U.S. patent application number 13/899513 was filed with the patent office on 2013-11-28 for d-amino acid compounds for liver disease.
The applicant listed for this patent is Idenix Pharmaceuticals, Inc.. Invention is credited to Benjamin Alexander MAYES, Adel M. MOUSSA, Alistair James STEWART.
Application Number | 20130315868 13/899513 |
Document ID | / |
Family ID | 48537028 |
Filed Date | 2013-11-28 |
United States Patent
Application |
20130315868 |
Kind Code |
A1 |
MAYES; Benjamin Alexander ;
et al. |
November 28, 2013 |
D-AMINO ACID COMPOUNDS FOR LIVER DISEASE
Abstract
Provided herein are compounds, compositions and methods for the
treatment of liver disease and conditions, including HCV
infections. In certain embodiments, compounds and compositions of
nucleoside derivatives are disclosed, which can be administered
either alone or in combination with other anti-viral agents.
Inventors: |
MAYES; Benjamin Alexander;
(Boston, MA) ; MOUSSA; Adel M.; (Burlington,
MA) ; STEWART; Alistair James; (Lincoln, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Idenix Pharmaceuticals, Inc. |
Cambridge |
MA |
US |
|
|
Family ID: |
48537028 |
Appl. No.: |
13/899513 |
Filed: |
May 21, 2013 |
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Current U.S.
Class: |
424/85.7 ;
424/278.1; 424/85.4; 514/4.3; 514/48; 514/51; 536/26.7;
536/26.8 |
Current CPC
Class: |
C07H 19/10 20130101;
A61K 31/497 20130101; A61K 31/708 20130101; A61K 45/06 20130101;
A61K 38/55 20130101; A61K 31/4709 20130101; A61K 31/7072 20130101;
C07H 19/16 20130101; A61K 38/21 20130101; C07H 19/20 20130101; A61K
31/7056 20130101; A61K 31/7076 20130101; A61P 31/14 20180101; A61P
31/12 20180101; A61K 31/403 20130101; A61P 35/00 20180101; A61P
1/16 20180101; A61K 31/403 20130101; A61K 2300/00 20130101; A61K
31/4709 20130101; A61K 2300/00 20130101; A61K 31/497 20130101; A61K
2300/00 20130101; A61K 31/7056 20130101; A61K 2300/00 20130101;
A61K 31/7072 20130101; A61K 2300/00 20130101; A61K 38/21 20130101;
A61K 2300/00 20130101; A61K 38/55 20130101; A61K 2300/00
20130101 |
Class at
Publication: |
424/85.7 ;
536/26.8; 514/51; 424/85.4; 424/278.1; 514/4.3; 536/26.7;
514/48 |
International
Class: |
C07H 19/20 20060101
C07H019/20; A61K 31/708 20060101 A61K031/708; A61K 45/06 20060101
A61K045/06; A61K 31/7076 20060101 A61K031/7076; C07H 19/10 20060101
C07H019/10; A61K 31/7072 20060101 A61K031/7072 |
Claims
1. A compound according to formula (I): ##STR00175## or a
pharmaceutically acceptable salt, solvate, stereoisomeric form,
tautomeric form or polymorphic form thereof, wherein: Base is a
nucleobase; W is S or O; X is a D-amino acid residue, or an ester
thereof; Y is hydrogen, --OR.sup.1, --SW, or --NR.sup.1R.sup.2;
R.sup.b1 is --CH.sub.3, --H, azido, cyano, or halogen; R.sup.b2 is
--OH, --Cl, --F, --H, azido, cyano, amino, or alkoxyl; R.sup.c is
--H or --OH, or, in the alternative, Y and R.sup.c join to form a
six-membered heterocyclic ring wherein Y and R.sup.c together
represent a single divalent --O--; R.sup.d is --H, --F, azido, or
allenyl; or, in the alternative, R.sup.b2 and R.sup.d join to form
alkylene or substituted alkylene; R.sup.e is --H or alkyl; each
R.sup.1 is independently alkyl, cycloalkyl, aryl, heteroaryl,
arylalkyl, heteroarylalkyl, substituted alkyl or hydantoinylalkyl;
and each R.sup.2 is independently hydrogen or alkyl.
2. The compound of claim 1 wherein each R.sup.1 is independently
alkyl, cycloalkyl, heterocyclylalkyl, aryl, heteroaryl, arylalkyl,
heteroarylalkyl, alkylcarbonylthioalkyl, alkoxycarbonylalkyl,
arylalkoxycarbonylalkyl, alkylcarbonylalkoxy(arylalkyl),
(alkoxycarbonyl)(alkoxycarbonylamino)alkyl,
cycloalkylcarbonylalkoxyl,
alkoxycarbonylaminoalkylcarbonylthioalkyl,
hydroxylalkylcarbonylthioalkyl,
aminoalkylcarbonylalkoxycarbonylthioalkyl, or hydantoinylalkyl.
3. The compound of claim 1 according to formula (II): ##STR00176##
wherein each Y is independently --OR.sup.1, --SR.sup.1, or
--NR.sup.1R.sup.2; each R.sup.10 is independently alkyl, arylalkyl,
heteroarylalkyl, cycloalkyl or a side chain of a naturally
occurring amino acid other than hydrogen; and each R.sup.11 is
independently alkyl or --H.
4. The compound of claim 1 wherein R.sup.e and R.sup.d are H.
5-10. (canceled)
11. The compound of claim 1 wherein W is O, and R.sup.b2 is Cl, F
or OH.
12. (canceled)
13. The compound of claim 1 wherein Y is ##STR00177## and R.sup.3
is alkyl, alkoxycarbonylaminoalkyl, hydroxylalkyl, or
aminoalkylcarbonylalkoxyl.
14. The compound claim 1 wherein: each Base is independently
##STR00178## or a tautomer thereof; each R.sup.4 is independently
hydrogen, hydroxyl, hydroxylamine, alkylamino, halogen, sulfanyl,
amino or alkoxyl; each R.sup.5 is independently hydrogen, halogen
or methyl; and each R.sup.6 is independently hydrogen, amino, or
halo.
15. The compound of claim 14 wherein R.sup.4 is alkylamino.
16. The compound of claim 1 according to any of the following
formulas: ##STR00179## ##STR00180##
17. The compound of claim 1 according to any of the following
formulas: ##STR00181##
18. The R.sub.P compound of claim 1.
19. The S.sub.P compound of claim 1.
20. A pharmaceutical composition comprising the compound of claim 1
and a pharmaceutically acceptable excipient, carrier or
diluent.
21. The pharmaceutical composition of claim 20, wherein the
composition is an oral formulation.
22. A method for the treatment of a host infected with a hepatitis
C virus, comprising the administration of an effective treatment
amount of a compound of claim 1.
23. The method of claim 22, wherein the host is a human.
24. The method of claim 22, wherein the administration directs a
substantial amount of the compound, or pharmaceutically acceptable
salt or stereoisomer thereof, to a liver of the host.
25. The method of claim 22, wherein the compound or composition is
administered in combination or alternation with a second anti-viral
agent selected from the group consisting of an interferon, a
nucleotide analogue, a polymerase inhibitor, an NS3 protease
inhibitor, an NS5A inhibitor, an entry inhibitor, a non-nucleoside
polymerase inhibitor, a cyclosporine immune inhibitor, an NS4A
antagonist, an NS4B-RNA binding inhibitor, a locked nucleic acid
mRNA inhibitor, a cyclophilin inhibitor, and combinations
thereof.
26. The method of claim 25, wherein the second anti-viral agent is
selected from the group consisting of telaprevir, bocepravir,
interferon alfacon-1, interferon alfa-2b, pegylated interferon
alpha 2a, pegylated interferon alpha 2b, ribavirin, and
combinations thereof.
27. The method of claim 25, wherein the second anti-viral agent is
selected from the group consisting of telaprevir, bocepravir,
interferon alfacon-1, interferon alfa-2b, pegylated interferon
alpha 2a, pegylated interferon alpha 2b, and combinations thereof,
and further wherein the administration is not in combination or
alternation with ribavirin.
28. A method of treating a liver disease or condition comprising
administering to a host in need thereof a compound comprising a
D-amino acid linked to a therapeutic moiety.
Description
FIELD
[0001] Provided herein are compounds, methods and pharmaceutical
compositions for use in treatment of liver diseases and conditions,
including viral infections such as hepatitis C virus infect ions in
hosts in need thereof. In certain embodiments, D-amino acids linked
to therapeutic nucleoside analogs are provided which display
remarkable efficacy and bioavailability for the treatment of, for
example, HCV infection in a human.
BACKGROUND
[0002] The hepatitis C virus (HCV) is the leading cause of chronic
liver disease worldwide. (Boyer, N. et al., J. Hepatol. 32:98-112,
2000). HCV causes a slow growing viral infection and is the major
cause of cirrhosis and hepatocellular carcinoma (Di Besceglie, A.
M. and Bacon, B. R., Scientific American, October: 80-85, 1999;
Boyer, N. et al., J. Hepatol. 32:98-112, 2000). It is estimated
there are about 130-170 million people with chronic hepatitis C
virus infection, and there are about 350,000 deaths from hepatitis
C-related liver diseases each year (Hepatitis C Fact Sheet, World
Health Organization Fact Sheet No. 164, June 2011). Cirrhosis
caused by chronic hepatitis C infection accounts for 8,000-12,000
deaths per year in the United States, and HCV infection is the
leading indication for liver transplantation.
[0003] HCV infection becomes chronic in about 75% of cases, with
many patients initially being asymptomatic. The first symptoms of
HCV infection are often those of chronic liver disease. About 20 to
30% of patients with chronic hepatitis due to HCV develop
cirrhosis, although this may take decades. Development of cirrhosis
due to HCV also increases the risk of hepatocellular cancer (The
Merck Manual Online, Chronic Hepatitis, available at
www.merckmanuals.com/professional/hepatic_and biliary
disorders/hepatitis/chronic hepatitis.html, last revision February
2007).
[0004] In light of the fact that HCV infection has reached epidemic
levels worldwide, and has tragic effects on the infected patient,
there remains a strong need to provide new effective pharmaceutical
agents to treat hepatitis C that have low toxicity to the host.
Further, given the rising threat of other flaviviridae infections,
there remains a strong need to provide new effective pharmaceutical
agents that have low toxicity to the host. Therefore, there is a
continuing need for effective treatments of flavivirus infections
and HCV infections.
SUMMARY
[0005] Provided herein are compounds useful for treatment of liver
diseases and conditions, for example, for the treatment of
flavivirus infections such as HCV infections.
[0006] The compounds comprise D-amino acids linked to therapeutic
moieties. In certain embodiments the D-amino acid compounds display
high tissue levels of active species, remarkable efficacy, or
bioavailability, or all, for the treatment of, for example, liver
disease and conditions in a human in need thereof. Some of the
compounds are based, in part, on the discovery that the active
component of certain therapeutic moieties linked to D-amino acids
can accumulate favorably in liver cells when the compounds are
administered to subjects.
[0007] In certain embodiments, the compounds provided herein are
useful in the prevention and treatment of Flaviviridae infections
and other related conditions such as anti-Flaviviridae antibody
positive and Flaviviridae-positive conditions, chronic liver
inflammation caused by HCV, cirrhosis, fibrosis, acute hepatitis,
fulminant hepatitis, chronic persistent hepatitis and fatigue.
These compounds or formulations can also be used prophylactically
to prevent or retard the progression of clinical illness in
individuals who are anti-Flaviviridae antibody or
Flaviviridae-antigen positive or who have been exposed to a
Flaviviridae. In particular embodiments, the Flaviviridae is
hepatitis C. In certain embodiments, the compounds are used to
treat any virus that replicates through an RNA-dependent RNA
polymerase.
[0008] A method for the treatment of a Flaviviridae infection in a
host, including a human, is also provided that includes
administering an effective amount of a compound provided herein,
administered either alone or in combination or alternation with
another anti-Flaviviridae agent, optionally in a pharmaceutically
acceptable carrier.
[0009] In certain embodiments, provided herein are compounds
according to formula (2001):
##STR00001##
or a pharmaceutically acceptable salt, solvate, stereoisomeric
form, tautomeric form or polymorphic form thereof, wherein: Base is
a nucleobase; A is S or O; W is S or O; X is a D-amino acid
residue, or an ester thereof; Y is hydrogen, --OR.sup.1,
--SR.sup.1, or --NR.sup.1R.sup.2; R.sup.b1 is alkyl, cycloalkyl,
--H, azido, cyano, or halogen; R.sup.b2 is --OH, --Cl, --F, --H,
azido, cyano, amino, or alkoxyl, or, in the alternative, R.sup.b1
and R.sup.b2, along with the carbon atom to which they are
attached, join to form a three-membered carbocyclic or heterocyclic
ring; R.sup.c is --H or --OH, or, in the alternative, Y and R.sup.c
join to form a six-membered heterocyclic ring wherein Y and R.sup.c
together represent a single divalent --O--; R.sup.d is --H, --F,
azido, or allenyl; or, in the alternative, R.sup.b2 and R.sup.d
join to form alkylene or substituted alkylene; R.sup.c is --H or
alkyl; each R.sup.1 is independently alkyl, cycloalkyl, aryl,
heteroaryl, arylalkyl, heteroarylalkyl, substituted alkyl or
hydantoinylalkyl; and each R.sup.2 is independently hydrogen or
alkyl.
[0010] In one aspect, the compounds provided herein are provided or
administered in combination with a second therapeutic agent, such
as one useful for the treatment or prevention of HCV infections.
Exemplary second therapeutic agents are provided in detail
elsewhere herein.
[0011] In another aspect, provided herein are pharmaceutical
compositions, single unit dosage forms, and kits suitable for use
in treating or preventing disorders such as HCV infections which
comprise a therapeutically or prophylactically effective amount of
a compound provided herein and a therapeutically or
prophylactically effective amount of a second therapeutic agent
such as one useful for the treatment or prevention of HCV
infections.
[0012] In certain embodiments, a method of treatment of a liver
disease or disorder is provided comprising administering to an
individual in need thereof a treatment effective amount of a
compound provided herein.
[0013] Flaviviridae which can be treated are, e.g., discussed
generally in Fields Virology, Fifth Ed., Editors: Knipe, D. M., and
Howley, P. M., Lippincott Williams & Wilkins Publishers,
Philadelphia, Pa., Chapters 33-35, 2006. In a particular embodiment
of the invention, the Flaviviridae is HCV. In an alternate
embodiment, the Flaviviridae is a flavivirus or pestivirus. In
certain embodiments, the Flaviviridae can be from any class of
Flaviviridae. In certain embodiments, the Flaviviridae is a
mammalian tick-borne virus. In certain embodiments, the
Flaviviridae is a seabird tick-borne virus. In certain embodiments,
the Flaviviridae is a mosquito-borne virus. In certain embodiments,
the Flaviviridae is an Aroa virus. In certain embodiments, the
Flaviviridae is a Dengue virus. In certain embodiments, the
Flaviviridae is a Japanese encephalitis virus. In certain
embodiments, the Flaviviridae is a Kokobera virus. In certain
embodiments, the Flaviviridae is a Ntaya virus. In certain
embodiments, the Flaviviridae is a Spondweni virus. In certain
embodiments, the Flaviviridae is a Yellow fever virus. In certain
embodiments, the Flaviviridae is a Entebbe virus. In certain
embodiments, the Flaviviridae is a Modoc virus. In certain
embodiments, the Flaviviridae is a Rio Bravo virus.
[0014] Specific flaviviruses include, without limitation:
Absettarov, Aedes, Alfuy, Alkhurma, Apoi, Aroa, Bagaza, Banzi,
Bukalasa bat, Bouboui, Bussuquara, Cacipacore, Calbertado, Carey
Island, Cell fusing agent, Cowbone Ridge, Culex, Dakar bat, Dengue
1, Dengue 2, Dengue 3, Dengue 4, Edge Hill, Entebbe bat, Gadgets
Gully, Hanzalova, Hypr, Ilheus, Israel turkey meningoencephalitis,
Japanese encephalitis, Jugra, Jutiapa, Kadam, Kamiti River, Karshi,
Kedougou, Kokobera, Koutango, Kumlinge, Kunjin, Kyasanur Forest
disease, Langat, Louping ill, Meaban, Modoc, Montana myotis
leukoencephalitis, Murray valley encephalitis, Nakiwogo, Naranjal,
Negishi, Ntaya, Omsk hemorrhagic fever, Phnom-Penh bat, Powassan,
Quang Binh, Rio Bravo, Rocio, Royal Farm, Russian spring-summer
encephalitis, Saboya, St. Louis encephalitis, Sal Vieja, San
Perlita, Saumarez Reef, Sepik, Sokuluk, Spondweni, Stratford,
Tembusu, Tick-borne encephalitis, Turkish sheep encephalitis,
Tyuleniy, Uganda S, Usutu, Wesselsbron, West Nile, Yaounde, Yellow
fever, Yokose, and Zika.
[0015] Pestiviruses which can be treated are discussed generally in
Fields Virology, Fifth Ed., Editors: Knipe, D. M., and Howley, P.
M., Lippincott Williams & Wilkins Publishers, Philadelphia,
Pa., Chapters 33-35, 2006. Specific pestiviruses include, without
limitation: bovine viral diarrhea virus ("BVDV"), classical swine
fever virus ("CSFV," also called hog cholera virus), and border
disease virus ("BDV").
DESCRIPTION OF EXEMPLARY EMBODIMENTS
[0016] Provided herein are compounds, compositions and methods
useful for treating liver disorders such as HCV infection in a
subject. Further provided are dosage forms useful for such
methods.
Definitions
[0017] When referring to the compounds provided herein, the
following terms have the following meanings unless indicated
otherwise. Unless defined otherwise, all technical and scientific
terms used herein have the same meaning as is commonly understood
by one of ordinary skill in the art. In the event that there is a
plurality of definitions for a term herein, those in this section
prevail unless stated otherwise.
[0018] The term "alkyl," as used herein, unless otherwise
specified, refers to a saturated straight or branched hydrocarbon.
In certain embodiments, the alkyl group is a primary, secondary, or
tertiary hydrocarbon. In certain embodiments, the alkyl group
includes one to ten carbon atoms, i.e., C.sub.1 to C.sub.10 alkyl.
In certain embodiments, the alkyl group is selected from the group
consisting of methyl, CF.sub.3, CCl.sub.3, CFCl.sub.2, CF.sub.2Cl,
ethyl, CH.sub.2CF.sub.3, CF.sub.2CF.sub.3, propyl, isopropyl,
butyl, isobutyl, secbutyl, t-butyl, pentyl, isopentyl, neopentyl,
hexyl, isohexyl, 3-methylpentyl, 2,2-dimethylbutyl, and
2,3-dimethylbutyl. The term includes both substituted and
unsubstituted alkyl groups, including halogenated alkyl groups. In
certain embodiments, the alkyl group is a fluorinated alkyl group.
Non-limiting examples of moieties with which the alkyl group can be
substituted are selected from the group consisting of halogen
(fluoro, chloro, bromo or iodo), hydroxyl, carbonyl, sulfanyl,
amino, alkylamino, arylamino, alkoxy, aryloxy, nitro, cyano,
sulfonic acid, sulfate, phosphonic acid, phosphate, or phosphonate,
either unprotected, or protected as necessary, as known to those
skilled in the art, for example, as taught in Greene, et al.,
Protective Groups in Organic Synthesis, John Wiley and Sons, Second
Edition, 1991, hereby incorporated by reference.
[0019] The term "lower alkyl," as used herein, and unless otherwise
specified, refers to a saturated straight or branched hydrocarbon
having one to six carbon atoms, i.e., C.sub.1 to C.sub.6 alkyl. In
certain embodiments, the lower alkyl group is a primary, secondary,
or tertiary hydrocarbon. The term includes both substituted and
unsubstituted moieties.
[0020] The term "upper alkyl," as used herein, and unless otherwise
specified, refers to a saturated straight or branched hydrocarbon
having seven to thirty carbon atoms, i.e., C.sub.7 to C.sub.30
alkyl. In certain embodiments, the upper alkyl group is a primary,
secondary, or tertiary hydrocarbon. The term includes both
substituted and unsubstituted moieties.
[0021] The term "cycloalkyl," as used herein, unless otherwise
specified, refers to a saturated cyclic hydrocarbon. In certain
embodiments, the cycloalkyl group may be a saturated, and/or
bridged, and/or non-bridged, and/or a fused bicyclic group. In
certain embodiments, the cycloalkyl group includes three to ten
carbon atoms, i.e., C.sub.3 to C.sub.10 cycloalkyl. In some
embodiments, the cycloalkyl has from 3 to 15 (C.sub.3-15), from 3
to 10 (C.sub.3-10), or from 3 to 7 (C.sub.3-7) carbon atoms. In
certain embodiments, the cycloalkyl group is cyclopropyl,
cyclobutyl, cyclopentyl, cyclohexyl, cyclohexylmethyl, cycloheptyl,
bicyclo[2.1.1]hexyl, bicyclo[2.2.1]heptyl, decalinyl or adamantyl.
The term includes both substituted and unsubstituted cycloalkyl
groups, including halogenated cycloalkyl groups. In certain
embodiments, the cycloalkyl group is a fluorinated cycloalkyl
group. Non-limiting examples of moieties with which the cycloalkyl
group can be substituted are selected from the group consisting of
halogen (fluoro, chloro, bromo or iodo), hydroxyl, carbonyl,
sulfanyl, amino, alkylamino, arylamino, alkoxy, aryloxy, nitro,
cyano, sulfonic acid, sulfate, phosphonic acid, phosphate, or
phosphonate, either unprotected, or protected as necessary.
[0022] "Cyclopropylene," as used herein, refers to a divalent
cyclopropane group. In certain embodiments, a cyclopropylene group
is of formula
##STR00002##
[0023] "Oxiranylene," as used herein, refers to a divalent oxirane
group. In certain embodiments, a oxiranylene group is of
formula
##STR00003##
[0024] "Alkylene" refers to divalent saturated aliphatic
hydrocarbon groups particularly having from one to eleven carbon
atoms which can be straight-chained or branched. In certain
embodiments, the alkylene group contains 1 to 10 carbon atoms. The
term includes both substituted and unsubstituted moieties. This
term is exemplified by groups such as methylene (--CH.sub.2--),
ethylene (--CH.sub.2CH.sub.2--), the propylene isomers (e.g.,
--CH.sub.2CH.sub.2CH.sub.2-- and --CH(CH.sub.3)CH.sub.2--) and the
like. The term includes groups having more than one double bond,
such as allenes comprising an allenylene (>C.dbd.C.dbd.C<) or
allenyl (>C.dbd.C=CH.sub.2) group. The term includes halogenated
alkylene groups. In certain embodiments, the alkylene group is a
fluorinated alkylene group. Non-limiting examples of moieties with
which the alkylene group can be substituted are selected from the
group consisting of halogen (fluoro, chloro, bromo or iodo),
hydroxyl, carbonyl, sulfanyl, amino, alkylamino, alkylaryl,
arylamino, alkoxy, aryloxy, nitro, cyano, sulfonic acid, sulfate,
phosphonic acid, phosphate, and phosphonate, either unprotected, or
protected as necessary.
[0025] "Alkenyl" refers to monovalent olefinically unsaturated
hydrocarbon groups, in certain embodiment, having up to about 11
carbon atoms, from 2 to 8 carbon atoms, or from 2 to 6 carbon
atoms, which can be straight-chained or branched and having at
least 1 or from 1 to 2 sites of olefinic unsaturation. The term
includes both substituted and unsubstituted moieties. Exemplary
alkenyl groups include ethenyl (i.e., vinyl, or --CH.dbd.CH.sub.2),
n-propenyl (--CH.sub.2CH.dbd.CH.sub.2), isopropenyl
(--C(CH.sub.3).dbd.CH.sub.2), and the like. The term includes
halogenated alkenyl groups. In certain embodiments, the alkenyl
group is a fluorinated alkenyl group. Non-limiting examples of
moieties with which the alkenyl group can be substituted are
selected from the group consisting of halogen (fluoro, chloro,
bromo or iodo), hydroxyl, carbonyl, sulfanyl, amino, alkylamino,
arylamino, alkoxy, aryloxy, nitro, cyano, sulfonic acid, sulfate,
phosphonic acid, phosphate, or phosphonate, either unprotected, or
protected as necessary.
[0026] The term "cycloalkenyl," as used herein, unless otherwise
specified, refers to an unsaturated cyclic hydrocarbon. In certain
embodiments, cycloalkenyl refers to mono- or multicyclic ring
systems that include at least one double bond. In certain
embodiments, the cycloalkenyl group may be a bridged, non-bridged,
and/or a fused bicyclic group. In certain embodiments, the
cycloalkyl group includes three to ten carbon atoms, i.e., C.sub.3
to C.sub.10 cycloalkyl. In some embodiments, the cycloalkenyl has
from 3 to 7 (C.sub.3-10), or from 4 to 7 (C.sub.3-7) carbon atoms.
The term includes both substituted and unsubstituted cycloalkenyl
groups, including halogenated cycloalkenyl groups. In certain
embodiments, the cycloalkenyl group is a fluorinated cycloalkenyl
group. Non-limiting examples of moieties with which the
cycloalkenyl group can be substituted are selected from the group
consisting of halogen (fluoro, chloro, bromo or iodo), hydroxyl,
carbonyl, sulfanyl, amino, alkylamino, arylamino, alkoxy, aryloxy,
nitro, cyano, sulfonic acid, sulfate, phosphonic acid, phosphate,
or phosphonate, either unprotected, or protected as necessary.
[0027] "Alkenylene" refers to divalent olefinically unsaturated
hydrocarbon groups, in certain embodiments, having up to about 11
carbon atoms or from 2 to 6 carbon atoms which can be
straight-chained or branched and having at least 1 or from 1 to 2
sites of olefinic unsaturation. This term is exemplified by groups
such as ethenylene (--CH.dbd.CH--), the propenylene isomers (e.g.,
--CH.dbd.CHCH.sub.2-- and --C(CH.sub.3).dbd.CH-- and
--CH.dbd.C(CH.sub.3)--) and the like. The term includes both
substituted and unsubstituted alkenylene groups, including
halogenated alkenylene groups. In certain embodiments, the
alkenylene group is a fluorinated alkenylene group. Non-limiting
examples of moieties with which the alkenylene group can be
substituted are selected from the group consisting of halogen
(fluoro, chloro, bromo or iodo), hydroxyl, carbonyl, sulfanyl,
amino, alkylamino, arylamino, alkoxy, aryloxy, nitro, cyano,
sulfonic acid, sulfate, phosphonic acid, phosphate, or phosphonate,
either unprotected, or protected as necessary.
[0028] "Alkynyl" refers to acetylenically unsaturated hydrocarbon
groups, in certain embodiments, having up to about 11 carbon atoms
or from 2 to 6 carbon atoms which can be straight-chained or
branched and having at least 1 or from 1 to 2 sites of alkynyl
unsaturation. Non-limiting examples of alkynyl groups include
acetylenic, ethynyl (--C.ident.CH), propargyl
(--CH.sub.2C.ident.CH), and the like. The term includes both
substituted and unsubstituted alkynyl groups, including halogenated
alkynyl groups. In certain embodiments, the alkynyl group is a
fluorinated alkynyl group. Non-limiting examples of moieties with
which the alkynyl group can be substituted are selected from the
group consisting of halogen (fluoro, chloro, bromo or iodo),
hydroxyl, carbonyl, sulfanyl, amino, alkylamino, arylamino, alkoxy,
aryloxy, nitro, cyano, sulfonic acid, sulfate, phosphonic acid,
phosphate, or phosphonate, either unprotected, or protected as
necessary.
[0029] The term "aryl," as used herein, and unless otherwise
specified, refers to phenyl, biphenyl or naphthyl. The term
includes both substituted and unsubstituted moieties. An aryl group
can be substituted with any described moiety, including, but not
limited to, one or more moieties selected from the group consisting
of halogen (fluoro, chloro, bromo or iodo), alkyl, haloalkyl,
hydroxyl, amino, alkylamino, arylamino, alkoxy, aryloxy, nitro,
cyano, sulfonic acid, sulfate, phosphonic acid, phosphate, or
phosphonate, either unprotected, or protected as necessary, as
known to those skilled in the art, for example, as taught in
Greene, et al., Protective Groups in Organic Synthesis, John Wiley
and Sons, Second Edition, 1991.
[0030] "Alkoxy" refers to the group --OR' where R' is alkyl or
cycloalkyl. Alkoxy groups include, by way of example, methoxy,
ethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, sec-butoxy,
n-pentoxy, n-hexoxy, 1,2-dimethylbutoxy, and the like.
[0031] "Alkoxycarbonyl" refers to a radical --C(O)-alkoxy where
alkoxy is as defined herein.
[0032] The term "heterocyclylalkyl" refers to a radical
-alkyl-heterocyclyl, where alkyl and heterocyclyl are as defined
herein.
[0033] The term "alkylcarbonylthioalkyl" refers to a radical
-alkyl-S--C(O)-alkyl, where alkyl is as defined herein.
[0034] The term "alkoxycarbonylalkyl" refers to a radical
-alkyl-C(O)-alkoxy, where alkyl and alkoxy are as defined
herein.
[0035] The term "arylalkoxycarbonylalkyl" refers to a radical
-alkyl-C(O)-alkoxy-aryl, where alkyl, alkoxy and aryl are as
defined herein.
[0036] The term "alkylcarbonylalkoxy(arylalkyl)" refers to a
radical -alkoxy(-alkyl-aryl)-C(O)-alkyl, where alkyl, alkoxy and
aryl are as defined herein.
[0037] The term "(alkoxycarbonyl)(alkoxycarbonylamino)alkyl" refers
to a radical -alkyl(-carbonyl-alkoxy)(-amino-carbonyl-alkoxy),
where alkyl, carbonyl, alkoxy, and amino are as defined herein.
[0038] The term "cycloalkylcarbonylalkoxyl" refers to a radical
-alkoxyl-C(O)-cycloalkyl, where alkoxyl and cycloalkyl are as
defined herein.
[0039] The term "alkoxycarbonylaminoalkylcarbonylthioalkyl" refers
to a radical -alkyl-S--C(O)--NH-alkyl-C(O)-alkoxy or
-alkyl-S--C(O)-alkyl-NH--C(O)-alkoxy, where alkyl and alkoxy are as
defined herein.
[0040] The term "hydroxylalkylcarbonylthioalkyl" refers to a
radical -alkyl-S--C(O)-alkyl-OH, where alkyl is as defined
herein.
[0041] The term "aminoalkylcarbonylalkoxycarbonylthioalkyl" refers
to a radical -alkyl-S--C(O)-alkoxy-C(O)--NH-alkyl or
-alkyl-S--C(O)-alkoxy-C(O)-alkyl-NH.sub.2, where alkyl and alkoxy
are as defined herein.
[0042] The term "alkoxycarbonylaminoalkyl" refers to a radical
-alkyl-NH--C(O)-alkoxy or --NH-alkyl-C(O)-alkoxy, where alkyl and
alkoxy are as defined herein.
[0043] The term "hydroxylalkyl" refers to a radical -alkyl-OH,
where alkyl is as defined herein.
[0044] The term "aminoalkylcarbonylalkoxyl" refers to a radical
-alkoxy-C(O)-alkyl-NH.sub.2 or -alkoxy-C(O)--NH-alkyl, where alkyl
and alkoxy are as defined herein.
[0045] "Amino" refers to the radical --NH.sub.2 or --NH--R, where
each R is independently alkyl, aryl, or cycloalkyl.
[0046] "Amino alcohol" refers to the radical --NHLOH, wherein L is
alkylene.
[0047] "Carboxyl" or "carboxy" refers to the radical --C(O)OH.
[0048] The term "alkylamino" or "arylamino" refers to an amino
group that has one or two alkyl or aryl substituents, respectively.
In certain embodiments, the alkyl substituent is upper alkyl. In
certain embodiments, the alkyl substituent is lower alkyl. In
another embodiment, the alkyl, upper alkyl, or lower alkyl is
unsubstituted.
[0049] "Halogen" or "halo" refers to chloro, bromo, fluoro or
iodo.
[0050] "Monoalkylamino" refers to the group alkyl-NR'--, wherein R'
is selected from hydrogen and alkyl or cycloalkyl.
[0051] "Thioalkoxy" refers to the group --SR' where R' is alkyl or
cycloalkyl.
[0052] The term "heterocyclyl" or "heterocyclic" refers to a
monovalent monocyclic non-aromatic ring system and/or multicyclic
ring system that contains at least one non-aromatic ring, wherein
one or more of the non-aromatic ring atoms are heteroatoms
independently selected from O, S, or N; and the remaining ring
atoms are carbon atoms. In certain embodiments, the heterocyclyl or
heterocyclic group has from 3 to 20, from 3 to 15, from 3 to 10,
from 3 to 8, from 4 to 7, or from 5 to 6 ring atoms. Heterocyclyl
groups are bonded to the rest of the molecule through the
non-aromatic ring. In certain embodiments, the heterocyclyl is a
monocyclic, bicyclic, tricyclic, or tetracyclic ring system, which
may include a fused or bridged ring system, and in which the
nitrogen or sulfur atoms may be optionally oxidized, the nitrogen
atoms may be optionally quaternized, and some rings may be
partially or fully saturated, or aromatic. The heterocyclyl may be
attached to the main structure at any heteroatom or carbon atom
which results in the creation of a stable compound. Examples of
such heterocyclic radicals include, but are not limited to,
azepinyl, benzodioxanyl, benzodioxolyl, benzofuranonyl,
benzopyranonyl, benzopyranyl, benzotetrahydrofuranyl,
benzotetrahydrothienyl, benzothiopyranyl, benzoxazinyl,
.beta.-carbolinyl, chromanyl, chromonyl, cinnolinyl, coumarinyl,
decahydroisoquinolinyl, dihydrobenzisothiazinyl,
dihydrobenzisoxazinyl, dihydrofuryl, dihydroisoindolyl,
dihydropyranyl, dihydropyrazolyl, dihydropyrazinyl,
dihydropyridinyl, dihydropyrimidinyl, dihydropyrrolyl, dioxolanyl,
1,4-dithianyl, furanonyl, imidazolidinyl, imidazolinyl, indolinyl,
isobenzotetrahydrofuranyl, isobenzotetrahydrothienyl, isochromanyl,
isocoumarinyl, isoindolinyl, isothiazolidinyl, isoxazolidinyl,
morpholinyl, octahydroindolyl, octahydroisoindolyl, oxazolidinonyl,
oxazolidinyl, oxiranyl, piperazinyl, piperidinyl, 4-piperidonyl,
pyrazolidinyl, pyrazolinyl, pyrrolidinyl, pyrrolinyl,
quinuclidinyl, tetrahydrofuryl, tetrahydroisoquinolinyl,
tetrahydropyranyl, tetrahydrothienyl, thiamorpholinyl,
thiazolidinyl, tetrahydroquinolinyl, and 1,3,5-trithianyl. In
certain embodiments, heterocyclic may also be optionally
substituted as described herein.
[0053] The term "heteroaryl" refers to refers to a monovalent
monocyclic aromatic group and/or multicyclic aromatic group that
contain at least one aromatic ring, wherein at least one aromatic
ring contains one or more heteroatoms independently selected from
O, S and N in the ring. Heteroaryl groups are bonded to the rest of
the molecule through the aromatic ring. Each ring of a heteroaryl
group can contain one or two O atoms, one or two S atoms, and/or
one to four N atoms, provided that the total number of heteroatoms
in each ring is four or less and each ring contains at least one
carbon atom. In certain embodiments, the heteroaryl has from 5 to
20, from 5 to 15, or from 5 to 10 ring atoms. Examples of
monocyclic heteroaryl groups include, but are not limited to,
furanyl, imidazolyl, isothiazolyl, isoxazolyl, oxadiazolyl,
oxadiazolyl, oxazolyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridyl,
pyrimidinyl, pyrrolyl, thiadiazolyl, thiazolyl, thienyl,
tetrazolyl, triazinyl and triazolyl. Examples of bicyclic
heteroaryl groups include, but are not limited to, benzofuranyl,
benzimidazolyl, benzoisoxazolyl, benzopyranyl, benzothiadiazolyl,
benzothiazolyl, benzothienyl, benzotriazolyl, benzoxazolyl,
furopyridyl, imidazopyridinyl, imidazothiazolyl, indolizinyl,
indolyl, indazolyl, isobenzofuranyl, isobenzothienyl, isoindolyl,
isoquinolinyl, isothiazolyl, naphthyridinyl, oxazolopyridinyl,
phthalazinyl, pteridinyl, purinyl, pyridopyridyl, pyrrolopyridyl,
quinolinyl, quinoxalinyl, quinazolinyl, thiadiazolopyrimidyl, and
thienopyridyl. Examples of tricyclic heteroaryl groups include, but
are not limited to, acridinyl, benzindolyl, carbazolyl,
dibenzofuranyl, perimidinyl, phenanthrolinyl, phenanthridinyl,
phenarsazinyl, phenazinyl, phenothiazinyl, phenoxazinyl and
xanthenyl. In certain embodiments, heteroaryl may also be
optionally substituted as described herein.
[0054] The term "alkylaryl" refers to an aryl group with an alkyl
substituent. The term "aralkyl" or "arylalkyl" refers to an alkyl
group with an aryl substituent.
[0055] The term "alkylheterocyclyl" refers to a heterocyclyl group
with an alkyl substituent. The term heterocyclylalkyl refers to an
alkyl group with a heterocyclyl substituent.
[0056] The term "alkylheteroaryl" refers to a heteroaryl group with
an alkyl substituent. The term heteroarylalkyl refers to an alkyl
group with a heteroaryl substituent.
[0057] The term "protecting group" as used herein and unless
otherwise defined refers to a group that is added to an oxygen,
nitrogen or phosphorus atom to prevent its further reaction or for
other purposes. A wide variety of oxygen and nitrogen protecting
groups are known to those skilled in the art of organic
synthesis.
[0058] "Pharmaceutically acceptable salt" refers to any salt of a
compound provided herein which retains its biological properties
and which is not toxic or otherwise undesirable for pharmaceutical
use. Such salts may be derived from a variety of organic and
inorganic counter-ions well known in the art. Such salts include,
but are not limited to: (1) acid addition salts formed with organic
or inorganic acids such as hydrochloric, hydrobromic, sulfuric,
nitric, phosphoric, sulfamic, acetic, trifluoroacetic,
trichloroacetic, propionic, hexanoic, cyclopentylpropionic,
glycolic, glutaric, pyruvic, lactic, malonic, succinic, sorbic,
ascorbic, malic, maleic, fumaric, tartaric, citric, benzoic,
3-(4-hydroxybenzoyl)benzoic, picric, cinnamic, mandelic, phthalic,
lauric, methanesulfonic, ethanesulfonic, 1,2-ethane-disulfonic,
2-hydroxyethanesulfonic, benzenesulfonic, 4-chlorobenzenesulfonic,
2-naphthalenesulfonic, 4-toluenesulfonic, camphoric,
camphorsulfonic, 4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic,
glucoheptonic, 3-phenylpropionic, trimethylacetic,
tert-butylacetic, lauryl sulfuric, gluconic, benzoic, glutamic,
hydroxynaphthoic, salicylic, stearic, cyclohexylsulfamic, quinic,
muconic acid and the like acids; or (2) base addition salts formed
when an acidic proton present in the parent compound either (a) is
replaced by a metal ion, e.g., an alkali metal ion, an alkaline
earth ion or an aluminum ion, or alkali metal or alkaline earth
metal hydroxides, such as sodium, potassium, calcium, magnesium,
aluminum, lithium, zinc, and barium hydroxide, ammonia or (b)
coordinates with an organic base, such as aliphatic, alicyclic, or
aromatic organic amines, such as ammonia, methylamine,
dimethylamine, diethylamine, picoline, ethanolamine,
diethanolamine, triethanolamine, ethylenediamine, lysine, arginine,
ornithine, choline, N,N'-dibenzylethylene-diamine, chloroprocaine,
diethanolamine, procaine, N-benzylphenethylamine, N-methylglucamine
piperazine, tris(hydroxymethyl)-aminomethane, tetramethylammonium
hydroxide, and the like.
