Et.sub.3N (0.31 mL) Ac.sub.2O (0.21 mL) and DMAP (5
mg, 0.25 eq.) was stirred at room temperature for 5 h and then
evaporated. The oily residue was dissolved in EtOAc (200 mL),
washed with water (2.times.20 mL), dried (Na.sub.2SO.sub.4) and
evaporated to yield pure title compound (1.12 g).
Step E:
2-Amino-7-[2-O-acetyl-.beta.-D-arabinofuranosyl]-4-chloro-7H-pyrro-
lo[2,3-d]pyrimidine
[1045] To an ice-cold solution of the compound from Step D (0.95 g,
1.63 mmol) in THF (10 mL) and AcOH (0.19 mL) was added dropwise 1.0
M tetrabutylammonium fluoride solution in THF (3.4 mL) and stirred
at 0.degree. C. for 15 min. The solution was concentrated and the
oily residue applied onto a silica gel column packed in
CH.sub.2Cl.sub.2 and eluted with CH.sub.2Cl.sub.2[MeOH: 50/1, 25/1
and 20/1. Appropriate fractions were pooled and evaporated to give
the title nucleoside (0.56 g) as a white solid.
Step F:
2-Amino-7-[2-O-acetyl-3,5-di-O-(tetrahydro-2-pyranyl)-.beta.-D-ara-
binofuranosyl]-4-chloro-7H-pyrrolo[2,3-d]pyrimidine
[1046] To a solution of the compound from Step E (0.5 g, 1.46 mmol)
in CH.sub.2Cl.sub.2 (10 mL) and 3,4-dihydro-2-H-pyrane (0.67 mL)
was added dropwise TMSI (30 .mu.L, 0.2 mmol). The reaction mixture
was stirred at room temperature for 1 h and then evaporated. The
oily residue was purified on a silica gel column packed in a
solvent system of hexanes/EtOAc/Et.sub.3N: 75/25/1 and eluted with
a solvent system of hexanes/EtOAc: 3/1. The fractions containing
the product were collected and evaporated to give the desired
compound (0.60 g).
Step G:
2-Amino-7-[3,5-di-O-(tetrahydro-2-pyranyl)-.beta.-D-arabinofuranos-
yl]-4-chloro-7H-pyrrolo[2,3-d]pyrimidine
[1047] A mixture of the compound from Step F (0.27 g, 0.53 mmol)
and methanolic ammonia (saturated at 0.degree. C.; 10 mL) was kept
overnight at 0.degree. C. Evaporation of the solvent yielded the
desired compound (0.25 g).
Step H:
2-Amino-7-[2-deoxy-2-fluoro-3,5-di-O-(tetrahydro-2-pyranyl)-(.beta-
.-D-ribofuranosyl]-4-chloro-7H-pyrrolo[2,3-d]pyrimidine
[1048] To a solution of the compound from Step G (0.24 g, 0.51
mmol) in CH.sub.2Cl.sub.2 (5 mL) and pyridine (0.8 mL) at
-60.degree. C. was added diethylaminosulfur trifluoride (DAST; 0.27
mL) dropwise under Ar. The solution was stirred at -60.degree. C.
for 0.5 h, at 0.degree. C. overnight and at room temperature for 3
h. The mixture was diluted with CH.sub.2Cl.sub.2 (25 mL) and poured
into saturated aqueous NaHCO.sub.3 (15 mL). The organic layer was
washed with water (10 mL), dried (Na.sub.2SO.sub.4) and evaporated.
The residue was purified on a silica gel column with a solvent
system of hexanes/EtOAc: 5/1 to give the title compound (45 mg) as
a pale yellow foam.
Step I:
2-Amino-7-(2-deoxy-2-fluoro-.beta.-D-ribofuranosyl)-4-chloro-7H-py-
rrolo[2,3-d]-pyrimidine
[1049] A solution of the compound from Step H (40 mg. 0.08 mmol) in
EtOH (2 mL) was stirred with pyridinium p-toluenesulfonate (40 mg,
0.16 mmol) at 60.degree. C. for 3 h. The mixture was then
evaporated and the residue purified on a silica gel column with a
solvent system of hexanes/EtOAc: 1/1 and 1/2 to give the desired
compound (24 mg).
Step J:
2-Amino-7-(2-deoxy-2-fluoro-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-
-d]pyrimidin-4(3H)-one
[1050] A mixture of the compound from Step I (4 mg, 0.08 mmol) in
2N aqueous NaOH (1.2 mL) was stirred at reflux temperature for 1.5
h. The solution was cooled in an ice-bath, neutralized with 2 N
aqueous HCl and evaporated to dryness. The residue was suspended in
MeOH, mixed with silica gel and evaporated. The solid residue was
placed onto a silica gel column (packed in a solvent system of
CH.sub.2Cl.sub.2/MeOH: 10/1) which was eluted with a solvent system
of CH.sub.2Cl.sub.2/MeOH: 10/1. The fractions containing the
product were collected and evaporated to dryness to yield the title
compound (20 mg) as a white solid.
[1051] .sup.1H NMR (CD.sub.3OD): .delta. 3.73, 3.88 (2dd, 2H,
J=12.4, 3.8, 2.6 Hz), 4.01 (m, 1H), 4.47 (ddd, 1H J=16.5, 6.6 Hz),
5.14 (ddd, 1H, J=5.3, 4.7 Hz), 6.19 (dd, 1H, J=17.8, 3.0 Hz), 6.39
(d, 1H, J=3.6 Hz), 6.95 (d, 1H). .sup.9F NMR (CD.sub.3OD): .delta.
-206.53 (dt).
EXAMPLE 95
2-Amino-7-(.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidin-4(3H)-one
[1052] 125
[1053] This compound was prepared following the procedures
described in J. Chem. Soc. Perkin Trans. 1, 2375 (1989).
EXAMPLE 96
2-Amino-7-(2-deoxy-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidin-4(3H-
)-one
[1054] 126
[1055] This compound was prepared following the procedures in
Tetrahedron Lett. 28:5107 (1987).
EXAMPLE 97
6-Amino-1-(2-O-methyl-.beta.-D-ribofuranosyl)-1H-imidazo[4,5-c]pyridin-4(5-
H)-one
[1056] 127
[1057] This compound was prepared in a manner similar to the
preparation of
2-amino-7-(3-deoxy-3-fluoro-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]p-
yrimidin-4(3H)-one (Example 23).
EXAMPLE 98
6-Amino-1-(2-deoxy-.beta.-D-ribofuranosyl)-1H-imidazo[4,5-c]pyridin-4(5H)--
one
[1058] 128
[1059] This compound was prepared following the procedures
described in J. Med. Chem. 26: 286 (1983).
EXAMPLE 99
6-Amino-1-(3-deoxy-3-methyl-.beta.-D-ribofuranosyl)-1H-imidazo[4,5-c]pyrid-
in-4(5H)-one
[1060] 129
[1061] This compound was prepared in a manner similar to the
preparation of
2-amino-7-(3-deoxy-3-fluoro-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]p-
yrimidin-4(3H)-one (Example 23).
EXAMPLE 100
6-Amino-1-(2-deoxy-2-fluoro-.beta.-D-ribofuranosyl)-1H-imidazo[4,5-c]pyrid-
in-4(5H)-one
[1062] 130
[1063] This compound was prepared in a manner similar to the
preparation of
2-amino-7-(3-deoxy-3-fluoro-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]p-
yrimidin-4(3H)-one (Example 23).
EXAMPLE 101
6-Amino-1-(.beta.-D-arabinofuranosyl)-1H-imidazo[4,5-c]pyridin-4(5H)-one
[1064] 131
[1065] A preparation of this compound is given in Eur. Pat. Appln.
43722 A1 (1982).
EXAMPLE 102
2'-O-[2-(N,N-diethylaminooxy)ethyl]-5-methyluridine
[1066] 132
Step A:
5'-O-tert-Butyldiphenylsilyl-2'-O-(2-hydroxyethyl)-5-methyluridine
[1067] In a 2 L stainless steel, unstirred pressure reactor was
added borane in tetrahydrofuran (1.0 M, 2.0 eq, 622 mL). In the
fume hood and with manual stirring, ethylene glycol (350 mL,
excess) was added cautiously at first until the evolution of
hydrogen gas subsided.
5'-O-tert-Butyldiphenylsilyl-O.sup.2-2'-anhydro-5-methyluridine
(149 g, 0.311 mol) and sodium bicarbonate (0.074 g) were added with
manual stirring. The reactor was sealed and heated in an oil bath
until an internal temperature of 160.degree. C. was reached and
then maintained for 16 h (pressure <100 psig). The reaction
vessel was cooled to ambient and opened. The reaction mixture was
concentrated under reduced pressure (10 to 1 mm Hg) in a warm water
bath (40-100.degree. C.) with the more extreme conditions used to
remove the ethylene glycol. The residue was purified by column
chromatography (2 kg silica gel, ethyl acetate:hexanes gradient 1:1
to 4:1). The appropriate fractions were combined, stripped and
dried to product as white crisp foam (84 g), contaminated starting
material (17.4 g) and pure reusable starting material (20 g). TLC
and NMR were consistent with 99% pure product.
[1068] .sup.1H NMR (DMSO-d.sub.6): .delta. 1.05 (s, 9H), 1.45 (s,
3H), 3.5-4.1 (m, 8H), 4.25 (m, 1H), 4.80 (t, 1H), 5.18 (d, 2H),
5.95 (d, 1H), 7.35-7.75 (m, 11H), 11.42 (s, 1H).
Step B:
2'-O-[2-(2-phthalimidoxy)ethyl]-5'-t-butyldiphenylsilyl-5-methylur-
idine
[1069]
5'-O-tert-Butyldiphenylsilyl-2'-O-(2-hydroxyethyl)-5-methyluridine
(20 g, 36.98 mmol) was mixed with triphenylphosphine (11.63 g,
44.36 mmol) and N-hydroxyphthalimide (7.24 g, 44.36 mmol). It was
then dried over P.sub.2O.sub.5 under high vacuum for two days at
40.degree. C. The reaction mixture was flushed with argon and dry
THF (369.8 mL) was added to get a clear solution. Diethyl
azodicarboxylate (6.98 mL, 44.36 mmol) was added dropwise to the
reaction mixture. The rate of addition was maintained such that
resulting deep red coloration is just discharged before adding the
next drop. After the addition was complete, the reaction was
stirred for 4 h. By that time TLC showed the completion of the
reaction (ethyl acetate/hexane, 60:40). The solvent was evaporated
under vacuum. Residue obtained was placed on a flash silica gel
column and eluted with ethyl acetate-hexane (60:40) to give the
title compound as a white foam (21.8 g).
[1070] .sup.1H NMR (DMSO-d.sub.6): .delta. 11.32 (s, 1H), 7.82 (m,
4H), 7.6-7.65 (m, 5H), 7.34-7.46 (m, 6H), 5.90 (d, 1H, J=6 Hz),
5.18 (d, J=5.6 Hz), 4.31 (bs, 2H), 4.25 (m, 1H), 4.09 (t, 1H, J=5.6
Hz), 3.81-3.94 (m, 5H), 1.44 (s, 3H), 1.1 (s, 9H); .sup.13C NMR
(CDCl.sub.3): .delta. 11.8, 19.40, 26.99, 62.62, 68.36, 68.56,
77.64, 83.04, 84.14, 87.50, 110.93, 123.59, 127.86, 129.89, 132.45,
134.59, 134.89, 135.17, 150.50, 163.63, 163.97; MS [FAB] m/z 684
[M-H].sup.-.
Step C:
5'-O-tert-Butyldiphenylsilyl-2'-O-[2-(acetaldoximinooxy)ethyl]-5-m-
ethyluridine
[1071]
2'-O-[2-(2-Phthalimidoxy)ethyl]-5'-t-butyldiphenylsilyl-5-methyluri-
dine (10 g, 14.6 mmol) was dissolved in CH.sub.2Cl.sub.2 (146 mL)
and cooled to -10.degree. C. in an isopropanol-dry ice bath. To
this methylhydrazine (1.03 mL, 14.6 mmol) was added dropwise.
Reaction mixture was stirred at -10.degree. C. to 0.degree. C. for
1 h. A white precipitate formed and was filtered and washed
thoroughly with CH.sub.2Cl.sub.2 (ice cold). The filtrate was
evaporated to dryness. Residue was dissolved in methanol (210 mL)
and acetaldehyde (0.89 mL, 16 mmol) was added and stirred at room
temperature for 12 h. Solvent was removed in vacuo and residue was
purified by silica gel column chromatography using and ethyl
acetate/hexane (6:4) as solvent system to yield the title compound
(4.64 g).
[1072] .sup.1H NMR (DMSO-d.sub.6): .delta. 1.02 (s, 9H), 1.44 (s,
3H), 1.69 (dd, 3H, J=5.6 Hz), 3.66 (m, 1H), 3.76 (m, 2H), 3.94 (m,
2H), 4.05 (s, 2H), 4.15 (m, 1H), 4.22 (m, 1H), 5.18 (d, 1H, J=6.0
Hz), 5.9 (dd, 1H, J=4.4 Hz), 7.36 (m, 1H), 7.40 (m, 7H), 7.63 (m,
5H), 11.38 (s, 1H), .sup.13C NMR (CDCl.sub.3): .delta. 11.84,
15.05, 19.38, 26.97, 63.02, 68.62, 70.26, 71.98, 72.14, 82.72,
84.34, 87.02, 111.07, 127.89, 130.02, 134.98, 135.13, 135.42,
147.85, 150.51, 164.12; HRMS (FAB) Calcd for
C.sub.30H.sub.39N.sub.3O.sub.7SiNa.sup..sym. 604.2455, found
604.2471.
Step D:
5'-O-tert-Butyldiphenylsilyl-2'-O-[2-(N,N-diethylaminooxy)ethyl]-5-
-methyluridine
[1073]
5'-O-tert-Butyldiphenylsilyl-2'-O-[2-(acetaldoximinooxy)ethyl]-5-me-
thyluridine (4.5 g, 7.74 mmol) was dissolved in 1M pyridinium
p-toluenesulfonate (PPTS) in MeOH (77.4 mL). It was then cooled to
10.degree. C. in an ice bath. To this mixture NaBH.sub.3CN (0.97 g,
15.5 mmol) was added and the mixture was stirred at 10.degree. C.
for 10 minutes. Reaction mixture was allowed to come to room
temperature and stirred for 4 h. Solvent was removed in vacuo to
give an oil. Diluted the oil with ethyl acetate (100 mL), washed
with water (75 m]L), 5% NaHCO.sub.3 (75 mL) and brine (75 mL). The
organic phase was dried over anhydrous Na.sub.2SO.sub.4 and
evaporated. Residue obtained was dissolved in 1M PPTS in MeOH (77.4
mL), acetaldehyde (0.48 mL, 8.52 mmol) was added and stirred at
ambient temperature for 10 minutes. Then reaction mixture was
cooled to 10.degree. C. in an ice bath and NaBH.sub.3CN (0.97 g,
15.50 mmol) was added and stirred at 10.degree. C. for 10 minutes.
Reaction mixture was allowed to come to room temperature and
stirred for 4 h. Solvent was removed in vacuo to get an oil. The
oil was dissolved in ethyl acetate (100 mL), washed with water (75
mL), 5% NaHCO.sub.3 (75 mL) and brine (75 mL). The organic phase
was dried over anhydrous Na.sub.2SO.sub.4 and evaporated to
dryness. The residue obtained was purified by silica gel column
chromatography and eluted with CH.sub.2Cl.sub.2/MeOH/NEt.sub.3,
94:5:1 to give title compound (3.55 g) as a white foam.
[1074] .sup.1H NMR (DMSO-d.sub.6): .delta. 0.95 (t, 6H, J=7.2 Hz),
1.03 (s, 9H), 1.43 (s, 3H), 2.58 (q, 4H, J=7.2 Hz), 3.59 (m, 1H),
3.73 (m, 3H), 3.81 (m, 1H), 3.88 (m, 1H), 3.96 (m, 2H), 4.23 (m,
1H), 5.21 (d, 1H, J=5.6 Hz), 5.95 (d, 1H, J=6.4 Hz), 7.43 (m, 7H),
7.76 (m, 4H), 11.39 (s, 1H); .sup.13C NMR (CDCl.sub.3): .delta.
