U.S. patent application number 10/900008 was filed with the patent office on 2005-04-07 for purine nucleoside analogues for treating flaviviridae including hepatitis c.
Invention is credited to Dukhan, David, Gosselin, Gilles, Leroy, Frederic, Storer, Richard.
Application Number | 20050075309 10/900008 |
Document ID | / |
Family ID | 34102969 |
Filed Date | 2005-04-07 |
United States Patent
Application |
20050075309 |
Kind Code |
A1 |
Storer, Richard ; et
al. |
April 7, 2005 |
Purine nucleoside analogues for treating Flaviviridae including
hepatitis C
Abstract
This invention is directed to a method for treating a host,
especially a human, infected with hepatitis C, flavivirus and/or
pestivirus, comprising administering to that host an effective
amount of an anti-HCV biologically active pentofuranonucleoside
where the pentofuranonucleoside base is an optionally substituted
2-azapurine. The optionally substituted pentofuranonucleoside, or a
salt or prodrug thereof, may be administered alone or in
combination with one or more optionally substituted
pentofuranonucleosides or other anti-viral agents.
Inventors: |
Storer, Richard;
(Folkestone, GB) ; Gosselin, Gilles; (Montpellier,
FR) ; Dukhan, David; (Montpellier, FR) ;
Leroy, Frederic; (Jonquieres, FR) |
Correspondence
Address: |
KING & SPALDING LLP
191 PEACHTREE STREET, N.E.
ATLANTA
GA
30303-1763
US
|
Family ID: |
34102969 |
Appl. No.: |
10/900008 |
Filed: |
July 26, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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60490216 |
Jul 25, 2003 |
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Current U.S.
Class: |
514/47 ;
514/263.23; 514/269; 514/50; 514/81; 514/86 |
Current CPC
Class: |
C07H 19/052 20130101;
C07H 19/044 20130101; A61P 31/14 20180101; A61K 31/00 20130101;
C07H 19/056 20130101; A61P 43/00 20180101; C07H 19/04 20130101;
C07H 19/23 20130101; A61K 31/706 20130101; A61K 45/06 20130101 |
Class at
Publication: |
514/047 ;
514/050; 514/081; 514/086; 514/263.23; 514/269 |
International
Class: |
A61K 031/7076; A61K
031/7072; A61K 031/513; A61K 031/52 |
Claims
We claim:
1. A method of treating a host infected with a flavivirus or
pestivirus, comprising administering an effective amount of an
anti-pestivirus or anti-flavivirus biologically active
ribofuranonucleoside of Formula (I): 68or a pharmacologically
acceptable salt or prodrug thereof, wherein: R is H, mono-, di-, or
triphosphate, a stabilized phosphate, or phosphonate; X is O,
S[O].sub.n, CH.sub.2, CHOH, CH-alkyl, CH-alkenyl, CH-alkynyl,
C-dialkyl, CH--O-alkyl, CH--O-alkenyl, CH--O-alkynyl, CH--S-alkyl,
CH--S-alkenyl, CH--S-alkynyl, NH, N-alkyl, N-alkenyl, N-alkynyl,
S(O)N-alkyl, S(O)N-alkenyl, S(O)N-alkynyl, SCH-halogen, or
C-(halogen).sub.2, wherein alkyl, alkenyl or alkynyl may optionally
be substituted; n is 0-2; such than when X is CH.sub.2, CHOH,
CH-alkyl, CH-alkenyl, CH-alkynyl, C-dialkyl, CH--O-alkyl,
CH--O-alkenyl, CH--O-alkynyl, CH--S-alkyl, CH--S-alkenyl,
CH--S-alkynyl, CH-halogen, or C-(halogen).sub.2, then each R.sup.1
and R.sup.1' is independently H, OH, optionally substituted alkyl,
lower alkyl, azido, cyano, optionally substituted alkenyl or
alkynyl, --C(O)O-(alkyl), --C(O)O(lower alkyl), --C(O)O-(alkenyl),
--C(O)O-(alkynyl), --O(acyl), --O(lower acyl), --O(alkyl),
--O(lower alkyl), --O(alkenyl), --O(alkynyl), halogen, halogenated
alkyl, --NO.sub.2, --NH.sub.2, --NH(lower alkyl), --N(lower
alkyl).sub.2, --NH(acyl), --N(acyl).sub.2, --C(O)NH.sub.2,
--C(O)NH(alkyl), --C(O)N(alkyl).sub.2, S(O)N-alkyl, S(O)N-alkenyl,
S(O)N-alkynyl, SCH-halogen, wherein alkyl, alkenyl, and/or alkynyl
may optionally be substituted; and such that when X is O,
S[O].sub.n, NH, N-alkyl, N-alkenyl, N-alkynyl, S(O)N-alkyl,
S(O)N-alkenyl, S(O)N-alkynyl, or SCH-halogen, then each R.sup.1 and
R.sup.1+ is independently H, optionally substituted alkyl, lower
alkyl, azido, cyano, optionally substituted alkenyl or alkynyl,
--C(O)O-(alkyl), --C(O)O(lower alkyl), --C(O)O-(alkenyl),
--C(O)O-(alkynyl), halogenated alkyl, --C(O)NH.sub.2,
--C(O)NH(alkyl), --C(O)N(alkyl).sub.2, --C(H).dbd.N--NH.sub.2,
C(S)NH.sub.2, C(S)NH(alkyl), or C(S)N(alkyl).sub.2, wherein alkyl,
alkenyl and/or alkynyl may optionally be substituted; each R.sup.2
and R.sup.3 independently is OH, NH.sub.2, SH, F, Cl, Br, I, CN,
NO.sub.2, --C(O)NH.sub.2, --C(O)NH(alkyl), and
--C(O)N(alkyl).sub.2, N.sub.3, optionally substituted alkyl, lower
alkyl, optionally substituted alkenyl or alkynyl, halogenated
alkyl, --C(O)O-(alkyl), --C(O)O(lower alkyl), --C(O)O-(alkenyl),
--C(O)O-(alkynyl), --O(acyl), --O(alkyl), --O(alkenyl),
--O(alkynyl), --OC(O)NH.sub.2, NC, C(O)OH, SCN, OCN, --S(alkyl),
--S(alkenyl), --S(alkynyl), --NH(alkyl), --N(alkyl).sub.2,
--NH(alkenyl), --NH(alkynyl), an amino acid residue or derivative,
a prodrug or leaving group that provides OH in vivo, or an
optionally substituted 3-7 membered heterocyclic ring having O, S
and/or N independently as a heteroatom taken alone or in
combination; each R.sup.2' and R.sup.3' independently is H;
optionally substituted alkyl, alkenyl, or alkynyl; --C(O)O(alkyl),
--C(O)O(lower alkyl), --C(O)O(alkenyl), --C(O)O(alkynyl),
--C(O)NH.sub.2, --C(O)NH(alkyl), --C(O)N(alkyl).sub.2, --O(acyl),
--O(lower acyl), --O(alkyl), --O(lower alkyl), --O(alkenyl),
halogen, halogenated alkyl and particularly CF.sub.3, azido, cyano,
NO.sub.2, --S(alkyl), --S(alkenyl), --S(alkynyl), NH.sub.2,
--NH(alkyl), --N(alkyl).sub.2, --NH(alkenyl), --NH(alkynyl),
--NH(acyl), or --N(acyl).sub.2, and R.sub.3 at 3'-C may also be OH;
and Base is selected from the group consisting of: 6970wherein each
R' and R" independently is H, C.sub.1-6 alkyl, C.sub.2-6 alkenyl,
C.sub.2-6 alkynyl, halogen, halogenated alkyl, OH, CN, N.sub.3,
carboxy, CN.sub.4alkoxycarbonyl, NH.sub.2, C.sub.1-4 alkylamino,
di(C.sub.1-4 alkyl)amino, C.sub.1-6 alkoxy, C.sub.1-6
alkylsulfonyl, (C.sub.1-4 alkyl).sub.0-2 aminomethyl; each W is Cl,
Br, I, F, halogenated alkyl, alkoxy, OH, SH, O-alkyl, S-alkyl,
O-alkenyl, O-alkynyl, S-alkenyl, S-alkynyl, --OC(O)NR.sup.4R.sup.4,
O-acyl, S-acyl, CN, SCN, OCN, NO.sub.2, N.sub.3, NH.sub.2,
NH(alkyl), N(alkyl).sub.2, NH-cycloalkyl, NH-acyl, NH.dbd.NH,
CONH.sub.2, CONH(alkyl), or CON(alkyl).sub.2; and each R.sup.4 is
independently H, acyl, or C.sub.1-6 alkyl; each Z is O, S, NH,
N--OH, N--NH.sub.2, NH(alkyl), N(alkyl).sub.2, N-cycloalkyl,
alkoxy, CN, SCN, OCN, SH, NO.sub.2, NH.sub.2, N.sub.3, NH.dbd.NH,
NH(alkyl), N(alkyl).sub.2, CONH.sub.2, CONH(alkyl), or
CON(alkyl).sub.2; with the caveat that when X is S, then the
compound is not
5-(4-amino-imidazo[4,5-d][1,2,3]triazin-7-yl)-2-hydroxymethyl-tetrahydro--
thiophen-3-ol or
7-(4-hydroxy-5-hydroxy-methyl-tetrahydro-thiophen-2-yl)-3-
,7-dihydro-imidazo[4,5-d][1,2,3]triazin-4-one.
2. A method of treating a host infected with a flavivirus or
pestivirus, comprising administering an effective amount of an
anti-pestivirus or anti-flavivirus biologically active
ribofuranonucleoside of Formula (II): 71or a pharmacologically
acceptable salt or prodrug thereof, wherein: X* is CY.sup.3;
Y.sup.3 is hydrogen, alkyl, bromo, chloro, fluoro, iodo, azido,
cyano, alkenyl, alkynyl, --C(O)O(alkyl), --C(O)O(lower alkyl),
CF.sub.3, --CONH.sub.2, --CONH(alkyl), or --CON(alkyl).sub.2; R is
H, mono-, di-, or triphosphate, a stabilized phosphate, or
phosphonate; R.sup.1 is H, OH, optionally substituted alkyl, lower
alkyl, azido, cyano, optionally substituted alkenyl or alkynyl.
--C(O)O-(alkyl), --C(O)O(lower alkyl), --C(O)O-(alkenyl),
--C(O)O-(alkynyl), --O(acyl), --O(lower acyl), --O(alkyl),
--O(lower alkyl), --O(alkenyl), --O(alkynyl), halogen, halogenated
alkyl, --NO.sub.2, --NH.sub.2, --NH(lower alkyl), --N(lower
alkyl).sub.2, --NH(acyl), --N(acyl).sub.2, --C(O)NH.sub.2,
--C(O)NH(alkyl), or --C(O)N(alkyl).sub.2, wherein an optional
substitution on alkyl, alkenyl, and/or alkynyl may be one or more
halogen, hydroxy, alkoxy or alkylthio groups taken in any
combination; each R.sup.2 and R.sup.3 independently is OH,
NH.sub.2, F, Cl, Br, I, CN, NO.sub.2, --C(O)NH.sub.2,
--C(O)NH(alkyl), --C(O)N(alkyl).sub.2, N.sub.3, optionally
substituted alkyl, lower alkyl, optionally substituted alkenyl or
alkynyl, halogenated alkyl, --C(O)O-(alkyl), --C(O)O(lower alkyl),
--C(O)O-(alkenyl), --C(O)O-(alkynyl), an amino acid residue or
derivative, a prodrug or leaving group that provides OH in vivo, or
an optionally substituted 3-7 membered heterocyclic ring having O,
S and/or N independently as a heteroatom taken alone or in
combination; each R.sup.2' and R.sup.3' independently is H;
optionally substituted alkyl, alkenyl, or alkynyl; --C(O)O(alkyl),
--C(O)O(lower alkyl), --C(O)O(alkenyl), --C(O)O(alkynyl),
--C(O)NH.sub.2, --C(O)NH(alkyl), and --C(O)N(alkyl).sub.2,
--O(acyl), --O(lower acyl), --O(alkyl), --O(lower alkyl),
--O(alkenyl), halogen, halogenated alkyl and particularly CF.sub.3,
azido, cyano, NO.sub.2, --S(alkyl), --S(alkenyl), --S(alkynyl),
NH.sub.2, --NH(alkyl), --N(alkyl).sub.2, --NH(alkenyl),
--NH(alkynyl), --NH(acyl), or --N(acyl).sub.2, and R.sub.3 at 3'-C
may also be OH; and Base is selected from the group consisting of:
7273wherein each R' and R" independently is H, C.sub.1-6 alkyl,
C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, halogen, halogenated alkyl,
OH, CN, N.sub.3, carboxy, C.sub.1-4alkoxycarbonyl, NH.sub.2,
C.sub.1-4 alkylamino, di(C.sub.1-4 alkyl)amino, C.sub.1-6 alkoxy,
C.sub.1-6 alkylsulfonyl, (C.sub.1-4 alkyl).sub.0-2 aminomethyl;
each W is Cl, Br, I, F, halogenated alkyl, alkoxy, OH, SH, O-alkyl,
S-alkyl, O-alkenyl, O-alkynyl, S-alkenyl, S-alkynyl,
--OC(O)NR.sup.4R.sup.4, O-acyl, S-acyl, CN, SCN, OCN, NO.sub.2,
N.sub.3, NH.sub.2, NH(alkyl), N(alkyl).sub.2, NH-cycloalkyl,
NH-acyl, NH.dbd.NH, CONH.sub.2, CONH(alkyl), or CON(alkyl).sub.2;
and each R.sup.4 is independently H, acyl, or C.sub.1-6 alkyl; each
Z is O, S, NH, N--OH, N--NH.sub.2, NH(alkyl), N(alkyl).sub.2,
N-cycloalkyl, alkoxy, CN, SCN, OCN, SH, NO.sub.2, NH.sub.2,
N.sub.3, NH.dbd.NH, NH(alkyl), N(alkyl).sub.2, CONH.sub.2,
CONH(alkyl), or CON(alkyl).sub.2.
3. A method of treating a host infected with a flavivirus or
pestivirus, comprising administering an effective amount of an
anti-pestivirus or anti-flavivirus biologically active
ribofuranonucleoside of Formula (III): 74or a pharmacologically
acceptable salt or prodrug thereof, wherein: each R, R.sup.2*, and
R.sup.3* independently is H, mono-, di-, or triphosphate, a
stabilized phosphate, or phosphonate; optionally substituted alkyl,
lower alkyl, optionally substituted alkenyl or alkynyl, acyl,
--C(O)-(alkyl), --C(O)(lower alkyl), --C(O)-(alkenyl),
--C(O)-(alkynyl), lipid, phospholipid, carbohydrate, peptide,
cholesterol, an amino acid residue or derivative, or other
pharmaceutically acceptable leaving group that is capable of
providing H or phosphate when administered in vivo; X is O,
S[O].sub.n, CH.sub.2, CHOH, CH-alkyl, CH-alkenyl, CH-alkynyl,
C-dialkyl, CH--O-alkyl, CH--O-alkenyl, CH--O-alkynyl, CH--S-alkyl,
CH--S-alkenyl, CH--S-alkynyl, NH, N-alkyl, N-alkenyl, N-alkynyl,
S(O)N-alkyl, S(O)N-alkenyl, S(O)N-alkynyl, SCH-halogen, or
C-(halogen).sub.2, wherein alkyl, alkenyl or alkynyl optionally may
be substituted; n is 0-2; each R.sup.2' independently is H;
optionally substituted alkyl, alkenyl, or alkynyl; --C(O)O(alkyl),
--C(O)O(lower alkyl), --C(O)O(alkenyl), --C(O)O(alkynyl),
--C(O)NH.sub.2, --C(O)NH(alkyl), --C(O)N(alkyl).sub.2, --OH,
--O(acyl), --O(lower acyl), --O(alkyl), --O(lower alkyl),
--O(alkenyl), halogen, halogenated alkyl and particularly CF.sub.3,
azido, cyano, NO.sub.2, --S(alkyl), --S(alkenyl), --S(alkynyl),
NH.sub.2, --NH(alkyl), --N(alkyl).sub.2, --NH(alkenyl),
--NH(alkynyl), --NH(acyl), or --N(acyl).sub.2; and Base is selected
from the group consisting of: 7576wherein each R' and R"
independently is H, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6
alkynyl, halogen, halogenated alkyl, OH, CN, N.sub.3, carboxy,
C.sub.1-4alkoxycarbonyl, NH.sub.2, C.sub.1-4 alkylamino,
di(C.sub.1-4 alkyl)amino, C.sub.1-6 alkoxy, C.sub.1-6
alkylsulfonyl, (C.sub.1-4 alkyl).sub.0-2 aminomethyl; each W is Cl,
Br, I, F, halogenated alkyl, alkoxy, OH, SH, O-alkyl, S-alkyl,
O-alkenyl, O-alkynyl, S-alkenyl, S-alkynyl, --OC(O)NR.sup.4R.sup.4,
O-acyl, S-acyl, CN, SCN, OCN, NO.sub.2, N.sub.3, NH.sub.2,
NH(alkyl), N(alkyl).sub.2, NH-cycloalkyl, NH-acyl, NH.dbd.NH,
CONH.sub.2, CONH(alkyl), or CON(alkyl).sub.2; and each R.sup.4 is
independently H, acyl, or C.sub.1-6 alkyl; each Z is O, S, NH,
N--OH, N--NH.sub.2, NH(alkyl), N(alkyl).sub.2, N-cycloalkyl,
alkoxy, CN, SCN, OCN, SH, NO.sub.2, NH.sub.2, N.sub.3, NH.dbd.NH,
NH(alkyl), N(alkyl).sub.2, CONH.sub.2, CONH(alkyl), or
CON(alkyl).sub.2.
4. The method of claim 3, wherein R.sup.2' is an optionally
substituted alkyl, alkenyl, or alkynyl; halogen, halogenated alkyl,
CH.sub.3, CF.sub.3, azido, or cyano.
5. The method of claim 3, wherein R.sup.2' is an optionally
substituted alkyl, alkenyl, or alkynyl; halogen, halogenated alkyl,
CH.sub.3, or CF.sub.3.
6. The method of claim 3, wherein R.sup.2' is CH.sub.3 or
CF.sub.3.
7. The method of claim 3, wherein each R, R.sup.2*, and R.sup.3* is
independently H, mono-, di-, or triphosphate, a stabilized
phosphate, or phosphonate.
8. The method of claim 3, wherein each R, R.sup.2*, and R.sup.3* is
independently H.
9. The method of claim 3, wherein each R, R.sup.2*, and R.sup.3* is
independently H, acyl, or an amino acid acyl residue.
10. The method of claim 3, wherein X is O or S.
11. The method of claim 3, wherein X is O.
12. A method of treating a host infected with a flavivirus or
pestivirus, comprising administering an effective amount of an
anti-pestivirus or anti-flavivirus biologically active
ribofuranonucleoside of Formula (IV): 77or a pharmacologically
acceptable salt or prodrug thereof, wherein: each R, R.sup.2*, and
R.sup.3* independently is H, mono, di, or triphosphate, a
stabilized phosphate, or phosphonate; optionally substituted alkyl,
lower alkyl, optionally substituted alkenyl or alkynyl, acyl,
--C(O)-(alkyl), --C(O)(lower alkyl), --C(O)-(alkenyl),
--C(O)-(alkynyl), lipid, phospholipid, carbohydrate, peptide,
cholesterol, an amino acid residue or derivative, or other
pharmaceutically acceptable leaving group that is capable of
providing H or phosphate when administered in vivo; X is O,
S[O].sub.n, CH.sub.2, CHOH, CH-alkyl, CH-alkenyl, CH-alkynyl,
C-dialkyl, CH--O-alkyl, CH--O-alkenyl, CH--O-alkynyl, CH--S-alkyl,
CH--S-alkenyl, CH--S-alkynyl, NH, N-alkyl, N-alkenyl, N-alkynyl,
S(O)N-alkyl, S(O)N-alkenyl, S(O)N-alkynyl, SCH-halogen, or
C-(halogen).sub.2, wherein alkyl, alkenyl or alkynyl optionally may
be substituted; n is 0-2; each R.sup.3' independently is H;
optionally substituted alkyl, alkenyl, or alkynyl; --C(O)O(alkyl),
--C(O)O(lower alkyl), --C(O)O(alkenyl), --C(O)O(alkynyl),
--C(O)NH.sub.2, --C(O)NH(alkyl), --C(O)N(alkyl).sub.2, --OH,
--O(acyl), --O(lower acyl), --O(alkyl), --O(lower alkyl),
--O(alkenyl), halogen, halogenated alkyl and particularly CF.sub.3,
azido, cyano, NO.sub.2, --S(alkyl), --S(alkenyl), --S(alkynyl),
NH.sub.2, --NH(alkyl), --N(alkyl).sub.2, --NH(alkenyl),
--NH(alkynyl), --NH(acyl), or --N(acyl).sub.2; and Base is selected
from the group consisting of: 7879wherein each R' and R"
independently is H, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6
alkynyl, halogen, halogenated alkyl, OH, CN, N.sub.3, carboxy,
C.sub.1-4alkoxycarbonyl, NH.sub.2, C.sub.1-4 alkylamino,
di(C.sub.1-4 alkyl)amino, C.sub.1-6 alkoxy, C.sub.1-6
alkylsulfonyl, (C.sub.1-4 alkyl).sub.0-2 aminomethyl; each W is Cl,
Br, I, F, halogenated alkyl, alkoxy, OH, SH, O-alkyl, S-alkyl,
O-alkenyl, O-alkynyl, S-alkenyl, S-alkynyl, --OC(O)NR.sup.4R.sup.4,
O-acyl, S-acyl, CN, SCN, OCN, NO.sub.2, N.sub.3, NH.sub.2,
NH(alkyl), N(alkyl).sub.2, NH-cycloalkyl, NH-acyl, NH.dbd.NH,
CONH.sub.2, CONH(alkyl), or CON(alkyl).sub.2; and each R.sup.4 is
independently H, acyl, or C.sub.1-6 alkyl; each Z is O, S, NH,
N--OH, N--NH.sub.2, NH(alkyl), N(alkyl).sub.2, N-cycloalkyl,
alkoxy, CN, SCN, OCN, SH, NO.sub.2, NH.sub.2, N.sub.3, NH.dbd.NH,
NH(alkyl), N(alkyl).sub.2, CONH.sub.2, CONH(alkyl), or
CON(alkyl).sub.2.
13. The method of claim 12, wherein R.sup.3' is an optionally
substituted alkyl, alkenyl, or alkynyl; halogen, halogenated alkyl,
CH.sub.3, CF.sub.3, azido, or cyano.
14. The method of claim 12, wherein R.sup.3' is an optionally
substituted alkyl, alkenyl, or alkynyl; halogen, halogenated alkyl,
CH.sub.3, or CF.sub.3.
15. The method of claim 12, wherein R.sup.3' is CH.sub.3 or
CF.sub.3.
16. The method of claim 12, wherein each R, R.sup.2*, and R.sup.3*
is independently H, mono-, di-, or triphosphate, a stabilized
phosphate, or phosphonate.
17. The method of claim 12, wherein each R, R.sup.2*, and R.sup.3*
is independently H.
18. The method of claim 12, wherein each R, R.sup.2*, and R.sup.3*
is independently H, acyl, or an amino acid acyl residue.
19. The method of claim 12, wherein X is O or S.
20. The method of claim 12, wherein X is O.
21. The method of one of claims 3 or 12 wherein the host is a
mammal.
22. The method of claim 21, wherein the mammal is a human.
23. The method of one of claims 3 or 12, further comprising
administering an antivirally effective amount of the compound, or a
pharmaceutically acceptable salt or prodrug thereof, in combination
or alternation with one or more additional antivirally effective
agents.
24. The method of claim 23 wherein the additional antivirally
effective agent is selected from the group consisting of an
interferon, ribavirin, an interleukin, an NS3 protease inhibitor, a
cysteine protease inhibitor, phenanthrenequinone, a thiazolidine
derivative, a thiazolidine and a benzanilide, a helicase inhibitor,
a polymerase inhibitor, a nucleotide analogue, gliotoxin,
cerulenin, an antisense phosphorothioate oligodeoxynucleotide, an
inhibitor of IRES-dependent translation, and a ribozyme.
25. The method of claim 24, wherein the additional antivirally
effective agent is an interferon.
26. The method of claim 25 wherein the additional antivirally
effective agent is selected from the group consisting of pegylated
interferon alpha 2a, interferon alphacon-1, natural interferon,
albuferon, interferon beta-1a, omega interferon, interferon alpha,
interferon gamma, interferon tau, interferon delta and interferon
gamma-1b.
27. The method of one of claims 3 and 12, wherein the compound is
in the form of a dosage unit.
28. The method of claim 27 wherein the dosage unit contains 50 to
1000 mg of the compound.
29. The method of claim 28, wherein the said dosage unit is a
tablet or capsule.
30. The method of one of claims 3 or 12, wherein the compound is in
substantially pure form.
31. The method of claim 30 wherein the compound is at least 90% by
weight of the .beta.-D-isomer.
32. The method of claim 30 wherein the compound is at least 95% by
weight of the .beta.-D-isomer.
33. The method of claim 30 wherein the compound is at least 90% by
weight of the .beta.-L-isomer.
34. The method of claim 30 wherein the compound is at least 95% by
weight of the .beta.-L-isomer.
