U.S. patent application number 12/142536 was filed with the patent office on 2009-01-01 for modified fluorinated nucleoside analogues.
This patent application is currently assigned to PHARMASSET, INC.. Invention is credited to Jeremy Clark.
Application Number | 20090004135 12/142536 |
Document ID | / |
Family ID | 33563735 |
Filed Date | 2009-01-01 |
United States Patent
Application |
20090004135 |
Kind Code |
A1 |
Clark; Jeremy |
January 1, 2009 |
MODIFIED FLUORINATED NUCLEOSIDE ANALOGUES
Abstract
The disclosed invention provides compositions and methods of
treating a Flaviviridae infection, including hepatitis C virus,
West Nile Virus, yellow fever virus, and a rhinovirus infection in
a host, including animals, and especially humans, using a
(2'R)-2'-deoxy-2'-fluoro-2'-C-methyl nucleosides, or a
pharmaceutically acceptable salt or prodrug thereof.
Inventors: |
Clark; Jeremy; (Birmingham,
AL) |
Correspondence
Address: |
DUANE MORRIS LLP - Atlanta;IP DEPARTMENT
ATLANTIC CENTER PLAZA, 1180 WEST PEACHTREE STREET, NW SUITE 700
ATLANTA
GA
30309-3348
US
|
Assignee: |
PHARMASSET, INC.
PRINCETON
NJ
|
Family ID: |
33563735 |
Appl. No.: |
12/142536 |
Filed: |
June 19, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10828753 |
Apr 21, 2004 |
7429572 |
|
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12142536 |
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60474368 |
May 30, 2003 |
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Current U.S.
Class: |
424/85.2 ;
424/184.1; 424/85.5; 424/85.7; 514/45; 536/27.21 |
Current CPC
Class: |
C07H 19/06 20130101;
A61P 1/16 20180101; A61K 31/7072 20130101; A61P 31/00 20180101;
Y02A 50/30 20180101; C07H 19/14 20130101; C07H 19/00 20130101; A61P
31/14 20180101; C07H 19/048 20130101; A61P 31/16 20180101; A61P
31/12 20180101; A61P 35/00 20180101; A61K 31/7068 20130101; A61P
43/00 20180101; A61P 31/04 20180101; C07H 19/16 20130101; C07H
19/04 20130101 |
Class at
Publication: |
424/85.2 ;
536/27.21; 514/45; 424/85.7; 424/85.5; 424/184.1 |
International
Class: |
A61K 38/20 20060101
A61K038/20; C07H 19/173 20060101 C07H019/173; A61K 31/7076 20060101
A61K031/7076; A61K 39/00 20060101 A61K039/00; A61P 31/12 20060101
A61P031/12; A61K 38/21 20060101 A61K038/21 |
Claims
1-129. (canceled)
130. A (2'R)-2'-deoxy-2'-fluoro-2'-C-methyl nucleoside (.beta.-D or
.beta.-L) or its pharmaceutically acceptable salt of the structure:
##STR00049## wherein the base is a purine base represented by the
following formula: ##STR00050## X is O, S, CH.sub.2, Se, NH,
N-alkyl, CHW (R, S, or racemic), C(W).sub.2, wherein W is F, Cl,
Br, or I; R.sup.1 and R.sup.7 are independently H, phosphate,
including monophosphate, diphosphate, triphosphate, or a stabilized
phosphate prodrug, H-phosphonate, including stabilized
H-phosphonates, acyl, including optionally substituted phenyl and
lower acyl, alkyl, including lower alkyl, O-substituted
carboxyalkylamino or its peptide derivatives, sulfonate ester,
including alkyl or arylalkyl sulfonyl, including methanesulfonyl
and benzyl, wherein the phenyl group is optionally substituted, a
lipid, including a phospholipid, an L or D-amino acid (or racemic
mixture), a carbohydrate, a peptide, a cholesterol, or other
pharmaceutically acceptable leaving group which when administered
in vivo is capable of providing a compound wherein R.sup.1 is H or
phosphate; R.sup.2 is H or phosphate; R.sup.1 and R.sup.2 or
R.sup.7 can also be linked with cyclic phosphate group; R.sup.2 and
R.sup.2' are independently H, C.sub.1-4 allyl, C.sub.1-4 alkenyl,
C.sub.1-4 alkynyl, vinyl, N.sub.3, CN, Cl, Br, F, I, NO2,
C(O)O(C.sub.1-4 alkyl), C(O)O(C.sub.1-4 alkyl), C(O)O(C.sub.1-4
alkynyl), C(O)O(C.sub.1-4 alkenyl), O(C.sub.1-4 acyl), O(C.sub.1-4
alkyl), O(C.sub.1-4 alkenyl), S(C.sub.1-4 acyl), S(C.sub.1-4
alkyl), S(C.sub.1-4 alkynyl), S(C.sub.1-4 alkenyl), SO(C.sub.1-4
acyl), SO(C.sub.1-4 alkyl), SO(C.sub.1-4 alkynyl), SO(C.sub.1-4
alkenyl), SO.sub.2(C.sub.1-4 acyl), SO.sub.2(C.sub.1-4 allyl),
SO.sub.2(C.sub.1-4 alkynyl), SO.sub.2(C.sub.1-4 alkenyl),
O.sub.3S(C.sub.1-4 acyl), O.sub.3S(C.sub.1-4 alkyl),
O.sub.3S(C.sub.1-4 alkenyl), NH.sub.2, NH(C.sub.1-4 alkyl),
NH(C.sub.1-4 alkenyl), NH(C.sub.1-4 alkynyl), NH(C.sub.1-4 acyl),
N(C.sub.1-4 alkyl).sub.2, N(C.sub.1-18 acyl).sub.2, wherein alkyl,
alkynyl, alkenyl and vinyl are optionally substituted by N.sub.3,
CN, one to three halogen (Cl, Br, F, I), NO.sub.2, C(O)O(C.sub.1-4
alkyl), C(O)O(C.sub.1-4 alkyl), C(O)O(C.sub.1-4 alkynyl),
C(O)O(C.sub.1-4 alkenyl), O(C.sub.1-4 acyl), O(C.sub.1-4 alkyl),
O(C.sub.1-4 alkenyl), S(C.sub.1-4 acyl), S(C.sub.1-4 allyl),
S(C.sub.1-4 alkynyl), S(C.sub.1-4 alkenyl), SO(C.sub.1-4 acyl),
SO(C.sub.1-4 alkyl), SO(C.sub.1-4 alkynyl), SO(C.sub.1-4 alkenyl),
SO.sub.2(C.sub.1-4 acyl), SO.sub.2(C.sub.1-4 alkyl),
SO.sub.2(C.sub.1-4 alkynyl), SO.sub.2(C.sub.1-4 alkenyl),
O.sub.3S(C.sub.1-4 acyl), O.sub.3S(C.sub.1-4 alkyl),
O.sub.3S(C.sub.1-4 alkenyl), NH.sub.2, NH(C.sub.1-4 alkyl),
NH(C.sub.1-4 alkenyl), NH(C.sub.1-4 alkynyl), NH(C.sub.1-4 acyl),
N(C.sub.1-4 alkyl).sub.2, N(C.sub.1-4 acyl).sub.2, OR.sup.7;
R.sup.2 and R.sup.2' can be linked together to form a vinyl
optionally substituted by one or two of N.sub.3, CN, Cl, Br, F, I,
NO.sub.2; R.sup.6 is an optionally substituted alkyl (including
lower alkyl), cyano (CN), CH.sub.3, OCH.sub.3, OCH.sub.2CH.sub.3,
hydroxy methyl (CH.sub.2OH), fluoromethyl (CH.sub.2F), azido
(N.sub.3), CHCN, CH.sub.2N.sub.3, CH.sub.2NH.sub.2,
CH.sub.2NHCH.sub.3, CH.sub.2N(CH.sub.3).sub.2, alkyne (optionally
substituted), or fluoro; R.sup.4 and R.sup.5 are independently H,
halogen including F, Cl, Br, I, OH, OR', SH, SR', NH.sub.2, NHR',
NR'.sub.2, lower alkyl of C.sub.1-C.sub.6, halogenated (F, Cl, Br,
I) lower allyl of C.sub.1-C.sub.6, lower alkenyl of
C.sub.2-C.sub.6, halogenated (F, Cl, Br, I) lower alkenyl of
C.sub.2-C.sub.6, lower alkynyl of C.sub.2-C.sub.6 such as
C.ident.CH, halogenated (F, Cl, Br, I) lower alkynyl of
C.sub.2-C.sub.6, lower alkoxy of C.sub.1-C.sub.6 such as CH.sub.2OH
and CH.sub.2CH.sub.2OH, halogenated (F, Cl, Br, I) lower alkoxy of
C.sub.1-C.sub.6, lower hydroxyalkyl, CO.sub.2H, CO.sub.2R',
CONH.sub.2, CONHR', CONR'.sub.2, CH.dbd.CHCO.sub.2H,
CH.dbd.CHCO.sub.2R'; and R' is an optionally substituted alkyl of
C.sub.1-C.sub.12, cycloalkyl, optionally substituted alkynyl of
C.sub.2-C.sub.6, optionally substituted lower alkenyl of
C.sub.2-C.sub.6 or optionally substituted acyl. when n is 1, then
R.sup.4 is .dbd.O. when n is 0, then a double bond exists between
position 1 and position 6.
131. The (2'R)-2'-deoxy-2'-fluoro-2'-C-methyl nucleoside (.beta.-D)
of claim 130 or its pharmaceutically acceptable salt thereof,
wherein the Base is represented by the following formula:
##STR00051## when n is 0, then R.sup.4 is NH.sub.2, R.sup.5 is H
and a double bond exists between position 1 and position 6; when n
is 1, then R.sup.4 is .dbd.O and R.sup.5 is NH.sub.2.
132. A (2'R)-2'-deoxy-2'-fluoro-2'-C-methyl nucleoside (.beta.-D or
.beta.-L) or its pharmaceutically acceptable salt of the structure:
##STR00052## wherein the base is a purine base; X is O, S,
CH.sub.2, Se, NH, N-alkyl, CHW (R, S, or racemic), C(W).sub.2,
wherein W is F, Cl, Br, or I; R.sup.1 and R.sup.7 are independently
H, phosphate, including monophosphate, diphosphate, triphosphate,
or a stabilized phosphate prodrug, H-phosphonate, including
stabilized H-phosphonates, acyl, including optionally substituted
phenyl and lower acyl, alkyl, including lower allyl, O-substituted
carboxyalkylamino or its peptide derivatives, sulfonate ester,
including alkyl or arylalkyl sulfonyl, including methanesulfonyl
and benzyl, wherein the phenyl group is optionally substituted, a
lipid, including a phospholipid, an L or D-amino acid (or racemic
mixture), a carbohydrate, a peptide, a cholesterol, or other
pharmaceutically acceptable leaving group which when administered
in vivo is capable of providing a compound wherein R.sup.1 is H or
phosphate; R.sup.2 is H or phosphate; R.sup.1 and R.sup.2 or
R.sup.7 can also be linked with cyclic phosphate group.
133. The (2'R)-2'-deoxy-2'-fluoro-2'-C-methyl nucleoside (.beta.-D)
of claim 132 or its pharmaceutically acceptable salt thereof,
wherein the Base is represented by the following formula:
##STR00053## when n is 0, then R.sup.4 is NH.sub.2, R.sup.5 is H
and a double bond exists between position 1 and position 6; when n
is 1, then R.sup.4 is .dbd.O and R.sup.5 is NH.sub.2.
134. A (2'R)-2'-deoxy-2'-fluoro-2'-C-methyl nucleoside (.beta.-D)
or its pharmaceutically acceptable salt thereof of the formula:
##STR00054## wherein R.sup.1 and R.sup.7 are independently H,
phosphate, including monophosphate, diphosphate, triphosphate, or a
stabilized phosphate prodrug, H-phosphonate, including stabilized
H-phosphonates, acyl, including optionally substituted phenyl and
lower acyl, alkyl, including lower alkyl, O-substituted
carboxyalkylamino or its peptide derivatives, sulfonate ester,
including alkyl or arylalkyl sulfonyl, including methanesulfonyl
and benzyl, wherein the phenyl group is optionally substituted, a
lipid, including a phospholipid, an L or D-amino acid (or racemic
mixture), a carbohydrate, a peptide, a cholesterol, or other
pharmaceutically acceptable leaving group which when administered
in vivo is capable of providing a compound wherein R.sup.1 or
R.sup.7 is independently H or phosphate; R.sup.1 and R.sup.7 can
also be linked with cyclic phosphate group; and R.sup.6 is an
optionally substituted alkyl (including lower allyl), cyano (CN),
CH.sub.3, OCH.sub.3, OCH.sub.2CH.sub.3, hydroxy methyl
(CH.sub.2OH), fluoromethyl (CH.sub.2F), azido (N.sub.3), CHCN,
CH.sub.2N.sub.3, CH.sub.2NH.sub.2, CH.sub.2NHCH.sub.3,
CH.sub.2N(CH.sub.3).sub.2, alkyne (optionally substituted), or
fluoro.
135. A (2'R)-2'-deoxy-2'-fluoro-2'-C-methyl nucleoside (.beta.-D)
or its pharmaceutically acceptable salt thereof of the formula:
##STR00055##
136. A (2'R)-2'-deoxy-2'-fluoro-2'-C-methyl nucleoside (.beta.-D)
or its pharmaceutically acceptable salt thereof of the formula:
##STR00056## wherein R.sup.1 is H, phosphate, including
monophosphate, diphosphate, triphosphate, or a stabilized phosphate
prodrug, H-phosphonate, including stabilized H-phosphonates, acyl,
including optionally substituted phenyl and lower acyl, alkyl,
including lower alkyl, O-substituted carboxyalkylamino or its
peptide derivatives, sulfonate ester, including alkyl or arylalkyl
sulfonyl, including methanesulfonyl and benzyl, wherein the phenyl
group is optionally substituted, a lipid, including a phospholipid,
an L or D-amino acid (or racemic mixture), a carbohydrate, a
peptide, a cholesterol, or other pharmaceutically acceptable
leaving group which when administered in vivo is capable of
providing a compound wherein R.sup.1 is H or phosphate.
137. A (2'R)-2'-deoxy-2'-fluoro-2'-C-methyl nucleoside (.beta.-D)
or its pharmaceutically acceptable salt thereof of the formula:
##STR00057##
138. A pharmaceutical composition comprising the nucleoside of
claim 130 or its pharmaceutically acceptable salt and a
pharmaceutically acceptable carrier.
139. A pharmaceutical composition comprising the nucleoside of
claim 131 or its pharmaceutically acceptable salt and a
pharmaceutically acceptable carrier.
140. A pharmaceutical composition comprising the nucleoside of
claim 132 or its pharmaceutically acceptable salt and a
pharmaceutically acceptable carrier.
141. A pharmaceutical composition comprising the nucleoside of
claim 133 or its pharmaceutically acceptable salt and a
pharmaceutically acceptable carrier.
142. A pharmaceutical composition comprising a
(2'R)-2'-deoxy-2'-fluoro-2'-C-methyl nucleoside (.beta.-D or
.beta.-L), or its pharmaceutically acceptable salt thereof, in a
pharmaceutically acceptable carrier, of the formula: ##STR00058##
wherein R.sup.1 and R.sup.7 are independently H, phosphate,
including monophosphate, diphosphate, triphosphate, or a stabilized
phosphate prodrug, H-phosphonate, including stabilized
H-phosphonates, acyl, including optionally substituted phenyl and
lower acyl, allyl, including lower alkyl, O-substituted
carboxyalkylamino or its peptide derivatives, sulfonate ester,
including alkyl or arylalkyl sulfonyl, including methanesulfonyl
and benzyl, wherein the phenyl group is optionally substituted, a
lipid, including a phospholipid, an L or D-amino acid (or racemic
mixture), a carbohydrate, a peptide, a cholesterol, or other
pharmaceutically acceptable leaving group which when administered
in vivo is capable of providing a compound wherein R.sup.1 or
R.sup.7 is independently H or phosphate; R.sup.1 and R.sup.7 can
also be linked with cyclic phosphate group; and R.sup.6 is an
optionally substituted allyl (including lower alkyl), cyano (CN),
CH.sub.3, OCH.sub.3, OCH.sub.2CH.sub.3, hydroxy methyl
(CH.sub.2OH), fluoromethyl (CH.sub.2F), azido (N.sub.3), CHCN,
CH.sub.2N.sub.3, CH.sub.2NH.sub.2, CH.sub.2NHCH.sub.3,
CH.sub.2N(CH.sub.3).sub.2, alkyne (optionally substituted), or
fluoro.
143. A pharmaceutical composition comprising a
(2'R)-2'-deoxy-2'-fluoro-2'-C-methyl nucleoside (.beta.-D or
.beta.-L), or its pharmaceutically acceptable salt thereof in a
pharmaceutically acceptable carrier of the formula:
##STR00059##
144. A pharmaceutical composition comprising a
(2'R)-2'-deoxy-2'-fluoro-2'-C-methyl nucleoside (.beta.-D), or its
pharmaceutically acceptable salt thereof in a pharmaceutically
acceptable carrier of the formula: ##STR00060## wherein R.sup.1 is
H, phosphate, including monophosphate, diphosphate, triphosphate,
or a stabilized phosphate prodrug, H-phosphonate, including
stabilized H-phosphonates, acyl, including optionally substituted
phenyl and lower acyl, alkyl, including lower alkyl, O-substituted
carboxyalkylamino or its peptide derivatives, sulfonate ester,
including alkyl or arylalkyl sulfonyl, including methanesulfonyl
and benzyl, wherein the phenyl group is optionally substituted, a
lipid, including a phospholipid, an L or D-amino acid (or racemic
mixture), a carbohydrate, a peptide, a cholesterol, or other
pharmaceutically acceptable leaving group which when administered
in vivo is capable of providing a compound wherein R.sup.1 is H or
phosphate.
145. A pharmaceutical composition comprising a
(2'R)-2'-deoxy-2'-fluoro-2'-C-methyl nucleoside (.beta.-D), or its
pharmaceutically acceptable salt thereof in a pharmaceutically
acceptable carrier of the formula: ##STR00061##
146. A method for the treatment or prophylaxis of Hepatitis C
infection comprising administering to a host an antivirally
effective amount of the nucleoside of claim 130 or its
pharmaceutically acceptable salt optionally in a pharmaceutically
acceptable carrier.
147. A method for the treatment or prophylaxis of Hepatitis C
infection comprising administering to a host an antivirally
effective amount of the nucleoside of claim 131 or its
pharmaceutically acceptable salt optionally in a pharmaceutically
acceptable carrier.
148. A method for the treatment or prophylaxis of Hepatitis C
infection comprising administering to a host an antivirally
effective amount of the nucleoside of claim 132 or its
pharmaceutically acceptable salt optionally in a pharmaceutically
acceptable carrier.
149. A method for the treatment or prophylaxis of Hepatitis C
infection comprising administering to a host an antivirally
effective amount of the nucleoside of claim 133 or its
pharmaceutically acceptable salt optionally in a pharmaceutically
acceptable carrier.
150. A method for the treatment or prophylaxis of Hepatitis C
infection comprising administering to a host an antivirally
effective amount of a (2'R)-2'-deoxy-2'-fluoro-2'-C-methyl
nucleoside (.beta.-D) or its pharmaceutically acceptable salt
thereof of the formula: ##STR00062## wherein R.sup.1 and R.sup.7
are independently H, phosphate, including monophosphate,
diphosphate, triphosphate, or a stabilized phosphate prodrug,
H-phosphonate, including stabilized H-phosphonates, acyl, including
optionally substituted phenyl and lower acyl, alkyl, including
lower alkyl, O-substituted carboxyalkylamino or its peptide
derivatives, sulfonate ester, including alkyl or arylalkyl
sulfonyl, including methanesulfonyl and benzyl, wherein the phenyl
group is optionally substituted, a lipid, including a phospholipid,
an L or D-amino acid (or racemic mixture), a carbohydrate, a
peptide, a cholesterol, or other pharmaceutically acceptable
leaving group which when administered in vivo is capable of
providing a compound wherein R.sup.1 or R.sup.7 is independently H
or phosphate; R.sup.1 and R.sup.7 can also be linked with cyclic
phosphate group; and R.sup.6 is an optionally substituted alkyl
(including lower alkyl), cyano (CN), CH.sub.3, OCH.sub.3,
OCH.sub.2CH.sub.3, hydroxy methyl (CH.sub.2OH), fluoromethyl
(CH.sub.2F), azido (N.sub.3), CHCN, CH.sub.2N.sub.3,
CH.sub.2NH.sub.2, CH.sub.2NHCH.sub.3, CH.sub.2N(CH.sub.3).sub.2,
alkyne (optionally substituted), or fluoro. optionally in a
pharmaceutically acceptable carrier.
151. A method for the treatment or prophylaxis of Hepatitis C
infection comprising administering to a host an antivirally
effective amount of a (2'R)-2'-deoxy-2'-fluoro-2'-C-methyl
nucleoside (.beta.-D) or its pharmaceutically acceptable salt
thereof of the formula: ##STR00063## optionally in a
pharmaceutically acceptable carrier.
152. A method for the treatment or prophylaxis of Hepatitis C
infection comprising administering to a host an antivirally
effective amount of a (2'R)-2'-deoxy-2'-fluoro-2'-C-methyl
nucleoside (.beta.-D) or its pharmaceutically acceptable salt
thereof of the formula: ##STR00064## wherein R.sup.1 is H,
phosphate, including monophosphate, diphosphate, triphosphate, or a
stabilized phosphate prodrug, H-phosphonate, including stabilized
H-phosphonates, acyl, including optionally substituted phenyl and
lower acyl, alkyl, including lower alkyl, O-substituted
carboxyalkylamino or its peptide derivatives, sulfonate ester,
including alkyl or arylalkyl sulfonyl, including methanesulfonyl
and benzyl, wherein the phenyl group is optionally substituted, a
lipid, including a phospholipid, an L or D-amino acid (or racemic
mixture), a carbohydrate, a peptide, a cholesterol, or other
pharmaceutically acceptable leaving group which when administered
in vivo is capable of providing a compound wherein R.sup.1 is H or
phosphate. optionally in a pharmaceutically acceptable carrier.
153. A method for the treatment or prophylaxis of Hepatitis C
infection comprising administering to a host an antivirally
effective amount of a (2'R)-2'-deoxy-2'-fluoro-2'-C-methyl
nucleoside (.beta.-D) or its pharmaceutically acceptable salt
thereof of the formula: ##STR00065## optionally in a
pharmaceutically acceptable carrier.
154. A method for the treatment or prophylaxis of Rhinovirus
infection comprising administering to a host an antivirally
effective amount of the nucleoside of claim 130 or its
pharmaceutically acceptable salt optionally in a pharmaceutically
acceptable carrier.
155. A method for the treatment or prophylaxis of Rhinovirus
infection comprising administering to a host an antivirally
effective amount of the nucleoside of claim 131 or its
pharmaceutically acceptable salt optionally in a pharmaceutically
acceptable carrier.
156. A method for the treatment or prophylaxis of Rhinovirus
infection comprising administering to a host an antivirally
effective amount of the nucleoside of claim 132 or its
pharmaceutically acceptable salt optionally in a pharmaceutically
acceptable carrier.
157. A method for the treatment or prophylaxis of Rhinovirus
infection comprising administering to a host an antivirally
effective amount of the nucleoside of claim 133 or its
pharmaceutically acceptable salt optionally in a pharmaceutically
acceptable carrier.
158. A method for the treatment or prophylaxis of Rhinovirus
infection comprising administering to a host an antivirally
effective amount of a (2'R)-2'-deoxy-2'-fluoro-2'-C-methyl
nucleoside (.beta.-D) or its pharmaceutically acceptable salt
thereof of the formula: ##STR00066## wherein R.sup.1 and R.sup.7
are independently H, phosphate, including monophosphate,
diphosphate, triphosphate, or a stabilized phosphate prodrug,
H-phosphonate, including stabilized H-phosphonates, acyl, including
optionally substituted phenyl and lower acyl, alkyl, including
lower alkyl, O-substituted carboxyalkylamino or its peptide
derivatives, sulfonate ester, including alkyl or arylalkyl
sulfonyl, including methanesulfonyl and benzyl, wherein the phenyl
group is optionally substituted, a lipid, including a phospholipid,
an L or D-amino acid (or racemic mixture), a carbohydrate, a
peptide, a cholesterol, or other pharmaceutically acceptable
leaving group which when administered in vivo is capable of
providing a compound wherein R.sup.1 or R.sup.7 is independently H
or phosphate; R.sup.1 and R.sup.7 can also be linked with cyclic
phosphate group; and R.sup.6 is an optionally substituted alkyl
(including lower alkyl), cyano (CN), CH.sub.3, OCH.sub.3,
OCH.sub.2CH.sub.3, hydroxy methyl (CH.sub.2OH), fluoromethyl
(CH.sub.2F), azido (N.sub.3), CHCN, CH.sub.2N.sub.3,
CH.sub.2NH.sub.2, CH.sub.2NHCH.sub.3, CH.sub.2N(CH.sub.3).sub.2,
alkyne (optionally substituted), or fluoro. optionally in a
pharmaceutically acceptable carrier.
159. A method for the treatment or prophylaxis of Rhinovirus
infection comprising administering to a host an antivirally
effective amount of a (2'R)-2'-deoxy-2'-fluoro-2'-C-methyl
nucleoside (.beta.-D) or its pharmaceutically acceptable salt
thereof of the formula: ##STR00067## optionally in a
pharmaceutically acceptable carrier.
160. A method for the treatment or prophylaxis of Rhinovirus
infection comprising administering to a host an antivirally
effective amount of a (2'R)-2'-deoxy-2'-fluoro-2'-C-methyl
nucleoside (.beta.-D) or its pharmaceutically acceptable salt
thereof of the formula: ##STR00068## wherein R.sup.1 is H,
phosphate, including monophosphate, diphosphate, triphosphate, or a
stabilized phosphate prodrug, H-phosphonate, including stabilized
H-phosphonates, acyl, including optionally substituted phenyl and
lower acyl, alkyl, including lower alkyl, O-substituted
carboxyalkylamino or its peptide derivatives, sulfonate ester,
including alkyl or arylalkyl sulfonyl, including methanesulfonyl
and benzyl, wherein the phenyl group is optionally substituted, a
lipid, including a phospholipid, an L or D-amino acid (or racemic
mixture), a carbohydrate, a peptide, a cholesterol, or other
pharmaceutically acceptable leaving group which when administered
in vivo is capable of providing a compound wherein R.sup.1 is H or
phosphate. optionally in a pharmaceutically acceptable carrier.
161. A method for the treatment or prophylaxis of Rhinovirus
infection comprising administering to a host an antivirally
effective amount of a (2'R)-2'-deoxy-2'-fluoro-2'-C-methyl
nucleoside (.beta.-D) or its pharmaceutically acceptable salt
thereof of the formula: ##STR00069## optionally in a
pharmaceutically acceptable carrier.
162. A method for the treatment or prophylaxis of Yellow Fever
Virus infection comprising administering to a host an antivirally
effective amount of the nucleoside of claim 130 or its
pharmaceutically acceptable salt optionally in a pharmaceutically
acceptable carrier.
163. A method for the treatment or prophylaxis of Yellow Fever
Virus infection comprising administering to a host an antivirally
effective amount of the nucleoside of claim 131 or its
pharmaceutically acceptable salt optionally in a pharmaceutically
acceptable carrier.
164. A method for the treatment or prophylaxis of Yellow Fever
Virus infection comprising administering to a host an antivirally
effective amount of the nucleoside of claim 132 or its
pharmaceutically acceptable salt optionally in a pharmaceutically
acceptable carrier.
165. A method for the treatment or prophylaxis of Yellow Fever
Virus infection comprising administering to a host an antivirally
effective amount of the nucleoside of claim 133 or its
pharmaceutically acceptable salt optionally in a pharmaceutically
acceptable carrier.
166. A method for the treatment or prophylaxis of Yellow Fever
Virus infection comprising administering to a host an antivirally
effective amount of a (2'R)-2'-deoxy-2'-fluoro-2'-C-methyl
nucleoside (.beta.-D) or its pharmaceutically acceptable salt
thereof of the formula: ##STR00070## wherein R.sup.1 and R.sup.7
are independently H, phosphate, including monophosphate,
diphosphate, triphosphate, or a stabilized phosphate prodrug,
H-phosphonate, including stabilized H-phosphonates, acyl, including
optionally substituted phenyl and lower acyl, alkyl, including
lower allyl, O-substituted carboxyalkylamino or its peptide
derivatives, sulfonate ester, including alkyl or arylalkyl
sulfonyl, including methanesulfonyl and benzyl, wherein the phenyl
group is optionally substituted, a lipid, including a phospholipid,
an L or D-amino acid (or racemic mixture), a carbohydrate, a
peptide, a cholesterol, or other pharmaceutically acceptable
leaving group which when administered in vivo is capable of
providing a compound wherein R.sup.1 or R.sup.7 is independently H
or phosphate; R.sup.1 and R.sup.7 can also be linked with cyclic
phosphate group; and R.sup.6 is an optionally substituted alkyl
(including lower allyl), cyano (CN), CH.sub.3, OCH.sub.3,
OCH.sub.2CH.sub.3, hydroxy methyl (CH.sub.2OH), fluoromethyl
(CH.sub.2F), azido (N.sub.3), CHCN, CH.sub.2N.sub.3,
CH.sub.2NH.sub.2, CH.sub.2NHCH.sub.3, CH.sub.2N(CH.sub.3).sub.2,
alkyne (optionally substituted), or fluoro. optionally in a
pharmaceutically acceptable carrier.
167. A method for the treatment or prophylaxis of Yellow Fever
Virus infection comprising administering to a host an antivirally
effective amount of a (2'R)-2'-deoxy-2'-fluoro-2'-C-methyl
nucleoside (.beta.-D) or its pharmaceutically acceptable salt
thereof of the formula: ##STR00071## optionally in a
pharmaceutically acceptable carrier.