[0059] Pharmaceutically acceptable salts further include, by way of
example only and without limitation, sodium, potassium, calcium,
magnesium, ammonium, tetraalkylammonium and the like, and when the
compound contains a basic functionality, salts of non-toxic organic
or inorganic acids, such as hydrohalides, e.g. hydrochloride and
hydrobromide, sulfate, phosphate, sulfamate, nitrate, acetate,
trifluoroacetate, trichloroacetate, propionate, hexanoate,
cyclopentylpropionate, glycolate, glutarate, pyruvate, lactate,
malonate, succinate, sorbate, ascorbate, malate, maleate, fumarate,
tartarate, citrate, benzoate, 3-(4-hydroxybenzoyl)benzoate,
picrate, cinnamate, mandelate, phthalate, laurate, methanesulfonate
(mesylate), ethanesulfonate, 1,2-ethane-disulfonate,
2-hydroxyethanesulfonate, benzenesulfonate (besylate),
4-chlorobenzenesulfonate, 2-naphthalenesulfonate,
4-toluenesulfonate, camphorate, camphorsulfonate,
4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylate, glucoheptonate,
3-phenylpropionate, trimethylacetate, tert-butylacetate, lauryl
sulfate, gluconate, benzoate, glutamate, hydroxynaphthoate,
salicylate, stearate, cyclohexylsulfamate, quinate, muconate and
the like.
[0060] As used herein, the term "nucleobase" refers to the base
portion of a nucleoside or nucleotide. In certain embodiments, a
nucleobase is a purine or pyrimidine base, as defined herein.
[0061] The term "purine" or "pyrimidine" base refers to, but is not
limited to, adenine, N.sup.6-alkylpurines, N.sup.6-acylpurines
(wherein acyl is C(O)(alkyl, aryl, alkylaryl, or arylalkyl),
N.sup.6-benzylpurine, N.sup.6-halopurine, N.sup.6-vinylpurine,
N.sup.6-acetylenic purine, N.sup.6-acyl purine,
N.sup.6-hydroxyalkyl purine, N.sup.6-alkylaminopurine,
N.sup.6-thioalkyl purine, N.sup.2-alkylpurines,
N.sup.2-alkyl-6-thiopurines, thymine, cytosine, 5-fluorocytosine,
5-methylcytosine, 6-azapyrimidine, including 6-azacytosine, 2-
and/or 4-mercaptopyrmidine, uracil, 5-halouracil, including
5-fluorouracil, C.sup.5-alkylpyrimidines,
C.sup.5-benzylpyrimidines, C.sup.5-halopyrimidines,
C.sup.5-vinylpyrimidine, C.sup.5-acetylenic pyrimidine,
C.sup.5-acyl pyrimidine, C.sup.5-hydroxyalkyl purine,
C.sup.5-amidopyrimidine, C.sup.5-cyanopyrimidine,
C.sup.5-iodopyrimidine, C.sup.6-iodo-pyrimidine, C.sup.5--Br-vinyl
pyrimidine, C.sup.6--Br-vinyl pyrimidine, C.sup.5-nitropyrimidine,
C.sup.5-amino-pyrimidine, N.sup.2-alkylpurines,
N.sup.2-alkyl-6-thiopurines, 5-azacytidinyl, 5-azauracilyl,
triazolopyridinyl, imidazolopyridinyl, pyrrolopyrimidinyl, and
pyrazolopyrimidinyl. Purine bases include, but are not limited to,
guanine, adenine, hypoxanthine, 7-deazaguanine, 7-deazaadenine,
2,6-diaminopurine, and 6-chloropurine. Functional oxygen and
nitrogen groups on the base can be protected as necessary or
desired. Suitable protecting groups are well known to those skilled
in the art, and include trimethylsilyl, dimethylhexylsilyl,
t-butyldimethylsilyl, and t-butyldiphenylsilyl, trityl, alkyl
groups, and acyl groups such as acetyl and propionyl,
methanesulfonyl, and p-toluenesulfonyl.
[0062] The term "acyl" or "O-linked ester" refers to a group of the
formula C(O)R', wherein R' is alkyl or cycloalkyl (including lower
alkyl), carboxylate reside of amino acid, aryl including phenyl,
alkaryl, arylalkyl including benzyl, alkoxyalkyl including
methoxymethyl, aryloxyalkyl such as phenoxymethyl; or substituted
alkyl (including lower alkyl), aryl including phenyl optionally
substituted with chloro, bromo, fluoro, iodo, C.sub.1 to C.sub.4
alkyl or C.sub.1 to C.sub.4 alkoxy, sulfonate esters such as alkyl
or arylalkyl sulphonyl including methanesulfonyl, the mono, di or
triphosphate ester, trityl or monomethoxy-trityl, substituted
benzyl, alkaryl, arylalkyl including benzyl, alkoxyalkyl including
methoxymethyl, aryloxyalkyl such as phenoxymethyl. Aryl groups in
the esters optimally comprise a phenyl group. In particular, acyl
groups include acetyl, trifluoroacetyl, methylacetyl,
cyclpropylacetyl, propionyl, butyryl, hexanoyl, heptanoyl,
octanoyl, neo-heptanoyl, phenylacetyl, 2-acetoxy-2-phenylacetyl,
diphenylacetyl,
.alpha.-methoxy-.alpha.-trifluoromethyl-phenylacetyl, bromoacetyl,
2-nitro-benzeneacetyl, 4-chloro-benzeneacetyl,
2-chloro-2,2-diphenylacetyl, 2-chloro-2-phenylacetyl,
trimethylacetyl, chlorodifluoroacetyl, perfluoroacetyl,
fluoroacetyl, bromodifluoroacetyl, methoxyacetyl,
2-thiopheneacetyl, chlorosulfonylacetyl, 3-methoxyphenylacetyl,
phenoxyacetyl, tert-butylacetyl, trichloroacetyl,
monochloro-acetyl, dichloroacetyl, 7H-dodecafluoro-heptanoyl,
perfluoro-heptanoyl, 7H-dodeca-fluoroheptanoyl,
7-chlorododecafluoro-heptanoyl, 7-chlorododecafluoro-heptanoyl,
7H-dodecafluoroheptanoyl, 7H-dodeca-fluoroheptanoyl,
nonafluoro-3,6-dioxa-heptanoyl, nonafluoro-3,6-dioxaheptanoyl,
perfluoroheptanoyl, methoxybenzoyl, methyl
3-amino-5-phenylthiophene-2-carboxyl,
3,6-dichloro-2-methoxy-benzoyl,
4-(1,1,2,2-tetrafluoro-ethoxy)-benzoyl, 2-bromo-propionyl,
omega-aminocapryl, decanoyl, n-pentadecanoyl, stearyl,
3-cyclopentyl-propionyl, 1-benzene-carboxyl, O-acetylmandelyl,
pivaloyl acetyl, 1-adamantane-carboxyl, cyclohexane-carboxyl,
2,6-pyridinedicarboxyl, cyclopropane-carboxyl,
cyclobutane-carboxyl, perfluorocyclohexyl carboxyl,
4-methylbenzoyl, chloromethyl isoxazolyl carbonyl,
perfluorocyclohexyl carboxyl, crotonyl,
1-methyl-1H-indazole-3-carbonyl, 2-propenyl, isovaleryl,
1-pyrrolidinecarbonyl, 4-phenylbenzoyl.
[0063] The term "amino acid" refers to naturally occurring and
synthetic .alpha., .beta. .gamma. or .delta. amino acids, and
includes but is not limited to, amino acids found in proteins, i.e.
glycine, alanine, valine, leucine, isoleucine, methionine,
phenylalanine, tryptophan, proline, serine, threonine, cysteine,
tyrosine, asparagine, glutamine, aspartate, glutamate, lysine,
arginine and histidine. In certain embodiments, the amino acid is
in the L-configuration. Alternatively, the amino acid can be a
derivative of alanyl, valinyl, leucinyl, isoleuccinyl, prolinyl,
phenylalaninyl, tryptophanyl, methioninyl, glycinyl, serinyl,
threoninyl, cysteinyl, tyrosinyl, asparaginyl, glutaminyl,
aspartoyl, glutaroyl, lysinyl, argininyl, histidinyl,
.beta.-alanyl, .beta.-valinyl, .beta.-leucinyl,
.beta.-isoleuccinyl, .beta.-prolinyl, .beta.-phenylalaninyl,
.beta.-tryptophanyl, .beta.-methioninyl, .beta.-glycinyl,
.beta.-serinyl, .beta.-threoninyl, .beta.-cysteinyl,
.beta.-tyrosinyl, .beta.-asparaginyl, .beta.-glutaminyl,
.beta.-aspartoyl, .beta.-glutaroyl, .beta.-lysinyl,
.beta.-argininyl or .beta.-histidinyl.
[0064] The term "amino acid derivative" refers to a group derivable
from a naturally or non-naturally occurring amino acid, as
described and exemplified herein. Amino acid derivatives are
apparent to those of skill in the art and include, but are not
limited to, ester, amino alcohol, amino aldehyde, amino lactone,
and N-methyl derivatives of naturally and non-naturally occurring
amino acids. In an embodiment, an amino acid derivative is provided
as a substituent of a compound described herein, wherein the
substituent is --NH-G(S.sub.C)--C(O)-Q or --OC(O)G(S.sub.C)-Q,
wherein Q is --SR, --NRR or alkoxyl, R is hydrogen or alkyl,
S.sub.C is a side chain of a naturally occurring or non-naturally
occurring amino acid and G is C.sub.1-C.sub.2 alkyl. In certain
embodiments, G is C.sub.1 alkyl and S.sub.C is selected from the
group consisting of hydrogen, alkyl, heteroalkyl, arylalkyl and
heteroarylalkyl.
[0065] As used herein, the term "hydantoinyl" refers to the
group
##STR00004##
where R.sup.XX and R.sup.YY are each independently hydrogen or
lower alkyl.
[0066] As used herein, the term "hydantoinylalkyl" refers to the
group -alkyl-hydantoinyl, where alkyl and hydantoinyl are as
described herein.
[0067] The term "substantially free of" or "substantially in the
absence of" with respect to a nucleoside composition refers to a
nucleoside composition that includes at least 85 or 90% by weight,
in certain embodiments 95%, 98%, 99% or 100% by weight, of the
designated enantiomer of that nucleoside. In certain embodiments,
in the methods and compounds provided herein, the compounds are
substantially free of enantiomers.
[0068] Similarly, the term "isolated" with respect to a nucleoside
composition refers to a nucleoside composition that includes at
least 85, 90%, 95%, 98%, 99% to 100% by weight, of the nucleoside,
the remainder comprising other chemical species or enantiomers.
[0069] "Solvate" refers to a compound provided herein or a salt
thereof, that further includes a stoichiometric or
non-stoichiometric amount of solvent bound by non-covalent
intermolecular forces. Where the solvent is water, the solvate is a
hydrate.
[0070] "Isotopic composition" refers to the amount of each isotope
present for a given atom, and "natural isotopic composition" refers
to the naturally occurring isotopic composition or abundance for a
given atom. Atoms containing their natural isotopic composition may
also be referred to herein as "non-enriched" atoms. Unless
otherwise designated, the atoms of the compounds recited herein are
meant to represent any stable isotope of that atom. For example,
unless otherwise stated, when a position is designated specifically
as "H" or "hydrogen," the position is understood to have hydrogen
at its natural isotopic composition.
[0071] "Isotopic enrichment" refers to the percentage of
incorporation of an amount of a specific isotope at a given atom in
a molecule in the place of that atom's natural isotopic abundance.
For example, deuterium enrichment of 1% at a given position means
that 1% of the molecules in a given sample contain deuterium at the
specified position. Because the naturally occurring distribution of
deuterium is about 0.0156%, deuterium enrichment at any position in
a compound synthesized using non-enriched starting materials is
about 0.0156%. The isotopic enrichment of the compounds provided
herein can be determined using conventional analytical methods
known to one of ordinary skill in the art, including mass
spectrometry and nuclear magnetic resonance spectroscopy.
[0072] "Isotopically enriched" refers to an atom having an isotopic
composition other than the natural isotopic composition of that
atom. "Isotopically enriched" may also refer to a compound
containing at least one atom having an isotopic composition other
than the natural isotopic composition of that atom.
[0073] As used herein, "alkyl," "cycloalkyl," "alkenyl,"
"cycloalkenyl," "alkynyl," "aryl," "alkoxy," "alkoxycarbonyl,"
"amino," "carboxyl," "alkylamino," "arylamino," "thioalkyoxy,"
"heterocyclyl," "heteroaryl," "alkylheterocyclyl,"
"alkylheteroaryl," "acyl," "aralkyl," "alkaryl," "purine,"
"pyrimidine," "carboxyl" and "amino acid" groups optionally
comprise deuterium at one or more positions where hydrogen atoms
are present, and wherein the deuterium composition of the atom or
atoms is other than the natural isotopic composition.
[0074] Also as used herein, "alkyl," "cycloalkyl," "alkenyl,"
"cycloalkenyl," "alkynyl," "aryl," "alkoxy," "alkoxycarbonyl,"
"carboxyl," "alkylamino," "arylamino," "thioalkyoxy,"
"heterocyclyl," "heteroaryl," "alkylheterocyclyl,"
"alkylheteroaryl," "acyl," "aralkyl," "alkaryl," "purine,"
"pyrimidine," "carboxyl" and "amino acid" groups optionally
comprise carbon-13 at an amount other than the natural isotopic
composition.
[0075] As used herein, EC.sub.50 refers to a dosage, concentration
or amount of a particular test compound that elicits a
dose-dependent response at 50% of maximal expression of a
particular response that is induced, provoked or potentiated by the
particular test compound.
[0076] As used herein, the IC.sub.50 refers to an amount,
concentration or dosage of a particular test compound that achieves
a 50% inhibition of a maximal response in an assay that measures
such response.
[0077] The term "host," as used herein, refers to any unicellular
or multicellular organism in which the virus can replicate,
including cell lines and animals, and in certain embodiments, a
human. Alternatively, the host can be carrying a part of the
Flaviviridae viral genome, whose replication or function can be
altered by the compounds of the present invention. The term host
specifically includes infected cells, cells transfected with all or
part of the Flaviviridae genome and animals, in particular,
primates (including chimpanzees) and humans. In most animal
applications of the present invention, the host is a human patient.
Veterinary applications, in certain indications, however, are
clearly anticipated by the present invention (such as
chimpanzees).
[0078] As used herein, the terms "subject" and "patient" are used
interchangeably herein. The terms "subject" and "subjects" refer to
an animal, such as a mammal including a non-primate (e.g., a cow,
pig, horse, cat, dog, rat, and mouse) and a primate (e.g., a monkey
such as a cynomolgous monkey, a chimpanzee and a human), and for
example, a human. In certain embodiments, the subject is refractory
or non-responsive to current treatments for hepatitis C infection.
In another embodiment, the subject is a farm animal (e.g., a horse,
a cow, a pig, etc.) or a pet (e.g., a dog or a cat). In certain
embodiments, the subject is a human.
[0079] As used herein, the terms "therapeutic agent" and
"therapeutic agents" refer to any agent(s) which can be used in the
treatment or prevention of a disorder or one or more symptoms
thereof. In certain embodiments, the term "therapeutic agent"
includes a compound provided herein. In certain embodiments, a
therapeutic agent is an agent which is known to be useful for, or
has been or is currently being used for the treatment or prevention
of a disorder or one or more symptoms thereof.
[0080] "Therapeutically effective amount" refers to an amount of a
compound or composition that, when administered to a subject for
treating a disease, is sufficient to effect such treatment for the
disease. A "therapeutically effective amount" can vary depending
on, inter alia, the compound, the disease and its severity, and the
age, weight, etc., of the subject to be treated.
[0081] "Treating" or "treatment" of any disease or disorder refers,
in certain embodiments, to ameliorating a disease or disorder that
exists in a subject. In another embodiment, "treating" or
"treatment" includes ameliorating at least one physical parameter,
which may be indiscernible by the subject. In yet another
embodiment, "treating" or "treatment" includes modulating the
disease or disorder, either physically (e.g., stabilization of a
discernible symptom) or physiologically (e.g., stabilization of a
physical parameter) or both. In yet another embodiment, "treating"
or "treatment" includes delaying the onset of the disease or
disorder.
[0082] As used herein, the terms "prophylactic agent" and
"prophylactic agents" as used refer to any agent(s) which can be
used in the prevention of a disorder or one or more symptoms
thereof. In certain embodiments, the term "prophylactic agent"
includes a compound provided herein. In certain other embodiments,
the term "prophylactic agent" does not refer a compound provided
herein. For example, a prophylactic agent is an agent which is
known to be useful for, or has been or is currently being used to
prevent or impede the onset, development, progression and/or
severity of a disorder.
[0083] As used herein, the phrase "prophylactically effective
amount" refers to the amount of a therapy (e.g., prophylactic
agent) which is sufficient to result in the prevention or reduction
of the development, recurrence or onset of one or more symptoms
associated with a disorder, or to enhance or improve the
prophylactic effect(s) of another therapy (e.g., another
prophylactic agent).
[0084] Compounds
[0085] Provided herein are D-amino acid compounds useful for the
treatment of liver diseases and conditions, for example,
Flaviviridae infections such as HCV infection. The D-amino acid
compounds can be formed as described herein and used for the
treatment of, for example, Flaviviridae infections such as HCV
infection.
[0086] In certain embodiments, provided herein are compounds
according to formula (2001):
##STR00005##
or a pharmaceutically acceptable salt, solvate, stereoisomeric
form, tautomeric form or polymorphic form thereof, wherein: Base is
a nucleobase; A is S or O; W is S or O; X is a D-amino acid
residue, or an ester thereof; Y is hydrogen, --OR.sup.1,
--SR.sup.1, or --NR.sup.1R.sup.2; R.sup.b1 is alkyl, cycloalkyl,
--H, azido, cyano, or halogen; R.sup.b2 is --OH, --Cl, --F, --H,
azido, cyano, amino, or alkoxyl, or, in the alternative, R.sup.b1
and R.sup.b2, along with the carbon atom to which they are
attached, join to form a three-membered carbocyclic or heterocyclic
ring; R.sup.c is --H or --OH, or, in the alternative, Y and R.sup.c
join to form a six-membered heterocyclic ring wherein Y and R.sup.c
together represent a single divalent --O--; R.sup.d is --H, --F,
azido, or allenyl; or, in the alternative, R.sup.b2 and R.sup.d
join to form alkylene or substituted alkylene; R.sup.e is --H or
alkyl; each R.sup.1 is independently alkyl, cycloalkyl, aryl,
heteroaryl, arylalkyl, heteroarylalkyl, substituted alkyl or
hydantoinylalkyl; and each R.sup.2 is independently hydrogen or
alkyl. In certain embodiments of Formula (2001), R.sup.b1 and
R.sup.b2, along with the carbon atom to which they are attached,
join to form a three-membered carbocyclic or heterocyclic ring. In
certain embodiments, R.sup.b1 and R.sup.b2, along with the carbon
atom to which they are attached, join to form cyclopropylene or
oxiranylene.
[0087] In certain embodiments, provided herein are compounds
according to Formula (I):
##STR00006##
or a pharmaceutically acceptable salt, solvate, stereoisomeric
form, tautomeric form or polymorphic form thereof.
[0088] In certain embodiments, provided herein are compounds
according to Formula (Ia) or (Ib):
##STR00007##
or a pharmaceutically acceptable salt, solvate, stereoisomeric
form, tautomeric form or polymorphic form thereof. In certain
embodiments, R.sub.P compounds are provided. In certain
embodiments, S.sub.P compounds are provided.
[0089] In Formula (I), (Ia) or (Ib), Base is any nucleobase known
to those of skill in the art. Base can be a naturally occurring
nucleobase, or it can be a non-natural nucleobase known to those of
skill in the art. In certain embodiments, Base is a purine or
pyrimidine nucleobase. In particular embodiments, Base is
guanosine, uracil, cytosine, adenine or a derivative thereof.
Exemplary nucleobases are described herein.
[0090] In Formula (I), (Ia) or (Ib), W is S or O. In certain
embodiments, W is S. In certain embodiments, W is O.
[0091] In Formula (I), (Ia) or (Ib), X is a D-amino acid residue,
or an ester thereof. X can be any D-amino acid residue known to
those of skill in the art. X can be the D-enantiomer of a naturally
occurring amino acid residue, or X can be the D-enantiomer of a
non-natural amino acid residue. In particular embodiments, X is
D-alanine, D-phenylalanine, D-valine or D-terleucine. In preferred
embodiments, X is D-alanine. The ester can be any ester known to
those of skill in the art. In particular embodiments, the ester is
an alkyl ester. In certain embodiments, the ester is selected from
the group consisting of ethyl ester, propyl ester, n-propyl ester,
isopropyl ester, butyl ester, t-butyl ester, n-butyl ester, and
cyclopentyl ester.
[0092] In Formula (I), (Ia) or (Ib), Y is hydrogen, --OR.sup.1,
--SR.sup.1, or --NR.sup.1R.sup.2. Each R.sup.1 is independently
alkyl, cycloalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl,
substituted alkyl or hydantoinylalkyl. In certain embodiments, each
R.sup.1 is independently alkyl, cycloalkyl, heterocyclylalkyl,
aryl, heteroaryl, arylalkyl, heteroarylalkyl,
alkylcarbonylthioalkyl, alkoxycarbonylalkyl,
arylalkoxycarbonylalkyl, alkylcarbonylalkoxy(arylalkyl),
(alkoxycarbonyl)(alkoxycarbonylamino)alkyl,
cycloalkylcarbonylalkoxyl,
alkoxycarbonylaminoalkylcarbonylthioalkyl,
hydroxylalkylcarbonylthioalkyl,
aminoalkylcarbonylalkoxycarbonylthioalkyl, or hydantoinylalkyl.
Each R.sup.2 is independently hydrogen or alkyl. In particular
embodiments, R.sup.2 is H.
[0093] In Formula (I), (Ia) or (Ib), R.sup.c is --H or --OH. In the
alternative, in certain embodiments, Y and R.sup.c join to form a
six-membered heterocyclic ring wherein Y and R.sup.c together
represent a single divalent --O--. In these embodiments, the
compounds comprise a cyclic phosphate group linking the 3' and 5'
carbons of the nucleoside sugar.
[0094] In Formula (I), (Ia) or (Ib), R.sup.b1 is --CH.sub.3, --H,
azido, cyano, or halogen. In certain embodiments, R.sup.b1 is
--CH.sub.3. Also in Formula (I), (Ia) or (Ib), R.sup.b2 is --OH,
--Cl, --F, --H, azido, cyano, amino, or alkoxyl. In certain
embodiments, R.sup.b2 is --OH. In certain embodiments, R.sup.b2 is
--Cl. In certain embodiments, R.sup.b2 is --F. In certain
embodiments of Formula (I), (Ia) or (Ib), R.sup.b1 and R.sup.b2,
along with the carbon atom to which they are attached, join to form
a three-membered carbocyclic or heterocyclic ring. In certain
embodiments, R.sup.b1 and R.sup.b2, along with the carbon atom to
which they are attached, join to form cyclopropylene or
oxiranylene.
[0095] In Formula (I), (Ia) or (Ib), R.sup.d is --H, --F, azido, or
allenyl. In certain embodiments, R.sup.d is --H. In the
alternative, in certain embodiments, R.sup.b2 and R.sup.d join to
form alkylene or substituted alkylene. In particular embodiments,
R.sup.b2 and R.sup.d form --CH.sub.2--O--. In particular
embodiments, the --CH.sub.2-- is linked to the 4' carbon of the
sugar, and the --O-- is linked to the 2' carbon of the sugar.
[0096] In Formula (I), (Ia) or (Ib), R.sup.e is --H or alkyl. In
particular embodiments, R.sub.e is --H.
[0097] In certain embodiments according to Formula (I), (Ia) or
(Ib), R.sup.e is --H; R.sup.d is --H; R.sup.b1 is --CH.sub.3;
R.sup.b2 is --OH, --Cl or --F; R.sup.c is --H or --OH; and R.sup.2
is H. In particular embodiments, R.sup.e is --H; R.sup.d is --H;
R.sup.b1 is --CH.sub.3; R.sup.b2 is --OH, --Cl or --F; R.sup.c is
--H or --OH; R.sup.2 is H; and Base is selected from guanosine,
uracil, cytosine, adenine or a derivative thereof. In particular
embodiments, R.sup.e is --H; R.sup.d is --H; R.sup.b1 is
--CH.sub.3; R.sup.b2 is --OH, --Cl or --F; R.sup.c is --H or --OH;
R.sup.2 is H; Base is selected from guanosine, uracil, cytosine,
adenine or a derivative thereof; and X is D-alanine, or an ester
thereof. In certain embodiments according to this paragraph, Y is
alkyl, aryl, arylalkyl, cycloalkyl or
##STR00008##
wherein R.sup.3 is alkyl, alkoxycarbonylaminoalkyl, hydroxylalkyl,
or aminoalkylcarbonylalkoxyl.
[0098] In certain embodiments according to Formula (I), (Ia) or
(Ib), R.sup.e is --H; R.sup.b2 and R.sup.d form --CH.sub.2--O--;
R.sup.b1 is --CH.sub.3; R.sup.c is --H or --OH; and R.sup.2 is H.
In certain embodiments, R.sup.e is --H; R.sup.b2 and R.sup.d form
--CH.sub.2--O--; R.sup.b1 is --CH.sub.3; R.sup.c is --H or --OH;
R.sup.2 is H; and Base is selected from guanosine, uracil,
cytosine, adenine or a derivative thereof. In certain embodiments,
R.sup.e is --H; R.sup.b2 and R.sup.d form --CH.sub.2--O--; R.sup.b1
is --CH.sub.3; R.sup.c is --H or --OH; R.sup.2 is H; Base is
selected from guanosine, uracil, cytosine, adenine or a derivative
thereof; and X is D-alanine, or an ester thereof. In certain
embodiments according to this paragraph, Y is alkyl, aryl,
arylalkyl, cycloalkyl or
##STR00009##
wherein R.sup.3 is alkyl, alkoxycarbonylaminoalkyl, hydroxylalkyl,
or aminoalkylcarbonylalkoxyl.
[0099] In certain embodiments according to Formula (I), (Ia) or
(Ib), R.sup.e is --H; R.sup.b2 and R.sup.d form
--CH.sub.2CH.sub.2--; R.sup.b1 is --CH.sub.3; R.sup.c is --H or
--OH; and R.sup.2 is H. In certain embodiments, R.sup.e is --H;
R.sup.b2 and R.sup.d form --CH.sub.2CH.sub.2--; R.sup.b1 is
--CH.sub.3; R.sup.c is --H or --OH; R.sup.2 is H; and Base is
selected from guanosine, uracil, cytosine, adenine or a derivative
thereof. In certain embodiments, R.sup.e is --H; R.sup.b2 and
R.sup.d form --CH.sub.2CH.sub.2--; R.sup.b1 is --CH.sub.3; R.sup.e
is --H or --OH; R.sup.2 is H; Base is selected from guanosine,
uracil, cytosine, adenine or a derivative thereof; and X is
D-alanine, or an ester thereof. In certain embodiments according to
this paragraph, Y is alkyl, aryl, arylalkyl, cycloalkyl or
##STR00010##
wherein R.sup.3 is alkyl, alkoxycarbonylaminoalkyl, hydroxylalkyl,
or aminoalkylcarbonylalkoxyl.
[0100] In certain embodiments according to Formula (I), (Ia) or
(Ib), R.sup.e is --H; R.sup.d is --H; R.sup.b1 is --CH.sub.3;
R.sup.b2 is --OH, --Cl or --F; R.sup.2 is H; and Y and R.sup.e
together represent a single divalent --O--. In particular
embodiments, R.sup.e is --H; R.sup.d is --H; R.sup.b1 is
--CH.sub.3; R.sup.b2 is --OH, --Cl or --F; R.sup.2 is H; Y and
R.sup.c together represent a single divalent --O--; and Base is
selected from guanosine, uracil, cytosine, adenine or a derivative
thereof. In particular embodiments, R.sup.e is --H; R.sup.d is --H;
R.sup.b1 is --CH.sub.3; R.sup.b2 is --OH, --Cl or --F; R.sup.2 is
H; Y and R.sup.c together represent a single divalent --O--; and
Base is selected from guanosine, uracil, cytosine, adenine or a
derivative thereof; and X is D-alanine, or an ester thereof. In
certain embodiments according to this paragraph, Y is alkyl, aryl,
arylalkyl, cycloalkyl or
##STR00011##
wherein R.sup.3 is alkyl, alkoxycarbonylaminoalkyl, hydroxylalkyl,
or aminoalkylcarbonylalkoxyl.
[0101] In certain embodiments, provided herein are compounds
according to Formula (II):
##STR00012##
or a pharmaceutically acceptable salt, solvate, stereoisomeric
form, tautomeric form or polymorphic form thereof.
[0102] In certain embodiments, provided herein are compounds
according to Formula (IIa) or (IIb):
##STR00013##
or a pharmaceutically acceptable salt, solvate, stereoisomeric
form, tautomeric form or polymorphic form thereof. In certain
embodiments, R.sub.P compounds are provided. In certain
embodiments, S.sub.P compounds are provided.
[0103] In Formulas (II), (IIa) and (IIb), the symbols R.sup.b1,
R.sup.b2, R.sup.c, R.sup.d, R.sup.e, W, Y and Base have the
meanings provided above.
[0104] In Formulas (II), (IIa) and (IIb), each R.sup.10 is
independently alkyl, arylalkyl, heteroarylalkyl or a side chain of
a naturally occurring amino acid, other than hydrogen. In
particular embodiments, R.sup.10 is methyl, isopropyl, t-butyl or
benzyl.
[0105] In Formulas (II), (IIa) and (IIb), each R.sup.11 is
independently alkyl, cycloalkyl or --H. In particular embodiments,
each R.sup.11 is ethyl, propyl, isopropyl, n-propyl, butyl,
n-butyl, t-butyl or cyclopentyl.
[0106] In certain embodiments of Formulas (II), (IIa) and (IIb),
R.sup.c is --H or --OH. In the alternative, in certain embodiments,
Y and R.sup.c join to form a six-membered heterocyclic ring wherein
Y and R.sup.c together represent a single divalent --O--. In these
embodiments, the compounds comprise a cyclic phosphate group
linking the 3' and 5' carbons of the nucleoside sugar.
[0107] In certain embodiments of Formulas (II), (IIa) and (IIb),
R.sup.b1 is --CH.sub.3. Also in certain embodiments, R.sup.b2 is
--OH, --Cl or --F. In certain embodiments, R.sup.b2 is --OH. In
certain embodiments, R.sup.b2 is --Cl. In certain embodiments,
R.sup.b2 is --F.
[0108] In certain embodiments of Formulas (II), (IIa) and (IIb),
R.sup.d is --H. In the alternative, in certain embodiments,
R.sup.b2 and R.sup.d join to form alkylene or substituted alkylene.
In particular embodiments, R.sup.b2 and R.sup.d form
--CH.sub.2--O--. In particular embodiments, the --CH.sub.2-- is
linked to the 4' carbon of the sugar, and the --O-- is linked to
the 2' carbon of the sugar.
[0109] In certain embodiments of Formulas (II), (IIa) and (IIb),
R.sup.e is --H or alkyl. In particular embodiments, R.sup.e is
--H.
[0110] In certain embodiments according to Formula (II), (IIa) or
(IIb), R.sup.e is --H; R.sup.d is --H; R.sup.b1 is --CH.sub.3;
R.sup.b2 is --OH, --Cl or --F; R.sup.c is --H or --OH; and R.sup.2
is H. In particular embodiments, R.sup.e is --H; R.sup.d is --H;
R.sup.b1 is --CH.sub.3; R.sup.b2 is --OH, --Cl or --F; R.sup.c is
--H or --OH; R.sup.2 is H; and Base is selected from guanosine,
uracil, cytosine, adenine or a derivative thereof. In particular
embodiments, R.sup.e is --H; R.sup.d is --H; R.sup.b1 is
--CH.sub.3; R.sup.b2 is --OH, --Cl or --F; R.sup.c is --H or --OH;
R.sup.2 is H; Base is selected from guanosine, uracil, cytosine,
adenine or a derivative thereof and X is D-alanine, or an ester
thereof. In certain embodiments according to this paragraph, Y is
alkyl, aryl, arylalkyl, cycloalkyl or
##STR00014##
wherein R.sup.3 is alkyl, alkoxycarbonylaminoalkyl, hydroxylalkyl,
or aminoalkylcarbonylalkoxyl. In particular embodiments according
to this paragraph, R.sup.10 is methyl, isopropyl, t-butyl or
benzyl; and R.sup.11 is ethyl, propyl, isopropyl, n-propyl, butyl,
n-butyl, t-butyl or cyclopentyl.
[0111] In certain embodiments according to Formula (II), (IIa) or
(IIb), R.sup.e is --H; R.sup.b2 and R.sup.d form --CH.sub.2--O--;
R.sup.b1 is --CH.sub.3; R.sup.c is --H or --OH; and R.sup.2 is H.
In certain embodiments, R.sup.e is --H; R.sup.b2 and R.sup.d form
--CH.sub.2--O--; R.sup.b1 is --CH.sub.3; R.sup.c is --H or --OH;
R.sup.2 is H; and Base is selected from guanosine, uracil,
cytosine, adenine or a derivative thereof. In certain embodiments,
R.sup.e is --H; R.sup.b2 and R.sup.d form --CH.sub.2--O--; R.sup.b1
is --CH.sub.3; R.sup.c is --H or --OH; R.sup.2 is H; Base is
selected from guanosine, uracil, cytosine, adenine or a derivative
thereof; and X is D-alanine, or an ester thereof. In certain
embodiments according to this paragraph, Y is alkyl, aryl,
arylalkyl, cycloalkyl or
##STR00015##
wherein R.sup.3 is alkyl, alkoxycarbonylaminoalkyl, hydroxylalkyl,
or aminoalkylcarbonylalkoxyl. In particular embodiments according
to this paragraph, R.sup.10 is methyl, isopropyl, t-butyl or
benzyl; and R.sup.11 is ethyl, propyl, isopropyl, n-propyl, butyl,
n-butyl, t-butyl or cyclopentyl.
[0112] In certain embodiments according to Formula (II), (IIa) or
(IIb), R.sup.e is --H; R.sup.b2 and R.sup.d form
--CH.sub.2CH.sub.2--; R.sup.b1 is --CH.sub.3; R.sup.c is --H or
--OH; and R.sup.2 is H. In certain embodiments, R.sup.e is --H;
R.sup.b2 and R.sup.d form --CH.sub.2CH.sub.2--; R.sup.b1 is
--CH.sub.3; R.sup.c is --H or --OH; R.sup.2 is H; and Base is
selected from guanosine, uracil, cytosine, adenine or a derivative
thereof. In certain embodiments, R.sup.e is --H; R.sup.b2 and
R.sup.d form --CH.sub.2CH.sub.2--; R.sup.b1 is --CH.sub.3; R.sup.c
is --H or --OH; R.sup.2 is H; Base is selected from guanosine,
uracil, cytosine, adenine or a derivative thereof; and X is
D-alanine, or an ester thereof. In certain embodiments according to
this paragraph, Y is alkyl, aryl, arylalkyl, cycloalkyl or
##STR00016##
wherein R.sup.3 is alkyl, alkoxycarbonylaminoalkyl, hydroxylalkyl,
or aminoalkylcarbonylalkoxyl. In particular embodiments according
to this paragraph, R.sup.10 is methyl, isopropyl, t-butyl or
benzyl; and R.sup.11 is ethyl, propyl, isopropyl, n-propyl, butyl,
n-butyl, t-butyl or cyclopentyl.
[0113] In certain embodiments according to Formula (II), (IIa) or
(IIb), R.sup.e is --H; R.sup.d is --H; R.sup.b1 is --CH.sub.3;
R.sup.b2 is --OH, --Cl or --F; R.sup.2 is H; and Y and R.sup.e
together represent a single divalent --O--. In particular
embodiments, R.sup.e is --H; R.sup.d is --H; R.sup.b1 is
--CH.sub.3; R.sup.b2 is --OH, --Cl or --F; R.sup.2 is H; Y and
R.sup.e together represent a single divalent --O--; and Base is
selected from guanosine, uracil, cytosine, adenine or a derivative
thereof. In particular embodiments, R.sup.e is --H; R.sup.d is --H;
R.sup.b1 is --CH.sub.3; R.sup.b2 is --OH, --Cl or --F; R.sup.2 is
H; Y and R.sup.c together represent a single divalent --O--; and
Base is selected from guanosine, uracil, cytosine, adenine or a
derivative thereof; and X is D-alanine, or an ester thereof. In
certain embodiments according to this paragraph, Y is alkyl, aryl,
arylalkyl, cycloalkyl or
##STR00017##
wherein R.sup.3 is alkyl, alkoxycarbonylaminoalkyl, hydroxylalkyl,
or aminoalkylcarbonylalkoxyl. In particular embodiments according
to this paragraph, R.sup.10 is methyl, isopropyl, t-butyl or
benzyl; and R.sup.11 is ethyl, propyl, isopropyl, n-propyl, butyl,
n-butyl, t-butyl or cyclopentyl.
[0114] In certain embodiments, a compound of any of Formulas (I),
(Ia), (Ib), (II), (IIa), or (Ith) is provided wherein: each Base is
independently
##STR00018##
or a tautomer thereof; each R.sup.4 is independently hydrogen,
hydroxyl, hydroxylamine, alkylamino, halogen, sulfanyl, amino or
alkoxy; each R.sup.5 is independently hydrogen, halogen or methyl;
and each R.sup.6 is independently hydrogen, amino, or halo.
[0115] In certain embodiments, a compound of any of Formulas (I),
(Ia), (Ib), (II), (IIa) or (IIIb) is provided wherein: each Base is
independently
##STR00019##
or a tautomer thereof; each R.sup.4 is independently hydrogen,
hydroxyl, hydroxylamine, halogen, sulfanyl, amino or alkoxy; and
each R.sup.5 is independently hydrogen, halogen or methyl. In an
embodiment, each R.sup.4 is alkylamino. In an embodiment, each
R.sup.4 is alkylamino having from seven to thirty carbon atoms. In
an embodiment, each R.sup.4 is alkylamino having from fifteen to
thirty carbon atoms. In an embodiment, each R.sup.4 is alkylamino
having from twenty to thirty carbon atoms. In an embodiment, each
R.sup.4 is alkylamino having from seven to fifteen carbon atoms. In
an embodiment, each R.sup.4 is alkylamino having from seven to
twenty carbon atoms. In an embodiment, each R.sup.4 is alkylamino
having from ten to twenty carbon atoms.
[0116] In certain embodiments, a compound of any of the following
Formulas is provided:
##STR00020## ##STR00021##
or pharmaceutically acceptable salts, solvates, stereoisomeric
forms or polymorphic forms thereof, wherein: R.sup.b1, R.sup.b2,
R.sup.c, R.sup.d, R.sup.e, W, X, A and Y are as defined in the
context of Formula (I); and each R.sup.5 is independently hydrogen,
halogen or methyl.
[0117] In certain embodiments, provided herein are compounds
according to any of the following Formulas:
##STR00022##
or a pharmaceutically acceptable salt, solvate, stereoisomeric
form, tautomeric form or polymorphic form thereof, wherein
R.sup.b1, R.sup.b2, R.sup.c, R.sup.d, R.sup.e, W, X, and Y are as
defined in the context of Formula (I). In certain embodiments, a
compound of Formula (XVII) is provided. In certain embodiments, a
compound of Formula (XVIII) is provided. In certain embodiments, a
compound of Formula (XIX) is provided. In certain embodiments, a
compound of Formula (XX) is provided.