35. A compound of the general structure of Formula (I): 80or a
pharmacologically acceptable salt or prodrug thereof, wherein: R is
H, mono-, di-, or triphosphate, a stabilized phosphate, or
phosphonate; X is O, S[O].sub.n, CH.sub.2, CHOH, CH-alkyl,
CH-alkenyl, CH-alkynyl, C-dialkyl, CH--O-alkyl, CH--O-alkenyl,
CH--O-alkynyl, CH--S-alkyl, CH--S-alkenyl, CH--S-alkynyl, NH,
N-alkyl, N-alkenyl, N-alkynyl, S(O)N-alkyl, S(O)N-alkenyl,
S(O)N-alkynyl, SCH-halogen, or C-(halogen).sub.2, wherein alkyl,
alkenyl or alkynyl may optionally be substituted; n is 0-2; such
than when X is CH.sub.2, CHOH, CH-alkyl, CH-alkenyl, CH-alkynyl,
C-dialkyl, CH--O-alkyl, CH--O-alkenyl, CH--O-alkynyl, CH--S-alkyl,
CH--S-alkenyl, CH--S-alkynyl, CH-halogen, or C-(halogen).sub.2,
then each R.sup.1 and R.sup.1' is independently H, OH, optionally
substituted alkyl, lower alkyl, azido, cyano, optionally
substituted alkenyl or alkynyl, --C(O)O-(alkyl), --C(O)O(lower
alkyl), --C(O)O-(alkenyl), --C(O)O-(alkynyl), --O(acyl), --O(lower
acyl), --O(alkyl), --O(lower alkyl), --O(alkenyl), --O(alkynyl),
halogen, halogenated alkyl, --NO.sub.2, --NH.sub.2, --NH(lower
alkyl), --N(lower alkyl).sub.2, --NH(acyl), --N(acyl).sub.2,
--C(O)NH.sub.2, --C(O)NH(alkyl), --C(O)N(alkyl).sub.2, S(O)N-alkyl,
S(O)N-alkenyl, S(O)N-alkynyl, SCH-halogen, wherein alkyl, alkenyl,
and/or alkynyl may optionally be substituted; and such that when X
is O, S[O].sub.n, NH, N-alkyl, N-alkenyl, N-alkynyl, S(O)N-alkyl,
S(O)N-alkenyl, S(O)N-alkynyl, or SCH-halogen, then each R.sup.1 and
R.sup.1' is independently H, optionally substituted alkyl, lower
alkyl, azido, cyano, optionally substituted alkenyl or alkynyl,
--C(O)O-(alkyl), --C(O)O(lower alkyl), --C(O)O-(alkenyl),
--C(O)O-(alkynyl), halogenated alkyl, --C(O)NH.sub.2,
--C(O)NH(alkyl), --C(O)N(alkyl).sub.2, --C(H).dbd.N--NH.sub.2,
C(S)NH.sub.2, C(S)NH(alkyl), or C(S)N(alkyl).sub.2, wherein alkyl,
alkenyl and/or alkynyl may optionally be substituted; each R.sup.2
and R.sup.3 independently is OH, NH.sub.2, SH, F, Cl, Br, I, CN,
NO.sub.2, --C(O)NH.sub.2, --C(O)NH(alkyl), and
--C(O)N(alkyl).sub.2, N.sub.3, optionally substituted alkyl, lower
alkyl, optionally substituted alkenyl or alkynyl, halogenated
alkyl, --C(O)O-(alkyl), --C(O)O(lower alkyl), --C(O)O-(alkenyl),
--C(O)O-(alkynyl), --O(acyl), --O(alkyl), --O(alkenyl),
--O(alkynyl), --OC(O)NH.sub.2, NC, C(O)OH, SCN, OCN, --S(alkyl),
--S(alkenyl), --S(alkynyl), --NH(alkyl), --N(alkyl).sub.2,
--NH(alkenyl), --NH(alkynyl), an amino acid residue or derivative,
a prodrug or leaving group that provides OH in vivo, or an
optionally substituted 3-7 membered heterocyclic ring having O, S
and/or N independently as a heteroatom taken alone or in
combination; each R.sup.2' and R.sup.3, independently is H;
optionally substituted alkyl, alkenyl, or alkynyl; --C(O)O(alkyl),
--C(O)O(lower alkyl), --C(O)O(alkenyl), --C(O)O(alkynyl),
--C(O)NH.sub.2, --C(O)NH(alkyl), --C(O)N(alkyl).sub.2, --O(acyl),
--O(lower acyl), --O(alkyl), --O(lower alkyl), --O(alkenyl),
halogen, halogenated alkyl and particularly CF.sub.3, azido, cyano,
NO.sub.2, --S(alkyl), --S(alkenyl), --S(alkynyl), NH.sub.2,
--NH(alkyl), --N(alkyl).sub.2, --NH(alkenyl), --NH(alkynyl),
--NH(acyl), or --N(acyl).sub.2, and R.sub.3 at 3'-C may also be OH;
and Base is selected from the group consisting of: 8182wherein each
R' and R" independently is H, C.sub.1-6 alkyl, C.sub.2-6 alkenyl,
C.sub.2-6 alkynyl, halogen, halogenated alkyl, OH, CN, N.sub.3,
carboxy, C.sub.1-4alkoxycarbonyl, NH.sub.2, C.sub.1-4 alkylamino,
di(C.sub.1-4 alkyl)amino, C.sub.1-6 alkoxy, C.sub.1-6
alkylsulfonyl, (C.sub.1-4 alkyl).sub.0-2 aminomethyl; each W is Cl,
Br, I, F, halogenated alkyl, alkoxy, OH, SH, O-alkyl, S-alkyl,
O-alkenyl, O-alkynyl, S-alkenyl, S-alkynyl, --OC(O)NR.sup.4R.sup.4,
O-acyl, S-acyl, CN, SCN, OCN, NO.sub.2, N.sub.3, NH.sub.2,
NH(alkyl), N(alkyl).sub.2, NH-cycloalkyl, NH-acyl, NH.dbd.NH,
CONH.sub.2, CONH(alkyl), or CON(alkyl).sub.2; and each R.sup.4 is
independently H, acyl, or C.sub.1-6 alkyl; each Z is O, S, NH,
N--OH, N--NH.sub.2, NH(alkyl), N(alkyl).sub.2, N-cycloalkyl,
alkoxy, CN, SCN, OCN, SH, NO.sub.2, NH.sub.2, N.sub.3, NH.dbd.NH,
NH(alkyl), N(alkyl).sub.2, CONH.sub.2, CONH(alkyl), or
CON(alkyl).sub.2; with the caveat that when X is S, then the
compound is not
5-(4-amino-imidazo[4,5-d][1,2,3]triazin-7-yl)-2-hydroxymethyl-tetrahydro--
thiophen-3-ol or
7-(4-hydroxy-5-hydroxy-methyl-tetrahydro-thiophen-2-yl)-3-
,7-dihydro-imidazo[4,5-d][1,2,3]triazin-4-one.
36. A compound of the general structure of Formula (II): 83or a
pharmacologically acceptable salt or prodrug thereof, wherein: X*
is CY.sup.3; Y.sup.3 is hydrogen, alkyl, bromo, chloro, fluoro,
iodo, azido, cyano, alkenyl, alkynyl, --C(O)O(alkyl), --C(O)O(lower
alkyl), CF.sub.3, --CONH.sub.2, --CONH(alkyl), or
--CON(alkyl).sub.2; R is H, mono-, di-, or triphosphate, a
stabilized phosphate, or phosphonate; R.sup.1 is H, OH, optionally
substituted alkyl, lower alkyl, azido, cyano, optionally
substituted alkenyl or alkynyl, --C(O)O-(alkyl), --C(O)O(lower
alkyl), --C(O)O-(alkenyl), --C(O)O-(alkynyl), --O(acyl), --O(lower
acyl), --O(alkyl), --O(lower alkyl), --O(alkenyl), --O(alkynyl),
halogen, halogenated alkyl, --NO.sub.2, --NH.sub.2, --NH(lower
alkyl), --N(lower alkyl).sub.2, --NH(acyl), --N(acyl).sub.2,
--C(O)NH.sub.2, --C(O)NH(alkyl), or --C(O)N(alkyl).sub.2, wherein
an optional substitution on alkyl, alkenyl, and/or alkynyl may be
one or more halogen, hydroxy, alkoxy or alkylthio groups taken in
any combination; each R.sup.2 and R.sup.3 independently is OH,
NH.sub.2, F, Cl, Br, I, CN, NO.sub.2, --C(O)NH.sub.2,
--C(O)NH(alkyl), --C(O)N(alkyl).sub.2, N.sub.3, optionally
substituted alkyl, lower alkyl, optionally substituted alkenyl or
alkynyl, halogenated alkyl, --C(O)O-(alkyl), --C(O)O(lower alkyl),
--C(O)O-(alkenyl), --C(O)O-(alkynyl), an amino acid residue or
derivative, a prodrug or leaving group that provides OH in vivo, or
an optionally substituted 3-7 membered heterocyclic ring having O,
S and/or N independently as a heteroatom taken alone or in
combination; each R.sup.2' and R.sup.3' independently is H;
optionally substituted alkyl, alkenyl, or alkynyl; --C(O)O(alkyl),
--C(O)O(lower alkyl), --C(O)O(alkenyl), --C(O)O(alkynyl),
--C(O)NH.sub.2, --C(O)NH(alkyl), and --C(O)N(alkyl).sub.2,
--O(acyl), --O(lower acyl), --O(alkyl), --O(lower alkyl),
--O(alkenyl), halogen, halogenated alkyl and particularly CF.sub.3,
azido, cyano, NO.sub.2, --S(alkyl), --S(alkenyl), --S(alkynyl),
NH.sub.2, --NH(alkyl), --N(alkyl).sub.2, --NH(alkenyl),
--NH(alkynyl), --NH(acyl), or --N(acyl).sub.2, and R.sub.3 at 3'-C
may also be OH; and Base is selected from the group consisting of:
8485wherein each R' and R" independently is H, C.sub.1-6 alkyl,
C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, halogen, halogenated alkyl,
OH, CN, N.sub.3, carboxy, C.sub.1-4alkoxycarbonyl, NH.sub.2,
C.sub.1-4 alkylamino, di(C.sub.1-4 alkyl)amino, C.sub.1-6 alkoxy,
C.sub.1-6 alkylsulfonyl, (C.sub.1-4 alkyl).sub.0-2 aminomethyl;
each W is Cl, Br, I, F, halogenated alkyl, alkoxy, OH, SH, O-alkyl,
S-alkyl, O-alkenyl, O-alkynyl, S-alkenyl, S-alkynyl,
--OC(O)NR.sup.4R.sup.4, O-acyl, S-acyl, CN, SCN, OCN, NO.sub.2,
N.sub.3, NH.sub.2, NH(alkyl), N(alkyl).sub.2, NH-cycloalkyl,
NH-acyl, NH.dbd.NH, CONH.sub.2, CONH(alkyl), or CON(alkyl).sub.2;
and each R.sup.4 is independently H, acyl, or C.sub.1-6 alkyl; each
Z is O, S, NH, N--OH, N--NH.sub.2, NH(alkyl), N(alkyl).sub.2,
N-cycloalkyl, alkoxy, CN, SCN, OCN, SH, NO.sub.2, NH.sub.2,
N.sub.3, NH.dbd.NH, NH(alkyl), N(alkyl).sub.2, CONH.sub.2,
CONH(alkyl), or CON(alkyl).sub.2.
37. A compound of the general structure of Formula (III): 86or a
pharmacologically acceptable salt or prodrug thereof, wherein: each
R, R.sup.2*, and R.sup.3* independently is H, mono-, di-, or
triphosphate, a stabilized phosphate, or phosphonate; optionally
substituted alkyl, lower alkyl, optionally substituted alkenyl or
alkynyl, acyl, --C(O)-(alkyl), --C(O)(lower alkyl),
--C(O)-(alkenyl), --C(O)-(alkynyl), lipid, phospholipid,
carbohydrate, peptide, cholesterol, an amino acid residue or
derivative, or other pharmaceutically acceptable leaving group that
is capable of providing H or phosphate when administered in vivo; X
is O, S[O].sub.n, CH.sub.2, CHOH, CH-alkyl, CH-alkenyl, CH-alkynyl,
C-dialkyl, CH--O-alkyl, CH--O-alkenyl, CH--O-alkynyl, CH--S-alkyl,
CH--S-alkenyl, CH--S-alkynyl, NH, N-alkyl, N-alkenyl, N-alkynyl,
S(O)N-alkyl, S(O)N-alkenyl, S(O)N-alkynyl, SCH-halogen, or
C-(halogen).sub.2, wherein alkyl, alkenyl or alkynyl optionally may
be substituted; n is 0-2; each R.sup.2' independently is H;
optionally substituted alkyl, alkenyl, or alkynyl; --C(O)O(alkyl),
--C(O)O(lower alkyl), --C(O)O(alkenyl), --C(O)O(alkynyl),
--C(O)NH.sub.2, --C(O)NH(alkyl), --C(O)N(alkyl).sub.2, --OH,
--O(acyl), --O(lower acyl), --O(alkyl), --O(lower alkyl),
--O(alkenyl), halogen, halogenated alkyl and particularly CF.sub.3,
azido, cyano, NO.sub.2, --S(alkyl), --S(alkenyl), --S(alkynyl),
NH.sub.2, --NH(alkyl), --N(alkyl).sub.2, --NH(alkenyl),
--NH(alkynyl), --NH(acyl), or --N(acyl).sub.2; and Base is selected
from the group consisting of: 8788wherein each R' and R"
independently is H, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6
alkynyl, halogen, halogenated alkyl, OH, CN, N.sub.3, carboxy,
C.sub.1-4alkoxycarbonyl, NH.sub.2, C.sub.1-4 alkylamino,
di(C.sub.1-4 alkyl)amino, C.sub.1-6 alkoxy, C.sub.1-6
alkylsulfonyl, (C.sub.1-4 alkyl).sub.0-2 aminomethyl; each W is Cl,
Br, I, F, halogenated alkyl, alkoxy, OH, SH, O-alkyl, S-alkyl,
O-alkenyl, O-alkynyl, S-alkenyl, S-alkynyl, --OC(O)NR.sup.4R.sup.4,
O-acyl, S-acyl, CN, SCN, OCN, NO.sub.2, N.sub.3, NH.sub.2,
NH(alkyl), N(alkyl).sub.2, NH-cycloalkyl, NH-acyl, NH.dbd.NH,
CONH.sub.2, CONH(alkyl), or CON(alkyl).sub.2; and each R.sup.4 is
independently H, acyl, or C.sub.1-6 alkyl; each Z is O, S, NH,
N--OH, N--NH.sub.2, NH(alkyl), N(alkyl).sub.2, N-cycloalkyl,
alkoxy, CN, SCN, OCN, SH, NO.sub.2, NH.sub.2, N.sub.3, NH.dbd.NH,
NH(alkyl), N(alkyl).sub.2, CONH.sub.2, CONH(alkyl), or
CON(alkyl).sub.2.
38. The compound of claim 37, wherein R.sup.2' is an optionally
substituted alkyl, alkenyl, or alkynyl; halogen, halogenated alkyl,
CH.sub.3, CF.sub.3, azido, or cyano.
39. The compound of claim 37, wherein R.sup.2' is an optionally
substituted alkyl, alkenyl, or alkynyl; halogen, halogenated alkyl,
CH.sub.3, or CF.sub.3.
40. The compound of claim 37, wherein R.sup.2' is CH.sub.3 or
CF.sub.3.
41. The compound of claim 37, wherein each R, R.sup.2*, and
R.sup.3* is independently H, mono-, di-, or triphosphate, a
stabilized phosphate, or phosphonate.
42. The compound of claim 37, wherein each R, R.sup.2*, and
R.sup.3* is independently H.
43. The compound of claim 37, wherein each R, R.sup.2*, and
R.sup.3* is independently H, acyl, or an amino acid acyl
residue.
44. The compound of claim 37, wherein X is O or S.
45. The compound of claim 37, wherein X is O.
46. A compound of the general structure of Formula (IV): 89or a
pharmacologically acceptable salt or prodrug thereof, wherein: each
R, R.sup.2*, and R.sup.3* independently is H, mono-, di-, or
triphosphate, a stabilized phosphate, or phosphonate; optionally
substituted alkyl, lower alkyl, optionally substituted alkenyl or
alkynyl, acyl, --C(O)-(alkyl), --C(O)(lower alkyl),
--C(O)-(alkenyl), --C(O)-(alkynyl), lipid, phospholipid,
carbohydrate, peptide, cholesterol, an amino acid residue or
derivative, or other pharmaceutically acceptable leaving group that
is capable of providing H or phosphate when administered in vivo; X
is O, S[O].sub.n, CH.sub.2, CHOH, CH-alkyl, CH-alkenyl, CH-alkynyl,
C-dialkyl, CH--O-alkyl, CH--O-alkenyl, CH--O-alkynyl, CH--S-alkyl,
CH--S-alkenyl, CH--S-alkynyl, NH, N-alkyl, N-alkenyl, N-alkynyl,
S(O)N-alkyl, S(O)N-alkenyl, S(O)N-alkynyl, SCH-halogen, or
C-(halogen).sub.2, wherein alkyl, alkenyl or alkynyl optionally may
be substituted; n is 0-2; each R.sup.3' independently is H;
optionally substituted alkyl, alkenyl, or alkynyl; --C(O)O(alkyl),
--C(O)O(lower alkyl), --C(O)O(alkenyl), --C(O)O(alkynyl),
--C(O)NH.sub.2, --C(O)NH(alkyl), --C(O)N(alkyl).sub.2, --OH,
--O(acyl), --O(lower acyl), --O(alkyl), --O(lower alkyl),
--O(alkenyl), halogen, halogenated alkyl and particularly CF.sub.3,
azido, cyano, NO.sub.2, --S(alkyl), --S(alkenyl), --S(alkynyl),
NH.sub.2, --NH(alkyl), --N(alkyl).sub.2, --NH(alkenyl),
--NH(alkynyl), --NH(acyl), or --N(acyl).sub.2; and Base is selected
from the group consisting of: 9091wherein each R' and R"
independently is H, C.sub.1-6 alkyl, C.sub.2.sub.6 alkenyl,
C.sub.2-6 alkynyl, halogen, halogenated alkyl, OH, CN, N.sub.3,
carboxy, C.sub.1-4alkoxycarbonyl, NH.sub.2, C.sub.1-4 alkylamino,
di(C.sub.1-4 alkyl)amino, C.sub.1-6 alkoxy, C.sub.1-6
alkylsulfonyl, (C.sub.1-4 alkyl).sub.0-2 aminomethyl; each W is Cl,
Br, I, F, halogenated alkyl, alkoxy, OH, SH, O-alkyl, S-alkyl,
O-alkenyl, O-alkynyl, S-alkenyl, S-alkynyl, --OC(O)NR.sup.4R.sup.4,
O-acyl, S-acyl, CN, SCN, OCN, NO.sub.2, N.sub.3, NH.sub.2,
NH(alkyl), N(alkyl).sub.2, NH-cycloalkyl, NH-acyl, NH.dbd.NH,
CONH.sub.2, CONH(alkyl), or CON(alkyl).sub.2; and each R.sup.4 is
independently H, acyl, or C.sub.1-6 alkyl; each Z is O, S, NH,
N--OH, N--NH.sub.2, NH(alkyl), N(alkyl).sub.2, N-cycloalkyl,
alkoxy, CN, SCN, OCN, SH, NO.sub.2, NH.sub.2, N.sub.3, NH.dbd.NH,
NH(alkyl), N(alkyl).sub.2, CONH.sub.2, CONH(alkyl), or
CON(alkyl).sub.2.
47. The compound of claim 46, wherein R.sup.3' is an optionally
substituted alkyl, alkenyl, or alkynyl; halogen, halogenated alkyl,
CH.sub.3, CF.sub.3, azido, or cyano.
48. The compound of claim 46, wherein R.sup.3' is an optionally
substituted alkyl, alkenyl, or alkynyl; halogen, halogenated alkyl,
CH.sub.3, or CF.sub.3.
49. The compound of claim 46, wherein R.sup.3' is CH.sub.3 or
CF.sub.3.
50. The compound of claim 46, wherein each R, R.sup.2*, and
R.sup.3* is independently H, mono-, di-, or triphosphate, a
stabilized phosphate, or phosphonate.
51. The compound of claim 46, wherein each R, R.sup.2*, and
R.sup.3* is independently H.
52. The compound of claim 46, wherein each R, R.sup.2*, and
R.sup.3* is independently H, acyl, or an amino acid acyl
residue.
53. The compound of claim 46, wherein X is O or S.
54. The compound of claim 46, wherein X is O.
55. A pharmaceutical composition comprising an anti-virally
effective amount of a compound of one of claims 37 or 46,
optionally with a pharmaceutically acceptable carrier, diluent or
excipient.
56. The pharmaceutical composition of claim 55 wherein the
compound, salt or prodrug thereof is in the form of a dosage
unit.
57. The pharmaceutical composition of claim 56 wherein the dosage
unit contains from about 0.01 to about 50 mg of the compound.
58. The pharmaceutical composition of claim 57, wherein said dosage
unit is a tablet or capsule.
59. The pharmaceutical composition of claim 55, further comprising
one or more additional anti-virally effective agents.
60. The pharmaceutical composition of claim 59, wherein the
additional anti-virally agent is selected from the group consisting
of an interferon, ribavirin, an interleukin, an NS3 protease
inhibitor, a cysteine protease inhibitor, a thiazolidine
derivative, a thiazolidine and a benzanilide, phenanthrenequinone,
a helicase inhibitor, a polymerase inhibitor, a nucleotide
analogue, gliotoxin, cerulenin, an antisense oligodeoxynucleotide,
an inhibitor of IRES-dependent translation, and a ribozyme.
61. The pharmaceutical composition of claim 60 wherein the
additional anti-virally effective agent is an interferon.
62. The pharmaceutical composition of claim 61, wherein the
additional anti-virally effective agent is selected from the group
consisting of pegylated interferon alpha 2a, interferon alphacon-1,
natural interferon, albuferon, interferon beta-1a, omega
interferon, interferon alpha, interferon gamma, interferon tau,
interferon delta and interferon gamma-1b.
63. The pharmaceutical composition of one of claims 37 or 46,
wherein the compound is in substantially pure form.
64. The pharmaceutical composition of claim 63, wherein the
compound is at least 90% by weight of the .beta.-D-isomer.
65. The pharmaceutical composition of claim 63, wherein the
compound is at least 95% by weight of the .beta.-D-isomer.
66. The pharmaceutical composition of claim 63 wherein the compound
is at least 90% by weight of the .beta.-L-isomer.
67. The pharmaceutical composition of claim 63 wherein the compound
is at least 95% by weight of the .beta.-L-isomer.
Description
[0001] This application claims priority to U.S. Provisional
Applicaton No. 60/490,216, filed Jul. 25, 2003.
FIELD OF THE INVENTION
[0002] This invention is in the area of pharmaceutical chemistry,
and, in particular, in the area of purine nucleosides, their
syntheses, and their use as anti-Flaviviridae agents in the
treatment of hosts infected with Flaviviridae and especially with
Hepatitis C.
BACKGROUND OF THE INVENTION
[0003] Flaviviridae Viruses
[0004] The Flaviviridae family of viruses comprises at least three
distinct genera: pestiviruses, which cause disease in cattle and
pigs; flaviviruses, which are the primary cause of diseases such as
dengue fever and yellow fever; and hepaciviruses such as hepatitis
C (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 Dengue virus, hemorrhagic
fever viruses such as Lassa, Ebola, and yellow fever virus, shock
syndrome, and Japanese encephalitis virus (Halstead, S. B., Rev.
Infect. Dis., 1984, 6, 251-264; Halstead, S. B., Science,
239:476-481, 1988; Monath, T. P., New Eng. J. Med., 1988, 319,
641-643).
[0005] 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.
[0006] Pestiviruses and hepaciviruses are closely related virus
groups within the Flaviviridae family. Other closely related
viruses in this family include the GB virus A, GB virus A-like
agents, GB virus-B and GB virus-C (also called hepatitis G virus,
HGV). The hepacivirus group (hepatitis C virus; HCV) consists of a
number of closely related but genotypically distinguishable viruses
that infect humans. There are approximately 6 HCV genotypes and
more than 50 subtypes. Due to the similarities between pestiviruses
and hepaciviruses, combined with the poor ability of hepaciviruses
to grow efficiently in cell culture, bovine viral diarrhea virus
(BVDV) is often used as a surrogate to study the HCV virus.
[0007] 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, NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5A, and
NS5B.
[0008] The NS proteins of pestiviruses and hepaciviruses share
sequence domains that are characteristic of specific protein
functions. For example, the NS1 glycoprotein is a cell-surface
protein that is translocated into the ER lumen. NS1 was
characterized initially as soluble complement-fixing antigen found
in sera and tissues of infected animals, and now is known to elicit
humoral immune responses in its extracellular form. Antibodies to
NS1 may be used to confer passive immunity to certain pestiviruses
and flaviviruses. NS1 has been implicated in the process of RNA
replication where it is believed to have a functional role in the
cytoplasmic processing of RNA. NS2A is a small (approximately 22
kd) protein of unknown function. Studies suggest that it binds to
NS3 and NS5, and so may be a recruiter of RNA templates to
membrane-bound replicase. NS2B also is a small (about 14 kd)
protein that is membrane-associated, and is a required cofactor for
the serine protease function of NS3, with which it forms a
complex.
[0009] The NS3 proteins of viruses in both groups are large (about
70 kd), membrane-associated proteins that 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). Thus, the NS3 proteins have
enzymatic activity needed for processing polyproteins for RNA
replication. The C-terminal end of the NS3 proteins have an RNA
triphosphotase activity that appears to modify the 5' end of the
genome prior to 5'-cap addition by guanylyltransferase.
[0010] NS4A and NS4B are membrane-associated, small (about 16 kd
and about 27 kd, respectively), hydrophobic proteins that appear to
function in RNA replication by anchoring replicase components to
cellular membranes (Fields, Virology, 4.sup.th Edition, 2001, p.