168. A method for the treatment or prophylaxis of Yellow Fever
Virus infection comprising administering to a host an antivirally
effective amount of a (2'R)-2'-deoxy-2'-fluoro-2'-C-methyl
nucleoside (.beta.-D) or its pharmaceutically acceptable salt
thereof of the formula: ##STR00072## wherein R.sup.1 is H,
phosphate, including monophosphate, diphosphate, triphosphate, or a
stabilized phosphate prodrug, H-phosphonate, including stabilized
H-phosphonates, acyl, including optionally substituted phenyl and
lower acyl, alkyl, including lower alkyl, O-substituted
Carboxyalkylamino or its peptide derivatives, sulfonate ester,
including alkyl or arylalkyl sulfonyl, including methanesulfonyl
and benzyl, wherein the phenyl group is optionally substituted, a
lipid, including a phospholipid, an L or D-amino acid (or racemic
mixture), a carbohydrate, a peptide, a cholesterol, or other
pharmaceutically acceptable leaving group which when administered
in vivo is capable of providing a compound wherein R.sup.1 is H or
phosphate. optionally in a pharmaceutically acceptable carrier.
169. A method for the treatment or prophylaxis of Yellow Fever
Virus infection comprising administering to a host an antivirally
effective amount of a (2'R)-2'-deoxy-2'-fluoro-2'-C-methyl
nucleoside (.beta.-D) or its pharmaceutically acceptable salt
thereof of the formula: ##STR00073## optionally in a
pharmaceutically acceptable carrier.
170. A method for the treatment or prophylaxis of West Nile Virus
infection comprising administering to a host an antivirally
effective amount of the nucleoside of claim 130 or its
pharmaceutically acceptable salt optionally in a pharmaceutically
acceptable carrier.
171. A method for the treatment or prophylaxis of West Nile Virus
infection comprising administering to a host an antivirally
effective amount of the nucleoside of claim 131 or its
pharmaceutically acceptable salt optionally in a pharmaceutically
acceptable carrier.
172. A method for the treatment or prophylaxis of West Nile Virus
infection comprising administering to a host an antivirally
effective amount of the nucleoside of claim 132 or its
pharmaceutically acceptable salt optionally in a pharmaceutically
acceptable carrier.
173. A method for the treatment or prophylaxis of West Nile Virus
infection comprising administering to a host an antivirally
effective amount of the nucleoside of claim 133 or its
pharmaceutically acceptable salt optionally in a pharmaceutically
acceptable carrier.
174. A method for the treatment or prophylaxis of West Nile Virus
infection comprising administering to a host an antivirally
effective amount of a (2'R)-2'-deoxy-2'-fluoro-2'-C-methyl
nucleoside (.beta.-D) or its pharmaceutically acceptable salt
thereof of the formula: ##STR00074## wherein R.sup.1 and R.sup.7
are independently H, phosphates including monophosphate,
diphosphate, triphosphate, or a stabilized phosphate prodrug,
H-phosphonate, including stabilized H-phosphonates, acyl, including
optionally substituted phenyl and lower acyl, alkyl, including
lower alkyl, O-substituted carboxyalkylamino or its peptide
derivatives, sulfonate ester, including alkyl or arylalkyl
sulfonyl, including methanesulfonyl and benzyl, wherein the phenyl
group is optionally substituted, a lipid, including a phospholipid,
an L or D-amino acid (or racemic mixture), a carbohydrate, a
peptide, a cholesterol, or other pharmaceutically acceptable
leaving group which when administered in vivo is capable of
providing a compound wherein R.sup.1 or R.sup.7 is independently H
or phosphate; R.sup.1 and R.sup.7 can also be linked with cyclic
phosphate group; and R.sup.6 is an optionally substituted alkyl
(including lower alkyl), cyano (CN), CH.sub.3, OCH.sub.3,
OCH.sub.2CH.sub.3, hydroxy methyl (CH.sub.2OH), fluoromethyl
(CH.sub.2F), azido (N.sub.3), CHCN, CH.sub.2N.sub.3,
CH.sub.2NH.sub.2, CH.sub.2NHCH.sub.3, CH.sub.2N(CH.sub.3).sub.2,
alkyne (optionally substituted), or fluoro. optionally in a
pharmaceutically acceptable carrier.
175. A method for the treatment or prophylaxis of West Nile Virus
infection comprising administering to a host an antivirally
effective amount of a (2'R)-2'-deoxy-2'-fluoro-2'-C-methyl
nucleoside (.beta.-D) or its pharmaceutically acceptable salt
thereof of the formula: ##STR00075## optionally in a
pharmaceutically acceptable carrier.
176. A method for the treatment or prophylaxis of West Nile Virus
infection comprising administering to a host an antivirally
effective amount of a (2'R)-2'-deoxy-2'-fluoro-2'-C-methyl
nucleoside (.beta.-D) or its pharmaceutically acceptable salt
thereof of the formula: ##STR00076## wherein R.sup.1 is H,
phosphate, including monophosphate, diphosphate, triphosphate, or a
stabilized phosphate prodrug, H-phosphonate, including stabilized
H-phosphonates, acyl, including optionally substituted phenyl and
lower acyl, alkyl, including lower alkyl, O-substituted
carboxyalkylamino or its peptide derivatives, sulfonate ester,
including alkyl or arylalkyl sulfonyl, including methanesulfonyl
and benzyl, wherein the phenyl group is optionally substituted, a
lipid, including a phospholipid, an L or D-amino acid (or racemic
mixture), a carbohydrate, a peptide, a cholesterol, or other
pharmaceutically acceptable leaving group which when administered
in vivo is capable of providing a compound wherein R.sup.1 is H or
phosphate. optionally in a pharmaceutically acceptable carrier.
177. A method for the treatment or prophylaxis of West Nile Virus
infection comprising administering to a host an antivirally
effective amount of a (2'R)-2'-deoxy-2'-fluoro-2'-C-methyl
nucleoside (.beta.-D) or its pharmaceutically acceptable salt
thereof of the formula: ##STR00077## optionally in a
pharmaceutically acceptable carrier.
178. A method for the treatment or prophylaxis of Dengue Virus
infection comprising administering to a host an antivirally
effective amount of the nucleoside of claim 130 or its
pharmaceutically acceptable salt optionally in a pharmaceutically
acceptable carrier.
179. A method for the treatment or prophylaxis of Dengue Virus
infection comprising administering to a host an antivirally
effective amount of the nucleoside of claim 131 or its
pharmaceutically acceptable salt optionally in a pharmaceutically
acceptable carrier.
180. A method for the treatment or prophylaxis of Dengue Virus
infection comprising administering to a host an antivirally
effective amount of the nucleoside of claim 132 or its
pharmaceutically acceptable salt optionally in a pharmaceutically
acceptable carrier.
181. A method for the treatment or prophylaxis of Dengue Virus
infection comprising administering to a host an antivirally
effective amount of the nucleoside of claim 133 or its
pharmaceutically acceptable salt optionally in a pharmaceutically
acceptable carrier.
182. A method for the treatment or prophylaxis of Dengue Virus
infection comprising administering to a host an antivirally
effective amount of a (2'R)-2'-deoxy-2'-fluoro-2'-C-methyl
nucleoside (.beta.-D) or its pharmaceutically acceptable salt
thereof of the formula: ##STR00078## wherein R.sup.1 and R.sup.7
are independently H, phosphate, including monophosphate,
diphosphate, triphosphate, or a stabilized phosphate prodrug,
H-phosphonate, including stabilized H-phosphonates, acyl, including
optionally substituted phenyl and lower acyl, alkyl, including
lower alkyl, O-substituted carboxyalkylamino or its peptide
derivatives, sulfonate ester, including allyl or arylalkyl
sulfonyl, including methanesulfonyl and benzyl, wherein the phenyl
group is optionally substituted, a lipid, including a phospholipid,
an L or D-amino acid (or racemic mixture), a carbohydrate, a
peptide, a cholesterol, or other pharmaceutically acceptable
leaving group which when administered in vivo is capable of
providing a compound wherein R.sup.1 or R.sup.7 is independently H
or phosphate; R.sup.1 and R.sup.7 can also be linked with cyclic
phosphate group; and R.sup.6 is an optionally substituted alkyl
(including lower allyl), cyano (CN), CH.sub.3, OCH.sub.3,
OCH.sub.2CH.sub.3, hydroxy methyl (CH.sub.2OH), fluoromethyl
(CH.sub.2F), azido (N.sub.3), CHCN, CH.sub.2N.sub.3,
CH.sub.2NH.sub.2, CH.sub.2NHCH.sub.3, CH.sub.2N(CH.sub.3).sub.2,
alkyne (optionally substituted), or fluoro. optionally in a
pharmaceutically acceptable carrier.
183. A method for the treatment or prophylaxis of Dengue Virus
infection comprising administering to a host an antivirally
effective amount of a (2'R)-2'-deoxy-2'-fluoro-2'-C-methyl
nucleoside (.beta.-D) or its pharmaceutically acceptable salt
thereof of the formula: ##STR00079## optionally in a
pharmaceutically acceptable carrier.
184. A method for the treatment or prophylaxis of Dengue Virus
infection comprising administering to a host an antivirally
effective amount of a (2'R)-2'-deoxy-2'-fluoro-2'-C-methyl
nucleoside (.beta.-D) or its pharmaceutically acceptable salt
thereof of the formula: ##STR00080## wherein R.sup.1 is H,
phosphate, including monophosphate, diphosphate, triphosphate, or a
stabilized phosphate prodrug, H-phosphonate, including stabilized
H-phosphonates, acyl, including optionally substituted phenyl and
lower acyl, alkyl, including lower alkyl, O-substituted
carboxyalkylamino or its peptide derivatives, sulfonate ester,
including alkyl or arylalkyl sulfonyl, including methanesulfonyl
and benzyl, wherein the phenyl group is optionally substituted, a
lipid, including a phospholipid, an L or D-amino acid (or racemic
mixture), a carbohydrate, a peptide, a cholesterol, or other
pharmaceutically acceptable leaving group which when administered
in vivo is capable of providing a compound wherein R.sup.1 is H or
phosphate. optionally in a pharmaceutically acceptable carrier.
185. A method for the treatment or prophylaxis of Dengue Virus
infection comprising administering to a host an antivirally
effective amount of a (2'R)-2'-deoxy-2'-fluoro-2'-C-methyl
nucleoside (.beta.-D) or its pharmaceutically acceptable salt
thereof of the formula: ##STR00081## optionally in a
pharmaceutically acceptable carrier.
186. The method of claim 146, wherein the antivirally effective
amount of (2'R)-2'-deoxy-2'-fluoro-2'-C-methyl nucleoside is
administered in combination or alternation with at least one
treatment selected from the group consisting of: interferon,
including interferon alpah 2a, interferon alpha 2b, a pegylated
interferon, interferon beta, interferon gamma, interferon tau and
interferon omega; an interleukin, including interleukin 10 and
interleukin 12; ribavirin; interferon alpha or pegylated interferon
alpha in combination with ribavirin or levovirin; levovirin; a
protease inhibitor including NS3 inhibitor, a NS3-4A inhibitor; a
helicase inhibitor; a polymerase inhibitor including HCV RNA
polymerase and NS5B polymerase inhibitor; gliotoxin; an IRES
inhibitor, and antisense oligonucleotide; a thiazolidine
derivative; a benzanilide, a ribozyme; another nucleoside,
nucleoside prodrug or nucleoside derivative; a
1-amino-alkylcyclohexane; an antioxidant including vitamin E;
squalene; amantadine; a bile acid; N-(phosphonoacetyl)-L-aspartic
acid; a benzenedicarboxamide; polyadenylic acid; a benzimidazole;
thymosin; a beta tubulin inhibitor; a prophylactic vaccine; an
immune modulator, an IMPDH inhibitor; silybin-phosphatidylcholine
phytosome; and mycophenolate.
187. The method of claim 151, wherein the antivirally effective
amount of (2'R)-2'-deoxy-2'-fluoro-2'-C-methyl nucleoside is
administered in combination or alternation with at least one
treatment selected from the group consisting of: interferon,
including interferon alpah 2a, interferon alpha 2b, a pegylated
interferon, interferon beta, interferon gamma, interferon tau and
interferon omega; an interleukin, including interleukin 10 and
interleukin 12; ribavirin; interferon alpha or pegylated interferon
alpha in combination with ribavirin or levovirin; levovirin; a
protease inhibitor including NS3 inhibitor, a NS3-4A inhibitor; a
helicase inhibitor; a polymerase inhibitor including HCV RNA
polymerase and NS5B polymerase inhibitor; gliotoxin; an IRES
inhibitor, and antisense oligonucleotide; a thiazolidine
derivative; a benzanilide, a ribozyme; another nucleoside,
nucleoside prodrug or nucleoside derivative; a
1-amino-alkylcyclohexane; an antioxidant including vitamin E;
squalene; amantadine; a bile acid; N-(phosphonoacetyl)-L-aspartic
acid; a benzenedicarboxamide; polyadenylic acid; a benzimidazole;
thymosin; a beta tubulin inhibitor; a prophylactic vaccine; an
immune modulator, an IMPDH inhibitor; silybin-phosphatidylcholine
phytosome; and mycophenolate.
188. The method of claim 153, wherein the antivirally effective
amount of (2'R)-2'-deoxy-2'-fluoro-2'-C-methyl nucleoside is
administered in combination or alternation with at least one
treatment selected from the group consisting of: interferon,
including interferon alpah 2a, interferon alpha 2b, a pegylated
interferon, interferon beta, interferon gamma, interferon tau and
interferon omega; an interleukin, including interleukin 10 and
interleukin 12; ribavirin; interferon alpha or pegylated interferon
alpha in combination with ribavirin or levovirin; levovirin; a
protease inhibitor including NS3 inhibitor, a NS3-4A inhibitor; a
helicase inhibitor; a polymerase inhibitor including HCV RNA
polymerase and NS5B polymerase inhibitor; gliotoxin; an IRES
inhibitor, and antisense oligonucleotide; a thiazolidine
derivative; a benzanilide, a ribozyme; another nucleoside,
nucleoside prodrug or nucleoside derivative; a
1-amino-alkylcyclohexane; an antioxidant including vitamin E;
squalene; amantadine; a bile acid; N-(phosphonoacetyl)-L-aspartic
acid; a benzenedicarboxamide; polyadenylic acid; a benzimidazole;
thymosin; a beta tubulin inhibitor; a prophylactic vaccine; an
immune modulator, an IMPDH inhibitor; silybin-phosphatidylcholine
phytosome; and mycophenolate.
189. The method of claim 154, wherein the antivirally effective
amount of (2'R)-2'-deoxy-2'-fluoro-2'-C-methyl nucleoside is
administered in combination or alternation with at least one
treatment selected from the group consisting of: interferon,
including interferon alpah 2a, interferon alpha 2b, a pegylated
interferon, interferon beta, interferon gamma, interferon tau and
interferon omega; an interleukin, including interleukin 10 and
interleukin 12; ribavirin; interferon alpha or pegylated interferon
alpha in combination with ribavirin or levovirin; levovirin; a
protease inhibitor including NS3 inhibitor, a NS3-4A inhibitor; a
helicase inhibitor; a polymerase inhibitor including HCV RNA
polymerase and NS5B polymerase inhibitor; gliotoxin; an IRES
inhibitor, and antisense oligonucleotide; a thiazolidine
derivative; a benzanilide, a ribozyme; another nucleoside,
nucleoside prodrug or nucleoside derivative; a
1-amino-alkylcyclohexane; an antioxidant including vitamin E;
squalene; amantadine; a bile acid; N-(phosphonoacetyl)-L-aspartic
acid; a benzenedicarboxamide; polyadenylic acid; a benzimidazole;
thymosin; a beta tubulin inhibitor; a prophylactic vaccine; an
immune modulator, an IMPDH inhibitor; silybin-phosphatidylcholine
phytosome; and mycophenolate.
190. The method of claim 159, wherein the antivirally effective
amount of (2'R)-2'-deoxy-2'-fluoro-2'-C-methyl nucleoside is
administered in combination or alternation with at least one
treatment selected from the group consisting of: interferon,
including interferon alpah 2a, interferon alpha 2b, a pegylated
interferon, interferon beta, interferon gamma, interferon tau and
interferon omega; an interleukin, including interleukin 10 and
interleukin 12; ribavirin; interferon alpha or pegylated interferon
alpha in combination with ribavirin or levovirin; levovirin; a
protease inhibitor including NS3 inhibitor, a NS3-4A inhibitor; a
helicase inhibitor; a polymerase inhibitor including HCV RNA
polymerase and NS5B polymerase inhibitor; gliotoxin; an IRES
inhibitor, and antisense oligonucleotide; a thiazolidine
derivative; a benzanilide, a ribozyme; another nucleoside,
nucleoside prodrug or nucleoside derivative; a
1-amino-alkylcyclohexane; an antioxidant including vitamin E;
squalene; amantadine; a bile acid; N-(phosphonoacetyl)-L-aspartic
acid; a benzenedicarboxamide; polyadenylic acid; a benzimidazole;
thymosin; a beta tubulin inhibitor; a prophylactic vaccine; an
immune modulator, an IMPDH inhibitor; silybin-phosphatidylcholine
phytosome; and mycophenolate.
191. The method of claim 161, wherein the antivirally effective
amount of (2'R)-2'-deoxy-2'-fluoro-2'-C-methyl nucleoside is
administered in combination or alternation with at least one
treatment selected from the group consisting of: interferon,
including interferon alpah 2a, interferon alpha 2b, a pegylated
interferon, interferon beta, interferon gamma, interferon tau and
interferon omega; an interleukin, including interleukin 10 and
interleukin 12; ribavirin; interferon alpha or pegylated interferon
alpha in combination with ribavirin or levovirin; levovirin; a
protease inhibitor including NS3 inhibitor, a NS3-4A inhibitor; a
helicase inhibitor; a polymerase inhibitor including HCV RNA
polymerase and NS5B polymerase inhibitor; gliotoxin; an IRES
inhibitor, and antisense oligonucleotide; a thiazolidine
derivative; a benzanilide, a ribozyme; another nucleoside,
nucleoside prodrug or nucleoside derivative; a
1-amino-alkylcyclohexane; an antioxidant including vitamin E;
squalene; amantadine; a bile acid; N-(phosphonoacetyl)-L-aspartic
acid; a benzenedicarboxamide; polyadenylic acid; a benzimidazole;
thymosin; a beta tubulin inhibitor; a prophylactic vaccine; an
immune modulator, an IMPDH inhibitor; silybin-phosphatidylcholine
phytosome; and mycophenolate.
192. The method of claim 162, wherein the antivirally effective
amount of (2'R)-2'-deoxy-2'-fluoro-2'-C-methyl nucleoside is
administered in combination or alternation with at least one
treatment selected from the group consisting of: interferon,
including interferon alpah 2a, interferon alpha 2b, a pegylated
interferon, interferon beta, interferon gamma, interferon tau and
interferon omega; an interleukin, including interleukin 10 and
interleukin 12; ribavirin; interferon alpha or pegylated interferon
alpha in combination with ribavirin or levovirin; levovirin; a
protease inhibitor including NS3 inhibitor, a NS3-4A inhibitor; a
helicase inhibitor; a polymerase inhibitor including HCV RNA
polymerase and NS5B polymerase inhibitor; gliotoxin; an IRES
inhibitor, and antisense oligonucleotide; a thiazolidine
derivative; a benzanilide, a ribozyme; another nucleoside,
nucleoside prodrug or nucleoside derivative; a
1-amino-alkylcyclohexane; an antioxidant including vitamin E;
squalene; amantadine; a bile acid; N-(phosphonoacetyl)-L-aspartic
acid; a benzenedicarboxamide; polyadenylic acid; a benzimidazole;
thymosin; a beta tubulin inhibitor; a prophylactic vaccine; an
immune modulator, an IMPDH inhibitor; silybin-phosphatidylcholine
phytosome; and mycophenolate.
193. The method of claim 167, wherein the antivirally effective
amount of (2'R)-2'-deoxy-2'-fluoro-2'-C-methyl nucleoside is
administered in combination or alternation with at least one
treatment selected from the group consisting of: interferon,
including interferon alpah 2a, interferon alpha 2b, a pegylated
interferon, interferon beta, interferon gamma, interferon tau and
interferon omega; an interleukin, including interleukin 10 and
interleukin 12; ribavirin; interferon alpha or pegylated interferon
alpha in combination with ribavirin or levovirin; levovirin; a
protease inhibitor including NS3 inhibitor, a NS3-4A inhibitor; a
helicase inhibitor; a polymerase inhibitor including HCV RNA
polymerase and NS5B polymerase inhibitor; gliotoxin; an IRES
inhibitor, and antisense oligonucleotide; a thiazolidine
derivative; a benzanilide, a ribozyme; another nucleoside,
nucleoside prodrug or nucleoside derivative; a
1-amino-alkylcyclohexane; an antioxidant including vitamin E;
squalene; amantadine; a bile acid; N-(phosphonoacetyl)-L-aspartic
acid; a benzenedicarboxamide; polyadenylic acid; a benzimidazole;
thymosin; a beta tubulin inhibitor; a prophylactic vaccine; an
immune modulator, an IMPDH inhibitor; silybin-phosphatidylcholine
phytosome; and mycophenolate.
194. The method of claim 169, wherein the antivirally effective
amount of (2'R)-2'-deoxy-2'-fluoro-2'-C-methyl nucleoside is
administered in combination or alternation with at least one
treatment selected from the group consisting of: interferon,
including interferon alpah 2a, interferon alpha 2b, a pegylated
interferon, interferon beta, interferon gamma, interferon tau and
interferon omega; an interleukin, including interleukin 10 and
interleukin 12; ribavirin; interferon alpha or pegylated interferon
alpha in combination with ribavirin or levovirin; levovirin; a
protease inhibitor including NS3 inhibitor, a NS3-4A inhibitor; a
helicase inhibitor; a polymerase inhibitor including HCV RNA
polymerase and NS5B polymerase inhibitor; gliotoxin; an IRES
inhibitor, and antisense oligonucleotide; a thiazolidine
derivative; a benzanilide, a ribozyme; another nucleoside,
nucleoside prodrug or nucleoside derivative; a
1-amino-alkylcyclohexane; an antioxidant including vitamin E;
squalene; amantadine; a bile acid; N-(phosphonoacetyl)-L-aspartic
acid; a benzenedicarboxamide; polyadenylic acid; a benzimidazole;
thymosin; a beta tubulin inhibitor; a prophylactic vaccine; an
immune modulator, an IMPDH inhibitor; silybin-phosphatidylcholine
phytosome; and mycophenolate.
195. The method of claim 170, wherein the antivirally effective
amount of (2'R)-2'-deoxy-2'-fluoro-2'-C-methyl nucleoside is
administered in combination or alternation with at least one
treatment selected from the group consisting of: interferon,
including interferon alpah 2a, interferon alpha 2b, a pegylated
interferon, interferon beta, interferon gamma, interferon tau and
interferon omega; an interleukin, including interleukin 10 and
interleukin 12; ribavirin; interferon alpha or pegylated interferon
alpha in combination with ribavirin or levovirin; levovirin; a
protease inhibitor including NS3 inhibitor, a NS3-4A inhibitor; a
helicase inhibitor; a polymerase inhibitor including HCV RNA
polymerase and NS5B polymerase inhibitor; gliotoxin; an IRES
inhibitor, and antisense oligonucleotide; a thiazolidine
derivative; a benzanilide, a ribozyme; another nucleoside,
nucleoside prodrug or nucleoside derivative; a
1-amino-alkylcyclohexane; an antioxidant including vitamin E;
squalene; amantadine; a bile acid; N-(phosphonoacetyl)-L-aspartic
acid; a benzenedicarboxamide; polyadenylic acid; a benzimidazole;
thymosin; a beta tubulin inhibitor; a prophylactic vaccine; an
immune modulator, an IMPDH inhibitor; silybin-phosphatidylcholine
phytosome; and mycophenolate.
196. The method of claim 175, wherein the antivirally effective
amount of (2'R)-2'-deoxy-2'-fluoro-2'-C-methyl nucleoside is
administered in combination or alternation with at least one
treatment selected from the group consisting of: interferon,
including interferon alpah 2a, interferon alpha 2b, a pegylated
interferon, interferon beta, interferon gamma, interferon tau and
interferon omega; an interleukin, including interleukin 10 and
interleukin 12; ribavirin; interferon alpha or pegylated interferon
alpha in combination with ribavirin or levovirin; levovirin; a
protease inhibitor including NS3 inhibitor, a NS3-4A inhibitor; a
helicase inhibitor; a polymerase inhibitor including HCV RNA
polymerase and NS5B polymerase inhibitor; gliotoxin; an IRES
inhibitor, and antisense oligonucleotide; a thiazolidine
derivative; a benzanilide, a ribozyme; another nucleoside,
nucleoside prodrug or nucleoside derivative; a
1-amino-alkylcyclohexane; an antioxidant including vitamin E;
squalene; amantadine; a bile acid; N-(phosphonoacetyl)-L-aspartic
acid; a benzenedicarboxamide; polyadenylic acid; a benzimidazole;
thymosin; a beta tubulin inhibitor; a prophylactic vaccine; an
immune modulator, an IMPDH inhibitor; silybin-phosphatidylcholine
phytosome; and mycophenolate.
197. The method of claim 177, wherein the antivirally effective
amount of (2'R)-2'-deoxy-2'-fluoro-2'-C-methyl nucleoside is
administered in combination or alternation with at least one
treatment selected from the group consisting of: interferon,
including interferon alpah 2a, interferon alpha 2b, a pegylated
interferon, interferon beta, interferon gamma, interferon tau and
interferon omega; an interleukin, including interleukin 10 and
interleukin 12; ribavirin; interferon alpha or pegylated interferon
alpha in combination with ribavirin or levovirin; levovirin; a
protease inhibitor including NS3 inhibitor, a NS3-4A inhibitor; a
helicase inhibitor; a polymerase inhibitor including HCV RNA
polymerase and NS5B polymerase inhibitor; gliotoxin; an IRES
inhibitor, and antisense oligonucleotide; a thiazolidine
derivative; a benzanilide, a ribozyme; another nucleoside,
nucleoside prodrug or nucleoside derivative; a
1-amino-alkylcyclohexane; an antioxidant including vitamin E;
squalene; amantadine; a bile acid; N-(phosphonoacetyl)-L-aspartic
acid; a benzenedicarboxamide; polyadenylic acid; a benzimidazole;
thymosin; a beta tubulin inhibitor; a prophylactic vaccine; an
immune modulator, an IMPDH inhibitor; silybin-phosphatidylcholine
phytosome; and mycophenolate.
198. The method of claim 178, wherein the antivirally effective
amount of (2'R)-2'-deoxy-2'-fluoro-2'-C-methyl nucleoside is
administered in combination or alternation with at least one
treatment selected from the group consisting of: interferon,
including interferon alpah 2a, interferon alpha 2b, a pegylated
interferon, interferon beta, interferon gamma, interferon tau and
interferon omega; an interleukin, including interleukin 10 and
interleukin 12; ribavirin; interferon alpha or pegylated interferon
alpha in combination with ribavirin or levovirin; levovirin; a
protease inhibitor including NS3 inhibitor, a NS3-4A inhibitor; a
helicase inhibitor; a polymerase inhibitor including HCV RNA
polymerase and NS5B polymerase inhibitor; gliotoxin; an IRES
inhibitor, and antisense oligonucleotide; a thiazolidine
derivative; a benzanilide, a ribozyme; another nucleoside,
nucleoside prodrug or nucleoside derivative; a
1-amino-alkylcyclohexane; an antioxidant including vitamin E;
squalene; amantadine; a bile acid; N-(phosphonoacetyl)-L-aspartic
acid; a benzenedicarboxamide; polyadenylic acid; a benzimidazole;
thymosin; a beta tubulin inhibitor; a prophylactic vaccine; an
immune modulator, an IMPDH inhibitor; silybin-phosphatidylcholine
phytosome; and mycophenolate.
199. The method of claim 183, wherein the antivirally effective
amount of (2'R)-2'-deoxy-2'-fluoro-2'-C-methyl nucleoside is
administered in combination or alternation with at least one
treatment selected from the group consisting of: interferon,
including interferon alpah 2a, interferon alpha 2b, a pegylated
interferon, interferon beta, interferon gamma, interferon tau and
interferon omega; an interleukin, including interleukin 10 and
interleukin 12; ribavirin; interferon alpha or pegylated interferon
alpha in combination with ribavirin or levovirin; levovirin; a
protease inhibitor including NS3 inhibitor, a NS3-4A inhibitor; a
helicase inhibitor; a polymerase inhibitor including HCV RNA
polymerase and NS5B polymerase inhibitor; gliotoxin; an IRES
inhibitor, and antisense oligonucleotide; a thiazolidine
derivative; a benzanilide, a ribozyme; another nucleoside,
nucleoside prodrug or nucleoside derivative; a
1-amino-alkylcyclohexane; an antioxidant including vitamin E;
squalene; amantadine; a bile acid; N-(phosphonoacetyl)-L-aspartic
acid; a benzenedicarboxamide; polyadenylic acid; a benzimidazole;
thymosin; a beta tubulin inhibitor; a prophylactic vaccine; an
immune modulator, an IMPDH inhibitor; silybin-phosphatidylcholine
phytosome; and mycophenolate.
200. The method of claim 185, wherein the antivirally effective
amount of (2'R)-2'-deoxy-2'-fluoro-2'-C-methyl nucleoside is
administered in combination or alternation with at least one
treatment selected from the group consisting of: interferon,
including interferon alpah 2a, interferon alpha 2b, a pegylated
interferon, interferon beta, interferon gamma, interferon tau and
interferon omega; an interleukin, including interleukin 10 and
interleukin 12; ribavirin; interferon alpha or pegylated interferon
alpha in combination with ribavirin or levovirin; levovirin; a
protease inhibitor including NS3 inhibitor, a NS3-4A inhibitor; a
helicase inhibitor; a polymerase inhibitor including HCV RNA
polymerase and NS5B polymerase inhibitor; gliotoxin; an IRES
inhibitor, and antisense oligonucleotide; a thiazolidine
derivative; a benzanilide, a ribozyme; another nucleoside,
nucleoside prodrug or nucleoside derivative; a
1-amino-alkylcyclohexane; an antioxidant including vitamin E;
squalene; amantadine; a bile acid; N-(phosphonoacetyl)-L-aspartic
acid; a benzenedicarboxamide; polyadenylic acid; a benzimidazole;
thymosin; a beta tubulin inhibitor; a prophylactic vaccine; an
immune modulator, an IMPDH inhibitor; silybin-phosphatidylcholine
phytosome; and mycophenolate.