11.84, 19.35, 26.97, 52.27, 63.27, 68.81, 70.27, 72.27, 82.64,
84.47, 86.77, 111.04, 127.87, 130.01, 135.11, 135.41, 141.32,
150.48, 164.04; HRMS (FAB), Calcd for
C.sub.32H.sub.45N.sub.3O.sub.7SiCs.sup..sym., 744.2081, found
744.2067.
Step E: 2'-O-[2-(N,N-diethylaminooxy)ethyl]-5-methyluridine
[1075] A mixture of triethlyamine trihydrogenfluoride (4.39 mL,
26.81 mmol) and triethylamine (1.87 mL, 13.41 mmol) in THF (53.6
mL) was added to
5'-O-tert-butyldiphenylsilyl-2'-O-[2-(N,N-diethylaminooxy)ethyl]-5-met-
hyluridine (3.28 g, 5.36 mmol). The reaction mixture was stirred at
room temperature for 18 h. Solvent was removed in vacuo. The
residue was placed on a silica gel column and eluted with
CH.sub.2Cl.sub.2/MeOH/NEt.s- ub.3, 89:10:1, to yield the title
compound (1.49 g).
[1076] .sup.1H NMR (DMSO-d.sub.6): .delta. 0.97 (t, 6H, J=7.2 Hz),
1.75 (s, 3H), 2.58 (q, 4H, J=7.2 Hz), 3.55 (m, 4H), 3.66 (m, 2H),
3.83 (bs, 1H), 3.95 (t, 1H, J=5.6 Hz), 4.11 (q, 1H, J=4.8 Hz and
5.6 Hz), 5.05 (d, 1H, J=5.6 Hz), 5.87 (d, 1H, J=6.0 Hz), 7.75 (s,
1H), 11.31 (s, 1H); .sup.13C NMR (CDCl.sub.3): .delta. 11.75,
12.27, 52.24, 61.31, 68.86, 70.19, 72.25, 81.49, 85.10, 90.29,
110.60, 137.79, 150.57, 164.37; HRMS (FAB) Calcd for
C.sub.16H.sub.28N.sub.3O.sub.7.sup..sym. 374.1927, found
374.1919.
EXAMPLE 103
1-(2-C-Methyl-.beta.-D-arabinofuranosyl)uracil
[1077] 133
[1078] This compound was prepared following the procedures
described in Chem. Pharm. Bull. 35: 2605 (1987).
EXAMPLE 104
5-Methyl-3'-deoxycytidine
[1079] 134
[1080] This compound was prepared following the procedures
described in Chem. Pharm. Bull. 30: 2223 (1982).
EXAMPLE 105
2-Amino-2'-O-methyladenosine
[1081] 135
[1082] This compound was obtained from commercial sources.
EXAMPLE 106
2'-Deoxy-2'-fluoroadenosine
[1083] 136
[1084] This compound was obtained from commercial sources.
EXAMPLE 107
3'-Deoxy-3'-fluoroadenosine
[1085] 137
[1086] This compound was prepared following the procedures
described in Nucleosides Nucleotides 10: 719 (1991).
EXAMPLE 108
3'-Deoxy-3'-methyladenosine
[1087] 138
[1088] This compound was prepared following the procedures
described in J. Med. Chem. 19: 1265 (1976).
EXAMPLE 109
2-Amino-7-(2-deoxy-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine
[1089] 139
[1090] This compound was prepared following the procedures
described in J. Am. Chem. Soc. 106: 6379 (1984).
EXAMPLE 110
4-Amino-7-(.beta.-D-arabinofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine
[1091] 140
[1092] This compound is described in U.S. Pat. No. 4,439,604, which
is incorporated by reference herein in its entirety.
EXAMPLE 111
4-Amino-1-(3-deoxy-3-fluoro-.beta.-D-ribofuranosyl)-1H-imidazo[4,5-c]pyrid-
ine
[1093] 141
[1094] This compound can be prepared readily by the similar method
described for the preparation of Example 24 except the nucleobase
is 3-deazaadenine.
EXAMPLE 112
4-Amino-7-(.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine
(tubercidin)
[1095] 142
[1096] This compound was obtained from commercial sources.
EXAMPLE 113
4-Amino-1-(3-deoxy-.beta.-D-ribofuranosyl)-1H-imidazo[4,5-c]pyridine
[1097] 143
[1098] This compound is described in Acta Crystallogr., Sect. C:
Cryst. Struct. Commun. C43: 1790 (1987).
EXAMPLE 114
4-Amino-1-(3-deoxy-3-methyl-.beta.-D-ribofuranosyl)-1H-imidazo[4,5-c]pyrid-
ine
[1099] 144
[1100] The procedure described earlier for Example 23 is used to
synthesize this example by reacting the appropriately substituted
3-C-methyl-sugar intermediate with a protected 3-deazaadenine
derivative.
EXAMPLE 115
4-Amino-1-.beta.-D-ribofuranosyl-1H-imidazo[4,5-c]pyridine
[1101] 145
[1102] This compound was obtained from commercial sources.
EXAMPLE 116
9-(2-C-Methyl-.beta.-D-arabinofuranosyl)adenine
[1103] 146
[1104] This compound is prepared from
4-amino-9-(3,5-bis-O-tert-butyldimet-
hylsilyi-.beta.-D-erythro-pentofuran-2-ulosyl)purine (J. Med. Chem.
1992, 35, 2283) by reaction with MeMgBr and deprotection as
described in Example 61.
EXAMPLE 117
4-Amino-7-(2-C-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine
[1105] 147
Step A:
3,5-Bis-O-(2,4-dichlorophenylmethyl)-2-C-ethyl-1-O-methyl-.alpha.--
D-ribofuranose
[1106] To diethyl ether (300 mL) at -78.degree. C. was slowly added
EtMgBr (3.0 M, 16.6 mL) and then dropwise the compound from Step B
of Example 62 (4.80 g, 10.0 mmol) in anhydrous Et.sub.2O (100 mL).
The reaction mixture was stirred at -78.degree. C. for 15 min,
allowed to warm to -15.degree. C. and stirred for another 2 h, and
then poured into a stirred mixture of water (300 mL) and Et.sub.2O
(600 mL). The organic phase was separated, dried (MgSO.sub.4), and
evaporated in vacuo. The crude product was purified on silica gel
using ethyl acetate/hexane (1:2) as eluent. Fractions containing
the product were pooled and evaporated in vacuo to give the desired
product (3.87 g) as a colorless oil.
Step B:
4-Chloro-7-[3,5-bis-O-(2,4-dichlorophenylmethyl)-2-C-ethyl-.beta.--
D-ribofuranosyl]-7H-pyrrolo[2,3-d]pyrimidine
[1107] To a solution of the compound from Step A (1.02 mg, 2.0
mmol) in dichloromethane (40 mL) was added HBr (5.7 M in acetic
acid) (1.75 mL, 10.0 mmol) dropwise at 0.degree. C. The resulting
solution was stirred at rt for 2 h, evaporated in vacuo and
co-evaporated twice from toluene (10 mL). The oily residue was
dissolved in acetonitrile (10 mL) and added to a vigorously stirred
mixture of 4-chloro-1H-pyrrolo[2,3-d]pyrimidine (307 mg, 2.0 mmol),
potassium hydroxide (337 mg, 6.0 mmol) and
tris[2-(2-methoxyethoxy)ethyl]amine (130 mg, 0.4 mmol) in
acetonitrile (10 mL). The resulting mixture was stirred at room
temperature overnight, and then poured into a stirred mixture of
saturated ammonium chloride (100 mL) and ethyl acetate (100 mL).
The organic layer was separated, washed with brine (100 mL), dried
over MgSO.sub.4, filtered and evaporated in vacuo. The crude
product was purified on silica gel using ethyl acetate/hexane (1:2)
as eluent to give the desired product (307 mg) as a colorless
foam.
Step C:
4-Chloro-7-(2-C-ethyl-(.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]py-
rimidine
[1108] To a solution of the compound from Step B (307 mg, 0.45
mmol) in dichloromethane (8 mL) was added boron trichloride (1M in
dichloromethane) (4.50 mL, 4.50 mmol) at -78.degree. C. The mixture
was stirred at -78.degree. C. for 1 h, then at -10.degree. C. for 3
h. The reaction was quenched by addition of
methanol/dichloromethane (1:1) (10 mL), stirred at -15.degree. C.
for 30 min, and neutralized by addition of aqueous ammonium
hydroxide. The mixture was evaporated in vacuo and the resulting
oil purified on silica gel using methanol/dichloromethane (1:9) as
eluent. Fractions containing the product were pooled and evaporated
in vacuo to give the desired product (112 mg) as a colorless
foam.
Step D:
4-Amino-7-(2-C-ethyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyri-
midine
[1109] To the compound from Step C (50 mg, 0.16 mmol) was added
saturated ammonia in methanol (4 mL). The mixture was stirred at
75.degree. C. for 72 h in a closed container, cooled and evaporated
in vacuo. The crude mixture was purified on silica gel using
methanol/dichloromethane (1:9) as eluent. Fractions containing the
product were pooled and evaporated in vacuo to give the desired
product (29 mg) as a colorless powder.
[1110] .sup.1H NMR (200 MHz, DMSO-d.sub.6): .delta. 0.52 (t, 3H),
1.02 (m, 2H), 4.01-3.24 (m, 6H), 5.06 (m, 1H), 6.01 (s, 1H), 6.51
(d, 1H), 6.95 (s br, 2H), 6.70 (d, 1H), 7.99 (s, 1H).
[1111] LC-MS: Found: 295.2 (M+H.sup.+); calc. for
C.sub.13H.sub.18N.sub.4O- .sub.4+H.sup.+: 295.14.
EXAMPLE 118
2-Amino-7-(2-C-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidin-4-
(3H)-one
[1112] 148
Step A:
2-Amino-4-chloro-7-[3,5-bis-O-(2,4-dichlorophenylmethyl)-2-C-methy-
l-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine
[1113] To an ice-cold solution of product from Step C of Example 62
(1.27 g, 2.57 mmol) in CH.sub.2Cl.sub.2 (30 mL) was added HBr (5.7
M in acetic acid; 3 mL) dropwise. The reaction mixture was stirred
at room temperature for 2 h, concentrated in vacuo and
co-evaporated with toluene (2.times.15 mL). The resulting oil was
dissolved in MeCN (15 mL) and added dropwise into a well-stirred
mixture of 2-amino-4-chloro-7H-pyrrolo- [2,3-d]pyrimidine [for
preparation see Heterocycles 35: 825 (1993)] (433 mg, 2.57 mmol),
KOH (85%, powdered) (0.51 g, 7.7 mmol),
tris-[2-(2-methoxyethoxy)ethyl]amine (165 .mu.L, 0.51 mmol) in
acetonitrile (30 mL). The resulting mixture was stirred at rt for 1
h, filtered and evaporated. The residue was purified on a silica
gel column using hexanes/EtOAc, 5/1, 3/1 and 2/1 as eluent to give
the title compound as a colorless foam (0.65 g).
Step B:
2-Amino-4-chloro-7-(2-C-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[-
2,3-d]pyrimidine
[1114] To a solution of the product from Step A (630 mg, 1.0 mmol)
in CH.sub.2Cl.sub.2 (20 mL) at -78.degree. C. was added boron
trichloride (1M in CH.sub.2Cl.sub.2) (10 mL, 10 mmol). The mixture
was stirred at -78.degree. C. for 2 h, then at -20.degree. C. for
2.5 h. The reaction was quenched with CH.sub.2Cl.sub.2/MeOH (1:1)
(10 mL), stirred at -20.degree. C. for 0.5 h, and neutralized at
0.degree. C. with aqueous ammonia. The solid was filtered, washed
with CH.sub.2Cl.sub.2/MeOH (1:1) and the combined filtrate
evaporated in vacuo. The residue was purified on a silica gel
column with CH.sub.2CH.sub.2MeOH, 50/1 and 20/1 as eluent to give
the title compound as a colorless foam (250 mg).
Step C:
2-Amino-7-(2-C-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyr-
imidin-4(3H)-one
[1115] A mixture of product from Step B (90 mg, 0.3 mmol) in
aqueous NaOH (2N, 9 mL) was heated at reflux temperature for 5 h,
then neutralized at 0.degree. C. with 2 N aqueous HCl and
evaporated to dryness. Purification on a silica gel column with
CH.sub.2Cl.sub.2/MeOH, 5/1 as eluent afforded the title compound as
a white solid (70 mg).
[1116] .sup.1H NMR (200 MHz, CD.sub.3OD): .delta. 0.86 (s, 3H),
3.79 (m 1H), 3.90-4.05 (m, 3H), 6.06 (s, 1H), 6.42 (d, J=3.7 Hz,
1H), 7.05 (d, J=3.7 Hz, 1H).
EXAMPLE 119
2-Amino-4-cyclopropylamino-7-(2-C-methyl-.beta.-D-ribofuranosyl)-7H-pyrrol-
o[2,3-d]pyrimidine
[1117] 149
[1118] A solution of
2-amino-4-chloro-7-(2-C-methyl-.beta.-D-ribofuranosyl-
)-7H-pyrrolo[2,3-d]pyrimidine (Example 118, Step B) (21 mg, 0.07
mmol) in cyclopropylamine (0.5 mL) was heated at 70.degree. C. for
two days, then evaporated to an oily residue and purified on a
silica gel column with CH.sub.2Cl.sub.2/MeOH, 20/1, as eluent to
give the title compound as a white solid (17 mg).
[1119] .sup.1H NMR (200 MHz, CD.sub.3CN): .delta. 0.61 (m, 2H),
0.81 (m, 2H), 0.85 (s, 3H), 2.83 (m, 1H), 3.74-3.86 (m, 1H),
3.93-4.03 (m, 2H), 4.11 (d, J=8.9 Hz, 1H), 6.02 (s, 1H), 6.49 (d,
J=3.7 Hz, 1H), 7.00 (d, J=3.7 Hz, 1H).
EXAMPLE 120
3',5'-Bis-[O-(1-oxooctyl)-2'-O-methylcytidine
[1120] 150
[1121] 1,3-Dicyclohexylcarbodiimide (21.48 g, 104 mmol) was
dissolved in anhydrous dichloromethane (100 mL). To the solution
was added octanoic acid (5.49 mL, 34.5 mmol, made anhydrous by
keeping over molecular sieves, 4 A.degree. overnight at room
temperature), and the resulting reaction mixture was stirred under
argon atmosphere for 6 h. The white precipitate which formed was
filtered, and the filtrate was concentrated under reduced pressure.
The residue obtained was dissolved in anhydrous pyridine and added
to N.sup.4-(4,4'-dimethoxytrityl)-2'-O-methylcytidine (0.43 g,
0.77). DMAP (0.09 g, 0.77 mmol) was added and the resulting mixture
was stirred at room temperature under argon atmosphere for 12 h.
The solvent was removed under reduced pressure and the residue
obtained was dissolved in ethyl acetate (100 mL). The organic phase
was washed with aqueous sodium bicarbonate (5%, 50 mL), dried over
anhydrous Na.sub.2SO.sub.4 and concentrated under reduced pressure.
The residue was purified by flash silica gel column chromatography
and eluted with 5% MeOH in dichloromethane. The product obtained
was dissolved in a mixture of acetic acid: MeOH: H.sub.2O (20 mL,
3:6:1). The resulting mixture was heated at 50.degree. C. for 24 h.
The solvent was removed under reduced pressure. The residue
obtained was purified by flash silica gel column chromatography and
eluted with dichloromethane containing 0 to 5% of MeOH to give the
title compound (0.22 g).
[1122] .sup.1H NMR (200 MHz, DMSO-d.sub.6) .delta. 0.83 (m, 6H),
1.23 (br s, 16H), 1.51 (m, 4H), 2.33 (m, 4H), 3.26 (s, 3H), 4.06
(t, J=5.2 Hz, 1H), 4.21 (m, 3H), 5.11 (t, J=5.2 Hz, 1H), 5.75 (d,
J=7.4 Hz, 1H), 5.84 (d, J=4.8 Hz, 1H), 7.26 (br s, 2H), 7.61 (d,
J=7.4 Hz, 1H).