[0118] In certain embodiments, provided herein are compounds
according to any of the following formulas:
##STR00023## ##STR00024## ##STR00025## ##STR00026## ##STR00027##
##STR00028## ##STR00029##
or a pharmaceutically acceptable salt, solvate, stereoisomeric
form, tautomeric form or polymorphic form thereof.
[0119] In certain embodiments, provided herein are compounds
according to any of the following formulas:
##STR00030## ##STR00031## ##STR00032## ##STR00033## ##STR00034##
##STR00035## ##STR00036## ##STR00037##
or a pharmaceutically acceptable salt, solvate, stereoisomeric
form, tautomeric form or polymorphic form thereof.
[0120] In certain embodiments, provided herein are compounds
according to any of the following formulas:
##STR00038## ##STR00039## ##STR00040## ##STR00041## ##STR00042##
##STR00043## ##STR00044## ##STR00045##
or a pharmaceutically acceptable salt, solvate, stereoisomeric
form, tautomeric form or polymorphic form thereof.
[0121] In certain embodiments, provided herein are compounds
according to any of the following formulas:
##STR00046## ##STR00047## ##STR00048## ##STR00049##
##STR00050##
or a pharmaceutically acceptable salt, solvate, stereoisomeric
form, tautomeric form or polymorphic form thereof.
[0122] In certain embodiments, provided herein are compounds
according to any of the following formulas:
##STR00051##
or a pharmaceutically acceptable salt, solvate, stereoisomeric
form, tautomeric form or polymorphic form thereof.
[0123] In certain embodiments, a compound of formula according to
formula 401 or 425 is provided:
##STR00052##
or a pharmaceutically acceptable salt, solvate, phosphate, prodrug,
stereoisomeric form, tautomeric form or polymorphic form
thereof.
[0124] In certain embodiments, provided herein are compounds
according to any of the following formulas:
##STR00053## ##STR00054## ##STR00055##
or a pharmaceutically acceptable salt, solvate, stereoisomeric
form, tautomeric form or polymorphic form thereof.
[0125] In certain embodiments, provided herein are compounds
according to any of the following formulas:
##STR00056##
or a pharmaceutically acceptable salt, solvate, stereoisomeric
form, tautomeric form or polymorphic form thereof.
[0126] In certain embodiments, provided herein are compounds
according to any of the following formulas:
##STR00057## ##STR00058##
or a pharmaceutically acceptable salt, solvate, stereoisomeric
form, tautomeric form or polymorphic form thereof.
[0127] In certain embodiments provided herein are compounds
according to any of the following formulas:
##STR00059## ##STR00060## ##STR00061## ##STR00062## ##STR00063##
##STR00064## ##STR00065## ##STR00066## ##STR00067## ##STR00068##
##STR00069## ##STR00070## ##STR00071## ##STR00072## ##STR00073##
##STR00074## ##STR00075## ##STR00076## ##STR00077## ##STR00078##
##STR00079## ##STR00080## ##STR00081##
or a pharmaceutically acceptable salt, solvate, stereoisomeric
form, tautomeric form or polymorphic form thereof.
[0128] In certain embodiments, provided herein are compounds
according to any of the following Formulas:
##STR00082## ##STR00083##
or a pharmaceutically acceptable salt, solvate, stereoisomeric
form, tautomeric form or polymorphic form thereof.
[0129] In an embodiment, provided herein are compounds comprising a
D-amino acid, or ester thereof, linked to a drug. In certain
embodiments, the drug is a drug for treating a liver disease or
condition. In certain embodiments, the liver disease or condition
is hepatitis, fatty liver disease, cirrhosis, liver cancer, biliary
cirrhosis, sclerosing cholangitis, Budd-Chiari syndrome,
hemochromatosis, Wilson's disease, Gilbert's syndrome, biliary
atresia, alpha-1 antitrypsin deficiency, alagille syndrome, or
progressive familial intrahepatic cholestasis. In certain
embodiments, the drug is a drug for treating hepatitis C. In
certain embodiments, the drug is an interferon, a nucleotide
analogue, a polymerase inhibitor, an NS3 protease inhibitor, an
NS5A inhibitor, an entry inhibitor, a non-nucleoside polymerase
inhibitor, a cyclosporine immune inhibitor, an NS4A antagonist, an
NS4B-RNA binding inhibitor, a locked nucleic acid mRNA inhibitor,
or a cyclophilin inhibitor.
[0130] In some embodiments, provided herein are: [0131] (a)
compounds as described herein and pharmaceutically acceptable salts
and compositions thereof; [0132] (b) compounds as described herein
and pharmaceutically acceptable salts and compositions thereof for
use in the treatment and/or prophylaxis of a liver disorder
including Flaviviridae infection, especially in individuals
diagnosed as having a Flaviviridae infection or being at risk of
becoming infected by hepatitis C; [0133] (c) processes for the
preparation of compounds as described herein as described in more
detail elsewhere herein; [0134] (d) pharmaceutical formulations
comprising a compound as described herein, or a pharmaceutically
acceptable salt thereof together with a pharmaceutically acceptable
carrier or diluent; [0135] (e) pharmaceutical formulations
comprising a compound as described herein or a pharmaceutically
acceptable salt thereof together with one or more other effective
anti-HCV agents, optionally in a pharmaceutically acceptable
carrier or diluent; [0136] (f) a method for the treatment and/or
prophylaxis of a host infected with Flaviviridae that includes the
administration of an effective amount of a compound as described
herein its pharmaceutically acceptable salt or composition; or
[0137] (g) a method for the treatment and/or prophylaxis of a host
infected with Flaviviridae that includes the administration of an
effective amount of a compounds as described herein, its
pharmaceutically acceptable salt or composition in combination
and/or alternation with one or more effective anti-HCV agent.
Optically Active Compounds
[0138] It is appreciated that compounds provided herein have
several chiral centers and may exist in and be isolated in
optically active and racemic forms. Some compounds may exhibit
polymorphism. It is to be understood that any racemic,
optically-active, diastereomeric, polymorphic, or stereoisomeric
form, or mixtures thereof, of a compound provided herein, which
possess the useful properties described herein is within the scope
of the invention. It being well known in the art how to prepare
optically active forms (for example, by resolution of the racemic
form by recrystallization techniques, by synthesis from
optically-active starting materials, by chiral synthesis, or by
chromatographic separation using a chiral stationary phase).
[0139] In particular, since the 1' and 4' carbons of a nucleoside
are chiral, their non-hydrogen substituents (the base and the CHOR
groups, respectively) can be either cis (on the same side) or trans
(on opposite sides) with respect to the sugar ring system. The four
optical isomers therefore are represented by the following
configurations (when orienting the sugar moiety in a horizontal
plane such that the oxygen atom is in the back): cis (with both
groups "up", which corresponds to the configuration of naturally
occurring B-D nucleosides), cis (with both groups "down", which is
a non-naturally occurring B-L configuration), trans (with the C2'
substituent "up" and the C4' substituent "down"), and trans (with
the C2' substituent "down" and the C4' substituent "up"). The
"D-nucleosides" are cis nucleosides in a natural configuration and
the "L-nucleosides" are cis nucleosides in the non-naturally
occurring configuration.
[0140] Likewise, most amino acids are chiral (designated as L or D,
wherein the L enantiomer is the naturally occurring configuration)
and can exist as separate enantiomers.
[0141] Examples of methods to obtain optically active materials are
known in the art, and include at least the following. [0142] i)
physical separation of crystals--a technique whereby macroscopic
crystals of the individual enantiomers are manually separated. This
technique can be used if crystals of the separate enantiomers
exist, i.e., the material is a conglomerate, and the crystals are
visually distinct; [0143] ii) simultaneous crystallization--a
technique whereby the individual enantiomers are separately
crystallized from a solution of the racemate, possible only if the
latter is a conglomerate in the solid state; [0144] iii) enzymatic
resolutions--a technique whereby partial or complete separation of
a racemate by virtue of differing rates of reaction for the
enantiomers with an enzyme; [0145] iv) enzymatic asymmetric
synthesis--a synthetic technique whereby at least one step of the
synthesis uses an enzymatic reaction to obtain an enantiomerically
pure or enriched synthetic precursor of the desired enantiomer;
[0146] v) chemical asymmetric synthesis--a synthetic technique
whereby the desired enantiomer is synthesized from an achiral
precursor under conditions that produce asymmetry (i.e., chirality)
in the product, which may be achieved using chiral catalysts or
chiral auxiliaries; [0147] vi) diastereomer separations--a
technique whereby a racemic compound is reacted with an
enantiomerically pure reagent (the chiral auxiliary) that converts
the individual enantiomers to diastereomers. The resulting
diastereomers are then separated by chromatography or
crystallization by virtue of their now more distinct structural
differences and the chiral auxiliary later removed to obtain the
desired enantiomer; [0148] vii) first- and second-order asymmetric
transformations--a technique whereby diastereomers from the
racemate equilibrate to yield a preponderance in solution of the
diastereomer from the desired enantiomer or where preferential
crystallization of the diastereomer from the desired enantiomer
perturbs the equilibrium such that eventually in principle all the
material is converted to the crystalline diastereomer from the
desired enantiomer. The desired enantiomer is then released from
the diastereomer; [0149] viii) kinetic resolutions--this technique
refers to the achievement of partial or complete resolution of a
racemate (or of a further resolution of a partially resolved
compound) by virtue of unequal reaction rates of the enantiomers
with a chiral, non-racemic reagent or catalyst under kinetic
conditions; [0150] ix) enantiospecific synthesis from non-racemic
precursors--a synthetic technique whereby the desired enantiomer is
obtained from non-chiral starting materials and where the
stereochemical integrity is not or is only minimally compromised
over the course of the synthesis; [0151] x) chiral liquid
chromatography--a technique whereby the enantiomers of a racemate
are separated in a liquid mobile phase by virtue of their differing
interactions with a stationary phase. The stationary phase can be
made of chiral material or the mobile phase can contain an
additional chiral material to provoke the differing interactions;
[0152] xi) chiral gas chromatography--a technique whereby the
racemate is volatilized and enantiomers are separated by virtue of
their differing interactions in the gaseous mobile phase with a
column containing a fixed non-racemic chiral adsorbent phase;
[0153] xii) extraction with chiral solvents--a technique whereby
the enantiomers are separated by virtue of preferential dissolution
of one enantiomer into a particular chiral solvent; [0154] xiii)
transport across chiral membranes--a technique whereby a racemate
is placed in contact with a thin membrane barrier. The barrier
typically separates two miscible fluids, one containing the
racemate, and a driving force such as concentration or pressure
differential causes preferential transport across the membrane
barrier. Separation occurs as a result of the non-racemic chiral
nature of the membrane which allows only one enantiomer of the
racemate to pass through.
[0155] In some embodiments, compositions of 2'-chloro nucleoside
analog compounds that are substantially free of a designated
enantiomer of that compound. In certain embodiments, in the methods
and compounds of this invention, the compounds are substantially
free of enantiomers. In some embodiments, the composition includes
that includes a compound that is at least 85, 90%, 95%, 98%, 99% to
100% by weight, of the compound, the remainder comprising other
chemical species or enantiomers.
[0156] Isotopically Enriched Compounds
[0157] Also provided herein are isotopically enriched compounds,
including but not limited to isotopically enriched 2'-chloro
nucleoside analog compounds.
[0158] Isotopic enrichment (for example, deuteration) of
pharmaceuticals to improve pharmacokinetics ("PK"),
pharmacodynamics ("PD"), and toxicity profiles, has been
demonstrated previously with some classes of drugs. See, for
example, Lijinsky et. al., Food Cosmet. Toxicol., 20: 393 (1982);
Lijinsky et. al., J. Nat. Cancer Inst., 69: 1127 (1982); Mangold
et. al., Mutation Res. 308: 33 (1994); Gordon et. al., Drug Metab.
Dispos., 15: 589 (1987); Zello et. al., Metabolism, 43: 487 (1994);
Gately et. al., J. Nucl. Med., 27: 388 (1986); Wade D, Chem. Biol.
Interact. 117: 191 (1999).
[0159] Isotopic enrichment of a drug can be used, for example, to
(1) reduce or eliminate unwanted metabolites, (2) increase the
half-life of the parent drug, (3) decrease the number of doses
needed to achieve a desired effect, (4) decrease the amount of a
dose necessary to achieve a desired effect, (5) increase the
formation of active metabolites, if any are formed, and/or (6)
decrees the production of deleterious metabolites in specific
tissues and/or create a more effective drug and/or a safer drug for
combination therapy, whether the combination therapy is intentional
or not.
[0160] Replacement of an atom for one of its isotopes often will
result in a change in the reaction rate of a chemical reaction.
This phenomenon is known as the Kinetic Isotope Effect ("KIE"). For
example, if a C--H bond is broken during a rate-determining step in
a chemical reaction (i.e. the step with the highest transition
state energy), substitution of a deuterium for that hydrogen will
cause a decrease in the reaction rate and the process will slow
down. This phenomenon is known as the Deuterium Kinetic Isotope
Effect ("DKIE"). (See, e.g., Foster et al., Adv. Drug Res., vol.
14, pp. 1-36 (1985); Kushner et al., Can. J. Physiol. Pharmacol.,
vol. 77, pp. 79-88 (1999)).
[0161] The magnitude of the DKIE can be expressed as the ratio
between the rates of a given reaction in which a C--H bond is
broken, and the same reaction where deuterium is substituted for
hydrogen. The DKIE can range from about 1 (no isotope effect) to
very large numbers, such as 50 or more, meaning that the reaction
can be fifty, or more, times slower when deuterium is substituted
for hydrogen. High DKIE values may be due in part to a phenomenon
known as tunneling, which is a consequence of the uncertainty
principle. Tunneling is ascribed to the small mass of a hydrogen
atom, and occurs because transition states involving a proton can
sometimes form in the absence of the required activation energy.
Because deuterium has more mass than hydrogen, it statistically has
a much lower probability of undergoing this phenomenon.
[0162] Tritium ("T") is a radioactive isotope of hydrogen, used in
research, fusion reactors, neutron generators and
radiopharmaceuticals. Tritium is a hydrogen atom that has 2
neutrons in the nucleus and has an atomic weight close to 3. It
occurs naturally in the environment in very low concentrations,
most commonly found as T.sub.2O. Tritium decays slowly
(half-life=12.3 years) and emits a low energy beta particle that
cannot penetrate the outer layer of human skin. Internal exposure
is the main hazard associated with this isotope, yet it must be
ingested in large amounts to pose a significant health risk. As
compared with deuterium, a lesser amount of tritium must be
consumed before it reaches a hazardous level. Substitution of
tritium ("T") for hydrogen results in yet a stronger bond than
deuterium and gives numerically larger isotope effects. Similarly,
substitution of isotopes for other elements, including, but not
limited to, .sup.13C or .sup.14C for carbon, .sup.33S, .sup.34S, or
.sup.36S for sulfur, .sup.15N for nitrogen, and .sup.17O or
.sup.18O for oxygen, may lead to a similar kinetic isotope
effect.
[0163] For example, the DKIE was used to decrease the
hepatotoxicity of halothane by presumably limiting the production
of reactive species such as trifluoroacetyl chloride. However, this
method may not be applicable to all drug classes. For example,
deuterium incorporation can lead to metabolic switching. The
concept of metabolic switching asserts that xenogens, when
sequestered by Phase I enzymes, may bind transiently and re-bind in
a variety of conformations prior to the chemical reaction (e.g.,
oxidation). This hypothesis is supported by the relatively vast
size of binding pockets in many Phase I enzymes and the promiscuous
nature of many metabolic reactions. Metabolic switching can
potentially lead to different proportions of known metabolites as
well as altogether new metabolites. This new metabolic profile may
impart more or less toxicity.
[0164] The animal body expresses a variety of enzymes for the
purpose of eliminating foreign substances, such as therapeutic
agents, from its circulation system. Examples of such enzymes
include the cytochrome P450 enzymes ("CYPs"), esterases, proteases,
reductases, dehydrogenases, and monoamine oxidases, to react with
and convert these foreign substances to more polar intermediates or
metabolites for renal excretion. Some of the most common metabolic
reactions of pharmaceutical compounds involve the oxidation of a
carbon-hydrogen (C--H) bond to either a carbon-oxygen (C-0) or
carbon-carbon (C--C) pi-bond. The resultant metabolites may be
stable or unstable under physiological conditions, and can have
substantially different pharmacokinetic, pharmacodynamic, and acute
and long-term toxicity profiles relative to the parent compounds.
For many drugs, such oxidations are rapid. These drugs therefore
often require the administration of multiple or high daily
doses.
[0165] Therefore, isotopic enrichment at certain positions of a
compound provided herein will produce a detectable KIE that will
affect the pharmacokinetic, pharmacologic, and/or toxicological
profiles of a compound provided herein in comparison with a similar
compound having a natural isotopic composition.
[0166] Preparation of Compounds
[0167] The compounds provided herein can be prepared, isolated or
obtained by any method apparent to those of skill in the art.
Compounds provided herein can be prepared according to the
Exemplary Preparation Schemes in the Examples provided below.
Reaction conditions, steps and reactants not provided in the
Exemplary Preparation Schemes would be apparent to, and known by,
those skilled in the art.
[0168] Pharmaceutical Compositions and Methods of
Administration
[0169] Compounds provided herein can be formulated into
pharmaceutical compositions using methods available in the art and
those disclosed herein. Any of the compounds disclosed herein can
be provided in the appropriate pharmaceutical composition and be
administered by a suitable route of administration.
[0170] The methods provided herein encompass administering
pharmaceutical compositions containing at least one compound as
described herein, if appropriate in the salt form, either used
alone or in the form of a combination with one or more compatible
and pharmaceutically acceptable carriers, such as diluents or
adjuvants, or with another anti-HCV agent.
[0171] In certain embodiments, the second agent can be formulated
or packaged with the compound provided herein. Of course, the
second agent will only be formulated with the compound provided
herein when, according to the judgment of those of skill in the
art, such co-formulation should not interfere with the activity of
either agent or the method of administration. In certain
embodiments, the compound provided herein and the second agent are
formulated separately. They can be packaged together, or packaged
separately, for the convenience of the practitioner of skill in the
art.
[0172] In clinical practice the active agents provided herein may
be administered by any conventional route, in particular orally,
parenterally, rectally or by inhalation (e.g. in the form of
aerosols). In certain embodiments, the compound provided herein is
administered orally.
[0173] Use may be made, as solid compositions for oral
administration, of tablets, pills, hard gelatin capsules, powders
or granules. In these compositions, the active product is mixed
with one or more inert diluents or adjuvants, such as sucrose,
lactose or starch.
[0174] These compositions can comprise substances other than
diluents, for example a lubricant, such as magnesium stearate, or a
coating intended for controlled release.
[0175] Use may be made, as liquid compositions for oral
administration, of solutions which are pharmaceutically acceptable,
suspensions, emulsions, syrups and elixirs containing inert
diluents, such as water or liquid paraffin. These compositions can
also comprise substances other than diluents, for example wetting,
sweetening or flavoring products.
[0176] The compositions for parenteral administration can be
emulsions or sterile solutions. Use may be made, as solvent or
vehicle, of propylene glycol, a polyethylene glycol, vegetable
oils, in particular olive oil, or injectable organic esters, for
example ethyl oleate. These compositions can also contain
adjuvants, in particular wetting, isotonizing, emulsifying,
dispersing and stabilizing agents. Sterilization can be carried out
in several ways, for example using a bacteriological filter, by
radiation or by heating. They can also be prepared in the form of
sterile solid compositions which can be dissolved at the time of
use in sterile water or any other injectable sterile medium.
[0177] The compositions for rectal administration are suppositories
or rectal capsules which contain, in addition to the active
principle, excipients such as cocoa butter, semi-synthetic
glycerides or polyethylene glycols.
[0178] The compositions can also be aerosols. For use in the form
of liquid aerosols, the compositions can be stable sterile
solutions or solid compositions dissolved at the time of use in
apyrogenic sterile water, in saline or any other pharmaceutically
acceptable vehicle. For use in the form of dry aerosols intended to
be directly inhaled, the active principle is finely divided and
combined with a water-soluble solid diluent or vehicle, for example
dextran, mannitol or lactose.
[0179] In certain embodiments, a composition provided herein is a
pharmaceutical composition or a single unit dosage form.
Pharmaceutical compositions and single unit dosage forms provided
herein comprise a prophylactically or therapeutically effective
amount of one or more prophylactic or therapeutic agents (e.g., a
compound provided herein, or other prophylactic or therapeutic
agent), and a typically one or more pharmaceutically acceptable
carriers or excipients. In a specific embodiment and in this
context, the term "pharmaceutically acceptable" means approved by a
regulatory agency of the Federal or a state government or listed in
the U.S. Pharmacopeia or other generally recognized pharmacopeia
for use in animals, and more particularly in humans. The term
"carrier" includes a diluent, adjuvant (e.g., Freund's adjuvant
(complete and incomplete)), excipient, or vehicle with which the
therapeutic is administered. Such pharmaceutical carriers can be
sterile liquids, such as water and oils, including those of
petroleum, animal, vegetable or synthetic origin, such as peanut
oil, soybean oil, mineral oil, sesame oil and the like. Water can
be used as a carrier when the pharmaceutical composition is
administered intravenously. Saline solutions and aqueous dextrose
and glycerol solutions can also be employed as liquid carriers,
particularly for injectable solutions. Examples of suitable
pharmaceutical carriers are described in "Remington's
Pharmaceutical Sciences" by E. W. Martin.
[0180] Typical pharmaceutical compositions and dosage forms
comprise one or more excipients. Suitable excipients are well-known
to those skilled in the art of pharmacy, and non-limiting examples
of suitable excipients include starch, glucose, lactose, sucrose,
gelatin, malt, rice, flour, chalk, silica gel, sodium stearate,
glycerol monostearate, talc, sodium chloride, dried skim milk,
glycerol, propylene, glycol, water, ethanol and the like. Whether a
particular excipient is suitable for incorporation into a
pharmaceutical composition or dosage form depends on a variety of
factors well known in the art including, but not limited to, the
way in which the dosage form will be administered to a subject and
the specific active ingredients in the dosage form. The composition
or single unit dosage form, if desired, can also contain minor
amounts of wetting or emulsifying agents, or pH buffering
agents.
[0181] Lactose free compositions provided herein can comprise
excipients that are well known in the art and are listed, for
example, in the U.S. Pharmocopia (USP)SP(XXI)/NF (XVI). In general,
lactose free compositions comprise an active ingredient, a
binder/filler, and a lubricant in pharmaceutically compatible and
pharmaceutically acceptable amounts. Exemplary lactose free dosage
forms comprise an active ingredient, microcrystalline cellulose,
pre gelatinized starch, and magnesium stearate.
[0182] Further encompassed herein are anhydrous pharmaceutical
compositions and dosage forms comprising active ingredients, since
water can facilitate the degradation of some compounds. For
example, the addition of water (e.g., 5%) is widely accepted in the
pharmaceutical arts as a means of simulating long term storage in
order to determine characteristics such as shelf life or the
stability of formulations over time. See, e.g., Jens T. Carstensen,
Drug Stability: Principles & Practice, 2d. Ed., Marcel Dekker,
New York, 1995, pp. 379 80. In effect, water and heat accelerate
the decomposition of some compounds. Thus, the effect of water on a
formulation can be of great significance since moisture and/or
humidity are commonly encountered during manufacture, handling,
packaging, storage, shipment, and use of formulations.
[0183] Anhydrous pharmaceutical compositions and dosage forms
provided herein can be prepared using anhydrous or low moisture
containing ingredients and low moisture or low humidity conditions.
Pharmaceutical compositions and dosage forms that comprise lactose
and at least one active ingredient that comprises a primary or
secondary amine can be anhydrous if substantial contact with
moisture and/or humidity during manufacturing, packaging, and/or
storage is expected.
[0184] An anhydrous pharmaceutical composition should be prepared
and stored such that its anhydrous nature is maintained.
Accordingly, anhydrous compositions can be packaged using materials
known to prevent exposure to water such that they can be included
in suitable formulary kits. Examples of suitable packaging include,
but are not limited to, hermetically sealed foils, plastics, unit
dose containers (e.g., vials), blister packs, and strip packs.
[0185] Further provided are pharmaceutical compositions and dosage
forms that comprise one or more compounds that reduce the rate by
which an active ingredient will decompose. Such compounds, which
are referred to herein as "stabilizers," include, but are not
limited to, antioxidants such as ascorbic acid, pH buffers, or salt
buffers.
[0186] The pharmaceutical compositions and single unit dosage forms
can take the form of solutions, suspensions, emulsion, tablets,
pills, capsules, powders, sustained-release formulations and the
like. Oral formulation can include standard carriers such as
pharmaceutical grades of mannitol, lactose, starch, magnesium
stearate, sodium saccharine, cellulose, magnesium carbonate, etc.
Such compositions and dosage forms will contain a prophylactically
or therapeutically effective amount of a prophylactic or
therapeutic agent, in certain embodiments, in purified form,
together with a suitable amount of carrier so as to provide the
form for proper administration to the subject. The formulation
should suit the mode of administration. In a certain embodiment,
the pharmaceutical compositions or single unit dosage forms are
sterile and in suitable form for administration to a subject, for
example, an animal subject, such as a mammalian subject, for
example, a human subject.
[0187] A pharmaceutical composition is formulated to be compatible
with its intended route of administration. Examples of routes of
administration include, but are not limited to, parenteral, e.g.,
intravenous, intradermal, subcutaneous, intramuscular,
subcutaneous, oral, buccal, sublingual, inhalation, intranasal,
transdermal, topical, transmucosal, intra-tumoral, intra-synovial
and rectal administration. In a specific embodiment, the
composition is formulated in accordance with routine procedures as
a pharmaceutical composition adapted for intravenous, subcutaneous,
intramuscular, oral, intranasal or topical administration to human
beings. In an embodiment, a pharmaceutical composition is
formulated in accordance with routine procedures for subcutaneous
administration to human beings. Typically, compositions for
intravenous administration are solutions in sterile isotonic
aqueous buffer. Where necessary, the composition may also include a
solubilizing agent and a local anesthetic such as lignocamne to
ease pain at the site of the injection.
[0188] Examples of dosage forms include, but are not limited to:
tablets; caplets; capsules, such as soft elastic gelatin capsules;
cachets; troches; lozenges; dispersions; suppositories; ointments;
cataplasms (poultices); pastes; powders; dressings; creams;
plasters; solutions; patches; aerosols (e.g., nasal sprays or
inhalers); gels; liquid dosage forms suitable for oral or mucosal
administration to a subject, including suspensions (e.g., aqueous
or non-aqueous liquid suspensions, oil in water emulsions, or a
water in oil liquid emulsions), solutions, and elixirs; liquid
dosage forms suitable for parenteral administration to a subject;
and sterile solids (e.g., crystalline or amorphous solids) that can
be reconstituted to provide liquid dosage forms suitable for
parenteral administration to a subject.
[0189] The composition, shape, and type of dosage forms provided
herein will typically vary depending on their use. For example, a
dosage form used in the initial treatment of viral infection may
contain larger amounts of one or more of the active ingredients it
comprises than a dosage form used in the maintenance treatment of
the same infection. Similarly, a parenteral dosage form may contain
smaller amounts of one or more of the active ingredients it
comprises than an oral dosage form used to treat the same disease
or disorder. These and other ways in which specific dosage forms
encompassed herein will vary from one another will be readily
apparent to those skilled in the art. See, e.g., Remington's
Pharmaceutical Sciences, 20th ed., Mack Publishing, Easton Pa.
(2000).
[0190] Generally, the ingredients of compositions are supplied
either separately or mixed together in unit dosage form, for
example, as a dry lyophilized powder or water free concentrate in a
hermetically sealed container such as an ampoule or sachette
indicating the quantity of active agent. Where the composition is
to be administered by infusion, it can be dispensed with an
infusion bottle containing sterile pharmaceutical grade water or
saline. Where the composition is administered by injection, an
ampoule of sterile water for injection or saline can be provided so
that the ingredients may be mixed prior to administration.
[0191] Typical dosage forms comprise a compound provided herein, or
a pharmaceutically acceptable salt, solvate or hydrate thereof lie
within the range of from about 0.1 mg to about 1000 mg per day,
given as a single once-a-day dose in the morning or as divided
doses throughout the day taken with food. Particular dosage forms
can have about 0.1, 0.2, 0.3, 0.4, 0.5, 1.0, 2.0, 2.5, 5.0, 10.0,
15.0, 20.0, 25.0, 50.0, 100, 200, 250, 500 or 1000 mg of the active
compound.
[0192] Oral Dosage Forms
[0193] Pharmaceutical compositions that are suitable for oral
administration can be presented as discrete dosage forms, such as,
but are not limited to, tablets (e.g., chewable tablets), caplets,
capsules, and liquids (e.g., flavored syrups). Such dosage forms
contain predetermined amounts of active ingredients, and may be
prepared by methods of pharmacy well known to those skilled in the
art. See generally, Remington's Pharmaceutical Sciences, 20th ed.,
Mack Publishing, Easton Pa. (2000).
[0194] In certain embodiments, the oral dosage forms are solid and
prepared under anhydrous conditions with anhydrous ingredients, as
described in detail herein. However, the scope of the compositions
provided herein extends beyond anhydrous, solid oral dosage forms.
As such, further forms are described herein.
[0195] Typical oral dosage forms are prepared by combining the
active ingredient(s) in an intimate admixture with at least one
excipient according to conventional pharmaceutical compounding
techniques. Excipients can take a wide variety of forms depending
on the form of preparation desired for administration. For example,
excipients suitable for use in oral liquid or aerosol dosage forms
include, but are not limited to, water, glycols, oils, alcohols,
flavoring agents, preservatives, and coloring agents. Examples of
excipients suitable for use in solid oral dosage forms (e.g.,
powders, tablets, capsules, and caplets) include, but are not
limited to, starches, sugars, micro crystalline cellulose,
diluents, granulating agents, lubricants, binders, and
disintegrating agents.
[0196] Because of their ease of administration, tablets and
capsules represent the most advantageous oral dosage unit forms, in
which case solid excipients are employed. If desired, tablets can
be coated by standard aqueous or non-aqueous techniques. Such
dosage forms can be prepared by any of the methods of pharmacy. In
general, pharmaceutical compositions and dosage forms are prepared
by uniformly and intimately admixing the active ingredients with
liquid carriers, finely divided solid carriers, or both, and then
shaping the product into the desired presentation if necessary.
[0197] For example, a tablet can be prepared by compression or
molding. Compressed tablets can be prepared by compressing in a
suitable machine the active ingredients in a free flowing form such
as powder or granules, optionally mixed with an excipient. Molded
tablets can be made by molding in a suitable machine a mixture of
the powdered compound moistened with an inert liquid diluent.
[0198] Examples of excipients that can be used in oral dosage forms
include, but are not limited to, binders, fillers, disintegrants,
and lubricants. Binders suitable for use in pharmaceutical
compositions and dosage forms include, but are not limited to, corn
starch, potato starch, or other starches, gelatin, natural and
synthetic gums such as acacia, sodium alginate, alginic acid, other
alginates, powdered tragacanth, guar gum, cellulose and its
derivatives (e.g., ethyl cellulose, cellulose acetate,
carboxymethyl cellulose calcium, sodium carboxymethyl cellulose),
polyvinyl pyrrolidone, methyl cellulose, pre gelatinized starch,
hydroxypropyl methyl cellulose, (e.g., Nos. 2208, 2906, 2910),
microcrystalline cellulose, and mixtures thereof.
[0199] Examples of fillers suitable for use in the pharmaceutical
compositions and dosage forms disclosed herein include, but are not
limited to, talc, calcium carbonate (e.g., granules or powder),
microcrystalline cellulose, powdered cellulose, dextrates, kaolin,
mannitol, silicic acid, sorbitol, starch, pre gelatinized starch,
and mixtures thereof. The binder or filler in pharmaceutical
compositions is typically present in from about 50 to about 99
weight percent of the pharmaceutical composition or dosage
form.
[0200] Suitable forms of microcrystalline cellulose include, but
are not limited to, the materials sold as AVICEL PH 101, AVICEL PH
103 AVICEL RC 581, AVICEL PH 105 (available from FMC Corporation,
American Viscose Division, Avicel Sales, Marcus Hook, Pa.), and
mixtures thereof. A specific binder is a mixture of
microcrystalline cellulose and sodium carboxymethyl cellulose sold
as AVICEL RC 581. Suitable anhydrous or low moisture excipients or
additives include AVICEL PH 103.TM. and Starch 1500 LM.
[0201] Disintegrants are used in the compositions to provide
tablets that disintegrate when exposed to an aqueous environment.
Tablets that contain too much disintegrant may disintegrate in
storage, while those that contain too little may not disintegrate
at a desired rate or under the desired conditions. Thus, a
sufficient amount of disintegrant that is neither too much nor too
little to detrimentally alter the release of the active ingredients
should be used to form solid oral dosage forms. The amount of
disintegrant used varies based upon the type of formulation, and is
readily discernible to those of ordinary skill in the art. Typical
pharmaceutical compositions comprise from about 0.5 to about 15
weight percent of disintegrant, specifically from about 1 to about
5 weight percent of disintegrant.
[0202] Disintegrants that can be used in pharmaceutical
compositions and dosage forms include, but are not limited to,
agar, alginic acid, calcium carbonate, microcrystalline cellulose,
croscarmellose sodium, crospovidone, polacrilin potassium, sodium
starch glycolate, potato or tapioca starch, pre gelatinized starch,
other starches, clays, other algins, other celluloses, gums, and
mixtures thereof.
[0203] Lubricants that can be used in pharmaceutical compositions
and dosage forms include, but are not limited to, calcium stearate,
magnesium stearate, mineral oil, light mineral oil, glycerin,
sorbitol, mannitol, polyethylene glycol, other glycols, stearic
acid, sodium lauryl sulfate, talc, hydrogenated vegetable oil
(e.g., peanut oil, cottonseed oil, sunflower oil, sesame oil, olive
oil, corn oil, and soybean oil), zinc stearate, ethyl oleate, ethyl
laureate, agar, and mixtures thereof. Additional lubricants
include, for example, a syloid silica gel (AEROSIL 200,
manufactured by W.R. Grace Co. of Baltimore, Md.), a coagulated
aerosol of synthetic silica (marketed by Degussa Co. of Plano,
Tex.), CAB 0 SIL (a pyrogenic silicon dioxide product sold by Cabot
Co. of Boston, Mass.), and mixtures thereof. If used at all,
lubricants are typically used in an amount of less than about 1
weight percent of the pharmaceutical compositions or dosage forms
into which they are incorporated.
[0204] Delayed Release Dosage Forms
[0205] Active ingredients such as the compounds provided herein can
be administered by controlled release means or by delivery devices
that are well known to those of ordinary skill in the art. Examples
include, but are not limited to, those described in U.S. Pat. Nos.
3,845,770; 3,916,899; 3,536,809; 3,598,123; and 4,008,719;
5,674,533; 5,059,595; 5,591,767; 5,120,548; 5,073,543; 5,639,476;
5,354,556; 5,639,480; 5,733,566; 5,739,108; 5,891,474; 5,922,356;
5,972,891; 5,980,945; 5,993,855; 6,045,830; 6,087,324; 6,113,943;
6,197,350; 6,248,363; 6,264,970; 6,267,981; 6,376,461; 6,419,961;
6,589,548; 6,613,358; and 6,699,500; each of which is incorporated
herein by reference in its entirety. Such dosage forms can be used
to provide slow or controlled release of one or more active
ingredients using, for example, hydropropylmethyl cellulose, other
polymer matrices, gels, permeable membranes, osmotic systems,
multilayer coatings, microparticles, liposomes, microspheres, or a
combination thereof to provide the desired release profile in
varying proportions. Suitable controlled release formulations known
to those of ordinary skill in the art, including those described
herein, can be readily selected for use with the active ingredients
provided herein. Thus encompassed herein are single unit dosage
forms suitable for oral administration such as, but not limited to,
tablets, capsules, gelcaps, and caplets that are adapted for
controlled release.
[0206] All controlled release pharmaceutical products have a common
goal of improving drug therapy over that achieved by their
non-controlled counterparts. Ideally, the use of an optimally
designed controlled release preparation in medical treatment is
characterized by a minimum of drug substance being employed to cure
or control the condition in a minimum amount of time. Advantages of
controlled release formulations include extended activity of the
drug, reduced dosage frequency, and increased subject compliance.
In addition, controlled release formulations can be used to affect
the time of onset of action or other characteristics, such as blood
levels of the drug, and can thus affect the occurrence of side
(e.g., adverse) effects.
[0207] Most controlled release formulations are designed to
initially release an amount of drug (active ingredient) that
promptly produces the desired therapeutic effect, and gradually and
continually release of other amounts of drug to maintain this level
of therapeutic or prophylactic effect over an extended period of
time. In order to maintain this constant level of drug in the body,
the drug must be released from the dosage form at a rate that will
replace the amount of drug being metabolized and excreted from the
body. Controlled release of an active ingredient can be stimulated
by various conditions including, but not limited to, pH,
temperature, enzymes, water, or other physiological conditions or
compounds.
[0208] In certain embodiments, the drug may be administered using
intravenous infusion, an implantable osmotic pump, a transdermal
patch, liposomes, or other modes of administration. In certain
embodiments, a pump may be used (see, Sefton, CRC Crit. Ref Biomed.
Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek
et al., N. Engl. J. Med. 321:574 (1989)). In another embodiment,
polymeric materials can be used. In yet another embodiment, a
controlled release system can be placed in a subject at an
appropriate site determined by a practitioner of skill, i.e., thus
requiring only a fraction of the systemic dose (see, e.g., Goodson,
Medical Applications of Controlled Release, vol. 2, pp. 115-138
(1984)). Other controlled release systems are discussed in the
review by Langer (Science 249:1527-1533 (1990)). The active
ingredient can be dispersed in a solid inner matrix, e.g.,
polymethylmethacrylate, polybutylmethacrylate, plasticized or
unplasticized polyvinylchloride, plasticized nylon, plasticized
polyethyleneterephthalate, natural rubber, polyisoprene,
polyisobutylene, polybutadiene, polyethylene, ethylene-vinylacetate
copolymers, silicone rubbers, polydimethylsiloxanes, silicone
carbonate copolymers, hydrophilic polymers such as hydrogels of
esters of acrylic and methacrylic acid, collagen, cross-linked
polyvinylalcohol and cross-linked partially hydrolyzed polyvinyl
acetate, that is surrounded by an outer polymeric membrane, e.g.,
polyethylene, polypropylene, ethylene/propylene copolymers,
ethylene/ethyl acrylate copolymers, ethylene/vinylacetate
copolymers, silicone rubbers, polydimethyl siloxanes, neoprene
rubber, chlorinated polyethylene, polyvinylchloride, vinylchloride
copolymers with vinyl acetate, vinylidene chloride, ethylene and
propylene, ionomer polyethylene terephthalate, butyl rubber
epichlorohydrin rubbers, ethylene/vinyl alcohol copolymer,
ethylene/vinyl acetate/vinyl alcohol terpolymer, and
ethylene/vinyloxyethanol copolymer, that is insoluble in body
fluids. The active ingredient then diffuses through the outer
polymeric membrane in a release rate controlling step. The
percentage of active ingredient in such parenteral compositions is
highly dependent on the specific nature thereof, as well as the
needs of the subject.
[0209] Parenteral Dosage Forms
[0210] In certain embodiments, provided are parenteral dosage
forms. Parenteral dosage forms can be administered to subjects by
various routes including, but not limited to, subcutaneous,
intravenous (including bolus injection), intramuscular, and
intraarterial. Because their administration typically bypasses
subjects' natural defenses against contaminants, parenteral dosage
forms are typically, sterile or capable of being sterilized prior
to administration to a subject. Examples of parenteral dosage forms
include, but are not limited to, solutions ready for injection, dry
products ready to be dissolved or suspended in a pharmaceutically
acceptable vehicle for injection, suspensions ready for injection,
and emulsions.