1001).
[0011] The NS5 proteins are the largest (about 103 kd) and most
conserved, with sequence homology to other (+)-stranded RNA
viruses. It also plays a pivotal role in viral replication. The
NS5B proteins of pestiviruses and hepaciviruses are the enzymes
necessary for synthesis of the negative-stranded RNA intermediate
that is complementary to the viral genome, and of the
positive-stranded RNA that is complementary to the
negative-stranded RNA intermediate. The NS5B gene product has
Gly-Asp-Asp (GDD) as a hallmark sequence, which it shares with
reverse transcriptases and other viral polymerases and which is
predictive of RNA dependent RNA polymerase (RdRP) activity
(DeFrancesco et al., Antiviral Research, 2003, 58:1-16).
Interestingly, it was found that the NS5B C-terminal 21 residue
long hydrophobic tail is needed to target NS5B to the ER membrane,
but its removal has no other effect and, in fact, leads to
increased enzymatic solubility and activity (Tomei et al., J. Gen.
Virol., 2000, 81:759-767; Lohmann et al., J. Virol., 1997,
71:8416-28; Ferrari et al., J. Virol., 1999, 73:1649-54).
[0012] The NS5B enzyme products have the motifs characteristic of
RNA-directed RNA polymerases, and in addition, share homology with
methyltransferase enzymes that are involved in RNA cap formation
(Koonin, E. V. and Dolja, V. V. (1993) Crit. Rev. Biochem. Molec.
Biol. 28:375-430; Behrens et al.(1996) EMBO J. 15:12-22; Lchmannet
al.(1997) J. Virol. 71:8416-8428; Yuan et al.(1997) Biochem.
Biophys. Res. Comm. 232:231-235; Hagedorn, PCT WO 97/12033; Zhong
et al.(1998) J. Virol. 72.9365-9369). The unliganded crystal
structure of NS5B shows the unique structural feature of folding in
a classic "right hand" shape, in which fingers, palm and thumb
subdomains can be recognized (a feature it shares with other
polymerases), but differs from other "half-open right hand"
polymerases by having a more compact shapes due to two extended
loops that span the finger and thumb domains at the top of the
active site cavity (DeFrancesco et al. at 9). The finger, thumb and
palm subdomains encircle the active site cavity to which the RNA
template and NTP substrates have access via two positively charged
tunnels (Bressanelli et al., J. Virol., 2002, 76, 3482-92). Finger
and thumb domains have strong interactions that limit their ability
to change conformation independently of one another, a structural
feature shared by other RdRPs. The thumb domain contains a
.beta.-hairpin loop that extends toward the cleft of the active
site and may play a role in restricting the binding of the
template/primer at the enzyme active site (DeFrancesco et al., at
10). Studies are in progress to determine the role of this loop in
the initiation mechanism of RNA synthesis (Id.)
[0013] Nucleotidyl transfer reaction residues are located in the
palm domain and contain the signature GDD motif (DeFrancesco et
al., at 9). Palm domain geometry is highly conserved in all
polymerases, and has a conserved two-metal-ion catalytic center
that is required for catalyzing a phosphory transfer reaction at
the polymerase active site.
[0014] It is believed that the de novo initiation model of RNA
polymerization, rather than a "copy back" mechanism, is utilized by
pesti-, flavi- and hepaciviruses. In the de novo initiation model,
complementary RNA synthesis is initiated at the 3'-end of the
genome by a nucleotide triphosphate rather than a nucleic acid or a
protein primer. Purified NS5B is capable of this type of
primer-independent action, and the C-terminal .beta.-loop is
believed to correctly position the 3'-end of the RNA template by
functioning as a gate that retards slippage of the RNA 3'-end
through the polymerase active site (Hong et al., Virology, 2001,
285:6-11. Bressanelli et al. reported the structure of NS5B
polymerase in complex with nucleotides in which three distinct
nucleotide-binding sites were observed in the catalytic center of
the HCV RdRP, and the complex exhibited a geometry similar to the
de novo initiation complex of phi 6 polymerase (Bressanelli et al.,
J. Virol., 2002, 76: 3482-92). Thus, de novo initiation occurs and
apparently is followed by RNA elongation, termination of
polymerization, and release of the new strand. At each of these
steps is the opportunity for intervention and inhibition of the
viral lifecycle.
[0015] The actual roles and functions of the NS proteins of
pestiviruses and hepaciviruses in the lifecycle of the viruses are
directly analogous. In both cases, the NS3 serine proteinase is
responsible for all proteolytic processing of polyprotein
precursors downstream of its position in the ORF (Wiskerchen and
Collett (1991) Virology 184:341-350; Bartenschlager et al. (1993)
J. Virol. 67:3835-3844; Eckart et al. (1993) Biochem. Biophys. Res.
Comm. 192:399-406; Grakoui et al. (1993) J. Virol. 67:2832-2843;
Grakoui et al. (1993) Proc. Natl. Acad. Sci. USA 90:10583-10587;
Hijikata et al. (1993) J. Virol. 67:4665-4675; Tome et al. (1993)
J. Virol. 67:4017-4026). The NS4A protein, in both cases, acts as a
cofactor with the NS3 serine protease (Bartenschlager et al. (1994)
J. Virol. 68:5045-5055; Failla et al. (1994) J. Virol. 68:
3753-3760; Lin et al. (1994) 68:8147-8157; Xu et al. (1997) J.
Virol. 71:5312-5322). The NS3 protein of both viruses also
functions as a helicase (Kim et al. (1995) Biochem. Biophys. Res.
Comm. 215: 160-166; Jin and Peterson (1995) Arch. Biochem.
Biophys., 323:47-53; Warrener and Collett (1995) J. Virol.
69:1720-1726). Finally, the NS5B proteins of pestiviruses and
hepaciviruses have the predicted RNA-directed RNA polymerases
activity (Behrens et al.(1996) EMBO J. 15:12-22; Lchmannet
al.(1997) J. Virol. 71:8416-8428; Yuan et al.(1997) Biochem.
Biophys. Res. Comm. 232:231-235; Hagedorn, PCT WO 97/12033; Zhong
et al.(1998) J. Virol. 72.9365-9369).
[0016] Hepatitis C Virus
[0017] The hepatitis C virus (HCV) is the leading cause of chronic
liver disease worldwide. (Boyer, N. et al. J. Hepatol. 32:98-112,
2000). HCV causes a slow growing viral infection and is the major
cause of cirrhosis and hepatocellular carcinoma (Di Besceglie, A.
M. and Bacon, B. R., Scientific American, Oct.: 80-85, (1999);
Boyer, N. et al. J. Hepatol. 32:98-112, 2000). An estimated 170
million persons are infected with HCV worldwide. (Boyer, N. et al.
J. Hepatol. 32:98-112, 2000). Cirrhosis caused by chronic hepatitis
C infection accounts for 8,000-12,000 deaths per year in the United
States, and HCV infection is the leading indication for liver
transplantation.
[0018] HCV is known to cause at least 80% of posttransfusion
hepatitis and a substantial proportion of sporadic acute hepatitis.
Preliminary evidence also implicates HCV in many cases of
"idiopathic" chronic hepatitis, "cryptogenic" cirrhosis, and
probably hepatocellular carcinoma unrelated to other hepatitis
viruses, such as Hepatitis B Virus (HBV). A small proportion of
healthy persons appear to be chronic HCV carriers, varying with
geography and other epidemiological factors. The numbers may
substantially exceed those for HBV, though information is still
preliminary; how many of these persons have subclinical chronic
liver disease is unclear. (The Merck Manual, ch. 69, p. 901, 16th
ed., (1992)).
[0019] HCV is an enveloped virus containing a positive-sense
single-stranded RNA genome of approximately 9.4 kb. The viral
genome consists of a 5' untranslated region (UTR), a long open
reading frame encoding a polyprotein precursor of approximately
3011 amino acids, and a short 3' UTR. The 5' UTR is the most highly
conserved part of the HCV genome and is important for the
initiation and control of polyprotein translation. Translation of
the HCV genome is initiated by a cap-independent mechanism known as
internal ribosome entry. This mechanism involves the binding of
ribosomes to an RNA sequence known as the internal ribosome entry
site (IRES). An RNA pseudoknot structure has recently been
determined to be an essential structural element of the HCV IRES.
Viral structural proteins include a nucleocapsid core protein (C)
and two envelope glycoproteins, E1 and E2.
[0020] HCV also encodes two proteinases, a zinc-dependent
metalloproteinase encoded by the NS2-NS3 region and a serine
proteinase encoded in the NS3 region. These proteinases are
required for cleavage of specific regions of the precursor
polyprotein into mature peptides: the junction between NS2 and NS3
is autocatalytically cleaved the NS2/NS3 protease, while the
remaining junctions are cleaved by the N-terminal serine protease
domain of NS3 complexed with NS4A. The NS3 protein contains the
NTP-dependent helicase activity that unwinds duplex RNA during
replication. The hydrophobic carboxy-terminal 21 amino acids of
nonstructural protein 5, NS5B, contains the RNA-dependent RNA
polymerase that is essential for viral replication (Fields
Virology, Fourth Edition, Editors: Fields, B. N., Knipe, D. M., and
Howley, P. M., Lippincott-Raven Publishers, Philadelphia, Pa.,
2001, Chapter 32, pp. 1014-1015). NS5B is known to bind RNAs
nonspecifically, and to interact directly with NS3 and NS4A that,
in turn, form complexes with NS4B and NS5A (Id. @ 1015; Ishido et
al., Biochem. Biophys. Res. Commun., 1998; 244:35-40). Certain in
vitro experiments using NS5B and guanosine 5'-mono-, di-, and
triphosphate as well as 5'-triphosphate of 2'-deoxy- and
2',3'-dideoxy-guanosine as HCV inhibitors suggest that HCV-RdRP may
have a strict specificity for 5'-triphosphates and 2'- and 3'-OH
groups (Watanabe et al., U.S. 2002/0055483). Otherwise, the
function(s) of the remaining nonstructural proteins, NS4A, NS4B,
and NS5A (the amino-terminal half of nonstructural protein 5)
remain unknown.
[0021] A significant focus of current antiviral research is
directed to the development of improved methods of treatment of
chronic HCV infections in humans (Di Besceglie, A. M. and Bacon, B.
R., Scientific American, Oct.: 80-85, (1999)).
[0022] Methods to Treat Flaviviridae Infections
[0023] The development of new antiviral agents for Flaviviridae
infections, especially hepatitis C, is currently underway. Specific
inhibitors of HCV-derived enzymes such as protease, helicase, and
polymerase inhibitors are being developed. Drugs that inhibit other
steps in HCV replication are also in development, for example,
drugs that block production of HCV antigens from the RNA (IRES
inhibitors), drugs that prevent the normal processing of HCV
proteins (inhibitors of glycosylation), drugs that block entry of
HCV into cells (by blocking its receptor) and nonspecific
cytoprotective agents that block cell injury caused by the virus
infection. Further, molecular approaches are also being developed
to treat hepatitis C, for example, ribozymes, which are enzymes
that break down specific viral RNA molecules, and antisense
oligonucleotides, which are small complementary segments of DNA
that bind to viral RNA and inhibit viral replication, are under
investigation. A number of HCV treatments are reviewed by Bymock et
al. in Antiviral Chemistry & Chemotherapy, 11:2; 79-95 (2000)
and De Francesco et al. in Antiviral Research, 58: 1-16 (2003).
[0024] Idenix Pharmaceuticals, Ltd. discloses branched nucleosides,
and their use in the treatment of HCV and flaviviruses and
pestiviruses in U.S. patent Publication Nos. 2003/0050229 A1,
2004/0097461 A1, 2004/0101535 A1, 2003/0060400 A1, 2004/0102414 A1,
2004/0097462 A1, and 2004/0063622 A1 which correspond to
International Publication Nos. WO 01/90121 and WO 01/92282. A
method for the treatment of hepatitis C infection (and flaviviruses
and pestiviruses) in humans and other host animals is disclosed in
the Idenix publications that includes administering an effective
amount of a biologically active 1', 2', 3' or 4'-branched .beta.-D
or .beta.-L nucleosides or a pharmaceutically acceptable salt or
prodrug thereof, administered either alone or in combination,
optionally in a pharmaceutically acceptable carrier. See also U.S.
patent Publication Nos. 2004/0006002 and 2004/0006007 as well as WO
03/026589 and WO 03/026675. Idenix Pharmaceuticals, Ltd. also
discloses in U.S. patent Publication No. 2004/0077587
pharmaceutically acceptable branched nucleoside prodrugs, and their
use in the treatment of HCV and flaviviruses and pestiviruses in
prodrugs. See also PCT Publication Nos. WO 04/002422, WO 04/002999,
and WO 04/003000. Further, Idenix Pharmaceuticals, Ltd. also
discloses in WO 04/046331 Flaviviridae mutations caused by
biologically active 2'-branched .beta.-D or .beta.-L nucleosides or
a pharmaceutically acceptable salt or prodrug thereof.
[0025] Biota Inc. discloses various phosphate derivatives of
nucleosides, including 1', 2', 3' or 4'-branched .beta.-D or
.beta.-L nucleosides, for the treatment of hepatitis C infection in
International Patent Publication WO 03/072757.
[0026] Emory University and the University of Georgia Research
Foundation, Inc. (UGARF) discloses the use of 2'-fluoronucleosides
for the treatment of HCV in U.S. Pat. No. 6,348,587. See also U.S.
patent Publication No. 2002/0198171 and International Patent
Publication WO 99/43691.
[0027] BioChem Pharma Inc. (now Shire Biochem, Inc.) discloses the
use of various 1,3-dioxolane nucleosides for the treatment of a
Flaviviridae infection in U.S. Pat. No. 6,566,365. See also U.S.
Pat. Nos. 6,340,690 and 6,605,614; U.S. patent Publication Nos.
2002/0099072 and 2003/0225037, as well as International Publication
No. WO 01/32153 and WO 00/50424.
[0028] BioChem Pharma Inc. (now Shire Biochem, Inc.) also discloses
various other 2'-halo, 2'-hydroxy and 2'-alkoxy nucleosides for the
treatment of a Flaviviridae infection in U.S. patent Publication
No. 2002/0019363 as well as International Publication No. WO
01/60315 (PCT/CA01/00197; filed Feb. 19, 2001).
[0029] ICN Pharmaceuticals, Inc. discloses various nucleoside
analogs that are useful in modulating immune response in U.S. Pat.
Nos. 6,495,677 and 6,573,248. See also WO 98/16184, WO 01/68663,
and WO 02/03997.
[0030] U.S. Pat. No. 6,660,721; U.S. patent Publication Nos.
2003/083307 A1, 2003/008841 A1, and 2004/0110718; as well as
International Patent Publication Nos. WO 02/18404; WO 02/100415, WO
02/094289, and WO 04/043159; filed by F. Hoffmann-La Roche A G,
discloses various nucleoside analogs for the treatment of HCV RNA
replication.
[0031] Pharmasset Limited discloses various nucleosides and
antimetabolites for the treatment of a variety of viruses,
including Flaviviridae, and in particular HCV, in U.S. patent
Publication Nos. 2003/0087873, 2004/0067877, 2004/0082574,
2004/0067877, 2004/002479, 2003/0225029, and 2002/00555483, as well
as International Patent Publication Nos. WO 02/32920, WO 01/79246,
WO 02/48165, WO 03/068162, WO 03/068164 and WO 2004/013298.
[0032] Merck & Co., Inc. and Isis Pharmaceuticals disclose in
U.S. patent Publication No. 2002/0147160, 2004/0072788,
2004/0067901, and 2004/0110717; as well as the corresponding
International Patent Publication Nos. WO 02/057425 (PCT/US02/01531;
filed Jan. 18, 2002) and WO 02/057287 (PCT/US02/03086; filed Jan.
18, 2002) various nucleosides, and in particular several
pyrrolopyrimidine nucleosides, for the treatment of viruses whose
replication is dependent upon RNA-dependent RNA polymerase,
including Flaviviridae, and in particular HCV. See also WO
2004/000858, WO 2004/003138, WO 2004/007512, and WO
2004/009020.
[0033] U.S. patent Publication No. 2003/028013 A1 as well as
International Patent Publication Nos. WO 03/051899, WO 03/061576,
WO 03/062255 WO 03/062256, WO 03/062257, and WO 03/061385, filed by
Ribapharm, also are directed to the use of certain nucleoside
analogs to treat hepatitis C virus.
[0034] Genelabs Technologies disclose in U.S. patent Publication
No. 2004/0063658 as well as International Patent Publication Nos.
WO 03/093290 and WO 04/028481 various base modified derivatives of
nucleosides, including 1', 2', 3' or 4'-branched .beta.-D or
.beta.-L nucleosides, for the treatment of hepatitis C
infection.
[0035] Eldrup et al. (Oral Session V, Hepatitis C Virus,
Flaviviridae; 16.sup.th International Conference on Antiviral
Research (Apr. 27, 2003, Savannah, Ga.) p. A75) 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.
[0038] Drug-resistant variants of viruses 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, and, for example, in the case of HIV,
reverse transcriptase, protease, or DNA polymerase. It has been
demonstrated that the efficacy of a drug against 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
pressures on the virus. One cannot predict, however, what mutations
will be induced in the viral genome by a given drug, whether the
mutation is permanent or transient, or how an infected cell with a
mutated viral sequence will respond to therapy with other agents in
combination or alternation. This is exacerbated by the fact that
there is a paucity of data on the kinetics of drug resistance in
long-term cell cultures treated with modern antiviral agents.
[0039] In view of the severity of diseases associated with
pestiviruses, flaviviruses, and hepatitis C virus, and their
pervasiveness in animals and humans, it is an object of the present
invention to provide a compound, method and composition for the
treatment of a host infected with any member of the family
Flaviviridae, including hepatitis C virus.
[0040] Further, it is an object of the present invention to provide
a compound, method and pharmaceutically-acceptable composition for
the prophylaxis and/or treatment of a host, and particularly a
human, infected with any member of the family Flaviviridae.
[0041] 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.
[0042] Therefore, it is an object of the present invention to
provide a compound, method and composition for the treatment of a
host infected with any member of the family Flaviviridae, including
hepatitis C virus, that have low toxicity to the host.
[0043] It is another object of the present invention to provide a
compound, method and composition generally for the treatment of
patients infected with pestiviruses, flaviviruses, or
hepaciviruses.
SUMMARY OF THE INVENTION
[0044] Methods and compositions for the treatment of pestivirus,
flavivirus and hepatitis C virus infection are described that
include administering an effective amount of a beta-D or
beta-L-nucleoside of the Formulae (I) and (II), or a
pharmaceutically acceptable salt or prodrug thereof.
[0045] In a first principal embodiment, a compound of the Formula
(I), or a pharmaceutically acceptable salt or prodrug thereof, is
provided: 1
[0046] wherein
[0047] each R is independently H, phosphate (including mono-, di-,
or triphosphate or a stabilized phosphate prodrug) or phosphonate;
optionally substituted alkyl including lower alkyl, optionally
substituted alkenyl or alkynyl, acyl, --C(O)-(alkyl), --C(O)(lower
alkyl), --C(O)-(alkenyl), --C(O)-(alkynyl), lipid, phospholipid,
carbohydrate, peptide, cholesterol, an amino acid residue or
derivative, or other pharmaceutically acceptable leaving group that
is capable of providing H or phosphate when administered in
vivo;
[0048] n is 0-2;
[0049] when X is CH.sub.2, CHOH, CH-alkyl, CH-alkenyl, CH-alkynyl,
C-dialkyl, CH--O-alkyl, CH--O-alkenyl, CH--O-alkynyl, CH--S-alkyl,
CH--S-alkenyl, CH--S-alkynyl, CH-halogen, or C-(halogen).sub.2,
[0050] then each R.sup.1 and R.sup.1' is independently H, OH,
optionally substituted alkyl including lower alkyl, azido, cyano,
optionally substituted alkenyl or alkynyl, --C(O)O-(alkyl),
--C(O)O(lower alkyl), --C(O)O-(alkenyl), --C(O)O-(alkynyl),
--O(acyl), --O(lower acyl), --O(alkyl), --O(lower alkyl),
--O(alkenyl), --O(alkynyl), halogen, halogenated alkyl, --NO.sub.2,
--NH.sub.2, --NH(lower alkyl), --N(lower alkyl).sub.2, --NH(acyl),
--N(acyl).sub.2, --C(O)NH.sub.2, --C(O)NH(alkyl),
--C(O)N(alkyl).sub.2, S(O)N-alkyl, S(O)N-alkenyl, S(O)N-alkynyl, or
SCH-halogen, wherein alkyl, alkenyl, and/or alkynyl may optionally
be substituted;
[0051] when X is O, S[O].sub.n, NH, N-alkyl, N-alkenyl, N-alkynyl,
S(O)N-alkyl, S(O)N-alkenyl, S(O)N-alkynyl, or SCH-halogen,
[0052] then each R.sup.1 and R.sup.1' is independently H,
optionally substituted alkyl including lower alkyl, azido, cyano,
optionally substituted alkenyl or alkynyl, --C(O)O-(alkyl),
--C(O)O(lower alkyl), --C(O)O-(alkenyl), --C(O)O-(alkynyl),
halogenated alkyl, --C(O)NH.sub.2, --C(O)NH(alkyl),
--C(O)N(alkyl).sub.2, --C(H).dbd.N--NH.sub.2, C(S)NH.sub.2,
C(S)NH(alkyl), or C(S)N(alkyl).sub.2, wherein alkyl, alkenyl,
and/or alkynyl may optionally be substituted;
[0053] each R.sup.2 and R.sup.3 is independently H, OH, NH.sub.2,
SH, F, Cl, Br, I, CN, NO.sub.2, --C(O)NH.sub.2, --C(O)NH(alkyl),
and --C(O)N(alkyl).sub.2, N.sub.3, optionally substituted alkyl
including lower alkyl, optionally substituted alkenyl or alkynyl,
halogenated alkyl, --C(O)O-(alkyl), --C(O)O(lower alkyl),
--C(O)O-(alkenyl), --C(O)O-(alkynyl), --O(acyl), --O(alkyl),
--O(alkenyl), --O(alkynyl), --OC(O)NH.sub.2, NC, C(O)OH, SCN, OCN,
--S(alkyl), --S(alkenyl), --S(alkynyl), --NH(alkyl),
--N(alkyl).sub.2, --NH(alkenyl), --NH(alkynyl), an amino acid
residue or derivative, a prodrug or leaving group that provides OH
in vivo, or an optionally substituted 3-7 membered heterocyclic
ring having O, S and/or N independently as a heteroatom taken alone
or in combination;
[0054] each R.sup.2' and R.sup.3' is independently H; optionally
substituted alkyl, alkenyl, or alkynyl; --C(O)O(alkyl),
--C(O)O(lower alkyl), --C(O)O(alkenyl), --C(O)O(alkynyl),
--C(O)NH.sub.2, --C(O)NH(alkyl), --C(O)N(alkyl).sub.2, --O(acyl),
--O(lower acyl), --O(alkyl), --O(lower alkyl), --O(alkenyl),
halogen, halogenated alkyl and particularly CF.sub.3, azido, cyano,
NO.sub.2, --S(alkyl), --S(alkenyl), --S(alkynyl), NH.sub.2,
--NH(alkyl), --N(alkyl).sub.2, --NH(alkenyl), --NH(alkynyl),
--NH(acyl), or --N(acyl).sub.2, and R.sub.3 at 3'-C may also be OH;
and
[0055] Base is selected from the group consisting of: 2
[0056] wherein:
[0057] each A independently is N or C--R.sup.5;
[0058] each W is H, OH, --O(acyl), --O(C.sub.14 alkyl),
--O(alkenyl), --O(alkynyl), --OC(O)R.sup.4R.sup.4, --OC(O)N
R.sup.4R.sup.4, SH, --S(acyl), --S(C.sub.1-4 alkyl), NH.sub.2,
NH(acyl), N(acyl).sub.2, NH(C.sub.1-4 alkyl), N(C.sub.1-4
alkyl).sub.2, --N(cycloalkyl) C.sub.1-4 alkylamino,
di(C.sub.1I.sub.4 alkyl)amino, C.sub.3-6 cycloalkylamino, halogen,
C.sub.1-4 alkyl, C.sub.1-4 alkoxy, CN, SCN, OCN, SH, N.sub.3,
NO.sub.2, NH.dbd.NH.sub.2, N.sub.3, NHOH, --C(O)NH.sub.2,
--C(O)NH(acyl), --C(O)N(acyl).sub.2, --C(O)NH(C.sub.1-4 alkyl),
--C(O)N(C.sub.1-4 alkyl).sub.2, --C(O)N(alkyl)(acyl), or
halogenated alkyl;
[0059] each Z is O, S, NH, N--OH, N--NH.sub.2, NH(alkyl),
N(alkyl).sub.2, N-cycloalkyl, alkoxy, CN, SCN, OCN, SH, NO.sub.2,
NH.sub.2, N.sub.3, NH.dbd.NH, NH(alkyl), N(alkyl).sub.2,
CONH.sub.2, CONH(alkyl), or CON(alkyl).sub.2;
[0060] each R.sup.4 is independently H, acyl, or C.sub.1-6
alkyl;
[0061] each R.sup.5 is independently H, Cl, Br, F, I, CN, OH,
optionally substituted alkyl, alkenyl or alkynyl, carboxy,
C(.dbd.NH)NH.sub.2, C.sub.1-4 alkoxy, C.sub.1-4 alkyloxycarbonyl,
N.sub.3, NH.sub.2, NH(alkyl), N(alkyl).sub.2, NO.sub.2, N.sub.3,
halogenated alkyl especially CF.sub.3, C.sub.1-4 alkylamino,
di(C.sub.1-4 alkyl)amino, C.sub.3-6 cycloalkylamino, C.sub.1-6
alkoxy, SH, --S(C.sub.1-4 alkyl), --S(C.sub.1-4 alkenyl),
--S(C.sub.1-4 alkynyl), C.sub.1-6 alkylthio, C.sub.1-6
alkylsulfonyl, (C.sub.1-4 alkyl).sub.0-2 aminomethyl, C.sub.3-6
cycloalkylamino-alkenyl, -alkynyl, --(O)alkyl, --(O)alkenyl,
--(O)alkynyl, --(O)acyl, --O(C.sub.1-4 alkyl), --O(C.sub.1-4
alkenyl), --O(C.sub.1-4 alkynyl), --O--C(O)NH.sub.2,
--OC(O)N(alkyl), --OC(O)R'R", --C(O)OH, C(O)O-alkyl, C(O)O-alkenyl,
C(O)O-alkynyl, S-alkyl, S-acyl, S-alkenyl, S-alkynyl, SCN, OCN, NC,
--C(O)--NH.sub.2, C(O)NH(alkyl), C(O)N(alkyl).sub.2, C(O)NH(acyl),
C(O)N(acyl).sub.2, (S)--NH.sub.2, NH-alkyl, N(dialkyl).sub.2,
NH-acyl, N-diacyl, or a 3-7 membered heterocycle having O, S, or N
taken independently in any combination;
[0062] each R' and R" independently is H, C.sub.1-6 alkyl,
C.sub.2.sub.6 alkenyl, C.sub.2-6 alkynyl, halogen, halogenated
alkyl, OH, CN, N.sub.3, carboxy, C.sub.1-4alkoxycarbonyl, NH.sub.2,
C.sub.1-4 alkylamino, di(C.sub.1-4 alkyl)amino, C.sub.1-6 alkoxy,
C.sub.1-6 alkylsulfonyl, or (C.sub.1-4 alkyl).sub.0-2 aminomethyl;
and
[0063] all tautomeric, enantiomeric and stereoisomeric forms
thereof;
[0064] with the caveat that when X is S in Formula (I), then the
compound is not
5-(4-amino-imidazo[4,5-d][1,2,3]triazin-7-yl)-2-hydroxymethyl-tetr-
ahydro-thiophen-3-ol or
7-(4-hydroxy-5-hydroxy-methyl-tetrahydro-thiophen--
2-yl)-3,7-dihydro-imidazo[4,5-d][1,2,3]triazin-4-one.