[1123] MS (ES): m/z 510.3 [M+H].sup.+; HRMS (FAB) Calcd for
C.sub.26H.sub.44N.sub.3O.sub.7: 510.3179; found 510.3170.
EXAMPLE 121
4-Amino-1-(.theta.-D-ribofuranosyl)-1H-pyrazolo[3,4-d]pyrimidine
[1124] 151
[1125] This compound was prepared following procedures described in
Nucleic Acids Res., 11: 871-872 (1983).
EXAMPLE 122
2'-C-Methyl-cytidine
[1126] 152
[1127] This compound was prepared following procedures described in
L. Beigelman et al., Carbohyd. Res. 166: 219-232 (1987) or X-Q
Tang, et al., J. Org. Chem. 64: 747-754 (1999).
EXAMPLE 123
4-Amino-7-(2-C-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine--
5-carbonitrile
[1128] 153
[1129] This compound was prepared following procedures described by
Y. Murai et al. in Heterocycles 33: 391-404 (1992).
EXAMPLE 124
4-Amino-7-(2-C-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine--
5-carboxamide
[1130] 154
[1131] This compound was prepared following procedures described by
Y. Murai et al. in Heterocycles 33: 391-404 (1992).
EXAMPLE 125
8-Aminoadenosine
[1132] 155
[1133] This compound was prepared following the procedure described
in M. Ikehara and S. Yamada, Chem. Pharm. Bull., 19: 104
(1971).
EXAMPLE 126
Mass Spectral Characterization of Nucleoside 5'-Triphosphates
[1134] Mass spectra of nucleoside 5'-triphosphates were determined
as described in Example 87. Listed in the following table are the
calculated and experimental masses for the nucleoside
5'-triphosphates prepared according to the procedures of Example
86. The example numbers correspond to the parent nucleoside of the
nucleoside 5'-triphosphate.
1 Example Calculated Found 1 507.0 506.9 2 525.0 524.9 5 537.0
537.0 6 539.0 539.0 7 565.0 565.0 8 547.0 546.9 9 550.0 550.0 10
506.0 505.9 11 536.0 535.9 12 536.0 536.0 13 561.0 560.9 14 550.0
550.0 15 524.0 524.0 16 522.0 521.9 17 547.0 546.9 18 536.0 536.0
20 531.0 530.9 21 522.0 522.0 22 536.0 536.0 23 506.0 506.1 24
524.0 524.0 25 508.0 508.0 26 508.0 508.0 27 552.0 552.0 28 506.0
506.0 29 579.0 578.9 30 582.0 582.0 31 568.0 567.9 32 554.0 553.9
33 540.0 539.9 34 554.0 553.9 35 568.0 567.9 36 541.0 541.0 37
565.0 564.9 38 542.0 541.9 39 554.0 553.9 41 481.0 481.0 42 467.0
467.0 43 485.0 484.8 46 482.0 482.0 47 486.0 485.8 48 482.0 482.0
49 554.0 554.0 51 468.0 468.1 52 521.0 521.0 53 491.0 491.2 55
584.9 585.1 56 521.0 521.2 58 506.0 506.0 61 520.0 519.9 62 520.0
520.0 63 547.0 547.0 64 533.0 533.0 65 549.0 549.0 67 551.0 551.0
68 515.0 514.9 69 520.0 520.1 71 490.0 489.9 89 523.0 522.9 90
521.0 520.9 91 645.1 645.0 94 524.0 523.9 95 522.0 521.8 98 536.0
535.9 99 520.0 520.0 102 613.1 613.0 103 498.0 497.9 104 481.0
481.0 105 536.0 536.2 106 509.0 508.9 108 505.0 505.0 112 506.0
506.1 113 490.0 490.0 117 534.0 534.0 118 536.0 536.0
EXAMPLE 127
[4-Amino-7-(2-C-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]-pyrimidin-
e]-5'-monophosphate
[1135] 156
[1136] To the compound from Step F of Example 62 (14 mg, 0.05 mmol)
(dried by coevaporation with pyridine and several times with
toluene) was added trimethyl phosphate (0.5 mL). The mixture was
stirred overnight in a sealed container. It was then cooled to
0.degree. C. and phosphorous oxychloride (0.0070 mL, 0.075 mmol)
was added via a syringe. The mixture was stirred for 3 h at
0.degree. C., then the reaction was quenched by addtion of
tetraethylammonium bicarbonate (TEAB) (1M) (0.5 mL) and water (5
mL). The reaction mixture was purified and analyzed according to
the procedure described in Example 87.
[1137] Electron spray mass spectrum (ES-MS): Found: 359.2
(M-H.sup.+), calc. for C.sub.12H.sub.17N.sub.4O.sub.7P--H.sup.+:
359.1.
EXAMPLE 128
[4-Amino-7-(2-C-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]-pyrimidin-
e]-5'-diphosphate
[1138] 157
[1139] To the compound from Step F of Example 62 (56 mg, 0.20 mmol)
(dried by coevaporation with pyridine and several times with
toluene) was added trimethyl phosphate (stored over sieves) (1.0
mL). The mixture was stirred overnight in a sealed container. It
was then cooled to 0.degree. C. and phosphorous oxychloride (0.023
mL, 0.25 mmol) was added via a syringe. The mixture was stirred for
2 h at 0.degree. C., then tributylamine (0.238 mL, 1.00 mmol) and
tributylammonium phosphate (generated from phosphoric acid and
tributylamine in pyridine, followed by repeated azeotropic
evaporation with pyridine and acetonitrile) (1.0 mmol in 3.30 mL
acetonitrile) was added. The mixture was stirred for an additional
30 min at 0.degree. C., the sealed vial was then opened and the
reaction quenched by addition of TEAB (1M) (1.0 mL) and water (5
mL). The reaction mixture was purified and analyzed according to
the procedure described in Example 87.
[1140] ES-MS: Found: 439.0 (M-H.sup.+), calc. for
C.sub.12H.sub.18N.sub.4O- .sub.1 OP.sub.2--H.sup.+: 439.04.
EXAMPLE 129
[4-Amino-7-(2-C-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]-pyrimidin-
e]-5'-triphosphate
[1141] 158
[1142] To the compound from Step F of Example 62 (20 mg, 0.07 mmol)
(dried by coevaporation with pyridine and several times with
toluene) was added trimethyl phosphate (stored over sieves) (0.4
mL). The mixture was stirred overnight in a sealed container. It
was then cooled to 0.degree. C. and phosphorous oxychloride (0.0070
mL, 0.075 mmol) was added via syringe. The mixture was stirred for
3 h at 0.degree. C., then tributylamine (0.083 mL, 0.35 mmol),
tributylammonium pyrophosphate (0.35 mmol, 127 mg) and acetonitrile
(stored over sieves) (0.25 mL) were added. The mixture was stirred
for an additional 30 min at 0.degree. C., the sealed vial was then
opened and the reaction quenched by addition of TEAB (1M) (0.5 mL)
and water (5 mL). The reaction mixture was purified and analyzed
according to the procedure described in Example 87.
[1143] ES-MS: Found: 519.0 (M-H.sup.+), calc. for
C.sub.12H.sub.19N.sub.4O- .sub.13P.sub.3-11.sup.+: 519.01.
EXAMPLE 130
7-(2-C-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidin-4(3H)-one
[1144] 159
[1145] To the compound from Step E of Example 62 (59 mg, 0.18 mmol)
was added aqueous sodium hydroxide (1M). The mixture was heated to
reflux for 1 hr, cooled, neutralized with aqueous HCl (2M) and
evaporated in vacuo. The residue was purified on silica gel using
dichloromethane/methanol (4:1) as eluent. Fractions containing the
product were pooled and evaporated in vacuo to give the desired
product (53 mg) as a colorless oil.
[1146] .sup.1H NMR (CD.sub.3CN): .delta. 0.70 (s, 3H), 3.34-4.15
(overlapping m, 7H), 6.16 (s, 1H), 6.57 (d, 3.6 Hz, 1H), 7.37 (d,
3.6 Hz, 1H), 8.83 (s, 1H).
EXAMPLE 131
4-Amino-5-chloro-7-(2-C-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]py-
rimidine
[1147] 160
[1148] To a pre-cooled solution (0.degree. C.) of the compound from
Step F of Example 62 (140 mg, 0.50 mmol) in DMF (2.5 mL) was added
N-chlorosuccinimide (0.075 g, 0.55 mmol) in DMF (0.5 mL) dropwise.
The solution was stirred at rt for 1 h and the reaction quenched by
addition of methanol (4 mL) and evaporated in vacuo. The crude
product was purified on silica gel using methanol/dichloromethane
(1:9) as eluent. Fractions containing the product were pooled and
evaporated in vacuo to give the desired product (55 mg) as a
colorless solid.
[1149] .sup.1H NMR (CD.sub.3CN): .delta. 0.80 (s, 3H), 3.65-4.14
(overlapping m, 7H), 5.97 (s br, 2H), 6.17 (s, 1H), 7.51 (s, 1H),
8.16 (s, 1H).
[1150] ES-MS: Found: 315.0 (M+H.sup.+), calc. for
C.sub.12H.sub.15ClN.sub.- 4O.sub.4+H.sup.+: 315.09.
EXAMPLE 132
4-Amino-5-bromo-7-(2-C-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyr-
imidine
[1151] 161
[1152] To a pre-cooled solution (0.degree. C.) of the compound from
Step F of Example 62 (28 mg, 0.10 mmol) in DMF (0.5 mL) was added
N-bromosuccinimide (0.018 g, 0.10 mmol) in DMF (0.5 mL) dropwise.
The solution was stirred at 0.degree. C. for 20 min, then at rt for
10 min. The reaction was quenched by addition of methanol (4 mL)
and evaporated in vacuo. The crude product was purified on silica
gel using methanol/dichloromethane (1:9) as eluent. Fractions
containing the product were pooled and evaporated in vacuo to give
the desired product (13.0 mg) as a colorless solid.
[1153] .sup.1H NMR (CD.sub.3CN): .delta. 0.69 (s, 3H), 3.46-4.00
(overlapping m, 7H), 5.83 (s br, 2H), 6.06 (s, 1H), 7.45 (s, 114),
8.05 (s, 1H).
[1154] ES-MS: Found: 359.1 (M+H.sup.+), calc. for
C.sub.12H.sub.15BrN.sub.- 4OO.sub.4+H.sup.+: 359.04.
EXAMPLE 133
2-Amino-7-(2-C-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine
[1155] 162
[1156] A mixture of
2-amino-4-chloro-7-(2-C-methyl-.beta.-D-ribofuranosyl)-
-7H-pyrrolo[2,3-d]pyrimidine (Example 118, Step B) (20 mg, 0.07
mmol) in EtOH (1.0 mL), pyridine (0.1 mL) and 10% Pd/C (6 mg) under
H.sub.2 (atmospheric pressure) was stirred overnight at room
temperature. The mixture was filtered through a Celite pad which
was thorougly washed with EtOH. The combined filtrate was
evaporated and purified on a silica gel column with
CH.sub.2Cl.sub.2/MeOH, 20/1 and 10/1, as eluent to give the title
compound as a white solid (16 mg).
[1157] .sup.1H NMR (200 MHz, CD.sub.3OD): .delta. 0.86 (s, 3H,
2'C--Me), 3.82 (dd, J.sub.5'4'=3.6 Hz, J.sub.5',5"=12.7 Hz, 1H,
H-5'), 3.94-4.03 (m, 2H, H-5', H-4'), 4.10 (d, J.sub.3'4'=8.8 Hz,
1H, H-3'), 6.02 (s, 1H, H-1'), 6.41 (d, J.sub.5,6=3.8 Hz, 1H, H-5),
7.39 (d, 1H, H-6), 8.43 (s, 1H, H-4). ES MS: 281.4 (MH.sup.+).
EXAMPLE 134
2-Amino-5-methyl-7-(2-C,2-O-dimethyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,-
3-d]pyrimidin-4(3H)-one
[1158] 163
Step A:
2-Amino-4-chloro-7-[3,5-bis-O-(2,4-dichlorophenylmethyl)-2-C-methy-
l-.beta.-D-ribofuranosyl]-5-methyl-7H-pyrrolo[2,3-d]pyrimidine
[1159] To an ice-cold solution of the product from Step C of
Example 62 (1.57 g, 3.16 mmol) in CH.sub.2Cl.sub.2 (50 mL) was
added HBr (5.7 M in acetic acid; 3.3 mL) dropwise. The reaction
mixture was stirred at 0.degree. C. for 1 h and then at room
temperature for 2 h, concentrated in vacuo and co-evaporated with
toluene (2.times.20 mL). The resulting oil was dissolved in MeCN
(20 mL) and added dropwise to a solution of the sodium salt of
2-amino-4-chloro-5-methyl-1H-pyrrolo[2,3-d]pyrimidine in
acetonitrile [generated in situ from
2-amino-4-chloro-5-methyl-1H-pyrrolo- [2,3-d]pyrimidine [for
preparation, see Liebigs Ann. Chem. 1984: 708-721] (1.13 g, 6.2
mmol) in anhydrous acetonitrile (150 mL), and NaH (60% in mineral
oil, 248 mg, 6.2 mmol), after 2 h of vigorous stirring at rt]. The
combined mixture was stirred at rt for 24 h and then evaporated to
dryness. The residue was suspended in water (100 mL) and extracted
with EtOAc (300+150 mL). The combined extracts were washed with
brine (100 mL), dried over Na.sub.2SO.sub.4, filtered and
evaporated. The crude product was purified on a silica gel column
(5.times.7 cm) using ethyl acetate/hexane (0 to 30% EtOAc in 5%
step gradient) as the eluent. Fractions containing the product were
combined and evaporated in vacuo to give the desired product (0.96
g) as a colorless foam.
Step B:
2-Amino-4-chloro-7-[3,5-bis-O-(2,4-dichlorophenylmethyl)-2-C,2-O-d-
imethyl-.beta.-D-ribofuranosyl]-5-methyl-7H-pyrrolo[2,3-d]pyrimidine
[1160] To an ice-cold mixture of the product from Step A (475 mg,
0.7 mmol) in THF (7 mL) was added NaH (60% in mineral oil, 29 mg)
and stirred at 0.degree. C. for 0.5 h. Then MeI (48 .mu.L) was
added and reaction mixture stirred at rt for 24 h. The reaction was
quenched with MeOH and the mixture evaporated. The crude product
was purified on a silica gel column (5.times.3.5 cm) using
hexane/ethyl acetate (9/1, 7/1, 5/1 and 3/1) as eluent. Fractions
containing the product were combined and evaporated to give the
desired compound (200 mg) as a colorless foam.
Step C:
2-Amino-7-[3,5-bis-O-(2,4-dichlorophenylmethyl)-2-C,2-O-dimethyl-.-
beta.-D-ribofuranosyl]-5-methyl-7H-pyrrolo[2,3-d]pyrimidine-4(3H)-one
[1161] A mixture of the product from Step B (200 mg, 0.3 mmol) in
1,4-dioxane (15 mL) and aqueous NaOH (2N, 15 mL) in a pressure
bottle was heated overnight at 135.degree. C. The mixture was then
cooled to 0.degree. C., neutralized with 2N aqueous HCl and
evaporated to dryness. The crude product was suspended in MeOH,
filtered, and the solid thoroughly washed with MeOH. The combined
filtrate was concentrated, and the residue purified on a silica gel
column (5.times.5 cm) using CH.sub.2Cl.sub.2/MeOH (40/1, 30/1 and
20/1) as eluent to give the desired compound (150 mg) as a
colorless foam.