[0211] Suitable vehicles that can be used to provide parenteral
dosage forms are well known to those skilled in the art. Examples
include, but are not limited to: Water for Injection USP; aqueous
vehicles such as, but not limited to, Sodium Chloride Injection,
Ringer's Injection, Dextrose Injection, Dextrose and Sodium
Chloride Injection, and Lactated Ringer's Injection; water miscible
vehicles such as, but not limited to, ethyl alcohol, polyethylene
glycol, and polypropylene glycol; and non-aqueous vehicles such as,
but not limited to, corn oil, cottonseed oil, peanut oil, sesame
oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.
[0212] Compounds that increase the solubility of one or more of the
active ingredients disclosed herein can also be incorporated into
the parenteral dosage forms.
[0213] Transdermal, Topical & Mucosal Dosage Forms
[0214] Also provided are transdermal, topical, and mucosal dosage
forms. Transdermal, topical, and mucosal dosage forms include, but
are not limited to, ophthalmic solutions, sprays, aerosols, creams,
lotions, ointments, gels, solutions, emulsions, suspensions, or
other forms known to one of skill in the art. See, e.g.,
Remington's Pharmaceutical Sciences, 16.sup.th, 18th and 20.sup.th
eds., Mack Publishing, Easton Pa. (1980, 1990 & 2000); and
Introduction to Pharmaceutical Dosage Forms, 4th ed., Lea &
Febiger, Philadelphia (1985). Dosage forms suitable for treating
mucosal tissues within the oral cavity can be formulated as
mouthwashes or as oral gels. Further, transdermal dosage forms
include "reservoir type" or "matrix type" patches, which can be
applied to the skin and worn for a specific period of time to
permit the penetration of a desired amount of active
ingredients.
[0215] Suitable excipients (e.g., carriers and diluents) and other
materials that can be used to provide transdermal, topical, and
mucosal dosage forms encompassed herein are well known to those
skilled in the pharmaceutical arts, and depend on the particular
tissue to which a given pharmaceutical composition or dosage form
will be applied. With that fact in mind, typical excipients
include, but are not limited to, water, acetone, ethanol, ethylene
glycol, propylene glycol, butane 1,3 diol, isopropyl myristate,
isopropyl palmitate, mineral oil, and mixtures thereof to form
lotions, tinctures, creams, emulsions, gels or ointments, which are
nontoxic and pharmaceutically acceptable. Moisturizers or
humectants can also be added to pharmaceutical compositions and
dosage forms if desired. Examples of such additional ingredients
are well known in the art. See, e.g., Remington's Pharmaceutical
Sciences, 16.sup.th, 18th and 20.sup.th eds., Mack Publishing,
Easton Pa. (1980, 1990 & 2000).
[0216] Depending on the specific tissue to be treated, additional
components may be used prior to, in conjunction with, or subsequent
to treatment with active ingredients provided. For example,
penetration enhancers can be used to assist in delivering the
active ingredients to the tissue. Suitable penetration enhancers
include, but are not limited to: acetone; various alcohols such as
ethanol, oleyl, and tetrahydrofuryl; alkyl sulfoxides such as
dimethyl sulfoxide; dimethyl acetamide; dimethyl formamide;
polyethylene glycol; pyrrolidones such as polyvinylpyrrolidone;
Kollidon grades (Povidone, Polyvidone); urea; and various water
soluble or insoluble sugar esters such as Tween 80 (polysorbate 80)
and Span 60 (sorbitan monostearate).
[0217] The pH of a pharmaceutical composition or dosage form, or of
the tissue to which the pharmaceutical composition or dosage form
is applied, may also be adjusted to improve delivery of one or more
active ingredients. Similarly, the polarity of a solvent carrier,
its ionic strength, or tonicity can be adjusted to improve
delivery. Compounds such as stearates can also be added to
pharmaceutical compositions or dosage forms to advantageously alter
the hydrophilicity or lipophilicity of one or more active
ingredients so as to improve delivery. In this regard, stearates
can serve as a lipid vehicle for the formulation, as an emulsifying
agent or surfactant, and as a delivery enhancing or penetration
enhancing agent. Different salts, hydrates or solvates of the
active ingredients can be used to further adjust the properties of
the resulting composition.
[0218] Dosage and Unit Dosage Forms
[0219] In human therapeutics, the doctor will determine the
posology which he considers most appropriate according to a
preventive or curative treatment and according to the age, weight,
stage of the infection and other factors specific to the subject to
be treated. In certain embodiments, doses are from about 1 to about
1000 mg per day for an adult, or from about 5 to about 250 mg per
day or from about 10 to 50 mg per day for an adult. In certain
embodiments, doses are from about 5 to about 400 mg per day or 25
to 200 mg per day per adult. In certain embodiments, dose rates of
from about 50 to about 500 mg per day are also contemplated.
[0220] In further aspects, provided are methods of treating or
preventing an HCV infection in a subject by administering, to a
subject in need thereof, an effective amount of a compound provided
herein, or a pharmaceutically acceptable salt thereof. The amount
of the compound or composition which will be effective in the
prevention or treatment of a disorder or one or more symptoms
thereof will vary with the nature and severity of the disease or
condition, and the route by which the active ingredient is
administered. The frequency and dosage will also vary according to
factors specific for each subject depending on the specific therapy
(e.g., therapeutic or prophylactic agents) administered, the
severity of the disorder, disease, or condition, the route of
administration, as well as age, body, weight, response, and the
past medical history of the subject. Effective doses may be
extrapolated from dose-response curves derived from in vitro or
animal model test systems.
[0221] In certain embodiments, exemplary doses of a composition
include milligram or microgram amounts of the active compound per
kilogram of subject or sample weight (e.g., about 10 micrograms per
kilogram to about 50 milligrams per kilogram, about 100 micrograms
per kilogram to about 25 milligrams per kilogram, or about 100
microgram per kilogram to about 10 milligrams per kilogram). For
compositions provided herein, in certain embodiments, the dosage
administered to a subject is 0.140 mg/kg to 3 mg/kg of the
subject's body weight, based on weight of the active compound. In
certain embodiments, the dosage administered to a subject is
between 0.20 mg/kg and 2.00 mg/kg, or between 0.30 mg/kg and 1.50
mg/kg of the subject's body weight.
[0222] In certain embodiments, the recommended daily dose range of
a composition provided herein for the conditions described herein
lie within the range of from about 0.1 mg to about 1000 mg per day,
given as a single once-a-day dose or as divided doses throughout a
day. In certain embodiments, the daily dose is administered twice
daily in equally divided doses. In certain embodiments, a daily
dose range should be from about 10 mg to about 200 mg per day, in
other embodiments, between about 10 mg and about 150 mg per day, in
further embodiments, between about 25 and about 100 mg per day. It
may be necessary to use dosages of the active ingredient outside
the ranges disclosed herein in some cases, as will be apparent to
those of ordinary skill in the art. Furthermore, it is noted that
the clinician or treating physician will know how and when to
interrupt, adjust, or terminate therapy in conjunction with subject
response.
[0223] Different therapeutically effective amounts may be
applicable for different diseases and conditions, as will be
readily known by those of ordinary skill in the art. Similarly,
amounts sufficient to prevent, manage, treat or ameliorate such
disorders, but insufficient to cause, or sufficient to reduce,
adverse effects associated with the composition provided herein are
also encompassed by the herein described dosage amounts and dose
frequency schedules. Further, when a subject is administered
multiple dosages of a composition provided herein, not all of the
dosages need be the same. For example, the dosage administered to
the subject may be increased to improve the prophylactic or
therapeutic effect of the composition or it may be decreased to
reduce one or more side effects that a particular subject is
experiencing.
[0224] In certain embodiment, the dosage of the composition
provided herein, based on weight of the active compound,
administered to prevent, treat, manage, or ameliorate a disorder,
or one or more symptoms thereof in a subject is 0.1 mg/kg, 1 mg/kg,
2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 10 mg/kg, or 15 mg/kg
or more of a subject's body weight. In another embodiment, the
dosage of the composition or a composition provided herein
administered to prevent, treat, manage, or ameliorate a disorder,
or one or more symptoms thereof in a subject is a unit dose of 0.1
mg to 200 mg, 0.1 mg to 100 mg, 0.1 mg to 50 mg, 0.1 mg to 25 mg,
0.1 mg to 20 mg, 0.1 mg to 15 mg, 0.1 mg to 10 mg, 0.1 mg to 7.5
mg, 0.1 mg to 5 mg, 0.1 to 2.5 mg, 0.25 mg to 20 mg, 0.25 to 15 mg,
0.25 to 12 mg, 0.25 to 10 mg, 0.25 mg to 7.5 mg, 0.25 mg to 5 mg,
0.5 mg to 2.5 mg, 1 mg to 20 mg, 1 mg to 15 mg, 1 mg to 12 mg, 1 mg
to 10 mg, 1 mg to 7.5 mg, 1 mg to 5 mg, or 1 mg to 2.5 mg.
[0225] In certain embodiments, treatment or prevention can be
initiated with one or more loading doses of a compound or
composition provided herein followed by one or more maintenance
doses. In such embodiments, the loading dose can be, for instance,
about 60 to about 400 mg per day, or about 100 to about 200 mg per
day for one day to five weeks. The loading dose can be followed by
one or more maintenance doses. In certain embodiments, each
maintenance does is, independently, about from about 10 mg to about
200 mg per day, between about 25 mg and about 150 mg per day, or
between about 25 and about 80 mg per day. Maintenance doses can be
administered daily and can be administered as single doses, or as
divided doses.
[0226] In certain embodiments, a dose of a compound or composition
provided herein can be administered to achieve a steady-state
concentration of the active ingredient in blood or serum of the
subject. The steady-state concentration can be determined by
measurement according to techniques available to those of skill or
can be based on the physical characteristics of the subject such as
height, weight and age. In certain embodiments, a sufficient amount
of a compound or composition provided herein is administered to
achieve a steady-state concentration in blood or serum of the
subject of from about 300 to about 4000 ng/mL, from about 400 to
about 1600 ng/mL, or from about 600 to about 1200 ng/mL. In some
embodiments, loading doses can be administered to achieve
steady-state blood or serum concentrations of about 1200 to about
8000 ng/mL, or about 2000 to about 4000 ng/mL for one to five days.
In certain embodiments, maintenance doses can be administered to
achieve a steady-state concentration in blood or serum of the
subject of from about 300 to about 4000 ng/mL, from about 400 to
about 1600 ng/mL, or from about 600 to about 1200 ng/mL.
[0227] In certain embodiments, administration of the same
composition may be repeated and the administrations may be
separated by at least 1 day, 2 days, 3 days, 5 days, 10 days, 15
days, 30 days, 45 days, 2 months, 75 days, 3 months, or 6 months.
In other embodiments, administration of the same prophylactic or
therapeutic agent may be repeated and the administration may be
separated by at least at least 1 day, 2 days, 3 days, 5 days, 10
days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months, or 6
months.
[0228] In certain aspects, provided herein are unit dosages
comprising a compound, or a pharmaceutically acceptable salt
thereof, in a form suitable for administration. Such forms are
described in detail herein. In certain embodiments, the unit dosage
comprises 1 to 1000 mg, 5 to 250 mg or 10 to 50 mg active
ingredient. In particular embodiments, the unit dosages comprise
about 1, 5, 10, 25, 50, 100, 125, 250, 500 or 1000 mg active
ingredient. Such unit dosages can be prepared according to
techniques familiar to those of skill in the art.
[0229] The dosages of the second agents are to be used in the
combination therapies provided herein. In certain embodiments,
dosages lower than those which have been or are currently being
used to prevent or treat HCV infection are used in the combination
therapies provided herein. The recommended dosages of second agents
can be obtained from the knowledge of those of skill. For those
second agents that are approved for clinical use, recommended
dosages are described in, for example, Hardman et al., eds., 1996,
Goodman & Gilman's The Pharmacological Basis Of Basis Of
Therapeutics 9.sup.th Ed, Mc-Graw-Hill, New York; Physician's Desk
Reference (PDR) 57.sup.th Ed., 2003, Medical Economics Co., Inc.,
Montvale, N.J., which are incorporated herein by reference in its
entirety.
[0230] In various embodiments, the therapies (e.g., a compound
provided herein and the second agent) are administered less than 5
minutes apart, less than 30 minutes apart, 1 hour apart, at about 1
hour apart, at about 1 to about 2 hours apart, at about 2 hours to
about 3 hours apart, at about 3 hours to about 4 hours apart, at
about 4 hours to about 5 hours apart, at about 5 hours to about 6
hours apart, at about 6 hours to about 7 hours apart, at about 7
hours to about 8 hours apart, at about 8 hours to about 9 hours
apart, at about 9 hours to about 10 hours apart, at about 10 hours
to about 11 hours apart, at about 11 hours to about 12 hours apart,
at about 12 hours to 18 hours apart, 18 hours to 24 hours apart, 24
hours to 36 hours apart, 36 hours to 48 hours apart, 48 hours to 52
hours apart, 52 hours to 60 hours apart, 60 hours to 72 hours
apart, 72 hours to 84 hours apart, 84 hours to 96 hours apart, or
96 hours to 120 hours apart. In various embodiments, the therapies
are administered no more than 24 hours apart or no more than 48
hours apart. In certain embodiments, two or more therapies are
administered within the same patient visit. In other embodiments,
the compound provided herein and the second agent are administered
concurrently.
[0231] In other embodiments, the compound provided herein and the
second agent are administered at about 2 to 4 days apart, at about
4 to 6 days apart, at about 1 week part, at about 1 to 2 weeks
apart, or more than 2 weeks apart.
[0232] In certain embodiments, administration of the same agent may
be repeated and the administrations may be separated by at least 1
day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2
months, 75 days, 3 months, or 6 months. In other embodiments,
administration of the same agent may be repeated and the
administration may be separated by at least at least 1 day, 2 days,
3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75
days, 3 months, or 6 months.
[0233] In certain embodiments, a compound provided herein and a
second agent are administered to a patient, for example, a mammal,
such as a human, in a sequence and within a time interval such that
the compound provided herein can act together with the other agent
to provide an increased benefit than if they were administered
otherwise. For example, the second active agent can be administered
at the same time or sequentially in any order at different points
in time; however, if not administered at the same time, they should
be administered sufficiently close in time so as to provide the
desired therapeutic or prophylactic effect. In certain embodiments,
the compound provided herein and the second active agent exert
their effect at times which overlap. Each second active agent can
be administered separately, in any appropriate form and by any
suitable route. In other embodiments, the compound provided herein
is administered before, concurrently or after administration of the
second active agent.
[0234] In certain embodiments, the compound provided herein and the
second agent are cyclically administered to a patient. Cycling
therapy involves the administration of a first agent (e.g., a first
prophylactic or therapeutic agents) for a period of time, followed
by the administration of a second agent and/or third agent (e.g., a
second and/or third prophylactic or therapeutic agents) for a
period of time and repeating this sequential administration.
Cycling therapy can reduce the development of resistance to one or
more of the therapies, avoid or reduce the side effects of one of
the therapies, and/or improve the efficacy of the treatment.
[0235] In certain embodiments, the compound provided herein and the
second active agent are administered in a cycle of less than about
3 weeks, about once every two weeks, about once every 10 days or
about once every week. One cycle can comprise the administration of
a compound provided herein and the second agent by infusion over
about 90 minutes every cycle, about 1 hour every cycle, about 45
minutes every cycle. Each cycle can comprise at least 1 week of
rest, at least 2 weeks of rest, at least 3 weeks of rest. The
number of cycles administered is from about 1 to about 12 cycles,
more typically from about 2 to about 10 cycles, and more typically
from about 2 to about 8 cycles.
[0236] In other embodiments, courses of treatment are administered
concurrently to a patient, i.e., individual doses of the second
agent are administered separately yet within a time interval such
that the compound provided herein can work together with the second
active agent. For example, one component can be administered once
per week in combination with the other components that can be
administered once every two weeks or once every three weeks. In
other words, the dosing regimens are carried out concurrently even
if the therapeutics are not administered simultaneously or during
the same day.
[0237] The second agent can act additively or synergistically with
the compound provided herein. In certain embodiments, the compound
provided herein is administered concurrently with one or more
second agents in the same pharmaceutical composition. In another
embodiment, a compound provided herein is administered concurrently
with one or more second agents in separate pharmaceutical
compositions. In still another embodiment, a compound provided
herein is administered prior to or subsequent to administration of
a second agent. Also contemplated are administration of a compound
provided herein and a second agent by the same or different routes
of administration, e.g., oral and parenteral. In certain
embodiments, when the compound provided herein is administered
concurrently with a second agent that potentially produces adverse
side effects including, but not limited to, toxicity, the second
active agent can advantageously be administered at a dose that
falls below the threshold that the adverse side effect is
elicited.
[0238] Kits
[0239] Also provided are kits for use in methods of treatment of a
liver disorder such as HCV infections. The kits can include a
compound or composition provided herein, a second agent or
composition, and instructions providing information to a health
care provider regarding usage for treating the disorder.
Instructions may be provided in printed form or in the form of an
electronic medium such as a floppy disc, CD, or DVD, or in the form
of a website address where such instructions may be obtained. A
unit dose of a compound or composition provided herein, or a second
agent or composition, can include a dosage such that when
administered to a subject, a therapeutically or prophylactically
effective plasma level of the compound or composition can be
maintained in the subject for at least 1 days. In some embodiments,
a compound or composition can be included as a sterile aqueous
pharmaceutical composition or dry powder (e.g., lyophilized)
composition.
[0240] In some embodiments, suitable packaging is provided. As used
herein, "packaging" includes a solid matrix or material customarily
used in a system and capable of holding within fixed limits a
compound provided herein and/or a second agent suitable for
administration to a subject. Such materials include glass and
plastic (e.g., polyethylene, polypropylene, and polycarbonate)
bottles, vials, paper, plastic, and plastic-foil laminated
envelopes and the like. If e-beam sterilization techniques are
employed, the packaging should have sufficiently low density to
permit sterilization of the contents.
[0241] Methods of Use
[0242] In certain embodiments, provided herein are methods for the
treatment and/or prophylaxis of a host infected with Flaviviridae
that includes the administration of an effective amount of a
compounds provided herein, or a pharmaceutically acceptable salt
thereof. In certain embodiments, provided herein are methods for
treating an HCV infection in a subject. In certain embodiments, the
methods encompass the step of administering to the subject in need
thereof an amount of a compound effective for the treatment or
prevention of an HCV infection in combination with a second agent
effective for the treatment or prevention of the infection. The
compound can be any compound as described herein, and the second
agent can be any second agent described in the art or herein. In
certain embodiments, the compound is in the form of a
pharmaceutical composition or dosage form, as described elsewhere
herein.
[0243] Flaviviridae that can be treated are discussed generally in
Fields Virology, Editors: Fields, B. N., Knipe, D. M., and Howley,
P. M., Lippincott-Raven Publishers, Philadelphia, Pa., Chapter 31,
1996. In a particular embodiment of the invention, the Flaviviridae
is HCV. In an alternate embodiment of the invention, the
Flaviviridae is a flavivirus or pestivirus. Specific flaviviruses
include, without limitation: Absettarov, Alfuy, Apoi, Aroa, Bagaza,
Banzi, Bouboui, Bussuquara, Cacipacore, Carey Island, Dakar bat,
Dengue 1, Dengue 2, Dengue 3, Dengue 4, Edge Hill, Entebbe bat,
Gadgets Gully, Hanzalova, Hypr, Ilheus, Israel turkey
meningoencephalitis, Japanese encephalitis, Jugra, Jutiapa, Kadam,
Karshi, Kedougou, Kokobera, Koutango, Kumlinge, Kunjin, Kyasanur
Forest disease, Langat, Louping ill, Meaban, Modoc, Montana myotis
leukoencephalitis, Murray valley encephalitis, Naranjal, Negishi,
Ntaya, Omsk hemorrhagic fever, Phnom-Penh bat, Powassan, Rio Bravo,
Rocio, Royal Farm, Russian spring-summer encephalitis, Saboya, St.
Louis encephalitis, Sal Vieja, San Perlita, Saumarez Reef, Sepik,
Sokuluk, Spondweni, Stratford, Tembusu, Tyuleniy, Uganda S, Usutu,
Wesselsbron, West Nile, Yaounde, Yellow fever, and Zika.
[0244] Pestiviruses that can be treated are discussed generally in
Fields Virology, Editors: Fields, B. N., Knipe, D. M., and Howley,
P. M., Lippincott-Raven Publishers, Philadelphia, Pa., Chapter 33,
1996. Specific pestiviruses include, without limitation: bovine
viral diarrhea virus ("BVDV"), classical swine fever virus ("CSFV,"
also called hog cholera virus), and border disease virus
("BDV").
[0245] In certain embodiments, the subject can be any subject
infected with, or at risk for infection with, HCV. Infection or
risk for infection can be determined according to any technique
deemed suitable by the practitioner of skill in the art. In certain
embodiments, subjects are humans infected with HCV.
[0246] In certain embodiments, the subject has never received
therapy or prophylaxis for an HCV infection. In further
embodiments, the subject has previously received therapy or
prophylaxis for an HCV infection. For instance, in certain
embodiments, the subject has not responded to an HCV therapy. For
example, under current interferon therapy, up to 50% or more HCV
subjects do not respond to therapy. In certain embodiments, the
subject can be a subject that received therapy but continued to
suffer from viral infection or one or more symptoms thereof. In
certain embodiments, the subject can be a subject that received
therapy but failed to achieve a sustained virologic response. In
certain embodiments, the subject has received therapy for an HCV
infection but has failed to show, for example, a 2 log.sub.10
decline in HCV RNA levels after 12 weeks of therapy. It is believed
that subjects who have not shown more than 2 log.sub.10 reduction
in serum HCV RNA after 12 weeks of therapy have a 97-100% chance of
not responding.
[0247] In certain embodiments, the subject is a subject that
discontinued an HCV therapy because of one or more adverse events
associated with the therapy. In certain embodiments, the subject is
a subject where current therapy is not indicated. For instance,
certain therapies for HCV are associated with neuropsychiatric
events. Interferon (IFN)-alfa plus ribavirin is associated with a
high rate of depression. Depressive symptoms have been linked to a
worse outcome in a number of medical disorders. Life-threatening or
fatal neuropsychiatric events, including suicide, suicidal and
homicidal ideation, depression, relapse of drug addiction/overdose,
and aggressive behavior have occurred in subjects with and without
a previous psychiatric disorder during HCV therapy.
Interferon-induced depression is a limitation for the treatment of
chronic hepatitis C, especially for subjects with psychiatric
disorders. Psychiatric side effects are common with interferon
therapy and responsible for about 10% to 20% of discontinuations of
current therapy for HCV infection.
[0248] Accordingly, provided are methods of treating or preventing
an HCV infection in subjects where the risk of neuropsychiatric
events, such as depression, contraindicates treatment with current
HCV therapy. In certain embodiments, provided are methods of
treating or preventing HCV infection in subjects where a
neuropsychiatric event, such as depression, or risk of such
indicates discontinuation of treatment with current HCV therapy.
Further provided are methods of treating or preventing HCV
infection in subjects where a neuropsychiatric event, such as
depression, or risk of such indicates dose reduction of current HCV
therapy.
[0249] Current therapy is also contraindicated in subjects that are
hypersensitive to interferon or ribavirin, or both, or any other
component of a pharmaceutical product for administration of
interferon or ribavirin. Current therapy is not indicated in
subjects with hemoglobinopathies (e.g., thalassemia major,
sickle-cell anemia) and other subjects at risk from the hematologic
side effects of current therapy. Common hematologic side effects
include bone marrow suppression, neutropenia and thrombocytopenia.
Furthermore, ribavirin is toxic to red blood cells and is
associated with hemolysis. Accordingly, in certain embodiments,
provided are methods of treating or preventing HCV infection in
subjects hypersensitive to interferon or ribavirin, or both,
subjects with a hemoglobinopathy, for instance thalassemia major
subjects and sickle-cell anemia subjects, and other subjects at
risk from the hematologic side effects of current therapy.
[0250] In certain embodiments, the subject has received an HCV
therapy and discontinued that therapy prior to administration of a
method provided herein. In further embodiments, the subject has
received therapy and continues to receive that therapy along with
administration of a method provided herein. The methods can be
co-administered with other therapy for HBC and/or HCV according to
the judgment of one of skill in the art. In certain embodiments,
the methods or compositions provided herein can be co-administered
with a reduced dose of the other therapy for HBC and/or HCV.
[0251] In certain embodiments, provided are methods of treating a
subject that is refractory to treatment with interferon. For
instance, in some embodiments, the subject can be a subject that
has failed to respond to treatment with one or more agents selected
from the group consisting of interferon, interferon .alpha.,
pegylated interferon .alpha., interferon plus ribavirin, interferon
.alpha. plus ribavirin and pegylated interferon .alpha. plus
ribavirin. In some embodiments, the subject can be a subject that
has responded poorly to treatment with one or more agents selected
from the group consisting of interferon, interferon .alpha.,
pegylated interferon .alpha., interferon plus ribavirin, interferon
.alpha. plus ribavirin and pegylated interferon .alpha. plus
ribavirin. A pro-drug form of ribavirin, such as taribavirin, may
also be used.
[0252] In certain embodiments, the subject has, or is at risk for,
co-infection of HCV with HIV. For instance, in the United States,
30% of HIV subjects are co-infected with HCV and evidence indicates
that people infected with HIV have a much more rapid course of
their hepatitis C infection. Maier and Wu, 2002, World J
Gastroenterol 8:577-57. The methods provided herein can be used to
treat or prevent HCV infection in such subjects. It is believed
that elimination of HCV in these subjects will lower mortality due
to end-stage liver disease. Indeed, the risk of progressive liver
disease is higher in subjects with severe AIDS-defining
immunodeficiency than in those without. See, e.g., Lesens et al.,
1999, J Infect Dis 179:1254-1258. In certain embodiments, compounds
provided herein have been shown to suppress HIV in HIV subjects.
Thus, in certain embodiments, provided are methods of treating or
preventing HIV infection and HCV infection in subjects in need
thereof.
[0253] In certain embodiments, the compounds or compositions are
administered to a subject following liver transplant. Hepatitis C
is a leading cause of liver transplantation in the U.S., and many
subjects that undergo liver transplantation remain HCV positive
following transplantation. In certain embodiments, provided are
methods of treating such recurrent HCV subjects with a compound or
composition provided herein. In certain embodiments, provided are
methods of treating a subject before, during or following liver
transplant to prevent recurrent HCV infection.
[0254] Assay Methods
[0255] Compounds can be assayed for HCV activity according to any
assay known to those of skill in the art.
[0256] Further, compounds can be assayed for accumulation in liver
cells of a subject according to any assay known to those of skill
in the art. In certain embodiments, a compound can be administered
to the subject, and a liver cell of the subject can be assayed for
the compound or a derivative thereof, e.g. a nucleoside, nucleoside
phosphate or nucleoside triphosphate derivative thereof.
[0257] In certain embodiments, a 2'-chloro nucleoside analog
compound is administered to cells, such as liver cells, in vivo or
in vitro, and the nucleoside triphosphate levels delivered
intracellularly are measured, to indicate delivery of the compound
and triphosphorylation in the cell. The levels of intracellular
nucleoside triphosphate can be measured using analytical techniques
known in the art. Methods of detecting ddATP are described herein
below by way of example, but other nucleoside triphosphates can be
readily detected using the appropriate controls, calibration
samples and assay techniques.
[0258] In certain embodiments, ddATP concentrations are measured in
a sample by comparison to calibration standards made from control
samples. The ddATP concentrations in a sample can be measured using
an analytical method such as HPLC LC MS. In certain embodiments, a
test sample is compared to a calibration curve created with known
concentrations of ddATP to thereby obtain the concentration of that
sample.
[0259] In certain embodiments, the samples are manipulated to
remove impurities such as salts (Na.sup.+, K.sup.+, etc.) before
analysis. In certain embodiments, the lower limit of quantitation
is about .about.0.2 pmol/mL for hepatocyte cellular extracts
particularly where reduced salt is present.
[0260] In certain embodiments, the method allows successfully
measuring triphosphate nucleotides formed at levels of 1-10,000
pmol per million cells in e.g. cultured hepatocytes and HepG2
cells.
[0261] Second Therapeutic Agents
[0262] In certain embodiments, the compounds and compositions
provided herein are useful in methods of treatment of a liver
disorder, that comprise further administration of a second agent
effective for the treatment of the disorder, such as HCV infection
in a subject in need thereof. The second agent can be any agent
known to those of skill in the art to be effective for the
treatment of the disorder, including those currently approved by
the FDA.
[0263] In certain embodiments, a compound provided herein is
administered in combination with one second agent. In further
embodiments, a second agent is administered in combination with two
second agents. In still further embodiments, a second agent is
administered in combination with two or more second agents.
[0264] As used herein, the term "in combination" includes the use
of more than one therapy (e.g., one or more prophylactic and/or
therapeutic agents). The use of the term "in combination" does not
restrict the order in which therapies (e.g., prophylactic and/or
therapeutic agents) are administered to a subject with a disorder.
A first therapy (e.g., a prophylactic or therapeutic agent such as
a compound provided herein) can be administered prior to (e.g., 5
minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4
hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1
week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12
weeks before), concomitantly with, or subsequent to (e.g., 5
minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4
hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1
week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12
weeks after) the administration of a second therapy (e.g., a
prophylactic or therapeutic agent) to a subject with a
disorder.
[0265] As used herein, the term "synergistic" includes a
combination of a compound provided herein and another therapy
(e.g., a prophylactic or therapeutic agent) which has been or is
currently being used to prevent, manage or treat a disorder, which
is more effective than the additive effects of the therapies. A
synergistic effect of a combination of therapies (e.g., a
combination of prophylactic or therapeutic agents) permits the use
of lower dosages of one or more of the therapies and/or less
frequent administration of said therapies to a subject with a
disorder. The ability to utilize lower dosages of a therapy (e.g.,
a prophylactic or therapeutic agent) and/or to administer said
therapy less frequently reduces the toxicity associated with the
administration of said therapy to a subject without reducing the
efficacy of said therapy in the prevention or treatment of a
disorder). In addition, a synergistic effect can result in improved
efficacy of agents in the prevention or treatment of a disorder.
Finally, a synergistic effect of a combination of therapies (e.g.,
a combination of prophylactic or therapeutic agents) may avoid or
reduce adverse or unwanted side effects associated with the use of
either therapy alone.
[0266] The active compounds provided herein can be administered in
combination or alternation with another therapeutic agent, in
particular an anti-HCV agent. In combination therapy, effective
dosages of two or more agents are administered together, whereas in
alternation or sequential-step therapy, an effective dosage of each
agent is administered serially or sequentially. The dosages given
will depend on absorption, inactivation and excretion rates of the
drug as well as other factors known to those of skill in the art.
It is to be noted that dosage values will also vary with the
severity of the condition to be alleviated. It is to be further
understood that for any particular subject, specific dosage
regimens and schedules should be adjusted over time according to
the individual need and the professional judgment of the person
administering or supervising the administration of the
compositions. In certain embodiments, an anti-HCV (or
anti-pestivirus or anti-flavivirus) compound that exhibits an
EC.sub.50 of 10-15 .mu.M. In certain embodiments, less than 1-5
.mu.M, is desirable.
[0267] It has been recognized that drug-resistant variants of
flaviviruses, pestiviruses or HCV can emerge after prolonged
treatment with an antiviral agent. Drug resistance most typically
occurs by mutation of a gene that encodes for an enzyme used in
viral replication. The efficacy of a drug against the viral
infection can be prolonged, augmented, or restored by administering
the compound in combination or alternation with a second, and
perhaps third, antiviral compound that induces a different mutation
from that caused by the principle drug. Alternatively, the
pharmacokinetics, biodistribution or other parameter of the drug
can be altered by such combination or alternation therapy. In
general, combination therapy is typically preferred over
alternation therapy because it induces multiple simultaneous
stresses on the virus.
[0268] Any of the viral treatments described in the Background of
the Invention can be used in combination or alternation with the
compounds described in this specification. Non-limiting examples of
second agents include:
[0269] HCV Protease inhibitors: Examples include Medivir HCV
Protease Inhibitor (HCV-PI or TMC435) (Medivir/Tibotec); MK-7009
(Merck), RG7227 (ITMN-191) (Roche/Pharmasset/InterMune), boceprevir
(SCH 503034) (Schering), SCH 446211 (Schering), narlaprevir
SCH900518 (Schering/Merck), ABT-450 (Abbott/Enanta), ACH-1625
(Achillion), BI 201335 (Boehringer Ingelheim), PHX1766 (Phenomix),
VX-500 (Vertex) and telaprevir (VX-950) (Vertex). Further examples
of protease inhibitors include substrate-based NS3 protease
inhibitors (Attwood et al., Antiviral peptide derivatives, PCT WO
98/22496, 1998; Attwood et al., Antiviral Chemistry and
Chemotherapy 1999, 10, 259-273; Attwood et al., Preparation and use
of amino acid derivatives as anti-viral agents, German Patent Pub.
DE 19914474; Tung et al., Inhibitors of serine proteases,
particularly hepatitis C virus NS3 protease, PCT WO 98/17679),
including alphaketoamides and hydrazinoureas, and inhibitors that
terminate in an electrophile such as a boronic acid or phosphonate
(Llinas-Brunet et al, Hepatitis C inhibitor peptide analogues, PCT
WO 99/07734); Non-substrate-based NS3 protease inhibitors such as
2,4,6-trihydroxy-3-nitro-benzamide derivatives (Sudo K. et al.,
Biochemical and Biophysical Research Communications, 1997, 238,
643-647; Sudo K. et al., Antiviral Chemistry and Chemotherapy,
1998, 9, 186), including RD3-4082 and RD3-4078, the former
substituted on the amide with a 14 carbon chain and the latter
processing a para-phenoxyphenyl group; and Sch 68631, a
phenanthrenequinone, an HCV protease inhibitor (Chu M. et al.,
Tetrahedron Letters 37:7229-7232, 1996).
[0270] SCH 351633, isolated from the fungus Penicillium
griseofulvum, was identified as a protease inhibitor (Chu M. et
al., Bioorganic and Medicinal Chemistry Letters 9:1949-1952). Eglin
c, isolated from leech, is a potent inhibitor of several serine
proteases such as S. griseus proteases A and B, a-chymotrypsin,
chymase and subtilisin. Qasim M. A. et al., Biochemistry
36:1598-1607, 1997.
[0271] U.S. patents disclosing protease inhibitors for the
treatment of HCV include, for example, U.S. Pat. No. 6,004,933 to
Spruce et al., which discloses a class of cysteine protease
inhibitors for inhibiting HCV endopeptidase 2; U.S. Pat. No.
5,990,276 to Zhang et al., which discloses synthetic inhibitors of
hepatitis C virus NS3 protease; U.S. Pat. No. 5,538,865 to Reyes et
a; WO 02/008251 to Corvas International, Inc., and U.S. Pat. No.
7,169,760, US2005/176648, WO 02/08187 and WO 02/008256 to Schering
Corporation. HCV inhibitor tripeptides are disclosed in U.S. Pat.
Nos. 6,534,523, 6,410,531, and 6,420,380 to Boehringer Ingelheim
and WO 02/060926 to Bristol Myers Squibb. Diaryl peptides as NS3
serine protease inhibitors of HCV are disclosed in WO 02/48172 and
U.S. Pat. No. 6,911,428 to Schering Corporation. Imidazoleidinones
as NS3 serine protease inhibitors of HCV are disclosed in WO
02/08198 and U.S. Pat. No. 6,838,475 to Schering Corporation and WO
02/48157 and U.S. Pat. No. 6,727,366 to Bristol Myers Squibb. WO
98/17679 and U.S. Pat. No. 6,265,380 to Vertex Pharmaceuticals and
WO 02/48116 and U.S. Pat. No. 6,653,295 to Bristol Myers Squibb
also disclose HCV protease inhibitors. Further examples of HCV
serine protease inhibitors are provided in U.S. Pat. No. 6,872,805
(Bristol-Myers Squibb); WO 2006000085 (Boehringer Ingelheim); U.S.
Pat. No. 7,208,600 (Vertex); US 2006/0046956 (Schering-Plough); WO
2007/001406 (Chiron); US 2005/0153877; WO 2006/119061 (Merck); WO
00/09543 (Boehringer Ingelheim), U.S. Pat. No. 6,323,180
(Boehringer Ingelheim) WO 03/064456 (Boehringer Ingelheim), U.S.
Pat. No. 6,642,204(Boehringer Ingelheim), WO 03/064416 (Boehringer
Ingelheim), U.S. Pat. No. 7,091,184 (Boehringer Ingelheim), WO
03/053349 (Bristol-Myers Squibb), U.S. Pat. No. 6,867,185, WO
03/099316 (Bristol-Myers Squibb), U.S. Pat. No. 6,869,964, WO
03/099274 (Bristol-Myers Squibb), U.S. Pat. No. 6,995,174, WO
2004/032827 (Bristol-Myers Squibb), U.S. Pat. No. 7,041,698, WO
2004/043339 and U.S. Pat. No. 6,878,722 (Bristol-Myers Squibb).
[0272] Thiazolidine derivatives which show relevant inhibition in a
reverse-phase HPLC assay with an NS3/4A fusion protein and NS5A/5B
substrate (Sudo K. et al., Antiviral Research, 1996, 32, 9-18),
especially compound RD-1-6250, possessing a fused cinnamoyl moiety
substituted with a long alkyl chain, RD4 6205 and RD4 6193;
[0273] Thiazolidines and benzanilides identified in Kakiuchi N. et
al., J. EBS Letters 421, 217-220; Takeshita N. et al., Analytical
Biochemistry, 1997, 247, 242-246;
[0274] A phenanthrenequinone possessing activity against protease
in a SDS-PAGE and autoradiography assay isolated from the
fermentation culture broth of Streptomyces sp., SCH 68631 (Chu M.
et al., Tetrahedron Letters, 1996, 37, 7229-7232), and SCH 351633,
isolated from the fungus Penicillium griseofulvum, which
demonstrates activity in a scintillation proximity assay (Chu M. et
al., Bioorganic and Medicinal Chemistry Letters 9, 1949-1952);
[0275] Helicase inhibitors (Diana G. D. et al., Compounds,
compositions and methods for treatment of hepatitis C, U.S. Pat.
No. 5,633,358; Diana G. D. et al., Piperidine derivatives,
pharmaceutical compositions thereof and their use in the treatment
of hepatitis C, PCT WO 97/36554);
[0276] HCV polymerase inhibitors, including nucleoside and
non-nucleoside polymerase inhibitors, such as ribavirin,
viramidine, clemizole, filibuvir (PF-00868554), HCV POL, NM 283
(valopicitabine), MK-0608, 7-Fluoro-MK-0608, MK-3281, IDX-375,
ABT-072, ABT-333, ANA598, BI 207127, GS 9190, PSI-6130, R1626,
PSI-6206, PSI-938, PSI-7851, PSI-7977, RG1479, RG7128, HCV-796
VCH-759 or VCH-916.
[0277] Gliotoxin (Ferrari R. et al., Journal of Virology, 1999, 73,
1649-1654), and the natural product cerulenin (Lohmann V. et al.,
Virology, 1998, 249, 108-118);
[0278] Interfering RNA (iRNA) based antivirals, including short
interfering RNA (siRNA) based antivirals, such as Sirna-034 and
others described in International Patent Publication Nos.
WO/03/070750 and WO 2005/012525, and US Patent Publication No. US
2004/0209831.