Description
CROSS-REFERENCE TO RELATED PATENT APPLICATION
[0001] This application claims the benefit, pursuant to 35 U.S.C.
.sctn.119(e), of provisional U.S. Patent Application Ser. No.
60/474,368, filed May 30, 2003, the disclosure of which is hereby
incorporated herein in its entirety by reference.
FIELD OF THE INVENTION
[0002] The present invention includes
(2'R)-2'-deoxy-2'-fluoro-2'-C-methyl nucleosides having the natural
.beta.-D configuration and methods for the treatment of
Flaviviridae infections, especially hepatitis C virus (HCV).
BACKGROUND OF THE INVENTION
[0003] Hepatitis C virus (HCV) infection is a major health problem
that leads to chronic liver disease, such as cirrhosis and
hepatocellular carcinoma, in a substantial number of infected
individuals, estimated to be 2-15% of the world's population. There
are an estimated 4.5 million infected people in the United States
alone, according to the U.S. Center for Disease Control. According
to the World Health Organization, there are more than 200 million
infected individuals worldwide, with at least 3 to 4 million people
being infected each year. Once infected, about 20% of people clear
the virus, but the rest can harbor HCV the rest of their lives. Ten
to twenty percent of chronically infected individuals eventually
develop liver-destroying cirrhosis or cancer. The viral disease is
transmitted parenterally by contaminated blood and blood products,
contaminated needles, or sexually and vertically from infected
mothers or carrier mothers to their offspring. Current treatments
for HCV infection, which are restricted to immunotherapy with
recombinant interferon-.alpha. alone or in combination with the
nucleoside analog ribavirin, are of limited clinical benefit as
resistance develops rapidly. Moreover, there is no established
vaccine for HCV. Consequently, there is an urgent need for improved
therapeutic agents that effectively combat chronic HCV
infection.
[0004] The HCV virion is an enveloped positive-strand RNA virus
with a single oligoribonucleotide genomic sequence of about 9600
bases which encodes a polyprotein of about 3,010 amino acids. The
protein products of the HCV gene consist of the structural proteins
C, E1, and E2, and the non-structural proteins NS2, NS3, NS4A and
NS4B, and NS5A and NS5B. The nonstructural (NS) proteins are
believed to provide the catalytic machinery for viral replication.
The NS3 protease releases NS5B, the RNA-dependent RNA polymerase
from the polyprotein chain. HCV NS5B polymerase is required for the
synthesis of a double-stranded RNA from a single-stranded viral RNA
that serves as a template in the replication cycle of HCV.
Therefore, NS5B polymerase is considered to be an essential
component in the HCV replication complex (K. Ishi, et al.,
"Expression of Hepatitis C Virus NS5B Protein: Characterization of
Its RNA Polymerase Activity and RNA Binding," Heptology, 29:
1227-1235 (1999); V. Lohmann, et al., "Biochemical and Kinetic
Analysis of NS5B RNA-Dependent RNA Polymerase of the Hepatitis C
Virus," Virology, 249: 108-118 (1998)). Inhibition of HCV NS5B
polymerase prevents formation of the double-stranded HCV RNA and
therefore constitutes an attractive approach to the development of
HCV-specific antiviral therapies.
[0005] HCV belongs to a much larger family of viruses that share
many common features.
Flaviviridae Viruses
[0006] The Flaviviridae family of viruses comprises at least three
distinct genera: pestiviruses, which cause disease in cattle and
pigs; flavivruses, which are the primary cause of diseases such as
dengue fever and yellow fever; and hepaciviruses, whose sole member
is HCV. The flavivirus genus includes more than 68 members
separated into groups on the basis of serological relatedness
(Calisher et al., J. Gen. Virol, 1993,70,37-43). Clinical symptoms
vary and include fever, encephalitis and hemorrhagic fever (Fields
Virology, Editors: Fields, B. N., Knipe, D. M., and Howley, P. M.,
Lippincott-Raven Publishers, Philadelphia, Pa., 1996, Chapter 31,
931-959). Flaviviruses of global concern that are associated with
human disease include the Dengue Hemorrhagic Fever viruses (DHF),
yellow fever virus, shock syndrome and Japanese encephalitis virus
(Halstead, S. B., Rev. Infect. Dis., 1984, 6, 251-264; Halstead, S.
B., Science, 239:476-481, 1988; Monath, T. P., New Eng. J. Med,
1988, 319, 64 1-643).
[0007] The pestivirus genus includes bovine viral diarrhea virus
(BVDV), classical swine fever virus (CSFV, also called hog cholera
virus) and border disease virus (BDV) of sheep (Moennig, V. et al.
Adv. Vir. Res. 1992, 41, 53-98). Pestivirus infections of
domesticated livestock (cattle, pigs and sheep) cause significant
economic losses worldwide. BVDV causes mucosal disease in cattle
and is of significant economic importance to the livestock industry
(Meyers, G. and Thiel, H. J., Advances in Virus Research, 1996, 47,
53-118; Moennig V., et al, Adv. Vir. Res. 1992, 41, 53-98). Human
pestiviruses have not been as extensively characterized as the
animal pestiviruses. However, serological surveys indicate
considerable pestivirus exposure in humans.
[0008] Pestiviruses and hepaciviruses are closely related virus
groups within the Flaviviridae family. Other closely related
viruses in this family include the GB virus A, GB virus A-like
agents, GB virus-B and GB virus-C (also called hepatitis G virus,
HGV). The hepacivirus group (hepatitis C virus; HCV) consists of a
number of closely related but genotypically distinguishable viruses
that infect humans. There are at least 6 HCV genotypes and more
than 50 subtypes. Due to the similarities between pestiviruses and
hepaciviruses, combined with the poor ability of hepaciviruses to
grow efficiently in cell culture, bovine viral diarrhea virus
(BVDV) is often used as a surrogate to study the HCV virus.
[0009] The genetic organization of pestiviruses and hepaciviruses
is very similar. These positive stranded RNA viruses possess a
single large open reading frame (ORF) encoding all the viral
proteins necessary for virus replication. These proteins are
expressed as a polyprotein that is co- and post-translationally
processed by both cellular and virus-encoded proteinases to yield
the mature viral proteins. The viral proteins responsible for the
replication of the viral genome RNA are located within
approximately the carboxy-terminal. Two-thirds of the ORF are
termed nonstructural (NS) proteins. The genetic organization and
polyprotein processing of the nonstructural protein portion of the
ORF for pestiviruses and hepaciviruses is very similar. For both
the pestiviruses and hepaciviruses, the mature nonstructural (NS)
proteins, in sequential order from the amino-terminus of the
nonstructural protein coding region to the carboxy-terminus of the
ORF, consist of p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B.
[0010] The NS proteins of pestiviruses and hepaciviruses share
sequence domains that are characteristic of specific protein
functions. For example, the NS3 proteins of viruses in both groups
possess amino acid sequence motifs characteristic of serine
proteinases and of helicases (Gorbalenya et al. (1988) Nature
333:22; Bazan and Fletterick (1989) Virology 171:637-639;
Gorbalenya et al. (1989) Nucleic Acid Res. 17.3889-3897).
Similarly, the NS5B proteins of pestiviruses and hepaciviruses have
the motifs characteristic of RNA-directed RNA polymerases (Koonin,
E. V. and Dolja, V. V. (1993) Crir. Rev. Biochem. Molec. Biol.
28:375-430).
[0011] The actual roles and functions of the NS proteins of
pestiviruses and hepaciviruses in the lifecycle of the viruses are
directly analogous. In both cases, the NS3 serine proteinase is
responsible for all proteolytic processing of polyprotein
precursors downstream of its position in the ORF (Wiskerchen and
Collett (1991) Virology 184:341-350; Bartenschlager et al. (1993)
J. Virol. 67:3835-3844; Eckart et al. (1993) Biochem. Biophys. Res.
Comm. 192:399-406; Grakoui et al. (1993) J. Virol. 67:2832-2843;
Grakoui et al. (1993) Proc. Natl. Acad. Sci. USA 90:10583-10587;
Hijikata et al. (1993) J. Virol. 67:4665-4675; Tome et al. (1993)
J. Virol. 67:4017-4026). The NS4A protein, in both cases, acts as a
cofactor with the NS3 serine protease (Bartenschlager et al. (1994)
J. Virol. 68:5045-5055; Failla et al. (1994) J. Virol. 68:
3753-3760; Xu et al. (1997) J. Virol. 71:53 12-5322). The NS3
protein of both viruses also functions as a helicase (Kim et al.
(1995) Biochem. Biophys. Res. Comm. 215: 160-166; Jin and Peterson
(1995) Arch. Biochem. Biophys., 323:47-53; Warrener and Collett
(1995) J. Virol. 69:1720-1726). Finally, the NS5B proteins of
pestiviruses and hepaciviruses have the predicted RNA-directed RNA
polymerases activity (Behrens et al. (1996) EMBO. 15:12-22;
Lechmann et al. (1997) J. Virol. 71:8416-8428; Yuan et al. (1997)
Biochem. Biophys. Res. Comm. 232:231-235; Hagedorn, PCT WO
97/12033; Zhong et al. (1998) J. Virol. 72.9365-9369).
Treatment of HCV Infection with Interferon
[0012] Interferons (IFNs) have been commercially available for the
treatment of chronic hepatitis for nearly a decade. IFNs are
glycoproteins produced by immune cells in response to viral
infection. IFNs inhibit replication of a number of viruses,
including HCV, and when used as the sole treatment for hepatitis C
infection, IFN can in certain cases suppress serum HCV-RNA to
undetectable levels. Additionally, IFN can normalize serum amino
transferase levels. Unfortunately, the effect of IFN is temporary
and a sustained response occurs in only 8%-9% of patients
chronically infected with HCV (Gary L. Davis. Gastroenterology
18:S104-S114, 2000). Most patients, however, have difficulty
tolerating interferon treatment, which causes severe flu-like
symptoms, weight loss, and lack of energy and stamina.
[0013] A number of patents disclose Flaviviridae, including HCV,
and treatments using interferon-based therapies. For example, U.S.
Pat. No. 5,980,884 to Blatt et al. discloses methods for
retreatment of patients afflicted with HCV using consensus
interferon. U.S. Pat. No. 5,942,223 to Bazer et al. discloses an
anti-HCV therapy using ovine or bovine interferon-tau. U.S. Pat.
No. 5,928,636 to Alber et al. discloses the combination therapy of
interleukin-12 and interferon alpha for the treatment of infectious
diseases including HCV. U.S. Pat. No. 5,849,696 to Chretien et al.
discloses the use of thymosins, alone or in combination with
interferon, for treating HCV. U.S. Pat. No. 5,830,455 to Valtuena
et al. discloses a combination HCV therapy employing interferon and
a free radical scavenger. U.S. Pat. No. 5,738,845 to Imakawa
discloses the use of human interferon tau proteins for treating
HCV. Other interferon-based treatments for HCV are disclosed in
U.S. Pat. No. 5,676,942 to Testa et al., U.S. Pat. No. 5,372,808 to
Blatt et al., and U.S. Pat. No. 5,849,696. A number of patents also
disclose pegylated forms of interferon, such as U.S. Pat. Nos.
5,747,646, 5,792,834 and 5,834,594 to Hoffmann-La Roche; PCT
Publication No. WO 99/32139 and WO 99/32140 to Enzon; WO 95/13090
and U.S. Pat. Nos. 5,738,846 and 5,711,944 to Schering; and U.S.
Pat. No. 5,908,621 to Glue et al.
[0014] Interferon alpha-2a and interferon alpha-2b are currently
approved as monotherapy for the treatment of HCV. ROFERON.RTM.-A
(Roche) is the recombinant form of interferon alpha-2a.
PEGASYS.RTM. (Roche) is the pegylated (i.e. polyethylene glycol
modified) form of interferon alpha-2a. INTRON.RTM.A (Schering
Corporation) is the recombinant form of Interferon alpha-2b, and
PEG-INTRON.RTM. (Schering Corporation) is the pegylated form of
interferon alpha-2b.
[0015] Other forms of interferon alpha, as well as interferon beta,
gamma, tau and omega are currently in clinical development for the
treatment of HCV. For example, INFERGEN (interferon alphacon-1) by
InterMune, OMNIFERON (natural interferon) by Viragen, ALBUFERON by
Human Genome Sciences, REBIF (interferon beta-1a) by Ares-Serono,
Omega Interferon by BioMedicine, Oral Interferon Alpha by Amarillo
Biosciences, and interferon gamma, interferon tau, and interferon
gamma-lb by InterMune are in development.
Ribivarin
[0016] Ribavirin
(1-.beta.-D-ribofuranosyl-1-1,2,4-triazole-3-carboxamide) is a
synthetic, non-interferon-inducing, broad spectrum antiviral
nucleoside analog sold under the trade name, Virazole (The Merck
Index, 11th edition, Editor: Budavari, S., Merck & Co., Inc.,
Rahway, N.J., p 1304, 1989). U.S. Pat. No. 3,798,209 and RE29,835
disclose and claim ribavirin. Ribavirin is structurally similar to
guanosine, and has in vitro activity against several DNA and RNA
viruses including Flaviviridae (Gary L. Davis. Gastroenterology
118: 5104-51 14,2000).
[0017] Ribavirin reduces serum amino transferase levels to normal
in 40% of patients, but it does not lower serum levels of HCV-RNA
(Gary L. Davis, 2000). Thus, ribavirin alone is not effective in
reducing viral RNA levels. Additionally, ribavirin has significant
toxicity and is known to induce anemia. Ribavirin is not approved
for monotherapy against HCV. It has been approved in combination
with interferon alpha-2a or interferon alpha-2b for the treatment
of HCV.
[0018] Ribavirin is a known inosine monophosphate dehydrogenase
inhibitor that does not have specific anti-HCV activity in the HCV
replicon system (Stuyver et al. Journal of Virology, 2003, 77,
10689-10694).
Combination of Interferon and Ribavirin
[0019] The current standard of care for chronic hepatitis C is
combination therapy with an alpha interferon and ribavirin. The
combination of interferon and ribavirin for the treatment of HCV
infection has been reported to be effective in the treatment of
interferon naive patients (Battaglia, A. M. et al., Ann.
Pharmacother. 34:487-494, 2000), as well as for treatment of
patients when histological disease is present (Berenguer, M. et al.
Antivir. Ther. 3(Suppl. 3):125-136, 1998). Studies have shown that
more patients with hepatitis C respond to pegylated
interferon-alpha/ribavirin combination therapy than to combination
therapy with unpegylated interferon alpha. However, as with
monotherapy, significant side effects develop during combination
therapy, including hemolysis, flu-like symptoms, anemia, and
fatigue. (Gary L. Davis, 2000). Combination therapy with
PEG-INTRON.RTM. (peginterferon alpha-2b) and REBETOL.RTM.
(Ribavirin, USP) capsules are available from Schering Corporation.
REBETOL.RTM. (Schering Corporation) has also been approved in
combination with INTRON.RTM. A (Interferon alpha-2b, recombinant,
Schering Corporation). Roche's PEGASYS.RTM. (pegylated interferon
alpha-2a) and COPEGUS.RTM. (ribavirin), as well as Three River
Pharmacetical's Ribosphere.RTM. are also approved for the treatment
of HCV.
[0020] PCT Publication Nos. WO 99/59621, WO 00/37110, WO 01/81359,
WO 02/32414 and WO 03/02446 1 by Schering Corporation disclose the
use of pegylated interferon alpha and ribavirin combination therapy
for the treatment of HCV. PCT Publication Nos. WO 99/15 194, WO
99/64016, and WO 00/24355 by Hoffmann-La Roche Inc. also disclose
the use of pegylated interferon alpha and ribavirin combination
therapy for the treatment of HCV.
Additional Methods to Treat Flaviviridae Infections
[0021] The development of new antiviral agents for Flaviviridae
infections, especially hepatitis C, is currently underway. Specific
inhibitors of HCV-derived enzymes such as protease, helicase, and
polymerase inhibitors are being developed. Drugs that inhibit other
steps in HCV replication are also in development, for example,
drugs that block production of HCV antigens from the RNA (IRES
inhibitors), drugs that prevent the normal processing of HCV
proteins (inhibitors of glycosylation), drugs that block entry of
HCV into cells (by blocking its receptor) and nonspecific
cytoprotective agents that block cell injury caused by the virus
infection. Further, molecular approaches are also being developed
to treat hepatitis C, for example, ribozymes, which are enzymes
that break down specific viral RNA molecules, antisense
oligonucleotides, which are small complementary segments of DNA
that bind to viral RNA and inhibit viral replication, and RNA
interference techniques are under investigation (Bymock et al.
Antiviral Chemistry & Chemotherapy, 11:2; 79-95 (2000); De
Francesco et al. in Antiviral Research, 58: 1-16 (2003); and Kronke
et al., J. Virol., 78:3436-3446 (2004).
[0022] Bovine viral diarrhea virus (BVDV) is a pestivirus belonging
to the family Flaviviridae and has been used as a surrogate for in
vitro testing of potential antiviral agents. While activity against
BVDV may suggest activity against other flaviviruses, often a
compound can be inactive against BVDV and active against another
flavivirus. Sommadossi and La Colla have revealed ("Methods and
compositions for treating flaviviruses and pestiviruses", PCT WO
01/92282) that ribonucleosides containing a methyl group at the 2'
"up" position have activity against BVDV. However, it is unclear
whether these compounds can inhibit other flaviviruses, including
HCV in cell culture or at the HCV NS5B level. Interestingly while
this publication discloses a large number of compounds that are
2'-methyl-2'-X-ribonucleosides, where X is a halogen, fluorine is
not considered. Furthermore, a synthetic pathway leading to
nucleosides halogenated at the 2' "down" position is not shown by
these inventors.
[0023] Dengue virus (DENV) is the causative agent of Dengue
hemorrhagic fever (DHF). According to the world Health Organization
(WHO), two fifths of the world population are now at risk for
infection with this virus. An estimated 500,000 cases of DHF
require hospitalization each year with a mortality rate of 5% in
children.
[0024] West Nile virus (WNV), a flavivirus previously known to
exist only in intertropical regions, has emerged in recent years in
temperate areas of Europe and North America, presenting a threat to
public health. The most serious manifestation of WNV infection is
fatal encephalitis in humans. Outbreaks in New York City and
sporadic occurrences in the Southern United States have been
reported since 1999.
[0025] There is currently no preventive treatment of HCV, Dengue
virus (DENV) or West Nile virus infection. Currently approved
therapies, which exist only against HCV, are limited. Examples of
antiviral agents that have been identified as active against the
hepatitis C flavivirus include:
1) Protease inhibitors:
[0026] Substrate-based NS3 protease inhibitors (Attwood et al., PCT
WO 98/22496, 1998; Attwood et al., Antiviral Chemistry and
Chemotherapy 1999, 10, 259-273; Attwood et al., Preparation and use
of amino acid derivatives as anti-viral agents, German Patent Pub.
DE 19914474; Tung et al. Inhibitors of serine proteases,
particularly hepatitis C virus NS3 protease, PCT WO 98/17679),
including alphaketoamides and hydrazinoureas, and inhibitors that
terminate in an electrophile such as a boronic acid or phosphonate
(Llinas-Brunet et al., Hepatitis C inhibitor peptide analogues, PCT
WO 99/07734) are being investigated.
[0027] Non-substrate-based NS3 protease inhibitors such as
2,4,6-trihydroxy-3-nitro-benzamide derivatives (Sudo K. et al.,
Biochemical and Biophysical Research Communications, 1997, 238,
643-647; Sudo K. et al. Antiviral Chemistry and Chemotherapy, 1998,
9, 186), including RD3-4082 and RD3-4078, the former substituted on
the amide with a 14 carbon chain and the latter processing a
para-phenoxyphenyl group are also being investigated.
[0028] SCH 68631, a phenanthrenequinone, is an HCV protease
inhibitor (Chu M. et al., Tetrahedron Letters 3 7:7229-7232, 1996).
In another example by the same authors, SCH 351633, isolated from
the fungus Penicillium griseofulvum, was identified as a protease
inhibitor (Chu M. et al., Bioorganic and Medicinal Chemistry
Letters 9:1949-1952). Nanomolar potency against the HCV NS3
protease enzyme has been achieved by the design of selective
inhibitors based on the macromolecule eglin c. Eglin c, isolated
from leech, is a potent inhibitor of several serine proteases such
as S. griseus proteases A and B, .alpha.-chymotrypsin, chymase and
subtilisin (Qasim M. A. et al., Biochemistry 36:1598-1607,
1997).
[0029] Several U.S. patents disclose protease inhibitors for the
treatment of HCV. For example, U.S. Pat. No. 6,004,933 to Spruce et
al. discloses a class of cysteine protease inhibitors for
inhibiting HCV endopeptidase 2. U.S. Pat. No. 5,990,276 to Zhang et
al. discloses synthetic inhibitors of hepatitis C virus NS3
protease. The inhibitor is a subsequence of a substrate of the NS3
protease or a substrate of the NS4A cofactor. The use of
restriction enzymes to treat HCV is disclosed in U.S. Pat. No.
5,538,865 to Reyes et al. Peptides as NS3 serine protease
inhibitors of HCV are disclosed in WO 02/008251 to Corvas
International, Inc. and WO 02/08187 and WO 02/008256 to Schering
Corporation. HCV inhibitor tripeptides are disclosed in U.S. Pat.
Nos. 6,534,523, 6,410,531, and 6,420,380 to Boehringer Ingelheim
and WO 02/060926 to Bristol Myers Squibb. Diaryl peptides as NS3
serine protease inhibitors of HCV are disclosed in WO 02/48172 to
Schering Corporation. Imidazoleidinones as NS3 serine protease
inhibitors of HCV are disclosed in WO 02/08198 to Schering
Corporation and WO 02/48157 to Bristol Myers Squibb. WO 98/17679 to
Vertex Pharmaceuticals and WO 02/48116 to Bristol Myers Squibb also
disclose HCV protease inhibitors.
2) Thiazolidine derivatives which show relevant inhibition in a
reverse-phase HPLC assay with an NS3/4A fusion protein and NS5A/5B
substrate (Sudo K. et al., Antiviral Research, 1996, 32, 9-18),
especially compound RD-1-6250, possessing a fused cinnamoyl moiety
substituted with a long alkyl chain, RD4 6205 and RD4 6193; 3)
Thiazolidines and benzanilides identified in Kakiuchi N. et al. J.
EBS Letters 421, 217-220; Takeshita N. et al. Analytical
Biochemistry, 1997, 247,242-246; 4) 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); 5) Helicase inhibitors
(Diana G. D. et al., Compounds, compositions and methods for
treatment of hepatitis C, U.S. Pat. No. 5,633,358; Diana G. D. et
al., Piperidine derivatives, pharmaceutical compositions thereof
and their use in the treatment of hepatitis C, PCT WO 97/36554); 6)
Nucleotide polymerase inhibitors and gliotoxin (Ferrari R. et al.
Journal of Virology, 1999, 73, 1649-1654), and the natural product
cerulenin (Lohmann V. et al, Virology, 1998, 249, 108-118); 7)
Antisense phosphorothioate oligodeoxynucleotides (S-ODN)
complementary to sequence stretches in the 5' non-coding region
(NCR) of the virus (Alt M. et al., Hepatology, 1995, 22, 707-717),
or nucleotides 326-348 comprising the 3' end of the NCR and
nucleotides 371-388 located in the core coding region of the HCV
RNA (Alt M. et al., Archives of Virology, 1997, 142, 589-599;
Galderisi U. et al., Journal of Cellular Physiology, 1999, 181,
251-257); 8) Inhibitors of IRES-dependent translation (Ikeda N. et
al., Agent for the prevention and treatment of hepatitis C,
Japanese Patent Pub. JP-8268890; Kai Y. et al. Prevention and
treatment of viral diseases, Japanese Patent Pub. JP-101 01591); 9)
Ribozymes, such as nuclease-resistant ribozymes (Maccjak, D. J. et
al., Hepatology 1999, 30, abstract 995) and those disclosed in U.S.
Pat. No. 6,043,077 to Barber et al., and U.S. Pat. Nos. 5,869,253
and 5,610,054 to Draper et al.; 10) Nucleoside analogs have also
been developed for the treatment of Flaviviridae infections.
[0030] Idenix Pharmaceuticals discloses the use of certain branched
nucleosides in the treatment of flaviviruses (including HCV) and
pestiviruses in International Publication Nos. WO 01/90121 and WO
01/92282. Specifically, a method for the treatment of hepatitis C
virus infection (and flaviviruses and pestiviruses) in humans and
other host animals is disclosed in the Idenix publications that
includes administering an effective amount of a biologically active
1', 2', 3' or 4'-branched .beta.-D or .beta.-L nucleosides or a
pharmaceutically acceptable salt or derivative thereof,
administered either alone or in combination with another antiviral
agent, optionally in a pharmaceutically acceptable carrier.
[0031] WO 2004/002422 to Idenix published Jan. 8, 2004 discloses a
family of 2'-methyl nucleosides for the treatment of flavivirus
infections. WO 2004/002999 to Idenix, published Jan. 8, 2004
discloses a series of 2' or 3' prodrugs of 1', 2', 3', or 4' branch
nucleosides for the treatment of flavivirus infections including
HCV infections.
[0032] Other patent applications disclosing the use of certain
nucleoside analogs to treat hepatitis C virus infection include:
PCT/CAOO/01316 (WO 01/32153; filed Nov. 3, 2000) and PCT/CAOI/00197
(WO 01/60315; filed Feb. 19, 2001) filed by BioChem Pharma, Inc.
(now Shire Biochem, Inc.); PCT/US02/01531 (WO 02/057425; filed Jan.
18, 2002) and PCT/U502/03086 (WO 02/057287; filed Jan. 18, 2002)
filed by Merck & Co., Inc., PCT/EPOT/09633 (WO 02/18404;
published Aug. 21, 2001) filed by Roche, and PCT Publication Nos.
WO 01/79246 (filed Apr. 13, 2001), WO 02/32920 (filed Oct. 18,
2001) and WO 02/48 165 by Pharmasset, Ltd.
[0033] WO 2004/007512 to Merck & Co. discloses a number of
nucleoside compounds disclosed as inhibitors of RNA-dependent RNA
viral polymerase. The nucleosides disclosed in this publication are
primarily 2'-methyl-2'-hydroxy substituted nucleosides. WO
02/057287 to Merck et al. published Jul. 25, 2002, discloses a
large genus of pyrimidine derivative nucleosides of the
2'-methyl-2'-hydroxy substitutions. WO 2004/009020 to Merck et al.
discloses a series of thionucleoside derivatives as inhibitors of
RNA dependent RNA viral polymerase. WO 03/105770 to Merck et al.
discloses a series of carbocyclic nucleoside derivatives that are
useful for the treatment of HCV infections.
[0034] PCT Publication No. WO 99/43691 to Emory University,
entitled "2'-Fluoronucleosides" discloses the use of certain
2'-fluoronucleosides to treat HCV. U.S. Pat. No. 6,348,587 to Emory
University entitled "2'-fluoronucleosides" discloses a family of
2'-fluoronucleosides useful for the treatment of hepatitis B, HCV,
HIV and abnormal cellular proliferation. The 2' substitutent is
disclosed to be in either the "up" or "down" position.
[0035] Eldrup et al. (Oral Session V, Hepatitis C Virus,
Flaviviridae; 16.sup.th International Conference on Antiviral
Research (Apr. 27, 2003, Savannah, Ga.)) described the structure
activity relationship of 2'-modified nucleosides for inhibition of
HCV.
[0036] Bhat et al. (Oral Session V, Hepatitis C Virus,
Flaviviridae; 16.sup.th International Conference on Antiviral
Research (Apr. 27, 2003, Savannah, Ga.); p A75) describe the
synthesis and pharmacokinetic properties of nucleoside analogues as
possible inhibitors of HCV RNA replication. The authors report that
2'-modified nucleosides demonstrate potent inhibitory activity in
cell-based replicon assays.
[0037] Olsen et al. (Oral Session V, Hepatitis C Virus,
Flaviviridae; 16.sup.th International Conference on Antiviral
Research (Apr. 27, 2003, Savannah, Ga.) p A76) also described the
effects of the 2'-modified nucleosides on HCV RNA replication.
11) Other miscellaneous compounds including
1-amino-alkylcyclohexanes (U.S. Pat. No. 6,034,134 to Gold et al.),
alkyl lipids (U.S. Pat. No. 5,922,757 to Chojkier et al.), vitamin
E and other antioxidants (U.S. Pat. No. 5,922,757 to Chojkier et
al.), squalene, amantadine, bile acids (U.S. Pat. No. 5,846,964 to
Ozeki et al.), N-(phosphonoacetyl)-L-aspartic acid, (U.S. Pat. No.
5,830,905 to Diana et al.), benzenedicarboxamides (U.S. Pat. No.
5,633,388 to Diana et al.), polyadenylic acid derivatives (U.S.
Pat. No. 5,496,546 to Wang et al.), 2,3-dideoxyinosine (U.S. Pat.
No. 5,026,687 to Yarchoan et al.), benzimidazoles (U.S. Pat. No.
5,891,874 to Colacino et al.), plant extracts (U.S. Pat. No.
5,837,257 to Tsai et al., U.S. Pat. No. 5,725,859 to Omer et al.,
and U.S. Pat. No. 6,056,961), and piperidenes (U.S. Pat. No.
5,830,905 to Diana et al.). 12) Other compounds currently in
preclinical or clinical development for treatment of hepatitis C
virus infection include: Interleukin-10 by Schering-Plough, IP-SOI
by Intemeuron, Merimebodib (VX-497) by Vertex, AMANTADINE.RTM.
(Symmetrel) by Endo Labs Solvay, HEPTAZYME.RTM. by RPI, IDN-6556 by
Idun Pharma., XTL-002 by XTL., HCV/MFS9 by Chiron, CIVACIR.RTM.