[0065] In a second principal embodiment, a compound of the Formula
(II), or a pharmaceutically acceptable salt or prodrug thereof, is
provided: 3
[0066] wherein:
[0067] R, R.sup.2, R.sup.2', R.sup.3, and R.sup.3' are all as
defined above;
[0068] X* is CY.sup.3;
[0069] Y.sup.3 is hydrogen, alkyl, bromo, chloro, fluoro, iodo,
azido, cyano, alkenyl, alkynyl, --C(O)O(alkyl), --C(O)O(lower
alkyl), CF.sub.3, --CONH.sub.2, --CONH(alkyl), or
--CON(alkyl).sub.2;
[0070] R.sup.1 is H, OH, optionally substituted alkyl including
lower alkyl, azido, cyano, optionally substituted alkenyl or
alkynyl, --C(O)O-(alkyl), --C(O)O(lower alkyl), --C(O)O-(alkenyl),
--C(O)O-(alkynyl), --O(acyl), --O(lower acyl), --O(alkyl),
--O(lower alkyl), --O(alkenyl), --O(alkynyl), halogen, halogenated
alkyl, --NO.sub.2, --NH.sub.2, --NH(lower alkyl), --N(lower
alkyl).sub.2, --NH(acyl), --N(acyl).sub.2, --C(O)NH.sub.2,
--C(O)NH(alkyl), or --C(O)N(alkyl).sub.2, wherein an optional
substitution on alkyl, alkenyl, and/or alkynyl may be one or more
halogen, hydroxy, alkoxy or alkylthio groups taken in any
combination;
[0071] Base is defined as above for formulae (A)-(G); and
[0072] all tautomeric, enantiomeric and stereoisomeric forms
thereof;
[0073] with the caveat that when X is S in Formula (I), then the
compound is not
5-(4-amino-imidazo[4,5-d][1,2,3]triazin-7-yl)-2-hydroxymethyl-tetr-
ahydro-thiophen-3-ol or
7-(4-hydroxy-5-hydroxy-methyl-tetrahydro-thiophen--
2-yl)-3,7-dihydro-imidazo[4,5-d][1,2,3]triazin-4-one.
[0074] In preferred embodiments, Bases (A)-(G) have a structure
selected from the group consisting of: 45
[0075] wherein
[0076] each R' and R" independently is H, C.sub.1-6 alkyl,
C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, halogen, halogenated alkyl,
OH, CN, N.sub.3, carboxy, CN.sub.4alkoxycarbonyl, NH.sub.2,
C.sub.1-4 alkylamino, di(C.sub.1-4 alkyl)amino, C.sub.1-6 alkoxy,
C.sub.1-6 alkylsulfonyl, (C.sub.1-4 alkyl).sub.0-2 aminomethyl, as
provided above in the definitions of A and Z for the Base Formulae
(A)-(G);
[0077] each W is independently H, Cl, Br, I, F, halogenated alkyl,
alkoxy, OH, SH, O-alkyl, S--alkyl, O-alkenyl, O-alkynyl, S-alkenyl,
S-alkynyl, --OC(O)NR.sup.4R.sup.4, O-acyl, S-acyl, CN, SCN, OCN,
NO.sub.2, N.sub.3, NH.sub.2, NH(alkyl), N(alkyl).sub.2,
NH-cycloalkyl, NH-acyl, NH.dbd.NH, CONH.sub.2, CONH(alkyl), or
CON(alkyl).sub.2; and
[0078] each R.sup.4 is independently H, acyl, or C.sub.1-6
alkyl;
[0079] each Z is O, S, NH, N--OH, N--NH.sub.2, NH(alkyl),
N(alkyl).sub.2, N-cycloalkyl, alkoxy, CN, SCN, OCN, SH, NO.sub.2,
NH.sub.2, N.sub.3, NH.dbd.NH, NH(alkyl), N(alkyl).sub.2,
CONH.sub.2, CONH(alkyl), or CON(alkyl).sub.2.
[0080] In its preferred embodiments, the compounds of the present
invention comprise nucleosides in which each variable in Formula
(I) is selected from the following, in any combination: X is O or
S; R is H or phosphate; R.sub.1 is H, CH.sub.2OH, or CONH.sub.2;
R.sub.2 is OH or F; R.sub.3 is alkyl, especially methyl or
propynyl, or H at the 3' position; A is H, CH or N; Z is O, S, or
NH; W is NH.sub.2, Cl, OMe, OH, NH-cyclopropyl, S-Me; and each R'
and R" independently is Cl, CN, CONH.sub.2 or Me.
[0081] In its preferred embodiments for Formula (II), the compounds
of the present invention comprise nucleosides in which each
variable in Formula (II) is selected from the following, in any
combination: X* is CH; R is H or phosphate; R.sub.1 is H,
CH.sub.2OH, or CONH.sub.2; R.sub.2 is OH or F; R.sub.3 is alkyl,
especially methyl or propynyl, or H at the 3' position; A is H, CH
or N; Z is O, S, or NH; W is NH.sub.2, Cl, OMe, OH, NH-cyclopropyl,
S-Me; and each R' and R" independently is Cl, CN, CONH.sub.2 or
Me.
[0082] In all embodiments, optional substituents are selected from
the group consisting of one or more halogen, amino, hydroxy,
carboxy and alkoxy groups or atoms, among others. It is to be
understood that all stereoisomeric and tautomeric forms of the
compounds shown are included herein.
[0083] The active compounds of the present invention can be
administered in combination, alternation or sequential steps with
another anti-HCV agent. In combination therapy, effective dosages
of two or more agents are administered together, whereas in
alternation or sequential-step therapy, an effective dosage of each
agent is administered serially or sequentially. The dosages given
will depend on absorption, inactivation and excretion rates of the
drug as well as other factors known to those of skill in the art.
It is to be noted that dosage values will also vary with the
severity of the condition to be alleviated. It is to be further
understood that for any particular subject, specific dosage
regimens and schedules should be adjusted over time according to
the individual need and the professional judgment of the person
administering or supervising the administration of the
compositions.
BRIEF DESCRIPTION OF THE DRAWINGS
[0084] FIG. 1 show generalized structural depictions for Formula
(I) and Formula (II) of the ribofuranosylnucleosides of the present
invention.
[0085] FIG. 2 shows generalized structures for the 2-azapurine
bases of the present invention.
[0086] FIG. 3 shows structural depictions for preferred bases of
the present invention.
DETAILED DESCRIPTION OF THE INVENTION
[0087] The present invention provides a compound, method and
composition for the treatment of a pestivirus, flavivirus and/or
hepatitis C in humans or other host animals that includes
administering an effective anti-pestivirus, anti-flavivirus or
anti-HCV treatment amount of a beta-D- or beta-L-nucleoside as
described herein, or a pharmaceutically acceptable salt or prodrug
thereof, optionally in a pharmaceutically acceptable carrier. The
compounds of this invention either possess antiviral activity, or
are metabolized to a compound that exhibits such activity.
[0088] Flaviviruses included within the scope of this invention are
discussed generally in Fields Virology, Editors: Fields, N., Knipe,
D. M. and Howley, P. M.; Lippincott-Raven Pulishers, Philadelphia,
Pa.; Chapter 31 (1996). Specific flaviviruses include, without
limitation: Absettarov; Alfuy; Apoi; Aroa; Bagaza; Banzi; Bououi;
Bussuquara; Cacipacore; Carey Island; Dakar bat; Dengue viruses 1,
2, 3 and 4; Edge Hill; Entebbe bat; Gadgets Gully; Hanzalova; Hypr;
Uheus; Israel turkey meningoencephalitis; Japanese encephalitis;
Jugra; Jutiapa; Kadam; Karshi; Kedougou; Kokoera; Koutango;
Kumlinge; Kunjin; Kyasanur Forest disease; Langat; Louping ill;
Meaban; Modoc; Montana myotis leukoencephalitis; Murray valley
encephalitis; Naranjal; Negishi; Ntaya; Omsk hemorrhagic fever;
Phnom-Penh bat; Powassan; Rio Bravo; Rocio; Royal Farm; Russian
spring-summer encephalitis; Saboya; St. Louis encephalitis; Sal
Vieja; San Perlita; Saumarez Reef; Sepik; Sokuluk; Spondweni;
Stratford; Temusu; Tyuleniy; Uganda S, Usutu, Wesselsbron; West
Nile; Yaounde; Yellow fever; and Zika.
[0089] Pestiviruses included within the scope of this invention are
also discussed generally in Fields Virology (Id.). Specific
pestiviruses include, without limitation: bovine viral diarrhea
virus ("VDV"); classical swine fever virus ("CSFV") also known as
hog cholera virus); and border disease virus ("DV").
[0090] HCV is a member of the family, Flaviviridae; however, HCV
now has been placed in a new monotypic genus, hepacivirus.
[0091] Active Compounds, Pharmaceutically Acceptable Salts and
Prodrugs Thereof
[0092] In a first principal embodiment, a compound of the Formula
(I), or a pharmaceutically acceptable salt or prodrug thereof, is
provided: 6
[0093] wherein
[0094] R is H, phosphate (including mono-, di-, or triphosphate or
a stabilized phosphate prodrug) or phosphonate; optionally
substituted alkyl including lower alkyl, optionally substituted
alkenyl or alkynyl, acyl, --C(O)-(alkyl), --C(O)(lower alkyl),
--C(O)-(alkenyl), --C(O)-(alkynyl), lipid, phospholipid,
carbohydrate, peptide, cholesterol, an amino acid residue or
derivative, or other pharmaceutically acceptable leaving group that
is capable of providing H or phosphate when administered in
vivo;
[0095] n is 0-2;
[0096] when X is CH.sub.2, CHOH, CH-alkyl, CH-alkenyl, CH-alkynyl,
C-dialkyl, CH--O-alkyl, CH--O-alkenyl, CH--O-alkynyl, CH--S-alkyl,
CH--S-alkenyl, CH--S-alkynyl, CH-halogen, or C-(halogen).sub.2,
[0097] then each R.sup.1 and R.sup.1' is independently H, OH,
optionally substituted alkyl including lower alkyl, azido, cyano,
optionally substituted alkenyl or alkynyl, --C(O)O-(alkyl),
--C(O)O(lower alkyl), --C(O)O-(alkenyl), --C(O)O-(alkynyl),
--O(acyl), --O(lower acyl), --O(alkyl), --O(lower alkyl),
--O(alkenyl), --O(alkynyl), halogen, halogenated alkyl, --NO.sub.2,
--NH.sub.2, --NH(lower alkyl), --N(lower alkyl).sub.2, --NH(acyl),
--N(acyl).sub.2, --C(O)NH.sub.2, --C(O)NH(alkyl),
--C(O)N(alkyl).sub.2, S(O)N-alkyl, S(O)N-alkenyl, S(O)N-alkynyl, or
SCH-halogen, wherein alkyl, alkenyl, and/or alkynyl may optionally
be substituted;
[0098] when X is O, S[O].sub.n, NH, N-alkyl, N-alkenyl, N-alkynyl,
S(O)N-alkyl, S(O)N-alkenyl, S(O)N-alkynyl, or SCH-halogen,
[0099] then each R.sup.1 and R.sup.1' is independently H,
optionally substituted alkyl including lower alkyl, azido, cyano,
optionally substituted alkenyl or alkynyl, --C(O)O-(alkyl),
--C(O)O(lower alkyl), --C(O)O-(alkenyl), --C(O)O-(alkynyl),
halogenated alkyl, --C(O)NH.sub.2, --C(O)NH(alkyl),
--C(O)N(alkyl).sub.2, --C(H).dbd.N--NH.sub.2, C(S)NH.sub.2,
C(S)NH(alkyl), or C(S)N(alkyl).sub.2, wherein alkyl, alkenyl,
and/or alkynyl may optionally be substituted;
[0100] each R.sup.2 and R.sup.3 is independently is OH, NH.sub.2,
SH, F, Cl, Br, I, CN, NO.sub.2, --C(O)NH.sub.2, --C(O)NH(alkyl),
--C(O)N(alkyl).sub.2, N.sub.3, optionally substituted alkyl
including lower alkyl, optionally substituted alkenyl or alkynyl,
halogenated alkyl, --C(O)O-(alkyl), --C(O)O(lower alkyl),
--C(O)O-(alkenyl), --C(O)O-(alkynyl), --O(acyl), --O(alkyl),
--O(alkenyl), --O(alkynyl), --OC(O)NH.sub.2, NC, C(O)OH, SCN, OCN,
--S(alkyl), --S(alkenyl), --S(alkynyl), --NH(alkyl),
--N(alkyl).sub.2, --NH(alkenyl), --NH(alkynyl), an amino acid
residue or derivative, a prodrug or leaving group that provides OH
in vivo, or an optionally substituted 3-7 membered heterocyclic
ring having O, S and/or N independently as a heteroatom taken alone
or in combination;
[0101] each R.sup.2' and R.sup.3' independently is H; optionally
substituted alkyl, alkenyl, or alkynyl; --C(O)O(alkyl),
--C(O)O(lower alkyl), --C(O)O(alkenyl), --C(O)O(alkynyl),
--C(O)NH.sub.2, --C(O)NH(alkyl), --C(O)N(alkyl).sub.2, --O(acyl),
--O(lower acyl), --O(alkyl), --O(lower alkyl), --O(alkenyl),
halogen, halogenated alkyl and particularly CF.sub.3, azido, cyano,
NO.sub.2, --S(alkyl), --S(alkenyl), --S(alkynyl), NH.sub.2,
--NH(alkyl), --N(alkyl).sub.2, --NH(alkenyl), --NH(alkynyl),
--NH(acyl), or --N(acyl).sub.2, and R.sub.3 at 3'-C may also be OH;
and
[0102] Base is selected from the group consisting of: 7
[0103] wherein
[0104] each A independently is N or C--R.sup.5;
[0105] W is H, OH, --O(acyl), --O(C.sub.1-4 alkyl), --O(alkenyl),
--O(alkynyl), --OC(O)R.sup.4R.sup.4, --OC(O)N R.sup.4R.sup.4, SH,
--S(acyl), --S(C.sub.1-4 alkyl), NH.sub.2, NH(acyl), N(acyl).sub.2,
NH(C.sub.1-4 alkyl), N(C.sub.1-4 alkyl).sub.2, --N(cycloalkyl)
C.sub.1-4 alkylamino, di(C.sub.1-4 alkyl)amino, C.sub.3-6
cycloalkylamino, halogen, C.sub.1-4 alkyl, C.sub.1-4 alkoxy, CN,
SCN, OCN, SH, N.sub.3, NO.sub.2, NH.dbd.NH.sub.2, N.sub.3, NHOH,
--C(O)NH.sub.2, --C(O)NH(acyl), --C(O)N(acyl).sub.2,
--C(O)NH(C.sub.1-4 alkyl), --C(O)N(C.sub.1-4 alkyl).sub.2,
--C(O)N(alkyl)(acyl), or halogenated alkyl;
[0106] Z is O, S, NH, N--OH, N--NH.sub.2, NH(alkyl),
N(alkyl).sub.2, N-cycloalkyl, alkoxy, CN, SCN, OCN, SH, NO.sub.2,
NH.sub.2, N.sub.3, NH.dbd.NH, NH(alkyl), N(alkyl).sub.2,
CONH.sub.2, CONH(alkyl), or CON(alkyl).sub.2;
[0107] each R.sup.4 is independently H, acyl, or C.sub.1-6
alkyl;
[0108] each R.sup.5 is independently H, Cl, Br, F, I, CN, OH,
optionally substituted alkyl, alkenyl or alkynyl, carboxy,
C(.dbd.NH)NH.sub.2, C.sub.1-4 alkoxy, C.sub.1-4 alkyloxycarbonyl,
N.sub.3, NH.sub.2, NH(alkyl), N(alkyl).sub.2, NO.sub.2, N.sub.3,
halogenated alkyl especially CF.sub.3, C.sub.1-4 alkylamino,
di(C.sub.1-4 alkyl)amino, C.sub.3-6 cycloalkylamino, C.sub.1-6
alkoxy, SH, -$(C.sub.1-4 alkyl), --S(C.sub.1-4 alkenyl),
--S(C.sub.1-4 alkynyl), C.sub.1-6 alkylthio, C.sub.1-6
alkylsulfonyl, (C.sub.1-4 alkyl).sub.0-2 aminomethyl, C.sub.3-6
cycloalkylamino-alkenyl, -alkynyl, --(O)alkyl, --(O)alkenyl,
--(O)alkynyl, --(O)acyl, --O(C.sub.1-4 alkyl), --O(C.sub.1-4
alkenyl), --O(C.sub.1-4 alkynyl), --O--C(O)NH.sub.2,
--OC(O)N(alkyl), --OC(O)R'R", --C(O)OH, C(O)O-alkyl, C(O)O-alkenyl,
C(O)O-alkynyl, S-alkyl, S-acyl, S-alkenyl, S-alkynyl, SCN, OCN, NC,
--C(O)--NH.sub.2, C(O)NH(alkyl), C(O)N(alkyl).sub.2, C(O)NH(acyl),
C(O)N(acyl).sub.2, (S)--NH.sub.2, NH-alkyl, N(dialkyl).sub.2,
NH-acyl, N-diacyl, or a 3-7 membered heterocycle having O, S, or N
taken independently in any combination;
[0109] each R' and R" independently is H, C.sub.1-6 alkyl, C.sub.26
alkenyl, C.sub.2-6 alkynyl, halogen, halogenated alkyl, OH, CN,
N.sub.3, carboxy, C.sub.1-4alkoxycarbonyl, NH.sub.2, C.sub.1-4
alkylamino, di(C.sub.1-4 alkyl)amino, C.sub.1-6 alkoxy, C.sub.1-6
alkylsulfonyl, or (C.sub.1-4 alkyl).sub.0-2 aminomethyl; and
[0110] all tautomeric, enantiomeric and stereoisomeric forms
thereof;
[0111] with the caveat that when X is S in Formula (I), then the
compound is not
5-(4-amino-imidazo[4,5-d][1,2,3]triazin-7-yl)-2-hydroxymethyl-tetr-
ahydro-thiophen-3-ol or
7-(4-hydroxy-5-hydroxy-methyl-tetrahydro-thiophen--
2-yl)-3,7-dihydro-imidazo[4,5-d][1,2,3]triazin-4-one.
[0112] In a second principal embodiment, a compound of the Formula
(II), or a pharmaceutically acceptable salt or prodrug thereof, is
provided: 8
[0113] wherein:
[0114] R, R.sup.2, R.sup.2', R.sup.3, and R.sup.3' are all as
defined above;
[0115] X* is CY.sup.3;
[0116] Y.sup.3 is hydrogen, alkyl, bromo, chloro, fluoro, iodo,
azido, cyano, alkenyl, alkynyl, --C(O)O(alkyl), --C(O)O(lower
alkyl), CF.sub.3, --CONH.sub.2, --CONH(alkyl), or
--CON(alkyl).sub.2;
[0117] R.sup.1 is H, OH, optionally substituted alkyl including
lower alkyl, azido, cyano, optionally substituted alkenyl or
alkynyl, --C(O)O-(alkyl), --C(O)O(lower alkyl), --C(O)O-(alkenyl),
--C(O)O-(alkynyl), --O(acyl), --O(lower acyl), --O(alkyl),
--O(lower alkyl), --O(alkenyl), --O(alkynyl), halogen, halogenated
alkyl, --NO.sub.2, --NH.sub.2, --NH(lower alkyl), --N(lower
alkyl).sub.2, --NH(acyl), --N(acyl).sub.2, --C(O)NH.sub.2,
--C(O)NH(alkyl), or --C(O)N(alkyl).sub.2, wherein an optional
substitution on alkyl, alkenyl, and/or alkynyl may be one or more
halogen, hydroxy, alkoxy or alkylthio groups taken in any
combination;
[0118] Base is defined as above for formulae (A)-(G); and
[0119] A and Z are as defined above,
[0120] with the caveat that when X is S in Formula (I), then the
compound is not
5-(4-amino-imidazo[4,5-d][1,2,3]triazin-7-yl)-2-hydroxymethyl-tetr-
ahydro-thiophen-3-ol or
7-(4-hydroxy-5-hydroxy-methyl-tetrahydro-thiophen--
2-yl)-3,7-dihydro-imidazo[4,5-d][1,2,3]triazin-4-one; and
[0121] all tautomeric, enantiomeric and stereoisomeric forms
thereof.
[0122] In preferred embodiments, Bases (A)-(G) have a structure
selected from the group consisting of: 910
[0123] wherein
[0124] each R' and R" independently is H, C.sub.1-6 alkyl, C.sub.26
alkenyl, C.sub.2-6 alkynyl, halogen, halogenated alkyl, OH, CN,
N.sub.3, carboxy, C.sub.1-4alkoxycarbonyl, NH.sub.2, C.sub.1-4
alkylamino, di(C.sub.1-4 alkyl)amino, C.sub.1-6 alkoxy, C.sub.1-6
alkylsulfonyl, (C.sub.1-4 alkyl).sub.0-2 aminomethyl, as provided
above in the definitions of A and Z for the Base Formulae
(A)-(G);
[0125] each W is Cl, Br, I, F, halogenated alkyl, alkoxy, OH, SH,
O-alkyl, S-alkyl, O-alkenyl, O-alkynyl, S-alkenyl, S-alkynyl,
--OC(O)NR.sup.4R.sup.4, O-acyl, S-acyl, CN, SCN, OCN, NO.sub.2,
N.sub.3, NH.sub.2, NH(alkyl), N(alkyl).sub.2, NH-cycloalkyl,
NH-acyl, NH.dbd.NH, CONH.sub.2, CONH(alkyl), or
CON(alkyl).sub.2;
[0126] each R.sup.4 is independently H, acyl, or C.sub.1-6 alkyl;
and
[0127] each Z is O, S, NH, N--OH, N--NH.sub.2, NH(alkyl),
N(alkyl).sub.2, N-cycloalkyl, alkoxy, CN, SCN, OCN, SH, NO.sub.2,
NH.sub.2, N.sub.3, NH.dbd.NH, NH(alkyl), N(alkyl).sub.2,
CONH.sub.2, CONH(alkyl), or CON(alkyl).sub.2.
[0128] In its preferred embodiments, the compounds of the present
invention comprise nucleosides in which each variable in Formula
(I) is selected from the following, in any combination: X is O or
S; R is H or phosphate; R.sub.1 is H, CH.sub.2OH, or CONH.sub.2;
R.sub.2 is OH or F; R.sub.3 is alkyl, especially methyl or
propynyl, or H at the 3' position; A is H, CH or N; Z is O, S, or
NH; W is NH.sub.2, Cl, OMe, OH, NH-cyclopropyl, S-Me; and each R'
and R" independently is Cl, CN, CONH.sub.2 or Me.
[0129] In its preferred embodiments for Formula (II), the compounds
of the present invention comprise nucleosides in which each
variable in Formula (II) is selected from the following, in any
combination: X* is CH; R is H or phosphate; R.sub.1 is H,
CH.sub.2OH, or CONH.sub.2; R.sub.2 is OH or F; R.sub.3 is alkyl,
especially methyl or propynyl, or H at the 3' position; A is H, CH
or N; Z is O, S, or NH; W is NH.sub.2, Cl, OMe, OH, NH-cyclopropyl,
S-Me; and each R' and R" independently is Cl, CN, CONH.sub.2 or
Me.