[0279] Antisense phosphorothioate oligodeoxynucleotides (S-ODN)
complementary to sequence stretches in the 5' non-coding region
(NCR) of the virus (Alt M. et al., Hepatology, 1995, 22, 707-717),
or nucleotides 326-348 comprising the 3' end of the NCR and
nucleotides 371-388 located in the core coding region of the HCV
RNA (Alt M. et al., Archives of Virology, 1997, 142, 589-599;
Galderisi U. et al., Journal of Cellular Physiology, 1999, 181,
251-257);
[0280] Inhibitors of IRES-dependent translation (Ikeda N et al.,
Agent for the prevention and treatment of hepatitis C, Japanese
Patent Pub. JP-08268890; Kai Y. et al., Prevention and treatment of
viral diseases, Japanese Patent Pub. JP-10101591);
[0281] HCV NS5A inhibitors, such as BMS-790052 (daclatasvir,
Bristol-Myers Squibb), PPI-461 (Presidio Pharmaceuticals), PPI-1301
(Presidio Pharmaceuticals), IDX-719 (Idenix Pharmaceuticals),
AZD7295 (Arrow Therapeutics, AstraZeneca), EDP-239 (Enanta),
ACH-2928 (Achillion), ACH-3102 (Achillion), ABT-267 (Abbott), or
GS-5885 (Gilead);
[0282] HCV entry inhibitors, such as celgosivir (MK-3253) (MIGENIX
Inc.), SP-30 (Samaritan Pharmaceuticals), ITX4520 (iTherX), ITX5061
(iTherX), PRO-206 (Progenics Pharmaceuticals) and other entry
inhibitors by Progenics Pharmaceuticals, e.g., as disclosed in U.S.
Patent Publication No. 2006/0198855.
[0283] Ribozymes, such as nuclease-resistant ribozymes (Maccjak, D.
J. et al., Hepatology 1999, 30, abstract 995) and those disclosed
in U.S. Pat. No. 6,043,077 to Barber et al., and U.S. Pat. Nos.
5,869,253 and 5,610,054 to Draper et al.; and
[0284] Nucleoside analogs have also been developed for the
treatment of Flaviviridae infections.
[0285] In certain embodiments, the compounds provided herein can be
administered in combination with any of the compounds described by
Idenix Pharmaceuticals in International Publication Nos. WO
01/90121, WO 01/92282, WO 2004/003000, 2004/002422 and WO
2004/002999.
[0286] Other patent applications disclosing the use of certain
nucleoside analogs that can be used as second agents to treat
hepatitis C virus include: PCT/CA00/01316 (WO 01/32153; filed Nov.
3, 2000) and PCT/CA01/00197 (WO 01/60315; filed Feb. 19, 2001)
filed by BioChem Pharma, Inc. (now Shire Biochem, Inc.);
PCT/US02/01531 (WO 02/057425; filed Jan. 18, 2002); PCT/US02/03086
(WO 02/057287; filed Jan. 18, 2002); U.S. Pat. No. 7,202,224; U.S.
Pat. No. 7,125,855; U.S. Pat. No. 7,105,499 and U.S. Pat. No.
6,777,395 by Merck & Co., Inc.; PCT/EP01/09633 (WO 02/18404;
published Aug. 21, 2001); US 2006/0040890; 2005/0038240;
2004/0121980; U.S. Pat. No. 6,846,810; U.S. Pat. No. 6,784,166 and
U.S. Pat. No. 6,660,721 by Roche; PCT Publication Nos. WO 01/79246
(filed Apr. 13, 2001), WO 02/32920 (filed Oct. 18, 2001) and WO
02/48165; US 2005/0009737; US 2005/0009737; U.S. Pat. No. 7,094,770
and U.S. Pat. No. 6,927,291 by Pharmasset, Ltd.
[0287] Further compounds that can be used as second agents to treat
hepatitis C virus are disclosed in PCT Publication No. WO 99/43691
to Emory University, entitled "2'-Fluoronucleosides". The use of
certain 2'-fluoronucleosides to treat HCV is disclosed.
[0288] Other compounds that can be used as second agents include
1-amino-alkylcyclohexanes (U.S. Pat. No. 6,034,134 to Gold et al.),
alkyl lipids (U.S. Pat. No. 5,922,757 to Chojkier et al.), vitamin
E and other antioxidants (U.S. Pat. No. 5,922,757 to Chojkier et
al.), squalene, amantadine, bile acids (U.S. Pat. No. 5,846,964 to
Ozeki et al.), N-(phosphonoacetyl)-L-aspartic acid, (U.S. Pat. No.
5,830,905 to Diana et al.), benzenedicarboxamides (U.S. Pat. No.
5,633,388 to Diana et al.), polyadenylic acid derivatives (U.S.
Pat. No. 5,496,546 to Wang et al.), 2',3'-dideoxyinosine (U.S. Pat.
No. 5,026,687 to Yarchoan et al.), benzimidazoles (U.S. Pat. No.
5,891,874 to Colacino et al.), plant extracts (U.S. Pat. No.
5,837,257 to Tsai et al., U.S. Pat. No. 5,725,859 to Omer et al.,
and U.S. Pat. No. 6,056,961), and piperidines (U.S. Pat. No.
5,830,905 to Diana et al.).
[0289] In certain embodiments, a compound of a formula provided
herein, or a composition comprising a compound of a formula
provided herein, is administered in combination or alternation with
a second anti-viral agent selected from the group consisting of an
interferon, a nucleotide analogue, a polymerase inhibitor, an NS3
protease inhibitor, an NS5A inhibitor, an entry inhibitor, a
non-nucleoside polymerase inhibitor, a cyclosporine immune
inhibitor, an NS4A antagonist, an NS4B-RNA binding inhibitor, a
locked nucleic acid mRNA inhibitor, a cyclophilin inhibitor, and
combinations thereof.
[0290] Exemplary Second Therapeutic Agents for Treatment of HCV
[0291] In certain embodiments, one or more compounds provided
herein can be administered in combination or alternation with an
anti-hepatitis C virus interferon, such as Intron A.RTM.
(interferon alfa-2b) and; Roferon A.RTM. (Recombinant interferon
alfa-2a), Infergen.RTM. (consensus interferon; interferon
alfacon-1), PEG-Intron.RTM. (pegylated interferon alfa-2b), and
Pegasys.RTM. (pegylated interferon alfa-2a). In certain
embodiments, one or more compounds provided herein can be
administered in combination or alternation with ribavirin and in
combination or alternation with an anti-hepatitis C virus
interferon. In certain embodiments, one or more compounds provided
herein can be administered in combination or alternation with
ribavirin, in combination or alternation with an anti-hepatitis C
virus interferon, and in combination or alternation with an
anti-hepatitis C virus protease inhibitor. In certain embodiments,
one or more compounds provided herein can be administered in
combination or alternation with ribavirin. In certain embodiments,
one or more compounds provided herein can be administered in
combination or alternation with an anti-hepatitis C virus
interferon and without ribavirin. In certain embodiments, one or
more compounds provided herein can be administered in combination
or alternation with an anti-hepatitis C virus interferon, in
combination or alternation with an anti-hepatitis C virus protease
inhibitor, and without ribavirin.
[0292] In certain embodiments, the anti-hepatitis C virus
interferon is infergen, IL-29 (PEG-Interferon lambda), R7025
(Maxy-alpha), Belerofon, Oral Interferon alpha, BLX-883 (Locteron),
omega interferon, multiferon, medusa interferon, Albuferon or
REBIF.RTM..
[0293] In certain embodiments, one or more compounds provided
herein can be administered in combination or alternation with an
anti-hepatitis C virus polymerase inhibitor, such as ribavirin,
viramidine, HCV POL, NM 283 (valopicitabine), MK-0608,
7-Fluoro-MK-0608, PSI-6130, R1626, PSI-6206, PSI-938, R1479,
HCV-796, VX-950 (Telaprevir, Vertex), GS 9190 NN (Gilead), GS 9256
(Gilead), PSI-7792 (BMS), BI 207127 (BI), R7128 (Roche), or
PSI-7977 (Pharmasset), PSI-938 (Pharmasset), VX-222 (Vertex),
ALS-2200 (Vertex), ALS-2158 (Vertex), MK-0608 (Merck), TMC649128
(Medivir), PF-868554 (Pfizer), PF-4878691 (Pfizer), ANA598 (Roche),
VCH-759 (Vertex), IDX184 (Idenix), IDX375 (Idenix), A-837093
(Abbott), GS 9190 (Gilead), GSK625433 (GlaxoSmithKline), ABT-072
(Abbott), ABT-333 (Abbott), INX-189 (Inhibitex), or EDP-239
(Enanta).
[0294] In certain embodiments, the one or more compounds provided
herein can be administered in combination with ribavarin and an
anti-hepatitis C virus interferon, such as Intron A.RTM.
(interferon alfa-2b) and Pegasys.RTM. (Peginterferon alfa-2a);
Roferon A.RTM. (Recombinant interferon alfa-2a), Infergen.RTM.
(consensus interferon; interferon alfacon-1), PEG-Intron.RTM.
(pegylated interferon alfa-2b), Zalbin (albinterferon alfa-2b),
omega interferon, pegylated interferon lambda, and Pegasys.RTM.
(pegylated interferon alfa-2a).
[0295] In certain embodiments, one or more compounds provided
herein can be administered in combination or alternation with an
anti-hepatitis C virus protease inhibitor such as ITMN-191, SCH
503034 (bocepravir), VX950 (telaprevir), VX985, VX500, VX813,
PHX1766, BMS-650032, GS 9256, BI 201335, IDX320, R7227, MK-7009
(vaniprevir), TMC435, BMS-791325, ACH-1625, ACH-2684, ABT-450, or
AVL-181.
[0296] In certain embodiments, one or more compounds provided
herein can be administered in combination or alternation with an
HCV NS5A inhibitor, such as BMS-790052 (daclatasvir, Bristol-Myers
Squibb), PPI-461 (Presidio Pharmaceuticals), PPI-1301 (Presidio
Pharmaceuticals), IDX-719 (Idenix Pharmaceuticals), AZD7295 (Arrow
Therapeutics, AstraZeneca), EDP-239 (Enanta), ACH-2928 (Achillion),
ACH-3102 (Achillion), ABT-267 (Abbott), or GS-5885 (Gilead).
[0297] In certain embodiments, one or more compounds provided
herein can be administered in combination or alternation with an
anti-hepatitis C virus vaccine, such as TG4040, PeviPRO.TM.,
CGI-5005, HCV/MF59, GV1001, IC.sub.41, GNI-103, GenPhar HCV
vaccine, C-Vaxin, CSL123, Hepavaxx C, ChronVac-C.RTM. or INNO0101
(E1).
[0298] In certain embodiments, one or more compounds provided
herein can be administered in combination or alternation with an
anti-hepatitis C virus monoclonal antibody, such as MBL-HCV1, AB68
or XTL-6865 (formerly HepX-C); or an anti-hepatitis C virus
polyclonal antibody, such as cicavir.
[0299] In certain embodiments, one or more compounds provided
herein can be administered in combination or alternation with an
anti-hepatitis C virus immunomodulator, such as Zadaxin.RTM.
(thymalfasin), SCV-07, NOV-205 or Oglufanide.
[0300] In certain embodiments, one or more compounds provided
herein can be administered in combination or alternation with
cyclophilin inhibitor, such as Enanta cyclophilin binder, SCY-635,
or Debio-025.
[0301] In certain embodiments, one or more compounds provided
herein can be administered in combination or alternation with
Nexavar, doxorubicin, PI-88, amantadine, JBK-122, VGX-410C, MX-3253
(Ceglosivir), Suvus (BIVN-401 or virostat), PF-03491390 (formerly
IDN-6556), G126270, UT-231B, DEBIO-025, EMZ702, ACH-0137171, MitoQ,
ANA975, AVI-4065, Bavituxinab (Tarvacin), Alinia (nitrazoxanide) or
PYN17.
[0302] In certain embodiments, one or more compounds provided
herein can be administered in combination or alternation with
telaprevir, bocepravir, interferon alfacon-1, interferon alfa-2b,
pegylated interferon alpha 2a, pegylated interferon alpha 2b,
ribavirin, or combinations thereof.
[0303] In certain embodiments, one or more compounds provided
herein can be administered in combination or alternation with a
protease inhibitor. In certain embodiments, one or more compounds
provided herein can be administered in combination or alternation
with telaprevir. In certain embodiments, one or more compounds
provided herein can be administered in combination or alternation
with bocepravir.
[0304] In certain embodiments, one or more compounds provided
herein can be administered in combination or alternation with a
protease inhibitor and in combination or alternation with
ribavirin. In certain embodiments, one or more compounds provided
herein can be administered in combination or alternation with
telaprevir and in combination or alternation with ribavirin. In
certain embodiments, one or more compounds provided herein can be
administered in combination or alternation with bocepravir and in
combination or alternation with ribavirin.
[0305] In certain embodiments, one or more compounds provided
herein can be administered in combination or alternation with a
protease inhibitor and not in combination or alternation with
ribavirin. In certain embodiments, one or more compounds provided
herein can be administered in combination or alternation with
telaprevir and not in combination or alternation with ribavirin. In
certain embodiments, one or more compounds provided herein can be
administered in combination or alternation with bocepravir and not
in combination or alternation with ribavirin.
[0306] In certain embodiments, one or more compounds provided
herein can be administered in combination or alternation with an
interferon. In certain embodiments, one or more compounds provided
herein can be administered in combination or alternation with
interferon alfacon-1. In certain embodiments, one or more compounds
provided herein can be administered in combination or alternation
with interferon alfa-2b. In certain embodiments, one or more
compounds provided herein can be administered in combination or
alternation with pegylated interferon alpha 2a. In certain
embodiments, one or more compounds provided herein can be
administered in combination or alternation with pegylated
interferon alpha 2b.
[0307] In certain embodiments, one or more compounds provided
herein can be administered in combination or alternation with an
interferon and in combination or alternation with ribavirin. In
certain embodiments, one or more compounds provided herein can be
administered in combination or alternation with interferon
alfacon-land in combination or alternation with ribavirin. In
certain embodiments, one or more compounds provided herein can be
administered in combination or alternation with interferon alfa-2b
and in combination or alternation with ribavirin. In certain
embodiments, one or more compounds provided herein can be
administered in combination or alternation with pegylated
interferon alpha 2a and in combination or alternation with
ribavirin. In certain embodiments, one or more compounds provided
herein can be administered in combination or alternation with
pegylated interferon alpha 2b and in combination or alternation
with ribavirin.
[0308] In certain embodiments, one or more compounds can be
administered in combination or alternation with one or more of the
second agents provided herein and not in combination or alternation
with ribavirin. In certain embodiments, one or more compounds
provided herein can be administered in combination or alternation
with an interferon and not in combination or alternation with
ribavirin. In certain embodiments, one or more compounds provided
herein can be administered in combination or alternation with
interferon alfacon-land not in combination or alternation with
ribavirin. In certain embodiments, one or more compounds provided
herein can be administered in combination or alternation with
interferon alfa-2b and not in combination or alternation with
ribavirin. In certain embodiments, one or more compounds provided
herein can be administered in combination or alternation with
pegylated interferon alpha 2a and not in combination or alternation
with ribavirin. In certain embodiments, one or more compounds
provided herein can be administered in combination or alternation
with pegylated interferon alpha 2b and not in combination or
alternation with ribavirin.
EXAMPLES
[0309] As used herein, the symbols and conventions used in these
processes, schemes and examples, regardless of whether a particular
abbreviation is specifically defined, are consistent with those
used in the contemporary scientific literature, for example, the
Journal of the American Chemical Society or the Journal of
Biological Chemistry. Specifically, but without limitation, the
following abbreviations may be used in the examples and throughout
the specification: g (grams); mg (milligrams); mL (milliliters);
.mu.L (microliters); mM (millimolar); .mu.M (micromolar); Hz
(Hertz); MHz (megahertz); mmol (millimoles); hr or hrs (hours); min
(minutes); MS (mass spectrometry); ESI (electrospray ionization);
TLC (thin layer chromatography); HPLC (high pressure liquid
chromatography); THF (tetrahydrofuran); CDCl.sub.3 (deuterated
chloroform); AcOH (acetic acid); DCM (dichloromethane); DMSO
(dimethylsulfoxide); DMSO-d.sub.6 (deuterated dimethylsulfoxide);
EtOAc (ethyl acetate); MeOH (methanol); and BOC
(t-butyloxycarbonyl).
[0310] For all of the following examples, standard work-up and
purification methods known to those skilled in the art can be
utilized. Unless otherwise indicated, all temperatures are
expressed in .degree. C. (degrees Centigrade). All reactions are
conducted at room temperature unless otherwise noted. Synthetic
methodologies illustrated herein are intended to exemplify the
applicable chemistry through the use of specific examples and are
not indicative of the scope of the disclosure.
Example 1
Preparation of 2'-Chloro Nucleoside Analogs
##STR00084## ##STR00085## ##STR00086##
[0311]
Ethyl(3R)-2-chloro-3-[(4R)-2,2-dimethyl-1,3-dioxolan-4-yl]-3-hydrox-
y-2-methylpropanoate (A2)
##STR00087##
[0313] A 5 L flange flask was fitted with a thermometer, nitrogen
inlet, pressure equalizing dropping funnel, bubbler, and a
suba.cndot.seal. Methyl lithium solution (1.06 L, 1.6 M in
diethylether, 1.7 equiv.) was added, and the solution was cooled to
about -25.degree. C. Diisopropyl amine (238 ml, 1.7 equiv.) was
added using the dropping funnel over about 40 minutes. The reaction
was left stirring, allowing to warm to ambient temperature
overnight. CO.sub.2(s)/acetone cooling was applied to the LDA
solution, cooling to about -70.degree. C.
[0314] R-Glyceraldehyde dimethylacetal solution (50% in DCM) was
evaporated down to .about.100 mbar at a bath temp of 35.degree. C.,
to remove the DCM, then azeotroped with anhydrous hexane (200 ml),
under the same Biichi conditions. .sup.1H NMR was used to confirm
that all but a trace of DCM remained.
[0315] The fresh aldehyde (130 g, 1 mol) and ethyl
2-chloropropionionate (191 ml, 1.5 equiv.) were placed in a 1 L
round bottom flask, which was filled with toluene (800 ml). This
solution was cooled in a CO.sub.2(s)/acetone bath, and added via
cannula to the LDA solution over about 50 minutes, keeping the
internal temperature of the reaction mixture cooler than
-60.degree. C. The mixture was stirred with cooling (internal temp.
slowly fell to .about.-72.degree. C.) for 90 min, then warmed to
room temperature over 30 minutes using a water bath. This solution
was added to a sodium dihydrogen phosphate solution equivalent to
360 g of NaH.sub.2PO.sub.4 in 1.5 L of ice/water, over about 10
minutes, with ice-bath cooling. The mixture was stirred for 20
minutes, then transferred to a sep. funnel, and partitioned. The
aqueous layer was further extracted with EtOAc (2.times.1 L), and
the combined organic extracts were dried over sodium sulfate. The
volatiles were removed in vacuo (down to 20 mbar). The resultant
oil was hydrolyzed crude.
(3R,4R,5R)-3-chloro-4-hydroxy-5-(hydroxymethyl)-3-methyloxolan-2-one
(A4)
##STR00088##
[0317] The crude oil A2 was taken up in acetic acid (1.5 L, 66% in
water) and heated to 90.degree. C. over one hour, then at held at
that temperature for one hour. Once the mixture had cooled to room
temperature, the volatiles were removed in vacuo, and azeotroped
with toluene (500 ml). The resultant oil was combined with some
mixed material from an earlier synthesis and columned in two
portions (each .about.1.25 L of silica, 38.fwdarw.75% EtOAc in
DCM). The lower of the two main spots is the desired material;
fractions containing this material as the major component were
combined and the solvent removed in vacuo to give 82 g of orange
solid whose .sup.1H NMR showed the material to be of about 57%
purity (of the remainder 29% was the indicated epimer). This
material was recrystallized from toluene/butanone (600
ml/.about.185 ml), the butanone being the `good` solvent. The
resultant solid was filtered washing with toluene and hexane, and
dried in vacuo to give product of about 92% purity (30 g).
(2R,3R,4R)-2-[(benzoyloxy)methyl]-4-chloro-4-methyl-5-oxooxolan-3-yl
benzoate (A5)
##STR00089##
[0319] A 2 L 3-neck round bottom flask was fitted with an overhead
stirrer, thermometer and pressure equalizing dropping funnel
(.fwdarw.N.sub.2). The intermediate A4 (160 mmol) in acetonitrile
(1 L) was added, followed by 4-dimethylaminopyridine (3.2 mmol) and
benzoyl chloride (352 mmol). Finally triethylamine (384 mmol) was
added over 10 minutes using the dropping funnel. The addition of
the triethylamine is accompanied by a mild exotherm, which obviated
the addition of a cold water bath to keep the internal temperature
below 25.degree. C. The reaction was stirred at ambient temperature
for 2.5 hours. The reaction mixture was transferred to a sep.
funnel with EtOAc (2 L) and half saturated brine (2 L), and
partitioned. The aqueous layer was re-extracted with EtOAc (1 L).
The combined organic layers were washed with 50% sodium
bicarbonate/25% brine (1.5 L) and dried over sodium sulfate, to
give 62 g of solid. This was recrystallized from 1.8 L of 1:1
toluene/trimethylpentane (95.degree. C.), to give 52.4 g of
product.
[0320] .sup.1H NMR (CDCl.sub.3, 400 MHz): .delta. (ppm) 1.91 (s,
3H), 4.57 (dd, J=5.12 Hz and J=12.57 Hz, 1H), 4.77 (dd, J=3.29 Hz
and J=12.68 Hz, 1H), 4.92-4.96 (m, 1H), 5.60 (d, J=8.36 Hz, 1H),
7.38-7.66 (m, 6H), 7.97-7.99 (m, 2H), 8.08-8.10 (m, 2H); MS (ESI)
m/z=411.1 (MNa.sup.+).
3,5-Di-O-benzoyl-2-C-chloro-2-C-methyl-D-ribofuranose (A6)
##STR00090##
[0322] To a solution of A5 (14.48 mmol) in anhydrous
tetrahydrofurane (70 ml) was added under inert atmosphere at
-35.degree. C., LiAlH(OtBu).sub.3 (1M in tetrahydrofurane, 21.7
mmol) over a 30 min period. The reaction mixture was stirred for 1
hour at -20.degree. C. and quenched by addition of a saturated
NH.sub.4Cl solution, keeping the temperature bellow 0.degree. C.
Ethyl acetate was added and the white suspension was filtered
through a pad of celite and washed with ethyl acetate. The filtrate
was extracted with ethyl acetate twice. The combined organic layers
were dried over anhydrous sodium sulfate, filtered and evaporated
under reduced pressure. The residue was purified by chromatography
on silica gel (eluent: petroleum ether/ethyl acetate 0 to 20%). The
product was dried in vacuum (50.degree. C.) overnight to afford
expected intermediate as a colorless oil in 96% yield (mixture
.alpha./.beta.: 45/55).
[0323] .sup.1H NMR (CDCl.sub.3, 400 MHz): .delta. (ppm) 1.74 (s,
1.75H.sub..beta.), 1.76 (s, 1.25H.sub..alpha.), 4.42-4.69 (m, 3H),
5.30 (d, J=12.8 Hz, 0.55H.sub..beta.), 5.43-5.47 (m,
0.45H.sub..alpha.), 5.60 (d, J=7.0 Hz, 0.55H.sub..beta.), 5.78 (d,
J=7.0 Hz, 0.45H.sub..alpha.), 7.35-7.41 (m, 2H), 7.45-7.56 (m, 3H),
7.59-7.65 (m, 1H), 7.96-8.04 (m, 2H), 8.06-8.14 (m, 2H); MS (ESI)
m/z=413 (MNa.sup.+).
3,5-Di-O-benzoyl-2-C-chloro-2-C-methyl-D-arabinofuranosyl bromide
(A7)
##STR00091##
[0325] To a solution of A6 (12.80 mmol) in anhydrous
dichloromethane (80 ml) was added under inert atmosphere at
-20.degree. C., triphenylphosphine (18.0 mmol). The reaction
mixture was stirred for 15 minutes at -20.degree. C. and CBr.sub.4
(19.20 mmol) was added. The reaction mixture was then stirred for 1
hour at -20.degree. C. The crude was partially concentrated under
reduced pressure (bath temperature bellow 30.degree. C.) and
directly purified by chromatography on silica gel (eluent:
petroleum ether/ethyl acetate 0 to 30%) to afford a mixture of 0
sugar A7a (1.67 g) and a sugar A7b (2.15 g) as a colorless gum in
66% global yield.
[0326] .sup.1H NMR (CDCl.sub.3, 400 MHz): .beta. sugar .delta.
(ppm) 1.93 (s, 3H), 4.60-4.88 (m, 3H), 6.08 (d, J=7.9 Hz, 1H), 6.62
(s, 1H), 7.31-7.38 (m, 2H), 7.41-7.55 (m, 3H), 7.59-7.65 (m, 1H),
8.00-8.05 (m, 2H), 8.06-8.12 (m, 2H); .alpha. sugar .delta. (ppm)
1.88 (s, 3H), 4.66-4.89 (m, 3H), 5.37 (d, J=4.88 Hz, 1H), 6.44 (s,
1H), 7.41-7.55 (m, 4H), 7.54-7.65 (m, 2H), 8.00-8.05 (m, 2H),
8.14-8.20 (m, 2H); MS (ESI) m/z=476/478 (MNa.sup.+).
3',5'-Di-O-benzoyl-2'-C-chloro-2'-C-methyl-4-benzoyl-cytidine
(A8)
##STR00092##
[0328] To a suspension of N-benzoyl cytosine (9.48 mmol), and a
catalytic amount of ammonium sulfate in 4-chlorobenzene (24 ml) was
added HMDS (28.44 mmol). The reaction mixture was heated during 2
hours at 140.degree. C. The solvent was removed under inert
atmosphere and the residue was taken in 4-chlorobenzene (15 ml).
Then, A7b (4.74 mmol) in chlorobenzene (10 ml) was added dropwise
to the reaction mixture followed by SnCl.sub.4 (14.22 mmol)
dropwise. The reaction mixture was stirred at 70.degree. C.
overnight, cooled to room temperature and diluted with
dichloromethane and a saturated NaHCO.sub.3 solution. The white
suspension was filtered through a pad of celite and washed with
dichloromethane. The filtrate was extracted with dichloromethane
twice. The combined organic layers were dried over anhydrous
Na.sub.2SO.sub.4, filtered and evaporated under reduced pressure to
afford expected intermediate as a white solid in 89% yield.
[0329] .sup.1H NMR (DMSO, 400 MHz): .delta. (ppm) 1.58 (s, 3H),
4.68-4.81 (m, 3H), 5.68 (brs, 1H), 6.55 (brs, 1H), 7.36 (d, J=7.84
Hz, 1H), 7.39-7.76 (m, 9H), 7.88-8.07 (m, 6H), 8.30 (d, J=7.84 Hz,
1H); MS (ESI) m/z=588 (MH.sup.+).
3',5'-Di-O-benzoyl-2'-C-chloro-2'-C-methyluridine (A9)
##STR00093##
[0331] A suspension of A8 (4.19 mmol) in an acetic acid/water
mixture (67 ml/17 ml, v/v), was heated at 110.degree. C. for 3
hours. The reaction mixture was evaporated to dryness and
co-evaporated with toluene (three times) to afford expected
intermediate in quantitative yield as an oil which was directly
used for the next step; MS (ESI) m/z=485 (MH.sup.+).
2'-C-Chloro-2'-C-methyluridine (301)
##STR00094##
[0333] Intermediate A9 (4.19 mmol) in 7 N methanolic ammonia (80
ml) was stirred at room temperature for 24 hours. The mixture was
evaporated to dryness, diluted with water and transferred into a
separatory funnel. The aqueous layer was extracted with
dichloromethane and water was removed under reduced pressure. The
residue was purified by flash RP18 gel chromatography (eluent:
water/acetonitrile 0 to 40%) to afford pure expected compound as a
white foam in 79% yield.
[0334] .sup.1H NMR (DMSO, 400 MHz): .delta. (ppm) 1.44 (s, 3H),
3.60-3.68 (m, 1H), 3.80-3.94 (m, 3H), 5.39 (t, J=4.45 Hz, 1H), 5.63
(d, J=8.26 Hz, 1H), 5.93 (d, J=5.72 Hz, 1H), 6.21 (s, 1H), 8.16 (d,
J=8.90 Hz, 1H), 11.44 (m, 1H); MS (ESI) m/z=277 (MH.sup.+).
General Method D
[0335] The following procedure was used to obtain intermediates
A22a, A22b, A22c and A22d.
##STR00095##
##STR00096##
[0336] To a stirred solution of 4-nitrophenyl dichlorophosphate
(Aldrich) (14.91 mmol) in DCM (30 mL) was added a solution of
phenol (Aldrich) (14.91 mmol) and TEA (16.40 mmol) in DCM (30 mL)
at -78.degree. C. over a period of 20 minutes. The reaction mixture
was stirred at -78.degree. C. during 30 minutes and then,
transferred into another round-bottom flask containing L- or
D-alanine isopropyl ethyl ester hydrochloride (14.91 mmol) in DCM
(30 mL) at 0.degree. C. To the mixture was added TEA (31.31 mmol)
over a period of 15 minutes. The reaction mixture was stirred at
0.degree. C. during 1 hour and then, the solvent was evaporated.
The residue was triturated with ethyl acetate (45 mL) and the white
solid was filtered-off. The filtrate was concentrated under reduced
pressure and the residue was purified by chromatography on silica
gel (eluent: petroleum ether-petroleum ether/ethyl acetate 20%) to
give the expected intermediate.
Isopropyl(2S)-2-[[(4-nitrophenoxy)-phenoxy-phosphoryl]amino]propanoate
(A22a)
##STR00097##
[0338] 60% yield; .sup.1H NMR (CDCl.sub.3, 400 MHz) .delta. (ppm)
1.15 (d, J=6.26 Hz, 3H), 1.16 (d, J=6.26 Hz, 3H), 1.33 (m, 3H),
3.83 (dd, J=9.7 and 11.76 Hz, 1H), 3.97-4.08 (m, 1H), 4.94
(heptuplet, J=6.26 Hz, 1H), 7.11-7.19 (m, 3H), 7.27-7.35 (m, 4H),
8.16 (dd, J=1.72 and 9.07 Hz, 2H); .sup.31P NMR (CDCl.sub.3, 161.98
MHz): .delta. (ppm)-3.21 (s, 0.45P), -3.18 (s, 0.55P); MS (ESI)
m/z=409.14 (MH.sup.+).
Isopropyl(2R)-2-[[(4-nitrophenoxy)-phenoxy-phosphoryl]amino]propanoate
(A22b)
##STR00098##
[0340] 80% yield; .sup.1H NMR (CDCl.sub.3, 400 MHz) .delta. (ppm)
1.22 (d, J=6.28 Hz, 3H), 1.23 (d, J=6.28 Hz, 3H), 1.40 (m, 3H),
3.91-3.96 (m, 1H), 4.05-4.13 (m, 1H), 5.01 (heptuplet, J=6.30 Hz,
1H), 7.19-7.25 (m, 3H), 7.33-7.41 (m, 4H), 8.22 (dd, J=1.74 Hz and
8.95 Hz, 2H); .sup.31P NMR (CDCl.sub.3, 161.98 MHz): .delta.
(ppm)-3.21 (s, 0.45P), -3.18 (s, 0.55P); MS (ESI) m/z=409.14
(MH.sup.+).
Butyl (2R)-2-[[(4-nitrophenoxy)-phenoxy-phosphoryl]amino]propanoate
(A22c)
##STR00099##
[0342] 72% yield; yellow oil; .sup.1H NMR (CDCl.sub.3, 400 MHz)
.delta. (ppm) 0.92 (t, J=7.35 Hz, 3H), 1.30-1.39 (m, 2H), 1.40-1.43
(m, 3H), 1.56-1.63 (m, 2H), 3.84-3.89 (m, 1H), 4.08-4.18 (m, 3H),
7.18-7.26 (m, 3H), 7.33-7.41 (m, 4H), 8.23 (dd, J=1.77 Hz and 9.01
Hz, 2H); MS (ESI) m/z=423 (MH.sup.+).
Benzyl
(2R)-2-[[(4-nitrophenoxy)-phenoxy-phosphoryl]amino]propanoate
(A22d)
##STR00100##
[0344] 89% yield; yellow oil; .sup.1H NMR (CDCl.sub.3, 400 MHz)
.delta. (ppm) 1.41-1.44 (m, 3H), 3.82-3.88 (m, 1H), 4.13-4.25 (m,
1H), 5.14-5.15 (m, 2H), 7.18-7.24 (m, 3H), 7.28-7.38 (m, 9H),
8.16-8.21 (m, 2H); MS (ESI) m/z=457 (MH.sup.+).
General Method F
[0345] The following procedure was used to obtain compounds 40i and
40ii.
[0346] To a solution of compound 301 (15 mmol) in THF (5 mL/mmol)
was added tert-butylmagnesium chloride (1M in THF) (31 mmol) over a
period of 10 minutes. Appropriate intermediate A22 (18 mmol) in THF
(20 mL) was added and the reaction mixture was stirred at room
temperature during 3 days. The reaction mixture was quenched with
saturated aqueous solution of ammonium chloride. The residue was
suspended in ethyl acetate and washed with water. The organic layer
was washed with aqueous sodium bicarbonate and brine, dried over
MgSO.sub.4, filtered and concentrated under reduced pressure. The
residue was purified by chromatography on silica gel (eluent:
DCM-DCM/MeOH 2%) to separate the diastereoisomers.
Compound 40ii
Diastereoisomer 2
##STR00101##
[0348] White solid; 13% yield; .sup.1H NMR (CDCl.sub.3, 400 MHz):
.delta. (ppm) 1.24-1.26 (m, 6H), 1.36 (d, J=7.04 Hz, 3H), 1.59 (s,
3H), 3.69-3.77 (m, 1H), 3.91-3.99 (m, 2H), 4.17-4.19 (m, 1H),
4.43-4.59 (m, 2H), 5.01-5.06 (m, 1H), 5.68 (d, J=8.20 Hz, 1H), 6.42
(s, 1H), 7.21-7.39 (m, 5H), 7.60 (d, J=8.20 Hz, 1H), 8.14 (s, 1H);
.sup.31P NMR (CDCl.sub.3, 161.98 MHz): .delta. (ppm) 3.47 (s, 1P);
MS (ESI) m/z=546.2 (MH.sup.+).
Compound 40i
Diastereoisomer 1
##STR00102##
[0350] In this case, after chromatography on silica gel, the
mixture of diastereoisomers was purified by preparative HPLC.
[0351] White solid; 3% yield; .sup.1H NMR (CDCl.sub.3, 400 MHz)
.delta. 1.25 (d, J=6.25 Hz, 6H), 1.38 (d, J=7.04 Hz, 3H), 1.51 (s,
3H), 3.66-3.74 (m, 2H), 3.82-3.96 (m, 2H), 4.15 (dd, J=1.62 and
9.24 Hz, 1H), 4.39-4.53 (m, 2H), 5.03 (heptuplet, J=6.26 Hz, 1H),
5.56 (dd, J=2.29 and 8.18 Hz, 1H), 6.39 (s, 1H), 7.19-7.26 (m, 3H),
7.34-7.43 (m, 3H), 8.06 (s, 1H); .sup.31P NMR (CDCl.sub.3, 161.98
MHz): .delta. (ppm) 3.35 (s, 1P); MS (ESI) m/z=546.20
(MH.sup.+).
##STR00103##
[0352] The following abbreviations are used in Scheme 8:
##STR00104##
General Method K
[0353] The following procedure was used to obtain compounds 202i
and 205i.
[0354] To as solution of compound 301 (0.72 mmol) in anhydrous THF
(7 mL/mmol) under nitrogen at room temperature was added
tert-butylmagnesium chloride (1M in THF) (1.52 mmol) followed by
compound A22c or A22d (0.795 mmol) solubilized in THF (4 mL/mmol).
DMSO (4 mL/mmol) was added and the mixture was stirred at room
temperature overnight. The reaction mixture was diluted with
dichloromethane and washed with H.sub.2O. The organic phase was
dried, filtered and concentrated under reduced pressure. The
residue was purified by chromatography on silica gel (eluent:
DCM/MeOH 0 to 2%) followed by purification by preparative HPLC to
give the expected pure diastereoisomers.
Compound 202i
Mixture of Diastereoisomers
##STR00105##
[0355] 202i P-Diastereoisomer 1
[0356] 15% yield; white solid; .sup.1H NMR (CDCl.sub.3, 400 MHz):
.delta. (ppm) 0.93 (t, J=7.37 Hz, 3H), 1.32-1.40 (m, 2H), 1.40 (d,
J=7.04 Hz, 3H), 1.51 (s, 3H), 1.56-1.65 (m, 2H), 3.63 (d, J=7.70
Hz, 1H), 3.70-3.75 (m, 1H), 3.82-3.86 (m, 1H), 3.92-4.02 (m, 1H),
4.08-4.19 (m, 3H), 4.39-4.52 (m, 2H), 5.56 (d, J=8.20 Hz, 1H), 6.39
(s, 1H), 7.19-7.26 (m, 3H), 7.34-7.38 (m, 2H), 7.41 (d, J=8.21 Hz,
1H), 8.10 (s, 1H); .sup.31P NMR (CDCl.sub.3, 161.98 MHz): .delta.
(ppm) 4.27 (s, 1P); MS (ESI, El.sup.+) m/z=560 (MH.sup.+).
202i P-Diastereoisomer 2
[0357] 18% yield; white solid; .sup.1H NMR (CDCl.sub.3, 400 MHz):
.delta. (ppm) 0.92 (t, J=7.35 Hz, 3H), 1.30-1.38 (m, 2H), 1.37 (d,
J=7.13 Hz, 3H), 1.57-1.62 (m, 2H), 1.61 (s, 3H), 3.45-3.53 (m, 2H),
4.00-4.20 (m, 5H), 4.46-4.59 (m, 2H), 5.63 (d, J=8.26 Hz, 1H), 6.44
(s, 1H), 7.19-7.22 (m, 3H), 7.34-7.38 (m, 2H), 7.66 (d, J=8.18 Hz,
1H), 8.04 (s, 1H); .sup.31P NMR (CDCl.sub.3, 161.98 MHz): .delta.
(ppm) 3.84 (s, 1P); MS (ESI, El.sup.+) m/z=560 (MH.sup.+)
Compound 205i
Mixture of Diastereoisomers
##STR00106##
[0358] 205i P-Diastereoisomer 1
[0359] 12% yield; white solid; .sup.1H NMR (CDCl.sub.3, 400 MHz):
.delta. (ppm) 1.40 (d, J=7.08 Hz, 3H), 1.49 (s, 3H), 3.55 (d,
J=7.83 Hz, 1H), 3.65-3.70 (m, 1H), 3.77-3.81 (m, 1H), 3.97-4.04 (m,
1H), 4.07-4.10 (m, 1H), 4.28-4.45 (m, 2H), 5.16 (s, 2H), 5.54 (d,
J=8.22 Hz, 1H), 6.36 (s, 1H), 7.18-7.23 (m, 3H), 7.31-7.38 (m, 8H),
7.99 (s, 1H); .sup.31P NMR (CDCl.sub.3, 161.98 MHz): .delta. (ppm)
4.07 (s, 1P); MS (ESI, El.sup.+) m/z=594 (MH.sup.+).
205i P-Diastereoisomer 2
[0360] 19% yield; white solid; .sup.1H NMR (CDCl.sub.3, 400 MHz):
.delta. (ppm) 1.38 (d, J=7.11 Hz, 3H), 1.59 (s, 3H), 3.41 (d,
J=7.94 Hz, 1H), 3.51-3.56 (m, 1H), 3.98-4.02 (m, 1H), 4.09-4.19 (m,
2H), 4.43-4.57 (m, 2H), 5.13 (s, 2H), 5.61 (d, J=8.27 Hz, 1H), 6.43
(s, 1H), 7.18-7.22 (m, 3H), 7.28-7.39 (m, 7H), 7.63 (d, J=8.16 Hz,
1H), 8.16 (s, 1H); .sup.31P NMR (CDCl3, 161.98 MHz): .delta. (ppm)
3.69 (s, 1P); MS (ESI, El.sup.+) m/z=594 (MH.sup.+).