(hepatitis C Immune Globulin) by NABI, LEVOVIRIN.RTM. by
ICN/Ribaphamm, VIRAMIDINE.RTM. by ICN/Ribapharm, ZADAXIN.RTM.
(thymosin alpha-1) by SciClone, thymosin plus pegylated interferon
by Sci Clone, CEPLENE.RTM. (histamine dihydrochloride) by Maxim, VX
950/LY 570310 by Vertex/Eli Lilly, ISIS 14803 by Isis
Pharmaceutical/Elan, IDN-6556 by Idun Pharmaceuticals, Inc., JTK
003 by AKROS Pharma, BILN-2061 by Boehringer Ingelheim, CellCept
(mycophenolate mofetil) by Roche, T67, a .beta.-tubulin inhibitor,
by Tularik, a therapeutic vaccine directed to E2 by Innogenetics,
FK788 by Fujisawa Healthcare, Inc., 1 dB 1016 (Siliphos, oral
silybin-phosphatdylcholine phytosome), RNA replication inhibitors
(VP50406) by ViroPharma/Wyeth, therapeutic vaccine by Intercell,
therapeutic vaccine by Epimmune/Genencor, IRES inhibitor by Anadys,
ANA 245 and ANA 246 by Anadys, immunotherapy (Therapore) by Avant,
protease inhibitor by Corvas/SChering, helicase inhibitor by
Vertex, fusion inhibitor by Trimeris, T cell therapy by CellExSys,
polymerase inhibitor by Biocryst, targeted RNA chemistry by PTC
Therapeutics, Dication by Immtech, Int., protease inhibitor by
Agouron, protease inhibitor by Chiron/Medivir, antisense therapy by
AVI BioPharma, antisense therapy by Hybridon, hemopurifier by
Aethlon Medical, therapeutic vaccine by Merix, protease inhibitor
by Bristol-Myers Squibb/Axys, Chron-VacC, a therapeutic vaccine, by
Tripep, UT 231 B by United Therapeutics, protease, helicase and
polymerase inhibitors by Genelabs Technologies, IRES inhibitors by
Immusol, R803 by Rigel Pharmaceuticals, INFERGEN.RTM. (interferon
alphacon-1) by InterMune, OMNIERON.RTM. (natural interferon) by
Viragen, ALBUFERON.RTM. by Human Genome Sciences, REBIF.RTM.
(interferon beta-1a) by Ares-Serono, Omega Interferon by
BioMedicine, Oral Interferon Alpha by Amarillo Biosciences,
interferon gamma, interferon tau, and Interferon gamma-1b by
InterMune. Rigel Pharmaceuticals is developing a non-nucleoside HCV
polymerase inhibitor, R803, that shows promise as being synergistic
with IFN and ribavirin. 13) A summary of several investigational
drugs, including several discussed above, that are currently in
various phases of development for the treatment of HCV, are
summarized below:
TABLE-US-00001 Drug Mechanism/Target Company U.S. Status BILN-2061
NS3 Serine-protease Boehringer Phase II inhibitor Ingelheim ISIS
14803 Antisense/Prevent ISIS/Elan Phase II Translation of RNA
Viramidine Prodrug of Ribavirin Ribapharm Phase II NM 283 Inhibitor
of HCV RNA Idenix Phase II/III Polymerase VX-497 IMPDH Inhibitor
Vertex Phase I/II JKT-003 Inhibitor of HCV RNA Japan Tobacco/ Phase
I/II Polymerase Akros Levovirin L-Ribavirin analog Ribapharm/Roche
Phase I/II Isatoribine; Nucleoside analog Anadys Phase I ANA245
Interact with TLR7 receptor Albuferon Immune modulator Human Genome
Phase I Sciences Peg-Infergen Immune modulator Intermune Phase I
VX-950 Inhibitor of HCV Vertex Preclinical NS3-4A protease SCH 6
Inhibitor of HCV Schering Plough Preclinical NS3-4A protease R803
Inhibitor of HCV RNA Rigel Phase I polymerase HCV-086 --
ViroPharma/Wyeth Phase I R1479 Inhibitor of HCV RNA Roche Phase I
polymerase
[0038] Nucleoside prodrugs have been previously described for the
treatment of other forms of hepatitis. WO 00/09531 and WO 01/96353
to Idenix Pharmaceuticals, discloses 2'-deoxy-.beta.-L-nucleosides
and their 3'-prodrugs for the treatment of HBV. U.S. Pat. No.
4,957,924 to Beauchamp discloses various therapeutic esters of
acyclovir.
[0039] 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.
[0040] 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.
SUMMARY OF THE INVENTION
[0041] There is currently no preventive treatment of Hepatitis C
virus (HCV), Dengue virus (DENV) or West Nile virus (WNV)
infection, and currently approved therapies, which exist only
against HCV, are limited. Design and development of pharmaceutical
compounds is essential, especially those that are synergistic with
other approved and investigational Flaviviridae, and in particular
HCV, therapeutics for the evolution of treatment standards,
including more effective combination therapies.
[0042] The present invention provides a
(2'R)-2'-deoxy-2'-fluoro-2'-C-methyl nucleoside (.beta.-D or
.beta.-L), or its pharmaceutically acceptable salt or prodrug
thereof, and the use of such compounds for the treatment of a host
infected with a virus belonging to the Flaviviridae family,
including hepatitis C, West Nile Virus and yellow fever virus. In
addition, the nucleosides of the present invention show actively
against rhinovirus. Rhinoviruses (RVs) are small (30 nm),
nonenveloped viruses that contain a single-strand ribonucleic acid
(RNA) genome within an icosahedral (20-sided) capsid. RVs belong to
the Picornaviridae family, which includes the genera Enterovirus
(polioviruses, coxsackieviruses groups A and B, echoviruses,
numbered enteroviruses) and Hepatovirus (hepatitis A virus).
Approximately 101 serotypes are identified currently. Rhinoviruses
are most frequently associated with the common cold,
nasopharyngitis, croup, pneumonia, otitis media and asthma
exacerbations.
[0043] The inventor has made the unexpected discovery that the 2'
substitutions on the .beta.-D or .beta.-L nucleosides of the
present invention impart greater specificity for hepatitis C virus
as well as exhibiting lower toxicity following administration to a
host. The invention also includes a method for treating a
Flaviviridae infection, including hepatitis C virus, West Nile
Virus and yellow fever virus and rhinovirus infection, that
includes the administration of an anti-virally effective amount of
a .beta.-D or .beta.-L nucleoside disclosed herein, or its
pharmaceutically acceptable salt or prodrug, optionally in a
pharmaceutically acceptable carrier or diluent, optionally in
combination or alternation with another effective antiviral
agent.
[0044] The nucleosides of the present invention, possess the unique
properties of having greater specificity for the hepatitis C virus
and lower toxicity in culture or when administered into an animal.
One potential, but non-limiting reason for this is the presence of
the 2'-fluoro substitution on the ribose ring. For example, U.S.
Pat. No. 6,348,587 to Schinazi et al., discloses a family of
2'-fluoro nucleoside compounds that are useful in the treatment of
hepatitis C virus infection. In contrast, are 2'-methyl
substitutions such as found in 2'-C-methylcytidine as shown in WO
2004/02999 to Idenix wherein the 2'-methyl substitution on the
nucleoside ring at the 2' position is not specific to hepatitis
C.
[0045] Thus, in one aspect, the antivirally effective nucleoside is
a (2'R)-2'-deoxy-2'-fluoro-2'-C-methyl nucleoside (.beta.-D or
.beta.-L) or its pharmaceutically acceptable salt or prodrug
thereof of the general formula:
##STR00001## [0046] wherein [0047] (a) Base is a naturally
occurring or modified purine or pyrimidine base; [0048] (b) X is O,
S, CH.sub.2, Se, NH, N-alkyl, CHW (R, S, or racemic), C(W).sub.2,
wherein W is F, Cl, Br, or I; [0049] (c) R.sup.1 and R.sup.7 are
independently H, phosphate, including 5'-monophosphate,
diphosphate, triphosphate, or a stabilized phosphate prodrug,
H-phosphonate, including stabilized H-phosphonates, acyl, including
optionally substituted phenyl and lower acyl, alkyl, including
lower alkyl, O-substituted carboxyalkylamino or its peptide
derivatives, sulfonate ester, including alkyl or arylalkyl
sulfonyl, including methanesulfonyl and benzyl, wherein the phenyl
group is optionally substituted, a lipid, including a phospholipid,
an L or D-amino acid, a carbohydrate, a peptide, a cholesterol, or
other pharmaceutically acceptable leaving group which when
administered in vivo is capable of providing a compound wherein
R.sup.1 is H or phosphate; R.sup.2 is OH or phosphate; R.sup.1 and
R.sup.2 or R.sup.7 can also be linked with cyclic phosphate group;
and [0050] (d) R.sup.2 and R.sup.2' are independently H, C.sub.1-4
alkyl, C.sub.1-4 alkenyl, C.sub.1-4 alkynyl, vinyl, N.sub.3, CN,
Cl, Br, F, I, NO.sub.2, C(O)O(C.sub.1-4 alkyl), C(O)O(C.sub.1-4
alkyl), C(O)O(C.sub.1-4 alkynyl), C(O)O(C.sub.1-4 alkenyl),
O(C.sub.1-4 acyl), O(C.sub.1-4 alkyl), O(C.sub.1-4 alkenyl),
S(C.sub.1-4 acyl), S(C.sub.1-4 alkyl), S(C.sub.1-4 alkynyl),
S(C.sub.1-4 alkenyl), SO(C.sub.1-4 acyl), SO(C.sub.1-4 alkyl),
SO(C.sub.1-4 alkynyl), SO(C.sub.1-4 alkenyl), SO.sub.2(C.sub.1-4
acyl), SO.sub.2(C.sub.1-4 alkyl), SO.sub.2(C.sub.1-4 alkynyl),
SO.sub.2(C.sub.1-4 alkenyl), O.sub.3S(C.sub.1-4 acyl),
O.sub.3S(C.sub.1-4 alkyl), O.sub.3S(C.sub.1-4 alkenyl), NH.sub.2,
NH(C.sub.1-4 alkyl), NH(C.sub.1-4 alkenyl), NH(C.sub.1-4 alkynyl),
NH(C.sub.1-4 acyl), N(C.sub.1-4 alkyl).sub.2, N(C.sub.1-18
acyl).sub.2, wherein alkyl, alkynyl, alkenyl and vinyl are
optionally substituted by N.sub.3, CN, one to three halogen (Cl,
Br, F, I), NO.sub.2, C(O)O(C.sub.1-4 alkyl), C(O)O(C.sub.1-4
alkyl), C(O)O(C.sub.1-4 alkynyl), C(O)O(C.sub.1-4 alkenyl),
O(C.sub.1-4 acyl), O(C.sub.1-4 alkyl), O(C.sub.1-4 alkenyl),
S(C.sub.1-4 acyl), S(C.sub.1-4 alkyl), S(C.sub.1-4 alkynyl),
S(C.sub.1-4 alkenyl), SO(C.sub.1-4 acyl), SO(C.sub.1-4 alkyl),
SO(C.sub.1-4 alkynyl), SO(C.sub.1-4 alkenyl), SO.sub.2(C.sub.1-4
acyl), SO.sub.2(C.sub.1-4 alkyl), SO.sub.2(C.sub.1-4 alkynyl),
SO.sub.2(C.sub.1-4 alkenyl), O.sub.3S(C.sub.1-4 acyl),
O.sub.3S(C.sub.1-4 alkyl), O.sub.3S(C.sub.1-4 alkenyl), NH.sub.2,
NH(C.sub.1-4 alkyl), NH(C.sub.1-4 alkenyl), NH(C.sub.1-4 alkynyl),
NH(C.sub.1-4 acyl), N(C.sub.1-4 alkyl).sub.2, N(C.sub.1-4
acyl).sub.2, R.sup.2 and R.sup.2' can be together to form a vinyl
optionally substituted by one or two of N.sub.3, CN, Cl, Br, F, I,
NO.sub.2; OR.sup.7 and [0051] (e) R.sup.6 is an optionally
substituted alkyl (including lower alkyl), cyano (CN), CH.sub.3,
OCH.sub.3, OCH.sub.2CH.sub.3, hydroxy methyl (CH.sub.2OH),
fluoromethyl (CH.sub.2F), azido (N.sub.3), CHCN, CH.sub.2N.sub.3,
CH.sub.2NH.sub.2, CH.sub.2NHCH.sub.3, CH.sub.2N(CH.sub.3).sub.2,
alkyne (optionally substituted), or fluoro.
[0052] In various aspects of the invention, the Base can be
selected from
##STR00002##
[0053] wherein [0054] (a) Y is N or CH. [0055] (b) R.sup.3, R.sup.4
and R.sup.5 are independently H, halogen (including F, Cl, Br, I),
OH, OR', SH, SR', NH.sub.2, NHR', NR'.sub.2, lower alkyl of
C.sub.1-C.sub.6, halogenated (F, Cl, Br, I) lower alkyl of
C.sub.1-C.sub.6 such as CF.sub.3 and CH.sub.2CH.sub.2F, lower
alkenyl of C.sub.2-C.sub.6 such as CH.dbd.CH.sub.2, halogenated (F,
Cl, Br, I) lower alkenyl of C.sub.2-C.sub.6 such as CH.dbd.CHCl,
CH.dbd.CHBr and CH.dbd.CHI, lower alkynyl of C.sub.2-C.sub.6 such
as C.ident.CH, halogenated (F, Cl, Br, I) lower alkynyl of
C.sub.2-C.sub.6, lower alkoxy of C.sub.1-C.sub.6 such as CH.sub.2OH
and CH.sub.2CH.sub.2OH, halogenated (F, Cl, Br, I) lower alkoxy of
C.sub.1-C.sub.6, CO.sub.2H, CO.sub.2R', CONH.sub.2, CONHR',
CONR'.sub.2, CH.dbd.CHCO.sub.2H, CH.dbd.CHCO.sub.2R'; [0056]
wherein R' is an optionally substituted alkyl of C.sub.1-C.sub.12
(particularly when the alkyl is an amino acid residue), cycloalkyl,
optionally substituted alkynyl of C.sub.2-C.sub.6, optionally
substituted lower alkenyl of C.sub.2-C.sub.6, or optionally
substituted acyl.
[0057] In still another aspect, the
(2'R)-2'-deoxy-2'-fluoro-2'-C-methyl nucleoside or its
pharmaceutically acceptable salt or prodrug thereof can be of the
formula:
##STR00003## [0058] wherein [0059] (a) Base, Y, R.sup.1, R.sup.2,
R.sup.2', R.sup.3, R.sup.4, R.sup.5, R.sup.6, R.sup.7 and R' are as
described above.
[0060] Various aspects of the present invention also include
pharmaceutical compositions comprising any of the
(2'R)-2'-deoxy-2'-fluoro-2'-C-methyl nucleoside (.beta.-D or
.beta.-L) described herein or their pharmaceutically acceptable
salts or prodrugs thereof and a pharmaceutically acceptable
carrier.
[0061] The present invention also provides in various aspects,
methods for the treatment or prophylaxis of hepatitis C virus
infection, West Nile virus infection, a yellow fever viral
infection or a rhinovirus infection comprising administering to a
host an antivirally effective amount of a
(2'R)-2'-deoxy-2'-fluoro-2'-C-methyl nucleoside disclosed herein.
The invention also includes methods for treating or preventing
Flaviviridae infection, including all members of the Hepacivirus
genus (HCV), Pestivirus genus (BVDV, CSFV, BDV), or Flavivirus
genus (Dengue virus, Japanese encephalitis virus group (including
West Nile Virus), and Yellow Fever virus).
[0062] In various aspects, the (2'R)-2'-deoxy-2'-fluoro-2'-C-methyl
.beta.-D-nucleoside has an EC.sub.50 (effective concentration to
achieve 50% inhibition) when tested in an appropriate cell-based
assay, of less than 15 micromolar, and more particularly, less than
10 or 5 micromolar. In other aspects, the nucleoside is
enantiomerically enriched.
[0063] The present invention also provides methods for the
treatment or prophylaxis of a hepatitis C virus infection, West
Nile virus infection, a yellow fever viral infection or a
rhinovirus infection in a host comprising administering an
effective amount of a (2'R)-2'-deoxy-2'-fluoro-2'-C-methyl
nucleosides (.beta.-D or .beta.-L) disclosed herein, or its
pharmaceutically acceptable salt or prodrug thereof, in combination
or alternation with one or more other effective antiviral agent(s),
optionally in a pharmaceutically acceptable carrier or diluent
thereof, as described herein. Nonlimiting examples of the types of
antiviral agents or their prodrugs that can be used in combination
with the compounds disclosed herein include, but are not limited
to: interferon, including interferon alpha 2a, interferon alpha 2b,
a pegylated interferon, interferon beta, interferon gamma,
interferon tau and interferon omega; an interleukin, including
interleukin 10 and interleukin 12; ribavirin; interferon in
combination with ribavirin; a protease inhibitor including NS3
inhibitor; a helicase inhibitor; a polymerase inhibitor; gliotoxin;
an IRES inhibitor; and antisense oligonucleotide; a thiazolidine
derivative; a benzanilide, a ribozyme; another nucleoside,
nucleoside prodrug or nucleoside derivative; a
1-amino-alkylcyclohexane; an antioxidant including vitamin E;
squalene; amantadine; a bile acid; N-(phosphonoacetyl)-L-aspartic
acid; a benzenedicarboxamide; polyadneylic acid; a benzimidazoles;
thymosin; a beta tubulin inhibitor; a prophylactic vaccine;
silybin-phosphatidlycholine phytosome; and mycophenolate.
[0064] The following non-limiting aspects illustrate some general
methodology to obtain the nucleosides of the present invention.
Specifically, the synthesis of the present nucleosides can be
achieved by either of two general means:
[0065] 1) alkylating the appropriately modified carbohydrate
building block, subsequent fluorination, followed by coupling to
form the nucleosides of the present invention (Scheme 1) or
[0066] 2) glycosylation to form the nucleoside followed by
alkylation and fluorination of the pre-formed nucleosides of the
present invention (Scheme 2).
[0067] In addition, the L-enantiomers corresponding to the
compounds of the invention can be prepared following the same
general methods (Schemes 1 or 2), beginning with the corresponding
L-carbohydrate building block or nucleoside L-enantiomer as the
starting material.
[0068] Thus, the present invention includes at least the following
general features: [0069] (a) .beta.-D and .beta.-L nucleosides of
the general formulas disclosed, or their pharmaceutically
acceptable salts or prodrugs thereof, as described herein; [0070]
(b) processes for the preparation of the .beta.-D and .beta.-L
nucleosides of the general formula disclosed, or their
pharmaceutically acceptable salts or prodrugs thereof, as described
herein; [0071] (c) pharmaceutical compositions comprising a
.beta.-D or .beta.-L nucleoside of the general formulas disclosed,
or its pharmaceutically acceptable salt or prodrug thereof, in a
pharmaceutically acceptable carrier or diluent thereof, as
described herein, for the treatment or prophylaxis of a viral
infection in a host; [0072] (d) pharmaceutical compositions
comprising a .beta.-D or .beta.-L nucleoside of the general
formulas disclosed, or its pharmaceutically acceptable salt or
prodrug thereof, in combination with one or more other effective
antiviral agent(s), optionally in a pharmaceutically acceptable
carrier or diluent thereof, as described herein, for the treatment
or prophylaxis of a viral infection in a host; [0073] (e) methods
for the treatment or prophylaxis of a Flaviviridae infection,
including hepatitis C virus, West Nile Virus and yellow fever virus
and rhinovirus infection in a host comprising administering an
effective amount of a .beta.-D or .beta.-L nucleoside of the
general formulas disclosed, or its pharmaceutically acceptable salt
or prodrug thereof, optionally in a pharmaceutically acceptable
carrier or diluent thereof, as described herein; [0074] (f) methods
for the treatment or prophylaxis of a Flaviviridae infection,
including hepatitis C virus, West Nile Virus and yellow fever virus
and rhinovirus infection in a host comprising administering an
effective amount of a .beta.-D or .beta.-L nucleoside of the
general formulas disclosed, or its pharmaceutically acceptable salt
or prodrug thereof, in combination or alternation with one or more
other effective antiviral agent(s), optionally in a
pharmaceutically acceptable carrier or diluent thereof, as
described herein; [0075] (g) use of a .beta.-D or .beta.-L
nucleoside of the general formulas disclosed, or its
pharmaceutically acceptable salt or prodrug thereof, optionally in
a pharmaceutically acceptable carrier, as described herein, for the
treatment or prophylaxis of a Flaviviridae infection, including
hepatitis C virus, West Nile Virus and yellow fever virus and
rhinovirus infection in a host; [0076] (h) use of a .beta.-D or
.beta.-L nucleoside of the general formulas disclosed, or its
pharmaceutically acceptable salt or prodrug thereof, in combination
or alternation with one or more other effective antiviral agent(s),
optionally in a pharmaceutically acceptable carrier, as described
herein, for the treatment or prophylaxis of a Flaviviridae
infection, including hepatitis C virus, West Nile Virus and yellow
fever virus and rhinovirus infection in a host; [0077] (i) use of a
.beta.-D or .beta.-L nucleoside of the general formulas disclosed,
or its pharmaceutically acceptable salt or prodrug thereof,
optionally in a pharmaceutically acceptable carrier, as described
herein, in the manufacture of a medicament for the treatment or
prophylaxis of a Flaviviridae infection, including hepatitis C
virus, West Nile Virus and yellow fever virus and rhinovirus
infection in a host; [0078] (j) use of a .beta.-D or .beta.-L
nucleoside of the general formulas disclosed, or its
pharmaceutically acceptable salt or prodrug thereof, in combination
or alternation with one or more other effective antiviral agent(s),
optionally in a pharmaceutically acceptable carrier, as described
herein, in the manufacture of a medicament for the treatment or
prophylaxis of a Flaviviridae infection, including hepatitis C
virus, West Nile Virus and yellow fever virus and rhinovirus
infection in a host; [0079] (k) use of a .beta.-D or .beta.-L
nucleoside of the general formulas disclosed, or its
pharmaceutically acceptable salt or prodrug thereof, optionally in
a pharmaceutically acceptable carrier or diluent, as described
herein, in a medical therapy, i.e. as antiviral for example for the
treatment or prophylaxis of a Flaviviridae infection, including
hepatitis C virus, West Nile Virus and yellow fever virus and
rhinovirus infection; [0080] (l) use of a .beta.-D or .beta.-L
nucleoside of the general formulas disclosed, as described herein,
or its pharmaceutically acceptable salt or prodrug thereof, i.e. as
antiviral agent, in combination or alternation with one or more
other effective therapeutic agent(s), i.e. another antiviral agent,
optionally in a pharmaceutically acceptable carrier or diluent, as
described herein, in a medical therapy, for example for the
treatment or prophylaxis of a Flaviviridae infection, including
hepatitis C virus, West Nile Virus and yellow fever virus and
rhinovirus infection in a host.
BRIEF DESCRIPTION OF THE FIGURES
[0081] FIG. 1 is a graphical depiction of the dose-dependant
reduction of the replicon HCV RNA based on the treatment with
.beta.-D-(2'R)-2'-deoxy-2'-fluoro-2'-C-methylcytidine. (A): The
viral reduction was compared to the reduction of cellular RNA
levels (ribosomal RNA) to obtain therapeutic index values.
EC.sub.90 which represents the effective concentration 90% at 96
hours following the dose dependant administration of
(2'R)-2'-deoxy-2'-fluoro-2'-C-methylcytidine was determined to be 5
.mu.M. (B): HCV RNA was significantly reduced in a dose-dependent
manner for 7 days following treatment with 25 .mu.M.
[0082] FIG. 2 depicts the average weight change (%) of female Swiss
mice in the toxicity study of
.beta.-D-(2'R)-2'-deoxy-2'-fluoro-2'-C-methylcytidine at various
doses. Intraperitneal injections were given on days 0 to day 5 of
the 0, 3.3, 10, 33, 100 mg/kg. Each dosing group contained 5 mice
and no mice died during the 30-day study.
[0083] FIG. 3 depicts the pharmacokinetics of
.beta.-D-(2'R)-2'-deoxy-2'-fluoro-2'-C-methylcytidine in Rhesus
monkeys given a single dose (33.3 mg/kg) oral or intravenous dose
of .beta.-D-(2'R)-2'-deoxy-2'-fluoro-2'-C-methylcytidine.
DETAILED DESCRIPTION OF THE INVENTION
[0084] Various embodiments of the invention are now described in
detail. As used in the description herein and throughout the claims
that follow, the meaning of "a," "an," and "the" includes plural
reference unless the context clearly dictates otherwise. Also, as
used in the description herein and throughout the claims that
follow, the meaning of "in" includes "in" and "on" unless the
context clearly dictates otherwise.
[0085] The terms used in this specification generally have their
ordinary meanings in the art, within the context of the invention,
and in the specific context where each term is used. Certain terms
that are used to describe the invention are discussed below, or
elsewhere in the specification, to provide additional guidance to
the practitioner in describing the compositions and methods of the
invention and how to make and use them. For convenience, certain
terms may be highlighted, for example using italics and/or
quotation marks. The use of highlighting has no influence on the
scope and meaning of a term; the scope and meaning of a term is the
same, in the same context, whether or not it is highlighted. It
will be appreciated that the same thing can be said in more than
one way. Consequently, alternative language and synonyms may be
used for any one or more of the terms discussed herein, nor is any
special significance to be placed upon whether or not a term is
elaborated or discussed herein. Synonyms for certain terms are
provided. A recital of one or more synonyms does not exclude the
use of other synonyms. The use of examples anywhere in this
specification, including examples of any terms discussed herein, is
illustrative only, and in no way limits the scope and meaning of
the invention or of any exemplified term. Likewise, the invention
is not limited to various embodiments given in this
specification.
[0086] As used herein, "about" or "approximately" shall generally
mean within 20 percent, preferably within 10 percent, and more
preferably within 5 percent of a given value or range. Numerical
quantities given herein are approximate, meaning that the term
"about" or "approximately" can be inferred if not expressly
stated.
[0087] The present invention provides
(2'R)-2'-deoxy-2'-fluoro-2'-C-methyl nucleosides and their
pharmaceutically acceptable salts and prodrugs for the treatment of
hepatitis C virus infection, West Nile virus infection, a yellow
fever viral infection or a rhinovirus infection in a host.
[0088] The disclosed compounds or their pharmaceutically acceptable
derivatives or salts or pharmaceutically acceptable formulations
containing these compounds are useful in the prevention and
treatment of HCV infections. In addition, these compounds or
formulations can be used prophylactically to prevent or retard the
progression of clinical illness in individuals who are anti-HCV
antigen positive or who have been exposed to HCV.
[0089] The compounds disclosed herein can be converted into a
pharmaceutically acceptable ester by reaction with an appropriate
esterifying agent, for example, an acid halide or anhydride. The
compound or its pharmaceutically acceptable derivative can be
converted into a pharmaceutically acceptable salt thereof in a
conventional manner, for example, by treatment with an appropriate
base. The ester or salt of the compound can be converted into the
parent compound, for example, by hydrolysis.
DEFINITIONS
[0090] The term "independently" is used herein to indicate that the
variable, which is independently applied, varies independently from
application to application. Thus, in a compound such as
R.sup.aXYR.sup.a, wherein R.sup.a is "independently carbon or
nitrogen", both R.sup.a can be carbon, both R.sup.a can be
nitrogen, or one R.sup.a can be carbon and the other R.sup.a
nitrogen.
[0091] As used herein, the terms "enantiomerically pure" or
"enantiomerically enriched" refers to a nucleoside composition that
comprises at least approximately 95%, and preferably approximately
97%, 98%, 99% or 100% of a single enantiomer of that
nucleoside.
[0092] As used herein, the term "substantially free of" or
"substantially in the absence of" refers to a nucleoside
composition that includes at least 85 or 90% by weight, preferably
95% to 98% by weight, and even more preferably 99% to 100% by
weight, of the designated enantiomer of that nucleoside. In a
preferred embodiment, in the methods and compounds of this
invention, the compounds are substantially free of enantiomers.
[0093] Similarly, the term "isolated" refers to a nucleoside
composition that includes at least 85 or 90% by weight, preferably
95% to 98% by weight, and even more preferably 99% to 100% by
weight, of the nucleoside, the remainder comprising other chemical
species or enantiomers.
[0094] The term "alkyl," as used herein, unless otherwise
specified, refers to a saturated straight, branched, or cyclic,
primary, secondary, or tertiary hydrocarbon of typically C.sub.1 to
C.sub.10, and specifically includes methyl, trifluoromethyl, ethyl,
propyl, isopropyl, cyclopropyl, butyl, isobutyl, t-butyl, pentyl,
cyclopentyl, isopentyl, neopentyl, hexyl, isohexyl, cyclohexyl,
cyclohexylmethyl, 3-methylpentyl, 2,2-dimethylbutyl, and
2,3-dimethylbutyl. The term includes both substituted and
unsubstituted alkyl groups. Alkyl groups can be optionally
substituted with one or more moieties selected from the group
consisting of hydroxyl, amino, alkylamino, arylamino, alkoxy,
aryloxy, nitro, cyano, sulfonic acid, sulfate, phosphonic acid,
phosphate, or phosphonate, or any other viable functional group
that does not inhibit the pharmacological activity of this
compound, either unprotected, or protected, as necessary, as known
to those skilled in the art, for example, as taught in T. W. Greene
and P. G. M. Wuts, "Protective Groups in Organic Synthesis," 3rd
ed., John Wiley & Sons, 1999, hereby incorporated by
reference.
[0095] The term "lower alkyl," as used herein, and unless otherwise
specified, refers to a C.sub.1 to C.sub.4 saturated straight,
branched, or if appropriate, a cyclic (for example, cyclopropyl)
alkyl group, including both substituted and unsubstituted forms.
Unless otherwise specifically stated in this application, when
alkyl is a suitable moiety, lower alkyl is preferred. Similarly,
when alkyl or lower alkyl is a suitable moiety, unsubstituted alkyl
or lower alkyl is preferred.