[0130] In all embodiments, optional substituents are selected from
the group consisting of one or more halogen, amino, hydroxy,
carboxy and alkoxy groups or atoms, among others. It is to be
understood that all stereoisomeric and tautomeric forms of the
compounds shown are included herein.
[0131] In one particular embodiment, a compound of the Formula
(III), or a pharmaceutically acceptable salt or prodrug thereof, is
provided: 11
[0132] each R, R.sup.2*, and R.sup.3* independently is H, phosphate
(including mono-, di-, or triphosphate or a stabilized phosphate
prodrug) or phosphonate; optionally substituted alkyl including
lower alkyl, optionally substituted alkenyl or alkynyl, acyl,
--C(O)-(alkyl), --C(O)(lower alkyl), --C(O)-(alkenyl),
--C(O)-(alkynyl), lipid, phospholipid, carbohydrate, peptide,
cholesterol, an amino acid residue or derivative, or other
pharmaceutically acceptable leaving group that is capable of
providing H or phosphate when administered in vivo;
[0133] X is O, S[O].sub.n, CH.sub.2, CHOH, CH-alkyl, CH-alkenyl,
CH-alkynyl, C-dialkyl, CH--O-alkyl, CH--O-alkenyl, CH--O-alkynyl,
CH--S-alkyl, CH--S-alkenyl, CH--S-alkynyl, NH, N-alkyl, N-alkenyl,
N-alkynyl, S(O)N-alkyl, S(O)N-alkenyl, S(O)N-alkynyl, SCH-halogen,
or C-(halogen).sub.2, wherein alkyl, alkenyl or alkynyl optionally
may be substituted;
[0134] n is 0-2;
[0135] each R.sup.2' independently is H; optionally substituted
alkyl, alkenyl, or alkynyl; --C(O)O(alkyl), --C(O)O(lower alkyl),
--C(O)O(alkenyl), --C(O)O(alkynyl), --C(O)NH.sub.2,
--C(O)NH(alkyl), --C(O)N(alkyl).sub.2, --OH, --O(acyl), --O(lower
acyl), --O(alkyl), --O(lower alkyl), --O(alkenyl), halogen,
halogenated alkyl and particularly CF.sub.3, azido, cyano,
NO.sub.2, --S(alkyl), --S(alkenyl), --S(alkynyl), NH.sub.2,
--NH(alkyl), --N(alkyl).sub.2, --NH(alkenyl), --NH(alkynyl),
--NH(acyl), or --N(acyl).sub.2; and
[0136] Base is defined as above for formulae (A)-(G); and
preferably is a Base as defined by structures (i)-(xi) above.
[0137] In one embodiment, the R.sup.2' is an optionally substituted
alkyl, alkenyl, or alkynyl; halogen, halogenated alkyl and
particularly CF.sub.3, azido, or cyano. In a particular embodiment,
R.sup.2' is an optionally substituted alkyl, alkenyl, or alkynyl;
halogen, halogenated alkyl and particularly CF.sub.3. In yet
another particular embodiment, R.sup.2' is CH.sub.3 or
CF.sub.3.
[0138] In one embodiment, each R, R.sup.2*, and R.sup.3* is
independently H, phosphate (including mono-, di-, or triphosphate
or a stabilized phosphate prodrug) or phosphonate. In anther
embodiment, each R, R.sup.2*, and R.sup.3* is independently H. In
yet another embodiment, each R, R.sup.2*, and R.sup.3* is
independently H, acyl, or an amino acid acyl residue.
[0139] In one embodiment, X is O or S. In another embodiment, X is
O.
[0140] In another particular embodiment, a compound of the Formula
(IV), or a pharmaceutically acceptable salt or prodrug thereof, is
provided: 12
[0141] each R, R.sup.2*, and R.sup.3* independently is H, phosphate
(including mono-, di-, or triphosphate or a stabilized phosphate
prodrug) or phosphonate; optionally substituted alkyl including
lower alkyl, optionally substituted alkenyl or alkynyl, acyl,
--C(O)-(alkyl), --C(O)(lower alkyl), --C(O)-(alkenyl),
--C(O)-(alkynyl), lipid, phospholipid, carbohydrate, peptide,
cholesterol, an amino acid residue or derivative, or other
pharmaceutically acceptable leaving group that is capable of
providing H or phosphate when administered in vivo;
[0142] X is O, S[O].sub.n, CH.sub.2, CHOH, CH-alkyl, CH-alkenyl,
CH-alkynyl, C-dialkyl, CH--O-alkyl, CH--O-alkenyl, CH--O-alkynyl,
CH--S-alkyl, CH--S-alkenyl, CH--S-alkynyl, NH, N-alkyl, N-alkenyl,
N-alkynyl, S(O)N-alkyl, S(O)N-alkenyl, S(O)N-alkynyl, SCH-halogen,
or C-(halogen).sub.2, wherein alkyl, alkenyl or alkynyl optionally
may be substituted;
[0143] n is 0-2;
[0144] each R.sup.3' independently is H; optionally substituted
alkyl, alkenyl, or alkynyl; --C(O)O(alkyl), --C(O)O(lower alkyl),
--C(O)O(alkenyl), --C(O)O(alkynyl), --C(O)NH.sub.2,
--C(O)NH(alkyl), --C(O)N(alkyl).sub.2, --OH, --O(acyl), --O(lower
acyl), --O(alkyl), --O(lower alkyl), --O(alkenyl), halogen,
halogenated alkyl and particularly CF.sub.3, azido, cyano,
NO.sub.2, --S(alkyl), --S(alkenyl), --S(alkynyl), NH.sub.2,
--NH(alkyl), --N(alkyl).sub.2, --NH(alkenyl), --NH(alkynyl),
--NH(acyl), or --N(acyl).sub.2; and
[0145] Base is defined as above for formulae (A)-(G); and
preferably is a Base as defined by structures (i)-(xi) above.
[0146] In one embodiment, the R.sup.3' is an optionally substituted
alkyl, alkenyl, or alkynyl; halogen, halogenated alkyl and
particularly CF.sub.3, azido, or cyano. In a particular embodiment,
R.sup.3' is an optionally substituted alkyl, alkenyl, or alkynyl;
halogen, halogenated alkyl and particularly CF.sub.3. In yet
another particular embodiment, R.sup.3' is CH.sub.3 or
CF.sub.3.
[0147] In one embodiment, each R, R.sup.2*, and R.sup.3* is
independently H, phosphate (including mono-, di-, or triphosphate
or a stabilized phosphate prodrug) or phosphonate.
[0148] In anther embodiment, each R, R.sup.2*, and R.sup.3* is
independently H. In yet another embodiment, each R, R.sup.2*, and
R.sup.3* is independently H, acyl, or an amino acid acyl
residue.
[0149] In one embodiment, X is O or S. In another embodiment, X is
O.
[0150] The beta-D- and beta-L-nucleosides of this invention belong
to a class of anti-pestivirus, anti-flavivirus and anti-HCV agents
that inhibit viral polymerase. Triphosphate nucleosides can be
screened for their ability to inhibit viral polymerase, whether
HCV, flavivirus or pestivirus, in vitro according to screening
methods set forth below. Chiron Corporation developed a replicon
system for testing potential anti-HCV compounds that utilizes a
particular peptide sequence having an HCV protease-recognition site
(U.S. Pat. No. 6,436,666; U.S. Pat. No. 6,416,946; U.S. Pat. No.
6,416,944; U.S. Pat. No. 6,379,886; and U.S. Pat. No. 6,326,151, to
Chiron Corporation). Other systems for assessing the ability of
compounds to inhibit HCV and related viruses include those of Rice
(see U.S. Pat. No. 5,874,565) and the polymerase inhibition assay
of Dr. Ralf Bartenschlager (see EP 1 043 399 A2).
[0151] An alternative means of assessing a compound's ability to
inhibit HCV, pestivirus and/or flavivirus is through the use of
predictive animal model systems. The model of choice for testing
HCV is the chimpanzee, which has been used by the applicants.
Chimpanzees provide an excellent mammalian system for study of
anti-HCV compounds and an insight into the predictability or
unpredictability of drug activity based on the closeness of their
species relationship to humans.
[0152] The active compounds of the present invention can be
administered in combination, alternation or sequential steps with
another anti-HCV agent. In combination therapy, effective dosages
of two or more agents are administered together, whereas in
alternation or sequential-step therapy, an effective dosage of each
agent is administered serially or sequentially. The dosages given
will depend on absorption, inactivation and excretion rates of the
drug as well as other factors known to those of skill in the art.
It is to be noted that dosage values will also vary with the
severity of the condition to be alleviated. It is to be further
understood that for any particular subject, specific dosage
regimens and schedules should be adjusted over time according to
the individual need and the professional judgment of the person
administering or supervising the administration of the
compositions.
[0153] In particular, the present invention provides the
following:
[0154] a) a beta-D- or beta-L-nucleoside compound of Formula
(I)-(IV), or a pharmaceutically acceptable salt or prodrug
thereof;
[0155] b) a pharmaceutical composition comprising a beta-D- or
beta-L-nucleoside compound of Formula (I)-(IV), or a
pharmaceutically acceptable salt or prodrug thereof, optionally
together with a pharmaceutically acceptable carrier, excipient or
diluent;
[0156] c) a pharmaceutical composition comprising a beta-D- or
beta-L-nucleoside compound of Formula (I)-(IV), or a
pharmaceutically acceptable salt or prodrug thereof, with one or
more other effective antiviral agents, optionally with a
pharmaceutically acceptable carrier or diluent;
[0157] d) a pharmaceutical composition for the treatment or
prophylaxis of a pestivirus, flavivirus or HCV infection in a host,
especially a host diagnosed as having or being at risk for such
infection, comprising a beta-D- or beta-L-nucleoside compound of
Formula (I)-(IV), or a pharmaceutically acceptable salt or prodrug
thereof, together with a pharmaceutically acceptable carrier or
diluent;
[0158] e) a pharmaceutical formulation comprising the beta-D- or
beta-L-nucleoside compound of Formula (I)-(IV), or a
pharmaceutically acceptable salt or prodrug thereof, together with
a pharmaceutically acceptable carrier, excipient or diluent;
[0159] f) a method for the treatment of a pestivirus, flavivirus or
HCV infection in a host comprising a beta-D- or beta-L-nucleoside
compound of Formula (I)-(IV), or a pharmaceutically acceptable salt
or prodrug thereof, optionally with a pharmaceutically acceptable
carrier, excipient or diluent;
[0160] g) a method for the treatment of a pestivirus, flavivirus or
HCV infection in a host comprising administering an effective
amount of a beta-D- or beta-L-nucleoside compound of Formula
(I)-(IV), or a pharmaceutically acceptable salt or prodrug thereof,
with one or more other effective antiviral agents, optionally with
a pharmaceutically acceptable carrier, excipient or diluent;
[0161] h) a method for the treatment of a pestivirus, flavivirus or
HCV infection in a host comprising administering an effective
amount of a beta-D- or beta-L-nucleoside compound of Formula
(I)-(IV), or a pharmaceutically acceptable salt or prodrug thereof,
with one or more other effective antiviral agents, optionally with
a pharmaceutically acceptable carrier, excipient or diluent;
[0162] i) a method for the treatment of a pestivirus, flavivirus or
HCV infection in a host comprising administering an effective
amount of a beta-D- or beta-L-nucleoside compound of Formula
(I)-(IV), or a pharmaceutically acceptable salt or prodrug thereof,
with one or more other effective antiviral agents, optionally with
a pharmaceutically acceptable carrier, excipient or diluent;
[0163] j) a method for the treatment of a pestivirus, flavivirus or
HCV infection in a host comprising administering an effective
amount of a beta-D- or beta-L-nucleoside compound of Formula
(I)-(IV), or a pharmaceutically acceptable salt or prodrug thereof,
with one or more other effective antiviral agents, optionally with
a pharmaceutically acceptable carrier, excipient or diluent;
[0164] k) use of a beta-D- or beta-L-nucleoside compound of Formula
(I)-(IV), or a pharmaceutically acceptable salt or prodrug thereof,
optionally with a pharmaceutically acceptable carrier or diluent,
for the treatment of a pestivirus, flavivirus or HCV infection in a
host;
[0165] l) use of a beta-D- or beta-L-nucleoside compound of Formula
(I)-(IV), or a pharmaceutically acceptable salt or prodrug thereof,
with one or more other effective antiviral agents, optionally with
a pharmaceutically acceptable carrier or diluent, for the treatment
of a pestivirus, flavivirus and/or HCV infection in a host;
[0166] m) use of a beta-D- or beta-L-nucleoside compound of Formula
(I)-(IV), or a pharmaceutically acceptable salt or prodrug thereof,
optionally with a pharmaceutically acceptable carrier or diluent,
in the manufacture of a medicament for the treatment of a
pestivirus, flavivirus and/or HCV infection in a host;
[0167] n) use of a beta-D- or beta-L-nucleoside compound of Formula
(I)-(IV), or a pharmaceutically acceptable salt or prodrug thereof,
with one or more other effective antiviral agents and optionally
with a pharmaceutically acceptable carrier, excipient or diluent,
in the manufacture of a medicament for the treatment of a
pestivirus, flavivirus and/or HCV infection in a host;
[0168] o) a beta-D- or beta-L-nucleoside compound of Formula
(I)-(IV), or a pharmaceutically acceptable salt or prodrug thereof,
substantially in the absence of enantiomers of the described
nucleoside, or substantially isolated from other chemical
entities;
[0169] p) a process for the preparation of a beta-D- or
beta-L-nucleoside compound of Formula (I)-(IV), or a
pharmaceutically acceptable salt or prodrug thereof, as provided in
more detail below; and
[0170] q) a process for the preparation of a beta-D- or
beta-L-nucleoside compound of Formula (I)-(IV), or a
pharmaceutically acceptable salt or prodrug thereof, substantially
in the absence of enantiomers of the described nucleoside or
substantially isolated from other chemical entities.
[0171] 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. Non-limiting examples are the
pharmaceutically acceptable salts, which are alternatively referred
to as "physiologically acceptable salts", and a compound that has
been alkylated or acylated at the 5'-position or on the purine or
pyrimidine base, thereby forming 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.
[0172] Stereochemistry
[0173] 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).
[0174] Examples of methods to obtain optically active materials are
known in the art, and include at least the following.
[0175] 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;
[0176] 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;
[0177] 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;
[0178] 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;
[0179] 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;
[0180] 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;
[0181] vii) first- and second-order asymmetric transformations--a
technique whereby diastereomers from the racemate equilibrate to
yield a preponderance in solution of the diastereomer from the
desired enantiomer or where preferential crystallization of the
diastereomer from the desired enantiomer perturbs the equilibrium
such that eventually in principle all the material is converted to
the crystalline diastereomer from the desired enantiomer. The
desired enantiomer is then released from the diastereomer;
[0182] 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;
[0183] ix) enantiospecific synthesis from non-racernic
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;
[0184] 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;
[0185] 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;
[0186] 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;
[0187] 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.
[0188] Definitions
[0189] 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-dimethybutyl, and
2,3-dimethylbutyl. The term includes both substituted and
unsubstituted alkyl groups. Moieties with which the alkyl group can
be substituted with one or more substituents are selected from the
group consisting of halo, including Cl, F, Br and I so as to form,
for eg., CF.sub.3, 2-Br-ethyl, CH.sub.2F, CH.sub.2Cl,
CH.sub.2CF.sub.3, or CF.sub.2CF.sub.3; hydroxyl, for eg.
CH.sub.2OH; amino, for eg., CH.sub.2NH.sub.2, CH.sub.2NHCH.sub.3,
or CH.sub.2N(CH.sub.3).sub.2; carboxylate; carboxamido; alkylamino;
arylamino; alkoxy; aryloxy; nitro; azido, for eg., CH.sub.2N.sub.3;
cyano, for eg., CH.sub.2CN; thio; sulfonic acid; sulfate;
phosphonic acid; phosphate; and phosphonate, either unprotected or
protected as necessary, known to those skilled in the art, for eg.,
as taught in Greene et al., Protective Groups in Organic Synthesis,
John Wiley and Sons, Second Edition (1991), incorporated herein by
reference.
[0190] The term "lower alkyl" as used herein, and unless otherwise
specified, refers to a C.sub.1 to C.sub.6 saturated straight,
branched, or if appropriate, cyclic as in cyclopropyl, for eg.,
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.
[0191] The terms "alkylamino" and "arylamino" refer to an amino
group that has one or two alkyl or aryl substituents,
respectively.
[0192] 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. Numerous oxygen and nitrogen protecting groups are known
to those skilled in the art of organic synthesis.
[0193] 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 alkyl, hydroxyl,
amino, alkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, thio,
alkylthio, carboxamido, carboxylate, sulfonic acid, sulfate,
phosphonic acid, phosphate, or phosphonate, either unprotected or
protected as necessary, as known to those skilled in the art, for
eg., as taught in Greene et al., Protective Groups in Organic
Synthesis, John Wiley and Sons, Second Edition (1991), incorporated
herein by reference.
[0194] The terms "alkaryl" and "akylaryl" refer to an alkyl group
with an aryl sustituent.
[0195] The terms "aralkyl" and "arylalkyl" refer to an aryl group
with an alkyl substituent.
[0196] The term "halo" as used herein includes bromo, chloro, iodo
and fluoro.
[0197] The term purine base includes, but is not limited to,
adenine, 2-azapurine bases that are optionally substituted
imidazo-triazines, imidazo-pyridazines, pyrrolo-pyridazines,
pyrrolo-triazines, triazolo-triazines including
triazolo[4,5-d]triazines, pyrazolo-triazines including
pyrazolo[4,5-d]triazines, 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-thioalkyl purine,
N.sup.2-alkylpurines, N.sup.2-alkyl-6-thiopurines,
C.sup.5-hydroxyalkyl purine, N.sup.2-alkylpurines,
N.sup.2-alkyl-6-thiopurines, triazolopyridinyl, imidazolopyridinyl,
pyrrolopyrimidinyl, and pyrazolopyrimidinyl.
[0198] The Base maybe selected from the group consisting of: 13
[0199] 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, C.sub.1-C.sub.6 alkyl or C.sub.1-C.sub.6 alkoxy;
sulfonate esters such as alkyl or aralkyl sulphonyl including
methanesulfonyl; the mono-, di- or triphosphate ester; trityl or
monomethoxytrityl; substituted benzyl; trialkylsilyl as, for eg.,
dimethyl-t-butylsilyl or diphenylmethylsilyl. Aryl groups in the
esters optimally comprise a phenyl group. The term "lower acyl"
refers to an acyl group in which the non-carbonyl moiety is lower
alkyl.
[0200] As used herein, the terms "substantially free of" and
"substantially in the absence of" refer to a nucleoside composition
that includes at least 85-90% by weight, preferably 95%-98% by
weight, and even more preferably 99%-100% by weight, of the
designated enantiomer of that nucleoside. In a preferred
embodiment, the compounds listed in the methods and compounds of
this invention are substantially free of enantiomers other than for
the one designated.
[0201] Similarly, the term "isolated" refers to a nucleoside
composition that includes at least 85%-90% by weight, preferably
95%-98% by weight, and even more preferably 99%-100% by weight, of
the nucleoside, the remainder comprising other chemical species or
enantiomers.
[0202] The term "independently" is used herein to indicate that a
variable is applied in any one instance without regard to the
presence or absence of a variable having that same or a different
definition within the same compound. Thus, in a compound in which
R" appears twice and is defined as "independently carbon or
nitrogen", both R"s can be carbon, both R"s can be nitrogen, or one
R" can be carbon and the other nitrogen.
[0203] 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 flavivirus or pestivirus genome,
whose replication or function can be altered by the compounds of
the present invention. The term host specifically refers to
infected cells, cells transfected with all or part of the
flavivirus or pestivirus genome and animals, in particular,
primates (including chimpanzees) and humans. In most animal
applications of the present invention, the host is a human patient.
Veterinary applications, in certain indications, however, are
clearly anticipated by the present invention such as in
chimpanzees.
[0204] The term "pharmaceutically acceptable salt or prodrug" is
used throughout the specification to describe any pharmaceutically
acceptable form (ester, phosphate ester, salt of an ester or a
related group) of a nucleoside compound, which, upon administration
to a patient, provides the nucleoside compound. Pharmaceutically
acceptable salts include those derived from pharmaceutically
acceptable inorganic or organic bases and acids. Suitable salts
include those derived from alkali metals such as potassium and
sodium, alkaline earth metals such as calcium and magnesium, among
numerous other acids well known in the pharmaceutical art.
Pharmaceutically acceptable prodrugs refer to a compound that is
metabolized, for example, hydrolyzed or oxidized, in the host to
form the compound of the present invention. Typical examples of
prodrugs include compounds that have biologically labile protecting
groups on a functional moiety of the active compound. Prodrugs
include compounds that can be oxidized, reduced, aminated,
deaminated, hydroxylated, dehydroxylated, hydrolyzed, dehydrolyzed,
alkylated, dealkylated, acylated, deacylated, phosphorylated,
dephosphorylated to produce the active compound. The compounds of
this invention possess antiviral activity against flavivirus,
pestivirus or HCV, or are metabolized to a compound that exhibits
such activity.
[0205] Nucleoside Prodrug Formulations
[0206] 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 reduces polarity and
allows passage into cells. Examples of substituent groups that can
replace one or more hydrogens on the phosphate moiety are alkyl,
aryl, steroids, carbohydrates, including sugars,
1,2-diacylglycerol, alcohols, acyl (including lower acyl); alkyl
(including lower alkyl); sulfonate ester including alkyl or
arylalkyl sulfonyl including methanesulfonyl and benzyl, wherein
the phenyl group is optionally substituted with one or more
substituents as provided in the definition of an aryl given herein;
optionally substituted arylsulfonyl; a lipid, including a
phospholipid; an amino acid residue or derivative; a carbohydrate;
a peptide; cholesterol; or other pharmaceutically acceptable
leaving group which, when administered in vivo, provides a compound
wherein R.sup.1 is independently H or phosphate. Many more are
described in R. Jones and N. Bischoferger, Antiviral Research,
1995, 27:1-17. Any of these can be used in combination with the
disclosed nucleosides to achieve a desired effect.
[0207] 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 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.
[0208] 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.
[0209] 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. Raen, 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.
[0210] Nonlimiting examples of U.S. patents that disclose suitable
lipophilic substituents that can be covalently incorporated into
the nucleoside, preferably at the 5'-OH position of the nucleoside
or lipophilic preparations, include U.S. Pat. No. 5,149,794 (Sep.
22, 1992, Yatvin et al.); U.S. Pat. No. 5,194,654 (Mar. 16, 1993,
Hostetler et al., and U.S. Pat. No. 5,223,263 (Jun. 29, 1993,
Hostetler et al.); all of which are incorporated herein by
reference. Foreign patent applications that disclose lipophilic
substituents that can be attached to the nucleosides of the present
invention, or lipophilic preparations, include WO 89/02733, WO
90/00555, WO 91/16920, WO 91/18914, WO 93/00910, WO 94/26273, WO
96/15132, EP 0 350 287, EP 93917054.4, and WO 91/19721.
[0211] Combination and Alternation Therapy
[0212] It has been recognized that drug-resistant variants of 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 HCV 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, biodistriution 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.
[0213] Any of the HCV treatments described in the Background of the
Invention can be used in combination or alternation with the
compounds described in this specification. Nonlimiting examples
include:
[0214] (1) Interferon
[0215] Interferons (IFNs) are compounds that 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 viral replication of many viruses,
including HCV, and when used as the sole treatment for hepatitis C
infection, IFN suppresses serum HCV-RNA to undetectable levels.
Additionally, IFN normalizes serum amino transferase levels.
Unfortunately, the effects of IFN are temporary and a sustained
response occurs in only 8%-9% of patients chronically infected with
HCV (Gary L. Davis. Gastroenterology 118:S104-S114, 2000).
[0216] A number of patents disclose HCV treatments using
interferon-based therapies. For example, U.S. Pat. No. 5,980,884 to
Blatt et al. discloses methods for re-treatment 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. 5,908,621 to Glue et al. discloses the use of polyethylene
glycol modified interferon for the treatment of 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.
[0217] (2) Ribavirin (Battaglia, A. M. et al., Ann. Pharmacother,
2000, 34, 487-494); Berenguer, M. et al. Antivir. Ther., 1998, 3
(Suppl. 3), 125-136).
[0218] Ribavirin
(1-.beta.-D-ribofuranosyl-1-1,2,4-triazole-3-carboxamide) is a
synthetic, non-interferon-inducing, broad spectrum antiviral
nucleoside analog. It is sold under the trade names Virazole.TM.
(The Merck Index, 11th edition, Editor: Budavari, S., Merck &
Co., Inc., Rahway, N.J., p1304, 1989); Rebetol (Schering Plough)
and Co-Pegasus (Roche). U.S. Pat. No. 3,798,209 and RE29,835 (ICN
Pharmaceuticals) disclose and claim ribavirin. Ribavirin is
structurally similar to guanosine, and has in vitro activity
against several DNA and RNA viruses including Flaviviridae (Gary L.
Davis. Gastroenterology 118:S104-S114, 2000). U.S. Pat. No.
4,211,771 (to ICN Pharmaceuticals) discloses the use of ribavirin
as an antiviral agent. Ribavirin reduces serum amino transferase
levels to normal in 40% of patients, but it does not lower serum
levels of HCV-RNA (Gary L. Davis. Gastroenterology 118:S104-S114,
2000). Thus, ribavirin alone is not effective in reducing viral RNA
levels. Additionally, ribavirin has significant toxicity and is
known to induce anemia.