Step D:
2-Amino-5-methyl-7-(2-C,2-O-dimethyl-.beta.-D-ribofuranosyl)-7H-py-
rrolo[2,3-d]pyrimidin-4(3H)-one
[1162] A mixture of the product from Step C (64 mg, 0.1 mmol) in
MeOH (5 mL) and Et.sub.3N (0.2 mL) and 10% Pd/C (24 mg) was
hydrogenated on a Parr hydrogenator at 50 psi at r.t. for 1.5 days,
then filtered through a Celite pad which was thoroughly washed with
MeOH. The combined filtrate was evaporated and the residue purified
on a silica gel column (3.times.4 cm) with CH.sub.2Cl.sub.2/MeOH
(30/1, 20/1) as eluent to yield
2-amino-5-methyl-7-(5-O-benzyl-2-C,2-O-dimethyl-.beta.-D-ribofuranosyl)-7-
H-pyrrolo[2,3-d]pyrimidin-4(3H)-one. The compound (37 mg) was
further hydrogenated in EtOH (2 mL) with 10% Pd/C and under
atmospheric pressure of hydrogen. After stirring 2 days at r.t.,
the reaction mixture was filtered through Celite, the filtrate
evaporated and the crude product purified on a silica gel column
(1.times.7 cm) with CH.sub.2Cl.sub.2/MeOH (30/1, 20/1 and 10/1) as
eluent to yield the title compound (12 mg) after freeze-drying.
[1163] .sup.1H NMR (200 MHz, CD.sub.3OD): .delta. 0.81 (s, 3H,
2'C--Me), 2.16 (d, J.sub.H-6,C5-Me=1.3 Hz, 3H, C5-Me), 3.41 (s, 3H,
2'-OMe), 3.67 (dd, J.sub.5'4'=3.4 Hz, J.sub.5',5"=12.6 Hz, 1H,
H-5'), 3.81-3.91 (m, 3H, H-5", H-4', H-3'), 6.10 (s, 1H, H-1'),
6.66 (d, 1H, H-6).
[1164] ES MS: 323.3 (M-H).sup.+.
EXAMPLE 135
4-Amino-5-methyl-7-(2-C-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]py-
rimidine
[1165] 164
Step A:
4-Chloro-7-[3,5-bis-O-(2,4-dichlorophenylmethyl)-2-C-methyl-.beta.-
-D-ribofuranosyl]-5-methyl-7H-pyrrolo[2,3-d]pyrimidine
[1166] To an ice-cold solution of the product from Step C of
Example 62 (1.06 g, 2.1 mmol) in CH.sub.2Cl.sub.2 (30 mL) was added
HBr (5.7 M in acetic acid; 2.2 mL) dropwise. The reaction mixture
was stirred at 0.degree. C. for 1 h and then at room temperature
for 2 h, concentrated in vacuo and co-evaporated with toluene
(2.times.15 mL). The resulting oil was dissolved in MeCN (10 mL)
and added dropwise into a solution of the sodium salt of
4-chloro-5-methyl-1H-pyrrolo[2,3-d]pyrimidine in acetonitrile
[generated in situ from 4-chloro-5-methyl-1H-pyrrolo[2,3-d]p-
yrimidine [for preparation, see J. Med. Chem. 33: 1984 (1990)]
(0.62 g, 3.7 mmol) in anhydrous acetonitrile (70 mL), and NaH (60%
in mineral oil, 148 mg, 3.7 mmol), after 2 h of vigorous stirring
at rt]. The combined mixture was stirred at rt for 24 h and then
evaporated to dryness. The residue was suspended in water (100 mL)
and extracted with EtOAc (250+100 mL). The combined extracts were
washed with brine (50 mL), dried over Na.sub.2SO.sub.4, filtered
and evaporated. The crude product was purified on a silica gel
column (5.times.5 cm) using hexane/ethyl acetate (9/1, 5/1, 3/1)
gradient as the eluent. Fractions containing the product were
combined and evaporated in vacuo to give the desired product (0.87
g) as a colorless foam.
Step B:
4-Chloro-5-methyl-7-(2-C-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo-
[2,3-d]pyrimidine
[1167] To a solution of the compound from Step A (0.87 g, 0.9 mmol)
in dichloromethane (30 mL) at -78.degree. C. was added boron
trichloride (1M in dichloromethane, 9.0 mL, 9.0 mmol) dropwise. The
mixture was stirred at -78.degree. C. for 2.5 h, then at
-30.degree. C. to -20.degree. C. for 3 h. The reaction was quenched
by addition of methanol/dichloromethane (1:1) (9 mL) and the
resulting mixture stirred at -15.degree. C. for 30 min., then
neutralized with aqueous ammonia at 0.degree. C. and stirred at rt
for 15 min. The solid was filtered and washed with
CH.sub.2Cl.sub.2/MeOH (1/1, 50 mL). The combined filtrate was
evaporated, and the residue was purified on a silica gel column
(5.times.5 cm) using CH.sub.2Cl.sub.2 and CH.sub.2Cl.sub.2/MeOH
(40/1 and 30/1) gradient as the eluent to furnish the desired
compound (0.22 g) as a colorless foam.
Step C:
4-Amino-5-methyl-7-(2-C-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[-
2,3-d]pyrimidine
[1168] To the compound from Step B (0.2 g, 0.64 mmol) was added
methanolic ammonia (saturated at 0.degree. C.; 40 mL). The mixture
was heated in a stainless steel autoclave at 100.degree. C. for 14
h, then cooled and evaporated in vacuo. The crude mixture was
purified on a silica gel column (5.times.5 cm) with
CH.sub.2Cl.sub.2/MeOH (50/1, 30/1, 20/1) gradient as eluent to give
the title compound as a white solid (0.12 g).
[1169] .sup.1H NMR (DMSO-d.sub.6): .delta. 0.60 (s, 3H, 2'C--Me),
2.26 (s, 3H, 5C--Me), 3.52-3.61 (m, 1H, H-5'), 3.70-3.88 (m, 3H,
H-5", H-4', H-3'), 5.00 (s, 1H, 2'-OH), 4.91-4.99 (m, 3H, 2'-OH,
3'-OH, 5'-OH), 6.04 (s, 1H, H-1'), 6.48 (br s, 2H, NH.sub.2), 7.12
(s, 1H, H-6), 7.94 (s, 1H, H-2). ES MS: 295.2 (MH.sup.+).
EXAMPLE 136
4-Amino-7-(2-C-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine--
S-carboxylic acid
[1170] 165
[1171] The compound of Example 123 (0.035 g, 0.11 mmol) was
dissolved in a mixture of aqueous ammonia (4 mL, 30 wt %) and
saturated methanolic ammonia (2 mL), and a solution of
H.sub.2O.sub.2 in water (2 mL, 35 wt %) was added. The reaction
mixture was stirred at room temperature for 18 h. Solvent was
removed under reduced pressure, and the residue obtained was
purified by HPLC on a reverse phase column (Altech Altima C-18,
10.times.299 mm, A=water, B=acetonitrile, 10 to 60% B in 50 min,
flow 2 mL/min) to yield the title compound (0.015 g, 41%) as a
white solid.
[1172] .sup.1H NMR (CD.sub.3OD): .delta. 0.85 (s, 3H, Me), 3.61 (m,
1H), 3.82 (m, 1H) 3.99-4.86 (m, 2H), 6.26 (s, 1H), 8.10 (s, 2H)
8.22 (s, 1H); .sup.13C NMR (CD.sub.3OD): 20.13, 61.37, 73.79,
80.42, 84.01, 93.00, 102.66, 112.07, 130.07, 151.40, 152.74,
159.12, 169.30.
[1173] HRMS (FAB) Calcd for C.sub.13H.sub.17N.sub.4O.sub.6.sup.+
325.1148, found 325.1143.
EXAMPLE 137
4-Amino-7-(2-C-vinyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine
[1174] 166
Step A:
3,5-Bis-O-(2,4-dichlorophenylmethyl)-2-C-vinyl-1-O-methyl-.alpha.--
D-ribofuranose
[1175] Cerium chloride heptahydrate (50 g, 134.2 mmol) was finely
crushed in a pre-heated mortar and transferred to a round-bottom
flask equipped with a mechanical stirrer. The flask was heated
under high vacuum overnight at 160.degree. C. The vacuum was
released under argon and the flask was cooled to room temperature.
Anhydrous THF (300 mL) was cannulated into the flask. The resulting
suspension was stirred at room temperature for 4 h and then cooled
to -78.degree. C. Vinylmagnesium bromide (1M in THF, 120 mL, 120
mmol) was added and stirring continued at -78.degree. C. for 2 h.
To this suspension was added a solution of
3,5-bis-O-(2,4-dichlorophenylmethyl)-1-O-methyl-.alpha.-D-erythro-pentofu-
ranose-2-ulose (14 g, 30 mmol) [from Example 2, Step B] in
anhydrous THF (100 mL), dropwise with constant stirring. The
reaction was stirred at -78.degree. C. for 4 h. The reaction was
quenched with saturated ammonium chloride solution and allowed to
come to room temperature. The mixture was filtered through a celite
pad and the residue washed with Et.sub.2O (2.times.500 mL). The
organic layer was separated and the aqueous layer extracted with
Et.sub.2O (2.times.200 mL). The combined organic layers were dried
over anhydrous Na.sub.2SO.sub.4 and concentrated to a viscous
yellow oil. The oil was purified by flash chromatography
(SiO.sub.2, 10% EtOAc in hexanes). The title compound (6.7 g, 13.2
mmol) was obtained as a pale yellow oil.
Step B:
4-Chloro-7-[3,5-bis-O-(2,4-dichlorophenylmethyl)-2-C-vinyl-.beta.--
D-ribofuranosyl]-7H-pyrrolo[2,3-d]pyrimidine
[1176] To a solution of the compound from Step A (6.4 g, 12.6 mmol)
in anhydrous dichloromethane (150 mL) at -20.degree. C. was added
HBr (30% solution in AcOH, 20 mL, 75.6 mmol) dropwise. The
resulting solution was stirred between -10.degree. C. and 0.degree.
C. for 4 h, evaporated in vacuo and co-evaporated with anhydrous
toluene (3.times.40 mL). The oily residue was dissolved in
anhydrous acetonitrile (100 mL) and added to a solution of the
sodium salt of 4-chloro-1H-pyrrolo[2,3-d]pyrimidine (5.8 g, 37.8
mmol) in acetonitrile (generated in situ as described in Example
62) at -20.degree. C. The resulting mixture was allowed to come to
room temperature and stirred at room temperature for 24 h. The
mixture was then evaporated to dryness, taken up in water and
extracted with EtOAc (2.times.300 mL). The combined extracts were
dried over Na.sub.2SO.sub.4, filtered and evaporated. The crude
mixture was purified by flash chromatography (SiO.sub.2, 10% EtOAc
in hexanes) and the title compound (1.75 g) isolated as a white
foam.
Step C:
4-Amino-7-[3,5-bis-O-(2,4-dichlorophenylmethyl)-2-C-vinyl-.beta.-D-
-ribofuranosyl]-7H-pyrrolo[2,3-d]pyrimidine
[1177] The compound from Step B (80, mg) was dissolved in the
minimum amount of 1,4-dioxane and placed in a stainless steel bomb.
The bomb was cooled to -78.degree. C. and liquid ammonia was added.
The bomb was sealed and heated at 90.degree. C. for 24 h. The
ammonia was allowed to evaporate and the residue concentrated to a
white solid which was used in the next step without further
purification.
Step D:
4-Amino-7-(2-C-vinyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyri-
midine
[1178] To a solution of the compound from Step C (60 mg) in
dichloromethane at -78.degree. C. was added boron trichloride (1M
in dichloromethane) dropwise. The mixture was stirred at
-78.degree. C. for 2.5 h, then at -30.degree. C. to -20.degree. C.
for 3 h. The reaction was quenched by addition of
methanol/dichloromethane (1:1) and the resulting mixture stirred at
-15.degree. C. for 0.5 h, then neutralized with aqueous ammonia at
0.degree. C. and stirred at room temperature for 15 min. The solid
was filtered and washed with methanol/dichloromethane (1:1). The
combined filtrate was evaporated and the residue purified by flash
chromatography (SiO.sub.2, 10% methanol in EtOAc containing 0.1%
triethylamine). The fractions containing the product were
evaporated to give the title compound as a white solid (10 mg).
[1179] .sup.1H NMR (DMSO-d.sub.6): .delta. 3.6 (m, 1H, H-5'), 3.8
(m, 1H, H-5"), 3.9 (m d, 1-H, H-4'), 4.3 (t, 1H, H-3'), 4.8-5.3 (m,
6H, CH.dbd.CH.sub.2, 2'-OH, 3'-OH, 5'-OH) 6.12 (s, 1H, H-1'), 6.59
(d, 1H, H-5), 7.1 (br s, 1H, NH.sub.2), 7.43 (d, 1H, H-6), 8.01 (s,
1H, H-2).
[1180] ES-MS: Found: 291.1 (M-H.sup.-); calc. for
C.sub.13H.sub.16N.sub.4O- .sub.4-H.sup.-: 291.2.
EXAMPLE 138
4-Amino-7-(2-C-hydroxymethyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyri-
midine
[1181] 167
Step A:
4-Chloro-7-[3,5-bis-O-(2,4-dichlorophenylmethyl)-2-C-hydroxymethyl-
-.beta.-D-ribofuranosyl-7H-pyrrolo[2,3-d]pyrimidine
[1182] To a solution of the compound from Example 137, Step B (300
mg, 0.48 mmol) in 1,4-dioxane (5 mL) were added
N-methylmorpholine-N-oxide (300 mg, 2.56 mmol) and osmium tetroxide
(4% solution in water, 0.3 mL). The mixture was stirred in the dark
for 14 h. The precipitate was removed by filtration through a
celite plug, diluted with water (3.times.), and extracted with
EtOAc. The EtOAc layer was dried over Na.sub.2SO.sub.4 and
concentrated in vacuo. The oily residue was taken up in
dichloromethane (5 mL) and stirred over NaIO.sub.4 on silica gel (3
g, 10% NaIO.sub.4) for 12 h. The silica gel was removed by
filtration and the residue was evaporated and taken up in absolute
ethanol (5 mL). The solution was cooled in an ice bath and sodium
borohydride (300 mg, 8 mmol) was added in small portions. The
resulting mixture was stirred at room temperature for 4 h and then
diluted with EtOAc. The organic layer was washed with water
(2.times.20 mL), brine (20 mL) and dried over Na.sub.2SO.sub.4. The
solvent was evaporated and the residue purified by flash
chromatography (SiO.sub.2, 2:1 hexanes/EtOAc) to give the title
compound (160 mg, 0.25 mmol) as white flakes.
Step B:
4-Amino-7-[3,5-bis-O-(2,4-dichlorophenylmethyl)-2-C-hydroxymethyl--
.beta.-D-ribofuranosyl]-7H-pyrrolo[2,3-d]pyrimidine
[1183] The compound from Step A (150 mg, 0.23 mmol) was dissolved
in the minimum amount of 1,4-dioxane (10 mL) and placed in a
stainless steel bomb. The bomb was cooled to -78.degree. C. and
liquid ammonia was added. The bomb was sealed and heated at
90.degree. C. for 24 h. The ammonia was allowed to evaporate and
the residue concentrated to a white solid which was used in the
next step without further purification.
Step C:
4-Amino-7-(2-C-hydroxymethyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,-
3-d]pyrimidine
[1184] The compound from Step B (120 mg, 0.2 mmol) was dissolved in
1:1 methanol/dichloromethane, 10% Pd--C was added, and the
suspension stirred under an H.sub.2 atmosphere for 12 h. The
catalyst was removed by filtration through a celite pad and washed
with copious amounts of methanol. The combined filtrate was
evaporated in vacuo and the residue was purified by flash
chromatography (SiO.sub.2, 10% methanol in EtOAc containing 0.1%
triethylamine) to give the title compound (50 mg) as a white
powder.
[1185] .sup.1H NMR (CD.sub.3OD): .delta. 3.12 (d, 1H, CH.sub.2'),
3.33 (d, 1H, CH.sub.2"), 3.82 (m, 1H, H-5'), 3.99-4.1 (m, 2H, H-4',
H-5"), 4.3 (d, 1H, H-3'), 6.2 (s, 1H, H-1'), 6.58 (d, 1H, H-5),
7.45 (d, 1H, H-6), 8.05 (s, 1H, H-2).
[1186] LC-MS: Found: 297.2 (M+H.sup.+); calc. for
C.sub.12H.sub.16N.sub.4O- .sub.5+H.sup.+: 297.3.