[0096] The terms "alkylamino" or "arylamino" refer to an amino
group that has one or two alkyl or aryl substituents,
respectively.
[0097] The term "protected," as used herein and unless otherwise
defined, refers to a group that is added to an oxygen, nitrogen, or
phosphorus atom to prevent its further reaction or for other
purposes. A wide variety of oxygen and nitrogen protecting groups
are known to those skilled in the art of organic synthesis.
Non-limiting examples include: C(O)-alkyl, C(O)Ph, C(O)aryl,
CH.sub.3, CH.sub.2-alkyl, CH.sub.2-alkenyl, CH.sub.2Ph,
CH.sub.2-aryl, CH.sub.2O-alkyl, CH.sub.2O-aryl, SO.sub.2-alkyl,
SO.sub.2-aryl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl,
and 1,3-(1,1,3,3-tetraisopropyldisiloxanylidene).
[0098] The term "aryl," as used herein, and unless otherwise
specified, refers to phenyl, biphenyl, or naphthyl, and preferably
phenyl. The term includes both substituted and unsubstituted
moieties. The aryl group can be substituted with one or more
moieties selected from the group consisting of 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 T. W. Greene and P. G. M.
Wuts, "Protective Groups in Organic Synthesis," 3rd ed., John Wiley
& Sons, 1999.
[0099] The terms "alkaryl" or "alkylaryl" refer to an alkyl group
with an aryl substituent. The terms "aralkyl" or "arylalkyl" refer
to an aryl group with an alkyl substituent.
[0100] The term "halo," as used herein, includes chloro, bromo,
iodo and fluoro.
[0101] The term "acyl" refers to a carboxylic acid ester in which
the non-carbonyl moiety of the ester group is selected from
straight, branched, or cyclic alkyl or lower alkyl, alkoxyalkyl
including methoxymethyl, aralkyl including benzyl, aryloxyalkyl
such as phenoxymethyl, aryl including phenyl optionally substituted
with halogen (F, Cl, Br, I), C.sub.1 to C.sub.4 alkyl or C.sub.1 to
C.sub.4 alkoxy, sulfonate esters such as alkyl or aralkyl sulphonyl
including methanesulfonyl, the mono, di or triphosphate ester,
trityl or monomethoxytrityl, substituted benzyl, trialkylsilyl
(e.g. dimethyl-t-butylsilyl) or diphenylmethylsilyl. Aryl groups in
the esters optimally comprise a phenyl group.
[0102] The term "lower acyl" refers to an acyl group in which the
non-carbonyl moiety is lower alkyl.
[0103] The term "purine" or "pyrimidine" base includes, but is not
limited to, adenine, N.sup.6-alkylpurines, N.sup.6-acylpurines
(wherein acyl is C(O)(alkyl, aryl, alkylaryl, or arylalkyl),
N.sup.6-benzylpurine, N.sup.6-halopurine, N.sup.6-vinylpurine,
N.sup.6-acetylenic purine, N.sup.6-acyl purine,
N.sup.6-hydroxyalkyl purine, N.sup.6-allylaminopurine,
N.sup.6-thioallyl 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-lodo-pyrimidine, C.sup.5-Br-vinyl
pyrimidine, C.sup.6-Br-vinyl pyrimidine, C.sup.5-nitropyrimidine,
C.sup.5-amino-pyrimidine, N.sup.2-alkylpurines,
N.sup.2-alkyl-6-thiopurines, 5-azacytidinyl, 5-azauracilyl,
triazolopyridinyl, imidazolopyridinyl, pyrrolopyrimidinyl, and
pyrazolopyrimidinyl. Purine bases include, but are not limited to,
guanine, adenine, hypoxanthine, 2,6-diaminopurine, and
6-chloropurine. Functional oxygen and nitrogen groups on the base
can be protected as necessary or desired. Suitable protecting
groups are well known to those skilled in the art, and include
trimethylsilyl, dimethylhexylsilyl, t-butyldimethylsilyl, and
t-butyldiphenylsilyl, trityl, alkyl groups, and acyl groups such as
acetyl and propionyl, methanesulfonyl, and p-toluenesulfonyl.
[0104] The term "acyl" or "O-linked ester" refers to a group of the
formula C(O)R', wherein R' is an straight, branched, or cyclic
alkyl (including lower alkyl), amino acid, aryl including phenyl,
ailcaryl, aralkyl including benzyl, alkoxyalkyl including
methoxymethyl, aryloxyalkyl such as phenoxymethyl; or substituted
ailcyl (including lower alkyl), aryl including phenyl optionally
substituted with chloro, bromo, fluoro, iodo, C.sub.1 to C.sub.4
alkyl or C.sub.1 to C.sub.4 alkoxy, sulfonate esters such as alkyl
or aralkyl sulphonyl including methanesulfonyl, the mono, di or
triphosphate ester, trityl or monomethoxy-trityl, substituted
benzyl, alkaryl, aralkyl including benzyl, alkoxyalicyl including
methoxymethyl, aryloxyalkyl such as phenoxymethyl. Aryl groups in
the esters optimally comprise a phenyl group. In particular, acyl
groups include acetyl, trifluoroacetyl, methylacetyl,
cyclopropylacetyl, cyclopropyl carboxy, propionyl, butyryl,
hexanoyl, heptanoyl, octanoyl, neo-heptanoyl, phenylacetyl,
2-acetoxy-2-phenylacetyl, diphenylacetyl,
.alpha.-methoxy-.alpha.-trifluoromethyl-phenylacetyl, bromoacetyl,
2-nitro-benzeneacetyl, 4-chloro-benzeneacetyl,
2-chloro-2,2-diphenylacetyl, 2-chloro-2-phenylacetyl,
trimethylacetyl, chlorodifluoroacetyl, perfluoroacetyl,
fluoroacetyl, bromodifluoroacetyl, methoxyacetyl,
2-thiopheneacetyl, chlorosulfonylacetyl, 3-methoxyphenylacetyl,
phenoxyacetyl, tert-butylacetyl, trichloroacetyl,
monochloro-acetyl, dichloroacetyl, 7H-dodecafluoro-heptanoyl,
perfluoro-heptanoyl, 7H-dodeca-fluoroheptanoyl,
7-chlorododecafluoro-heptanoyl, 7-chloro-dodecafluoro-heptanoyl,
7H-dodecafluoroheptanoyl, 7H-dodeca-fluoroheptanoyl,
nona-fluoro-3,6-dioxa-heptanoyl, nonafluoro-3,6-dioxaheptanoyl,
perfluoroheptanoyl, methoxybenzoyl, methyl
3-amino-5-phenylthiophene-2-carboxyl,
3,6-dichloro-2-methoxy-benzoyl,
4-(1,1,2,2-tetrafluoro-ethoxy)-benzoyl, 2-bromo-propionyl,
omega-aminocapryl, decanoyl, n-pentadecanoyl, stearyl,
3-cyclopentyl-propionyl, 1-benzene-carboxyl, O-acetylmandelyl,
pivaloyl acetyl, 1-adamantane-carboxyl, cyclohexane-carboxyl,
2,6-pyridinedicarboxyl, cyclopropane-carboxyl,
cyclobutane-carboxyl, perfluorocyclohexyl carboxyl,
4-methylbenzoyl, chloromethyl isoxazolyl carbonyl,
perfluorocyclohexyl carboxyl, crotonyl,
1-methyl-1H-indazole-3-carbonyl, 2-propenyl, isovaleryl,
1-pyrrolidinecarbonyl, 4-phenylbenzoyl. When the term acyl is used,
it is meant to be a specific and independent disclosure of acetyl,
trifluoroacetyl, methylacetyl, cyclopropylacetyl, propionyl,
butyryl, hexanoyl, heptanoyl, octanoyl, neo-heptanoyl,
phenylacetyl, diphenylacetyl, ct-trifluoromethyl-phenylacetyl,
bromoacetyl, 4-chloro-benzeneacetyl, 2-chloro-2,2-diphenylacetyl,
2-chloro-2-phenylacetyl, trimethylacetyl, chlorodifluoroacetyl,
perfluoroacetyl, fluoroacetyl, bromodifluoroacetyl,
2-thiopheneacetyl, tert-butylacetyl, trichloroacetyl,
monochloro-acetyl, dichloroacetyl, methoxybenzoyl,
2-bromo-propionyl, decanoyl, n-pentadecanoyl, stearyl,
3-cyclopentyl-propionyl, 1-benzene-carboxyl, pivaloyl acetyl,
1-adamantane-carboxyl, cyclohexane-carboxyl,
2,6-pyridinedicarboxyl, cyclopropane-carboxyl,
cyclobutane-carboxyl, 4-methylbenzoyl, crotonyl,
1-methyl-1H-indazole-3-carbonyl, 2-propenyl, isovaleryl,
4-phenylbenzoyl.
[0105] The term "amino acid" includes naturally occurring and
synthetic .alpha., .beta. .gamma. or .delta. amino acids, and
includes but is not limited to, amino acids found in proteins, i.e.
glycine, alanine, valine, leucine, isoleucine, methionine,
phenylalanine, tryptophan, proline, serine, threonine, cysteine,
tyrosine, asparagine, glutamine, aspartate, glutamate, lysine,
arginine and histidine. In a preferred embodiment, the amino acid
is in the L-configuration. Alternatively, the amino acid can be a
derivative of alanyl, valinyl, leucinyl, isoleucinyl, prolinyl,
phenylalaninyl, tryptophanyl, methioninyl, glycinyl, serinyl,
threoninyl, cysteinyl, tyrosinyl, asparaginyl, glutaminyl,
aspartoyl, glutaroyl, lysinyl, argininyl, histidinyl,
.beta.-alanyl, .beta.-valinyl, .beta.-leucinyl, .beta.-isoleucinyl,
.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. When the term amino acid is used, it is
considered to be a specific and independent disclosure of each of
the esters of .alpha., .beta. .gamma. or .delta. glycine, alanine,
valine, leucine, isoleucine, methionine, phenylalanine, tryptophan,
proline, serine, threonine, cysteine, tyrosine, asparagine,
glutamine, aspartate, glutamate, lysine, arginine and histidine in
the D and L-configurations.
[0106] The term "host," as used herein, refers to a unicellular or
multicellular organism in which the virus can replicate, including
cell lines and animals, and preferably a human. Alternatively, the
host can be carrying a part of the viral genome, whose replication
or functions can be altered by the compounds of the present
invention. The term host specifically refers to infected cells,
cells transfected with all or part of the viral genome, and
animals, in particular, primates 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.
[0107] The term "pharmaceutically acceptable salt or prodrug" is
used throughout the specification to describe any pharmaceutically
acceptable form (such as an ester, phosphate ester, salt of an
ester or a related group) of a compound which, upon administration
to a patient, provides the active compound. Pharmaceutically
acceptable salts include those derived from pharmaceutically
acceptable inorganic or organic bases and acids. Suitable salts
include those derived from alkali metals such as potassium and
sodium, alkaline earth metals such as calcium and magnesium, among
numerous other acids well known in the pharmaceutical art.
Pharmaceutically acceptable prodrugs refer to a compound that is
metabolized, for example hydrolyzed or oxidized, in the host to
form the compound of the present invention. Typical examples of
prodrugs include compounds that have biologically labile protecting
groups on a functional moiety of the active compound. Prodrugs
include compounds that can be oxidized, reduced, aminated,
deaminated, hydroxylated, dehydroxylated, hydrolyzed, dehydrolyzed,
alkylated, dealkylated, acylated, deacylated, phosphorylated,
dephosphorylated to produce the active compound.
I. Active Compound, and Physiologically Acceptable Derivatives and
Salts Thereof
[0108] A (2'R)-2'-deoxy-2'-fluoro-2'-C-methyl nucleoside or its
pharmaceutically acceptable salt or prodrug thereof is provided of
the structure:
##STR00004## [0109] wherein Base refers to a naturally occurring or
modified purine or pyrimidine base; X is O, S, CH.sub.2, Se, NH,
N-alkyl, CHW, C(W).sub.2, wherein W is F, Cl, Br, or I; [0110]
R.sup.1 and R.sup.7 are independently H, phosphate, including
monophosphate, diphosphate, triphosphate, or a stabilized phosphate
prodrug, H-phosphonate, including stabilized H-phosphonates, acyl,
including optionally substituted phenyl and lower acyl, alkyl,
including lower alkyl, O-substituted carboxyalkylamino or its
peptide derivatives, sulfonate ester, including alkyl or arylalkyl
sulfonyl, including methanesulfonyl and benzyl, wherein the phenyl
group is optionally substituted, a lipid, including a phospholipid,
an L or D-amino acid, a carbohydrate, a peptide, a cholesterol, or
other pharmaceutically acceptable leaving group which when
administered in vivo is capable of providing a compound wherein
R.sup.1 is H or phosphate; R.sup.2 is OH or phosphate; R.sup.1 and
R.sup.2 or R.sup.7 can also be linked with cyclic phosphate group;
and [0111] R.sup.2 and R.sup.2' are independently H, C.sub.1-4
alkyl, C.sub.1-4 alkenyl, C.sub.1-4 alkynyl, vinyl, N.sub.3, CN,
Cl, Br, F, I, NO.sub.2, C(O)O(C.sub.1-4 alkyl), C(O)O(C.sub.1-4
alkyl), C(O)O(C.sub.1-4 alkynyl), C(O)O(C.sub.1-4 alkenyl),
O(C.sub.1-4 acyl), O(C.sub.1-4 alkyl), O(C.sub.1-4 alkenyl),
S(C.sub.1-4 acyl), S(C.sub.1-4 alkyl), S(C.sub.1-4 alkynyl),
S(C.sub.1-4 alkenyl), SO(C.sub.1-4 acyl), SO(C.sub.1-4 alkyl),
SO(C.sub.1-4 alkynyl), SO(C.sub.1-4 alkenyl), SO.sub.2(C.sub.1-4
acyl), SO.sub.2(C.sub.1-4 alkyl), SO.sub.2(C.sub.1-4 alkynyl),
SO.sub.2(C.sub.1-4 alkenyl), O.sub.3S(C.sub.1-4 acyl),
O.sub.3S(C.sub.1-4 alkyl), O.sub.3S(C.sub.1-4 alkenyl), NH.sub.2,
NH(C.sub.1-4 alkyl), NH(C.sub.1-4 alkenyl), NH(C.sub.1-4 alkynyl),
NH(C.sub.1-4 acyl), N(C.sub.1-4 alkyl).sub.2, N(C.sub.1-18
acyl).sub.2, wherein alkyl, alkynyl, alkenyl and vinyl are
optionally substituted by N.sub.3, CN, one to three halogen (Cl,
Br, F, I), NO.sub.2, C(O)O(C.sub.1-4 alkyl), C(O)O(C.sub.1-4
alkyl), C(O)O(C.sub.1-4 alkynyl), C(O)O(C.sub.1-4 alkenyl),
O(C.sub.1-4 acyl), O(C.sub.1-4 alkyl), O(C.sub.1-4 alkenyl),
S(C.sub.1-4 acyl), S(C.sub.1-4 alkyl), S(C.sub.1-4 alkynyl),
S(C.sub.1-4 alkenyl), SO(C.sub.1-4 acyl), SO(C.sub.1-4 alkyl),
SO(C.sub.1-4 alkynyl), SO(C.sub.1-4 alkenyl), SO.sub.2(C.sub.1-4
acyl), SO.sub.2(C.sub.1-4 alkyl), SO.sub.2(C.sub.1-4 alkynyl),
SO.sub.2(C.sub.1-4 alkenyl), O.sub.3S(C.sub.1-4 acyl),
O.sub.3S(C.sub.1-4 alkyl), O.sub.3S(C.sub.1-4 alkenyl), NH.sub.2,
NH(C.sub.1-4 alkyl), NH(C.sub.1-4 alkenyl), NH(C.sub.1-4 alkynyl),
NH(C.sub.1-4 acyl), N(C.sub.1-4 alkyl).sub.2, N(C.sub.1-4
acyl).sub.2, OR.sup.7, R.sup.2 and R.sup.2' can be linked together
to form a vinyl optionally substituted by one or two of N.sub.3,
CN, Cl, Br, F, I, NO.sub.2; and [0112] R.sup.6 is an optionally
substituted alkyl (including lower alkyl), cyano (CN), CH.sub.3,
OCH.sub.3, OCH.sub.2CH.sub.3, hydroxy methyl (CH.sub.2OH),
fluoromethyl (CH.sub.2F), azido (N.sub.3), CHCN, CH.sub.2N.sub.3,
CH.sub.2NH.sub.2, CH.sub.2NHCH.sub.3, CH.sub.2N(CH.sub.3).sub.2,
alkyne (optionally substituted), or fluoro.
[0113] In a second embodiment, a
(2'R)-2'-deoxy-2'-fluoro-2'-C-methyl nucleoside or its
pharmaceutically acceptable salt or prodrug thereof is provided of
the structure:
##STR00005## [0114] wherein Base, R.sup.1, R.sup.2, R.sup.2',
R.sup.6 and R.sup.7 are as defined above.
[0115] A third embodiment provides a
(2'R)-2'-deoxy-2'-fluoro-2'-C-methyl nucleoside or its
pharmaceutically acceptable salt or prodrug thereof of the
structure:
##STR00006## [0116] wherein X, R.sup.1, R.sup.2, R.sup.2', R.sup.6
and R.sup.7 are as defined above, and [0117] Base is selected
from
[0117] ##STR00007## [0118] Y is N or CH; [0119] R.sup.3, R.sup.4
and R.sup.5 are independently H, halogen (including F, Cl, Br, I),
OH, OR', SH, SR', NH.sub.2, NHR', NR'.sub.2, lower alkyl of
C.sub.1-C.sub.6, halogenated (F, Cl, Br, I) lower alkyl of
C.sub.1-C.sub.6 such as CF.sub.3 and CH.sub.2CH.sub.2F, lower
alkenyl of C.sub.2-C.sub.6 such as CH.dbd.CH.sub.2, halogenated (F,
Cl, Br, I) lower alkenyl of C.sub.2-C.sub.6 such as CH.dbd.CHCl,
CH.dbd.CHBr and CH.dbd.CHI, lower alkynyl of C.sub.2-C.sub.6 such
as C.ident.CH, halogenated (F, Cl, Br, I) lower alkynyl of
C.sub.2-C.sub.6, lower alkoxy of C.sub.1-C.sub.6 such as CH.sub.2OH
and CH.sub.2CH.sub.2OH, halogenated (F, Cl, Br, I) lower alkoxy of
C.sub.1-C.sub.6, CO.sub.2H, CO.sub.2R', CONH.sub.2, CONHR',
CONR'.sub.2, CH.dbd.CHCO.sub.2H, CH.dbd.CHCO.sub.2R'; [0120] R' is
an optionally substituted alkyl of C.sub.1-C.sub.12 (particularly
when the alkyl is an amino acid residue), cycloalkyl, optionally
substituted alkynyl of C.sub.2-C.sub.6, optionally substituted
lower alkenyl of C.sub.2-C.sub.6, or optionally substituted
acyl.
[0121] In a fourth embodiment, a
(2'R)-2'-deoxy-2'-fluoro-2'-C-methyl nucleoside or its
pharmaceutically acceptable salt or prodrug thereof is provided of
the structure:
##STR00008## [0122] wherein Base is selected from
[0122] ##STR00009## [0123] and, wherein R.sup.1, R.sup.2, R.sup.2',
R.sup.3, R.sup.4, R.sup.5, R.sup.6 and Y are as defined above.
[0124] A fifth embodiment provides a
(2'R)-2'-deoxy-2'-fluoro-2'-C-methyl nucleoside or its
pharmaceutically acceptable salt or prodrug thereof of the
structure:
##STR00010## [0125] wherein Base refers to a naturally occurring or
modified purine or pyrimidine base; [0126] R.sup.7 is independently
H, phosphate, including monophosphate, diphosphate, triphosphate,
or a stabilized phosphate prodrug, H-phosphonate, including
stabilized H-phosphonates, acyl, including optionally substituted
phenyl and lower acyl, alkyl, including lower alkyl, O-substituted
carboxyalkylamino or its peptide derivatives, sulfonate ester,
including alkyl or arylalkyl sulfonyl, including methanesulfonyl
and benzyl, wherein the phenyl group is optionally substituted, a
lipid, including a phospholipid, an L or D-amino acid, a
carbohydrate, a peptide, a cholesterol, or other pharmaceutically
acceptable leaving group which when administered in vivo is capable
of providing a compound wherein R.sup.1 or R.sup.7 is independently
H or phosphate; R.sup.1 and R.sup.7 can also be linked with cyclic
phosphate group; and [0127] wherein X and R.sup.1 are as defined
above.
[0128] In a sixth embodiment, a
(2'R)-2'-deoxy-2'-fluoro-2'-C-methyl nucleoside or its
pharmaceutically acceptable salt or prodrug thereof is provided of
the structure:
##STR00011## [0129] wherein Base refers to a naturally occurring or
modified purine or pyrimidine base; and [0130] wherein R.sup.1 and
R.sup.7 are as defined above.
[0131] A seventh embodiment provides a
(2'R)-2'-deoxy-2'-fluoro-2'-C-methyl nucleoside or its
pharmaceutically acceptable salt or prodrug thereof of the
structure:
##STR00012## [0132] wherein Base is selected from
[0132] ##STR00013## [0133] and wherein X, Y, R.sup.1, R.sup.3,
R.sup.4, R.sup.5, R.sup.7 and R' are as defined above.
[0134] In an eighth embodiment, a
(2'R)-2'-deoxy-2'-fluoro-2'-C-methyl nucleoside or its
pharmaceutically acceptable salt or prodrug thereof is provided of
the structure:
##STR00014## [0135] wherein Base is selected from
[0135] ##STR00015## [0136] and, wherein Y, R.sup.1, R.sup.3,
R.sup.4, R.sup.5, R.sup.7 and R' are as defined above.
[0137] A ninth embodiment provides a
(2'R)-2'-deoxy-2'-fluoro-2'-C-methyl nucleoside or its
pharmaceutically acceptable salt or prodrug thereof of the
structure:
##STR00016## [0138] wherein Base is:
[0138] ##STR00017## [0139] and wherein X is defined as above,
R.sup.1 is H, R.sup.2 is OH, R.sup.2' is H, R.sup.3 is H, R.sup.4
is NH.sub.2 or OH, and R.sup.6 is H.
[0140] In a tenth embodiment, a
(2'R)-2'-deoxy-2'-fluoro-2'-C-methyl nucleoside or its
pharmaceutically acceptable salt or prodrug thereof is provided of
the structure:
##STR00018## [0141] wherein Base is:
[0141] ##STR00019## [0142] and wherein R.sup.1 is H, R.sup.2 is OH,
R.sup.2' is H, R.sup.3 is H, R.sup.4 is NH.sub.2 or OH, and R.sup.6
is H.
[0143] An eleventh embodiment provides a
(2'R)-2'-deoxy-2'-fluoro-2'-C-methyl nucleoside or its
pharmaceutically acceptable salt or prodrug thereof of the
structure:
##STR00020## [0144] wherein Base is:
[0144] ##STR00021## [0145] and wherein X is defined as above,
R.sup.1 is H, R.sup.3 is H, R.sup.4 is NH.sub.2 or OH, R.sup.6 is
H, and R.sup.7 is H.
[0146] In a twelfth embodiment, a
(2'R)-2'-deoxy-2'-fluoro-2'-C-methyl nucleoside or its
pharmaceutically acceptable salt or prodrug thereof is provided of
the structure:
##STR00022## [0147] wherein Base is:
[0147] ##STR00023## [0148] and wherein R.sup.1 is H, R.sup.3 is H,
R.sup.4 is NH.sub.2 or OH, and R.sup.7 is H.
[0149] A thirteenth embodiment provides a
(2'R)-2'-deoxy-2'-fluoro-2'-C-methyl nucleoside or its
pharmaceutically acceptable salt or prodrug thereof of the
structure:
##STR00024##
[0150] In a fourteenth embodiment, a
(2'R)-2'-deoxy-2'-fluoro-2'-C-methyl nucleoside, its
pharmaceutically acceptable salt or product thereof is provided by
the structure:
##STR00025## [0151] wherein X, R.sup.1, R.sup.6 and R.sup.7 are as
defined above.
[0152] In a fifteenth embodiment, a
(2'R)-2'-deoxy-2'-fluoro-2'-C-methyl nucleoside, its
pharmaceutically acceptable salt or product thereof is provided by
the structure:
##STR00026## [0153] wherein R.sup.1, R.sup.6 and R.sup.7 are as
defined above.
[0154] In a sixteenth embodiment, a
(2'R)-2'-deoxy-2'-fluoro-2'-C-methyl nucleoside nucleoside, its
pharmaceutically acceptable salt or product thereof is provided by
the structure:
##STR00027##
[0155] In a seventeenth embodiment, a
(2'R)-2'-deoxy-2'-fluoro-2'-C-methyl nucleoside, its
pharmaceutically acceptable salt or product thereof is provided by
the structure:
##STR00028## [0156] wherein X and R.sup.1 are as defined above.
[0157] In an eighteenth embodiment, a
(2'R)-2'-deoxy-2'-fluoro-2'-C-methyl nucleoside, its
pharmaceutically acceptable salt or product thereof is provided by
the structure:
##STR00029##
[0158] In a nineteenth embodiment, a
(2'R)-2'-deoxy-2'-fluoro-2'-C-methyl nucleoside, its
pharmaceutically acceptable salt or product thereof is provided by
the structure:
##STR00030## [0159] wherein X and R.sup.1 are as defined above.
[0160] In a twentieth embodiment, a
(2'R)-2'-deoxy-2'-fluoro-2'-C-methyl nucleoside, its
pharmaceutically acceptable salt or product thereof is provided by
the structure:
##STR00031##
[0161] The present invention also contemplates 5'-triphosphate
triphosphoric acid ester derivates of the 5'-hydroxyl group of a
nucleoside compound of the present invention having the following
general structural formula:
##STR00032## [0162] wherein Base, X, R.sup.2, R.sup.2', and R.sup.6
are as defined as above.
[0163] The compounds of the present invention are also intended to
include pharmaceutically acceptable salts of the triphosphate ester
as well as pharmaceutically acceptable salts of 5'-diphosphate and
5'-monophosphate ester derivatives of the following structural
formulas, respectively.
##STR00033## [0164] wherein Base, X, R.sup.2, R.sup.2' and R.sup.6
are as defined above.
[0165] Further non-limiting examples of phosphoric acid derivatives
are the nucleosides of the present invention are shown below:
##STR00034##
##STR00035## ##STR00036##
[0166] The present invention also contemplates that any phosphate
nucleoside derivative can include a
5'-(S-acyl-2-thioethyl)phosphate or "SATE" mono or di-ester
derivative of the 5'-monophosphates.
[0167] Alternative embodiments are also contemplated wherein the
N-4 amino group on a phosphate nucleoside derivative can be
replaced with H, F, Cl, Br or I.
[0168] Additional embodiments include 3' and/or 5' produrgs as
described in more detail herein.
[0169] In the various embodiments, the fluorinated derivatives are
preferred. Fluorine is viewed as "isosteric" with hydrogen because
of its size (Van der Waals radii for H is 1.20 A and for F 1.35 A).
However, the atomic weight (18.998) and electronegativity of
fluorine (4.0 [Pauling's scale], 4.000 [Sanderson's scale]) are
more similar to oxygen (3.5 [Pauling]. 3.654 [Sanderson]) than
hydrogen (2.1 [Pauling], 2.592 [Sanderson]) (March, J., "Advances
in Organic Chemistry Reactions, Mechanisms, and Structure" Third
edition, 1985, p. 14., Wiley Interscience, New York). Fluorine is
known to be capable of forming a hydrogen bond, but unlike a
hydroxyl group (which can act both as proton acceptor and proton
donor) fluorine acts only as a proton acceptor. On the other hand,
2'-fluoro-ribonucleosides can be viewed as analogues of both
ribonucleosides and deoxynucleosides. They may be better recognized
by viral RNA polymerase at the triphosphate level than by the host
RNA polymerase thus selectively inhibiting the viral enzyme.
II. Pharmaceutically Acceptable Salts and Prodrugs
[0170] In cases where compounds are sufficiently basic or acidic to
form stable nontoxic acid or base salts, administration of the
compound as a pharmaceutically acceptable salt may be appropriate.
Pharmaceutically acceptable salts include those derived from
pharmaceutically acceptable inorganic or organic bases and acids.
Suitable salts include those derived from alkali metals such as
potassium and sodium, alkaline earth metals such as calcium and
magnesium, among numerous other acids well known in the
pharmaceutical art. In particular, examples of pharmaceutically
acceptable salts are organic acid addition salts formed with acids,
which form a physiological acceptable anion, for example, tosylate,
methanesulfonate, acetate, citrate, malonate, tartarate, succinate,
benzoate, ascorbate, .alpha.-ketoglutarate, and
.alpha.-glycerophosphate. Suitable inorganic salts may also be
formed, including, sulfate, nitrate, bicarbonate, and carbonate
salts.
[0171] Pharmaceutically acceptable salts may be obtained using
standard procedures well known in the art, for example by reacting
a sufficiently basic compound such as an amine with a suitable acid
affording a physiologically acceptable anion. Alkali metal (for
example, sodium, potassium or lithium) or alkaline earth metal (for
example calcium) salts of carboxylic acids can also be made.
[0172] Any of the nucleosides described herein can be administered
as a nucleotide prodrug to increase the activity, bioavailability,
stability or otherwise alter the properties of the nucleoside. A
number of nucleotide prodrug ligands are known. In general,
alkylation, acylation or other lipophilic modification of the mono,
di or triphosphate of the nucleoside will increase the stability of
the nucleotide. Examples of substituent groups that can replace one
or more hydrogens on the phosphate moiety are alkyl, aryl,
steroids, carbohydrates, including sugars, 1,2-diacylglycerol and
alcohols. Many are described in R. Jones and N. Bischofberger,
Antiviral Research, 27 (1995) 1-17. Any of these can be used in
combination with the disclosed nucleosides to achieve a desired
effect.