[0219] Combination of Interferon and Ribavirin
[0220] Schering-Plough sells ribavirin as Rebetol.RTM. capsules
(200 mg) for administration to patients with HCV. The U.S. FDA has
approved Rebetol capsules to treat chronic HCV infection in
combination with Schering's alpha interferon-2b products
Intron.RTM. A and PEG-Intron.TM.. Rebetol capsules are not approved
for monotherapy (i.e., administration independent of Intron.RTM.A
or PEG-Intron), although Intron A and PEG-Intron are approved for
monotherapy (i.e., administration without ribavirin). Hoffman La
Roche is selling ribavirin under the name Co-Pegasus in Europe and
the United States, also for use in combination with interferon for
the treatment of HCV. Other alpha interferon products include
Roferon-A (Hoffmann-La Roche), Infergen.RTM. (Intermune, formerly
Amgen's product), and Weliferon.RTM. (Wellcome Foundation) are
currently FDA-approved for HCV monotherapy. Interferon products
currently in development for HCV include: Roferon-A (interferon
alfa-2a) by Roche, PEGASYS (pegylated interferon alfa-2a) by Roche,
INFERGEN (interferon alfacon-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-1b by InterMune.
[0221] The combination of IFN and ribavirin for the treatment of
HCV infection has been reported to be effective in the treatment of
IFN nave patients (for example, Battaglia, A. M. et al., Ann.
Pharmacother. 34:487-494, 2000). Combination treatment is effective
both before hepatitis develops and when histological disease is
present (for example, Berenguer, M. et al. Antivir. Ther. 3(Suppl.
3):125-136, 1998). Currently, the most effective therapy for HCV is
combination therapy of pegylated interferon with ribavirin (2002
NIH Consensus Development Conference on the Management of Hepatitis
C). However, the side effects of combination therapy can be
significant and include hemolysis, flu-like symptoms, anemia, and
fatigue (Gary L. Davis. Gastroenterology 118:S104-S114, 2000).
[0222] (3) Protease inhibitors have been developed for the
treatment of Flaviviridae infections. Examples, include, but are
not limited to the following
[0223] Substrate-based NS3 protease inhibitors (see, for example,
Attwood et al., Antiviral peptide derivatives, PCT WO 98/22496,
1998; Attwood et al., Antiviral Chemistry and Chemotherapy 1999,
10, 259-273; Attwood et al., Preparation and use of amino acid
derivatives as anti-viral agents, German Patent Pub. DE 19914474;
Tung et al. Inhibitors of serine proteases, particularly hepatitis
C virus NS3 protease, PCT WO 98/17679), including alphaketoamides
and hydrazinoureas, and inhibitors that terminate in an
electrophile such as a boronic acid or phosphonate (see, for
example, Llinas-Brunet et al, Hepatitis C inhibitor peptide
analogues, PCT WO 99/07734);
[0224] Non-substrate-based inhibitors such as
2,4,6-trihydroxy-3-nitro-ben- zamide derivatives (see, for example,
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;
[0225] Phenanthrenequinones possessing activity against protease,
for example in a SDS-PAGE and/or autoradiography assay, such as,
for example, Sch 68631, isolated from the fermentation culture
broth of Streptomyces sp., (see, for example, 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 (see, for example, Chu
M. et al., Bioorganic and Medicinal Chemistry Letters 9,
1949-1952); and
[0226] Selective NS3 inhibitors, for example, based on the
macromolecule elgin c, isolated from leech (see, for example, Qasim
M. A. et al., Biochemistry, 1997, 36, 1598-1607). 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.
[0227] Several U.S. patents disclose protease inhibitors for the
treatment of HCV. Non-limiting examples include, but are not
limited to the following. U.S. Pat. No. 6,004,933 to Spruce et al.
discloses a class of cysteine protease inhibitors for inhibiting
HCV endopeptidase. 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. Iridazoleidinones
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.
[0228] (4) Thiazolidine derivatives, for example, that show
relevant inhibition in a reverse-phase HPLC assay with an NS3/4A
fusion protein and NS5A/5B substrate (see, for example, 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;
[0229] (5) Thiazolidines and benzanilides, for example, as
identified in Kakiuchi N. et al. J. EBS Letters 421, 217-220;
Takeshita N. et al. Analytical Biochemistry, 1997, 247,
242-246;
[0230] (6) Helicase inhibitors (see, for example, 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);
[0231] (7) Polymerase inhibitors such as
[0232] i) nucleotide analogues, such as gliotoxin (see, for
example, Ferrari R. et al. Journal of Virology, 1999, 73,
1649-1654);
[0233] ii) the natural product cerulenin (see, for example, Lohmann
V. et al., Virology, 1998, 249, 108-118); and
[0234] iii) non-nucleoside polymerase inhibitors, including, for
example, compound R803 (see, for example, WO 04/018463 A2 and WO
03/040112 A1, both to Rigel Pharmaceuticals, Inc.); substituted
diamine pyrimidines (see, for example, WO 03/063794 A2 to Rigel
Pharmaceuticals, Inc.); benzimidazole derivatives (see, for
example, Bioorg. Med. Chem. Lett., 2004, 14:119-124 and Bioorg.
Med. Chem. Lett., 2004, 14:967-971, both to Boehringer Ingelheim
Corporation); N,N-disubstituted phenylalanines (see, for example,
J. Biol. Chem., 2003, 278:9495-98 and J. Med. Chem., 2003,
13:1283-85, both to Shire Biochem, Inc.); substituted
thiophene-2-carboxylic acids (see, for example, Bioorg. Med. Chem.
Lett., 2004, 14:793-796 and Bioorg. Med. Chem. Lett., 2004,
14:797-800, both to Shire Biochem, Inc.);
.alpha.,.gamma.-diketoacids (see, for example, J. Med. 5 Chem.,
2004, 14-17 and WO 00/006529 A1, both to Merck & Co., Inc.);
and meconic acid derivatives (see, for example, Bioorg. Med. Chem.
Lett., 2004, 3257-3261, WO 02/006246 A1 and WO03/062211 A1, all to
IRBM Merck & Co., Inc.);
[0235] (8) Antisense phosphorothioate oligodeoxynucleotides
(S--ODN) complementary, for example, to sequence stretches in the
5' non-coding region (NCR) of the virus (see, for example, Alt M.
et al., Hepatology, 1995, 22, 707-717), or to nucleotides 326-348
comprising the 3' end of the NCR and nucleotides 371-388 located in
the core coding region of the HCV RNA (see, for example, Alt M. et
al., Archives of Virology, 1997, 142, 589-599; Galderisi U. et al.,
Journal of Cellular Physiology, 1999, 181, 251-257).
[0236] (9) Inhibitors of IRES-dependent translation (see, for
example, Ikeda N et al., Agent for the prevention and treatment of
hepatitis C, Japanese Patent Pub. JP-08268890; Kai Y. et al.
Prevention and treatment of viral diseases, Japanese Patent Pub.
JP-10101591).
[0237] (10) Nuclease-resistant ribozymes (see, for example,
Maccjak, D. J. et al., Hepatology 1999, 30, abstract 995; 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.).
[0238] (11) Nucleoside analogs have also been developed for the
treatment of Flaviviridae infections.
[0239] Idenix Pharmaceuticals, Ltd. discloses branched nucleosides,
and their use in the treatment of HCV and flaviviruses and
pestiviruses in U.S. patent Publication Nos. 2003/0050229 A1,
2004/0097461 A1, 2004/0101535 A1, 2003/0060400 A1, 2004/0102414 A1,
2004/0097462 A1, and 2004/0063622 A1 which correspond to
International Publication Nos. WO 01/90121 and WO 01/92282. A
method for the treatment of hepatitis C infection (and flaviviruses
and pestiviruses) in humans and other host animals is disclosed in
the Idenix publications that includes administering an effective
amount of a biologically active 1', 2', 3' or 4'-branched .beta.-D
or .beta.-L nucleosides or a pharmaceutically acceptable salt or
prodrug thereof, administered either alone or in combination,
optionally in a pharmaceutically acceptable carrier. See also U.S.
patent Publication Nos. 2004/0006002 and 2004/0006007 as well as WO
03/026589 and WO 03/026675. Idenix Pharmaceuticals, Ltd. also
discloses in U.S. patent Publication No. 2004/0077587
pharmaceutically acceptable branched nucleoside prodrugs, and their
use in the treatment of HCV and flaviviruses and pestiviruses in
prodrugs. See also PCT Publication Nos. WO 04/002422, WO 04/002999,
and WO 04/003000. Further, Idenix Pharmaceuticals, Ltd. also
discloses in WO 04/046331 Flaviviridae mutations caused by
biologically active 2'-branched .beta.-D or .beta.-L nucleosides or
a pharmaceutically acceptable salt or prodrug thereof.
[0240] Biota Inc. discloses various phosphate derivatives of
nucleosides, including 1', 2', 3' or 4'-branched .beta.-D or
.beta.-L nucleosides, for the treatment of hepatitis C infection in
International Patent Publication WO 03/072757.
[0241] Emory University and the University of Georgia Research
Foundation, Inc. (UGARF) discloses the use of 2'-fluoronucleosides
for the treatment of HCV in U.S. Pat. No. 6,348,587. See also U.S.
patent Publication No. 2002/0198171 and International Patent
Publication WO 99/43691.
[0242] BioChem Pharma Inc. (now Shire Biochem, Inc.) discloses the
use of various 1,3-dioxolane nucleosides for the treatment of a
Flaviviridae infection in U.S. Pat. No. 6,566,365. See also U.S.
Pat. Nos. 6,340,690 and 6,605,614; U.S. patent Publication Nos.
2002/0099072 and 2003/0225037, as well as International Publication
No. WO 01/32153 and WO 00/50424.
[0243] BioChem Pharma Inc. (now Shire Biochem, Inc.) also discloses
various other 2'-halo, 2'-hydroxy and 2'-alkoxy nucleosides for the
treatment of a Flaviviridae infection in U.S. patent Publication
No. 2002/0019363 as well as International Publication No. WO
01/60315 (PCT/CA01/00197; filed Feb. 19, 2001).
[0244] ICN Pharmaceuticals, Inc. discloses various nucleoside
analogs that are useful in modulating immune response in U.S. Pat.
Nos. 6,495,677 and 6,573,248. See also WO 98/16184, WO 01/68663,
and WO 02/03997.
[0245] U.S. Pat. No. 6,660,721; U.S. patent Publication Nos.
2003/083307 A1, 2003/008841 A1, and 2004/0110718; as well as
International Patent Publication Nos. WO 02/18404; WO 02/100415, WO
02/094289, and WO 04/043159; filed by F. Hoffmann-La Roche AG,
discloses various nucleoside analogs for the treatment of HCV RNA
replication.
[0246] Pharmasset Limited discloses various nucleosides and
antimetabolites for the treatment of a variety of viruses,
including Flaviviridae, and in particular HCV, in U.S. patent
Publication Nos. 2003/0087873, 2004/0067877, 2004/0082574,
2004/0067877, 2004/002479, 2003/0225029, and 2002/00555483, as well
as International Patent Publication Nos. WO 02/32920, WO 01/79246,
WO 02/48165, WO 03/068162, WO 03/068164 and WO 2004/013298.
[0247] Merck & Co., Inc. and Isis Pharmaceuticals disclose in
U.S. patent Publication Nos. 2002/0147160, 2004/0072788,
2004/0067901, and 2004/0110717; as well as the corresponding
International Patent Publication Nos. WO 02/057425 (PCT/US02/01531;
filed Jan. 18, 2002) and WO 02/057287 (PCT/US02/03086; filed Jan.
18, 2002) various nucleosides, and in particular several
pyrrolopyrimidine nucleosides, for the treatment of viruses whose
replication is dependent upon RNA-dependent RNA polymerase,
including Flaviviridae, and in particular HCV. See also WO
2004/000858, WO 2004/003138, WO 2004/007512, and WO
2004/009020.
[0248] U.S. patent Publication No. 2003/028013 A1 as well as
International Patent Publication Nos. WO 03/051899, WO 03/061576,
WO 03/062255 WO 03/062256, WO 03/062257, and WO 03/061385, filed by
Ribapharm, also are directed to the use of certain nucleoside
analogs to treat hepatitis C virus.
[0249] Genelabs Technologies disclose in U.S. patent Publication
No. 2004/0063658 as well as International Patent Publication Nos.
WO 03/093290 and WO 04/028481 various base modified derivatives of
nucleosides, including 1', 2', 35' or 4'-branched .beta.-D or
.beta.-L nucleosides, for the treatment of hepatitis C
infection.
[0250] Eldrup et al. (Oral Session V, Hepatitis C Virus,
Flaviviridae; 16.sup.th International Conference on Antiviral
Research (Apr. 27, 2003, Savannah, Ga.) p. A75) described the
structure activity relationship of 2'-modified nucleosides for
inhibition of HCV.
[0251] 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.
[0252] 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.
[0253] (12) Other miscellaneous compounds including
1-amino-alkylcyclohexanes (for example, U.S. Pat. No. 6,034,134 to
Gold et al.), alkyl lipids (for example, U.S. Pat. No. 5,922,757 to
Chojkier et al.), vitamin E and other antioxidants (for example,
U.S. Pat. No. 5,922,757 to Chojkier et al.), squalene, amantadine,
bile acids (for example, U.S. Pat. No. 5,846,964 to Ozeki et al.),
N-(phosphonoacetyl)-L-aspartic acid (for example, U.S. Pat. No.
5,830,905 to Diana et al.), benzenedicarboxamides (for example,
U.S. Pat. No. 5,633,388 to Diana et al.), polyadenylic acid
derivatives (for example, U.S. Pat. No. 5,496,546 to Wang et al.),
2',3'-dideoxyinosine (for example, U.S. Pat. No. 5,026,687 to
Yarchoan et al.), benzimidazoles (for example, U.S. Pat. No.
5,891,874 to Colacino et al.), plant extracts (for example, 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 (for example,
U.S. Pat. No. 5,830,905 to Diana et al.).
[0254] (13) Other compounds currently in clinical development for
treatment of hepatitis C virus include, for example: Interleukin-10
by Schering-Plough, IP-501 by Interneuron, Merimebodib VX-497 by
Vertex, AMANTADINE.RTM. (Symmetrel) by Endo Labs Solvay,
HEPTAZYME.RTM. by RPI, IDN-6556 by Idun Pharma., XTL-002 by XTL.,
HCV/MF59 by Chiron, CIVACIR.RTM. (Hepatitis C Immune Globulin) by
NABI, LEVOVIRIN.RTM. by ICN/Ribapharm, VIRAMIDINE.RTM. by
ICN/Ribapharm, ZADAXIN.RTM. (thymosin alfa-1) by Sci Clone,
thymosin plus pegylated interferon by Sci Clone, CEPLENE.RTM.)
(histamine dihydrochloride) by Maxim, VX 950/LY 570310 by
Vertex/Eli Lilly, ISIS 14803 by Isis Pharmaceutical/Elan, IDN-6556
by Idun Pharmaceuticals, Inc., JTK 003 by AKROS Pharma, BILN-2061
by Boehringer Ingelheim, CellCept (mycophenolate mofetil) by Roche,
T67, a .beta.-tubulin inhibitor, by Tularik, a therapeutic vaccine
directed to E2 by Innogenetics, FK788 by Fujisawa Healthcare, Inc.,
IdB 1016 (Siliphos, oral silybin-phosphatdylcholine phytosome), RNA
replication inhibitors (VP50406) by ViroPharma/Wyeth, therapeutic
vaccine by Intercell, therapeutic vaccine by Epimmune/Genencor,
IRES inhibitor by Anadys, ANA 245 and ANA 246 by Anadys,
immunotherapy (Therapore) by Avant, protease inhibitor by
Corvas/SChering, helicase inhibitor by Vertex, fusion inhibitor by
Trimeris, T cell therapy by CellExSys, polymerase inhibitor by
Biocryst, targeted RNA chemistry by PTC Therapeutics, Dication by
Immtech, Int., protease inhibitor by Agouron, protease inhibitor by
Chiron/Medivir, antisense therapy by AVI BioPharma, antisense
therapy by Hybridon, hemopurifier by Aethlon Medical, therapeutic
vaccine by Merix, protease inhibitor by Bristol-Myers Squibb/Axys,
Chron-VacC, a therapeutic vaccine, by Tripep, UT 231B by United
Therapeutics, protease, helicase and polymerase inhibitors by
Genelabs Technologies, IRES inhibitors by Immusol, R803 by Rigel
Pharmaceuticals, INFERGEN.RTM. (interferon alphacon-1) by
InterMune, OMNIEFERON.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.
[0255] Pharmaceutical Compositions
[0256] Hosts, including humans, infected with pestivirus,
flavivirus, HCV or another organism replicating through a
RNA-dependent RNA viral polymerase, 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 diluent. The active
materials can be administered by any appropriate route, for
example, orally, parenterally, intravenously, intradermally,
subcutaneously, or topically, in liquid or solid form.
[0257] A preferred dose of the compound for pestivirus, flavivirus
or HCV will be in the range from about 1 to 50 mg/kg, preferably 1
to 20 mg/kg, of body weight per day, more generally 0.1 to about
100 mg per kilogram body weight of the recipient per day. The
effective dosage range of the pharmaceutically acceptable salts and
prodrugs can be calculated based on the weight of the parent
nucleoside to e 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.
[0258] The compound is conveniently administered in unit any
suitable dosage form, including but not limited to one containing 7
to 3000 mg, or 70 to 1400 mg of active ingredient per unit dosage
form. An oral dosage in one embodiment is 50-1000 mg. In another
embodiment, the dosage form contains 0.5-500 mg; or 0.5-100 mg; or
0.5-50 mg; or 0.5-25 mg; or 1.0-10 mg.
[0259] Ideally the active ingredient should be administered to
achieve peak plasma concentrations of the active compound of from
about 0.2 to 70 .mu.M, preferably about 1.0 to 10 .mu.M. This may
be achieved, for example, by the intravenous injection of a 0.1 to
5% solution of the active ingredient, optionally in saline, or
administered as a bolus of the active ingredient.
[0260] 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.
[0261] 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 e included as part of the composition.
[0262] 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.
[0263] 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.
[0264] The compound or a pharmaceutically acceptable prodrug or
salts thereof can also be mixed with other active materials that do
not impair the desired action, or with materials that supplement
the desired action, such as antibiotics, antifungals,
anti-inflammatories, or other antivirals, including other
nucleoside compounds. Solutions or suspensions used for parenteral,
intradermal, sucutaneous, or topical application can include the
following components: a sterile diluent such as water for
injection, saline solution, fixed oils, polyethylene glycols,
glycerine, propylene glycol or other synthetic solvents;
antibacterial agents such as benzyl alcohol or methyl parabens;
antioxidants such as ascorbic acid or sodium bisulfite; chelating
agents such as ethylenediaminetetraacetic acid; buffers such as
acetates, citrates or phosphates and agents for the adjustment of
tonicity such as sodium chloride or dextrose. The parental
preparation can be enclosed in ampoules, disposable syringes or
multiple dose vials made of glass or plastic.
[0265] If administered intravenously, preferred carriers are
physiological saline or phosphate buffered saline (PBS).
[0266] 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.
[0267] Liposomal suspensions (including liposomes targeted to
infected cells with monoclonal antibodies to viral antigens) are
also preferred as pharmaceutically acceptable carriers. These may
be prepared according to methods known to those skilled in the art,
for example, as described in U.S. Pat. No. 4,522,811 (which is
incorporated herein by reference in its entirety). For example,
liposome formulations may be prepared by dissolving appropriate
lipid(s) (such as stearoyl phosphatidyl ethanolamine, stearoyl
phosphatidyl choline, arachadoyl phosphatidyl choline, and
cholesterol) in an inorganic solvent that is then evaporated,
leaving behind a thin film of dried lipid on the surface of the
container. An aqueous solution of the active compound or its
monophosphate, diphosphate, and/or triphosphate derivatives is then
introduced into the container. The container is then swirled by
hand to free lipid material from the sides of the container and to
disperse lipid aggregates, thereby forming the liposomal
suspension.
[0268] Processes for the Preparation of Active Compounds
[0269] The nucleosides of the present invention can be synthesized
by any means known in the art. In particular, the synthesis of the
present nucleosides can be achieved by either alkylating the
appropriately modified sugar, followed by glycosylation or
glycosylation followed by alkylation of the nucleoside, though
preferably alkylating the appropriately modified sugar, followed by
glycosylation. The following non-limiting embodiments illustrate
some general methodology to obtain the nucleosides of the present
invention.
[0270] A. General Synthesis of 1'-C-Branched Nucleosides
[0271] 1'-C branched ribonucleosides of the following structures:
14
[0272] wherein
[0273] R is H, phosphate (including mono-, di-, or triphosphate or
a stabilized phosphate prodrug) or phosphonate;
[0274] n is 0-2;
[0275] when X is CH.sub.2, CHOH, CH-alkyl, CH-alkenyl, CH-alkynyl,
C-dialkyl, CH--O-alkyl, CH--O-alkenyl, CH--O-alkynyl, CH--S-alkyl,
CH--S-alkenyl, CH--S-alkynyl, CH-halogen, or C-(halogen).sub.2,
[0276] then each R.sup.1 and R.sup.1' is independently H, OH,
optionally substituted alkyl including lower alkyl, azido, cyano,
optionally substituted alkenyl or alkynyl, --C(O)O-(alkyl),
--C(O)O(lower alkyl), --C(O)O-(alkenyl), --C(O)O-(alkynyl),
--O(acyl), --O(lower acyl), --O(alkyl), --O(lower alkyl),
--O(alkenyl), --O(alkynyl), halogen, halogenated alkyl, --NO.sub.2,
--NH.sub.2, --NH(lower alkyl), --N(lower alkyl).sub.2, --NH(acyl),
--N(acyl).sub.2, --C(O)NH.sub.2, --C(O)NH(alkyl),
--C(O)N(alkyl).sub.2, S(O)N-alkyl, S(O)N-alkenyl, S(O)N-alkynyl,
SCH-halogen, wherein alkyl, alkenyl, and/or alkynyl maybe
optionally substituted;
[0277] when X is O, S[O].sub.n, NH, N-alkyl, N-alkenyl, N-alkynyl,
S(O)N-alkyl, S(O)N-alkenyl, S(O)N-alkynyl, or SCH-halogen,
[0278] then each R.sup.1 and R.sup.1' is independently H,
optionally substituted alkyl including lower alkyl, azido, cyano,
optionally substituted alkenyl or alkynyl, --C(O)O-(alkyl),
--C(O)O(lower alkyl), --C(O)O-(alkenyl), --C(O)O-(alkynyl),
halogenated alkyl, --C(O)NH.sub.2, --C(O)NH(alkyl),
--C(O)N(alkyl).sub.2, --C(H).dbd.N--NH.sub.2, C(S)NH.sub.2,
C(S)NH(alkyl), or C(S)N(alkyl).sub.2, wherein alkyl, alkenyl,
and/or alkynyl maybe optionally substituted;
[0279] when X* is CY.sup.3;
[0280] then each R.sup.1 is independently H, OH, optionally
substituted alkyl including lower alkyl, azido, cyano, optionally
substituted alkenyl or alkynyl, --C(O)O-(alkyl), --C(O)O(lower
alkyl), --C(O)O-(alkenyl), --C(O)O-(alkynyl), --O(acyl), --O(lower
acyl), --O(alkyl), --O(lower alkyl), --O(alkenyl), --O(alkynyl),
halogen, halogenated alkyl, --NO.sub.2, --NH.sub.2, --NH(lower
alkyl), --N(lower alkyl).sub.2, --NH(acyl), --N(acyl).sub.2,
--C(O)NH.sub.2, --C(O)NH(alkyl), and --C(O)N(alkyl).sub.2, wherein
an optional substitution on alkyl, alkenyl, and/or alkynyl may be
one or more halogen, hydroxy, alkoxy or alkylthio groups taken in
any combination; and
[0281] Y.sup.3 is hydrogen, alkyl, bromo, chloro, fluoro, iodo,
azido, cyano, alkenyl, alkynyl, --C(O)O(alkyl), --C(O)O(lower
alkyl), CF.sub.3, --CONH.sub.2, --CONH(alkyl),
--CON(alkyl).sub.2;
[0282] each R.sup.2 and R.sup.3 independently is OH, NH.sub.2, SH,
F, Cl, Br, I, CN, NO.sub.2, --C(O)NH.sub.2, --C(O)NH(alkyl),
--C(O)N(alkyl).sub.2, N.sub.3, optionally substituted alkyl
including lower alkyl, optionally substituted alkenyl or alkynyl,
halogenated alkyl, --C(O)O-(alkyl), --C(O)O(lower alkyl),
--C(O)O-(alkenyl), --C(O)O-(alkynyl), --O(acyl), --O(alkyl),
--O(alkenyl), --O(alkynyl), --OC(O)NH.sub.2, NC, C(O)OH, SCN, OCN,
--S(alkyl), --S(alkenyl), --S(alkynyl), --NH(alkyl),
--N(alkyl).sub.2, --NH(alkenyl), --NH(alkynyl), an amino acid
residue or derivative, a prodrug or leaving group that provides OH
in vivo, or an optionally substituted 3-7 membered heterocyclic
ring having O, S and/or N independently as a heteroatom taken alone
or in combination;
[0283] each R.sup.2' and R.sup.3' independently is H; optionally
substituted alkyl, alkenyl, or alkynyl; --C(O)O(alkyl),
--C(O)O(lower alkyl), --C(O)O(alkenyl), --C(O)O(alkynyl),
--C(O)NH.sub.2, --C(O)NH(alkyl), --C(O)N(alkyl).sub.2, --O(acyl),
--O(lower acyl), --O(alkyl), --O(lower alkyl), --O(alkenyl),
halogen, halogenated alkyl and particularly CF.sub.3, azido, cyano,
NO.sub.2, --S(alkyl), --S(alkenyl), --S(alkynyl), NH.sub.2,
--NH(alkyl), --N(alkyl).sub.2, --NH(alkenyl), --NH(alkynyl),
--NH(acyl), or --N(acyl).sub.2, and R.sub.3 at 3'-C may also be
OH;
[0284] Base is selected from the group consisting of: 15
[0285] wherein
[0286] each A independently is N or C--R.sup.5;
[0287] W is H, OH, --O(acyl), --O(C.sub.1-4 alkyl), --O(alkenyl),
--O(alkynyl), --OC(O)R.sup.4R.sup.4, --OC(O)N R.sup.4R.sup.4, SH,
--S(acyl), --S(C.sub.1-4 alkyl), NH.sub.2, NH(acyl), N(acyl).sub.2,
NH(C.sub.1-4 alkyl), N(C.sub.1-4 alkyl).sub.2, --N(cycloalkyl)
C.sub.1-4 alkylamino, di(C.sub.1-4 alkyl)amino, C.sub.3-6
cycloalkylamino, halogen, C.sub.1-4 alkyl, C.sub.1-4 alkoxy, CN,
SCN, OCN, SH, N.sub.3, NO.sub.2, NH.dbd.NH.sub.2, N.sub.3, NHOH,
--C(O)NH.sub.2, --C(O)NH(acyl), --C(O)N(acyl).sub.2,
--C(O)NH(C.sub.1-4 alkyl), --C(O)N(C.sub.1-4 alkyl).sub.2,
--C(O)N(alkyl)(acyl), or halogenated alkyl;
[0288] Z is O, S, NH, N--OH, N--NH.sub.2, NH(alkyl),
N(alkyl).sub.2, N-cycloalkyl, alkoxy, CN, SCN, OCN, SH, NO.sub.2,
NH.sub.2, N.sub.3, NH.dbd.NH, NH(alkyl), N(alkyl).sub.2,
CONH.sub.2, CONH(alkyl), or CON(alkyl).sub.2.