Example 1B
Preparation of Bridged Nucleosides
##STR00107## ##STR00108##
[0362] Isomer separation was carried out using a preparative SFC
system equipped with an AS-H chiral column and using
methanol/CO.sub.2 as the mobile phase. Synthesis of single
diastereomers was performed as provided in Scheme 2.
##STR00109##
[0363] DMSO (163.7 mL, 2.31 mol) was added drop-wise to a solution
of oxalyl chloride (97.5 mL, 1.15 mol) in DCM (1.5 L) at
-78.degree. C. After 15 min at this temperature a solution of A1
(200 g, 0.77 mol) in DCM (500 mL) was added drop-wise. After
additional 15 min at -78.degree. C. triethylamine (536 mL, 3.84
mol, 5 eq) was added drop-wise. The reaction mixture was allowed to
warm to -20.degree. C. then ethanol (1 L) and water (0.5 L) were
added followed by portion-wise addition of NaBH4 (30.2 g, 0.8 mol,
1.04 eq). The reaction mixture was allowed to warm to room
temperature and stirred for 18 hrs. The reaction mixture was poured
into 1M HCl aqueous solution and extracted with DCM. The organic
layers were washed with water, brine, dried (MgSO4) and evaporated
to give A2 (200 g, 100%) as an off-white solid. .sup.1H NMR (300
MHz, CDCl3) .delta. 5.80 (d, 1H), 4.60 (dd, 1H), 4.30 (dt, 1H),
4.10-3.99 (m, 3H), 3.80 (dd, 1H), 2.57 (d, 1H), 1.56 (s, 3H), 1.45
(s, 3H), 1.39 (s, 3H), 1.38 (s, 3H).
Preparation of compound A3
##STR00110##
[0365] NaH (60% in mineral oil, 14.4 g, 0.36 mol) was suspended in
acetonitrile (600 mL) and cooled to 0.degree. C. A solution of A2
(78.0 g, 0.3 mol) in acetonitrile (600 mL) was added drop-wise
followed by a solution of benzyl bromide (42.8 mL, 0.36 mol) in
acetonitrile (100 mL). The reaction mixture was stirred for 4 h
before careful addition of methanol (100 mL). The reaction mixture
was partitioned between EtOAc and water. The aqueous layer was
extracted with EtOAc. The organic layers were combined and dried
(MgSO4) and evaporated to give A3 (-115 g, 100%) as a white solid.
.sup.1H NMR .delta. (300 MHz, CDCl3) .delta. 7.41-7.28 (m, 5H),
5.74 (d, 1H), 4.77 (d, 1H), 4.59 (d, 1H), 4.57 (d, 1H), 4.37 (ddd,
1H), 4.13 (dd, 1H), 4.04-3.92 (m, 2H), 3.88 (dd, 1H), 1.58 (s, 3H),
1.36 (s, 3H), 1.35 (s, 3H).
Preparation of compound A4
##STR00111##
[0367] Acetic acid in water (80%, 1 L) was added to A3 (100 g, 0.29
mol) and the mixture stirred at r.t. for 42 h. The reaction mixture
was poured into a solution of NaOH solution (540 g in 3 L water)
with vigorous stirring then extracted with EtOAc (.times.3). The
organic layers were combined and dried (MgSO4) then evaporated to
give A4 (86.7 g, 98%) as yellow oil. .sup.1H NMR .delta. (300 MHz,
CDCl3) .delta. 7.47-7.28 (m, 5H), 5.77 (d, 1H), 4.78 (d, 1H),
4.65-4.50 (m, 2H), 4.16-4.10 (m, 1H), 4.03-3.97 (m, 1H), 3.92 (dd,
1H), 3.75-3.61 (m, 2H), 2.50-2.38 (m, 2H), 1.59 (s, 3H), 1.36 (s,
3H).
Preparation of compound A5
##STR00112##
[0369] A solution of A4 (23.66 g, 76.2 mmol) in water (250 mL) was
slowly added to a solution of sodium periodate (19.08 g, 89.2 mmol)
in water (125 mL) at 0.degree. C. After 30 min., ethylene glycol
(2.5 mL) was added and the reaction mixture extracted with EtOAc.
The organic layers were combined, dried (MgSO4) and evaporated to
give AS (20.44 g, 96%) as yellow oil. .sup.1H NMR .delta. (300 MHz,
CDCl3) .delta. 9.61 (d, 1H), 7.42-7.24 (m, 5H), 5.81 (d, 1H), 4.75
(d, 1H), 4.63 (d, 1H), 4.60 (t, 1H), 4.49 (dd, 1H), 3.85 (dd, 1H),
1.60 (s, 3H), 1.36 (s, 3H).
Preparation of compound A6
##STR00113##
[0371] Aqueous 37% formaldehyde (40 mL) followed by 1N NaOH (200
mL) were added to a solution of A5 (20.44 g, 73.45 mmol) in water
(150 mL) and dioxane (50 mL) at 0.degree. C. The reaction mixture
was stirred at r.t. for 7 days and then partitioned between EtOAc
and brine. The organic layers were combined, dried (MgSO4) and
evaporated to give A6 (22.79 g, 100%) as a pale yellow oil. .sup.1H
NMR .delta. (400 MHz, CDCl3) .delta. 7.41-7.28 (m, 5H), 5.76 (d,
1H), 4.80 (d, 1H), 4.62 (dd, 1H), 4.52 (d, 1H), 4.21 (d, 1H), 3.90
(dd, 2H), 3.78 (dd, 1H), 3.55 (dd, 1H), 2.37 (t, 1H), 1.89 (dd,
1H), 1.63 (s, 3H), 1.33 (s, 3H).
Preparation of compound A7
##STR00114##
[0373] A solution of A6 (84.98 g, 273.83 mmol) in pyridine (360 mL)
was cooled to 0.degree. C. and MsCl (63.89 mL, 825.47 mmol) was
added portion wise. After addition, the reaction slurry was stirred
at room temperature for 3 hrs before cooled back to 14.degree. C.
55 mL water was added drop-wise over 23 min and temperature rose up
to 53.degree. C. Then pyridine was mostly removed using rotary
evaporator (bath temperature <40.degree. C.). The mixture was
then partitioned into EtOAc 550 mL and water 500 mL. Organic layer
was further washed with brine and the first aqueous layer was also
back extracted with EtOAc (200 mL). Organic layers were combined
and dried over Na2SO4, evaporated and chased with acetonitrile (100
mL.times.2) and gave yellow solid A7 (136.61 g, 91.83%). .sup.1H
NMR .delta. (400 MHz, CDCl3) .delta. 7.42-7.28 (m, 5H), 5.80 (d,
1H), 4.90 (d, 1H), 4.80 (d, 1H), 4.67 (m, 1H), 4.60 (d, 1H), 4.33
(2, 1H), 4.20 (d, 1H), 4.16 (d, 1H), 3.10 (s, 3H), 3.00 (s, 3H),
1.70 (s, 3H), 1.36 (s, 3H) LCMS [M] 485.2.
Preparation of compound A8
##STR00115##
[0375] Compound A7 (136.61 g, 268.9 mmol) was slowly dissolved into
acetic acid (1.25 L) and the solution was cooled to 7.degree. C.
before acetic anhydride (190 mL, 2010 mmol, 7.5 eq) and
concentrated H2SO4 (1.72 mL, 33 mmol, 0.12 eq) were added. The
solution was stirred for another 10 min. before warmed up to room
temperature. The solution was stirred at room temperature for 18
hrs and then cooled to 9.degree. C. 120 mL of water was added over
2 min and the mixture was stirred at room temperature for 1 hr,
before it was partitioned between water 964 mL and DCM 1140 mL.
Organic layer was isolated and evaporated in order to remove acetic
acid and gave yellow oil. The oil was re-dissolved into DCM (820
mL) and washed twice with saturated NaHCO3 solution. The solution
was dried over Na2SO4 and evaporated to give yellow oil A8 (127.3
g, 94.7%). .sup.1H NMR .delta. (400 MHz, CDCl3) .delta. 7.41-7.29
(m, 5H), 6.20 (S, 1H), 5.40 (d, 1H), 4.64 (d, 1H), 4.52 (m, 2H),
4.44 (m, 1H), 4.39 (d, 1H), 4.31 (d, 1H), 4.21 (m, 1H). LCMS
[M+CH3COO--] 569.0.
Preparation of compound A9
##STR00116##
[0377] At room temperature, into the mixture of A8 (113.04 g, 221.4
mmol) and chloro-purine (41.31 g, 243.6 mmol) in 1,2-dichloroethane
(1.36 L), was added N,O-Bis(trimethylsilyl)acetamide (108.4 mL,
443.3 mmol). The slurry was then heated to 81.degree. C. for 40
min. and cooled back to 29.degree. C. TMSOTf (81.34 mL, 444.9 mmol)
was added all at once and temperature rose to 36.degree. C. The
mixture was then heated to 81.degree. C. again for 2 hrs before it
was cooled down to room temperature. 1,2-dichloroethane was then
removed using rotary evaporator and remaining mixture was
partitioned into DCM (1.15 L) and saturated NaHCO3 solution (0.64
L). Solid crashed and slurry was filtered and solid rinsed with 65
mL of DCM. Filtrate and rinse were combined and the organic layer
was washed again with sat NaHCO3 solution, 5% brine solution and
dried over Na2SO4, evaporated to give a foamy solid A9 (136.79 g,
94.7%). .sup.1H NMR .delta. (400 MHz, CDCl3) .delta. 7.69 (S, 1H),
7.34-7.27 (m, 5H), 5.92 (d, 1H), 5.57 (t, 1H), 5.32 (b, 2H), 5.13
(d, 1H), 4.73 (d, 1H), 4.60 (m, 3H), 4.33 (t, 2H), 2.97 (s, 3H),
2.94 (s, 3H), 2.04 (s, 3H) LCMS [M] 620.15
Preparation of compound A10
##STR00117##
[0379] Compound A9 (136.79 g, 207.4 mmol) was mixed with 1.3 L THF
and 1.3 L EtOH. The solution was cooled to 0.degree. C., before
NaOEt (95%, 81.71 g, 1140.6 mmol) was added in portions. The
mixture was stirred and warmed up to room temperature over 18 hrs.
The mixture was then cooled to 0.degree. C. before HC12N (650 mL)
was added in portions. Organic solvents were removed and remaining
crude oil was partitioned into 1.0 L EtOAc and 150 mL water.
Aqueous layer was back extracted with EtOAc (200 mL) and all
organic layers were combined and washed with 5% brine solution (400
mL.times.4). Organic layer was isolated and dried over Na2SO4,
evaporated and gave brown powder A10 (100.0 g, 93.9%). .sup.1H NMR
.delta. (400 MHz, CDCl3) .delta. 7.63 (S, 1H), 7.37-7.30 (m, 5H),
5.93 (d, 1H), 4.93 (b, 2H), 4.76 (S, 1H), 4.73-4.54 (m, 6H), 4.40
(S, 1H), 4.20 (d, 1H), 4.01 (d, 1H), 3.04 (S, 3H), 1.49 (t, 3H).
LCMS [M+H] 492.19.
Preparation of compound A11
##STR00118##
[0381] Compound A10 (113.5 g, 219.7 mmol) was dissolved into DMSO
(114 mL). Then NaOBz powder (99.41 g, 689.9 mmol) was added. The
slurry was heated to 97.degree. C. for 2.5 hrs before it was cooled
down and mixed with water (1 L), and then with EtOAc (1 L). Aqueous
layer was further washed with EtOAc (0.7 L) and all EtOAc layers
were combined and washed with saturated NaHCO3 solution (0.72 L),
and with 5% brine solution (0.75 L.times.2). EtOAc layer was dried
over Na2SO4, evaporated and gave powder A11 (118.6 g, 93.6%).
.sup.1H NMR .delta. (400 MHz, CDCl3) .delta. 7.93 (t, 2H), 7.87 (s,
1H), 7.69 (m, 1H), 7.53 (m, 2H), 7.34-7.28 (m, 5H), 6.54 (b, 2H),
5.92 (S, 1H), 4.82 (S, 1H), 4.76 (d, 2H), 4.71 (d, 2H), 4.59 (s,
1H), 4.46 (m, 2H), 4.12 (m, 1H), 4.05 (m, 1H), 1.36 (t, 3H). LCMS
[M+H] 518.27.
Preparation of compound A12
##STR00119##
[0383] Compound A11 (118.6 g, 214.5 mmol) was dissolved into THF
(1.1 L). Then into the solution was added NaOH aq (NaOH 30.89 g,
772.2 mmol, 3.6 eq, with water 0.5 L) at room temperature. The
mixture was stirred over 16 hrs and then heated to 35.degree. C.
for 6.5 hrs. The reaction mixture was cooled to 1.degree. C. and
HCl (1N 550 mL) was added. Organic layer and aqueous layer were
separated. The aqueous layer was back extracted with EtOAc (0.5 L)
and both organic layers were combined and washed with saturated
NaHCO3 (450 mL) and then with 5% brine (450 mL.times.2). Brine
washes were combined and washed with EtOAc (200 mL). All organic
layers were combined and dried over Na2SO4, evaporated and gave
crude solid (103 g). The crude solid was purified on column (1.5 kg
Gold combiflash column, with solvents DCM and EtOAc), and gave pure
solid compound A12 (56.49 g, 96%). .sup.1H NMR .delta. (400 MHz,
CDCl3) .delta. 7.93 (s, 1H), 7.34-7.27 (m, 5H), 6.53 (br, 2H), 5.84
(s, 1H), 5.15 (t, 1H), 4.65 (d, 3H), 4.46 (q, 2H), 4.29 (s, 1H),
3.95 (d, 1H), 3.81 (m, 3H), 3.18 (d, 1H), 1.36 (t, 3H). LCMS [M+H]
414.20.
Preparation of compound A15
##STR00120##
[0385] To a stirred solution of D-alanine isopropyl ester HCl A13
(14.2 g, 84.66 mmol) and phenyl dichlorophosphate 14 (12.6 mL,
84.66 mmol) in DCM (142 mL) at -70.degree. C. was added a solution
of triethylamine (24.7 mL) in DCM (142 mL) over 50 min. The mixture
was stirred at this temperature for additional 1.5 hrs. The mixture
was filtered through a sintered glass funnel and the filtrate was
concentrated under reduced pressure. The residue was triturated
with TBME (120 mL), filtered off and rinsed with TBME (2.times.120
mL). The combined filtrate was concentrated under reduced pressure
to give A15 (25.9 g, 100%), which was used for the following
coupling reaction without further purification.
Preparation of compound A16
##STR00121##
[0387] To a stirred solution of nucleoside A12 (10 g, 24.19 mmol)
and N-methylimidazole (15.4 mL, 193.52 mmol) in DCM (200 mL) at
5.degree. C., was added a solution of compound A15 (25.9 g, 84.66
mmol) in DCM (45 mL) over 1 hr. The mixture was allowed to warm to
rt overnight and then concentrated under reduced pressure to give
yellow oil. This oil was diluted with EtOAc (200 mL) and water (200
mL). The organic layer was separated, washed with 5% aqueous
ammonium chloride solution (2.times.200 mL) and 5% brine solution
(200 mL), dried (sodium sulphate), filtered and concentrated under
reduced pressure to give crude product (29.9 g). The crude compound
was chromatographed using EtOAc/dichloromethane 3:2 gradient to
give product A16 (13.8 g, 83%) as off-white solid. .sup.1H NMR
(DMSO-d6) .delta. 7.92 (s, 1H), 7.17-7.34 (m, 10H), 6.54 (br s,
2H), 6.07 (q, 1H), 5.88 (d, 1H), 4.84 (m, 1H), 4.76 (d, 1H), 4.68
(d, 1H), 4.47 (m, 5H), 4.03 (m, 1H), 3.84 (m, 2H), 1.37 (t, 3H),
1.19 (m, 4H), 1.13 (m, 6H); .sup.31P NMR 3.70, 3.48; HPLC (test20)
5.42 min; LCMS 16.35 min (M.sup.++H) 683.33.
Preparation of 425 isomer mixture
##STR00122##
[0389] To a stirred mixture of Pd/C (5.4 g) in EtOH (140 mL) at
22.degree. C. was added a solution of nucleoside A16 (13.8 g, 20.21
mmol) in EtOH (560 mL), and the reaction mixture was heated to
50.degree. C. for 45 min. The crude mixture was filtered through a
Celite pad and rinsed with MeOH (4.times.250 mL). The combined
filtrate was concentrated under reduced pressure to give 12.4 g of
crude product. The crude compound was chromatographed using 0-5%
MeOH/dichloromethane gradient to give 425 (mixture of isomers, 9.3
g, 77% yield) as an off-white solid. .sup.1H NMR (DMSO-d6) .delta.
7.96 (s, 1H), 7.17-7.37 (m, 5H), 6.54 (br s, 2H), 6.06 (q, 1H),
5.90 (d, 1H), 5.81 (d, 1H), 4.86 (m, 1H), 4.32-4.47 (m, 6H), 3.97
(d, 1H), 3.79 (m, 2H), 1.37 (t, 3H), 1.34 (m, 3H), 1.16 (m, 6H);
.sup.31P NMR 3.83, 3.63; HPLC (test20) 4.34 min; LCMS 11.72 min
(M++H) 593.27.
Preparation of the compound A17
##STR00123##
[0391] To a stirred solution of D-alanine isopropyl ester
hydrochloride A13 (20 g, 119.3 mmol) and phenyl dichlorophosphate
(25.3 g, 17.9 mL, 118.8 mmol) in anhydrous dichloromethane (150 mL)
was added a solution of triethylamine (25.4 g, 35 mL, 251.3 mmol)
in anhydrous dichloromethane (150 mL) at -70.degree. C. over 45 min
dropwise. The reaction mixture was stirred at this temperature for
additional 30 min and then allowed to warm to 0.degree. C. over 2 h
and stirred for 1 h. To this mixture was added a solution of
2,3,4,5,6-pentafluoro phenol (22 g, 119.5 mmol) and triethylamine
(1.3 g, 17 mL, 122 mmol) in anhydrous dichloromethane (75 mL) over
40 min. The crude mixture was stirred at 0.degree. C. for 2 h and
then stored at 5.degree. C. over night. The white solid
(triethylamine hydrochloride) was filtered off and washed with
dichloromethane (1.times.25 mL). The filtrate was concentrated
under reduced pressure, the residue was triturated with TBME (300
mL) and the triethylamine hydrochloride salt was removed by
filtration. The cake was washed with dichloromethane (2.times.25
mL) and the combined filtrate was concentrated under reduced
pressure to give the crude solid containing even mixture of
diastereomers. The mixture was triturated with 20% EtOAc in hexanes
(200 mL) to give 29.5 g of compound A17 as a white solid. This was
further purified using a mixture of IPA (240 mL) and water (290 mL)
to give the desired compound A17 (21.5 g, 40%). .sup.31P NMR
(CDCl3, 162 MHz) .delta.-1.56; .sup.1H NMR (CDCl3, 400 MHz) .delta.
7.40-7.36 (m, 2H), 7.29-7.21 (m, 3H), 5.10-5.011H), 4.21-4.02 (m,
2H), 1.47 (d, J=7.2 Hz, 3H), 1.29-1.24 (m, 6H).
Preparation of compound A18
##STR00124##
[0393] To the stirred solution of compound A17 (1.5 g, 3.63 mmol)
in dry THF (35 mL) was added a 1.0 M solution of
tert-butylmagnesium chloride in THF (4.5 mL, 5.4 mmol) over 7 min
at -9.degree. C. The reaction mixture was stirred at that
temperature for 10 min and a solution of compound A2 (2 g, 4.4
mmol) in THF (10 mL) was added over 10 min at -9.degree. C. The
crude reaction mixture was stirred at that for additional 40 min,
warmed to rt over a period of 1 h, and then quenched with 2 N HCl
(20 mL). Toluene (100 mL) was added and the layers separated,
aqueous layer re-extracted with toluene (50 mL). The combined
toluene layer was washed with brine (1.times.50 mL), dried over
anhydrous sodium sulfate, filtered and concentrated under reduced
pressure to give the crude product (4.2 g). The crude compound was
chromatographed using 0-5% MeOH/dichloromethane gradient to give
product A18 (2.2 g, yield -88%). .sup.31P NMR (CDCl3, 162 MHz)
.delta. 2.43; .sup.1H NMR (CDCl3, 400 MHz) .delta. 7.68 (s, 1H),
7.29-7.22 (m, 9H), 7.16-7.12 (m, 1H), 5.90 (s, 1H), 5.02-4.98 (m,
2H), 4.64-4.55 (m, 2H), 4.46-4.45 (m, 3H), 4.13-4.11 (m, 2H),
4.02-3.92 (m, 2H), 3.83-3.80 (m, 1H), 1.48 (t, J=7.2 Hz, 3H),
1.39-1.37 (m, 4H), 1.27-1.19 (m, 6H); LCMS: 683.33 (MH+).
Preparation of 425
##STR00125##
[0395] To a stirred solution of compound A18 (2.0 g, 2.93 mmol) in
ethanol (40 mL) was added Pd/C (10%, 1.1 g). The crude mixture was
heated at 50.degree. C. and ammonium formate (0.96 g, 15.24 mmol)
was added. The reaction mixture was heated for additional 1.5 hrs
and filtered over a pad of celite. The celite bed washed with MeOH
(30 mL) and the filtrate was concentrated to give 3 g of crude
product. The crude product was chromatographed using 0-5%
MeOH/dichloromethane gradient to give 25 (1.0 g, yield 58%).
.sup.31P NMR (CDCl3, 162 MHz) .delta. 3.61; .sup.1H NMR (DMSO-d6,
400 MHz) .delta. 7.93 (s, 1H), 7.38-7.34 (m, 2H), 7.23-7.15 (m,
3H), 6.15 (bs, 2H), 6.09-6.04 (m, 1H), 5.95 (d, J=4 Hz, 1H), 5.80
(s, 1H), 4.89-4.86 (m, 1H), 4.51-4.29 (m, 6H), 3.99 (d, J=8 Hz,
1H), 3.82-3.73 (m, 2H), 1.35 (t, J=7.2 Hz, 3H), 1.23 (d, J=7.2 Hz,
3H), 1.16-1.14 (m, 6H); LCMS: 593.23 (MH+).
Preparation of 401
##STR00126##
[0396] (2S)-isopropyl
2-(((((1R,3R,4R,7S)-3-(2-amino-6-oxo-1H-purin-9(6H)-yl)-7-(benzyloxy)-2,5-
-dioxabicyclo[2.2.1] heptan-1-yl)methoxy)(phenoxy)phosphoryl)amino)
propanoate (B6)
[0397] To a solution of phenyl dichlorophosphate (7394, 4.7 mmol)
in THF (10 mL) at -55.6.degree. C. under Argon, was added L-alanine
isopropyl ester (827 mg, 4.93 mmol, 1.05 eq.) dissolved in 8.3 mL
of DCM over 5 min. (-44.3.degree. C.), Triethylamine (1.38 mL, 9.87
mmol, 2.1 eq.) was added over 3 min (-48.6.degree.
C..fwdarw.-40.degree. C.). The reaction was kept <-30.degree. C.
and reaction followed by LCMS, .sup.1H and .sup.31P NMR that
indicated reaction completion after 25 min. to give compound
B4.
[0398] To a suspension of B5 (1 g, 2.35 mmol, 0.5 eq.) in THF/DCM
(10/5 mL) at -40.degree. C. under Argon, was added t-BuMgCl (5.17
mL, 5.17 mmol, 1.1 eq.) over 4 min. (-32.1.degree. C.). The
reaction mixture was kept stirring at 0.degree. C. for 45 min (B5
completely solubilized). To this solution cooled at -50.degree. C.
was added the previous chlorophosphoramidate solution (compound 4)
over 7 min (-36.8.degree. C.) and 5 mL of THF was used to rinse the
remaining phosphoramidate compound. The reaction was kept stirring
at 0.degree. C. for 30 min., the reaction was followed by LCMS.
[0399] To the reaction mixture was added 25 mL 5% brine and 25 mL
ethyl acetate. The organic was separated then washed with 20 mL 5%
brine. The organic layer was then dried over Na.sub.2SO.sub.4 and
concentrated to give 2.47 g of a yellow oil. The crude product was
purified by Combiflash (80 g gold column, DCM 100%.fwdarw.DCM/MeOH
90/10). Compound B6 was isolated as a white solid (818.8 mg, 51%).
.sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 7.90 (d, J=4 Hz, 1H),
7.36-7.30 (m, 7H), 7.19-7.15 (m, 3H), 6.54 (br s, 2H), 6.08 (m,
1H), 5.88 (d, J=8 Hz, 1H), 5.01 (m, 1H), 4.77 (d, J=12 Hz, 1H),
4.66 (m, 2H), 4.50-4.38 (m, 5H), 4.02 (dd, J=8 Hz, 20 Hz, 1H),
3.86-3.74 (m, 2H), 1.39 (t, J=8 Hz, 3H), 1.23 (m, 9H). .sup.31P NMR
(400 MHz, DMSO-d.sub.6) .delta. 3.72, 3.65. (M+H.sup.+) 709.
(2S)-isopropyl
2-(((((1R,3R,4R,7S)-3-(2-amino-6-oxo-1H-purin-9(6H)-yl)-7-hydroxy-2,5-dio-
xabicyclo[2.2.1]
heptan-1-yl)methoxy)(phenoxy)phosphoryl)amino)proapnoate (401)
[0400] To a solution of B6 (350 mg, 0.484 mmol) in ethanol (17.5
mL) was added Pd/C (128.7 mg, 0.121 mmol, 0.25 eq.). The mixture
was heated at 50.degree. C. and ammonium formate (152.6 mg, 2.42
mmol, Seq.) was added. The reaction mixture was kept stirring at
50.degree. C. for 30 min. After cooling down to room temperature,
the mixture was filtered through a celite pad and solid rinsed with
3.times.10 mL of methanol. The filtrate was concentrated and
dissolved in 10 mL DCM, washed with 10 mL 1/4 saturated.
NaHCO.sub.3 solution, 10 mL water. The second organic solution was
extracted with 10 mL DCM. The combined organic phases were dried
over Na.sub.2SO.sub.4 and concentrated to give 311 mg of a white
solid. This crude product was purified using Combiflash (4 g gold
column DCM 100%.fwdarw.DCM/MeOH 90/10). Compound 7 was isolated as
a white solid (237 mg, 79%). .sup.1H NMR (400 MHz, DMSO-d.sub.6)
.delta. 7.95 (s, 1H), 7.37-7.35 (m, 2H), 7.22-7.18 (m, 3H), 6.54
(br s, 2H), 6.04 (m, 1H), 5.90-5.76 (m, 2H), 4.87 (sept, J=4 Hz,
1H), 4.49-4.4 (m, 5H), 4.30 (t, J=4 Hz, 1H), 4.01 (dd, J=8 Hz, 24
Hz, 1H), 3.85 (m, 2H), 1.38 (t, J=8 Hz, 3H), 1.25 (m, 3H), 1.16 (m,
6H). .sup.31P NMR (400 MHz, DMSO-d.sub.6) .delta. 3.85, 3.73.
(M+H.sup.+).
Example 1C
Compound 502a
##STR00127##
[0401] Intermediate B2
##STR00128##
[0403] To a stirred solution of 4-nitrophenyl dichlorophosphate
(Aldrich) (35.97 mmol) in DCM (2 mL/mmol) was added a solution of
phenol (Aldrich) (35.97 mmol) and TEA (39.57 mmol) in DCM (2
mL/mmol) at -78.degree. C. over a period of 20 minutes. The
reaction mixture was stirred at -78.degree. C. during 30 minutes
and then, transferred into another round-bottom flask containing
D-alanine isopropyl ester hydrochloride (35.97 mmol) in DCM (2
mL/mmol) at 0.degree. C. To the mixture was added TEA (31.31 mmol)
over a period of 15 minutes. The reaction mixture was stirred at
0.degree. C. during 1 hour and then, the solvent was evaporated.
The residue was triturated with ethyl acetate (45 mL) and the white
solid was filtered-off. The filtrate was concentrated under reduced
pressure and the residue was purified by chromatography on silica
gel (eluent: petroleum ether-petroleum ether/ethyl acetate 20%) to
give the expected compound in 80% yield; .sup.1H NMR (CDCl3, 400
MHz) .delta. (ppm) 1.22 (d, J=6.28 Hz, 3H), 1.23 (d, J=6.28 Hz,
3H), 1.40 (m, 3H), 3.91-3.96 (m, 1H), 4.05-4.13 (m, 1H), 5.01
(heptuplet, J=6.30 Hz, 1H), 7.19-7.25 (m, 3H), 7.33-7.41 (m, 4H),
8.22 (dd, J=1.74 and 8.95 Hz, 2H); .sup.31P NMR (CDCl3, 161.98
MHz): .delta. (ppm)-3.21 (s, 0.45P), -3.18 (s, 0.55P); MS (ESI)
m/z=409.14 (MH.sup.+).
Compound 502a
##STR00129##
[0405] To a solution of 3'-deoxy nucleoside (0.803 mmol) in
anhydrous THF (4 mL) at room temperature under nitrogen was added
dropwise tert-butylmagnesium chloride (1M in THF) (1.69 mmol)
followed by DMSO (0.6 mL). The heterogeneous reaction mixture was
stirred during 30 minutes at room temperature. Compound B2 (0.964
mmol) in THF (2.4 mL) was added dropwise and the reaction mixture
was abandoned at room temperature all the weekend. The reaction
mixture was quenched with saturated aqueous solution of NH.sub.4Cl
and diluted with ethyl acetate. The mixture was extracted with
ethyl acetate and the organic layer was washed with H.sub.2O and
NaHCO.sub.3. The organic layer was dried over MgSO.sub.4, filtered
and concentrated under reduced pressure. The residue was purified
by chromatography on silica gel (eluent:
CH.sub.2Cl.sub.2--CH.sub.2Cl.sub.2/CH.sub.3OH) and by preparative
HPLC to give two pure diastereoisomers.
Compound 502a, Diastereoisomer 1
[0406] white solid; 14% yield; .sup.1H NMR (DMSO-d.sub.6, 400 MHz)
.delta. (ppm) 1.09 (d, J=6.24 Hz, 3H), 1.12 (d, J=6.24 Hz, 3H),
1.13 (d, J=21.79 Hz, 3H), 1.19 (d, J=7.11 Hz, 3H), 1.35 (t, J=7.11
Hz, 3H), 2.28-2.37 (m, 1H), 3.70-3.80 (m, 1H), 4.23-4.29 (m, 1H),
4.35-4.40 (m, 1H), 4.45 (q, J=7.11 Hz, 2H), 4.47-4.51 (m, 1H), 4.83
(heptuplet, J=6.24 Hz, 1H), 6.04-6.09 (m, 1H), 6.06 (d, J=18.25 Hz,
1H), 6.56 (s, 2H), 7.14-7.17 (m, 1H), 7.20-7.22 (m, 2H), 7.32-7.36
(m, 2H), 7.94 (s, 1H); .sup.31P NMR (DMSO-d.sub.6, 161.98 MHz)
.delta. (ppm) 3.6 (s, 1P); MS (ESI) m/z=581.12 (MH.sup.+).
Compound 502a, Diastereoisomer 2
[0407] white solid; 6% yield; .sup.1H NMR (DMSO-d.sub.6, 400 MHz)
.delta. (ppm) 1.10 (d, J=6.21 Hz, 3H), 1.11 (d, J=6.21 Hz, 3H),
1.13 (d, J=6.95 Hz, 3H), 1.16 (d, J=21.98 Hz, 3H), 1.35 (t, J=7.10
Hz, 3H), 2.28-2.37 (m, 1H), 3.70-3.80 (m, 1H), 4.26-4.32 (m, 1H),
4.38-4.43 (m, 1H), 4.44 (q, J=7.12 Hz, 2H), 4.47-4.54 (m, 1H), 4.81
(heptuplet, J=6.23 Hz, 1H), 5.98 (dd, J=9.96 Hz and 12.72 Hz, 1H),
6.09 (d, J=18.27 Hz, 1H), 6.55 (s, 2H), 7.14-7.18 (m, 3H),
7.33-7.37 (m, 2H), 7.99 (s, 1H); .sup.31P NMR (DMSO-d.sub.6, 161.98
MHz) .delta. (ppm) 3.97 (s, 1P); MS (ESI) m/z=581.08
(MH.sup.+).
Example 1D
Preparation of 4'-fluoro nucleosides
Compound 602b
Two Diastereomers
##STR00130## ##STR00131## ##STR00132##
[0409] Step 1:
##STR00133##
[0410] Uridine (10 grams, 40.1 mmol) was dissolved in acetone (100
mL) containing sulfuric acid (conc., 1.0 mL). After stirring at
room temperature overnight, the mixture was concentrated under
reduced pressure. The crude product was purified by flash column
chromatography (Silica Gel, 100% DCM to 4% MeOH/DCM) to afford 11.0
grams of the acetonide A2 (98%).
[0411] HPLC (Method A, 254 nm) split peak at 2.3 and 2.49, 99.6A %;
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 1.34 (s, 3H), 1.56 (s,
3H), 2.50-2.60 (br-s, 1H), 3.79-3.90 (m, 2H), 4.27 (m, 1H), 4.95
(m, 1H), 5.02 (m, 1H), 5.53 (d, 1H), 5.71 (dd, 1H), 7.33 (d, 1H),
8.03 (br s, 1H).
[0412] Step 2:
##STR00134##
[0413] The acetonide A2 (11.0 g, 38.7 mmol) was suspended in
dichloromethane (110 mL). Dimethylaminopyridine (DMAP, 11.8 g, 96.8
mmol, 2.5 eq) was added and the mixture stirred at room temperature
until the acetonide had fully dissolved. The mixture was cooled to
ca. 0.degree. C. (ice-bath) and tosyl chloride (8.85 g, 46.4 mmol,
1.2 eq) was added in 5 portions. After the addition was complete,
the ice bath was removed and the mixture stirred for 1 hour. HPLC
analysis showed the reaction to be complete. The mixture was
transferred to a reparatory funnel and was washed with aqueous HCl
(1N, 2.times.100 mL), aqueous sodium bicarbonate (saturated, 100
mL), and brine (100 mL). The organic solution was dried over
magnesium sulfate and was concentrated under reduced pressure
affording the crude tosylate. (15.47 g, 91%). The crude product A3
(purity; ca 86% by NMR) was used without purification for
Step-3.
[0414] HPLC (Method A, 254 nm), 4.86 min; LCMS (m/e 327.05,
M.sup.+-Uracil); .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 1.29 (s,
3H), 1.50 (s, 3H), 2.40 (s, 3H), 4.22 (m, 2H), 4.30 (m, 1H), 4.76
(dd, 1H), 4.90 (dd, 1H), 5.61 (d, 1H), 5.67 (d, 1H), 7.21 (d, 1H),
7.29 (d, 2H), 7.72 (d, 2H), 9.39 (br s, 1H).
[0415] Step 3:
##STR00135##
[0416] The crude tosylate A3 (39.5 g, 78.7 mmol) was dissolved in
THF (100 mL) and was cooled to -10.degree. C. Potassium t-butoxide
(26.5 g, 236 mmol, 3 eq) was added forming a solid mass. An
additional 250 mL of THF was added to ensure adequate stirring. The
mixture was stirred for 30 minutes and HPLC analysis showed that
the reaction was complete. Silica gel (60 g) was added and the
mixture was concentrated under reduced pressure. The crude product
was purified by flash column chromatography (Silica Gel, 100% DCM
to 4% MeOH/DCM) to afford 13.2 g (62%) of the enol ether A4.
[0417] HPLC (Method A, 272 nm), 3.24 min, 98% A; LCMS (M.sup.++1
m/e 267.09); .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 1.41 (s,
3H); 1.53 (s, 3H), 4.42 (m, 1H), 4.60 (m, 1H), 5.05 (m, 1H), 5.33
(m, 1H), 5.67 (s, 1H), 5.75 (dd, 1H), 7.20 (d, 1H), 9.60 (br s,
1H).
[0418] Step 4:
##STR00136##
[0419] The nucleosidic enol-ether A4 (7.34 g, 27.6 mmol, 1 eq) and
finely crushed silver fluoride (17.5 g, 138 mmol, 5 eq) were added
to a flask containing dichloromethane (520 mL, DCM was needed to
ensure adequate stirring of the heterogeneous mixture.) The
suspension was stirred rapidly and cooled to 0.degree. C. In a
separate flask, iodine (14.0 g, 55.2 mmol, 2 eq) was dissolved in
THF (40 mL). (The limited solubility of iodine in DCM resulted in
incomplete reaction when DCM was used for preparing the iodine
solution.) The iodine solution was transferred to a slow-addition
funnel and was added to the reaction mixture over 70 minutes. This
addition rate provided a 7:1 ratio of the desired isomer (R) to
undesired isomer (S). The mixture was stirred for 10 min at which
point HPLC analysis showed the reaction to be complete. The
reaction mixture was quenched by the addition of an aqueous
solution of NaS.sub.2O.sub.3 and NaHCO.sub.3 (5 wt % each, 300 mL
total volume). The mixture was filtered through Celite.TM. and the
filter pad washed with DCM. The biphasic mixture was transferred to
a reparatory funnel and the phases were separated. The organic
phase was dried with magnesium sulfate and the mixture concentrated
under reduced pressure affording ca. 11 g of crude product. The
crude product was purified by flash column chromatography (Silica
Gel, 0 to 60% EtOAc/heptane) to provide AS as a beige colored
solid. The crude solid was dissolved in DCM (20 mL) which was then
added to heptane (200 mL) giving AS as a white-colored solid. (A5,
10.4 g, 82%).
[0420] HPLC (Method A, 254 nm); AS (4.18 and 4.38 min) 97% A, 7:1
R:S; .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 9.16 (br s, 1H),
7.20 (d, 1H), 5.77 (d, 1H), 5.65 (s, 1H), 5.16 (m, 1H), 5.10 (m,
1H), 3.53 (m, 1H), 3.48 (m, 1H), 1.59 (s, 3H), 1.38 (s, 3H);
.sup.19F NMR (376 MHz, CDCl.sub.3) .delta.-101.91 (1F, A5-R,
Major), -94.16 (0.165 F, Minor, A5-S).
[0421] Step 5:
##STR00137##
[0422] The iodofluorinated nucleoside AS (2.4 g, 5.8 mmol, 1 eq)
was dissolved in DMF (24 mL). Sodium azide (1.9 g, 29 mmol, 5 eq)
was added and the mixture stirred and heated at 100.degree. C.
overnight. HPLC analysis indicated that the reaction was
incomplete. Additional sodium azide (378 mg, 5.8 mmol, 1 eq) was
added and the reaction continued for another 105 minutes. HPLC
analysis showed that the reaction was nearly complete. The mixture
was allowed to cool to room temperature and ethyl acetate (75 mL)
and water (50 mL) were added. The mixture was then transferred to a
separatory funnel and the phases were split. The aqueous phase was
extracted with ethyl acetate (25 mL). The combined organic layers
were washed with water (4.times.50 mL), dried over magnesium
sulfate, and concentrated under reduced pressure. The crude product
was purified by flash column chromatography (Silica Gel, 0 to 60%
EtOAc/heptane) to provide 1.63 g of the desired azide A6 (86%).
[0423] HPLC (Method A, 254 nm); A6, 3.96 min, 4.09 min; .sup.1H NMR
(400 MHz, CDCl.sub.3) .delta. 8.90 (br s, 1H), 7.18 (d, 1H), 5.77
(dd, 1H), 5.68 (s, 1H), 5.10 (m, 2H), 3.57 (d, 1H), 3.54 (s, 1H),
1.60 (s, 3H), 1.38 (s, 3H); .sup.19F NMR (376 MHz, CDCl.sub.3)
.delta.-109.70 (1F, A6-R, Major), -102.10 (0.280 F, A6-S,
Minor).