[0173] The active nucleoside can also be provided as a
5'-phosphoether lipid or a 5'-ether lipid, as disclosed in the
following references, which are incorporated by reference herein:
Kucera, L. S., N. Iyer, E. Leake, A. Raben, Modest E. K., D. L. W.,
and C. Piantadosi. 1990. "Novel membrane-interactive ether lipid
analogs that inhibit infectious HIV-1 production and induce
defective virus formation." AIDS Res. Hum. Retro Viruses.
6:491-501; Piantadosi, C., J. Marasco C. J., S. L. Morris-Natschke,
K. L. Meyer, F. Gumus, J. R. Surles, K. S. Ishaq, L. S. Kucera, N.
Iyer, C. A. Wallen, S. Piantadosi, and E. J. Modest. 1991.
"Synthesis and evaluation of novel ether lipid nucleoside
conjugates for anti-HIV activity." J. Med. Chem. 34:1408.1414;
Hosteller, K. Y., D. D. Richman, D. A. Carson, L. M. Stuhmiller, G.
M. T. van Wijk, and H. van den Bosch. 1992. "Greatly enhanced
inhibition of human immunodeficiency virus type 1 replication in
CEM and HT4-6C cells by 3'-deoxythymidine diphosphate
dimyristoylglycerol, a lipid prodrug of 3'-deoxythymidine."
Antimicrob. Agents Chemother. 36:2025.2029; Hosetler, K. Y., L. M.
Stuhmiller, H. B. Lenting, H. van den Bosch, and D. D. Richman,
1990. "Synthesis and antiretroviral activity of phospholipid
analogs of azidothymidine and other antiviral nucleosides." J.
Biol. Chem. 265:61127.
[0174] Nonlimiting examples of U.S. patents that disclose suitable
lipophilic substituents that can be covalently incorporated into
the nucleoside, preferably at the 5'-OH position of the nucleoside
or lipophilic preparations, include U.S. Pat. Nos. 5,149,794;
5,194,654; 5,223,263; 5,256,641; 5,411,947; 5,463,092; 5,543,389;
5,543,390; 5,543,391; and 5,554,728, all of which are incorporated
herein by reference. Foreign patent applications that disclose
lipophilic substituents that can be attached to the nucleosides of
the present invention, or lipophilic preparations, include WO
89/02733, WO 90/00555, WO 91/16920, WO 91/18914, WO 93/00910, WO
94/26273, WO 96/15132, EP 0 350 287, EP 93917054.4, and WO
91/19721.
III. Pharmaceutical Compositions
[0175] Pharmaceutical compositions based upon a .beta.-D or
.beta.-L compound disclosed herein or its pharmaceutically
acceptable salt or prodrug can be prepared in a therapeutically
effective amount for treating a Flaviviridae infection, including
hepatitis C virus, West Nile Virus, yellow fever virus, and a
rhinovirus infection, optionally in combination with a
pharmaceutically acceptable additive, carrier or excipient. The
therapeutically effective amount may vary with the infection or
condition to be treated, its severity, the treatment regimen to be
employed, the pharmacokinetics of the agent used, as well as the
patient treated.
[0176] In one aspect according to the present invention, the
compound according to the present invention is formulated
preferably in a mixture with a pharmaceutically acceptable carrier.
In general, it is preferable to administer the pharmaceutical
composition in orally administrable form, but formulations may be
administered via parenteral, intravenous, intramuscular,
transdermal, buccal, subcutaneous, suppository or other route.
Intravenous and intramuscular formulations are preferably
administered in sterile saline. One of ordinary skill in the art
may modify the formulation within the teachings of the
specification to provide numerous formulations for a particular
route of administration without rendering the compositions of the
present invention unstable or compromising its therapeutic
activity. In particular, a modification of a desired compound to
render it more soluble in water or other vehicle, for example, may
be easily accomplished by routine modification (salt formulation,
esterification, etc.).
[0177] In certain pharmaceutical dosage forms, the prodrug form of
the compound, especially including acylated (acetylated or other)
and ether derivatives, phosphate esters and various salt forms of
the present compounds, is preferred. One of ordinary skill in the
art will recognize how to readily modify the present compound to a
prodrug form to facilitate delivery of active compound to a
targeted site within the host organism or patient. The artisan also
will take advantage of favorable pharmacokinetic parameters of the
prodrug form, where applicable, in delivering the desired compound
to a targeted site within the host organism or patient to maximize
the intended effect of the compound in the treatment of a
Flaviviridae infection, including hepatitis C virus, West Nile
Virus, yellow fever virus, and a rhinovirus infection.
[0178] The amount of compound included within therapeutically
active formulations, according to the present invention, is an
effective amount for treating the infection or condition, in
preferred embodiments, a Flaviviridae infection, including
hepatitis C virus, West Nile Virus, yellow fever virus, and a
rhinovirus infection. In general, a therapeutically effective
amount of the present compound in pharmaceutical dosage form
usually ranges from about 50 mg to about 2,000 mg or more,
depending upon the compound used, the condition or infection
treated and the route of administration. For purposes of the
present invention, a prophylactically or preventively effective
amount of the compositions, according to the present invention,
falls within the same concentration range as set forth above for
therapeutically effective amount and is usually the same as a
therapeutically effective amount.
[0179] Administration of the active compound may range from
continuous (intravenous drip) to several oral administrations per
day (for example, Q.I.D., B.I.D., etc.) and may include oral,
topical, parenteral, intramuscular, intravenous, subcutaneous,
transdermal (which may include a penetration enhancement agent),
buccal and suppository administration, among other routes of
administration. Enteric-coated oral tablets may also be used to
enhance bioavailability and stability of the compounds from an oral
route of administration. The most effective dosage form will depend
upon the pharmacokinetics of the particular agent chosen, as well
as the severity of disease in the patient. Oral dosage forms are
particularly preferred, because of ease of administration and
prospective favorable patient compliance.
[0180] To prepare the pharmaceutical compositions according to the
present invention, a therapeutically effective amount of one or
more of the compounds according to the present invention is
preferably mixed with a pharmaceutically acceptable carrier
according to conventional pharmaceutical compounding techniques to
produce a dose. A carrier may take a wide variety of forms
depending on the form of preparation desired for administration,
e.g., oral or parenteral. In preparing pharmaceutical compositions
in oral dosage form, any of the usual pharmaceutical media may be
used. Thus, for liquid oral preparations such as suspensions,
elixirs and solutions, suitable carriers and additives including
water, glycols, oils, alcohols, flavoring agents, preservatives,
coloring agents and the like may be used. For solid oral
preparations such as powders, tablets, capsules, and for solid
preparations such as suppositories, suitable carriers and additives
including starches, sugar carriers, such as dextrose, mannitol,
lactose and related carriers, diluents, granulating agents,
lubricants, binders, disintegrating agents and the like may be
used. If desired, the tablets or capsules may be enteric-coated for
sustained release by standard techniques. The use of these dosage
forms may significantly impact the bioavailability of the compounds
in the patient.
[0181] For parenteral formulations, the carrier will usually
comprise sterile water or aqueous sodium chloride solution, though
other ingredients, including those that aid dispersion, also may be
included. Where sterile water is to be used and maintained as
sterile, the compositions and carriers must also be sterilized.
Injectable suspensions may also be prepared, in which case
appropriate liquid carriers, suspending agents and the like may be
employed.
[0182] Liposomal suspensions (including liposomes targeted to viral
antigens) may also be prepared by conventional methods to produce
pharmaceutically acceptable carriers. This may be appropriate for
the delivery of free nucleosides, acyl nucleosides or phosphate
ester prodrug forms of the nucleoside compounds according to the
present invention.
[0183] In particularly preferred embodiments according to the
present invention, the compounds and compositions are used to
treat, prevent or delay the onset of a Flaviviridae infection,
including hepatitis C virus, West Nile Virus, yellow fever virus,
and a rhinovirus infection. The present compounds are preferably
administered orally, but may be administered parenterally,
topically or in suppository form.
[0184] The compounds according to the present invention, because of
their low toxicity to host cells in certain instances, may be
advantageously employed prophylactically to prevent a Flaviviridae
infection, including hepatitis C virus, West Nile Virus, yellow
fever virus, and a rhinovirus infection or to prevent the
occurrence of clinical symptoms associated with the viral infection
or condition. Thus, the present invention also encompasses methods
for the prophylactic treatment of viral infection, and in
particular a Flaviviridae infection, including hepatitis C virus,
West Nile Virus, yellow fever virus, and a rhinovirus infection. In
this aspect, according to the present invention, the present
compositions are used to prevent or delay the onset of a
Flaviviridae infection, including hepatitis C virus, West Nile
Virus, yellow fever virus, and a rhinovirus infection. This
prophylactic method comprises administration to a patient in need
of such treatment, or who is at risk for the development of the
virus or condition, an amount of a compound according to the
present invention effective for alleviating, preventing or delaying
the onset of the viral infection or condition. In the prophylactic
treatment according to the present invention, it is preferred that
the antiviral compound utilized should be low in toxicity and
preferably non-toxic to the patient. It is particularly preferred
in this aspect of the present invention that the compound that is
used should be maximally effective against the virus or condition
and should exhibit a minimum of toxicity to the patient. In the
case of a Flaviviridae infection, including hepatitis C virus, West
Nile Virus, yellow fever virus, and a rhinovirus infection,
compounds according to the present invention, which may be used to
treat these disease states, may be administered within the same
dosage range for therapeutic treatment (i.e., about 250 micrograms
up to 1 gram or more from one to four times per day for an oral
dosage form) as a prophylactic agent to prevent the proliferation
of the viral infection, or alternatively, to prolong the onset of
the viral infection, which manifests itself in clinical
symptoms.
[0185] In addition, compounds according to the present invention
can be administered in combination or alternation with one or more
antiviral agents, including other compounds of the present
invention. Certain compounds according to the present invention may
be effective for enhancing the biological activity of certain
agents according to the present invention by reducing the
metabolism, catabolism or inactivation of other compounds and as
such, are co-administered for this intended effect.
IV. Stereoisomerism and Polymorphism
[0186] It is appreciated that nucleosides of the present invention
have several chiral centers and may exist in and be isolated in
optically active and racemic forms. Some compounds may exhibit
polymorphism. It is to be understood that the present invention
encompasses any racemic, optically active, diastereomeric,
polymorphic, or stereoisomeric form, or mixtures thereof, of a
compound of the invention, which possess the useful properties
described herein. It being well known in the art how to prepare
optically active forms (for example, by resolution of the racemic
form by recrystallization techniques, by synthesis from
optically-active starting materials, by chiral synthesis, or by
chromatographic separation using a chiral stationary phase).
[0187] Carbons of the nucleoside are chiral, their nonhydrogen
substituents (the base and the CHOR groups, respectively) can be
either cis (on the same side) or trans (on opposite sides) with
respect to the sugar ring system. The four optical isomers
therefore are represented by the following configurations (when
orienting the sugar moiety in a horizontal plane such that the
oxygen atom is in the back): cis (with both groups "up", which
corresponds to the configuration of naturally occurring .beta.-D
nucleosides), cis (with both groups "down", which is a nonnaturally
occurring .beta.-L configuration), trans (with the C2' substituent
"up" and the C4' substituent "down"), and trans (with the C2'
substituent "down" and the C4' substituent "up"). The
"D-nucleosides" are cis nucleosides in a natural configuration and
the "L-nucleosides" are cis nucleosides in the nonnaturally
occurring configuration.
[0188] 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.
[0189] Examples of methods to obtain optically active materials are
known in the art, and include at least the following. [0190] 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; [0191] 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; [0192] 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; [0193] 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;
[0194] 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; [0195] 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; [0196] vii) first- and second-order asymmetric
transformations--a technique whereby diastereomers from the
raceniate 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; [0197] 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; [0198] 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; [0199] 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;
[0200] 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;
[0201] 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; [0202] 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. Chiral chromatography, including
simulated moving bed chromatography, is used in one embodiment. A
wide variety of chiral stationary phases are commercially
available.
[0203] Some of the compounds described herein contain olefinic
double bonds and unless otherwise specified, are meant to include
both E and Z geometric isomers.
[0204] In addition, some of the nucleosides described herein, may
exist as tautomers, such as, keto-enol tautomers. The individual
tautomers as well as mixtures thereof are intended to be
encompassed within the compounds of the present invention as
illustrated below.
[0205] A (2'R)-2'-deoxy-2'-fluoro-2'-C-methylcytidine:
##STR00037##
[0206] A (2'R)-2'-deoxy-2'-fluoro-2'-C-methylguanosine:
##STR00038##
[0207] A (2'R)-2-amino-2'-deoxy-2'-fluoro-2'-C-methyladenosine:
##STR00039##
[0208] In each example above, the first drawn structure is the
preferred form.
V. Prodrugs and Derivatives
[0209] The active compound can be administered as any salt or
prodrug that upon administration to the recipient is capable of
providing directly or indirectly the parent compound, or that
exhibits activity itself. Nonlimiting examples are the
pharmaceutically acceptable salts (alternatively referred to as
"physiologically acceptable salts"), and a compound, which has been
alkylated, acylated, or otherwise modified at the 5'-position, or
on the purine or pyrimidine base (a type of "pharmaceutically
acceptable prodrug"). Further, the modifications can affect the
biological activity of the compound, in some cases increasing the
activity over the parent compound. This can easily be assessed by
preparing the salt or prodrug and testing its antiviral activity
according to the methods described herein, or other methods known
to those skilled in the art.
Pharmaceutically Acceptable Salts
[0210] In cases where compounds are sufficiently basic or acidic to
form stable nontoxic acid or base salts, administration of the
compound as a pharmaceutically acceptable salt may be appropriate.
Examples of pharmaceutically acceptable salts are organic acid
addition salts formed by addition of acids, which form a
physiological acceptable anion, for example, tosylate,
methanesulfonate, acetate, citrate, malonate, tartarate, succinate,
benzoate, ascorate, a-ketoglutarate, a-glycerophosphate, formate,
fumarate, propionate, glycolate, lactate, pyruvate, oxalate,
maleate, and salicylate. Suitable inorganic salts may also be
formed, including, sulfate, nitrate, bicarbonate, carbonate salts,
hydrobromate and phosphoric acid. In a preferred embodiment, the
salt is a mono- or di-hydrochloride salt.
[0211] Pharmaceutically acceptable salts may be obtained using
standard procedures well known in the art, for example by reacting
a sufficiently basic compound such as an amine with a suitable acid
affording a physiologically acceptable anion. Alkali metal (for
example, sodium, potassium or lithium) or alkaline earth metal (for
example calcium) salts of carboxylic acids can also be made. In one
embodiment, the salt is a hydrochloride, hydrobromide, or mesylate
salt of the compound. In another embodiment, the pharmaceutically
acceptable salt is a dihydrochloride, dihydrobromide, or dimesylate
salt.
Nucleotide Prodrug Formulations
[0212] The nucleosides described herein can be administered as a
nucleotide prodrug to increase the activity, bioavailability,
stability or otherwise alter the properties of the nucleoside. A
number of nucleotide prodrug ligands are known. In general,
alkylation, acylation or other lipophilic modification of the
mono-, di- or triphosphate of the nucleoside reduces polarity and
allows passage into cells. Examples of substituent groups that can
replace one or more hydrogens on the phosphate moiety are ailcyl,
aryl, steroids, carbohydrates, including sugars, 1,2-diacylglycerol
and alcohols. Many are described in R. Jones and N. Bisehoferger,
Antiviral Research, 1995, 27:1-17. Any of these can be used in
combination with the disclosed nucleosides to achieve a desired
effect.
[0213] In an alternative embodiment, the nucleoside is delivered as
a phosphonate or a SATE derivative.
[0214] The active nucleoside can also be provided as a 2'-, 3'-
and/or 5'-phosphoether lipid or a 2'-, 3'- and/or 5'-ether lipid.
Non-limiting examples are described include the following
references, which are incorporated by reference herein: Kucera, L.
S., N. Iyer, E. Leake, A. Raben, Modest E. K., D. L. W., and C.
Piantadosi. 1990. "Novel membrane-interactive ether lipid analogs
that inhibit infectious HIV-1 production and induce defective virus
formation." AIDS Res. Hum. Retro Viruses. 6:491-501; Piantadosi,
C., J. Marasco C. J., S. L. Morris-Natschke, K. L. Meyer, F. Gumus,
J. R. Surles, K. S. Ishaq, L. S. Kucera, N. Iyer, C A. Wallen, S.
Piantadosi, and E. J. Modest. 1991. "Synthesis and evaluation of
novel ether lipid nucleoside conjugates for anti-HIV activity." J.
Med. Chem. 34:1408.1414; Hosteller, K. Y., D. D. Richman, D. A.
Carson, L. M. Stuhmiller, G. M. T. van Wijk, and H. van den Bosch.
1992. "Greatly enhanced inhibition of human immunodeficiency virus
type 1 replication in CEM and HT4-6C cells by 3'-deoxythymine
diphosphate dimyristoylglycerol, a lipid prodrug of
3-deoxythymine." Antlnzicrob. Agents Chemother. 36:2025.2029;
Hosetler, K. Y., L. M. Stuhmiller, H. B. Lenting, H. van den Bosch,
and D. D. Richman, 1990. "Synthesis and antiretroviral activity of
phospholipid analogs of azidothymidine and other antiviral
nucleosides." J. Biol. Chem. 265:61127.
[0215] Nonlimiting examples of U.S. patents that disclose suitable
lipophilic substituents that can be covalently incorporated into
the nucleoside, preferably at the 2'-, 3'- and/or 5'-OH position of
the nucleoside or lipophilic preparations, include U.S. Pat. Nos.
5,149,794 (Sep. 22, 1992, Yatvin et al.); 5,194,654 (Mar. 16, 1993,
Hostetler et al., 5,223,263 (Jun. 29, 1993, Hostetler et al.);
5,256,641 (Oct. 26, 1993, Yatvin et al.); 5,411,947 (May 2, 1995,
Hostetler et al.); 5,463,092 (Oct. 31, 1995, Hostetler et al.);
5,543,389 (Aug. 6, 1996, Yatvin et al.); 5,543,390 (Aug. 6, 1996,
Yatvin et al.); 5,543,391 (Aug. 6, 1996, Yatvin et al.); and
5,554,728 (Sep. 10, 1996; Basava et al.), all of which are
incorporated herein by reference. Foreign patent applications that
disclose lipophilic substituents that can be attached to the
nucleosides of the present invention, or lipophilic preparations,
include WO 89/02733, WO 90/00555, WO 91/16920, WO 91/18914, WO
93/00910, WO 94/26273, WO 96/15132, EP 0350287, EP 93917054.4, and
WO 91/19721.
[0216] Aryl esters, especially phenyl esters, are also provided.
Nonlimiting examples are disclosed in DeLambert et al., J. Med.
Chem. 37: 498 (1994). Phenyl esters containing a carboxylic ester
ortho to the phosphate are also provided. Khaninei and Torrence, J.
Med. Chem.; 39:41094115 (1996). In particular, benzyl esters, which
generate the parent compound, in some cases using substituents at
the ortho- or para-position to accelerate hydrolysis, are provided.
Examples of this class of prodrugs are described by Mitchell et
al., J. Chem. Soc. Perkin Trans. 12345 (1992); Brook, et al. WO
91/19721; and Glazier et al. WO 91/1 9721.
[0217] Cyclic and noncyclic phosphonate esters are also provided.
Nonlimiting examples are disclosed in Hunston et al., J. Med. Chem.
27: 440-444 (1984) and Starrett et al. J. Med. Chem. 37: 1857-1864
(1994). Additionally, cyclic 3',5'-phosphate esters are provided.
Nonlimiting examples are disclosed in Meier et al. J. Med. Chem.
22: 811-815 (1979). Cyclic 1',3'-propanyl phosphonate and phosphate
esters, such as ones containing a fused aryl ring, i.e. the
cyclosaligenyl ester, are also provided (Meier et al., Bioorg. Med.
Chem. Lett. 7: 99-104 (1997)). Unsubstituted cyclic 1',3'-propanyl
esters of the monophosphates are also provided (Farquhar et al., J.
Med. Chem. 26: 1153 (1983); Farquhar et al., J. Med. Chem. 28: 1358
(1985)) were prepared. In addition, cyclic 1',3'-propanyl esters
substituted with a pivaloyloxy methyloxy group at C-1' are provided
(Freed et al., Biochem. Pharmac. 38: 3193 (1989); Biller et al.,
U.S. Pat. No. 5,157,027).
[0218] Cyclic phosphoramidates are known to cleave in vivo by an
oxidative mechanism. Therefore, in one embodiment of the present
invention, a variety of substituted 1',3' propanyl cyclic
phosphoramidates are provided. Non-limiting examples are disclosed
by Zon, Progress in Med. Chem. 19, 1205 (1982). Additionally, a
number of 2'- and 3'-substituted proesters are provided.
2'-Substituents include methyl, dimethyl, bromo, trifluoromethyl,
chloro, hydroxy, and methoxy; 3'-substituents including phenyl,
methyl, trifluoromethyl, ethyl, propyl, i-propyl, and cyclohexyl. A
variety of 1'-substituted analogs are also provided.
[0219] Cyclic esters of phosphorus-containing compounds are also
provided. Non-limiting examples are described in the following:
[0220] di and tri esters of phosphoric acids as reported in
Nifantyev et al., Phosphorus, Sulfur Silicon and Related Elements,
113: 1 (1996); Wijnberg et al., EP-180276 A1; [0221] phosphorus
(III) acid esters. Kryuchkov et al., Izy. Akad. Nauk SSSR, Ser.
Khim. 6:1244 (1987). Some of the compounds were claimed to be
useful for the asymmetric synthesis of L-Dopa precursors. Sylvain
et al., DE3S 12781 A1; [0222] phosphoramidates. Shili et al., Bull.
Inst. Chem. Acad. Sin, 41: 9 (1994); Edmundson et al., J. Chem.
Res. Synop. 5:122 (1989); and [0223] phosphonates. Neidlein et al.,
Heterocycles 35: 1185 (1993). N.sup.4-acyl Prodrugs
[0224] The invention also provides N.sup.4-acyl prodrugs. A
non-limiting example of an N.sup.4-acyl derivative of
(2'R)-2'-F-2'-C-methylcytidine is shown below:
##STR00040## [0225] wherein R can be any acyl group as described
herein.
[0226] The invention also contemplates other embodiments, wherein
the prodrug of a (2'R)-2'-deoxy-2'-fluoro-2'-C-methyl nucleoside
(.beta.-D or .beta.-L) includes biologically cleavable moieties at
the 3' and/or 5' positions. Preferred moieties are natural of
synthetic D or L amino acid esters, including D or L-valyl, though
preferably L-amino acids esters, such as L-valyl, and alkyl esters
including acetyl. Therefore, this invention specifically includes
3'-L or D-amino acid ester and 3',5'-L or D-diamino acid ester of
(2'R)-2'-deoxy-2'-fluoro-2'-C-methyl nucleoside (.beta.-D or
.beta.-L) nucleosides, preferably L-amino acid, with any desired
purine or pyrimidine base, wherein the parent drug optionally has
an EC.sub.50 of less than 15 micromolar, and even more preferably
less than 10 micromolar; 3'-(alkyl or aryl) ester or
3',5'-L-di(alkyl or aryl) ester of
(2'R)-2'-deoxy-2'-fluoro-2'-C-methyl nucleoside (.beta.-D or
.beta.-L) with any desired purine or pyrimidine base, wherein the
parent drug optionally has an EC.sub.50 of less than 10 or 15
micromolar; and prodrugs of 3',5'-diesters of
(2'R)-2'-deoxy-2'-fluoro-2'-C-methyl nucleosides (.beta.-D or
.beta.-L) wherein (i) the 3' ester is an amino acid ester and the
5'-ester is an alkyl or aryl ester; (ii) both esters are amino acid
esters; (iii) both esters are independently alkyl or aryl esters;
and (iv) the 3' ester is independently an alkyl or aryl ester and
the 5'-ester is an amino acid ester, wherein the parent drug
optionally has an EC.sub.50 of less than 10 or 15 micromolar.
[0227] Non-limiting examples of prodrugs falling within the
invention are:
##STR00041##
VI. Combination or Alternation Therapy
[0228] In another embodiment, for the treatment, inhibition,
prevention and/or prophylaxis of any viral infection described
herein, the active compound or its derivative or salt can be
administered in combination or alternation with another antiviral
agent. In general, in combination therapy, effective dosages of two
or more agents are administered together, whereas during
alternation therapy, an effective dosage of each agent is
administered serially. The dosage 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.
[0229] 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.
[0230] For example, one skilled in the art will recognize that any
antiviral drug or therapy can be used in combination or alternation
with any nucleoside of the present invention. Any of the viral
treatments described in the Background of the Invention can be used
in combination or alternation with the compounds described in this
specification. Nonlimiting examples of the types of antiviral
agents or their prodrugs that can be used in combination with the
compounds disclosed herein include: interferon, including
interferon alpha 2a, interferon alpha 2b, a pegylated interferon,
interferon beta, interferon gamma, interferon tau and interferon
omega; an interleukin, including interleukin 10 and interleukin 12;
ribavirin; interferon alpha or pegylated interferon alpha in
combination with ribavirin or levovirin; levovirin; a protease
inhibitor including an NS3 inhibitor, a NS3-4A inhibitor; a
helicase inhibitor; a polymerase inhibitor including HCV RNA
polymerase and NS5B polymerase inhibitor; gliotoxin; an IRES
inhibitor; and antisense oligonucleotide; a thiazolidine
derivative; a benzanilide, a ribozyme; another nucleoside,
nucleoside prodrug or nucleoside derivative; a
1-amino-alkylcyclohexane; an antioxidant including vitamin E;
squalene; amantadine; a bile acid; N-(phosphonoacetyl)-L-aspartic
acid; a benzenedicarboxamide; polyadenylic acid; a benzimidazoles;
thymosin; a beta tubulin inhibitor; a prophylactic vaccine; an
immune modulator, an IMPDH inhibitor; silybin-phosphatidylcholine
phytosome; and mycophenolate.
[0231] Further nonlimiting examples of the types of drugs or their
prodrugs described above include: acyclovir (ACV), ganciclovir (GCV
or DHPG) and its prodrugs (e.g. valyl-ganciclovir),
E-5-(2-bromovinyl)-2'-deoxyuridine (BVDU),
(E)-5-vinyl-1-.beta.-D-arabonosyluracil (VaraU),
(E)-5-(2-bromovinyl)-1-.beta.-D-arabinosyluracil (BV-araU),
1-(2-deoxy-2-fluoro-.beta.-D-arabinosyl)-5-iodocytosine (D-FIAC),
1-(2-deoxy-2-fluoro-.beta.-L-arabinosyl)-5-methyluracil (L-FMAU, or
clevudine), (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine
[(S)-HPMPA],
(S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)-2,6-diaminopurine
[(S)-HPMPDAP], (S)-1-(3-hydroxy-2-phosphonyl-methoxypropyl)cytosine
[(S)-HPMPC, or cidofivir], and
(2S,4S)-1-[2-(hydroxymethyl)-1,3-dioxolan-4-yl]-5-iodouracil
(L-5-IoddU), entecavir, lamivudine (3TC), LdT, LdC, tenofovir, and
adefovir, the (-)-enantiomer of
2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane ((-)-FTC);
the (-)-enantiomer of
2-hydroxymethyl-5-(cytosin-1-yl)-1,3-oxathiolane (3TC); carbovir,
acyclovir, famciclovir, penciclovir, AZT, DDI, DDC, L-(-)-FMAU,
D4T, amdoxovir, Reverset, Racivir, abacavir, L-DDA phosphate
prodrugs, and .beta.-D-dioxolanyl-6-chloropurine (ACP),
non-nucleoside RT inhibitors such as nevirapine, MKC-442, DMP-226
(sustiva), protease inhibitors such as indinavir, saquinavir,
Kaletra, atazanavir; and anti-HIV compounds such as BILN-2061, ISIS
14803; viramidine, NM 283, VX-497, JKT-003, levovirin, isatoribine,
albuferon, Peg-infergen, VX-950, R803, HCV-086, R1479 and
DMP45.
Pharmaceutical Compositions
[0232] Hosts, including humans, infected with pestivirus,
flavivirus, HCV infection, or any other condition described herein,
or another organism replicating through a RNA-dependent RNA viral
polymerase, or for treating any other disorder described herein,
can be treated by administering to the patient an effective amount
of the active compound or a pharmaceutically acceptable prodrug or
salt thereof in the presence of a pharmaceutically acceptable
carrier or dilutent. The active materials can be administered by
any appropriate route, for example, orally, parenterally,
intravenously, intradermally, subcutaneously, or topically, in
liquid or solid form.
[0233] A preferred dose of the compound for a Flaviviridae
infection, including hepatitis C virus, West Nile Virus and yellow
fever virus and rhinovirus infection will be in the range from
about 50 to about 2000 mg one to four times per day. Lower doses
may be useful, and thus ranges can include from 50-1,000 mg one to
four times per day. The effective dosage range of the
pharmaceutically acceptable salts and prodrugs can be calculated
based on the weight of the parent nucleoside to be delivered. If
the salt or prodrug exhibits activity in itself the effective
dosage can be estimated as above using the weight of the salt or
prodrug, or by other means known to those skilled in the art.
[0234] The compound is conveniently administered in unit any
suitable dosage form, including but not limited to one containing
25 to 3000 mg, preferably 50 to 2000 mg of active ingredient per
unit dosage form. An oral dosage of 50-1000 mg is usually
convenient, including in one or multiple dosage forms of 50, 100,
200, 250, 300, 400, 500, 600, 700, 800, 900 or 1000 mgs. Also
contemplated are doses of 0.1-50 mg, or 0.1-20 mg or 0.1-10.0 mg.
Furthermore, lower doses may be utilized in the case of
administration by a non-oral route, as, for example, by injection
or inhalation.
[0235] Ideally the active ingredient should be administered to
achieve peak plasma concentrations (C.sub.max) of the active
compound of from about 5.0 to 70 .mu.M, preferably about 5.0 to 15
.mu.M. This may be achieved, for example, by the intravenous
injection of a 0.1 to 5% solution of the active ingredient,
optionally in saline, or administered as a bolus of the active
ingredient.
[0236] The concentration of active compound in the drug composition
will depend on absorption, inactivation and excretion rates of the
drug as well as other factors known to those of skill in the art.