[0289] each R.sup.4 is independently H, acyl, or C.sub.1-6
alkyl;
[0290] each R.sup.5 is independently H, Cl, Br, F, I, CN, OH,
optionally substituted alkyl, alkenyl or alkynyl, carboxy,
C(.dbd.NH)NH.sub.2, C.sub.1-4 alkoxy, C.sub.1-4 alkyloxycarbonyl,
N.sub.3, NH.sub.2, NH(alkyl), N(alkyl).sub.2, NO.sub.2, N.sub.3,
halogenated alkyl especially CF.sub.3, C.sub.1-4 alkylamino,
di(C.sub.1-4 alkyl)amino, C.sub.3-6 cycloalkylamino, C.sub.1-6
alkoxy, SH, --S(C.sub.1-4 alkyl), --S(C.sub.1-4 alkenyl),
--S(C.sub.1-4 alkynyl), C.sub.1-6 alkylthio, C.sub.1-6
alkylsulfonyl, (C.sub.1-4 alkyl).sub.0-2 aminomethyl, C.sub.3-6
cycloalkylamino -alkenyl, -alkynyl, --(O)alkyl, --(O)alkenyl,
--(O)alkynyl, --(O)acyl, --O(C.sub.1-4 alkyl), --O(C.sub.1-4
alkenyl), --O(Cl.sub.4 alkynyl), --O--C(O)NH.sub.2,
--OC(O)N(alkyl), --OC(O)R'R", --C(O)OH, C(O)O-alkyl, C(O)O-alkenyl,
C(O)O-alkynyl, S-alkyl, S-acyl, S-alkenyl, S-alkynyl, SCN, OCN, NC,
--C(O)--NH.sub.2, C(O)NH(alkyl), C(O)N(alkyl).sub.2, C(O)NH(acyl),
C(O)N(acyl).sub.2, (S)--NH.sub.2, NH-alkyl, N(dialkyl).sub.2,
NH-acyl, N-diacyl, or a 3-7 membered heterocycle having O, S, or N
taken independently in any combination;
[0291] each R' and R" independently is H, C.sub.1-6 alkyl,
C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, halogen, halogenated alkyl,
OH, CN, N.sub.3, carboxy, C.sub.1-4alkoxycarbonyl, NH.sub.2,
C.sub.1-4 alkylamino, di(C.sub.1-4 alkyl)amino, C.sub.1-6 alkoxy,
C.sub.1-6 alkylsulfonyl, or (C.sub.1-4 alkyl).sub.0-2 aminomethyl;
and all tautomeric, enantiomeric and stereoisomeric forms
thereof;
[0292] with the caveat that when X is S in Formula (I), then the
compound is not
5-(4-amino-imidazo[4,5-d][1,2,3]triazin-7-yl)-2-hydroxymethyl-tetr-
ahydro-thiophen-3-ol or
7-(4-hydroxy-5-hydroxy-methyl-tetrahydro-thiophen--
2-yl)-3,7-dihydro-imidazo[4,5-d][1,2,3]triazin-4-one, can be
prepared according to Schemes 1, 2 or 7 below.
[0293] Modification from the Lactone
[0294] The key starting material for this process is an
appropriately substituted lactone. The lactone may be purchased or
can be prepared by any known means including standard
epimerization, substitution and cyclization techniques. The lactone
optionally can be protected with a suitable protecting group,
preferably with an acyl or silyl group, by methods well known to
those skilled in the art, as taught by Greene et al., Protective
Groups in Organic Synthesis, John Wiley and Sons, Second Edition,
1991. The protected lactone can then be coupled with a suitable
coupling agent, such as an organometallic carbon nucleophile like a
Grignard reagent, an organolithium, lithium dialkylcopper or
R.sup.6--SiMe.sub.3 in TAF with the appropriate non-protic solvent
at a suitable temperature, to give the 1'-alkylated sugar.
[0295] The optionally activated sugar can then be coupled to the
base by methods well known to those skilled in the art, as taught
by Townsend, Chemistry of Nuceleotides, Plenum Press, 1994. For
example, an acylated sugar can be coupled to a silylated base with
a Lewis acid such as tin tetrachloride, titanium tetrachloride, or
trimethylsilyltriflate in the appropriate solvent at a suitable
temperature.
[0296] Subsequently, the nucleoside can be deprotected by methods
well known to those skilled in the art, as taught by Greene et al.,
Protective Groups in Organic Synthesis, John Wiley and Sons, Second
Edition, 1991.
[0297] In a particular embodiment, the 1'-C-branched ribonucleoside
is desired. The synthesis of a ribonucleoside is shown in Scheme 1.
Alternatively, deoxyribonucleoside is desired. To obtain these
nucleosides, the formed ribonucleoside an optionally be protected
by methods well known to those skilled in the art, as taught by
Greene et al., Protective Groups in Organic Synthesis, John Wiley
and Sons, Second Edition, 1991, and then the 2'-OH can be reduced
with a suitable reducing agent. Optionally, the 2'-OH can be
activated to facilitate reduction as, for example, via the Barton
reduction. 16
[0298] Alternative Method for the Preparation of 1'-C-Branched
Nucleosides
[0299] The key starting material for this process is an
appropriately substituted hexose. The hexose can be purchased or
can be prepared by any known means including standard epimerization
(as, for example, via alkaline treatment), substitution and
coupling techniques. The hexose can be protected selectively to
give the appropriate hexa-furanose, as taught by Townsend,
Chemistry of Nucleosides and Nucleotides, Plenum Press, 1994.
[0300] The 1'-OH optionally can be activated to a suitable leaving
group such as an acyl group or a halogen via acylation or
halogenation, respectively. The optionally activated sugar can then
be coupled to the base by methods well known to those skilled in
the art, as taught by Townsend, Chemistry of Nucleosides and
Nucleotides, Plenum Press, 1994. For example, an acylated sugar can
be coupled to a silylated base with a Lewis acid, such as tin
tetrachloride, titanium tetrachloride, or trimethylsilyltriflate in
an appropriate solvent at a suitable temperature. Alternatively, a
halo-sugar can be coupled to a silylated base in the presence of
trimethylsilyltriflate.
[0301] The 1'-CH.sub.2--OH, if protected, selectively can be
deprotected by methods well known in the art. The resultant primary
hydroxyl can be reduced to give the methyl, using a suitable
reducing agent. Alternatively, the hydroxyl can be activated prior
to reduction to facilitate the reaction, i.e., via the Barton
reduction. In an alternate embodiment, the primary hydroxyl can be
oxidized to the aldehyde, then coupled with a carbon nucleophile
such as a Grignard reagent, an organolithium, lithium dialkylcopper
or R.sup.6--SiMe.sub.3 in TAF with an appropriate non-protic
solvent at a suitable temperature.
[0302] In a particular embodiment, the 1'-C-branched ribonucleoside
is desired. The synthesis of a ribonucleoside is shown in Scheme 2.
Alternatively, deoxyribonucleoside is desired. To obtain these
nucleosides, the formed ribonucleoside optionally can be protected
by methods well known to those skilled in the art, as taught by
Greene et al., Protective Groups in Organic Synthesis, John Wiley
and Sons, Second Edition, 1991, and then the 2'-OH can be reduced
with a suitable reducing agent. Optionally, the 2'-OH can be
activated to facilitate reduction as, for example, via the Barton
reduction. 17
[0303] In addition, the L-enantiomers corresponding to the
compounds of the invention can be prepared following the same
general methods (1 or 2), beginning with the corresponding L-sugar
or nucleoside L-enantiomer as the starting material.
[0304] General Synthesis of 2'-C-Branched Nucleosides
[0305] 2'-C-branched ribonucleosides of the following structures:
18
[0306] wherein R, R.sup.1, R.sup.1', R.sup.2, R.sup.2', R.sup.3,
R.sup.3', X, X*, and Base are all as described above, can be
prepared according to Schemes 3 or 4 below.
[0307] Glycosylation of the Nucleboase with an Appropriately
Modified Sugar
[0308] The key starting material for this process is an
appropriately substituted sugar with a 2'-OH and 2'-H, with an
appropriate leaving group (LG), such as an acyl or halogen group,
for example. The sugar can be purchased or can be prepared by any
known means including standard epimerization, substitution,
oxidation and/or reduction techniques. The substituted sugar can
then be oxidized with an appropriate oxidizing agent in a
compatible solvent at a suitable temperature to yield the
2'-modified sugar. Possible oxidizing agents are Jones' reagent (a
mixture of chromic and sulfuric acids), Collins' reagent
(dipyridine Cr(VI)oxide), Corey's reagent (pyridinium
chlorochromate), pyridinium dichromate, acid dichromate, potassium
permanganate, MnO.sub.2, ruthenium tetroxide, phase transfer
catalysts such as chromic acid or permanganate supported on a
polymer, Cl.sub.2-pyridine, H.sub.2O.sub.2-ammonium molydate,
NarO.sub.2--CAN, NaOCl in HOAc, copper chromate, copper oxide,
Raney nickel, palladium acetate, Meerwin-Pondorf-Verley reagent
(aluminum t-butoxide with another ketone) and
N-bromosuccinimide.
[0309] Then coupling of an organometallic carbon nucleophile such
as a Grignard reagent, an organolithium, lithium dialkylcopper or
R.sup.6--SiMe.sub.3 in TAF with the ketone and an appropriate
non-protic solvent at a suitable temperature, yields the
2'-alkylated sugar. The alkylated sugar optionally can be protected
with a suitable protecting group, preferably with an acyl or silyl
group, by methods well known to those skilled in the art, as taught
by Greene et al., Protective Groups in Organic Synthesis, John
Wiley and Sons, Second Edition, 1991.
[0310] The optionally protected sugar can then be coupled to the
base by methods well known to those skilled in the art, as taught
by Townsend, Chemistry of Nucleosides and Nucleotides, Plenum
Press, 1994. For example, an acylated sugar can be coupled to a
silylated base with a Lewis acid, such as tin tetrachloride,
titanium tetrachloride, or trimethylsilyltriflate in an appropriate
solvent at a suitable temperature. Alternatively, a halo-sugar can
e coupled to a silylated base in the presence of
trimethylsilyltriflate.
[0311] Subsequently, the nucleoside can be deprotected by methods
well known to those skilled in the art, as by Greene et al.,
Protective Groups in Organic Synthesis, John Wiley and Sons, Second
Edition, 1991.
[0312] In a particular embodiment, the 2'-C-branched ribonucleoside
is desired, the synthesis of which is shown in Scheme 3.
Alternatively, a deoxyribonucleoside is desired. To obtain these
nucleosides, the formed ribonucleoside can optionally be protected
by methods well known to those skilled in the art, as by Greene et
al., Protective Groups in Organic Synthesis, John Wiley and Sons,
Second Edition, 1991, and then the 2'-OH can e reduced with a
suitable reducing agent. Optionally, the 2'-OH can be activated to
facilitate reduction, such as, for example, by the Barton
reduction. 19
[0313] Modification of a Pre-Formed Nucleoside
[0314] The key starting material for this process is an
appropriately substituted nucleoside with a 2'-OH and 2'-H. The
nucleoside can be purchased or can be prepared by any known means
including standard coupling techniques. The nucleoside optionally
can be protected with suitable protecting groups, preferably with
acyl or silyl groups, by methods well known to those skilled in the
art, as described in Greene et al., Protective Groups in Organic
Synthesis, John Wiley and Sons, Second Edition, 1991.
[0315] The appropriately protected nucleoside then can be oxidized
with an appropriate oxidizing agent in a compatible solvent at a
suitable temperature to yield the 2'-modified sugar. Possible
oxidizing agents include Jones' reagent (a mixture of chromic and
sulfuric acids), Collins' reagent (dipyridine Cr(VI)oxide), Corey's
reagent (pyridinium chlorochromate), pyridinium dichromate, acid
dichromate, potassium permanganate, MnO.sub.2, ruthenium tetroxide,
phase transfer catalysts such as chromic acid or permanganate
supported on a polymer, Cl.sub.2-pyridine, H.sub.2O.sub.2-ammonium
molydate, NarO.sub.2--CAN, NaOCl in HOAc, copper chromate, copper
oxide, Raney nickel, palladium acetate, Meerwin-Pondorf-Verley
reagent (aluminum t-butoxide with another ketone) and
N-bromosuccinimide.
[0316] Subsequently, the nucleoside can be deprotected by methods
well known to those skilled in the art, as by Greene et al.,
Protective Groups in Organic Synthesis, John Wiley and Sons, Second
Edition, 1991.
[0317] In a particular embodiment, a 2'-C-branched ribonucleoside
is desired, the synthesis of which is shown in Scheme 4.
Alternatively, the deoxyribonucleoside may be desired. To obtain
these nucleosides, the formed ribonucleoside optionally may be
protected by methods well known to those skilled in the art, as by
Greene et al., Protective Groups in Organic Synthesis, John Wiley
and Sons, Second Edition, 1991, and then the 2'-OH can be reduced
with a suitable reducing agent. Optionally, the 2'-OH can be
activated to facilitate reduction such as, for example, by the
Barton reduction. 20
[0318] In another embodiment of the invention, the L-enantiomers
are desired. These L-enantiomers corresponding to the compounds of
the invention may be prepared following the same general methods
given above, but beginning with the corresponding L-sugar or
nucleoside L-enantiomer as the starting material.
[0319] C. General Synthesis of 3'-C-Branched Nucleosides
[0320] 3'-C-branched ribonucleosides of the following structures:
21
[0321] wherein R, R.sup.1, R.sup.1', R.sup.2, R.sup.2', R.sup.3,
R.sup.3' X, X*, and Base are all as described above, can be
prepared according to Schemes 5 or 6 below.
[0322] Glycosylation of the Nucleobase with an Appropriately
Modified Sugar (Scheme 5)
[0323] The key starting material for this process is an
appropriately substituted sugar with a 3'-OH and a 3'-H, with an
appropriate leaving group (LG) such as, for example, an acyl group
or a halogen. The sugar can be purchased or can be prepared by any
known means including standard epimerization, substitution,
oxidation and/or reduction techniques. The substituted sugar then
can be oxidized by an appropriate oxidizing agent in a compatible
solvent at a suitable temperature to yield the 3'-modified
sugar.
[0324] Possible oxidizing agents include Jones' reagent (a mixture
of chromic and sulfuric acids), Collins' reagent (dipyridine
Cr(VI)oxide), Corey's reagent (pyridinium chlorochromate),
pyridinium dichromate, acid dichromate, potassium permanganate,
MnO.sub.2, ruthenium tetroxide, phase transfer catalysts such as
chromic acid or permanganate supported on a polymer,
Cl.sub.2-pyridine, H.sub.2O.sub.2-ammonium molydate,
NarO.sub.2--CAN, NaOCl in HOAc, copper chromate, copper oxide,
Raney nickel, palladium acetate, Meerwin-Pondorf-Verley reagent
(aluminum t-butoxide with another ketone) and
N-bromosuccinimide.
[0325] Then coupling of an organometallic carbon nucleophile such
as a Grignard reagent, an organolithium, lithium dialkylcopper or
R.sup.6--SiMe.sub.3 in TAF with the ketone and an appropriate
non-protic solvent at a suitable temperature, yields the
3'-C-branched sugar. The 3'-C-branched sugar optionally can e
protected with a suitable protecting group, preferably with an acyl
or silyl group, by methods well known to those skilled in the art,
as taught by Greene et al., Protective Groups in Organic Synthesis,
John Wiley and Sons, Second Edition, 1991.
[0326] The optionally protected sugar can then be coupled to the
base by methods well known to those skilled in the art, as taught
in Townsend, Chemistry of Nucleosides and Nucleotides, Plenum
Press, 1994. For example, an acylated sugar can be coupled to a
silylated base with a Lewis acid, such as tin tetrachloride,
titanium tetrachloride, or trimethylsilyltriflate in an appropriate
solvent at a suitable temperature. Alternatively, a halo-sugar can
be coupled to a silylated base in the presence of
trimethylsilyltriflate.
[0327] Subsequently, the nucleoside can be deprotected by methods
well known to those skilled in the art, as by Greene et al.,
Protective Groups in Organic Synthesis, John Wiley and Sons, Second
Edition, 1991.
[0328] In a particular embodiment, the 3'-C-branched ribonucleoside
is desired, the synthesis of which is shown in Scheme 5.
Alternatively, a deoxyribonucleoside is desired. To obtain these
nucleosides, the formed ribonucleoside can optionally be protected
by methods well known to those skilled in the art, as by Greene et
al., Protective Groups in Organic Synthesis, John Wiley and Sons,
Second Edition, 1991, and then the 2'-OH can be reduced with a
suitable reducing agent. Optionally, the 2'-OH can be activated to
facilitate reduction, such as, for example, by the Barton
reduction. 22
[0329] Modification of a Preformed Nucleoside.
[0330] The key starting material for this process is an
appropriately substituted nucleoside with a 3'-OH and 3'-H. The
nucleoside can be purchased or can be prepared by any known means
including standard coupling techniques. The nucleoside can be
optionally protected with suitable protecting groups, preferably
with acyl or silyl groups, by methods well known to those skilled
in the art, as taught by Greene et al., Protective Groups in
Organic Synthesis, John Wiley and Sons, Second Edition, 1991.
[0331] The appropriately protected nucleoside can then be oxidized
with the appropriate oxidizing agent in a compatible solvent at a
suitable temperature to yield the 2'-modified sugar. Possible
oxidizing agents include Jones' reagent (a mixture of chromic and
sulfuric acids), Collins' reagent (dipyridine Cr(VI)oxide), Corey's
reagent (pyridinium chlorochromate), pyridinium dichromate, acid
dichromate, potassium permanganate, MnO.sub.2, ruthenium tetroxide,
phase transfer catalysts such as chromic acid or permanganate
supported on a polymer, Cl.sub.2-pyridine, H.sub.2O.sub.2-ammonium
molydate, NarO.sub.2--CAN, NaOCl in HOAc, copper chromate, copper
oxide, Raney nickel, palladium acetate, Meerwin-Pondorf-Verley
reagent (aluminum t-butoxide with another ketone) and
N-bromosuccinimide.
[0332] Subsequently, the nucleoside can be deprotected by methods
well known to those skilled in the art, as by Greene et al.,
Protective Groups in Organic Synthesis, John Wiley and Sons, Second
Edition, 1991.
[0333] In a particular embodiment, the 3'-C-branched ribonucleoside
is desired, the synthesis of which is shown in Scheme 6.
Alternatively, a deoxyribonucleoside is desired. To obtain these
nucleosides, the formed ribonucleoside can optionally be protected
by methods well known to those skilled in the art, as by Greene et
al., Protective Groups in Organic Synthesis, John Wiley and Sons,
Second Edition, 1991, and then the 2'-OH can be reduced with a
suitable reducing agent. Optionally, the 2'-OH can be activated to
facilitate reduction, such as, for example, by the Barton
reduction. 23
[0334] In another embodiment of the invention, the L-enantiomers
are desired. These L-enantiomers corresponding to the compounds of
the invention may be prepared following the same general methods
given above, but beginning with the corresponding L-sugar or
nucleoside L-enantiomer as the starting material.
[0335] General Synthesis of 4'-C-Branched Nucleosides
[0336] 4'-C-branched ribonucleosides of the following structures:
24
[0337] wherein R, R.sup.1, R.sup.1', R.sup.2, R.sup.2', R.sup.3,
R.sup.3', X, X*, and Base are all as described above, can be
prepared according to the following general methods.
[0338] Modification from the pentodialdo-furanose.
[0339] The key starting material for this process is an
appropriately substituted pentodialdo-furanose. The
pentodialdo-furanose can be purchased or can be prepared by any
known means including standard epimerization, substitution and
cyclization techniques.
[0340] In a preferred embodiment, the pentodialdo-furanose is
prepared from the appropriately substituted hexose. The hexose can
be purchased or can be prepared by any known means including
standard epimerization (for eg., via alkaline treatment),
substitution, and coupling techniques. The hexose can be in either
the furanose form or cyclized by any means known in the art, such
as methodology taught by Townsend in Chemistry of Nucleosides and
Nucleotides, Plenum Press, 1994, preferably by selectively
protecting the hexose, to give the appropriate hexafuranose.
[0341] The 4'-hydroxymethylene of the hexafuranose then can be
oxidized with an appropriate oxidizing agent in a compatible
solvent at a suitable temperature to yield the 4'-aldo-modified
sugar. Possible oxidizing agents are Swern reagents, Jones' reagent
(a mixture of chromic and sulfuric acids), Collins' reagent
(dipyridine Cr(VI)oxide), Corey's reagent (pyridinium
chlorochromate), pyridinium dichromate, acid dichromate, potassium
permanganate, MnO.sub.2, ruthenium tetroxide, phase transfer
catalysts such as chromic acid or permanganate supported on a
polymer, Cl.sub.2-pyridine, H.sub.2O.sub.2-ammonium molybdate,
NarO.sub.2--CAN, NaOCl in HOAc, copper chromate, copper oxide,
Raney nickel, palladium acetate, Meerwin-Pondorf-Verley reagent
(aluminum t-butoxide with another ketone) and N-bromosuccinimide,
although using H.sub.3PO.sub.4, DMSO and DCC in a mixture of
benzene/pyridine at room temperature is preferred.
[0342] Then the pentodialdo-furanose optionally can be protected
with a suitable protecting group, preferably with an acyl or silyl
group, by methods well known to those skilled in the art, as taught
by Greene et al., Protective Groups in Organic Synthesis, John
Wiley and Sons, Second Edition, 1991. In the presence of a base,
such as sodium hydroxide, the protected pentodialdo-furanose then
can be coupled with a suitable electrophilic alkyl, halogeno-alkyl
(such as CF.sub.3), alkenyl or alkynyl (i.e., allyl), to obtain the
4'-alkylated sugar. Alternatively, the protected
pentodialdo-furanose can be coupled with a corresponding carbonyl,
such as formaldehyde, in the presence of a base like sodium
hydroxide and with an appropriate polar solvent like dioxane, at a
suitable temperature, and then reduced with an appropriate reducing
agent to provide the 4'-alkylated sugar. In one embodiment, the
reduction is carried out using PhOC(S)Cl and DMAP in acetonitrile
at room temperature, followed by reflux treatment with ACCN and
TMSS in toluene.
[0343] The optionally activated sugar can be coupled to the base by
methods well known to those skilled in the art, as taught by
Townsend in Chemistry of Nucleosides and Nucleotides, Plenum Press,
1994. For example, an acylated sugar can be coupled to a silylated
base with a Lewis acid, such as tin tetrachloride, titanium
tetrachloride, or trimethylsilyltriflate in an appropriate solvent
at room temperature.
[0344] Subsequently, the nucleoside can be deprotected by methods
well known to those skilled in the art, as by Greene et al.,
Protective Groups in Organic Synthesis, John Wiley and Sons, Second
Edition, 1991.
[0345] In a particular embodiment, the 4'-C-branched ribonucleoside
is desired. Alternatively, a deoxyribonucleoside is desired. To
obtain these nucleosides, the formed ribonucleoside can optionally
be protected by methods well known to those skilled in the art, as
by Greene et al., Protective Groups in Organic Synthesis, John
Wiley and Sons, Second Edition, 1991, and then the 2'-OH can be
reduced with a suitable reducing agent. Optionally, the 2'-OH can
be activated to facilitate reduction, such as, for example, by the
Barton reduction.
[0346] In another embodiment of the invention, the L-enantiomers
are desired. These L-enantiomers corresponding to the compounds of
the invention may be prepared following the same general methods
given above, but beginning with the corresponding L-sugar or
nucleoside L-enantiomer as the starting material.
[0347] Methods for Ribofuranosyl-2-azapurine Synthesis
Preparation of 1'-C-methyl-ribofuranosyl-2-azapurine via
6-amino-9-(1-deoxy-beta-D-psicofuranosyl)purine
[0348] As an alternative method of preparation, the title compound
can be prepared according to the published procedure of Farkas and
Sorm (J. Farkas and F. Sorm, "Nucleic acid components and their
analogues. XCIV. Synthesis of
6-amino-9-(1-deoxy-beta-D-psicofuranosyl)purine," Collect. Czech.