EXAMPLE 139
4-Amino-7-(2-C-fluoromethyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrim-
idine
[1187] 168
Step A:
4-Chloro-7-[3,5-bis-O-(2,4-dichlorophenylmethyl)-2-C-fluoromethyl--
.beta.-D-ribofuranosyl]-7H-pyrrolo[2,3-d]pyrimidine
[1188] To a solution of the compound from Example 138, Step A (63
mg, 0.1 mmol) in anhydrous dichloromethane (5 mL) under argon, were
added 4-dimethylaminopyridine (DMAP) (2 mg, 0.015 mmol) and
triethylamine (62 .mu.L, 0.45 mmol). The solution was cooled in an
ice bath and p-toluenesulfonyl chloride (30 mg, 0.15 mmol) was
added. The reaction was stirred at room temperature overnight,
washed with NaHCO.sub.3 (2.times.10 mL), water (10 mL), brine (10
mL), dried over Na.sub.2SO.sub.4 and concentrated to a pink solid
in vacuo. The solid was dissolved in anhydrous THF (5 mL) and
cooled in an icebath. Tetrabutylammonium fluoride (1M solution in
THF, 1 mL, 1 mmol) was added and the mixture stirred at room
temperature for 4 h. The solvent was removed in vacuo, the residue
taken up in dichloromethane, and washed with NaHCO.sub.3
(2.times.10 mL), water (10 mL) and brine (10 mL). The
dichloromethane layer was dried over anhydrous Na.sub.2SO.sub.4,
concentrated in vacuo, and purified by flash chromatography
(SiO.sub.2, 2:1 hexanes/EtOAc) to afford the title compound (20 mg)
as a white solid.
Step B:
4-Amino-7-(3,5-bis-O-(2,4-dichlorophenylmethyl)-2-C-fluoromethyl-.-
beta.-D-ribofuranosyl]-7H-pyrrolo[2,3-d]pyrimidine
[1189] The compound from Step A (18 mg, 0.03 mmol) was dissolved in
the minimum amount of 1,4-dioxane and placed in a stainless steel
bomb. The bomb was cooled to -78.degree. C. and liquid ammonia was
added. The bomb was sealed and heated at 90.degree. C. for 24 h.
The ammonia was allowed to evaporate and the residue concentrated
to a white solid which was used in the next step without further
purification.
Step C:
4-Amino-7-(2-C-fluoromethyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-
-d]pyrimidine
[1190] The compound from Step B (16 mg) was dissolved in 1:1
methanol/dichloromethane, 10% Pd--C was added, and the suspension
stirred under an H.sub.2 atmosphere for 12 h. The catalyst was
removed by filtration through a celite pad and washed with copious
amounts of methanol. The combined filtrate was evaporated in vacuo
and the residue was purified by flash chromatography (SiO.sub.2,
10% methanol in EtOAc containing 0.1% triethylamine) to give the
title compound (8 mg) as a white powder.
[1191] .sup.1H NMR (DMSO-d.sub.6): .delta. 3.6-3.7 (m, 1H, H-5'),
3.8-4.3 (m, 5H, H-5", H-4', H-3', CH.sub.2) 5.12 (t, 1H, 5'-OH),
5.35 (d, 1H, 3'-OH), 5.48 (s, 1H, 2'-OH), 6.21 (s, 1H, H-1'), 6.52
(d, 1H, H-5), 6.98 (br s, 2H, NH.sub.2), 7.44 (d, 1H, H-6), 8.02
(s, 1H, H-2).
[1192] .sup.19F NMR (DMSO-d.sub.6): .delta. -230.2 (t).
[1193] ES-MS: Found: 299.1 (M+H.sup.+), calc. for
C.sub.12H.sub.15FN.sub.4- O.sub.4+H.sup.+: 299.27.
EXAMPLES 140 AND 141
4-Amino-7-(3-deoxy-2-C-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyr-
imidine and
4-amino-7-(3-deoxy-2-C-methyl-.beta.-D-arabinofuranosyl)-7H-py-
rrolo[2,3-d]-pyrimidine
[1194] 169
Step A:
7-[2,5-Bis-O-(tert-butyldimethylsilyl)-.beta.-D-ribofuranosyl]-7H--
pyrrolo[2,3-d]pyrimidine and
7-[3,5-Bis-O-(tert-butyldimethylsilyl)-D-D-ri-
bofuranosyl]-7H-pyrrolo[2,3-d]pyrimidine
[1195] To a stirred solution of tubercidin (5.0 g, 18.7 m-mol) in a
mixture of pyridine (7.5 mL) and DMF (18.5 mL) was added silver
nitrate (6.36 g, 38.8 mmol). This mixture was stirred at room
temperature for 2 h. It was cooled in an ice bath and THF (37.4 mL)
and tert-butyldimethylsilyl chloride (5.6 g, 37 mmol) was added and
the mixture was stirred at room temperature for 2 h. The mixture
was then filtered through a pad of celite and washed with THF. The
filtrate and washings were diluted with ether containing a small
amount of chloroform. The organic layer was washed successively
with sodium bicarbonate and water (3.times.50 mL), dried over
anhydrous sodium sulfate and concentrated. The pyridine was removed
by coevaporation with toluene and the residue was purified by flash
chromatography on silica gel using 5-7% MeOH in CH.sub.2Cl.sub.2 as
the eluent; yield 3.0 g.
Step B:
7-[2,5-Bis-O-(tert-butyldimethylsilyl)-.beta.-D-ribofuranosyl)]-4--
[di-(4-methoxyphenyl)phenylmethyl]amino-7H-pyrrolo[2,3-d]pyrimidine
and
7-[3,5-bis-O-(tert-butyldimethylsilyl)-.beta.-D-ribofuranosyl]-4-[di-(4-m-
ethoxyphenyl)phenylmethyl]amino-7H-pyrrolo[2,3-d]pyrimidine
[1196] To a solution of mixture of the compounds from Step A (3.0
g, 6.0 mmol) in anhydrous pyridine (30 mL) was added
4,4'-dimethoxytrityl chloride (2.8 g, 8.2 mmol) and the reaction
mixture was stirred at room temperature overnight. The mixture was
then triturated with aqueous pyridine and extracted with ether. The
organic layer was washed with water, dried over anhydrous sodium
sulfate and concentrated to a yellow foam (5.6 g). The residue was
purified by flash chromatography over silica gel using 20-25% EtOAc
in hexanes as the eluent. The appropriate fractions were collected
and concentrated to furnish
2',5'-O-bis-O-(tert-butyldimethylsilyl)- and
3',5'-bis-O-(tert-butyldimet- hylsilyl) protected nucleosides as
colorless foams (2.2 g and 1.0 g, respectively).
Step C:
7-[2,5-Bis-O-(tert-butyldimethylsilyl)-3-O-tosyl-.beta.-D-ribofura-
nosyl)]-4-[di-(4-methoxyphenyl)phenylmethyl]amino-7H-pyrrolo[2,3-d]pyrimid-
ine
[1197] To an ice-cooled solution of
2',5'-bis-O-(tert-butyldimethylsilyl)-- protected nucleoside from
Step B (2.0 g, 2.5 mmol) in pyridine (22 mL) was added
p-toluenesulfonyl chloride (1.9 g, 9.8 mmol). The reaction mixture
was stirred at room temperature for four days. It was then
triturated with aqueous pyridine (50%, 10 mL) and extracted with
ether (3.times.50 mL) containing a small amount of CH.sub.2Cl.sub.2
(10 mL). The organic layer was washed with sodium bicarbonate and
water (3.times.30 mL). The organic layer was dried over anhydrous
Na.sub.2SO.sub.4 and concentrated. Pyridine was removed by
co-evaporation with toluene (3.times.25 mL). The residual oil was
filtered through a pad of silica gel using hexane:ethyl acetate
(70:30) as eluent; yield 1.4 g.
Step D:
4-[di-(4-methoxyphenyl)phenylmethyl]amino-7-[3-O-tosyl-.beta.-D-ri-
bofuranosyl-7H-pyrrolo[2,3-d]pyrimidine
[1198] A solution of the compound from Step C (1.0 g, 1.1 mmol) and
THF (10 mL) was stirred with tetrabutylammonium fluoride (1M
solution in THF, 2.5 mL) for 0.5 h. The mixture was cooled and
diluted with ether (50 mL). The solution was washed with water
(3.times.50 mL), dried over anhydrous Na.sub.2SO.sub.4, and
concentrated to an oil. The residue was purified by passing through
a pad of silica gel using hexane:ethyl acetate (1:1) as eluent;
yield 780 mg.
Step E:
4-Amino-7-(3-deoxy-2-C-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2-
,3-dl-pyrimidine and
4-amino-7-(3-deoxy-2-C-methyl-.beta.-D-arabinofuranos-
yl)-7H-pyrrolo-[2,3-d]pyrimidine
[1199] A solution of CH.sub.3MgI (3.0 M solution in ether, 3.0 mL)
in anhydrous toluene (3.75 mL) was cooled in an ice bath. To this
was added a solution of the compound from Step D (500 mg, 0.8 mmol)
in anhydrous toluene (3.7 mL). The resulting mixture was stirred at
room temperature for 3.5 h. It was cooled and treated with aqueous
NH.sub.4Cl solution and extracted with ether (50 mL containing 10
mL of CH.sub.2Cl.sub.2). The organic layer was separated and washed
with brine (2.times.30 mL) and water (2.times.25 mL), dried over
anhydrous Na.sub.2SO.sub.4 and concentrated to an oil which was
purified by flash chromatography on silica gel using 4% MeOH in
CH.sub.2Cl.sub.2 to furnish the 2-C-.alpha.-methyl compound (149
mg) and the 2-C-.beta.-methyl compound (34 mg). These derivatives
were separately treated with 80% acetic acid and the reaction
mixture stirred at room temperature for 2.5 h. The acetic acid was
removed by repeated co-evaporation with ethanol and toluene. The
residue was partitioned between chloroform and water. The aqueous
layer was washed with chloroform and concentrated. The evaporated
residue was purified on silica gel using 5-10% MeOH in
CH.sub.2Cl.sub.2 as the eluent to furnish the desired compounds as
white solids.
4-Amino-7-(3-deoxy-2-C-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyr-
imidine (9.0 mg):
[1200] .sup.1H-NMR (DMSO-d.sub.6): .delta. 0.74 (s, 3H, CH.sub.3),
1.77 (dd, 1H, H-3'), 2.08 (t, 1H, H-3"), 3.59 (m, 1H, H-5'), 3.73
(m, 1H, H-5"), 4.15 (m, 1H, H-4'), 5.02 (t, 1H, OH-5'), 5.33 (s,
1H, OH-2'), 6.00 (s, 1H, H-1'), 6.54 (d, 1H, H-7), 6.95 (br s, 2H,
NH.sub.2), 7.47 (d, 1H, H-8), 8.00 (s, 1H, H-2); ES-MS: 263.1
[M-H].
4-Amino-7-(3-deoxy-2-C-methyl-.beta.-D-arabinofuranosyl)-7H-pyrrolo[2,3-d]-
pyrimidine (15 mg)
[1201] .sup.1H NMR (DMSO-d.sub.6): .delta. 1.23 (s, 3H, CH.sub.3),
2.08 (ddd, 2H, H-3' and 3"), 3.57 (m, 2H, H-5' and 5"), 4.06 (m,
1H, H-4), 5.10 (s, 1H, OH-2'), 5.24 (t, 1H, OH-5'), 6.01 (s, 1H,
H-1'), 6.49 (d, 1H, H-7), 6.89 (br s, 2H, NH.sub.2), 7.35 (d, 1H,
H-8), 8.01 (s, 1H,H-2).
[1202] ES-MS: 265.2[M+H].
EXAMPLE 142
4-Amino-7-(2,4-C-dimethyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimid-
ine
[1203] 170
Step A: 5-Deoxy-1,2-O-isopropylidene-D-xylofuranose
[1204] 1,2-O-Isopropylidene-D-xylofuranose (38.4 g, 0.2 mol),
4-dimethylaminopyridine (5 g), triethylamine (55.7 mL, 0.4 mol)
were dissolved in dichloromethane (300 mL). p-Toluenesulfonyl
chloride (38.13 g, 0.2 mol) was added and the reaction mixture was
stirred at room temperature for 2 h. The reaction mixture was then
poured into saturated aqueous sodium bicarbonate (500 mL) and the
two layers were separated. The organic layer was washed with
aqueous citric acid solution (20%, 200 mL), dried
(Na.sub.2SO.sub.4) and evaporated to give a solid (70.0 g). The
solid was dissolved in dry THF (300 mL) and LiAlH.sub.4 (16.0 g,
0.42 mol) was added in portions over 30 min. The mixture was
stirred at room temperature for 15 h. Ethyl acetate (100 mL) was
added dropwise over 30 min and the mixture was filtered through a
silica gel bed. The filtrate was concentrated and the resulting oil
was chromatographed on silica gel (EtOAc/hexane 1/4) to afford the
product as a solid (32.5 g).
Step B:
3,5-Bis-O-(2,4-dichlorophenylmethyl)-1-O-methyl-4-methyl-.alpha.-D-
-ribofuranose
[1205] Chromium oxide (50 g, 0.5 mol), acetic anhydride (50 m]L,
0.53 mol) and pyridine (100 mL, 1.24 mol) were added to
dichloromethane (1 L) in an ice water bath and the mixture was
stirred for 15 min. 5-Deoxy-1,2-O-isopropylidene-D-xylofuranose (32
g, 0.18 mol) in dichloromethane (200 mL) was added, and the mixture
was stirred at the same temperature for 30 min. The reaction
solution was diluted with ethyl acetate (1 L) and filtered through
a silica gel bed. The filtrate was concentrated to give a yellow
oil. The oil was dissolved in 1,4-dioxane (1 L) and formaldehyde
(37%, 200 mL). The solution was cooled to 0.degree. C. and solid
KOH (50 g) was added. The mixture was stirred at room temperature
overnight and was then extracted with ethyl acetate (6.times.200
mL). After concentration, the residue was chromatographed on silica
gel (EtOAc) to afford the product as an oil (1.5 g). The oil was
dissolved in 1-methyl-2-pyrrolidinone (20 mL) and
2,4-dichlorophenylmethy- l chloride (4 g, 20.5 mmol) and NaH (60%,
0.8 g) were added. The mixture was stirred overnight and diluted
with toluene (100 mL). The mixture was then washed with saturated
aqueous sodium bicarbonate (3.times.50 mL), dried
(Na.sub.2SO.sub.4) and evaporated. The residue was dissolved in
methanol (50 mL) and HCl in dioxane (4 M, 2 mL) was added. The
solution was stirred overnight and evaporated. The residue was
chromatographed on silica gel (EtOAc/hexane: 1/4) to afford the
desired product as an oil (2.01 g).
Step C:
3,5-Bis-O-(2,4-dichlorophenylmethyl)-2,4-di-C-methyl-1-O-methyl-.a-
lpha.-D-ribofuranose
[1206] The product (2.0 g, 4.0 mmol) from Step B and Dess-Martin
periodinane (2.0 g) in dichloromethane (30 mL) were stirred
overnight at room temperature and was then concentrated under
reduced pressure. The residue was triturated with ether ether (50
mL) and filtered. The filtrate was washed with a solution of
Na.sub.2S.sub.2O.sub.3.5H.sub.2O (2.5 g) in saturated aqueous
sodium bicarbonate solution (50 mL), dried (MgSO.sub.4), filtered
and evaporated. The residue was dissolved in anhydrous Et.sub.2O
(20 m]L) and was added dropwise to a solution of MeMgBr in
Et.sub.2O (3 M, 10 mL) at -78-C. The reaction mixture was allowed
to warm to -30.degree. C. and stirred at -30.degree. C. to
-15.degree. C. for 5 h, then poured into saturated aqueous ammonium
chloride (50 mL). The two layers were separated and the organic
layer was dried (MgSO.sub.4), filtered and concentrated. The
residue was chromatographed on silica gel (EtOAc/hexane: 1/9) to
afford the title compound as a syrup (1.40 g).
Step D:
4-Chloro-7-[3,5-bis-O-(2,4-dichlorophenylmethyl)-2,4-di-C-methyl-(-
3-D-ribofuranosyl]-7H-pyrrolo[2,3-d]pyrimidine
[1207] To the compound from Step C (0.70 g, 1.3 mmol) was added HBr
(5.7 M in acetic acid, 2 mL). The resulting solution was stirred at
room temperature for 1 h, evaporated in vacuo and co-evaporated
with anhydrous toluene (3.times.10 mL).