It is to be noted that dosage values will also vary with the
severity of the condition to be alleviated. It is to be further
understood that for any particular subject, specific dosage
regimens should be adjusted over time according to the individual
need and the professional judgment of the person administering or
supervising the administration of the compositions, and that the
concentration ranges set forth herein are exemplary only and are
not intended to limit the scope or practice of the claimed
composition. The active ingredient may be administered at once, or
may be divided into a number of smaller doses to be administered at
varying intervals of time.
[0237] A preferred mode of administration of the active compound is
oral. Oral compositions will generally include an inert diluent or
an edible carrier. They may be enclosed in gelatin capsules or
compressed into tablets. For the purpose of oral therapeutic
administration, the active compound can be incorporated with
excipients and used in the form of tablets, troches, or capsules.
Pharmaceutically compatible binding agents, and/or adjuvant
materials can be included as part of the composition.
[0238] The tablets, pills, capsules, troches and the like can
contain any of the following ingredients, or compounds of a similar
nature: a binder such as microcrystalline cellulose, gum tragacanth
or gelatin; an excipient such as starch or lactose, a
disintegrating agent such as alginic acid, Primogel, or corn
starch; a lubricant such as magnesium stearate or Sterotes; a
glidant such as colloidal silicon dioxide; a sweetening agent such
as sucrose or saccharin; or a flavoring agent such as peppermint,
methyl salicylate, or orange flavoring. When the dosage unit form
is a capsule, it can contain, in addition to material of the above
type, a liquid carrier such as a fatty oil. In addition, dosage
unit forms can contain various other materials which modify the
physical form of the dosage unit, for example, coatings of sugar,
shellac, or other enteric agents.
[0239] The compound can be administered as a component of an
elixir, suspension, syrup, wafer, chewing gum or the like. A syrup
may contain, in addition to the active compounds, sucrose as a
sweetening agent and certain preservatives, dyes and colorings and
flavors.
[0240] The compound or a pharmaceutically acceptable prodrug or
salts thereof can also be mixed with other active materials that do
not impair the desired action, or with materials that supplement
the desired action, such as antibiotics, antifungals,
anti-inflammatories, or other antivirals, including other
nucleoside compounds. Solutions or suspensions used for parenteral,
intradermal, subutaneous, or topical application can include the
following components: a sterile diluent such as water for
injection, saline solution, fixed oils, polyethylene glycols,
glycerine, propylene glycol or other synthetic solvents;
antibacterial agents such as benzyl alcohol or methyl parabens;
antioxidants such as ascorbic acid or sodium bisulfite; chelating
agents such as ethylenediaminetetraacetic acid; buffers such as
acetates, citrates or phosphates and agents for the adjustment of
tonicity such as sodium chloride or dextrose. The parental
preparation can be enclosed in ampoules, disposable syringes or
multiple dose vials made of glass or plastic.
[0241] If administered intravenously, preferred carriers are
physiological saline or phosphate buffered saline (PBS).
[0242] In a preferred embodiment, the active compounds are prepared
with carriers that will protect the compound against rapid
elimination from the body, such as a controlled release
formulation, including implants and microencapsulated delivery
systems. Biodegradable, biocompatible polymers can be used, such as
ethylene vinyl acetate, polyanhydrides, polyglycolic acid,
collagen, polyorthoesters and polylactic acid. Methods for
preparation of such formulations will be apparent to those skilled
in the art. The materials can also be obtained commercially from
Alza Corporation.
[0243] Liposomal suspensions (including liposomes targeted to
infected cells with monoclonal antibodies to viral antigens) are
also preferred as pharmaceutically acceptable carriers. These may
be prepared according to methods known to those skilled in the art.
For example, liposome formulations may be prepared by dissolving
appropriate lipid(s) (such as stearoyl phosphatidyl ethanolamine,
stearoyl phosphatidyl choline, arachadoyl phosphatidyl choline, and
cholesterol) in an inorganic solvent that is then evaporated,
leaving behind a thin film of dried lipid on the surface of the
container. An aqueous solution of the active compound or its
monophosphate, diphosphate, and/or triphosphate derivatives is then
introduced into the container. The container is then swirled by
hand to free lipid material from the sides of the container and to
disperse lipid aggregates, thereby forming the liposomal
suspension.
VII. Biological Methods
[0244] Antiviral Testing of Candidate Compounds with HCV Replicon
System in Huh7 Cells.
[0245] Huh7 cells harboring the HCV replicon can be cultivated in
DMEM media (high glucose, no pyruvate) containing 10% fetal bovine
serum, 1.times. non-essential Amino Acids, Pen-Strep-Glu (100
units/liter, 100 microgram/liter, and 2.92 mg/liter, respectively)
and 500 to 1000 microgram/milliliter G418. Antiviral screening
assays can be done in the same media without G418 as follows: in
order to keep cells in logarithmic growth phase, cells are seeded
in a 96-well plate at low density, for example 1000 cells per well.
The test compound is added immediately after seeding the cells and
incubate for a period of 3 to 7 days at 37.degree. C. in an
incubator. Media is then removed, and the cells are prepared for
total nucleic acid extraction (including replicon RNA and host
RNA). Replicon RNA can then be amplified in a Q-RT-PCR protocol,
and quantified accordingly. The observed differences in replicon
HCV RNA levels compared to the untreated control is one way to
express the antiviral potency of the test compound.
[0246] In another typical setting, a compound might reduce the
viral RNA polymerase activity, but not the host RNA polymerase
activity. Therefore, quantification of rRNA or beta-actin mRNA (or
any other host RNA fragment) and comparison with RNA levels of the
no-drug control is a relative measurement of the inhibitory effect
of the test compound on cellular RNA polymerases.
Phosphorylation Assay of Nucleoside to Active Triphosphate
[0247] To determine the cellular metabolism of the compounds, Huh-7
cells are obtained from the American Type Culture Collection
(Rockville, Md.), and are grown in 225 cm.sup.2 tissue culture
flasks in minimal essential medium supplemented with non-essential
amino acids, 1% penicillin-streptomycin. The medium is renewed
every three days, and the cells are sub cultured once a week. After
detachment of the adherent monolayer with a 10 minute exposure to
30 mL of trypsin-EDTA and three consecutive washes with medium,
confluent Huh-7 cells are seeded at a density of 2.5.times.10.sup.6
cells per well in a 6-well plate and exposed to 10 .mu.M of
[.sup.3H] labeled active compound (500 dpm/pmol) for the specified
time periods. The cells are maintained at 37.degree. C. under a 5%
CO.sub.2 atmosphere. At the selected time points, the cells are
washed three times with ice-cold phosphate-buffered saline (PBS).
Intracellular active compound and its respective metabolites are
extracted by incubating the cell pellet overnight at -20.degree. C.
with 60% methanol followed by extraction with an additional 20
.mu.L of cold methanol for one hour in an ice bath. The extracts
are then combined, dried under gentle filtered air flow and stored
at -20.degree. C. until HPLC analysis.
Bioavailability Assay in Cynomolgus Monkeys
[0248] Within 1 week prior to the study initiation, the cynomolgus
monkey is surgically implanted with a chronic venous catheter and
subcutaneous venous access port (VAP) to facilitate blood
collection and underwent a physical examination including
hematology and serum chemistry evaluations and the body weight was
recorded. Each monkey (six total) receives approximately 250 .mu.Ci
of .sup.3H-labeled compound combined with each dose of active
compound at a dose level of 10 mg/kg at a dose concentration of 5
mg/mL, either via an intravenous bolus (3 monkeys, IV), or via oral
gavage (3 monkeys, PO). Each dosing syringe is weighed before
dosing to gravimetrically determine the quantity of formulation
administered. Urine samples are collected via pan catch at the
designated intervals (approximately 18-0 hours pre-dose, 0-4, 4-8
and 8-12 hours post-dosage) and processed. Blood samples are
collected as well (pre-dose, 0.25, 0.5, 1, 2, 3, 6, 8, 12 and 24
hours post-dosage) via the chronic venous catheter and VAP or from
a peripheral vessel if the chronic venous catheter procedure should
not be possible. The blood and urine samples are analyzed for the
maximum concentration (C.sub.max), time when the maximum
concentration is achieved (T.sub.max), area under the curve (AUC),
half life of the dosage concentration (T.sub.1/2), clearance (CL),
steady state volume and distribution (V.sub.ss) and bioavailability
(F).
Bone Marrow Toxicity Assay
[0249] Human bone marrow cells are collected from normal healthy
volunteers and the mononuclear population are separated by
Ficoll-Hypaque gradient centrifugation as described previously by
Sommadossi J-P, Carlisle R. "Toxicity of 3'-azido-3'-deoxythymidine
and 9-(1,3-dihydroxy-2-propoxymethyl)guanine for normal human
hematopoietic progenitor cells in vitro" Antimicrobial Agents and
Chemotherapy 1987; 31:452-454; and Sommadossi J-P, Schinazi R F,
Chu C K, Xie M-Y. "Comparison of cytotoxicity of the (-)- and
(+)-enantiomer of 2',3'-dideoxy-3'-thiacytidine in normal human
bone marrow progenitor cells" Biochemical Pharmacology 1992;
44:1921-1925. The culture assays for CFU-GM and BFU-E are performed
using a bilayer soft agar or methylcellulose method. Drugs are
diluted in tissue culture medium and filtered. After 14 to 18 days
at 37.degree. C. in a humidified atmosphere of 5% CO.sub.2 in air,
colonies of greater than 50 cells are counted using an inverted
microscope. The results are presented as the percent inhibition of
colony formation in the presence of drug compared to solvent
control cultures.
Mitochondria Toxicity Assay
[0250] Fifty microliters of 2.times. drug dilutions were added per
well in a 96 well plate. A "no drug" (media only) control was used
to determine maximum amount of mitochondrial DNA produced and
ribosomal DNA. 3TC @ 10 .mu.M was used as a negative control, and
ddC @ 10 .mu.M was used as a toxic control. Ribosomal DNA levels
were used to determine specific toxicity to mitochondria or
generally cytotoxicity. HepG2 cells (5,000 cells/well at 50 .mu.l)
were added to the plate. The plate was incubated at 37.degree. C.
in a humidified 5% CO.sub.2 atmosphere for 7 days. After
incubation, the supernatant was removed and stored for lactic acid
quantification, and total DNA was extracted from cells as described
in the RNeasy 96 handbook (February 1999), pages 22-23. No DNA
digestions were performed, therefore total RNA and DNA were
extracted.
[0251] The extracted DNA was amplified and the change in
mitochondrial DNA and ribosomal DNA for each sample was determined.
The fold difference in mitochondrial DNA normalized for ribosomal
DNA relative to control was calculated.
[0252] Lactic acid quantification was performed by the D-Lactic
Acid/L-Lactic acid test kit (Boehringer Mannheim/R-Biopharm/Roche).
The total amount of lactic acid produced for each sample was found
as well as the fold change in lactic acid production (% of lactic
acid/% of rDNA) as described in the manufacturers instructions.
Cytotoxicity Assay
[0253] 50 .mu.l of 2.times. drug dilutions were added per well in a
96 well plate. Final concentrations of drug ranged from 1 to 100
.mu.M. A "no drug" (media only) control was used to determine the
minimum absorbance values and a "cells+media only" control was used
for maximum absorbance value. A solvent control was also used.
Cells were then added (PBM: 5.times.10.sup.4 cells/well; CEM:
2.5.times.10.sup.3 cells/well; Vero, HepG2, Huh-7, and Clone A:
5.times.10.sup.3 cells/well) and incubated at 37.degree. C. in a
humidified 5% CO.sub.2 atmosphere for 3-5 days (PBM: 5 days; CEM: 3
days, all others: 4 days). After incubation, 20 .mu.l of MTS dye
was added from Cell Titer Aqueous One Solution Cell Proliferation
Assay to each well and the plate was re-incubated for 2-4 hours.
The absorbance (490 nm) was then read on an ELISA plate reader
using the media only/no cell wells as blanks. Percent inhibition
was found and used to calculate the CC.sub.50.
In Vivo Toxicity in Mice
[0254] In vivo toxicity was also determined following injections
into female Swiss mice of the various nucleosides declosed in the
present invention. Intraperitenal injections were given on days 0,
day 1, day 2, day 3, and day 5 of varying doses of the particular
nucleoside. Separate animals were injected with vehicle as control
groups. In these studies, each dosing group contained 5-10 mice.
The average weight change in each of the mice was measured as a
sign of toxicity of the compound.
(BVDV) Yield Reduction Assay]
[0255] Madin-Darby Bovine Kidney (MDBK) cells were grown in
Dulbecco's modified eagle medium supplemented with 10% horse serum
and 100 .mu.g/ml penicillin-streptomycin. Cells were seeded in a
96-well plate at 5.times.10.sup.3 cells/well and incubated for 72 h
at 37.degree. C. in a humidified 5% CO.sub.2 atmosphere. Cells were
infected with either cytopathic (NADL strain) or noncytopathic
(SD-1 strain) BVDV at a virus dilution of 10.sup.-2 and incubated
for 45 min. Cell monolayers were washed three times with medium.
Fresh medium-containing test compounds in dose response
concentrations or ribavirn, as a positive control, were added to
cultures and medium containing no drug was added to the no-drug
controls. After 72 h incubation, supernatant was collected and
viral RNA was extracted using the QIAmp Viral RNA Mini Kit (Qiagen,
CA). Viral load was determined by Q-RT-PCR using primers specific
for either NADL or SD-1 (1).
VIII. Synthetic Protocol
[0256] The following non-limiting embodiments illustrate some
general methodologies to obtain the nucleosides of the present
invention. Two representative general methods for the preparation
of compounds of the present invention are outlined in Schemes 1 and
2 while more specific examples of these general methods are
provided in Scheme 3 (Example 1), Scheme 4 (Example 2), Scheme 5
(Example 3), and Scheme 6 (Example 4). Scheme 1 represents a
generalized process starting from a (2R)
2-deoxy-2-methyl-2-fluoro-carbohydrate and forms the nucleosides of
the present invention by condensing with a nucleobase. Scheme 2
starts from a pre-formed, purine or pyrimidine nucleoside,
optionally substituted at C-4' and constructs the C-2' (R) methyl,
fluoro nucleosides of the present invention. While these schemes
illustrate the syntheses of compounds of the present invention of
general formulas (I) and (II) wherein there is a furanose ring in
the .beta.-D-ribo configuration, this is not intended to be a
limitation on the scope of the process invention in any way, and
they should not be so construed. Those skilled in the art of
nucleoside and nucleotide synthesis will readily appreciate that
known variations of the conditions and processes of the following
preparative procedures and known manipulations of the nucleobase
can be used to prepare these and other compounds of the present
invention. Additionally, the L-enantiomers corresponding to the
compounds of the invention can be prepared following the same
methods, beginning with the corresponding L-carbohydrate building
block or nucleoside L-enantiomer as the starting material.
1. Glycosylation of the Nucleobase with an Appropriately Modified
Sugar
##STR00042##
[0257] Step 1 in Scheme 1 introduces the 2-methyl group by using an
appropriate alkylating agent such as methyllithium,
trimethylaluminum, or methylmagnesium bromide in an anhydrous
solvent such as tetrahydrofuran (THF), chloroform, or diethyl
ether. Compounds 1-1 through 1-4 can be purely .alpha. or .beta. or
they may exist as an anomeric mixture containing both .alpha. and
.beta. anomers in any ratio. However, the preferred anomeric
configuration of structure 1-1 is .beta..
[0258] Step 2 introduces the fluorine atom at the 2-position of the
alkyl furanoside. This can be achieved by treatment of the tertiary
alcohol, 1-2, with a commercially available fluorinating reagent
such as (diethylamino)sulfur trifluoride (DAST) or Deoxofluor in an
anhydrous, aprotic solvent such as tetrahydrofuran, chloroform,
dichloromethane, or toluene. Preferably the stereochemistry
proceeds with inversion of configuration at C-2. That is, starting
from a C-2 hydroxyl "up" (or arabinofuranoside) in structure 1-2,
the C-2 fluorine is "down" in the intermediate ribofuranoside
1-3.
[0259] In step 3, the optional protecting groups (Pg) can be
deprotected and reprotected to groups more suitable for the
remaining manipulations (T. W. Greene and P. G. M. Wuts,
"Protective Groups in Organic Synthesis," 3rd ed., John Wiley &
Sons, 1999). For example, benzyl ethers (Bn) may be difficult to
remove in the protected nucleoside, 1-5 and may be deprotected and
replaced with a group more facile to remove from the nucleoside of
structural type 1-5. Furthermore, the anomeric position (C-1) can
also be optionally manipulated to a suitable group for the coupling
reaction with the nucleobase (step 4). Several methods for anomeric
manipulations are established to those skilled in the art of
nucleoside synthesis. Some non-limiting examples by treatment of
the alkyl furanoside (1-3, R=alkyl) with a mixture of acetic
anhydride, acetic acid, and a catalytic amount of sulfuric acid
(acetolysis) to provide structure 1-4 where R=Ac, with optional
protecting groups. Also, the alkyl group in 1-3 may be converted to
an acetate, benzoate, mesylate, tosylate, triflate, or tosylate,
for example, by first hydrolyzing the 1-Oalkyl group to a
1-hydroxyl group by using a mineral acid consisting of but not
limited to sulfuric acid, hydrochloric acid, and hydrobromic acid
or an organic acid consisting of but not limited to trifluoroacetic
acid, acetic acid, and formic acid (at ambient temperature or
elevated temperature). The reducing sugar could then be converted
to the desired carbohydrate by treatment with acetyl chloride,
acetic anhydride, benzoyl chloride, benzoic anhydride,
methanesulfonyl chloride, triflic anhydride, trifyl chloride, or
tosyl chloride in the presence of a suitable base such as
triethylamine, pyridine, or dimethylaminopyridine.
[0260] The nucleosidic linkage is constructed by treatment of
intermediate 1-3 or 1-4 with the appropriate persilylated
nucleobase in the presence of a lewis acid such as tin
tetrachloride, titanium tetrachloride, trimethylsilyltriflate, or a
mercury (II) reagent (HgO/HgBr.sub.2) usually at an elevated
temperature in an aprotic solvent such as toluene, acetonitrile,
benzene, or a mixture of any or all of these solvents.
[0261] The optional protecting groups in the protected nucleosides
or structural formula 1-5 can be cleaved following established
deprotection methodologies (T. W. Greene and P. G. M. Wuts,
"Protective Groups in Organic Synthesis," 3rd ed., John Wiley &
Sons, 1999).
2. Modification of a Pre-Formed Nucleoside
##STR00043##
[0263] The starting material for this process is an appropriately
substituted purine or pyrimidine nucleoside with a 2'-OH and 2'-H.
The nucleoside can be purchased or can be prepared by any known
means including standard coupling techniques. The nucleoside can be
optionally protected with suitable protecting groups, preferably
with acyl or silyl groups, by methods well known to those skilled
in the art, as taught by T. W. Greene and P. G. M. Wuts,
"Protective Groups in Organic Synthesis," 3rd ed., John Wiley &
Sons, 1999.
[0264] The purine or pyrimidine nucleoside can then be oxidized at
the 2'-position with the appropriate oxidizing agent in a
compatible solvent at a suitable temperature to yield the
2'-modified nucleoside. Possible oxidizing agents are a mixture of
dimethylsulfoxide, trifluoroacetic anhydride or acetic anhydride (a
Swern/Moffat oxidation), chromium trioxide or other chromate
reagent, Dess-Martin periodinane, or by ruthenium tetroxide/sodium
periodate.
[0265] The optionally protected nucleoside 2'-ketone is then
alkylated using such alkylating agents methyllithium,
trimethylaluminum, methylmagnesium bromide, or similar reagents in
an anhydrous solvent such tetrahydrofuran (THF), chloroform, or
diethyl ether usually at temperatures below 0.degree. C. Compounds
of the structural formula 2-3 are preferred to have the 2'(S) or
2'-methyl "down", 2'-OH "up" configuration.
[0266] The nucleoside of structure 2-3 can be deprotected and
reprotected with a number of protecting groups such as an O-acyl
(alkyl or aryl), O-sulfonyl, or an N-acyl (alkyl or aryl) for the
base. This optional reprotection step need not be limited to
protecting groups that function as chemical protecting groups.
Other protecting groups such as long chain acyl groups of between 6
and 18 carbon units or amino acids can be introduced independently
on the nucleobase or the sugar. The protecting groups can serve as
prodrugs of the active substance.
[0267] Step 5 introduces the fluorine atom at the 2' position of
the pre-formed nucleoside. This can be achieved by treatment of the
tertiary alcohol, 2-4, with a commercially available fluorinating
reagent such as (diethylamino)sulfur trifluoride (DAST) or
Deoxofluor in an anhydrous, aprotic solvent such as
tetrahydrofuran, chloroform, dichloromethane, or toluene.
Preferably the stereochemistry proceeds with inversion of
configuration at the 2' position. That is, starting from a C-2'
hydroxyl "up" (or arabinonucleoside) in structure 2-4, the C-2'
flourine is "down" in the intermediate nucleoside 2-5. The absolute
configuration of a nucleoside of structure 2-4 is (2'S) while the
absolute configuration of a nucleoside of structure 2-5 is
(2'R).
[0268] Subsequently, the nucleosides of structural type 2-5 can be
deprotected by methods well known to those skilled in the art, as
taught by T. W. Greene and P. G. M. Wuts, "Protective Groups in
Organic Synthesis," 3rd ed., John Wiley & Sons, 1999.
[0269] The following working examples provide a further
understanding of the method of the present invention and further
exemplify the general examples in Schemes 1 and 2 above. These
examples are of illustrative purposes, and are not meant to limit
the scope of the invention. Equivalent, similar or suitable
solvents, reagents or reaction conditions may be substituted for
those particular solvents, reagents or reaction conditions
described without departing from the general scope of the
method.
EXAMPLES
Example 1
Synthesis of (2'R)-2'-Deoxy-2'-Fluoro-2'-C-Methylcytidine Starting
from a Carbohydrate
##STR00044##
[0271] Step 1: Compound 3-1 (7.7 g, 0.022 mmol) was dissolved in
anhydrous diethyl ether and cooled to -78.degree. C. To this
solution was added MeLi (30 mL, 1.6 M in diethyl ether). After the
reaction was complete, the mixture was treated with ammonium
chloride (1 M, 65 mL) and the organic phase was separated, dried
(Na.sub.2SO.sub.4), filtered, and concentrated to dryness. Silica
gel chromatography followed by crystallization from diethyl
ether-hexanes afforded pure compound 3-2 (6.31 g). .sup.1H NMR (400
MHz, CDCl.sub.3): .delta. 1.40 (s, 3H), 3.41 (s, 3H), 3.49 (dd, 1H,
J=10.3, 6.89 Hz), 3.57 (dd, 1H, J=10.3, 3.88 Hz), 3.84 (d, 1H,
J=7.3 Hz), 4.03 (m, 1H), 4.48 (s, 1H), 4.58 (m, 3H), 4.83 (d, 1H,
J=11.6 Hz), 7.31-7.36 (m, 10H); .sup.13C NMR (100 MHz, CDCl.sub.3):
.delta. 18.4, 55.4, 72.2, 73.4, 79.5, 80.2, 84.7, 107.4, 127.7,
127.8, 127.83, 128.5, 138.2, 138.3.
[0272] Step 2: Compound 3-2 was dissolved in CH.sub.2Cl.sub.2 and
was treated with DAST (4.0 mL, 30.3 mmol) at room temperature. The
solution was stirred at room temp overnight. The so-obtained
mixture was poured into sat NaHCO.sub.3 (100 mL) and washed with
sat NaHCO.sub.3 (1.times.15 mL). The organic layer was further
worked up in the usual manner. Silica gel:chromatography (1:5
EtOAc-hexanes) gave crude compound 3-3 (0.671 g) that was
sufficiently pure for the next step. .sup.1H NMR (400 MHz,
CDCl.sub.3): .delta. 1.43 (d, 3H, J=22.8 Hz), 3.35 (s, 3H), 3.49
(dd, 1H, J=10.5, 5.4 Hz), 3.55 (dd, 1H, J=10.5, 4.1 Hz), 3.87 (dd,
1H, J=23.5, 7.5 Hz), 4.26 (m, 1H), 4.56 (d, 2H, J=6.9 Hz), 4.66 (d,
2H, J=8.2 Hz), 4.72 (d, 1H, J=10.8 Hz), 7.29-7.36 (m, 10H);
.sup.13C NMR (100 MHz, CDCl.sub.3): .delta. 17.0 (d, J=24.4 Hz),
55.2, 77.1, 73.4, 73.8, 77.3, 80.3, 81.2 (d, J=16 Hz), 99.7 (d,
J=178.9 Hz), 106.8 (d, J=32.0 Hz), 127.7, 127.8, 128.1, 128.3,
128.5, 128.6, 137.8, 138.3; .sup.19F NMR (100 MHz, CDCl.sub.3):
.delta. -8.2 (m, 1F).
[0273] Step 3: Compound 3-3 (0.39 g, 1.1 mmol) was dissolved in 1:2
EtOH-EtOAc and treated with Pd/C (.about.0.1 g) and cyclohexene
(.about.1 mL). The mixture was heated to reflux overnight and then
filtered through celite. The solvent was removed in vacuo and the
residue was dissolved in pyridine (.about.5 mL). To this solution
was added benzoyl chloride (0.22 mL, 1.83 mmol) and the mixture was
stirred at room temp overnight. The pyridine was removed in vacuo
and the residue was partitioned between CH.sub.2Cl.sub.2 and sat
NaHCO.sub.3 (10.0 mL). The organic phase was dried
(Na.sub.2SO.sub.4), filtered, and the solution was concentrated to
dryness. Column chromatography provided 0.350 g of pure compound
3-4. .sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 1.53 (d, 3H, J=22.4
Hz), 3.39 (s, 3H), 4.46 (dd, 1H, J=11.6, 4.7 Hz), 4.58 (m, 1H),
4.65 (dd, 1H, J=11.6, 3.9 Hz), 4.87 (d, 1H, J=9.9 Hz), 5.64 (dd,
2H, J=24.1, 7.8 Hz), 7.29-7.36 (m, 10H); .sup.19F NMR (100 MHz,
CDCl.sub.3): .delta. -7.5 (m, 1F).
[0274] Step 4: A solution of bis(trimethylsilyl)-N-benzoylcytosine
(0.28 g, 0.77 mmol) and compound 3-4 (0.20 g, 0.5 mmol) in 1,2
dichloroethane (2 mL) and toluene (2 mL) was treated with TMSOTf
(0.15 mL, 0.77 mmol). After most of the starting material
disappeared as judged by TLC, the solution was cooled to room temp,
washed with water (1.times.5 mL), brine (1.times.5 mL), dried
(Na.sub.2SO.sub.4), filtered, and concentrated to dryness. Flash
chromatography followed by crystallization from
CH.sub.2Cl.sub.2-hexanes afforded compound 3-5 (68 mg). mp
241.degree. C.; .sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 1.49 (d,
3H, J=22.4 Hz), 4.64 (dd, 1H, J=12.9, 3.4 Hz), 4.73 (app d, 1H,
J=9.5 Hz), 4.89 (dd, 1H, J=12.7, 2.2 Hz), 5.56 (dd, 1H, J=20.7, 8.6
Hz), 6.52 (d, 1H, J=15.9 Hz), 7.38-7.67 (m, 10H), 7.89 (d, 2H,
J=6.9 Hz), 8.07-8.11 (m, 5H), 8.67 (s, 1H); .sup.19F NMR (100 MHz,
CDCl.sub.3): .delta. 2.85 (m, 1F).
[0275] Step 5: Compound 3-5 (40 mg, 0.05 mmol) was dissolved in
methanolic ammonia and stirred at room temp for 48 h. The solution
was concentrated to dryness and chromatographed (SiO.sub.2) eluting
with 1:4 EtOH--CH.sub.2Cl.sub.2. The yield was about 12 mg of pure
(2'R)-2'-deoxy-2'-fluoro-2'-C-methylcytidine, 3-6. .sup.1H NMR (400
MHz, DMSO-d.sub.6): .delta. 1.16 (d, 3H, J=22.0 Hz), 3.61 (dd, 1H,
J=11.6, 5.2 Hz), 3.60-3.83 (m, 3H, J=10.5, 5.4 Hz), 5.24 (s, 1H,
exchangeable with D.sub.2O), 5.59 (s, 1H, exchangeable with
D.sub.2O), 5.71 (d, 1H, J=7.3 Hz), 6.08 (d, 1H, J=19.0 Hz), 7.24
(d, 1H, J=17.7 Hz, exchangeable with D.sub.2O), 7.87 (d, 1H);
.sup.19F NMR (100 MHz, DMSO-d.sub.6): .delta. 4.13 (m, 1F).
Example 2
Synthesis of (2'R)-2'-Deoxy-2'-Fluoro-2'-C-Methylcytidine Starting
from Cytidine
##STR00045## ##STR00046##
[0277] Step 1: To a suspension of cytidine (100 g, 0.411 mol) in
DMF (2.06 L) is added benzoic anhydride (102.4 g, 0.452 mol). The
mixture was stirred at room temperature for 20 h. The DMF was
removed in vacuo and the residue was triturated with diethyl ether.
The resulting solid was collected by suction filtration and washed
with diethyl ether (2.times.200 mL). Further drying in vacuo at
room temperature gave the N.sup.4 benzamide (140.6 g, 98.3%). A
portion of this material (139.3 g, 0.401 mol) was dissolved in
anhydrous pyridine (1.2 L) and was treated with
1,3-dichloro-1,1,3,3-tetraisopropyl-disiloxane (141.4 mL, 0.441
mol) at room temp. The solution was stirred at room temperature
overnight. The mixture was concentrated to near dryness in vacuo
and coevaporated with toluene (3.times.200 mL). The residue was
treated with EtOAc (1.8 L) and washed with HCl (2.times.200 mL,
0.05 N), NaHCO.sub.3 (5%, 2.times.400 mL). The organic layer was
washed dried (Na.sub.2SO.sub.4), filtered, and evaporated to
dryness. Compound 4-1 (256.5 g, >100%) was isolated as a white
foam and used without further purification.