[0424] Step 6:
##STR00138##
[0425] The azido nucleoside A6 (0.988 g, 3.2 mmol, 1 eq) was
dissolved in acetonitrile (10 mL). The mixture was cooled to
0.degree. C. (ice-bath) and nitrosyl tetrafluoroborate (1.06 g,
9.06 mmol, 3 eq) was added in a single portion. The mixture was
stirred for 30 minutes at 0.degree. C. The ice-bath was removed and
the mixture stirred for 1 hour at room temperature. HPLC analysis
showed the reaction to be complete. The reaction was quenched by
the addition of 50% brine/50% Na.sub.2HPO.sub.4 (20 mL). The
mixture was transferred to a separatory funnel and was extracted
with dichloromethane (3.times.20 mL). The combined organic extracts
were dried with magnesium sulfate and concentrated under reduced
pressure affording 0.699 g (81%) of crude A7. The crude material
was used in Step 7 without further purification.
[0426] HPLC (Method A, 254 nm); A7, 2.77 min; LCMS (M.sup.++1,
m/e=285).
[0427] Step 7:
##STR00139##
[0428] The nucleoside A7 (699 mg, 2.5 mmol, 1 eq) was dissolved in
THF (6.3 mL) and water (0.7 mL). TFA (35 .mu.L) was added and the
mixture stirred for 1 hour at room temperature. HPLC analysis
showed that the reaction was complete. The mixture was concentrated
under reduced pressure. The crude product was purified by flash
column chromatography (Silica Gel, 100% DCM to 4% MeOH/DCM) to
provide 308 mg (41%) of the hydroxymethyl nucleoside A8.
[0429] HPLC (Method A, 254 nm); A8, 2.74 min; LCMS (M.sup.--1,
m/e=301); .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 1.38 (s, 3H),
1.59 (s, 3H), 2.41 (br s, 1H), 3.82 (d, 2H), 5.10 (d, 1H), 5.24 (m,
1H), 5.72 (s, 1H), 5.77 (d, 1H), 7.23 (d, 1H), 9.06 (br s, 1H);
.sup.19F NMR (376 MHz, CDCl.sub.3) .delta.-115.65.
[0430] Step 8a:
##STR00140##
[0431] Phenyl dichlorophosphate (495 .mu.L, 3.31 mmol, 1 eq) was
dissolved in THF. The mixture was cooled to -66.degree. C. In a
separate flask, a solution of isopropyl alanine (583 mg, 3.48 mmol,
1.05 eq) in DCM (6 mL) was prepared. This solution was added to the
solution of the dichlorophosphate over 5 minutes. Triethylamine
(966 .mu.L, 6.95 mmol, 2.1 eq) was then added over 3 minutes
maintaining the temperature at -66.degree. C. The mixture was
stirred for 25 minutes and this solution was used for Step 8
without further purification.
[0432] Step 8:
##STR00141##
[0433] The nucleoside A8 (500 mg, 1.65 mmol, 0.5 eq) was dissolved
in THF (5 mL) forming a clean solution. The mixture was stirred and
cooled to -43.degree. C. t-Butyl magnesium chloride (1M in THF,
3.64 mL, 3.64 mmol, 1.1 eq) was added drop-wise over 5 minutes. The
mixture was cooled to 50.degree. C. and the solution of the
chlorophosphamidate A13 (3.31 mmol, 1 eq) was added drop-wise via a
syringe over 7 minutes. (The solution became brown-colored and
cloudy.) The mixture was stirred for 30 minutes and analyzed by
HPLC. The mixture was warmed to 0.degree. C. and stirred for 30
minutes. LCMS analysis indicated the reaction to be complete. Brine
(5%, 10 mL) was added, the mixture was transferred to a reparatory
funnel and was extracted with ethyl acetate (3.times.15 mL). The
organic extracts were dried over magnesium sulfate and were
concentrated under reduced pressure. The crude product was purified
by flash column chromatography (Silica Gel, 100% DCM to 4%
MeOH/DCM) to afford 324 mg (34%) of the mixture of the
phosphoramidate diastereomers A9.
[0434] HPLC (Method A, 254 nm); A9, 4.87 min, 4.95 min 1.8:1 ratio
of diastereomers; LCMS (M.sup.--1, m/e=570); .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta. 1.18 (m, 6H), 1.31 (m, 3H), 1.35 (m, 3H), 1.55
(s, 3H), 3.98 (m, 2H), 4.28 (m, 2H), 4.98 (m, 2H), 5.20 (m, 1H),
5.69 (m, 1H), 5.78 (s, 1H), 7.20, 7.28 (m, 6H), 9.22, 9.41 (2 s,
1H); .sup.19F NMR (376 MHz, CDCl.sub.3) .delta.-113.99 (m, 1F),
-113.53 (m, 0.6F); .sup.31P NMR (162 MHz, CDCl.sub.3), 2.33, 2.32
(2 s, 1P).
[0435] Step 9:
##STR00142##
[0436] The nucleoside A9 (548 mg, 0.959 mmol, 1 eq) was dissolved
in formic acid (80%, 35 mL). The mixture was stirred a room
temperature for 3 hour and 45 minutes. HPLC analysis showed the
reaction to be complete. The reaction mixture was transferred to a
reparatory funnel, was diluted with brine (35 mL) and was extracted
with ethyl acetate (3.times.40 mL). The combined organic extracts
were dried over magnesium sulfate and were concentrated under
reduced pressure. The crude product was purified by flash column
chromatography (Silica Gel, 100% DCM to 10% MeOH/DCM) to afford 296
mg (58%) of the mixture of the phosphoramidate diastereomers
602b.
[0437] HPLC (Method A, 254 nm); 2b, 3.80 min; LCMS (m/e=532
(M.sup.++1), 512(M.sup.+-F); .sup.1H NMR (400 MHz, CD.sub.3OD)
.delta. 1.18 (m, 6H), 1.28 (m, 3H), 3.27 (s, 1H), 3.85 (m, 1H),
4.28 (m, 3H), 4.47 (dd, 1H), 4.93 (m, 1H), 5.60 (d, 0.3H), 5.65 (d,
0.67H), 5.96 (m, 1H), 7.18 (m, 3H), 7.32 (m, 2H), 7.51 (d, 1H);
.sup.19F NMR (376 MHz, CD.sub.3OD) .delta.-123.73 (m, 2.2 F),
-123.96 (m, 1F); .sup.31P NMR (162 MHz, CD.sub.3OD), 3.43 (m,
2.2P), 3.59 (m, 1P).
Step 10: Semi-Preparative HPLC Separation of the Diastereomers of
602b
##STR00143##
[0439] The mixture of diastereomers 602b was separated using a
Phenomenex Luna C18 (2) and PrepMethod A. Approximately 290 mg of
602b was dissolved in 2 mL of methanol/heptanes (80:20) to give a
145 mg/mL solution. Four 500 .mu.L injections were made. The
fractions from the separations were analyzed by analytical HPLC
(Method B). The suitable fractions were combined and concentrated
providing 50 mg (34%) of 602b diastereomer 1 (13.99 min, 97.6 A %,
>99.9% de) and 30 mg (20%) of 602b diastereomer 2 (19.50 min,
96.8 A %, 94.2% de).
[0440] PrepMethod A:
[0441] Gilson prep HPLC system with GX-281 liquid handler and 322
pump. Phenomenex Luna C18(2) column, 150.times.21.20 mm, 5 .mu.m.
Mobile phase 40/60 MeOH/water. Flow=22 ml/min.
[0442] HPLC Method B:
[0443] Luna C18 (2), 5 .mu.m, 3.0.times.150 mm. Mobile Phase: 45%
Methanol:Water (Isocratic). Flow=0.6 mL min.sup.-1, 25 min runtime.
DAD detector monitored at 214 and 260 nm.
[0444] HPLC Method A:
[0445] Agilent Technologies 1100 Series HPLC with diode array
detector. Mobile Phase: ACN/NH.sub.4OAc pH 4.4 buffer (5% to 80%
over 10 min); Flow=1.4 ml min.sup.-1. DAD detector monitored at 254
and 272 nm.
Compound 603a
Single Diastereomer
##STR00144## ##STR00145## ##STR00146##
[0447] Step 1:
##STR00147##
[0448] The nucleoside C1 (10 g, 28.3 mmol) was dissolved in a 1:1
mixture of dimethoxypropane (50 mL, 408 mmol, 14.4 eq) and
dimethylformamide (DMF, 50 mL). p-Toluenesulfonic acid monohydrate
(p-TSA, 2.05 g, 10.77 mmol, 0.380 eq) was added and the mixture was
stirred at room temperature for 48 hours. Initially, 0.1 eq of
p-TSA was added; after 24 hours, the reaction was only 50%
complete. Additional aliquots of p-TSA (0.28 eq total) were needed
to drive the reaction to completion. The reaction mixture was
concentrated on a rotary evaporator and the residue was dissolved
in dichloromethane (DCM, 300 mL). The mixture was transferred to a
separatory funnel and was washed with saturated sodium bicarbonate
solution (300 mL). The aqueous phase was back-extracted with
2.times.100 mL of DCM and the combined organic phases were dried
over magnesium sulfate and were concentrated under reduced pressure
affording the crude product C2 (1.2 g, 108%). (.sup.1H NMR analysis
showed that the crude product contained DMF).
[0449] HPLC (Method A, 254 nm), RT 3.4 min; LCMS (M.sup.--1
m/e=392) .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 12.11 (br s,
1H), 7.95 (s, 1H), 7.82 (s, 1H), 5.80 (d, 1H), 5.08 (dd, 1H), 4.94
(dd, 1H), 4.31 (m, 1H), 3.84 (m, 1H), 3.70 (m, 1H), 2.65 (sept,
1H), 2.37 (br s, 1H), 1.51 (s, 3H), 1.28 (s, 3H), 1.18 (a-t,
6H).
[0450] Step 2:
##STR00148##
[0451] The crude nucleoside C2 (12.1 g, 28.3 mmol) was dissolved in
dichloromethane (DCM, 125 mL) under argon. Dimethylaminopyridine
(DMAP, 8.6 g, 70.8 mmol, 2.5 eq) was added and the mixture was
cooled in an ice-bath. Tosyl chloride (TsC1, 7.0 g, 36.8 mmol, 1.3
eq) was added and the mixture was stirred at 0.degree. C. for 30
minutes. The ice-bath was removed and the mixture was allowed to
stir at room temperature for an additional 30 minutes. HPLC
analysis showed that the reaction was complete. The mixture was
transferred to a separatory funnel and was diluted with DCM (125
mL). The DCM solution was washed with 1M HCl (2.times.100 mL),
saturated bicarbonate solution (100 mL), and brine (100 mL). The
mixture was dried over magnesium sulfate and was concentrated under
reduced pressure affording 15.73 g of the desired product C3 (101%,
contains DMF).
[0452] HPLC (Method A, 254 nm), RT 4.78 min; LCMS (M.sup.++1,
m/e=548); .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 12.11 (br s,
1H) 9.20 (br s, 1H), 7.66 (d, 2H), 7.58 (s, 1H), 7.27 (d, 2H), 5.79
(d, 1H), 5.22 (dd, 1H), 5.12 (dd, 1H), 4.49 (dd, 1H), 4.33 (m, 1H),
4.05 (dd, 1H), 2.61 (sept, 1H), 2.38 (s, 3H), 1.52 (s, 3H), 1.31
(s, 3H), 1.18 (d, 3H), 1.14 (d, 3H).
[0453] Step 3:
##STR00149##
[0454] The nucleoside C3 (8.0 g, 14.6 mmol) was dissolved in
pyridine (80 mL) under an argon atmosphere. Diisopropylethylamine
(DIPEA, 5.08 mL, 29.2 mmol, 2 eq)) was added followed by
diphenylcarbamoyl chloride (DPC-Cl, 3.72 g, 1.1 eq). The mixture
was stirred at room temperature under an argon atmosphere for 1
hour. HPLC analysis indicated the reaction to be complete. The
mixture was quenched by the addition of water (15 mL) and was
concentrated under reduced pressure. The residue was transferred to
a separatory funnel with DCM (150 mL). The DCM solution was washed
with aqueous HCl (1M, 100 mL), dried over magnesium sulfate, and
was concentrated under reduced pressure. The crude product was
purified by flash column chromatography (silica gel, 0.fwdarw.50%
EtOAc/heptanes) to provide 9.5 g of C4 (87%).
[0455] HPLC (Method A, 254 nm), RT 6.53 min; LCMS (M.sup.++1,
m/e=743); .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.99 (br s,
1H), 7.81 (s, 1H), 7.35 (m, 12H), 6.92 (d, 2H), 5.91 (d, 1H), 5.42
(dd, 1H), 5.14 (dd, 1H), 4.40 (m, 1H), 4.29 (m, 2H), 2.61 (sept,
1H), 2.10 (s, 3H), 1.50 (s, 3H), 1.29 (s, 3H), 1.18 (2d, 6H).
[0456] Step 4:
##STR00150##
[0457] The nucleoside C4 (9.5 g, 12.8 mmol) was dissolved in
acetone (100 mL) under an argon atmosphere. Sodium iodide (13.4 g,
89.6 mmol, 7 eq) was added and the mixture was refluxed overnight.
LCMS analysis indicated that the reaction was complete. The mixture
was allowed to cool and was concentrated under reduced pressure.
The mixture was transferred to a reparatory funnel with DCM (100
mL) and was washed with a mixture of 5% sodium bicarbonate and 5%
sodium thiosulfate (75 mL total). The organic phase was dried over
magnesium sulfate and concentrated under reduced pressure affording
9 grams of a dark-colored foam. The crude material was purified by
flash column chromatography (silica gel, 0.fwdarw.50%
EtOAc/heptanes) to provide 7.82 g of C5 (88%).
[0458] HPLC (Method A, 254 nm), RT 6.39 min; LCMS (M.sup.++1,
m/e=699); .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.96 (s, 1H),
7.95 (br s, 1H), 7.30 (m, 10H), 6.00 (d, 1H), 5.40 (m, 2H), 4.40
(m, 1H), 3.45 (m, 1H), 3.20 (dd, 1H), 2.67 (m, 1H), 1.54 (s, 3H),
1.34 (s, 3H), 1.19 (m, 6H).
[0459] Step 5:
##STR00151##
[0460] The nucleoside C5 (7.82 g, 11.2 mmol) was dissolved in
toluene. 1,8-Diazabicyclo[5.4.0] undec-7-ene (DBU, 5.0 mL, 33.6
mmol, 3 eq) was added dropwise over 3 minutes. The mixture was
stirred at room temperature for ca 64 hours. HPLC analysis
indicated the reaction to be complete. The reaction mixture was
diluted with DCM (50 mL) and saturated sodium bicarbonate solution
(50 mL). This mixture was transferred to a reparatory funnel along
with additional portions of DCM (100 mL) and saturated sodium
bicarbonate solution (50 mL). The layers were separated and the
organic phase dried over magnesium sulfate and was concentrated
under reduced pressure. The crude product was purified by flash
column chromatography (silica gel, 0.fwdarw.4% MeOH/DCM) affording
3.83 g of the desired product C6 (60%).
[0461] HPLC (Method A, 254 nm), RT 6.04 min; LCMS (M.sup.++1,
m/e=571); .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.88 (s, 1H),
7.87 (br s, 1H), 7.27 (m, 10H), 6.11 (s, 1H), 5.88 (d, 1H), 5.27
(d, 1H), 4.48 (m, 1H), 4.39 (m, 1H), 2.75 (m, 1H), 1.50 (s, 3H),
1.38 (s, 3H), 1.19 (m, 6H).
[0462] Step 6:
##STR00152##
[0463] The nucleoside C6 (1.1 g, 1.9 mmol) was dissolved in DCM (10
mL). Freshly crushed silver fluoride (1.22 g, 9.6 mmol, 5 eq) was
added. In a separate flask, iodine (627 mg, 2.5 mmol, 1.3 eq) was
dissolved in DCM (10 mL). The iodine solution was added drop-wise
to the solution of the nucleoside over 30 minutes. After stirring
for 5 minutes, HPLC analysis indicated that the reaction was
incomplete. An additional 5 eq of crushed silver fluoride (1.22 g,
9.6 mmol) was added followed by the portion-wise addition of solid
iodide (0.5 eq, 125 mg) over 5 minutes. After stirring at room
temperature for 5 minutes, HPLC analysis showed that the reaction
was complete. The mixture was quenched by the addition of 20 mL of
a mixture of 5% sodium bicarbonate and 5% sodium thiosulfate. The
mixture was filtered through Celite.TM. and was transferred to a
reparatory funnel. (Some finely divided solids were not removed by
the Celite.TM. filtration and were present in the organic phase.)
The organic solution was dried over magnesium sulfate and was
concentrated under reduced pressure providing the crude product
(1.45 g). The crude product was a 2:1 mixture of C7a and C7b. The
crude product was purified by flash column chromatography (silica
gel, 0->50% EtOAc) affording 396 mg of the desired diastereomer
C7a (29%). In another reaction, the desired diastereomer, C7a, was
obtained in 57% yield after chromatography accompanied by a 21%
isolated yield of the "S diastereomer".
[0464] HPLC (Method A, 254 nm), RT 6.47 min; LCMS (M.sup.++1,
m/e=717); .sup.1H NMR (400 MHz, CDCl.sub.3) 8.01 (br s, 1H), 7.89
(s, 1H), 7.34 (m, 10H), 6.27 (s, 1H), 6.10 (dd, 1H), 5.10 (d, 1H),
3.70 (m, 1H), 3.66 (s, 1H), 2.61 (sept, 1H), 1.58 (s, 3H), 1.32 (s,
3H), 1.20 (m, 6H); .sup.19F NMR (376 MHz, CDCl.sub.3);
.delta.-101.14 (m, 1F).
[0465] Step 7:
##STR00153##
[0466] The nucleoside C5 (837 mg, 1.17 mmol) was dissolved in DCM
(17 mL). In a separate flask, a solution of potassium hydrogen
phosphate (306 mg, 1.76 mmol, 1.5 eq) in water (1 mL) was prepared.
This solution along with bis(tetrabutylammonium)sulfate (50% in
water, 2.34 mL, 1.17 mmol, 131) were added to the solution of the
nucleoside. m-Chloroperbenzoic acid (mCPBA, 1.21 g, 7.02 mmol, 6
eq) was added and the mixture was stirred rapidly at room
temperature overnight. HPLC analysis indicated the reaction to be
complete. The mixture was transferred to a reparatory funnel and
was washed with a mixture of 5% sodium bicarbonate and 5% sodium
thiosulfate (20 mL total volume). The layers were separated and the
organic phase was dried over magnesium sulfate and was concentrated
under reduced pressure. The crude product was purified by flash
column chromatography (silica gel, 0.fwdarw.50% EtOAc/heptane)
providing the desired product C8 (525 mg, 60%).
[0467] HPLC (Method A, 254 nm), RT 6.93 min; LCMS (M.sup.++1,
m/e=745); .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 8.15 (br s,
1H), 7.93 (m, 1H), 7.91 (s, 1H), 7.82 (m, 1H), 7.20-7.55 (m, 12H),
6.25 (s, 1H), 6.11 (dd, 1H), 5.11 (d, 1H), 4.66 (dd, 1H), 4.49
(a-t, 1H), 2.49 (m, 1H), 1.59 (s, 3H), 1.35 (s, 3H), 1.07 (m, 6H);
.sup.19F NMR (376 MHz, CDCl.sub.3); .delta.-110.89 (m, 1F).
[0468] Step 8:
##STR00154##
[0469] The nucleoside C8 (1.92 g, 2.58 mmol) was dissolved in
n-butyl amine (19 mL) forming a green-colored solution. The mixture
was stirred and heated to 80.degree. C. for 30 minutes. (The color
of the solution had turned red). HPLC analysis indicated the
reaction to be complete. The mixture was concentrated under reduced
pressure. DCM (20 mL) was added to the red oil forming a thick
precipitate. The precipitate was removed by filtration and was
washed with copious amounts of cold DCM providing a white-colored
solid. This solid was dried in a vacuum oven overnight affording
538 mg of the desired product C9 (61%)
[0470] HPLC (Method A, 254 nm), RT 2.66 min; LCMS (M.sup.--1,
m/e=340); .sup.1H NMR (400 MHz, MeOD) .delta. 7.87 (s, 1H), 6.32
(s, 1H), 5.44 (dd, 1H), 5.17 (dd, 1H), 3.75 (s, 1H), 3.73 (s, 1H),
1.58 (s, 3H), 1.38 (s, 3H); .sup.19F NMR (376 MHz, CDCl.sub.3);
.delta.-116.90 (m, 1F).
[0471] Step 9:
##STR00155##
[0472] Preparation of (2R)-isopropyl
2-((chloro(phenoxy)phosphoryl)amino)propanoate, C12. Phenyl
dichlorophosphate (437 .mu.L, 2.93 mmol) was dissolved in THF (4
mL) and was cooled to -66.degree. C. with dry-ice/acetone. In a
separate flask, D-Ala isopropyl ester (516 mg, 3.08 mmol, 1.05 eq)
was dissolved in DCM (5 mL). This solution was added to the
solution of the dichlorophosphate drop wise over 5 minutes.
Triethylamine (855 .mu.L, 6.15 mmol, 2.1 eq) was added drop wise
over 5 minutes and the mixture was stirred for 30 minutes at
-66.degree. C. The formation of the chlorophosphoramidate reagent
C12 was shown to be complete by .sup.1H NMR, .sup.31P NMR and
LCMS.
[0473] LCMS (M.sup.--Cl+OH-1, m/e=286); .sup.31P NMR (162 MHz,
CDCl.sub.3), .delta. 8.08 (1P), 7.72 (1P).
[0474] The nucleoside C9 (500 mg, 1.46 mmol, 0.5 eq) was suspended
in THF (5 mL) and was cooled to -66.degree. C. t-Butyl magnesium
chloride (1 M in THF, 3.22 mL, 3.22 mmol, 1.1 eq) was slowly added
over 5 minutes. The mixture was stirred for 5 minutes followed by
the addition of the chlorophosphate C12 (prepared above) over 8
minutes. The dry-ice bath was replaced with an ice-bath and the
reaction mixture was stirred at 0.degree. C. for 30 minutes. HPLC
analysis indicated the reaction to be complete. The mixture was
quenched by the addition of 20% sodium chloride (NaCl, 25 mL) and
was extracted with DCM (2.times.10 mL). The organic solution was
washed with brine (25 mL), dried over MgSO4 and was concentrated.
The crude product was purified by flash column chromatography
(0.fwdarw.10% MeOH/DCM) to provide 317 mg of C10 (36%) as a single
diastereomer. Later column cuts provided an additional 365 mg (41%)
of product which was 85% pure.
[0475] HPLC (Method A, 254 nm), RT 6.26 min; LCMS (M.sup.++1,
m/e=611).
[0476] Step 10:
##STR00156##
[0477] The nucleoside C10 (315 mg, 0.52 mmol) was dissolved in 80%
formic acid (15 mL) and was allowed to stir at room temperature for
15 hours. HPLC analysis showed the reaction to be complete. The
mixture was concentrated under reduced pressure and the crude
material was purified by flash column-chromatography (silica gel,
0.fwdarw.10% MeOH/DCM) to afford 168 mg of 3a (57%) as a single
diastereomer.
[0478] HPLC (Method A, 254 nm), RT 3.52 min; LCMS (M.sup.++1,
m/e=571); .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 10.70 (br s,
1H), 7.84 (s, 1H), 7.33 (m, 2H), 7.16 (m, 3H), 6.56 (br s, 2H),
6.03 (m, 2H), 5.92 (br s, 1H), 5.35 (br s, 1H), 4.83 (m, 1H), 4.65
(dd, 1H), 4.44 (m, 1H), 4.19 (m, 2H), 3.71 (m, 1H), 1.21 (m, 3H),
1.14 (m, 6H); .sup.19F NMR (DMSO-d.sub.6,376 MHz,); 6-120.7 (m,
1F); .sup.31P NMR (162 MHz, DMSO-d.sub.6), .delta. 3.53 (1P).
[0479] HPLC Method A:
[0480] Agilent Technologies 1100 Series HPLC with diode array
detector. Mobile Phase: ACN/NH.sub.4OAc pH 4.4 buffer (5% to 80%
over 10 min). Flow=1.4 ml min.sup.-1. DAD detector monitored at 254
and 272 nm.
Example 1E
Preparation of Diastereomerically Pure D-Alanine,
N-((R.sub.P,2'R)-2'-deoxy-2'-fluoro-2'-methyl-P-phenyl-5'-uridylyl)-,
1-methylethyl ester Compound (804ai)
##STR00157##
##STR00158##
[0482] To a stirred solution of D-Alanine isopropyl ester
hydrochloride (47.7 mmol) in anhydrous CH.sub.2Cl.sub.2 (1.05
mL/mmol) was added TEA (98.30 mmol) at -70.degree. C. over 15
minutes dropwise. To this mixture was added a solution of phenyl
dichlorophosphate (47.7 mmol) in anhydrous CH.sub.2Cl.sub.2 (1.05
mL/mmol) over 1 hour. The reaction mixture was stirred at this
temperature for additional 30 minutes and then allowed to warm to
0.degree. C. over 2 hours. To this mixture was added a solution of
pentafluorophenol (47.7 mmol) and TEA (52 mmol) in CH.sub.2Cl.sub.2
(50 mL). The reaction mixture was stirred at 0.degree. C. during 1
hour. The triethylamine salt was filtered washed with
CH.sub.2Cl.sub.2. The filtrate was concentrated under reduced
pressure, the residue was triturated with TBME (150 mL). The
heterogeneous mixture was filtered and the solid was rinsed with
TBME. The filtrate was concentrated and the residue was triturated
with a mixture of hexane/ethyl acetate 20% (100 mL). The suspension
was filtered and the solid was rinsed with a mixture of
hexane/ethyl acetate 20% and dried to give the expected compound 1
in 11% yield as a single isomer.
[0483] .sup.1H NMR (400 MHz, CDCl.sub.3): .delta. (ppm) 1.23-1.26
(m, 6H), 1.46 (d, J=7.02 Hz, 3H), 3.94 (dd, J=9.47 Hz and 12. Hz,
1H), 4.09-4.19 (m, 1H), 4.99-5.09 (m, 1H), 7.19-7.27 (m, 3H),
7.34-7.38 (m, 2H).
D-Alanine,
N-((R.sub.P,2'R)-2'-deoxy-2'-fluoro-2'-methyl-P-phenyl-5'-uridy-
lyl)-, 1-methylethyl ester (804ai)
##STR00159##
[0485] Compound 2 was prepared according to published procedures.
To a solution of compound 2 (4.23 mmol) in THF (3.92 mL/mmol) at
-5.degree. C. under nitrogen was added dropwise tert-butylmagnesium
chloride (1M in THF) (8.92 mmol). The heterogeneous reaction
mixture was stirred during 30 minutes at -5.degree. C. and 30
minutes at room temperature. The reaction mixture was cooled down
to -5.degree. C. under nitrogen and compound 1 (5.07 mmol) in THF
(18 mL) was added dropwise. The reaction mixture was stirred at
-5.degree. C. to 0.degree. C. overnight. The reaction mixture was
quenched with aqueous solution of HCl 1N (20 mL) at -5.degree. C.
and extracted with CH.sub.2Cl.sub.2. The organic layer was washed
with H.sub.2O, Na.sub.2CO.sub.3 aq 5%, H.sub.2O and brine. The
organic layer was dried over Na.sub.2SO.sub.4, filtered and
concentrated under reduced pressure. The residue was purified by
chromatography on silica gel (eluent: 100% CH.sub.2Cl.sub.2 to
CH.sub.2Cl.sub.2:CH.sub.3OH 95:5) to give the desired pure isomer
as a white powder in 77% yield.
[0486] The crystal structure of pure isomer was obtained. The
crystal structure showed the pure isomer corresponds to the R.sub.P
isomer of Formula 804ai.
[0487] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. (ppm) 1.25 (d,
J=6.26 Hz, 6H), 1.33 (d, J=22.32 Hz, 3H), 1.38 (d, J=6.97 Hz, 3H),
3.61-3.63 (m, 1H), 3.72-3.98 (m, 3H), 4.06-4.10 (m, 1H), 4.39-4.51
(m, 2H), 5.03 (sept, J=6.22 Hz, 1H), 5.58 (dd, J=2.29 Hz and 8.19
Hz, 1H), 6.16 (d, J=19.05 Hz, 1H), 7.19-7.26 (m, 4H), 7.34-7.38 (m,
2H), 8.43 (brs, 1H); .sup.31P NMR (161.98 MHz, CDCl.sub.3) .delta.
(ppm) 4.29 (s, 1P). LCMS (ESI+) m/z 530.2 [M+H].sup.+ 100%. LCMS
(ESI-) m/z 528.2 [M-H].sup.- 100%.
Example 1F
Preparation of 2'-cyano, azido and amino nucleosides
##STR00160## ##STR00161##
##STR00162## ##STR00163##
[0488] Ethyl
2-cyano-3-(2,2-dimethyl-1,3-dioxolan-4-yl)-2-methylbutanoate
(A2)
##STR00164##
[0490] A 5 L flange flask was fitted with a thermometer, nitrogen
inlet, pressure equalizing dropping funnel, bubbler, and a
subaseal. Methyl lithium solution (956 mL, 1.6M in diethylether,
1.7 equiv.) was added, and the solution was cooled to about
-25.degree. C. Diisopropyl amine (214 mL, 1.7 equiv.) was added
using the dropping funnel over about 40 minutes. The reaction was
left stirring, allowing to warm to ambient temperature overnight.
CO.sub.2(s)/acetone cooling was applied to the LDA solution,
cooling to about -70.degree. C. R-Glyceraldehyde dimethylacetal
solution (50% in DCM) was evaporated down to .about.100 mbar at a
bath temp of 35.degree. C., to remove the DCM, then azeotroped with
anhydrous hexane (2.times.100 mL), under vacuum. The fresh aldehyde
(120 g, 0.9 mol) and ethyl 2-cyanopropionionate (170 mL, 1.5
equiv.) were placed in a 1 L round bottom flask, which was filled
with toluene (800 mL). This solution was cooled in a
CO.sub.2(s)/acetone bath, and added via cannula to the LDA solution
over about 50 minutes, keeping the internal temperature of the
reaction mixture cooler than -55.degree. C. The mixture was stirred
with cooling (internal temp. slowly fell to .about.-72.degree. C.)
for 90 min, then warmed to room temperature over 30 minutes using a
water bath. This solution was added to a sodium dihydrogen
phosphate solution 300 g of NaH.sub.2PO.sub.4 in 1.5 L of
ice/water, over about 10 minutes, with ice-bath cooling. The
mixture was stirred for 20 minutes, then filtered and transferred
to a reparatory funnel, and partitioned. The solid was further
washed with EtOAc (2.times.1 L) and the washings were used to
extract the aqueous. The combined organic extracts were dried over
sodium sulfate. The volatiles were removed in vacuo. The resultant
oil was hydrolyzed crude.
3-Cyano-4-hydroxy-5-(hydroxymethyl)-3-methyloxolan-2-one
##STR00165##
[0492] The crude oil was taken up in acetic acid (1.5 L, 66% in
water) and heated to 90.degree. C. over one hour, then at held at
that temperature for one hour. Once the mixture had cooled to room
temperature, the volatiles were removed in vacuo, and azeotroped
with toluene (2.times.500 mL). The resultant oil was combined with
some mixed material from an earlier synthesis and columned in two
portions (each .about.1.25 L of silica,
0.fwdarw.12.5%.fwdarw.25.fwdarw.50% EtOAc in DCM). The lower of the
two main spots is the desired material; fractions containing this
material as the major component were combined and the solvent
removed in vacuo to give 85.4 g of a brown oil as a mixture of 3
diastereomers (15:8:2).
((2R,3
S,4R)-3-(benzoyloxy)-4-cyano-4-methyl-5-oxotetrahydrofuran-2-yl)met-
hyl benzoate (A5)
##STR00166##
[0494] A 2 L 3-neck round bottom flask was fitted with an overhead
stirrer, thermometer and pressure equalizing dropping funnel under
nitrogen. 3-Cyano-4-hydroxy-5-(hydroxymethyl)-3-methyloxolan-2-one
(85.4 g, 0.50 mol) in acetonitrile (1.5 L) was added, followed by
4-dimethylaminopyridine (700 mg) and benzoyl chloride (128 mL, 2.2
equiv.). Finally triethylamine (167 mL, 2.4 equiv.) was added over
10 minutes using the dropping funnel. The addition of the
triethylamine is accompanied by a mild exotherm, which obviated the
addition of a cold water bath to keep the internal temperature
below 25.degree. C. The reaction was stirred at ambient temperature
for 2.5 hours. The reaction mixture was transferred to a separating
funnel with EtOAc (2.5 L) and half saturated brine (2.5 L), and
partitioned. The aqueous layer was re-extracted with EtOAc (1.5 L).
The combined organic layers were washed with 50% Sodium
bicarbonate/25% Brine (1.5 L) and dried over sodium sulphate. The
resultant brown solid was twice recrystallized from
hexane/chloroform, to give .about.15 g of product of the desired
purity. The mother liquors from the recrystallizations were further
recrystallized from chloroform/hexanes several times to give a
further 15 g of product.
[0495] .sup.1H NMR (CDCl.sub.3, 400 MHz) .delta. (ppm) 8.11 (dm,
J=8.3 Hz, 2H), 7.98 (dm, J=8.4 Hz, 2H), 7.66 (tm, J=7.5 Hz, 1H),
7.59 (tm, J=7.5 Hz, 1H), 7.49 (tm, J=7.6 Hz, 2H), 7.43 (tm, J=7.6
Hz, 2H), 5.54 (d, J=6.5 Hz, 1H), 4.97-5.02 (m, 1H), 4.77 (dd,
J=12.7, 3.5 Hz, 1H), 4.66 (dd, J=12.7, 4.7 Hz, 1H), 1.88 (s,
3H).
3,5-Di-O-benzoyl-2-C-cyano-2-C-methyl-D-ribofuranose (A6)
##STR00167##
[0497] To a solution of A5 (81.08 mmol) in anhydrous
tetrahydrofuran (650 mL) was added under inert atmosphere at
-35.degree. C., LiAlH(OtBu).sub.3 (1.0 M in tetrahydrofuran, 21.7
mmol) over a 20 minutes period. The reaction mixture was stirred
for 1 hour at -20.degree. C. and quenched by addition of a
saturated NH.sub.4Cl solution, keeping the temperature bellow
0.degree. C. Ethyl acetate was added and the white suspension was
filtered through a pad of celite and washed with ethyl acetate. The
filtrate was extracted with ethyl acetate twice. The combined
organic layers were dried over anhydrous sodium sulfate, filtered
and evaporated under reduced pressure. The expected intermediate
was used without further purification for the next step. MS (ESI)
m/z=404 (MNa.sup.+).
1-O-Acetyl-3,5-di-O-benzoyl-2-C-cyano-2-C-methyl-D-arabinofuranose
(A7)
##STR00168##
[0499] To a solution of A6 (81.0 mmol) in anhydrous tetrahydrofuran
(420 mL) was added dropwise under inert atmosphere (nitrogen) at
0.degree. C., acetic anhydride (405.0 mmol) followed by
4-dimethylaminopyridine (8.1 mmol). The reaction mixture was
allowed to warm-up to room temperature and was stirred for 1 hour.
The crude was partially concentrated under reduced pressure,
partitioned with dichloromethane and a saturated NaHCO.sub.3
solution, then transferred into a separatory funnel. The organic
layer was extracted, dried, filtered and evaporated under reduced
pressure. The residue was purified by flash chromatography on
silica gel [eluent: petroleum ether/ethyl acetate: 0 to 100%] to
afford a .alpha.,.beta. sugar mixture A7 in 96% overall yield (2
steps). MS (ESI) m/z=869.2 (2MNa.sup.+).
3',5'-Di-O-benzoyl-2'-C-cyano-2'-C-methyl-4-benzoyl-.alpha.,.beta.-cytidin-
e (A8)
##STR00169##
[0501] To a suspension of N-benzoyl cytosine (23.62 mmol), and a
catalytic amount of ammonium sulfate in 4-chlorobenzene (60 mL) was
added HMDS (70.85 mmol). The reaction mixture was heated at
140.degree. C. overnight. The solvent was removed under inert
atmosphere and the residue was taken in 4-chlorobenzene (20 ml).
Then, 7 (11.81 mmol) in chlorobenzene (40 mL) was added dropwise to
the rectional mixture followed by SnCl.sub.4 (23.62 mmol) dropwise.
The reaction mixture was stirred at 70.degree. C. overnight, cooled
to room temperature and diluted with dichloromethane and a
saturated NaHCO.sub.3 solution. The white suspension was filtered
through a pad of celite and washed with dichloromethane. The
filtrate was extracted with dichloromethane twice. The combined
organic layers were dried over anhydrous Na.sub.2SO.sub.4, filtered
and evaporated under reduced pressure to afford expected nucleoside
as an .alpha.,.beta. mixture. Crude material was used without
further purification for the next step. MS (ESI) m/z=598.2
(MH.sup.+).
2'-C-Cyano-2'-C-methyl-.alpha.,.beta.-cytidine, hydrochloride form
(A9)
##STR00170##
[0503] A suspension of A8 (11.8 mmol) in 7N methanolic ammonia (150
mL) was stirred at room temperature for 3 days in a stainless steel
pressure reactor. The mixture was evaporated to dryness, diluted
with water and transferred into a reparatory funnel. The aqueous
layer was extracted with dichloromethane and water was removed
under reduced pressure. Crude residue was diluted with ethanol (50
mL) and 10 mL of 1.25 N HCl in dioxan were added. Concentration of
the reaction mixture under reduced pressure followed by 3
co-evaporations with absolute ethanol afforded a precipitate which
was filtrated-off and washed with absolute ethanol to give pure
expected compound as a white solid in 41% overall yield (2 steps)
(57/43 .alpha.,.beta. mixture).
[0504] .sup.1H NMR (DMSO, 400 MHz) .delta. (ppm) 1.15 (s,
3H.beta.), 1.51 (s, 3H.alpha.), 3.45-3.95 (m, 3H.alpha.,.beta.),
4.00-4.10 (m, 1H.alpha.,.beta.), 4.98 (brs, 1H.alpha.), 5.29 (brs,
1H.beta.), 5.80 (d, J=7.40 Hz, 1H.beta.), 5.89 (d, J=7.40 Hz,
1H.alpha.), 5.95 (s, 1H.alpha.), 6.22 (s, 1H.beta.), 6.42 (brd,
1H.alpha.,.beta.), 7.53 (brs, 1H.alpha.,.beta.), 7.76 (d, J=7.40
Hz, 1H.alpha.), 7.89 (brs, 1H.alpha.,.beta.), 7.96 (d, J=7.40 Hz,
1H.beta.); MS (ESI) m/z=267 (MH.sup.+)
[0505] Compound A9b: The white solid A9 was triturated with a
mixture of methanol/triethylamine/water; and filtered to afford an
off-white solid A9a as .alpha.-anomer, and a filtrate. The filtrate
was concentrated under reduced pressure and purified by flash
chromatography on silica gel [eluent: DCM/methanol:80/20, with 1%
of Et3N] to afford the expected .beta.-anomer A9b. Off-white solid,
.sup.1H NMR (DMSO, 400 MHz) .delta. (ppm) 1.13 (s, 3H), 3.60-3.65
(m, 1H), 3.77-3.90 (m, 3H), 5.26 (brt, 1H), 5.73 (d, J=7.42 Hz,
1H), 6.24 (s, 1H), 6.38 (brd, 1H), 7.29 (brd, 2H), 7.88 (d, J=7.42
Hz, 1H); MS (ESI) m/z=267 (MH.sup.+).
Compound A10
##STR00171##
[0507] To a solution of compound A9 (2.31 mmol) in dry pyridine (16
mL) and DMF (was added dropwise TIPSCl.sub.2 (2.54 mmol) under
nitrogen atmosphere. The reaction was stirred for 5 hours at room
temperature. Then, DMAP (2.31 mmol) and mMTrCl (2.77 mmol) were
added at room temperature and the reaction mixture was stirred at
55.degree. C. overnight. The reaction mixture was slowly added to a
saturated solution of NaHCO.sub.3. The aqueous layer was extracted
with DCM and the combined organic layers were dried over
Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure.