4-Chloro-1H-pyrrolo[2,3-d]pyrimidine (0.5 g, 3.3 mmol) and powdered
KOH (85%, 150 mg, 2.3 mmol) were stirred in
1-methyl-2-pyrrolidinone (5 mL) for 30 min and the mixture was
co-evaporated with toluene (10 mL). The resulting solution was
poured into the above bromo sugar residue and the mixture was
stirred overnight. The mixture was diluted with toluene (50 mL),
washed with water (3.times.50 mL) and concentrated under reduced
pressure. The residue was chromatographed on silica gel eluting
with (EtOAc/Hexane 15/85) to afford a solid (270 mg).
Step E:
4-Amino-7-(2,4-di-C-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3--
d]pyrimidine
[1208] The compound from Step D (270 mg) was dissolved in dioxane
(2 mL) and liquid ammonia (20 g) was added in a stainless steel
autoclave. The mixture was heated at 100.degree. C. for 15 h, then
cooled and evaporated. The residue was chromatographed on silica
gel (EtOAc) to afford a solid (200 mg). The solid (150 mg) and Pd/C
(10% 150 mg) in methanol (20 mL) were shaken under H.sub.2 (30 psi)
for 3 h, filtered and evaporated. The residue was chromatographed
on silica gel (MeOH/CH.sub.2Cl.sub.2: 1/9) to afford the desired
product as a solid (35 mg).
[1209] .sup.1H NMR (DMSO-d.sub.6): .delta. 0.65 (s, 3H), 1.18 (s,
3H), 3.43 (m, 2H), 4.06 (d, 1H, J=6.3 Hz), 4.87 (s, 1H), 5.26 (br,
1H), 5.08 (d, 1H, J=6.3 Hz), 5.25 (t, 1H, J=3.0 Hz), 6.17 (s, 1H),
6.54 (d, 1H, J=3.5 Hz), 6.97 (s, br, 2H), 7.54 (d, 1H, J=3.4 Hz),
8.02 (s, 1H).
[1210] .sup.13C NMR (DMSO-d.sub.6): .delta. 18.19, 21.32, 65.38,
73.00, 79.33, 84.80, 90.66, 99.09, 102.41, 121.90, 149.58, 151.48,
157.38. LC-MS: Found: 295.1 (M+H.sup.+); calculated for
C.sub.13H.sub.18N.sub.4O.- sub.4+H.sup.+: 295.1
EXAMPLE 143
4-Amino-7-(3-deoxy-3-fluoro-2-C-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[-
2,3-d]pyrimidine
[1211] 171
Step A:
3-Deoxy-3-fluoro-1-O-methyl-5-O-toluoyl-.alpha.-D-ribofuranose
[1212] 1,2-O-Isopropylidene-D-xylofuranose (9.0 g, 50 mmol) and
p-toluoyl chloride (7.0 mL, 50 mmol) in pyridine (50 mL) were
stirred for 30 min. Water (10 mL) was added and the mixture was
concentrated under reduced pressure. The residue was dissolved in
toluene (500 mL) and the solution was washed with water (200 mL)
and saturated aqueous sodium bicarbonate (200 mL). The two layers
were separated and the organic layer was evaporated. The residue
was dissolved in methanol (100 mL) and HCl in dioxane (4 M, 10 mL)
was added. The mixture was stirred at room temperature overnight
and was then evaporated under reduced pressure. The resulting oil
was chromatographed on silica gel (EtOAc/hexane: 1/1) to afford an
oil (10.1 g). The oil was dissolved in dichloromethane (100 mL) and
diethylaminosulfur trifluoride (DAST) (5.7 mL) was added. The
mixture was stirred overnight and was then poured into saturated
aqueous sodium bicarbonate solution (100 mL). The mixture was
extracted with toluene (2.times.50 mL) and the combined organic
layers were concentrated. The residue was chromatographed on silica
gel (EtOAc/hexane: 15/85) to afford the title compound as an oil
(1.50 g).
Step B:
3-Deoxy-3-fluoro-2-C-methyl-1-O-methyl-5-O-toluoyl-.alpha.-D-ribof-
uranose
[1213] The product from Step A (1.0 g, 3.5 mmol) and Dess-Martin
periodinane (2.5 g) in dichloromethane (20 mL) were stirred
overnight at room temperature and was then concentrated under
reduced pressure. The residue was triturated with diethyl ether (50
mL) and filtered. The filtrate was washed with a solution of
Na.sub.2S.sub.2O.sub.3.5H.sub.2O (12.5 g) in saturated aqueous
sodium bicarbonate (100 mL), dried (MgSO.sub.4), filtered and
evaporated. The residue was dissolved in anhydrous THF (50 mL).
TiCl.sub.4 (3 mL) and methyl magnesium bromide in ethyl ether (3 M,
10 mL) were added at -78.degree. C. and the mixture was stirred at
-50 to -30.degree. C. for 2 h. The mixture was poured into
saturated aqueous sodium bicarbonate solution (100 mL) and filtered
through Celite. The filtrate was extracted with toluene (100 mL)
and evaporated. The residue was chromatographed on silica gel
(EtOAc/hexane: 15/85) to afford the title compound as an oil (150
mg).
Step C:
4-Amino-7-(3-deoxy-3-fluoro-2-C-methyl-.beta.-D-ribofuranosyl)-7H--
pyrrolo[2,3-d]pyrimidine
[1214] The product from Step B (150 mg, 0.5 mmol) was dissolved in
HBr (30%) in acetic acid (2 mL). After one hour, the mixture was
evaporated under reduced pressure and co-evaporated with toluene
(10 n-L). 4-Chloro-1H-pyrrolo[2,3-d]pyrimidine (0.5 g, 3.3 mmol)
and powdered KOH (85%, 150 mg, 2.3 mmol) were stirred in DMF (3 mL)
for 30 min and the mixture was co-evaporated with toluene (2 mL).
The resulting solution was poured into the above bromo sugar and
the mixture was stirred overnight. The mixture was diluted with
toluene (50 mL), washed with water (3.times.50 mL) and concentrated
under reduced pressure. The residue was chromatographed on silica
gel (EtOAc/hexane 15/85) to afford an oil (60 mg). The oil was
dissolved in dioxane (2 mL) and liquid ammonia (20 g) was added in
a stainless steel autoclave. The mixture was heated at 85.degree.
C. for 18 h, then cooled and evaporated. The residue was
chromatographed on silica gel (methanol/dichloromethane: 1/9) to
afford the title compound as a solid (29 mg).
[1215] .sup.1H NMR (DMSO-d.sub.6): .delta. 0.81 (s, 3H), 3.75 (m,
2H), 4.16 (m, 1H), 5.09 (dd, 1H, J 53.2, 7.8 Hz), 5.26 (br, 1H),
5.77 (s, 1H), 6.15 (d, 1H, J=2.9 Hz), 6.59 (d, 1H, J=3.4 Hz), 7.02
(s br, 2H), 7.39 (d, 1H, J=3.4 Hz), 8.06 (s, 1H).
[1216] .sup.13C NMR (DMSO-d.sub.6): 19.40, 59.56, 77.24, 79.29,
90.15, 91.92, 99.88, 102.39, 121.17, 149.80, 151.77, 157.47.
[1217] .sup.19F NMR (DMSO-d.sub.6): .delta. 14.66 (m).
[1218] ES-MS: Found: 283.1 (M+H.sup.+); calculated for
C.sub.12H.sub.15FN.sub.4O.sub.3+H.sup.30 : 283.1.
EXAMPLE 144
8-Amino-2'-C-methyladenosine
[1219] 172
Step A: 8-Bromo-2'-C-methyladenosine
[1220] To a solution of 2'-C-methyladenosine [for preparation, see
J. Med. Chem. 41: 1708 (1998)] (138 mg, 0.5 mmol) in DMF (4 mL) was
added N-bromosuccinimide (231 mg, 1.35 mmol). The solution was
stirred protected from light at rt for 2 d and then evaporated in
vacuo. The crude product was purified on a silica gel column
(3.times.9 cm) using dichloromethane/methanol (25/1, 20/1 and 15/1)
as eluent. Fractions containing the product were pooled and
evaporated in vacuo to give the desired product (38 mg) as a white
solid.
Step B: 8-Amino-2'-C-methyladenosine
[1221] A solution of the compound from Step A (38 mg, 0.11 mmol) in
liquid ammonia (10 mL) was heated in a stainless steel autoclave at
105.degree. C. for 1 d, then cooled and evaporated. The residue was
purified by HPLC [C-18 Phenomenex Luna (10 .mu.l; 250.times.21.2
mm) RP-column; solvents: (A) water, (B) acetonitrile; Linear
gradient: 2-35% B in 76 min.] to yield the title compound (12 mg)
as a white fluffy material after freeze-drying.
[1222] .sup.1H NMR (DMSO-d.sub.6): .delta. 0.70 (s, 3H, Me),
3.55-3.75 (m, 3H, H-5', H-5", H-4'), 4.03 (m, 1H, H-3'), 4.81 (s,
1H, 2'-OH), 5.10 (d, 1H, 3'-OH), 5.45 (t, 1H, 5'-OH), 5.86 (s, 1H,
H-1'), 6.30, 6.39 (2s, 6H, 2 NH.sub.2), 7.78 (s, 1H, H-2).
[1223] ES-MS: Found: 295.0 (M-H.sup.+).
EXAMPLE 145
4-Amino-7-(2-C,2-O-dimethyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrim-
idine
[1224] 173
Step A:
4-chloro-7-[3,5-bis-O-(2,4-dichlorophenylmethyl)-2-C,2-O-dimethyl--
.beta.-D-ribofuranosyl]-7H-pyrrolo[2,3-d]pyrimidine
[1225] To a pre-cooled (0.degree. C.) solution of the compound from
Example 62, Step D (618 mg, 1.0 mmol) in THF (8 mL) was added
methyl iodide (709 mg, 5.0 mmol) and NaH (60% in mineral oil) (44
mg, 1.1 mmol). The resulting mixture was stirred overnight at rt
and then poured into a stirred mixture of saturated aqueous
ammonium chloride (50 mL) and dichloromethane (50 mL). The organic
layer was washed with water (50 mL), dried (MgSO.sub.4) and
evaporated in vacuo. The resulting crude product was purified on
silica gel using ethyl acetate/hexane as the eluent. Fractions
containing the product were pooled and evaporated in vacuo to give
the desired product (735 mg) as a colorless foam.
Step B:
4-amino-7-[3,5-bis-O-(2,4-dichlorophenylmethyl)-2-C,2-O-dimethyl-.-
gamma.-D-ribofuranosyl]-7H-pyrrolo[2,3-d]pyrimidine
[1226] To the compound from Step A (735 mg, 1.16 mmol) was added
methanolic ammonia (saturated at 0.degree. C.) (20 mL). The mixture
was heated in a stainless steel autoclave at 80.degree. C.
overnight, then cooled and the content evaporated in vacuo. The
crude mixture was purified on silica gel using ethyl acetate/hexane
as the eluent. Fractions containing the product were pooled and
evaporated in vacuo to give the desired product (504 mg) as
colorless foam.
Step C:
4-amino-7-(2-C,2-O-dimethyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-
-d]pyrimidine
[1227] A mixture of the product from Step C (64 mg, 0.1 mmol), MeOH
(5 mL), Et.sub.3N (0.2 mL) and 10% Pd/C (61 mg) was hydrogenated on
a Parr hydrogenator at 50 psi at room temperature overnight. The
mixture was filtered throught celite, evaporated in vacuo and
filtered through a pad of silica gel using 2% methanol in
dichloromethane as eluent. The desired product was collected and
evaporated in vacuo. The compound was redissolved in methanol (10
mL) and 10% Pd/C (61 mg) was added. The mixture was hydrogenated on
a Parr hydrogenator at 55 psi at room temperature for two weeks.
The mixture was filtered through celite, evaporated in vacuo and
purified on silica gel using 10% methanol in dichloromethane as
eluent. Fractions containing the product were pooled and evaporated
in vacuo to give the desired product (110 mg) as a colorless
foam.
[1228] .sup.1H NMR (DMSO-d.sub.6): .delta. 0.68 (s, 3H,), 3.40 (s,
3H), 3.52-3.99 (overlapping m, 4H), 4.92 (d, 1H), 5.07 (t, 1H),
6.26 (s, 1H), 6.55 (d, 1H), 7.00s br, 2H), 7.46 (d, 1H), 8.05 (s,
1H).
[1229] LC-MS: Found: 293.1 (M-H.sup.+); calc. for
C.sub.12H.sub.16N.sub.4O- .sub.4--H.sup.+: 293.12.
EXAMPLE 146
4-Methylamino-7-(2-C-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrim-
idine
[1230] 174
[1231] The compound from Step E of Example 62 (200 mg, 0.67 mmol)
was added to methylamine (5 mL condensed in a small stainless steel
autoclave) and warmed at 85.degree. C. for 48 h, then cooled and
evaporated in vacuo. The crude mixture was purified on a silica gel
with ethanol as the eluent to give the title compound which
separated as an amorphous solid after treatment with MeCN. The
amorphous solid was dissolved in water and lyophilized to give a
colorless powder (144 mg).
[1232] .sup.1H NMR (DMSO-d.sub.6): .delta. 0.63 (s, 3H, CH.sub.3),
3.32 (s, 3H, N CH.sub.3), 3.58-3.67 (m, 1H, H-5'), 3.79-3.39 (m,
3H, H-5", H-4', H-3'), 5.03 (s, 1H, 2'-OH), 5.04-5.11 (1H,3'-OH,
1H, 5'-OH), 6.14 (s, 1H, H-1'), 6.58 (d, 1H, J.sub.5,6=3.6 Hz,
H-5), 7.46 (d, 1H, H-6), 7.70 (br s, 1H, NH), 8.14 (s, 1H,
H-2).
[1233] LC-MS: Found: 295.1 (M-H.sup.+); calc. for
C.sub.13H.sub.18N.sub.4O- .sub.4+H.sup.+: 294.3.
EXAMPLE 147
4-Dimethylamino-7-(2-C-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyr-
imidine
[1234] 175
[1235] The compound from Step E of Example 62 (200 mg, 0.67 mmol)
was added to dimethylamine (5 mL condensed in a small stainless
steel autoclave) and warmed at 85.degree. C. for 48 h, then cooled
and evaporated in vacuo. The crude mixture was purified on a silica
gel with ethanol as the eluent to give the title compound which
separated as an amorphous solid after treatment with MeCN. The
amorphous solid was dissolved in water and lyophilized to give a
colorless powder (164 mg).
[1236] .sup.1H NMR (DMSO-d.sub.6): .delta. 0.64 (s, 3H, CH.sub.3),
3.29 (s, 3H, N CH.sub.3), 3.32 (s, 3H, N CH.sub.3), 3.60-3.66 (m,
1H, H-5'), 3.77-3.97 (m, 3H, H-5", H-4', H-3'), 5.04 (s, 1H,
2'-OH), 5.06-5.11 (1H, 3'-OH, 1H, 5'-OH), 6.21 (s, 1H, H-1'), 6.69
(d, 1H, J.sub.5,6=3.6 Hz, H-5), 7.55 (d, 1H, H-6), 8.13 (s, 1H,
H-2).
[1237] LC-MS: Found: 309.3 (M-H.sup.+); calc. for
C.sub.14H.sub.20N.sub.4O- .sub.4+H.sup.+: 308.33.
EXAMPLE 148
4-Cyclopropylamino-7-(2-C-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]-
pyrimidine
[1238] 176
[1239] The compound from Step E of Example 62 (200 mg, 0.67 mmol)
was added to cyclopropylamine (5 mL condensed in a small stainless
steel autoclave) and warmed at 85.degree. C. for 48 h, then cooled
and evaporated in vacuo. The crude mixture was purified on a silica
gel with ethanol as the eluent to give the title compound which
separated as an amorphous solid after treatment with MeCN. The
amorphous solid was dissolved in water and lyophilized to give a
colorless powder (148 mg).