[0278] Step 2: Compound 4-1 (236.5 g, 0.40 mol) was dissolved in
dry THF (1.22 L). Anhydrous dmso (180.8 mL, 2.1 mol) was added and
the resulting solution was cooled to between -20.degree. C. and
-15.degree. C. Trifluoroacetic anhydride (90.6 mL, 0.64 mol) was
added dropwise over 45 minutes and the solution was stirred between
-20.degree. C. and -15.degree. C. for 2 hrs after which anhydrous
triethylamine (223.5 mL, 1.6 mol) was added over 20 min. The crude
reaction containing ketone 4-2 was dissolved in EtOAc (500 mL), and
the resulting solution was washed with H.sub.2O (3.times.400 mL),
dried (Na.sub.2SO.sub.4) and the solvents were removed in vacuo to
give a yellow solid that was purified on a silica gel column
eluting with a stepwise gradient of Et.sub.2O (0-60%) in hexanes
followed by a stepwise gradient of EtOAc (50-100%) in hexanes. The
crude ketone so-obtained (.about.192 g) was crystallized from
petroleum ether to give ketone 4-2 (138.91 g, 57.5% from cytidine)
as a white solid and 22 g of unreacted starting material, 4-1, as a
yellow solid.
[0279] Step 3: Compound 4-2 (48.57 g, 8.26 mmol) was dissolved in
anhydrous toluene (.about.400 mL) and the solvent was removed in
vacuo with exclusion of moisture. The residue was then further
dried in vacuo (oil pump) for another 2 h. With strict exclusion of
moisture, the residual foam was dissolved in anhydrous diethyl
ether (1.03 L) under argon. The resulting solution was cooled to
-78.degree. C. under argon and MeLi (1.6 M, 258.0 mL, 0.413 mol)
was added dropwise via additional funnel. After the addition was
complete, the mixture was stirred for 2 h at -78.degree. C. Aqueous
1 M NH.sub.4Cl (500 mL) was added slowly. After warming to room
temperature, the mixture was washed with H.sub.2O (2.times.500 mL),
dried (Na.sub.2SO.sub.4), and then concentrated to dryness to give
a brown foam (.about.60 g, >100%).
[0280] The reaction was performed two more times using 37.62 g and
56.4 g of compound 4-2. The combined crude products (128.0 g, 0.212
mol) were dissolved in THF (1.28 L) and treated with concd HOAc (23
mL, 0.402 mol). To the solution was added TBAF (384.0 mL, 1 M in
THF). The solution was stirred at room temp for 0.75 h and the
mixture was treated with silica gel (750 g) and concentrated to
dryness. The powder was placed on a silica gel column packed in
CH.sub.2Cl.sub.2. Elution with 1:7 EtOH--CH.sub.2Cl.sub.2 afforded
a dark waxy solid that was pre-adsorbed on silica gel (300 g) and
chromatographed as before. Compound 4-3 (46.4 g, 53.0% from 4-2)
was isolated as an off-white solid. .sup.1H NMR (DMSO-d.sub.6):
.delta. 1.20 (s, 3H, CH.sub.3), 3.62-3.69 (m, 2H), 3.73-3.78 (m,
2H), 5.19 (t, 1H, J=5.4 Hz, OH-5'), 5.25 (s, 1H, OH-2'), 5.52 (d,
1H, J=5.0 Hz, OH-3'), 5.99 (s, 1H, H-1'), 7.32 (d, 1H, J=5.8 Hz),
7.50 (.PSI.t, 2H, J=7.7 Hz), 7.62 (.PSI.t, 1H, J=7.3 Hz), 8.00 (d,
2H, J=7.3 Hz), 8.14 (d, 1H, J=6.9 Hz), 11.22 (s, 1H, NH). Anal.
Calcd for C.sub.17H.sub.19N.sub.3O.sub.6.0.5H.sub.2O: C, 55.13; H,
5.44; N, 11.35. Found: C, 55.21; H, 5.47; N, 11.33.
[0281] Step 4: Compound 4-3 (46.0 g, 0.13 mol) was dissolved in
anhydrous pyridine and concentrated to dryness in vacuo. The
resulting syrup was dissolved in anhydrous pyridine under argon and
cooled to 0.degree. C. with stirring. The brown solution was
treated with benzoyl chloride (30 mL, 0.250 mol) dropwise over 10
min. The ice bath was removed and stirring continued for 1.5 h
whereby TLC showed no remaining starting material. The mixture was
quenched by the addition of water (5 mL) and concentrated to
dryness. The residue was dissolved in a minimal amount of
CH.sub.2Cl.sub.2 and washed with satd NaHCO.sub.3 (1.times.500 mL)
and H.sub.2O (1.times.500 mL). The organic phase was dried
(Na.sub.2SO.sub.4) and filtered, concentrated to dryness and
chromatographed on silica gel eluting with a stepwise gradient of
EtOAc-hexanes (25-60%) to provide compound 4-4 as yellow foam (48.5
g, 67%). .sup.1H NMR (CDCl.sub.3): .delta. 1.64 (s, 3H, CH.sub.3),
4.50 (m, 1H, H-4), 4.78-4.85 (m, 2H, H-5',5a'), 5.50 (d, 1H, J=3.4
Hz, H-3'), 6.42 (s, 1H, H-1'), 7.44-7.54 (m, 7H, Ar), 7.57-7.66 (m,
3H, Ar), 7.94 (d, 2H, J=7.8 Hz), 8.05-8.09 (m, 4H, Ar), 8.21 (d,
1H, J=7.3 Hz). Anal. Calcd for C.sub.31H.sub.27N.sub.3O.sub.8: C,
65.37; H, 4.78; N, 7.38. Found: C, 65.59; H, 4.79; N, 7.16.
[0282] Step 5: Compound 4-4 (7.50 g, 0.013 mol) was dissolved in
anhydrous toluene (150 mL) under argon and cooled to -20.degree. C.
DAST (2.5 mL, 18.9 mmol) was added slowly and the cooling bath was
removed after the addition was complete. Stirring was continued for
1 h and the mixture was poured into satd NaHCO.sub.3 (100 mL) and
washed until gas evolution ceased. The organic phase was dried
(Na.sub.2SO.sub.4), concentrated, and purified by silica gel
chromatography eluting with 1:1 EtOAc-hexanes. Yield was 1.22 g
(16.3%) of pure 4-5 as a white solid. mp 241.degree. C.
(CH.sub.2Cl.sub.2-hexanes); .sup.1H NMR (CDCl.sub.3): .delta. 1.49
(d, 3H, J=22.4 Hz, CH.sub.3), 4.64 (dd, 1H, J=3.44, 12.9 Hz, H-5'),
4.73 (d, 1H, J=9.5 Hz, H-4'), 4.90 (dd, 1H, J=2.4, 12.7 Hz, H-5a'),
5.56 (dd, 1H, J=8.6, 20.7 Hz, H-3'), 6.52 (d, 1H, J=18.0 Hz, H-1'),
7.47-7.57 (m, 7H, Ar), 7.62-7.71 (m, 3H, Ar), 7.89 (d, 2H, J=6.9
Hz), 8.07-8.11 (m, 5H, Ar), 8.67 (bs, 1H, NH). .sup.19F NMR
(CDCl.sub.3): .delta. 3.3 (m). Anal. Calcd for
C.sub.31H.sub.26FN.sub.3O.sub.7.0.7H.sub.2O: C, 63.74; H, 4.72; N,
7.20. Found: C, 63.71; H, 4.54; N, 7.20.
[0283] Step 6: Compound 4-5 (6.30 g, 0.011 mol) was suspended in
methanolic ammonia (ca 7 N, 150 mL) and stirred at room temperature
overnight. The solvent was removed in vacuo, co-evaporated with
methanol (1.times.20 mL), and pre-adsorbed onto silica gel. The
white powder was placed onto a silica gel column (packed in
CHCl.sub.3) and the column was eluted with 9% EtOH in CHCl.sub.3,
then 17% EtOH and finally 25% EtOH in CHCl.sub.3. Concentration of
the fractions containing the product, filtration through a 0.4
.mu.m disk, and lyophilization from water afforded compound 4-6,
2.18 g (76%). .sup.1H NMR (DMSO-d.sub.6): .delta. 1.17 (d, 3H,
J=22.3 Hz, CH.sub.3), 3.63 (dd, 1H, J=2.7, 13.7 Hz, H-5'),
3.70-3.84 (m, 3H, H-3', H-4', H-5a'), 5.24 (app s, 1H, OH-3'), 5.60
(d, 1H, J=5.4 Hz, H-5'), 5.74 (d, 1H, J=7.71 Hz, H-5), 6.07 (d, 1H,
J=18.9 Hz, H-1'), 7.31 (s, 1H, NH.sub.2), 7.42 (s, 1H, NH.sub.2),
7.90 (d, 1H, J=7.3 Hz, H-6). .sup.19F NMR (DMSO-d.sub.6): .delta.
2.60 (m). Anal. Calcd for
C.sub.10H.sub.14FN.sub.3O.sub.4.1.4H.sub.2O: C, 44.22; H, 5.95; N,
14.77. Found: C, 42.24; H, 5.63; N, 14.54. Compound 4-6 (0.10 g,
0.386 mmol) was converted to the hydrochloride salt by dissolving
in water (2 mL) and adjusting the pH to approximately 3.0 with 1 M
HCl. The water was removed in vacuo and the residue was
crystallized from aqueous EtOH to give 4-6 as the hydrochloride
salt (71.0 mg). mp 243.degree. C. (dec); .sup.1H NMR
(DMSO-d.sub.6): .delta. 1.29 (d, 3H, J=22.6 Hz, CH.sub.3), 3.65
(dd, 1H, J=2.3, 12.7 Hz, H-5'), 3.76-3.90 (m, 3H, H-3', H-4',
H-5a'), 5.96 (d, 1H, J=17.3 Hz, H-1'), 6.15 (d, 1H, J=7.9 Hz, H-5),
8.33 (d, 1H, J=7.9 Hz, H-6), 8.69 (s, 1.5H, NH), 9.78 (s, 1.5H,
NH). .sup.19F NMR (DMSO-d.sub.6): .delta. 1.69 (m). Anal. Calcd for
C.sub.10H.sub.14FN.sub.3O.sub.4. HCl: C, 40.62; H, 5.11; N, 14.21.
Found: C, 40.80; H, 5.09; N, 14.23.
Example 3
Synthesis of (2'R)-6-Chloro-2'-Deoxy-2'-Fluoro-2'-C-Methylpurine
Starting from 6-Chloropurine Riboside
##STR00047##
[0285] Step 1: The nucleoside, 6-chloropurine riboside, (3.18 g,
11.09 mmol) was dissolved in anhydrous pyridine (300 mL) and was
treated dropwise with
1,3-dichloro-1,1,3,3-tetraisopropyl-disiloxane (4.08 mL, 12.75
mmol) at 0.degree. C. under an argon atmosphere. The solution was
brought to room temp and stirred overnight. The mixture was
concentrated to near dryness in vacuo, dissolved in a minimal
amount of chloroform, and washed with HCl (100 mL, 0.05 N) and
NaHCO.sub.3 (5%, 100 mL). The organic layer was dried
(Na.sub.2SO.sub.4), filtered, and evaporated to dryness to afford
compound 5-1 as an amber glass (6.10 g, >100%) that was used
without further purification. .sup.1H NMR (CDCl.sub.3): .delta.
1.01-1.13 (m, 24H), 4.03-4.18 (m, 3H), 4.58 (d, 1H, J=5.2 Hz), 5.01
(m, 1H), 6.07 (s, 1H), 8.31 (s, 1H), 8.71 (s, 1H).
[0286] Step 2: Compound 5-1 (7.13 g, 13.47 mmol) was dissolved in
dry THF (35 mL). Anhydrous DMSO (5.11 mL, 72.06 mmol) was added and
the resulting solution was cooled to between -20.degree. C. and
-15.degree. C. Trifluoroacetic anhydride (3.06 mL, 21.69 mmol) was
added dropwise over 45 minutes and the solution was stirred between
-20.degree. C. and -15.degree. C. for 2 hrs after which anhydrous
triethylamine (8.08 mL, 57.92 mmol) was added over 20 min. The
crude reaction containing ketone 5-2 was dissolved in Et.sub.2O (25
mL), and the resulting solution was washed with H.sub.2O
(2.times.50 mL), dried (Na.sub.2SO.sub.4) and the solvents were
removed in vacuo to give a yellow solid that was purified on a
silica gel column eluting with a stepwise stepwise gradient of
0-50% petroleum ether-diethyl ether afforded compound 5-2 as a
mixture with the corresponding geminal diol. The glass was
dissolved in CH.sub.2Cl.sub.2 and stirred over an excess of
MgSO.sub.4 for 36 h. The mixture, free from the geminal diol, was
filtered, and evaporated to dryness to afford compound 5-2 as an
amber glass (7.0 g, 97%). .sup.1H NMR (CDCl.sub.3): .delta.
1.01-1.13 (m, 24H), 4.09-4.22 (m, 3H), 5.55 (d, 1H, J=9.6 Hz), 5.80
(s, 1H), 8.19 (s, 1H), 8.61 (s, 1H).
[0287] Step 3: A solution of compound 5-2 (7.0 g, 13.26 mmol) in
anhydrous tetrahydrofuran (45 mL) was cooled to -78.degree. C. with
stirring under an argon atmosphere. To the solution was added
methylmagnesium bromide (15.85 mL, 3.0 M in ethyl ether) dropwise
over a 30 min period. After stirring for an additional 3 h at
-78.degree. C., the reaction was quenched by the careful addition
of aqueous 1 M NH.sub.4Cl (50.0 mL). After warming to room
temperature, the mixture was washed with H.sub.2O (2.times.500 mL),
dried (Na.sub.2SO.sub.4), and concentrated to dryness to give a
brown foam (3.8 g) that was dissolved in tetrahydrofuran (50 mL)
and treated with a solution of TBAF (18.9 mL, 1 M solution in THF)
and glacial acetic acid (0.85 mL) at room temp. The solution was
stirred at room temp for 2 h, concentrated to dryness, and purified
by silica gel chromatography to give compound 5-3 (2.0 g, 50%).
[0288] Step 4: Compound 5-3 (0.491 g, 1.63 mmol) was dissolved in
pyridine (3 mL) and treated with acetic anhydride (0.38 mL, 4.08
mL) at room temp. The solution was stirred at room temp for 2 h
after which time, the solution was concentrated to dryness and
treated with diethyl ether (10 mL) and water (5 mL). The organic
layer was further washed with water (2.times.10 mL), dried
(Na.sub.2SO.sub.4), filtered, and evaporated to dryness to give
compound 5-4 as a foam (0.450 g, 91.0%). .sup.1H NMR (CDCl.sub.3):
.delta. 1.39 (s, 3H), 2.15 (s, 3H), 2.21 (s, 3H), 4.27 (m, 1H),
4.49 (dd, 1H, J=4.2, 11.9 Hz), 4.57 (dd, 1H, J=6.16, 11.9 Hz), 5.14
(d, 1H, J=3.1 Hz), 6.25 (s, 1H), 8.54 (s, 1H), 8.75 (s, 1H).
[0289] Step 5: Compound 5-4 (0.100 g, 0.259 mmol) was dissolved in
anhydrous toluene (3.0 mL) under argon and cooled to -20.degree. C.
DAST (0.2 mL, 1.55 mmol) was added slowly and the cooling bath was
removed after the addition was complete. Stirring was continued for
1 h and the mixture was poured into satd NaHCO.sub.3 (100 mL) and
washed until gas evolution ceased. The organic phase was dried
(Na.sub.2SO.sub.4), concentrated, and purified by silica gel
chromatography eluting with 30% Et.sub.2O-petroleum ether gave pure
5-5 (0.028 g, 27.9%). .sup.1H NMR (CDCl.sub.3): .delta. 1.24 (d,
3H, J=22.8 Hz), 2.20 (s, 3H), 2.22 (s, 3H), 4.41-4.55 (m, 3H), 4.47
(dd, 1H, J=9.2, 22.0 Hz), 6.37 (d, 1H, J=17.6 Hz), 8.45 (s, 1H),
8.82 (s, 1H).
[0290] Step 6: Compound 5-5 (0.018 g, 0.047 mmol) was dissolved in
methanol (5 mL) and treated with a solution of sodium methoxide
(3.6 mg, 0.67 mmol) in methanol (5 mL). The solution was stirred at
room temp for 1 h, neutralized with concd acetic acid and
chromatographed on silica gel eluting with a stepwise gradient of
Et.sub.2O/methanol (0-5%) to afford compound 5-6 (0.010 g, 70.9%).
.sup.1H NMR (CDCl.sub.3): .delta. 1.23 (d, 3H, J=22.4 Hz), 4.04
(dd, 1H, J=2.11, 12.5 Hz), 4.17 (dd, 1H, J=1.5, 9.2 Hz), 4.25 (dd,
1H, J=1.9, 12.3 Hz), 4.61 (dd, 1H, J=9.2, 22.3 Hz), 6.37 (d, 1H,
J=17.3 Hz), 8.70 (s, 1H), 8.78 (s, 1H).
Example 4
Synthesis of (2'R)-2'-Deoxy-2'-Fluoro-2'-C-Methyladenosine Starting
from (2'R)-6-Chloro-2'-Deoxy-2'-Fluoro-2'-C-Methylpurine
##STR00048##
[0292] Step 1: Compound 5-5 (0.100 g, 0.26 mmol) was heated in a
pressure tube with methanolic ammonia (ca. 7 N, 25 mL) at
80.degree. C. for 12 h. The crude reaction was pre-adsorbed onto
silica gel and purified by column chromatography eluting with a
stepwise gradient of Et.sub.2O-MeOH (0-5%). The impure product was
converted to the hydrochloride salt by dissolving the compound in a
minimal amount of ethanol and treating the solution with 0.5 mL of
a 0.6 M HCl solution. Concentration to near dryness gave compound
6-1 as off-white crystals (0.020 g, 24.2%). .sup.1H NMR
(CD.sub.3OD): .delta. 1.19 (d, 3H, J=22.3 Hz), 3.88 (dd, 1H, J=2.7,
12.7 Hz), 4.06 (dd, 1H, J=2.1, 12.5 Hz), 4.11 (app d, 1H, J=9.2
Hz), 4.35 (dd, 1H, J=9.4, 24.5 Hz), 6.35 (d, 1H, J=16.5 Hz), 8.43
(s, 1H), 8.85 (s, 1H).
Example 5
Antiviral Activity of
(2'R)-2'-Deoxy-2'-Fluoro-2'-C-Methylcytidine
HCV Replicon Assay
[0293] The anti-flavivirus activity of the compounds was determined
as described by Stuyver, et al. ("Ribonucleoside analogue that
blocks replication of bovine viral diarrhea and hepatitis C viruses
in culture", Antimicrobial Agents and Chemotherapy 47:244-254
(2003)). The compound was dissolved in DMSO and added to the
culture media at final concentrations ranging from 3 to 100 .mu.M.
A 4-days incubation resulted in dose-dependant reduction of the
replicon HCV RNA (FIG. 1A). A 1-log reduction of replicon RNA (or
EC.sub.90 value) was reached at approximately 2.5 .mu.M.
Measurement of the reduction of rRNA gave an indication of the
inhibitory effect on cellular polymerases. Subtraction of this
cellular toxicity value from the antiviral values resulted in the
therapeutic index line and EC.sub.90 value. Based on these
calculations, an average EC.sub.90 value, corrected for cellular
toxicity, of approximately 2.5 .mu.M was obtained. FIG. 1A shows
the dose-dependant reduction of the replicon HCV RNA based on the
treatment with (2'R)-2'-deoxy-2'-fluoro-2'-C-methylcytidine. The
viral reduction was compared to the reduction of cellular RNA
levels (ribosomal RNA) to obtain therapeutic index values.
EC.sub.90 represents the effective concentration 90% at 96 hours
following the dose dependant administration of
(2'R)-2'-deoxy-2'-fluoro-2'-C-methylcytidine. FIG. 1B shows the
prolonged reduction in replicon HCV RNA up to 7 days following
treatment with 5 and 25 .mu.M.
[0294] The activity of (2'R)-2'-deoxy-2'-fluoro-2'-C-methylcytidine
in the replicon system is summarized in Table 1. The EC.sub.90
values for (2'R)-2'-deoxy-2'-fluoro-2'-C-methylcytidine as well as
2'-C-methylcytidine and 2'-C-methyladenosine are shown for three
separate replicon clones (HCV-WT (Wild Type), 9-13 and 21-5) as
well as two other clones (S282T and rRNA). The EC.sub.90 values for
(2'R)-2'-deoxy-2'-fluoro-2'-C-methylcytidine were in the range of
1.6 to 4.6 .mu.M for the replicon clones. In contrast the EC.sub.90
values for 2'-C-methylcytidine were in the range of 6.6-37.4 .mu.M.
Interestingly, the EC.sub.90 values for 2'-C-methyladenosine were
comparable to those of
(2'R)-2'-deoxy-2'-fluoro-2'-C-methylcytidine. The activity of
(2'R)-2'-deoxy-2'-fluoro-2'-C-methylcytidine and
2'-C-methylcytidine in other replicons tested is shown in Table
2.
Polymerase Assay
[0295] Table 3 shows the potency of
(2'R)-2'-deoxy-2'-fluoro-2'-C-methylcytidine-5'-triphosphate (TP)
in the NS5B polymerase assay. The inhibitory concentration 50% was
determined to be in the range of 1.7 to 7.7 .mu.M.
Toxicity
[0296] A summary of the toxicity data for
(2'R)-2'-deoxy-2'-fluoro-2'-C-methylcytidine using the
mitochondrial toxicity assay is shown in Tables 6 and 7. Table 7
shows the lack of effects of
(2'R)-2'-deoxy-2'-fluoro-2'-C-methylcytidine and
2'-C-methylcytidine on mitochondrial DNA synthesis and lack of
effects on lactic acid increase in this assay. Results shows the
relative lack of toxicity of
(2'R)-2'-deoxy-2'-fluoro-2'-C-methylcytidine. Table 6 shows a
cytotoxicity analysis in various cell lines (Clone A, Huh7, HepG2,
MDBK, PBM, CEM, Vero, MRC-5). Cytotoxic concentration 50%
(CC.sub.50) was greater than 75-100 .mu.M in all clones tested for
(2'R)-2'-deoxy-2'-fluoro-2'-C-methylcytidine as well as
2'-C-methylcytidine. In contrast is the relative toxicity of
2'-C-methyladenosine.
[0297] The effects the nucleoside analogs tested on human bone
marrow cells is depicted in Table 9. As shown, the IC.sub.50 values
for 2'-methyl-2'-fluorocytidine were significantly higher (98.2,
BFU-E) and 93.9 (CFU-GM) as compared to 2'-methylcytadine or AZT.
Results show that 2'-methyl-2'-fluorocytidine was significantly
less toxic than compared to the other nucleoside compounds.
Animal Studies
[0298] FIG. 2 depicts the average weight change (%) of female Swiss
mice in vivo the toxicity analysis of
(2'R)-2'-deoxy-2'-fluoro-2'-C-methylcytidine at various doses.
Intraperitneal injections were given on days 0 to day 5 of the 0,
3.3, 10, 33, 100 mg/kg. Each dosing group contained 5 mice and no
mice died during the 30-day study. No significant toxicity was
observed in the mice.
[0299] FIG. 3 and Table 6 summarize the pharmacokinetic parameters
of (2'R)-2'-deoxy-2'-fluoro-2'-C-methylcytidine in Rhesus monkeys
given a single dose (33.3 mg/kg) oral (Table 6, FIG. 3) or
intravenous dose (FIG. 3) of
(2'R)-2'-deoxy-2'-fluoro-2'-C-methylcytidine.
Other Antiviral Activity
[0300] Summary of the range of antiviral activity of
(2'R)-2'-deoxy-2'-fluoro-2'-C-methylcytidine is shown in Table 4.
Table shows that in addition to HCV virus
(2'R)-2'-deoxy-2'-fluoro-2'-C-methylcytidine shows activity against
Rhinovirus, West Nile virus, Yellow Fever virus, and Dengue
virus.
[0301] Table 5 shows the lack of activity of
(2'R)-2'-deoxy-2'-fluoro-2'-C-methylcytidine on HCV surrogate
models BVDV as well as other viruses including HIV, HBV and Corona
virus. In contrast, 2'-C-methylcytidine and 2'-C-methyladenosine
show greater activity in the HCV surrogate model, BVDV. These
results show the necessity for screening this series of compounds
against the HCV replicon system versus surrogate HCV systems.
TABLE-US-00002 TABLE 1 Summary of the Anti-HCV Replicon Activity of
(2'R)-2'-deoxy- 2'-fluoro-2'-C-methylcytidine* (2'R)-2'-deoxy-
2'-fluoro-2'-C- 2'-C- 2'-C- Replicon methylcytidine methylcytidine
methyladenosine HCV-WT 1b 4.6 .+-. 2.0 21.9 .+-. 4.3 2.1 .+-. 0.27
S282T mut. 1b 30.7 .+-. 11.7 37.4 .+-. 12.1 >100 9-13
(subgenomic) 4.6 .+-. 2.3 13.0 0.7 21-5 (full-length) 1.6 .+-. 0.7
6.6 0.6 *Values represent EC.sub.90 (.mu.M)
TABLE-US-00003 TABLE 2 Activity of
(2'R)-2'-deoxy-2'-fluoro-2'-C-methylcytidine and 2'-C-
methylcytidine in other Replicons (2'R)-2'-deoxy-2'-fluoro-
2'-C-methylcytidine 2'-C-methylcytidine IC.sub.90 IC.sub.90
EC.sub.90 (.mu.M) EC.sub.90 (.mu.M) Replicon (.mu.M) GAPDH MTT
(.mu.M) GAPDH MTT 1b (Ntat) 3.8 >100 >100 27.2 >100
>100 1b (Btat) 11.5 >100 >100 31.1 >100 >100 1a 34.7
>100 >100 35.0 >100 >100 (pp1aSI-7)
TABLE-US-00004 TABLE 3 HCV 1b NS5B Polymerase Assay (IC.sub.50,
.mu.M) (2'R)-2'-deoxy-2'- 2'-C- fluoro-2'-C- 2'-C-methylcytidine
methyladenosine methylcytidine TP TP TP Wild-Type 1.7 .+-.
0.4.sup.a 6.0 .+-. 0.5 20.6 .+-. 5.2 NS5B 7.7 .+-. 1.2.sup.b S282T
2.0.sup.a 26.9 .+-. 5.5 >100 8.3 .+-. 2.4.sup.c .sup.aValues
determined using batch 1; .sup.bValue determined using batch 2 and
3; and .sup.cValue determined using batch 2.
TABLE-US-00005 TABLE 4 Summary of Antiviral Activity of
(2'R)-2'-deoxy-2'-fluoro-2'-C- methylcytidine EC.sub.50, CPE
EC.sub.50, NR.sup.a CC.sub.50, CPE CC.sub.50, NR.sup.a Virus Cell
(.mu.M) (.mu.M) (.mu.M) (.mu.M) West Nile Vero 32 12 >100 32
Dengue Vero 32/55 >100/>100 >100 >100 Type 2 Yellow
Vero 19/3.2 32/12 >100 >100 Fever Influenza A MDCK >100
>100 >100 >100 (H1N1) Influenza A MDCK >100 >100
>100 >100 (H3N2) Influenza B MDCK >100 >100 >100
>100 Rhinovirus KB 25 20 >100 >100 Type 2 VEE Vero >100
>100 >100 >100 SARSCoV Vero >100 >100 >100
>100 .sup.aNR = Neutral Red.
TABLE-US-00006 TABLE 5 Summary of Antiviral Activity of
(2'R)-2'-deoxy-2'-fluoro- 2'-C-methylcytidine (2'R)-2'-deoxy-2'-
fluoro-2'-C- 2'-C- 2'-C- methylcytidine methylcytidine
methyladenosine Virus (EC.sub.90, .mu.M) (EC.sub.90, .mu.M)
(EC.sub.90, .mu.M) BVDVncp >22 0.5 1.2 BVDVcp >100 2 1.5 RSV
>100 >100 >100 HIV.sup.a >100 ND ND HBV >10 >10
ND Coronavirus 229E >100 ND ND ND = Not determined.
TABLE-US-00007 TABLE 6 Cytotoxicity Studies.sup.a
(2'R)-2'-deoxy-2'- fluoro-2'-C- 2'-C- methylcytidine
2'-C-methylcytidine methyladenosine Cell Line CC.sub.50, .mu.M
CC.sub.50, .mu.M CC.sub.50, .mu.M CloneA >100 >100 37 Huh7
>100 >100 30 HepG2 75 >100 58 MDBK >100 >100 PBM
>100 CEM >100 Vero >100 MRC-5 >100 .sup.aResults
determined using MTS assay.
TABLE-US-00008 TABLE 7 Mitochondrial Toxicity Study mtDNA Synthesis
Compound (IC.sub.50, .mu.M) Lactic Acid Increase
(2'R)-2'-deoxy-2'-fluoro-2'- >25 No effect .gtoreq. 33 .mu.M
C-methylcytidine 2'-C-methylcytidine >25 No effect .gtoreq. 33
.mu.M
TABLE-US-00009 TABLE 8 Preliminary PK Parameters in Rhesus Monkeys
Following a Single Oral Dose of
(2'R)-2'-deoxy-2'-fluoro-2'-C-methylcytidine at 33.3 mg/kg
Parameter Units Mean .+-. SD C.sub.max .mu.M 9.6 .+-. 2.7 T.sub.max
hours 2 .+-. 1 AUC.sub.0-last .mu.M.times.h 44.2 .+-. 22.2 T1/2
hours 3.9 .+-. 0.1 Bioavailability F % 21 .+-. 11
TABLE-US-00010 TABLE 9 Effect of Nucleoside Analogs on Human Bone
Marrow Cells BFU-E CFU-GM Compound (.beta.-D-analog) IC.sub.50
(.mu.M) 2'-fluoro-2'-C- 98.2 93.9 methylcytidine
2'-C-methylcytidine 20.1 13.2 AZT 0.08 0.95
* * * * *