The crude was diluted in MeOH (16 mL) and NH.sub.4F (11.55 mmol)
was added. The reaction mixture was stirred at 60.degree. C. during
1.5 hour and concentrated under reduced pressure. The residue was
purified by flash chromatography on silica gel [eluent: DCM to
DCM/MeOH 95/5] to afford a mixture of expected .beta. nucleoside 10
(270 mg, beige foam, 22% overall yield) and a nucleoside A11 (416
mg).
[0508] Compound A10: .beta. (ppm) 0.97 (s, 3H), 3.51-3.87 (m, 4H),
3.71 (s, 3H), 5.23 (brt, 1H), 6.06 (s, 1H), 6.26 (d, J=7.50 Hz,
1H), 6.35 (brd, J=4.50 Hz, 1H), 5.80 (d, J=7.40 Hz, 1H.beta.),
6.80-7.40 (m, 14H), 7.80 (d, J=7.50 Hz, 1H), 8.51 (brs, 1H); MS
(ESI) m/z=537.2 (MH.sup.-).
Compound A12
##STR00172##
[0510] To as solution of compound A10 (0.39 mmol) in anhydrous THF
(2 mL) under nitrogen at -5.degree. C. was added dropwise
tert-butylmagnesium chloride (1.0M in THF) (0.82 mmol). The white
suspension was stirred at this temperature for 15 minutes and then
warmed to ambient temperature and stirred for an additional 20
minutes. The reaction mixture was cooled down to 0.degree. C. and
compound A12.0 (0.47 mmol) solubilized in THF (2 mL) was added
dropwise. DMSO (0.4 mL) was added and the mixture was stirred at
7.degree. C. overnight. The reaction mixture was diluted with
dichloromethane and washed with H.sub.2O. The organic phase was
dried, filtered and concentrated under reduced pressure. The
residue was purified by flash chromatography on silica gel [eluent:
DCM/MeOH 0 to 4%] to give the expected compound in 68% yield. MS
(ESI) m/z=806.2 (MH).
Compound 901
##STR00173##
[0512] To a solution of compound A12 (0.27 mmol) in DCM (10 mL) was
added dropwise TFA (2.67 mmol) under nitrogen. The reaction mixture
was stirred at room temperature overnight. The reaction mixture was
purified directly by flash chromatography on silica gel and by
preparative HPLC to give the expected compound as a white
powder.
[0513] .sup.1H NMR (DMSO, 400 MHz) .delta. (ppm) 1.15 (d, J=3.06
Hz, 3H), 1.17 (d, J=3.06 Hz, 3H), 1.25 (brd, 6H), 3.65 (brd, J=13.0
Hz, 1H), 3.77-3.91 (m, 2H), 4.00 (brd, J=7.22 Hz, 1H), 4.70 (t,
J=8.30 Hz, 1H), 4.88 (heptuplet, J=6.30 Hz, 1H), 5.28 (brs, 1H),
5.76 (d, J=7.52 Hz, 1H), 6.28 (s, 1H), 6.34 (q, J=10.30 Hz, J=10.36
Hz), 7.17-7.27 (m, 3H), 7.31-7.42 (m, 4H), 7.83 (d, J=7.57 Hz, 1H);
.sup.31P NMR (DMSO, 161.98 MHz): .delta. (ppm) 3.48 (s, 1P); MS
(ESI) m/z=536.2 (MH.sup.+).
Example 2
HCV Replicon Assay
[0514] Huh-7-derived cell line (Zluc) that harbors an HCV genotype
1b replicon and a luciferase reporter gene was grown in Dulbecco's
Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine
serum, 2 mM GlutaMAX, 1% MEM nonessential amino acids, 100 IU/mL
penicillin, 100 .mu.g/mL streptomycin, and 0.5 mg/mL Geneticin.RTM.
(G418). For dose response testing the cells were seeded in 96-well
plates at 7.5.times.10.sup.3 cells per well in a volume of 50
.mu.L, and incubated at 37.degree. C./5% CO.sub.2. Drug solutions
were made up freshly in Huh-7 media as 2.times. stocks. Ten
additional 5-fold dilutions were prepared from these stocks in DMEM
without G418. At least three hours after Zluc cells were seeded,
drug treatment was initiated by adding 50 .mu.L of drug dilutions
to the plates in duplicate. Final concentrations of drug ranged
from 100 .mu.M to 0.0000512 .mu.M. Cells were then incubated at
37.degree. C./5% CO.sub.2. Alternatively, compounds were tested at
two concentrations (1 .mu.M and 10 .mu.M). In all cases, Huh-7
(which do not harbors the HCV replicon) served as negative control.
After 72 hours of incubation, the inhibition of HCV replication was
measured by quantification of photons emitted after
mono-oxygenation of 5'-fluoroluciferin to oxyfluoroluciferin by
firefly luciferase. For this, media was removed from the plates via
gentle tapping. Fifty microliters of ONE-glo luciferase assay
reagent was added to each well. The plates were shaken gently for 3
min at room temperature and luminescence was measured on a
Victor.sup.3 V 1420 multilabel counter (Perkin Elmer) with a 1
second read time using a 700 nm cut-off filter. The EC.sub.50
values were calculated from dose response curves from the resulting
best-fit equations determined by Microsoft Excel and XLfit 4.1
software. When screening at two fixed concentrations, the results
were expressed as % inhibition at 1 .mu.M and 10 .mu.M.
[0515] For cytotoxicity evaluation, Zluc cells were treated with
compound as described herein, and cell viability was monitored
using the CellTiter-Blue Cell Viability Assay (Promega) by adding
20 .mu.L of the assay solution to each well. The plates were then
incubated at 37.degree. C./5% CO.sub.2 for at least 3 hours.
Fluorescence was detected in plates using excitation and emission
wavelengths of 560 and 590 nm, respectively, in a Victor.sup.3 V
1420 multilabel counter (Perkin Elmer) and CC.sub.50 values were
determined using Microsoft Excel and XLfit 4.1 software.
[0516] Compounds presented in Table 2 below were assayed according
to the replicon assay described herein.
TABLE-US-00001 TABLE 2 HCV Replicon Activity Compound HCV Replicon
Compound HCV Replicon Reference EC.sub.50 CC.sub.50 Reference
EC.sub.50 CC.sub.50 Compound 40ii ++++ + Compound 40ii +++ +
Diastereomer 1 Diastereomer 2 Compound 40i + + Compound 202i +++ +
Diastereomer 1 Diastereomer 2 Compound 202i ++ + Compound 205i +++
+ Diastereomer 1 Diastereomer 2 Compound 205i ++ + Compound 603a
+++ ++ Diastereomer 1 Single Diastereomer Compound 401 ++ ++
Compound 401 +++ ++ Diastereomer 1 Diastereomer 2 Compound 425 + ++
Compound 425 + ++ Diastereomer 1 Diastereomer 2 Compound 502a ++ ++
Compound 502a + ++ Diastereomer 1 Diastereomer 2 Compound 602b + ++
Compound 602b ++ ++ Diastereomer 1 Diastereomer 2 EC.sub.50 is
provided as follows: ++++ .ltoreq. 250 nM, 250 nM < +++ .ltoreq.
1 .mu.M, 1 .mu.M < ++ .ltoreq. 10 .mu.M, and + > 10 .mu.M
CC.sub.50 is provided as follows: ++ .ltoreq. 50 .mu.M, + > 50
.mu.M
Example 3
Metabolism Assays
[0517] Assay for the Release of Active Metabolite in Huh-7
Cells.
[0518] Huh-7 cells were plated in 1 mL culture medium (DMEM,
containing glucose, L-glutamine and sodium pyruvate, 10% FBS, 100
IU/mL penicillin, 100 .mu.g/mL streptomycin, 2 mM GlutaMAX, 1% MEM
non-essential amino acids) at the concentration 0.8, 0.4 and 0.2
million cells per well on 6 well plates for 24, 48 and 72 hr
treatment, respectively. Plated cells were incubated overnight at
37.degree. C. in an incubator.
[0519] The following morning test compound was diluted to 20 .mu.M
from a stock solution in DMSO in fresh culture medium pre-warmed to
37.degree. C. and 1 mL of the solution/well was added to cells. A
final medium volume per well was 2.0 mL, test compound
concentration in well was 10 .mu.M and final DMSO concentration was
0.1%.
[0520] After 24, 48 or 72 hr, the medium was carefully removed and
cell monolayers were washed twice with 2 mL ice-cold PBS per well.
Following the last wash, all PBS was carefully removed and 1.0 mL
of extraction solution (ice-cold 70% methanol) added. The plate was
tightly covered with Parafilm, plastic plate cover and Parafilm
again and an intracellular content was extracted at -20.degree. C.
for 24 hr.
[0521] After 24 hr the extracts were transferred into polypropylene
microfuge tubes and dry on a refrigerated centrivap concentrator.
Dry residues were reconstituted in 250 .mu.L of HPLC-grade water
and centrifuged at 16,000.times.g for 10 min. Aliquots (100 .mu.L
each) of the supernatants were transferred into a 96 well plate and
internal standard (4 ng/mL final concentration) was added as the
internal standard (IS) for LC-MS/MS analysis.
[0522] Abbreviations:
[0523] FHH=fresh human hepatocytes; Ms=Mouse; MsH=fresh mouse
hepatocyte.
[0524] Assay for the Release of Active Metabolite in Primary
Hepatocytes:
[0525] Plates of fresh human and mouse hepatocytes were obtained on
ice. The medium was removed and replaced with hepatocyte culture
medium (William's E supplemented with penicillin-streptomycin, 1%
L-glutamine, 1% insulin-transferrin-selenium and 0.1 .mu.M
Dexamethasone (Invitrogen) or with Invitro GRO HI medium
complemented with Torpedo antibiotics (Celsis)). Cells were left
overnight in an incubator at 37.degree. C. to acclimatize to
culture and the medium.
[0526] Hepatocyte incubations were conducted at a final volume of
0.5 mL hepatocyte culture medium/well (0.8 million cells/well for
human and 0.5 million cells/well for mouse; 12 well plate no
overlay, collagen coat). Culture medium from overnight incubation
of cells was removed and replaced with fresh medium, pre-warmed to
37.degree. C., containing 10 .mu.M of test compound from a stock
solution in DMSO (final DMSO concentration was 0.1%). At each
specific time point, incubation medium was removed and cell
monolayers were carefully washed two times with ice-cold PBS.
Following the last wash, all PBS was carefully removed and 1.0 mL
of extraction solution (ice-cold 70% methanol/30% water) added.
Cells were scraped off and suspended in the extraction solution,
transferred to 2 mL polypropylene microfuge tubes and intracellular
contents extracted overnight at -20.degree. C.
[0527] After the overnight treatment the cellular extracts were
prepared by centrifugation at 16,000.times.g for 10 min to remove
cellular debris. The remaining sample was then dried using a
refrigerated centrivap concentrator. Dry extracts were
reconstituted in 1000 .mu.L of HPLC-grade water and centrifuged at
16,000.times.g for 10 min. Aliquots (100 .mu.L each) of the
supernatant were transferred into a 96 well plate and internal
standard (4 ng/mL final concentration) was added as the internal
standard (IS) for LC-MS/MS analysis.
[0528] The incubation time points were 6, 24 and 48 hours for human
hepatocytes and 1, 4, 8, 12 and 24 hours for mouse hepatocytes.
Results are provided in Table 4 below.
TABLE-US-00002 TABLE 4 Formation of Active Metabolite in Huh-7
cells and Hepatocytes Compound 40ii Compound 40ii Compound 40i
Compound 40i Cells Diastereomer 2 Diastereomer 1 Diastereomer 1
Diastereomer 2 Huh-7 TP C.sub.max 294 123 ND ND (pmol/mill cells)
Huh-7 TP 240 25 ND ND (24 hr) Huh-7 TP 294 96 ND ND (48 hr) Huh-7
TP 144 123 ND ND (72 hr) Huh-7 TP AUC 14544 4380 ND ND (pmol
hr/mill cells) FHH TP AUC 4934 ND ND ND (pmol hr/mill cells) FHH TP
C.sub.max 197 ND ND ND (pmol/mill cells) FHH TP (6 hr) 197 ND ND ND
FHH TP (24 hr) 89 ND ND ND FHH TP (48 hr) 59 ND ND ND MsH AUC 1794
ND 4052 1073 (pmol hr/mill cells) MsH C.sub.max 87 198 89
(pmol/mill cells) .sup.aND = not determined .sup.bBLD = below limit
of detection
Example 4
Pharmacokinetics of Plasma Nucleoside and Liver Triphosphate
Following a Single Oral Dose in CD-1 Mice
[0529] Abbreviations:
[0530] Ms=Mouse; TP=triphosphate.
[0531] A single oral dose of Compound 1 at 10 mg/kg in PEG 200
(dose volume 5 mL/kg) was administered to nine CD-1 male mice. Five
untreated animals were used for the collection of control plasma
and liver. Terminal plasma and liver samples were collected from
three animals per time point at 4, 12 and 24 hours post dose. Liver
specimens were collected from all animals immediately after the
incision. Freezing forceps stored in liquid nitrogen were used to
freeze the liver before excision.
[0532] Plasma samples were analyzed for nucleoside by LC-MS/MS. The
internal standard (IS) was either 2'-MeG-D3 or tiapride. For
protein precipitation and extraction, each plasma sample (50 .mu.L)
was treated with 500 .mu.L of 0.2% formic acid in acetonitrile and
20 .mu.L of the internal standard working solution. After vortexing
and centrifugation, 5004 of the sample extracts were transferred to
a new plate, dried under N.sub.2 at .about.28.degree. C. and
reconstituted with 75 .mu.L of 0.2% FA in water. The extracts were
chromatographed on an Aquasil C18 column using a gradient system of
0.2% formic acid in water and acetonitrile. The analytes were
detected and quantified by tandem mass spectrometry in positive ion
mode on an MDS Sciex API5000 equipped with a Turbo Ionspray.RTM.
interface. The calibration range was 0.500 (LLOQ) to 200 ng/mL in
mouse plasma.
[0533] Liver samples were analyzed for the active species
nucleoside triphosphate by LC-MS/MS. The triphosphate levels were
assayed by homogenizing (on ice) a known weight of mouse liver with
4.times. volume of 0.95 M trichloroacetic acid (TCA). Internal
standard solution was added to the homogenate followed by
neutralization with 20% ammonium hydroxide solution and addition of
500 .mu.L 1% formic acid. The tissue samples were extracted by weak
anion exchange solid phase extraction (SPE). Post extraction, the
eluates were evaporated under nitrogen, followed by reconstitution
before injection onto the LC-MS/MS system. The samples were
chromatographed on a Luna NH.sub.2 column using a gradient system
of ammonium acetate (1 mM to 20 mM and pH 8.0 to pH 10.0) in water
and acetonitrile (70:30). The analyte was detected and quantified
by tandem mass spectrometry in positive ion mode on an API4000
equipped with a Turbo Ionspray.RTM. interface.
[0534] Results are provided in Table 5 below.
TABLE-US-00003 TABLE 4 Mouse plasma and liver pharmacokinetic
parameters Ms Plasma Ms Plasma nucleoside C.sub.max nucleoside AUC
Ms Liver TP Ms Liver TP (pmol/mL at (pmol hr/mL at C.sub.max
(pmol/g at AUC (pmol hr/g Compound 1 .mu.mol/kg) 1 .mu.mol/kg) 1
.mu.mol/kg) at 1 .mu.mol/kg) Compound 40ii 86 970 71 840
Diastereomer 1 Compound 40ii 99 1300 34 560 Diastereomer 2 Compound
40i 94 1400 520 6200 Diastereomer 1 Compound 40i 64 1000 430 4400
Diastereomer 2 Compound 202i -- 1700 -- 8400 Diastereomer 1
Compound 202i -- 1700 -- 7200 Diastereomer 2 Compound 202ii -- --
160 1200 Diastereomer 1 Compound 202ii -- -- 84 850 Diastereomer 2
Compound 205i -- 1400 -- 5600 Diastereomer 1 Compound 205i -- 1700
-- 6900 Diastereomer 2 ND = Not determined; BLQ = below limit of
quantitation.
Example 4A
Pharmacokinetics of Plasma Nucleoside and Liver Triphosphate
Following a Single Oral Dose in CD-1 Mice
[0535] Abbreviations:
[0536] Ms=Mouse; TP=triphosphate.
[0537] A single oral dose of test compound at 2 mg/kg and 10 mg/kg
in PEG 200 (dose volume 1 mL/kg and 5 mL/kg) was administered to
nine CD-1 male mice. Five untreated animals were used for the
collection of control plasma and liver. Terminal plasma and liver
samples were collected from three animals per time point at 4, 12
and 24 hours post dose. Liver specimens were collected from all
animals immediately after the incision. Freezing forceps stored in
liquid nitrogen were used to freeze the liver before excision.
[0538] Plasma samples were analyzed for nucleoside by LC-MS/MS. The
internal standard (IS) was either 2'-MeG-D3 or tiapride. For
protein precipitation and extraction, each plasma sample (50 .mu.L)
was treated with 500 .mu.L of 0.2% formic acid in acetonitrile and
20 .mu.L of the internal standard working solution. After vortexing
and centrifugation, 500 .mu.L of the sample extracts were
transferred to a new plate, dried under N.sub.2 at
.about.28.degree. C. and reconstituted with 75 .mu.L of 0.2% FA in
water. The extracts were chromatographed on an Aquasil C18 column
using a gradient system of 0.2% formic acid in water and
acetonitrile. The analytes were detected and quantified by tandem
mass spectrometry in positive ion mode on an MDS Sciex API5000
equipped with a Turbo Ionspray.RTM. interface. The calibration
range was 0.500 (LLOQ) to 200 ng/mL in mouse plasma.
[0539] Liver samples were analyzed for the active species
nucleoside triphosphate by LC-MS/MS. The triphosphate levels were
assayed by homogenizing (on ice) a known weight of mouse liver with
4.times. volume of 0.95 M trichloroacetic acid (TCA). Internal
standard solution was added to the homogenate followed by
neutralization with 20% ammonium hydroxide solution and addition of
500 .mu.L 1% formic acid. The tissue samples were extracted by weak
anion exchange solid phase extraction (SPE). Post extraction, the
eluates were evaporated under nitrogen, followed by reconstitution
before injection onto the LC-MS/MS system. The samples were
chromatographed on a Luna NH.sub.2 column using a gradient system
of ammonium acetate (1 mM to 20 mM and pH 8.0 to pH 10.0) in water
and acetonitrile (70:30). The analyte was detected and quantified
by tandem mass spectrometry in positive ion mode on an API4000
equipped with a Turbo Ionspray.RTM. interface.
[0540] Results are provided in Table 3 below.
TABLE-US-00004 TABLE 4A Mouse plasma and liver pharmacokinetic
parameters Compound 425 Compound 425 Cells Diastereomer 1
Diastereomer 2 Ms Plasma nucleoside AUC 46 26 (pmol hr/mL after 2
mg/kg dose) Ms Liver TP AUC (pmol hr/g at 3900 1900 after 2 mg/kg
dose) Ms Plasma nucleoside AUC 130 75 (pmol hr/mL after 10 mg/kg
dose) Ms Liver TP AUC (pmol hr/g at after 7500 2500 10 mg/kg
dose)
Example 4B
Pharmacokinetics of Plasma Nucleoside and Liver Triphosphate
Following a Single Oral Dose in CD-1 Mice
[0541] Abbreviations:
[0542] Ms=Mouse; TP=triphosphate;
[0543] A single oral dose of test compound at 10 mg/kg and/or 2
mg/kg in PEG 200 (dose volume 5 mL/kg) was administered to nine
CD-1 male mice. Five untreated animals were used for the collection
of control plasma and liver. Terminal plasma and liver samples were
collected from three animals per time point at 4, 12 and 24 hours
post dose. Liver specimens were collected from all animals
immediately after the incision. Freezing forceps stored in liquid
nitrogen were used to freeze the liver before excision. Only liver
samples were analyzed for triphosphate levels.
[0544] Liver samples were analyzed for the active species
nucleoside triphosphate by LC-MS/MS. The triphosphate levels were
assayed by homogenizing (on ice) a known weight of mouse liver with
4.times. volume of 0.95 M trichloroacetic acid (TCA) in water.
Internal standard solution was added to the homogenate and mixed.
Sample homogenates were centrifuged at 16.1 krpm for 5 minutes.
Supernatants were transferred to 96 well plates and injected onto
the LC-MS/MS system. The samples were chromatographed on a Luna
NH.sub.2 column using a gradient system of ammonium acetate (1 mM
to 20 mM and pH 8.0 to pH 10.0) in water and acetonitrile (70:30).
The analyte was detected and quantified by tandem mass spectrometry
using the analyte specific MRM transition on an API4000 equipped
with a Turbo Ionspray.RTM. interface.
[0545] Results are provided in Table 3 below.
TABLE-US-00005 TABLE 4B Mouse liver pharmacokinetic parameters
Compound 602b Compound 602b Compound 603a Compound 603b Compound
603b Cells Diastereomer 1 Diastereomer 2 Single Diastereomer
Diastereomer 1 Diastereomer 2 Ms Liver TP AUC 5500 3600 120 110 --
(pmol hr/g at 1 .mu.mol/kg) following a single 10 mg/kg dose Ms
Liver TP AUC 4100 3200 -- -- -- (pmol hr/g at 1 .mu.mol/kg)
following a single 2 mg/kg dose
Example 4C
Plasma and Liver Pharmacokinetics Following a Single Oral Dose in
CD-1 Mice
##STR00174##
[0547] Abbreviations:
[0548] Ms=Mouse; 2'-Me-2'-F-U=2'-methyl-2'-fluorouridine;
2'-Me-2'-F-U TP=2'-methyl-2'-fluorouridine triphosphate;
2'-F-2'-Me-G=2'-fluoro-2'-methyl-guanosine.
[0549] A single oral dose of test compound at 10 mg/kg for 804a or
25 mg/kg for 804b in PEG 200 (dose volume 5 mL/kg) was administered
to nine CD-1 male mice. Five untreated animals were used for the
collection of control plasma and liver. Terminal plasma and liver
samples were collected from three animals per time point at 4, 12
and 24 hours post dose. Liver specimens were collected from all
animals immediately after the incision. Freezing forceps stored in
liquid nitrogen were used to freeze the liver before excision.
[0550] Plasma samples were analyzed for 2'-methyl-2'-fluorouridine
(2'-Me-2'-F-U) by LC-MS/MS. The internal standard (IS) was
D.sub.3-2'-F-2'-Me-G. For protein precipitation and extraction,
each plasma sample (50 .mu.L) was treated with 500 .mu.L of 0.2%
formic acid in acetonitrile and 20 .mu.L of the internal standard
working solution. After vortexing and centrifugation, 500 .mu.L of
the sample extracts were transferred to a new plate, dried under
N.sub.2 at .about.28.degree. C. and reconstituted with 75 .mu.L of
0.2% FA in water. The extracts were chromatographed on an Aquasil
C18 column using a gradient system of 0.2% formic acid in water and
acetonitrile. The analytes were detected and quantified by tandem
mass spectrometry in positive ion mode on an MDS Sciex API5000
equipped with a Turbo Ionspray.RTM. interface. The calibration
range was 0.500 (LLOQ) to 200 ng/mL in mouse plasma. The
corresponding range for molar units is 1.92 to 769 pmol/mL.
[0551] Liver samples were analyzed for the active species
2'-methyl-2'-fluorouridine triphosphate (2'-Me-2'-F-U TP) by
LC-MS/MS. 2'-Me-2'-F-U TP levels were assayed by homogenizing (on
ice) a known weight of mouse liver with 4.times. volume of 0.95 M
trichloroacetic acid (TCA). Internal standard solution was added to
the homogenate followed by neutralization with 20% ammonium
hydroxide solution and addition of 500 .mu.L 1% formic acid. The
tissue samples were extracted by weak anion exchange solid phase
extraction (SPE). Post extraction, the eluates were evaporated
under nitrogen, followed by reconstitution before injection onto
the LC-MS/MS system. The samples were chromatographed on a Luna NH2
column using a gradient system of ammonium acetate (1 mM to 20 mM
and pH 8.0 to pH 10.0) in water and acetonitrile (70:30). The
analyte was detected and quantified by tandem mass spectrometry in
positive ion mode on an API4000 equipped with a Turbo Ionspray.RTM.
interface. The calibration range was 10 to 10000 pmol/mL in mouse
liver homogenate (50 to 50000 pmol/g of mouse liver).
[0552] Results are provided in Table 5 below.
TABLE-US-00006 TABLE 4C Mouse plasma and liver pharmacokinetic
parameters Compound (804b) Compound (804a) Diastereomer 1
Diastereomer 2 Cells Ia (R.sub.P isomer) Ib (S.sub.P isomer)
(S.sub.P isomer) (R.sub.P isomer) Ms Plasma 2'-Me-2'-F-U 480 260
320 320 AUC (pmol hr/mL at 1 .mu.mol/kg) Ms Liver 2'-Me-2'-F-U TP
3200 430 250 310 AUC (pmol hr/g at 1 .mu.mol/kg)
Example 5
Pharmacokinetics of Liver Triphosphate and Plasma Prodrug and
Nucleoside Following a Single Oral Dose in Cynomolgus Monkeys
[0553] Abbreviations:
[0554] Mo=Monkey; TP=triphosphate; AUC=area under the concentration
curve.
[0555] For each compound, a single oral dose at 10 mg/kg in PEG 200
(dose volume 3 mL/kg) was administered to cynomolgus monkeys.
Untreated animals were used for the collection of control liver.
Plasma samples were collected at 0.5, 1, 2, 4, 6, 8, 12 and 24
hours for compound 37, diastereomer 2. Terminal liver samples were
collected from three animals per time point at 6, 12 and 24 hours
post dose for compound 37, diastereomer 2 and at 6 hours post dose
for compound 44, diastereomer 2. Liver specimens were collected
from all animals immediately after the incision. Freezing forceps
stored in liquid nitrogen were used to freeze the liver before
excision.
[0556] Plasma samples were analyzed for the prodrug and nucleoside
by LC-MS/MS. For protein precipitation and extraction, each plasma
sample (50 .mu.L) was treated with 500 .mu.L of 0.2% formic acid in
acetonitrile and 20 .mu.L of an appropriate internal standard
working solution. After vortexing and centrifugation, 5004 of the
sample extracts were transferred to a new plate, dried under
N.sub.2 at .about.28.degree. C. and reconstituted with 75 .mu.L of
0.2% FA in water. The extracts were chromatographed on an Aquasil
C18 column using a gradient system of 0.2% formic acid in water and
acetonitrile. The analytes were detected and quantified by tandem
mass spectrometry in positive ion mode on an MDS Sciex API4000
equipped with a Turbo Ionspray.RTM. interface.
[0557] Liver samples were analyzed for the relevant nucleoside
triphosphate by LC-MS/MS. The triphosphate levels were assayed by
homogenizing (on ice) a known weight of liver with 4.times. volume
of 0.95 M trichloroacetic acid (TCA). Appropriate internal standard
solution was added to the homogenate followed by neutralization
with 20% ammonium hydroxide solution and addition of 500 .mu.L 1%
formic acid. The tissue samples were extracted by weak anion
exchange solid phase extraction (SPE). Post extraction, the eluates
were evaporated under nitrogen, followed by reconstitution before
injection onto the LC-MS/MS system. The samples were
chromatographed on a Luna NH.sub.2 column using a gradient system
of ammonium acetate (1 mM to 20 mM and pH 8.0 to pH 10.0) in water
and acetonitrile (70:30). The analyte was detected and quantified
by tandem mass spectrometry in positive ion mode on an API4000
equipped with a Turbo Ionspray.RTM. interface. Results are provided
in Table 5 below.
TABLE-US-00007 TABLE 5 Pharmacokinetics of the prodrug and
nucleoside in plasma and triphosphate in liver of Cynomolgus
monkeys Compound 37 Compound 40ii Compound 40i Compound
Diastereomer 2 Diastereomer 2 Diastereomer 1 Dose (mg/kg) 10 10 10
Dose-normalized parameters.sup.a Plasma prodrug C.sub.max (pmol/mL)
840 ND.sup.b T.sub.max (hr) 4 ND AUC.sub.0-24 4000 ND (pmol hr/mL)
Plasma nucleoside C.sub.max (pmol/mL) 51 ND T.sub.max (hr) 4 ND
AUC.sub.0-24 650 ND (pmol hr/mL) Nucleoside triphosphate in Liver
C.sub.6 (pmol/g) 1500 120 270 C.sub.max (pmol/g) 1700 ND T.sub.max
(hr) 12 ND AUC.sub.0-24 (pmol hr/g) 29000 ND .sup.aThe C.sub.max,
C.sub.6 and AUC.sub.0-24 data are normalized to 1 .mu.mol/kg dose
.sup.bND = not determined
Example 6
Hydrolysis of D-Alanine Prodrugs by Cathepsin a (CatA) and/or
Carboxylesterase 1 (CES1)
Introduction
[0558] The HCV NS5B RNA-dependent RNA polymerase is essential for
the viral life cycle and thus, is a target for antiviral therapy.
The active site of NS5B is well conserved among the six genotypes
of HCV and therefore, nucleos(t)ide analogs can act
pan-genotypically. Furthermore, nucleotide inhibitors are typically
not cross-resistant to other classes of direct acting antivirals
and can have a higher barrier to resistance compared to
non-nucleoside, protease and non-structural protein 5A (NS5A)
inhibitors of HCV, making this class of HCV antivirals useful in a
of combination HCV antiviral therapy.
[0559] Nucleoside analogs are typically competitive inhibitors of
endogenous nucleosides and may act through chain termination upon
incorporation into the nascent HCV RNA chain during replication
(Eldrup, et al. 2004, Structure-Activity Relationship of Purine
Ribonucleosides for Inhibition of Hepatitis C Virus RNA-Dependent
RNA Polymerase. J. Med. Chem. 47: 2283-2295). However, upon cell
entry a nucleoside analog must first be phosphorylated to the
active triphosphate species (Gardelli, et al 2009, Phosphoramidate
prodrugs of 2'-C-methylcytidine for therapy of hepatitis C virus
infection. J. Med. Chem. 52:5394-5407; Stein and Moore, 2001,
Phosphorylation of nucleoside analog antiretrovirals: a review for
clinicians. Pharmacotherapy 21:11-34; Tomassini, et al 2005,
Inhibitory effect of 2'-substituted nucleosides on hepatitis C
virus replication correlates with metabolic properties in replicon
cells. Antimicrob. Agents Chemother. 49:2050-2058; Murakami, et al
2007, Mechanism of activation of
.beta.-D-2'-deoxy-2'-fluoro-2'-C-methylcytidine and inhibition of
hepatitis C virus NS5B RNA polymerase. Antimicrob. Agents
Chemother. 51:503-509). A barrier to first generation nucleoside
inhibitors was the often inefficient conversion of the nucleoside
to a nucleotide monophosphate (NMP) by cellular kinases (Gardelli,
et al 2009, Phosphoramidate prodrugs of 2'-C-methylcytidine for
therapy of hepatitis C virus infection. J. Med. Chem. 52:5394-5407;
Stein and Moore, 2001, Phosphorylation of nucleoside analog
antiretrovirals: a review for clinicians. Pharmacotherapy 21:11-34;
Murakami, et al 2007, Mechanism of activation of
.beta.-D-2'-deoxy-2'-fluoro-2'-C-methylcytidine and inhibition of
hepatitis C virus NS5B RNA polymerase. Antimicrob. Agents
Chemother. 51:503-509).
[0560] Second generation nucleoside analogs have been designed as
liver-targeted nucleotide prodrugs, which bypass the rate-limiting
NMP conversion to active species by delivering the nucleoside as a
monophosphate prodrug. As GS-7977, Z4 and Z2 are pyrimidine
nucleotide prodrugs that act by inhibition of the HCV NS5B
RNA-dependent RNA polymerase through a 2' modified UTP
metabolite.
[0561] The intracellular metabolism (anabolism) of nucleotide
analogs is critical to their antiviral activity. A first step in
the metabolism of nucleotide prodrugs is the removal of the prodrug
moiety by cellular enzymes followed by the activation of the
nucleoside monophosphate analog by host cell kinases for the
sequential phosphorylation of the parent nucleos(t)ide analog to
the 5'-triphosphate form, the biologically active metabolite.
Removal of the prodrug moiety often involves sequential or
independent work of different cellular enzymes.
[0562] In vivo Z4 and Z2 appear to be effectively liver-targeted
with a high liver:plasma ratio of drug metabolites. Both prodrugs
are readily converted to the triphosphate (TP) metabolite in the
liver of mice and monkey producing more TP than GS-7977. The TP
derivatives of Z4 and Z2 selectively inhibit wild-type HCV NS5B
enzyme in vitro with submicromolar IC.sub.50 values. When tested in
a genotype 1b HCV replicon-bearing human hepatoma cell line
(Huh-7), however, Z4 and Z2 were largely inactive and failed to
inhibit replicon reproduction (EC.sub.50>50 .mu.M). The in vitro
antiviral inactivity of Z4 and Z2 is thought to reflect an
inability of Huh-7 replicon cells to metabolize the prodrug
moiety.
[0563] The first step of GS-7977 activation includes hydrolysis of
the carboxyl ester by cathepsin A (CatA) and/or carboxylesterase 1
(CES1) (Saboulard et al, 2009, Characterization of the Activation
Pathway of Phosphoramidate Triester Prodrugs of Stavudine and
Zidovudine. Molecular Pharmacology. 56:693-704; Murakami et al,
2010, Mechanism of Activation of PSI-7851 and Its Diastereoisomer
PSI-7977, JBC, 285(45):34337-34347; Sofia et al, 2010, Discovery of
PSI-35366, a novel purine nucleotide prodrug for the treatment of
hepatitis C virus. J Med. Chem. 53:7202-7218). Since CES1 is
reported to be underexpressed in Huh-7 replicon cells, CatA appears
to be the major enzyme that hydrolyzes GS-7977 in these cells
(Murakami et al, 2010, Mechanism of Activation of PSI-7851 and Its
Diastereoisomer PSI-7977, JBC, 285(45):34337-34347).
Methods
[0564] In this example the hydrolysis of the two D-ala-McGuigan
prodrugs Z2 (2'-C1,2'-MeUMP, diastereoisomer R.sub.P) and Z4 (2'-F,
2'-MeUTP, diastereoisomer R.sub.P) using CatA and CES1 was compared
with activation of the L-ala-McGuigan prodrugs Y1 (2'-MeUTP,
S.sub.P stereoisomer), GS-7977 (X1, diastereoisomer S.sub.P) and
PSI-7976 (X2, diastereoisomer R.sub.P).
[0565] CatA, cathepsin L (CatL) and CES1 were purchased from R
& D Systems (Minneapolis, Minn.). Prior to the enzymatic
hydrolysis reactions, CatA was activated according to the
manufacturer's instruction. Briefly, CatA (0.05 .mu.g/.mu.L) was
incubated with CatL (0.005 .mu.g/.mu.L) for 30 min at 37.degree. C.
in 25 mM MES pH 6.0 containing 5 mM DTT. The reaction was stopped
by addition of the CatL specific inhibitor E64 (10 .mu.M).
[0566] The CatA assay was performed at 37.degree. C. The reaction
mixture contained 25 mM MES buffer pH 6.0, 100 mM NaCl, 4 mM DTT
and 100 .mu.M of the compound. The reaction was started by addition
of the activated CatA enzyme to a final concentration of 0.005
.mu.g/.mu.L. One hundred-.mu.L aliquots were taken after 0.5 min, 3
hrs and 18 hrs of incubation. Reactions were stopped by mixing the
sample with an equal volume of ice-cold methanol, and were loaded
on a HPLC for analysis.
[0567] CES1 assay was performed at 37.degree. C. in the reaction
mixture containing 50 mM Tris/HCl buffer pH 7.5 and 100 .mu.M of
the compound. Reaction was started by addition of the CES1 to the
final concentration 0.01 .mu.g/mL. 100 .mu.L aliquots were taken
after 0.5 min, 3 hrs and 21 hrs of the incubation and the reaction
was stopped by mixing with 100 .mu.l of the ice-cold methanol prior
to HPLC analysis.
[0568] Samples were analyzed by HPLC using 5 .mu.C-18,
4.6.times.250 mm Phenomenex.RTM. Columbus column (Phenomenex USA,
CA). The mobile phase consisted of buffer A (25 mM potassium
phosphate with 5 mM tetrabutylammonium dihydrogen phosphate pH 6.3)
and buffer B (100% methanol). HPLC gradient conditions are shown in
Table 6.
TABLE-US-00008 TABLE 6 Time (min) % A % B Flow (mL/min) 0 100 0 1
15 70 30 1 30 50 50 1 65 50 50 1 70 95 5 1
Results
[0569] As shown in Table 7, both CatA and CES1 hydrolyzed GS-7977
and its diastereoisomer PSI-7076. However, CatA cleaved GS-7977
(S.sub.P configuration) 10 times more efficiently than its R.sub.P
diastereoisomer, while CES 1 preferentially hydrolyzed the R.sub.P
diastereoisomer PSI-7976. These results are in good agreement with
the literature (Murakami, et al 2010, Mechanism of Activation of
PSI-7851 and Its Diastereoisomer PSI-7977, JBC,
285(45):34337-34347).
TABLE-US-00009 TABLE 7 Huh-7 Reference EC.sub.50, pmol*hr/ Liver TP
Compound number S.sub.P/R.sub.P (.mu.M) 10.sup.6cellsAUC.sub.0-72
pmol*hr/g CatA CES1 GS-7977 X1 S.sub.P 0.25 63555 250 100% @ 18 h
12%/3 h; L-Ala- 15%/21 h 2'F,2'MeUTP PSI-7976 X2 R.sub.P 2.08 6527
310 .sup. 10% @ 18 h 56%/3 h; L-Ala- 94%/21 h 2'F,2'MeUTP L-Ala- Y1
S.sub.P 0.17 63740 420 100 @ 3 h Not tested 2'MeUTP D-Ala- Z1
S.sub.P 7 4400 0% 4.5% @ 21 h 2'Cl,2'MeUTP Z2 R.sub.P 5.9; 14; 47
436.9 6200 0% 23% @ 3 h; 49% @ 21 h D-Ala- Z3 S.sub.P 17 430 0% 0%
2'F,2'MeUTP Z4 R.sub.P >50 720.4 3200 0% 10% @ 3 h; 26% @ 21
h
[0570] In contrast, CatA was unable to hydrolyze any of the
D-Ala-prodrugs tested. However, both Z2 and Z4 were processed by
CES 1.
[0571] Since Huh-7 replicon-bearing cells have been found to
express little or no CES1, CatA is the major enzyme that hydrolyzes
GS-7977 in these cells (Murakami et al, 2010, Mechanism of
Activation of PSI-7851 and Its Diastereoisomer PSI-7977, JBC,
285(45):34337-34347). The inability of CatA to activate the
D-Ala-prodrugs Z2 and Z4 may explain the inactivity of these
compounds in Huh-7 replicon-bearing cells, since the lack of in
vitro activity is believed to reflect low production of the active
TP moiety in Huh-7 replicon cells.
[0572] In vivo, high expression of CES1 in the liver coupled with
high catalytic efficiency and possible involvement of other liver
enzyme appears to result in efficient conversion of Z2 and Z4 to
their corresponding triphosphate metabolites.
[0573] All publications, patents, and patent applications cited in
this specification are herein incorporated by reference as if each
individual publication, patent, or patent application were
specifically and individually indicated to be incorporated by
reference. While the claimed subject matter has been described in
terms of various embodiments, the skilled artisan will appreciate
that various modifications, substitutions, omissions, and changes
may be made without departing from the spirit thereof. Accordingly,
it is intended that the scope of the claimed subject matter is
limited solely by the scope of the following claims, including
equivalents thereof.
* * * * *
References