[1240] .sup.1H NMR (DMSO-d.sub.6): .delta. 0.51-0.58 (m, 2H), 0.64
(s, 3H, CH.sub.3), 0.74-0.076 (m, 2H), 3.62-3.67 (m, 1H, H-5'),
3.79-3.82 (m, 3H, H-5"), 3.92-3.96 (m, H-4', H-3'), 5.03 (s, 1H,
2'-OH), 5.05-5.10 (1H, 3'-OH, 1H, 5'-OH), 6.15 (s, 1H, H-1'), 7.48
(d, 1H, J.sub.5,6=3.6 Hz, H-5), 7.59 (d, 1H, H-6), 8.13 (s, 1H,
H-2).
[1241] LC-MS: Found: 321.1 (M-H.sup.+); calc. for
C.sub.15H.sub.20N.sub.4O- .sub.4+H.sup.+: 320.3.
EXAMPLE 149
4-Amino-7-(3-C-methyl-.beta.-D-xylofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine
[1242] 177
Step A:
7-[2,5-Bis-O-(tert-butyldimethylsilyl)-.beta.-D-ribofuranosyl)]-4--
(4-methoxyphenyl)diphenylmethyl]amino-7H-pyrrolo[2,3-d]pyrimidine
and
7-[3,5-bis-O-(tert-butyldimethylsilyl)-.beta.-D-ribofuranosyl]-4-[(4-meth-
oxyphenyl)diphenylmethyl]amino-7H-pyrrolo[2,3-d]pyrimidine
[1243] To a solution of mixture of the compounds from Step A of
Examples 140 and 141 (0.32 g, 0.65 mmol) in anhydrous pyridine (6
mL) was added monomethoxytrityl chloride (0.30 g, 0.98 mmol) and
the reaction mixture was stirred at room temperature overnight. The
mixture was then concentrated and the residue was partitioned
between CH.sub.2Cl.sub.2 (70 mL) and water (20 mL). The organic
layer was washed with water and brine, dried (Na.sub.2SO.sub.4) and
concentrated. The residue was purified on silica gel column using
5-13% EtOAc in hexanes as the eluent. The appropriate fractions
were collected and concentrated to furnish
2',5'-bis-O-(tert-butyldimethylsilyl)- and
3',5'-bis-O-(tert-butyldimethy- lsilyl) protected nucleosides as
colorless foams (343 mg and 84 mg, respectively).
Step B:
7-[2,5-Bis-O-(tert-butyldimethylsilyl)-.beta.-D-erythro-pentofuran-
os-3-ulosyl]-4-[(4-methoxyphenyl)diphenylmethyl]amino-7H-pyrrolo[2,3-d]pyr-
imidine
[1244] To a well-stirred suspension of chromium trioxide (91 mg,
0.91 mmol) in CH.sub.2Cl.sub.2 (4 mL) at 0.degree. C. were added
pyridine (147 .mu.L, 1.82 mmol) and then acetic anhydride (86
.mu.L, 0.91 mmol). The mixture was stirred at room temperature for
0.5 h. Then the 2',5'-bis-O-(tert-butyldimethylsilyl) protected
nucleoside from step A (343 mg 0.45 mmol) in CH.sub.2Cl.sub.2 (2.5
mL) was added and the mixture stirred at room temperature 2 h. The
mixture was then poured into ice-cold EtOAc (10 mL) and filtered
through a short silica gel column using EtOAc as the eluent. The
filtrate was evaporated and the residue-purified on a silica gel
column with hexanes and hexanes/EtOAc (7/1) as the eluent to give
the title compound (180 mg).
Step C:
7-[2,5-Bis-O-(tert-butyldimethylsilyl)-3-C-methyl-.beta.-D-ribofur-
anosyl)-4-[(4-methoxyphenyl)diphenylmethyl]amino-7H-pyrrolo[2,3-d]pyrimidi-
ne and
7-[2,5-Bis-O-(tert-butyldimethylsilyl)-3-C-methyl-O-.beta.-xylofura-
nosyl)-4-[(4-methoxyphenyl)diphenylmethyl]amino-7H-pyrrolo[2,3-d]pyrimidin-
e
[1245] To a mixture of MeMgBr (3.0 M solution in ether; 0.17 mL,
0.5 mmol) in anhydrous hexanes (1.5 mL) at room temperature was
added dropwise a solution of the compound from Step B (78 mg, 0.1
mmol) in anhydrous hexanes (0.5 mL). After 2 h stirring at room
temperature, the reaction mixture was poured into ice-cold water
(10 mL) and diluted with EtOAc (20 mL), then filtered through
Celite which was then thoroughly washed with EtOAc. The layers were
separated and the organic layer was washed with brine, dried
(Na.sub.2SO.sub.4) and concentrated. The residue was purified on a
silica gel column using 8 to 25% EtOAc in hexanes as eluent to give
the 3-C-methyl xylo- (60 mg) and the 3-C-methyl ribo-isomer (20
mg).
Step D:
4-Amino-7-(3-C-methyl-.beta.-D-xylofuranosyl)-7H-pyrrolo[2,3-d]pyr-
imidine
[1246] To an ice-cold solution of 3-C-methyl-xylo isomer from Step
C (60 mg, 0.08 mmol) in THF (2 mL) was added TBAF (1 M in THF; 0.32
mL, 0.32 mmol). The reaction mixture was stirred at room
temperature for 5 h, then diluted with CH.sub.2Cl.sub.2 (50 mL),
washed with water (3.times.15 mL), dried, and evaporated. The
residue was dissolved in dioxane (0.3 mL) and 80% acetic acid (3
mL) was added. The reaction mixture was stirred at room temperature
for 1 d and then evaporated. The residue was co-evaporated with
dioxane, taken up in water (50 mL) and washed with CH.sub.2Cl.sub.2
(2.times.10 mL). The aqueous layer was concentrated and then
freeze-dried. The residue was purified on silica gel column with
CH.sub.2Cl.sub.2/MeOH (20/1 and 10/1) as the eluent to give the
title compound as a white fluffy compound after freeze drying (10
mg).
[1247] .sup.1H NMR (CD.sub.3CN): .delta. 1.28 (s, 3H, CH.sub.3),
3.56 (br s, 1H, OH), 3.78 (m, 3H, H-4', H-5', H-5"), 4.10 (br s,
1H, OH), 4.44 (d, 1H, J.sub.2'1'=3.9 Hz, H-2'), 5.58 (d, 1H, H-1'),
85 (br s, 2H, NH.sub.2), 6.15 (br s, 1H, OH), 6.48 (d, 1H,
J.sub.5,6=3.7 Hz, H-5), 7.23 (d, H, H-6), 8.11 (s, 1H, H-2). ES-MS:
281 [MH].sup.+.
EXAMPLE 150
4-Amino-7-(3-C-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine
[1248] 178
[1249] The ribo-isomer (20 mg) from Step C of Example 149 was
deprotected using the procedure described in Step D of Example 32
to yield the title compound (4 mg).
[1250] .sup.1H NMR (CD.sub.3CN): .delta. 1.43 (s, 3H, CH.sub.3),
3.28 (br s, 1H, OH), 3.58 (m, 2H, H-5', H-5"), 3.99 (m, 1H, H-4'),
4.10 (br s, 1H, OH), 4.62 (d, 1H, J.sub.2'1'=8.1 Hz, H-2'), 5.69
(d, 1H, H-1'), 5.88 (br s, 3H, OH, NH.sub.2), 6.45 (br s, 1H, OH),
6.51 (d, 1H, J.sub.5,6=3.7 Hz, H-5), 7.19 (d, 1H, H-6), 8.12 (s,
1H, H-2). ES-MS: 281 [MH].sup.+.
EXAMPLE 151
2,4-Diamino-7-(2-C-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimid-
ine
[1251] 179
[1252] A mixture of the product from Step B of Example 118 (24 mg)
in aqueous ammonia (30%, 10 mL) was heated in a stainless steel
autoclave at 100.degree. C. overnight, then cooled and evaporated.
The residue was purified on a silica gel column with
CH.sub.2Cl.sub.2/MeOH (10/1 and 5/1) as the eluent to afford the
title compound 15 mg).
[1253] .sup.1H NMR (DMSO-d.sub.6): .delta. 0.68 (s, 3H, CH.sub.3),
3.48-3.58 (m 1H, H-5'), 3.68-3.73 (m, 2H, H-5", H-4'), 3.84 (m, 1H,
H-3'), 4.72 (s, 1H, 2'-OH), 4.97-5.03 (m, 2H, 3'-OH, 5'-OH), 5.45
(br s, 2H, NH.sub.2), 6.00 (s, 1H, H-1'), 6.28 (d, 1H, J=3.7 Hz,
H-5), 6.44 (br s, 2H, NH.sub.2) 6.92 (d, 1H J=3.7 Hz, H-6).
[1254] ES MS: 294.1 (M-H.sup.+).
EXAMPLE 152
4-Amino-2-fluoro-7-(2-C-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]py-
rimidine
[1255] 180
[1256] To a solution of HF/pyridine (70%, 2 mL) diluted with
pyridine (1 mL) at -30.degree. C. is added the compound of Example
151 (60 mg, 0.2 mmol) in 0.5 mL pyridine followed by tert-butyl
nitrite (36 .mu.L, 0.3 mmol). Stirring is continued for 5 min
-25.degree. C. Then the solution is poured into ice-water (5 mL),
neutralized with 2 N aqueous NaOH, and evaporated to dryness. The
residue is purified on a silica gel column with
CH.sub.2Cl.sub.2MeOH (20/1 and 10/1) as the eluent to afford the
title compound.
EXAMPLE 153
4-Amino-5-fluoro-7-(2-C-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[2,3-d]py-
rimidine
[1257] 181
Step A:
4-Acetylamino-7-(2,3,5-tri-O-acetyl-2-C-methyl-.beta.-D-ribofurano-
syl)-7H-pyrrolo[2,3-d]pyrimidine
[1258] To a solution of the compound from step F of Example 62 (280
mg, 1.00 mmol) in pyridine is added acetic anhydride (613 mg, 6.0
mmol). The resulting solution is stirred overnight at ambient
temperature evaporated in vacuo and the resulting crude mixture is
purified on silica gel using ethyl acetate/hexane as the eluent.
Fractions containing the desired product are pooled and evaporated
in vacuo to give the desired product.
Step B:
4-Acetylamino-5-bromo-7-(2,3,5-tri-O-acetyl-2-C-methyl-.beta.-D-ri-
bofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine
[1259] To a pre-cooled (0.degree. C.) solution of the compound from
Step A (460 mg, 1.00 mmol) in DMF is added N-bromosuccinimide (178
mg, 1.0 mmol) in DMF. The resulting solution is stirred at
0.degree. C. for 30 min then at room temperature for another 30
min. The reaction is quenched by addition of methanol and
evaporated in vacuo. The resulting crude mixture is purified on
silica gel using ethyl acetate/hexane as the eluent. Fractions
containing the desired product are pooled and evaporated in vacuo
to give the desired product.
Step C:
4-Amino-5-fluoro-7-(2-C-methyl-.beta.-D-ribofuranosyl)-7H-pyrrolo[-
2,3-d]pyrimidine
[1260] To a pre-cooled (-78.degree. C.) solution of the compound
from Step B (529 mg, 1.00 mmol) in THF is added butyl lithium (2M
in hexanes) (0.5 mL, 1.00 mmol). The resulting solution is stirred
at -78.degree. C. for 30 min and then quenched with
N-fluorobenzensulfonimide (315 mg, 1.00 mmol) in THF. The resulting
solution is very slowly allowed to come to ambient temperature and
then poured into a stirred mixture of saturated aqueous ammonium
chloride and dichloromethane. The organic phase is evaporated in
vacuo and treated with ammonium hydroxide at 55.degree. C. in a
closed container overnight. The resulting crude mixture is purified
on silica gel using dichloromethane/methanol as the eluent.
Fractions containing the desired product are pooled and evaporated
in vacuo to give the desired product.
EXAMPLE 154
4-Amino-1-(2-C-methyl-.beta.-D-ribofuranosyl)-1H-pyrazolo[3,4-d]pyrimidine
[1261] 182
Step A:
4-Amino-1-[3,5-bis-O-(2,4-dichlorophenylmethyl)-2-C-methyl-.beta.--
D-ribofuranosyl]-1H-pyrazolo[3,4-d]pyrimidine
[1262] To the compound from Step C of Example 62 (1.00 g, 2.02
mmol) in dichloromethane (20 mL) was bubbled HBr gas for 5 min
until it was saturated. The resulting solution was stirred at room
temperature for 10 min, evaporated in vacuo and coevaporated with
anhydrous toluene (10 mL). 4-Amino-1H-pyrazolo[3,4-d]pyrimidine
(Aldrich, 0.43 g, 3.18 mmol) and NaH (60%, 150 mg, 3.8 mmol) were
stirred in 1-methyl-2-pyrrolidinone (10 mL) for 30 min. The
resulting solution was poured into the above bromo sugar residue
and the mixture was stirred overnight. The mixture was diluted with
toluene (50 mL), washed with brine (10%, 3.times.50 mL) and
concentrated under reduced pressure. The residue was
chromatographed on silica gel (EtOAc as eluent) to afford a solid
(400 mg).
Step B:
4-Amino-1-(2-C-methyl-.beta.-D-ribofuranosyl)-1H-pyrazolo[3,4-d]py-
rimidine
[1263] To a solution of the compound from Step A (0.20 g, 0.33
mmol) in dichloromethane (10 mL) at -78.degree. C. was added boron
trichloride (1M in dichloromethane) (3 mL, 3 mmol) dropwise. The
mixture was stirred at -78.degree. C. for 0.5 h, then at
-45.degree. C. to -30.degree. C. for 2 h. The reaction was quenched
by addition of sodium acetate (1.0 g) and methanol (10 mL). The
solution was evaporated and the residue was purified by flash
chromatography over silica gel using CH.sub.2Cl.sub.2 and
CH.sub.2Cl.sub.2/MeOH (95:5-90:10) gradient as the eluent to
furnish the desired compound (60 mg) as a solid, which was
recrystallized from methanol and acetonitrile to give the title
compound as an off-white solid (40 mg).
[1264] .sup.1H NMR (DMSO-d.sub.6): .delta. 0.75 (s, 3H), 3.59 (m,
1H), 3.69 (m, 1H), 3.91 (m, 1H), 4.12 (m, 1H), 4.69 (t, 1H, J=5.1
Hz), 5.15 (m, 2H), 6.13 (s, 1H), 7.68 (s, br, 1H), 7.96 (s, br,
1H), 8.18 (s, 1H), 8.21 (s, 1H).
[1265] .sup.13C NMR (DMSO-d.sub.6): 19.32, 62.78, 74.11, 78.60,
83.65, 90.72, 99.79, 133.50, 153.89, 156.21, 158.05.
[1266] LC-MS: Found: 282.1 (M+H.sup.+); calculated for C,
H.sub.15N.sub.5O.sub.4+H.sup.+: 282.1.
Biological Assays
[1267] The assays employed to measure the inhibition of HCV NS5B
polymerase and HCV replication are described below.
[1268] The effectiveness of the compounds of the present invention
as inhibitors of HCV NS5B RNA-dependent RNA polymerase (RdRp) was
measured in the following assay.
[1269] A. Assay for Inhibition of HCV NS5B Polymerase:
[1270] This assay was used to measure the ability of the nucleoside
derivatives of the present invention to inhibit the enzymatic
activity of the RNA-dependent RNA polymerase (NSSB) of the
hepatitis C virus (HCV) on a heteromeric RNA template.
[1271] Procedure:
[1272] Assay Buffer Conditions: (50 .mu.L-total/reaction)
[1273] 20 mM Tris, pH 7.5
[1274] 50 .mu.M EDTA
[1275] 5 mM DTT
[1276] 2 mM MgCl.sub.2
[1277] 80 mM KCl
[1278] 0.4 U/.mu.L RNAsin (Promega, stock is 40 units/.mu.L)
[1279] 0.75 .mu.g t500 (a 500-nt RNA made using T7 runoff
transcription with a sequence from the NS2/3 region of the
hepatitis C genome)
[1280] 1.6 .mu.g purified hepatitis C NS5B (form with 21 amino
acids C-terminally truncated)
[1281] 1 .mu.M A,C,U,GTP (Nucleoside triphosphate mix)
[1282] [alpha-.sup.32P]-GTP or [alpha-.sup.33P]-GTP
[1283] The compounds were tested at various concentrations up to
100 .mu.M final concentration.
[1284] An appropriate volume of reaction buffer was made including
enzyme and template t500. Nucleoside derivatives of the present
invention were pipetted into the wells of a 96-well plate. A
mixture of nucleoside triphosphates (NTP's), including the
radiolabeled GTP, was made and pipetted into the wells of a 96-well
plate. The reaction was initiated by addition of the
enzyme-template reaction solution and allowed to proceed at room
temperature for 1-2 h.
[1285] The reaction was quenched by addition of 20 .mu.L 0.5M EDTA,
pH 8.0. Blank reactions in which the quench solution was added to
the NTPs prior to the addition of the reaction buffer were
included.
[1286] 50 .mu.L of the quenched reaction were spotted onto DE81
filter disks (Whatman) and allowed to dry for 30 min. The filters
were washed with 0.3 M ammonium formate, pH 8 (150 mL/wash until
the cpm in 1 mL wash is less than 100, usually 6 washes). The
filters were counted in 5-mL scintillation fluid in a scintillation
counter.
[1287] The percentage of inhibition was calculated according to the
following equation: % Inhibition=[1-(cpm in test reaction-cpm in
blank)/(cpm in control reaction-cpm in blank)].times.100.
[1288] Representative compounds tested in the HCV NS5B polymerase
assay exhibited IC.sub.50's less than 100 micromolar.
[1289] B. Assay for Inhibition of HCV RNA Replication:
[1290] The compounds of the present invention were also evaluated
for their ability to affect the replication of Hepatitis C Virus
RNA in cultured hepatoma (HuH-7) cells containing a subgenomic HCV
Replicon. The details of the assay are described below. This
Replicon assay is a modification of that described in V. Lohmann,
F. Korner, J-O. Koch, U. Herian, L. Theilmann, and R.
Bartenschlager, "Replication of a Sub-genomic Hepatitis C Virus
RNAs in a Hepatoma Cell Line," Science 285:110 (1999).
[1291] Protocol:
[1292] The assay was an in situ Ribonuclease protection,
Scintillation Proximity based-plate assay (SPA). 10,000-40,000
cells were plated in 100-200 .mu.L of media containing 0.8 mg/mL
G418 in 96-well cytostar plates (Amersham). Compounds were added to
cells at various concentrations up to 100 .mu.M in 1% DMSO at time
0 to 18 h and then cultured for 24-96 h. Cells were fixed (20 min,
10% formalin), permeabilized (20 min, 0.25% Triton X-100/PBS) and
hybridized (overnight, 50.degree. C.) with a single-stranded
.sup.33P RNA probe complementary to the (+) strand NS5B (or other
genes) contained in the RNA viral genome. Cells were washed,
treated with RNAse, washed, heated to 65.degree. C. and counted in
a Top-Count.
[1293] Inhibition of replication was read as a decrease in counts
per minute (cpm). Human HuH-7 hepatoma cells, which were selected
to contain a subgenomic replicon, carry a cytoplasmic RNA
consisting of an HCV 5' non-translated region (NTR), a neomycin
selectable marker, an EMCV IRES (internal ribosome entry site), and
HCV non-structural proteins NS3 through NSSB, followed by the 3'
NTR.
[1294] Representative compounds tested in the replication assay
exhibited EC.sub.50's less than 100 micromolar.
[1295] The nucleoside derivatives of the present invention were
also evaluated for cellular toxicity and anti-viral specificity in
the counterscreens described below.
[1296] C. Counterscreens:
[1297] The ability of the nucleoside derivatives of the present
invention to inhibit human DNA polymerases was measured in the
following assays.
[1298] a. Inhibition of Human DNA Polymerases Alpha and Beta:
[1299] Reaction Conditions:
[1300] 50 mL reaction volume
[1301] Reaction Buffer Components:
[1302] 20 mM Tris-HCl, pH 7.5
[1303] 200 .mu.g/mL bovine serum albumin
[1304] 100 mM KCl
[1305] 2 mM .beta.-mercaptoethanol
[1306] 10 mM MgCl.sub.2
[1307] 1.6 .mu.M dA, dG, dC, dTTP
[1308] .alpha.-.sup.33P-dATP
[1309] Enzyme and Template:
[1310] 0.05 mg/mL gapped fish sperm DNA template
[1311] 0.01 U/.mu.L DNA polymerase .alpha. or .beta.
[1312] Preparation of Gapped Fish Sperm DNA Template:
[1313] Add 5 .mu.L 1M MgCl.sub.2 to 500 .mu.L activated fish sperm
DNA (USB 70076);
[1314] Warm to 37.degree. C. and add 30 .mu.L of 65 U/.mu.L of
exonuclease III (GibcoBRL 18013-011);
[1315] Incubate 5 min at 37.degree. C.;
[1316] Terminate reaction by heating to 65.degree. C. for 10
min;
[1317] Load 50-100 .mu.L aliquots onto Bio-spin 6 chromatography
columns (Bio-Rad 732-6002) equilibrated with 20 mM Tris-HCl, pH
7.5;
[1318] Elute by centrifugation at 1,000.times.g for 4 min;
[1319] Pool eluate and measure absorbance at 260 nm to determine
concentration.
[1320] The DNA template was diluted into an appropriate volume of
20 mM Tris-HCl, pH 7.5 and the enzyme was diluted into an
appropriate volume of 20 mM Tris-HCl, containing 2 mM
.beta.-mercaptoethanol, and 100 mM KCl. Template and enzyme were
pipetted into microcentrifuge tubes or a 96 well plate. Blank
reactions excluding enzyme and control reactions excluding test
compound were also prepared using enzyme dilution buffer and test
compound solvent, respectively. The reaction was initiated with
reaction buffer with components as listed above. The reaction was
incubated for 1 hour at 37.degree. C. The reaction was quenched by
the addition of 20 .mu.L 0.5M EDTA. 50 .mu.L of the quenched
reaction was spotted onto Whatman DE81 filter disks and air dried.
The filter disks were repeatedly washed with 150 mL 0.3M ammonium
formate, pH 8 until 1 mL of wash is <100 cpm. The disks were
washed twice with 150 mL absolute ethanol and once with 150 mL
anhydrous ether, dried and counted in 5 mL scintillation fluid.
[1321] The percentage of inhibition was calculated according to the
following equation: % inhibition=[1-(cpm in test reaction-cpm in
blank)/(cpm in control reaction-cpm in blank)].times.100.
[1322] b. Inhibition of Human DNA Polymerase Gamma:
[1323] The potential for inhibition of human DNA polymerase gamma
was measured in reactions that included 0.5 ng/.mu.L enzyme; 10
.mu.M dATP, dGTP, dCTP, and TTP; 2 .mu.Ci/reaction
[.alpha.-.sup.33P]-dATP, and 0.4 .mu.g/.mu.L activated fish sperm
DNA (purchased from US Biochemical) in a buffer containing 20 mM
Tris pH8, 2 mM .beta.-mercaptoethanol, 50 mM KCl, 10 mM MgCl.sub.2,
and 0.1 .mu.g/.mu.L BSA. Reactions were allowed to proceed for 1 h
at 37.degree. C. and were quenched by addition of 0.5 M EDTA to a
final concentration of 142 mM. Product formation was quantified by
anion exchange filter binding and scintillation counting. Compounds
were tested at up to 50 .mu.M.
[1324] The percentage of inhibition was calculated according to the
following equation: % inhibition=[1-(cpm in test reaction-cpm in
blank)/(cpm in control reaction-cpm in blank)].times.100.
[1325] The ability of the nucleoside derivatives of the present
invention to inhibit HIV infectivity and HIV spread was measured in
the following assays.
[1326] c. HIV Infectivity Assay
[1327] Assays were performed with a variant of HeLa Magi cells
expressing both CXCR4 and CCR5 selected for low background
.beta.-galactosidase (.beta.-gal) expression. Cells were infected
for 48 h, and .beta.-gal production from the integrated HIV-1 LTR
promoter was quantified with a chemiluminescent substrate
(Galactolight Plus, Tropix, Bedford, Mass.). Inhibitors were
titrated (in duplicate) in twofold serial dilutions starting at 100
.mu.M; percent inhibition at each concentration was calculated in
relation to the control infection.
[1328] d. Inhibition of HIV Spread
[1329] The ability of the compounds of the present invention to
inhibit the spread of the human immunedeficiency virus (HIV) was
measured by the method described in U.S. Pat. No. 5,413,999 (May 9,
1995), and J. P. Vacca, et al., Proc. Natl. Acad. Sci., 91:
4096-4100 (1994), which are incorporated by reference herein in
their entirety.
[1330] The nucleoside derivatives of the present invention were
also screened for cytotoxicity against cultured hepatoma (HuH-7)
cells containing a subgenomic HCV Replicon in an MTS cell-based
assay as described in the assay below. The HuH-7 cell line is
described in H. Nakabayashi, et al., Cancer Res., 42: 3858
(1982).
[1331] e. Cytotoxicity Assay:
[1332] Cell cultures were prepared in appropriate media at
concentrations of approximately 1.5.times.10.sup.5 cells/mL for
suspension cultures in 3 day incubations and 5.0.times.10.sup.4
cells/mL for adherent cultures in 3 day incubations. 99 .mu.L of
cell culture was transferred to wells of a 96-well tissue culture
treated plate, and 1 .mu.L of 100-times final concentration of the
test compound in DMSO was added. The plates were incubated at
37.degree. C. and 5% CO.sub.2 for a specified period of time. After
the incubation period, 20 .mu.L of CellTiter 96 Aqueous One
Solution Cell Proliferation Assay reagent (MTS) (Promega) was added
to each well and the plates were incubated at 37.degree. C. and 5%
CO.sub.2 for an additional period of time up to 3 h. The plates
were agitated to mix well and absorbance at 490 nm was read using a
plate reader. A standard curve of suspension culture cells was
prepared with known cell numbers just prior to the addition of MTS
reagent. Metabolically active cells reduce MTS to formazan.
Formazan absorbs at 490 nm. The absorbance at 490 nm in the
presence of compound was compared to absorbance in cells without
any compound added. Reference: Cory, A. H. et al., "Use of an
aqueous soluble tetrazolium/formazan assay for cell growth assays
in culture," Cancer Commun. 3: 207 (1991).
[1333] The following assays were employed to measure the activity
of the compounds of the present invention against other
RNA-dependent RNA viruses:
[1334] a. Determination of In Vitro Antiviral Activity of Compounds
Against Rhinovirus (Cytopathic Effect Inhibition Assay):
[1335] Assay conditions are described in the article by Sidwell and
Huffman, "Use of disposable microtissue culture plates for
antiviral and interferon induction studies," Appl. Microbiol. 22:
797-801 (1971).
[1336] Viruses:
[1337] Rhinovirus type 2 (RV-2), strain HGP, was used with KB cells
and media (0.1% NaHCO.sub.3, no antibiotics) as stated in the
Sidwell and Huffman reference. The virus, obtained from the ATCC,
was from a throat swab of an adult male with a mild acute febrile
upper respiratory illness.
[1338] Rhinovirus type 9 (RV-9), strain 211, and rhinovirus type 14
(RV-14), strain Tow, were also obtained from the American Type
Culture Collection (ATCC) in Rockville, Md. RV-9 was from human
throat washings and RV-14 was from a throat swab of a young adult
with upper respiratory illness. Both of these viruses were used
with HeLa Ohio-1 cells (Dr. Fred Hayden, Univ. of VA) which were
human cervical epitheloid carcinoma cells. MEM (Eagle's minimum
essential medium) with 5% Fetal Bovine serum (FBS) and 0.1%
NaHCO.sub.3 was used as the growth medium.
[1339] Antiviral test medium for all three virus types was MEM with
5% FBS, 0.1% NaHCO.sub.3, 50 .mu.g gentamicin/mL, and 10 mM
MgCl.sub.2.
[1340] 2000 .mu.g/mL was the highest concentration used to assay
the compounds of the present invention. Virus was added to the
assay plate approximately 5 min after the test compound. Proper
controls were also run. Assay plates were incubated with humidified
air and 5% CO.sub.2 at 37.degree. C. Cytotoxicity was monitored in
the control cells microscopically for morphologic changes.
Regression analysis of the virus CPE data and the toxicity control
data gave the ED50 (50% effective dose) and CC50 (50% cytotoxic
concentration). The selectivity index (SI) was calculated by the
formula: SI=CC50.div.ED50.
[1341] b. Determination of In Vitro Antiviral Activity of Compounds
Against Dengue, Banzi, and Yellow Fever (CPE Inhibition Assay)
[1342] Assay details are provided in the Sidwell and Huffman
reference above.
[1343] Viruses:
[1344] Dengue virus type 2, New Guinea strain, was obtained from
the Center for Disease Control. Two lines of African green monkey
kidney cells were used to culture the virus (Vero) and to perform
antiviral testing (MA-104). Both Yellow fever virus, 17D strain,
prepared from infected mouse brain, and Banzi virus, H 336 strain,
isolated from the serum of a febrile boy in South Africa, were
obtained from ATCC. Vero cells were used with both of these viruses
and for assay.
[1345] Cells and Media:
[1346] MA-104 cells (BioWhittaker, Inc., Walkersville, Md.) and
Vero cells (ATCC) were used in Medium 199 with 5% FBS and 0.1%
NaHCO.sub.3 and without antibiotics. Assay medium for dengue,
yellow fever, and Banzi viruses was MEM, 2% FBS, 0.18% NaHCO.sub.3
and 50 .mu.g gentamicin/mL.
[1347] Antiviral testing of the compounds of the present invention
was performed according to the Sidwell and Huffman reference and
similar to the above rhinovirus antiviral testing. Adequate
cytopathic effect (CPE) readings were achieved after 5-6 days for
each of these viruses.
[1348] c. Determination of In Vitro Antiviral Activity of Compounds
Against West Nile Virus (CPE Inhibition Assay)
[1349] Assay details are provided in the Sidwell and Huffman
reference cited above. West Nile virus, New York isolate derived
from crow brain, was obtained from the Center for Disease Control.
Vero cells were grown and used as described above. Test medium was
MEM, 1% FBS, 0.1% NaHCO.sub.3 and 50 .mu.g gentamicin/mL.
[1350] Antiviral testing of the compounds of the present invention
was performed following the methods of Sidwell and Huffman which
are similar to those used to assay for rhinovirus activity.
Adequate cytopathic effect (CPE) readings were achieved after 5-6
days.
[1351] d. Determination of In Vitro Antiviral Activity of Compounds
Against rhino, yellow Fever, Dengue, Banzi, and West Nile Viruses
(Neutral Red Uptake Assay)
[1352] After performing the CPE inhibition assays above, an
additional cytopathic detection method was used which is described
in "Microtiter Assay for Interferon: Microspectrophotometric
Quantitation of Cytopathic Effect," Appl. Environ. Microbiol. 31:
35-38 (1976). A Model EL309 microplate reader (Bio-Tek Instruments
Inc.) was used to read the assay plate. ED50's and CD50's were
calculated as above.
EXAMPLE OF A PHARMACEUTICAL FORMULATION
[1353] As a specific embodiment of an oral composition of a
compound of the present invention, 50 mg of Example 61 or 62 is
formulated with sufficient finely divided lactose to provide a
total amount of 580 to 590 mg to fill a size O hard gelatin
capsule.
[1354] While the invention has been described and illustrated in
reference to specific embodiments thereof, those skilled in the art
will appreciate that various changes, modifications, and
substitutions can be made therein without departing from the spirit
and scope of the invention. For example, effective dosages other
than the preferred doses as set forth heeinabove may be applicable
as a consequence of variations in the responsiveness of the human
being treated for severity of the HCV infection. Likewise, the
pharmacologic response observed may vary according to and depending
upon the particular active compound selected or whether there are
present pharmaceutical carriers, as well as the type of formulation
and mode of administration employed, and such expected variations
or differences in the results are contemplated in accordance with
the objects and practices of the present invention. It is intended
therefore that the invention be limited only by the scope of the
claims which follow and that such claims be interpreted as broadly
as is reasonable.
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