Chem. Commun., 1967, 32:2663-7; and J. Farkas, Collect. Czech.
Chem. Commun., 1966, 31:1535 (Scheme 7).
[0349] In a similar manner, but using the appropriate sugar and
2-azapurine base corresponding to the desired product compound, a
variety of Formula (I) and/or Formula (II) compounds can be
prepared. 25
[0350] Alternative Methods for Ribofuranosyl-Purine Analogue
Synthesis
Preparation of ribofuranosyl-purine Analogues:
2-aza-3,7-dideazaadenosine Derivative Compounds
[0351] Preparation of 2-aza-3,7-dideazaadenosine derivative
compounds may be prepared according to the published synthesis of
L. Towsend et al., Bioorganic & Med. Chem. Letters, 1991, 1(2):
111-114, where the starting material,
ethyl-3-cyanopyrrole-2-carboxylate 4 was synthesized by Huisgen
& Laschtuvka, according to the procedure provided in Chemische
Berichte, 1960, 93:65-81, as shown in Scheme 8: 26
Preparation of ribofuranosyl-purine Analogues:
2-aza-3-deazaadenosine Derivative Compounds
[0352] Preparation of 2-aza-3-deazaadenosine derivative compounds
may be prepared according to the published synthesis of B. Otter et
al., J.Heterocyclic Chem., 1984, 481-89 shown in Scheme 9. The
commercially available starting material used is the
4,5-dichloro-6-pyridazone 12. 27
[0353] An alternative preparation of 2-aza-3-deazaadenosine
derivative compounds that utilizes a chlorination step is that
according to R. Panzica, J. Chem. Soc. Perkin Trans I, 1989,
1769-1774 and J.Med. Chem., 1993, 4113-4120, shown in Scheme 10:
28
Preparation of Purine Analogues for Nucleosides: Optionally
Substituted 2,8-diaza-3,7-dideazaadenine Derivative Compounds
[0354] Preparation of certain 2,8-diaza-3,7-dideazaadenosine
derivative compounds may be prepared according to the published
synthesis by Oda et al. in J.Heterocyclic Chem., 1984, 21:1241-55
and Chem. Pharm. Bull., 1984, 32(11):4437-46, as shown in Scheme
11. The starting material is commercially available
4,5-dichloro-6-pyridazone 12. 29
Preparation of Purine Analogues for Nucleosides:
2,8-diaza-3-deazaadenosin- e Derivative Compounds
[0355] Preparation of certain 2,8-diaza-3-deazaadenosine derivative
compounds may be prepared according to the published synthesis by
Panzica et al. in J.Heterocyclic Chem., 1982, 285-88, J. Med.
Chem., 1993, 4113-20, and Bioorg. & Med. Chem. Letters., 1996,
4(10):1725-31, as provided in Scheme 12. The key intermediate 27
was prepared via a 1,3-dipolar cycloaddition reaction between the
2,3,5-tri-O-benzoyl-.beta.- -D-ribofuranosyl azide 26 and
methyl-hydroxy-2-butylnoate 25. A ribofuranosyl azide 26 synthesis
was described by A. Stimac et al., Carbohydrate Res., 1992,
232(2):359-65, using SnCl.sub.4 catalyzed azidolysis of
1-O-Acetyl-2,3,5-tri-O-benzoyl-.beta.-D-ribofuranose with
Me.sub.3SiN.sub.3 in CH.sub.2Cl.sub.2 at room temperature. 30
Preparation of Purine Analogues for Nucleosides: Alternative
Preparation of 2,8-diaza-3-deazaadenine Derivative Compounds
[0356] 2,8-diaza-3-deazaadenine derivative compounds may be
prepared (see Scheme 13) according to the published synthesis by
Chen et al. in J.Heterocyclic Chem., 1982, 285-88; however, no
condensation of this compound with ribofuranose is found. 31
Preparation of ribofuranosyl-2-azapurines via Use of Protective
Groups
[0357] As an alternative method of preparation, the compounds of
the present invention can also be prepared by synthetic methods
well known to those skilled in the art of nucleoside and nucleotide
chemistry, such as taught by Townsend in Chemistry of Nucleosides
and Nucleotides, Plenum Press, 1994.
[0358] A representative general synthetic method is provided in
Scheme 14. The starting material is a 3,5-is-O-protected
beta-D-alkyl ribofuranoside, but it will be understood that any 2',
3', or 5'-position may carry a protecting group to shield it from
reacting. The 2'-C--OH then is oxidized with a suitable oxidizing
agent in a compatible solvent at a suitable temperature to yield
the 2'-keto-modified sugar. Possible oxidizing agents are Swern
reagents, Jones' reagent (a mixture of chromic and sulfuric acids),
Collins' reagent (dipyridine Cr(VI)oxide), Corey's reagent
(pyridinium chlorochromate), pyridinium dichromate, acid
dichromate, potassium permanganate, MnO.sub.2, ruthenium tetroxide,
phase transfer catalysts such as chromic acid or permanganate
supported on a polymer, Cl.sub.2-pyridine, H.sub.2O.sub.2-ammonium
molydate, NarO.sub.2--CAN, NaOCl in HOAc, copper chromate, copper
oxide, Raney nickel, palladium acetate, Meerwin-Pondorf-Verley
reagent (aluminum t-butoxide with another ketone) and
N-bromosuccinimide.
[0359] Next, addition of a Grignard reagent, such as, for example,
an alkyl-, alkenyl- or alkynyl-magnesium halide like CH.sub.3MgBr,
CH.sub.3CH.sub.2MgBr, vinylMgBr, allylMgBr and ethynylMgBr, or an
alkyl-, alkenyl- or alkynyl-lithium, such as CH.sub.3Li, in a
suitable organic solvent, such as, for example, diethyl ether or
THF, across the double bond of the 2'-carbonyl group provides a
tertiary alcohol at this position. The addition of a hydrogen
halide in a suitable solvent, such as, for example, Hr in HOAc, in
the subsequent step provides a leaving group (LG) such as, for
example, a chloro, bromo or iodo, at the C-1 anomeric carbon of the
sugar ring that later generates a nucleosidic linkage. Other
suitable LGs include C-1 sulfonates such as, for example,
methanesulfonate, trifluoromethanesulfonate and/or
p-toluenesulfonate.
[0360] The introduction in the next step of a metal salt (Li, Na or
K) of an appropriately substituted 2-azapurine in a suitable
organic solvent such as, for example, THF, acetonitrile of DMF,
results in the formation of the desired nucleosidic linkage and
addition of the desired 2-azapurine base. This displacement
reaction may be catalyzed by a phase transfer catalyst like TDA-1
or triethylbenzylammonium chloride. The introduction of a "Z"
substituent on any of base formulae (i)-(vi) optionally may be
performed subsequent to the initial addition of protecting groups.
For example, the introduction of an amino group for "Z" is
accomplished by the addition of an appropriate amine in an
appropriate solvent to the 2'-C-halo intermediate just prior to the
last step of removal of the protecting groups. Appropriate amines
include alcoholic or liquid ammonia to generate a primary amine
(--NH.sub.2), an alkylamine to generate a secondary amine (--NHR),
or a dialkylamine to generate a tertiary amine (--NRR').
[0361] Finally, the nucleoside can be deprotected by methods well
known to those skilled in the art, as by Greene et al., Protective
Groups in Organic Synthesis, John Wiley and Sons, Second Edition,
1991. It is to be noted that this reaction scheme can be used for
joining any of the purine nucleoside analogue bases provided for in
Schemes 8-13 with a ribofuranosyl moiety. 32
[0362] The present invention is described by way of illustration in
the following examples. It will be understood by one of ordinary
skill in the art that these examples are in no way limiting and
that variations of detail can be made without departing from the
spirit and scope of the present invention.
EXAMPLES
[0363] The test compounds were dissolved in DMSO at an initial
concentration of 200 .mu.M and then were serially diluted in
culture medium.
[0364] Unless otherwise stated, bay hamster kidney (HK-21) (ATCC
CCL-10) and bos Taurus (T) (ATCC CRL 1390) cells were grown at
37.degree. C. in a humidified CO.sub.2 (5%) atmosphere. HK-21 cells
were passaged in Eagle MEM additioned of 2 mM L-glutamine, 10%
fetal ovine serum (FS, Gibco) and Earle's SS adjusted to contain
1.5 g/L sodium bicarbonate and 0.1 mM non-essential amino acids. T
cells were passaged in Dulbecco's modified Eagle's medium with 4 mM
L-glutamine and 10% horse serum (HS, Gibco), adjusted to contain
1.5 g/L sodium bicarbonate, 4.5 g/L glucose and 1.0 mM sodium
pyruvate. The vaccine strain 17D (YFV-17D) (Stamaril.RTM., Pasteur
Merieux) and Bovine Viral Diarrhea virus (BVDV) (ATCC VR-534) were
used to infect HK and T cells, respectively, in 75 cm.sup.2
bottles. After a 3 day incubation period at 37.degree. C.,
extensive cytopathic effect was observed. Cultures were
freeze-thawed three times, cell debris were removed by
centrifugation and the supernatant was aliquoted and stored at
-70.degree. C. YFV-17D and VDV were titrated in HK-21 and T cells,
respectively, that were grown to confluency in 24-well plates.
[0365] The following examples are derived by selection of an
appropriate, optionally substituted sugar or cyclopentane ring
coupled with an optionally substituted 2-azapurine base, and
prepared according to the following synthetic schemes:
[0366] Example 1: Synthesis of optionally substituted
1'-C-branched-ribofuranosyl, -sulfonyl, -thiophenyl or
cyclopentanyl-2-azapurines;
[0367] Example 2: Synthesis of optionally substituted
2'-C-branched-ribofuranosyl, -sulfonyl, -thiophenyl or
cyclopentanyl-2-azapurines;
[0368] Example 3: Synthesis of optionally substituted
3'-C-branched-ribofuranosyl, -sulfonyl, -thiophenyl or
cyclopentanyl-2-azapurines;
[0369] Example 4: Synthesis of optionally substituted
4'-C-branched-ribofuranosyl, -sulfonyl, -thiophenyl or
cyclopentanyl-2-azapurines;
[0370] Examples 5-13: Synthesis of specific compounds of the
present invention; and
[0371] Examples 14-18: Biologic test results of representative
examples of compounds of the present invention.
Example 1
1'-C-Branched ribofuranosyl, -sulfonyl or
cyclopentanyl-2-azapurine, Optionally Substituted
[0372] The title compound is prepared according to Schemes 1, 2, or
7. In a similar manner but using the appropriate sugar or
cyclopentane ring and optionally substituted 2-azapurine base, the
following nucleosides of Formulae (I) or (II) may be prepared:
33
[0373] wherein: base may be any of the Formulae (A)-(G) as
described herein where R in each instance may exist in mono-, di-
or triphosphate form.
[0374] Alternatively, the Dimroth rearrangement may be used for
making 2-azapurines from the corresponding purine base. In this
reaction, an N-alkylated or N-arylated imino heterocycle undergoes
rearrangement to its corresponding alkylamino or arylamino
heterocycle.
Example 1a
1'-C-hydroxymethyl-2-azaadenosine
[0375] 34
[0376] Step 1: 2-azaadenine, NaH, ACN, rt, 24 hrs; Step 2:
MeONa/MeOH
[0377] The starting material 2-azaadenine may be prepared starting
from malonitrile by the synthesis taught by D. W. Wooley, Journal
of Biological Chemistry, (1951), 189:401.
Example 2
2'-C-Branched ribofuranosyl, -sulfonyl or
cyclopentanyl-2-azapurine, Optionally Substituted
[0378] The title compound is prepared according to Schemes 3, 4, or
through protection of appropriately selected substituent groups in
Schemes 7 or 8. In a similar manner but using the appropriate sugar
or cyclopentane ring and optionally substituted 2-azapurine base,
the following nucleosides of Formulae (I) or (II) may be prepared:
35
[0379] wherein: base may be any of the Formulae (A)-(G) as
described herein where R in each instance may exist in mono-, di-
or triphosphate form.
[0380] Alternatively, the Dimroth rearrangement may be used for
making 2-azapurines from the corresponding purine base. In this
reaction, an N-alkylated or N-arylated imino heterocycle undergoes
rearrangement to its corresponding alkylamino or arylamino
heterocycle.
Example 2a
2'-C-methyl-2-azaadenosine
[0381] 36
[0382] (Synthesis according to the procedure of J. A. Montgomery,
Nucleic Acid Chemistry, 1978, Part II, 681-685 starting with
2'-C-methyladenosine.)
[0383] Step 1: H.sub.2O.sub.2, AcOH, 80%; Step 2: BnBr, DMAc, Step
3: NaOH, H.sub.2O, EtOH, 30%, Step 4: NH.sub.3/MeOH, 80.degree. C.,
2 days, 60%; Step 5: H.sub.2/Pd/C, 3 atm, MeOH, 30% Step 6:
.NaNO.sub.2, AcOH, H.sub.2O, 50%.
[0384]
4-Amino-7-(2-C-methyl-.beta.-D-ribofuranosyl)imidazo[4,5-d]-.nu.-tr-
iazine (2'-C-methyl-2-azaadenosine): .sup.1H NMR (DMSO-d.sub.6)
.delta. 8.82 (s, 1H, H8), 7.97 (br, 2H, NH.sub.2), 6.12 (s, 1H,
H1'), 5.22-5.51 (m, 3H, 3OH), 3.70-4.17 (m, 4H, H3', H4', 2H5'),
0.80 (s, 3H, CH.sub.3).
[0385] .sup.13C NMR (DMSO-d.sub.6) .delta. 153, 146, 142, 116, 92,
83, 79, 72, 60, 20. m/z (FAB>0) 565 (2M+H).sup.+, 283
(M+H).sup.+, (FAB<0) 563 (2M-H).sup.-.
[0386] Alternatively, 2-azaadenosine shown as the final product in
Example 2.a. may be prepared starting with adenosine, according to
the procedure of J. A. Montgomery, Nucleic Acid Chemistry, 1978,
Part II, 681-685 starting with 2'-C-methyladenosine, or via
2-azainosine in a synthetic procedure taught by R. P. Panzica,
Journal of Heterocyclic Chemistry, 1972, 9:623-628 starting with
AICA riboside.
Example 2b
2'-C-methyl-pyrrolo-4-amino-1,2,3-triazine
[0387] 37
[0388] Step 1: NCS, DMF; Step 2: mcPBA, AcOH; Step 3: a) BnBr,
DMAc; b) NaOH, H.sub.2O, EtOH, Step 4: NH.sub.3/MeOH, 80.degree.
C., Step 5: H.sub.2/Pd/C, MeOH; Step 6: NaNO.sub.2, AcOH,
H.sub.2O.
Example 3
3'-C-Branched ribofuranosyl, -sulfonyl or
cyclopentanyl-2-azapurine, Optionally Substituted
[0389] The title compound is prepared according to Schemes 5, 6, or
through protection of appropriately selected sustituent groups in
Scheme 8. In a similar manner ut using the appropriate sugar or
cyclopentane ring and optionally substituted 2-azapurine base, the
following nucleosides of Formulae (I) and (II)may be prepared:
38
[0390] wherein: base may be any of the Formulae (A)-(G) as
described herein where R in each instance may exist in mono-, di-
or triphosphate form.
[0391] Alternatively, the Dimroth rearrangement may be used for
making 2-azapurines from the corresponding purine base. In this
reaction, an N-alkylated or N-arylated imino heterocycle undergoes
rearrangement to its corresponding alkylamino or arylamino
heterocycle.
Example 4
4'-C-Branched ribofuranosyl, -sulfonyl or
cyclopentanyl-2-azapurine, Optionally Substituted
[0392] The title compound is prepared according to modification
from the corresponding pentodialdo-furanose. In a similar manner
but using the appropriate sugar or cyclopentane ring and optionally
substituted 2-azapurine base, the following nucleosides of Formulae
(I) or (II) may be prepared: 39
[0393] wherein: base may be any of the Formulae (A)-(G) as
described herein where R in each instance may exist in mono-, di-
or triphosphate form.
[0394] Alternatively, the Dimroth rearrangement may be used for
making 2-azapurines from the corresponding purine base. In this
reaction, an N-alkylated or N-arylated imino heterocycle undergoes
rearrangement to its corresponding alkylamino or arylamino
heterocycle.
Example 5
Synthesis of
4-amino-1-(.quadrature.-D-ribofuranosyl)imdazo[4,5-d]pyridazi-
ne
[0395] 40
Step A:
1-(2,3,5-tri-O-Benzoyl-.beta.-D-ribofuranosyl)-5-benzyloxymethylim-
idazo[4,5-d]pyridazin-4-one
[0396] The 5-benzyloxymethylimidazo[4,5-d]pyridazine (500 mg, 1.95
mmol) [for preparation see Journal of Heterocyclic Chemistry, 1984,
Vol 21, 481] was heated at reflux in hexamethyldisilazane (6 mL)
for 1 hour. The mixture was evaporated to dryness to give a slight
yellow syrup which was dissolved in dry 1,2-dichloroethane (20 mL).
The 1-O-acetyl -2,3,5-tri-O-benzoyl-.beta.-D-ribofuranose (1.04 g,
2.06 mmol) and stannic chloride (0.4 mL, 3.44 mmol) were added at
20.degree. C. and the mixture was stirred for 3 hours. The reaction
mixture was poured into an aqueous solution of sodium
hydrogenocarbonate, filtrated through a pad of celite and washed by
dichloromethane. The organic layer was evaporated to dryness to
give a yellow foam. The crude product was purified on silica gel
using n-hexane/ethyl acetate (3/2) as eluant to give the title
compound (703 mg) as a white powder.
[0397] .sup.1H NMR (DMSO-d.sub.6) .delta. ppm: 4.39 (s, 2H,
CH.sub.2), 4.60 (m, 2H), 4.73 (m, 1H), 5.34 (dd, 2H, CH.sub.2),
5.77-5.88 (m, 2H, H2' and H3'), 6.56 (m, 1H, H1'), 6.98-7.10 (m,
5H), 7.23-7.32 (m, 6H), 7.41-7.51 (m, 3H), 7.68-7.73 (m, 2H),
7.74-7.8 (m, 4H), 8.51 (s, 1H), 8.52 (s, 1H).
Step B:
1-(2,3,5-tri-O-Benzoyl-.beta.-D-ribofuranosyl)imidazo[4,5-d]pyrida-
zin-4-one
[0398] To a solution containing the compound from Step B (500 mg,
0.7 mmol), in dry dichloromethane (25 mL) was added a pre-cooled
(-78.degree. C.) solution of boron trichloride 1M (5 mL) at
-78.degree. C. and stirred for 2 hours at -78.degree. C. A mixture
of methanol/dichloromethane (1/1) was added to the mixture at
-78.degree. C. and then at 20.degree. C. The reaction mixture was
evaporated to dryness to give a yellow powder. The crude product
was purified on silica gel using n-hexane/ethyl acetate (3/2) as
eluant to give the title compound (400 mg) as a yellow powder.
[0399] .sup.1H NMR (DMSO-d.sub.6) .delta. ppm: 4.77-4.98 (m, 3H,
H4', 2H5'), 5.95-6.12 (m, 2H, H2' and H3'), 6.65 (m, 1H, H1'),
7.39-7.76 (m, 9H), 7.84-8.06 (m, 6H), 8.64-5.79 (m, 2H, H3 and H8),
12.84 (br, 1H, NH).
[0400] Mass spectrum: m/z (FAB>0) 581 (M+H).sup.+, (FAB<0)
579 (M-H).sup.-
Step C:
4-chloro-1-(2,3,5-tri-O-Benzoyl-.beta.-D-ribofuranosyl)imidazo[4,5-
-d]pyridazine
[0401] A solution containing the compound from Step B (1.32 g, 2.27
mmol), the N,N-diethylaniline (365 .mu.L), tetrabutylammonium
chloride (1.2 g), freshly distilled phosphorus chloride (1.3 .mu.L)
and anhydrous acetonitrile (17 mL) was stirred at 90.degree. C. for
1 hour. The reaction mixture was poured over cracked ice/water. The
aqueous layer was extracted with dichloromethane (3.times.60 mL).
The organic layer was washed with sodium hydrogenocarbonate 5%,
water and was evaporated to dryness. The crude product was purified
on silica gel using n-hexane/ethyl acetate (3/1) as eluant to give
the title compound (404 mg) as a yellow powder.
[0402] .sup.1H NMR (DMSO-d.sub.6) .delta. ppm: 4.82-6.87 (m, 2H),
4.9-6.95 (m, 1H), 6.0-6.08 (m, 1H), 6.12-6.19 (m, 1H), 6.90 (d, 1H,
J=5.2 Hz, H1'), 7.47-7.73 (m, 9H), 7.88-8.12 (m, 6H), 9.10 (s, 1H,
H8), 9.90 (s, 1H, H3)
Step D:
4-amino-1-.beta.-D-ribofuranosyl)imidazo[4,5-d]pyridazine
[0403] The compound from Step C (420 mg, 0.7 mmol) was added to a
solution of ammonia in methanol and stirred in a steel bomb at
150.degree. C. for 6 hours. The reaction mixture was evaporated to
dryness to afford a brown oil which was purified on silica gel
reverse-phase (C18) using water as eluant to give the title
compound (50 mg) as a yellow powder.
[0404] .sup.1H NMR (DMSO-d.sub.6) .delta. ppm: 3.58-4.48 (m, 5H,
H2', H3', H4', 2H5'), 5.14-5.68 (m, 3H, 3.times.OH), 5.90 (s, 1H,
H1'), 6.61 (br, 2H, NH.sub.2), 8.59 (s, 1H, H8), 9.12 (s, 1H,
3H)
Example 6
Synthesis of
1-(.beta.-D-ribofuranosyl)imidazo[4,5-d]pyridazin-4-one
[0405] 41
[0406]
1-(2,3,5-tri-O-Benzoyl-.beta.-D-ribofuranosyl)imidazo[4,5-d]pyridaz-
in-4-one (555 mg, 0.9 mmol) was added to a solution of sodium
methylate (205 mg) in methanol (25 mL) and stirred at 20.degree. C.
for 2 hours. The reaction mixture was evaporated to dryness. The
residue was dissolved in water and washed with ethyl acetate. The
aqueous layer was concentrated under pressure. The crude product
was purified on silica gel reverse-phase (C18) using water as
eluant to give the title compound (220 mg) as a white powder.
[0407] .sup.1H NMR (DMSO-d.sub.6) .delta. ppm: 3.59-3.62 (m, 2H),
4.02 (m, 1H), 4.11 (m, 1H), 4.22 (m, 1H), 5.16-5.72 (m, 3H,
3.times.OH), 5.91 (s, 1H, H1'), 8.52 (s, 1H, H8), 8.68 (s, 1H, H3),
12.75 (br, 1H, NH).
[0408] Mass spectrum: m/z (FAB>0) 537 (2M+H).sup.+, 269
(M+H).sup.+, (FAB<0) 535 (2M+H).sup.+, 267 (M-H).sup.-
Example 7
Synthesis of
4-amino-1-(2-C-methyl-.beta.-D-ribofuranosyl)imidazo[4,5-d]py-
ridazine
[0409] 42
Step A:
1-(2-C-methyl-2,3,5-tri-O-Benzoyl-.beta.-D-ribofuranosyl)imidazo[4-
,5-d]pyridazin-4-one
[0410] To a suspension of imidazo[4,5-d]pyridazine (3.48 g, 25.5
mmol) [for preparation see Journal of Heterocyclic Chemistry, 1969,
Vol 6, 93] in dry acetonitrile (35 mL) was added
1,2,3,5-tetra-O-benzoyl-2-C-methyl-- .beta.-D-ribofuranose (14.48
g, 25.0 mmol) at 20.degree. C. and stirred for 15 mn. DBU (11.5 mL,
76.3 mmol) was added at 0.degree. C. and the solution was stirred
for 15 mn at 0.degree. C. TMSOTf (24.7 mL, 127.8 mmol) was added at
0.degree. C. and the mixture was heated at 80.degree. C. for 20
hours. The reaction mixture was poured into an aqueous solution of
sodium hydrogenocarbonate and extracted by ethyl acetate. The
organic layer was evaporated to dryness to give a yellow powder.
The crude product was purified on silica gel using
dichloromethane/methanol (99.3/0.7) as eluant to give a slight
yellow powder which was crystallized from isopropanol to give the
title compound (2.45 g) as a white powder.
[0411] .sup.1H NMR (DMSO-d.sub.6) .delta. ppm: 1.48 (s, 3H,
CH.sub.3), 4.75-4.96 (m, 3H, H4', 2H5'), 5.81 (d, 1H, J=5.5 Hz,
H3'), 6.99 (s, 1H, H1'), 7.39-7.72 (m, 9H), 7.92-8.08 (m, 6H), 8.64
(s, 1H, H8), 8.71 (s, 1H, H3), 12.89 (br, 1H, NH).
[0412] Mass spectrum: m/z (FAB>0) 1189 (2M+H).sup.+, 585
(M+H).sup.+, (FAB<0) 593 (M-H).sup.-
Step B:
4-chloro-1-(2-C-methyl-2,3,5-tri-O-Benzoyl-.beta.-D-ribofuranosyl)-
imidazo[4,5-d]pyridazine
[0413] A solution containing the compound from Step A (300 mg, 0.50
mmol), the N,N-diethylaniline (1.2 mL) and freshly distilled
phosphorus chloride (24 mL) was stirred at reflux for 1 hour. The
reaction mixture was evaporated to dryness. Dichloromethane was
added to the residue and the organic layer poured over cracked
ice/water. The aqueous layer was extracted with dichloromethane.
The organic layer was washed with sodium hydrogenocarbonate 5%,
water and was evaporated to dryness. The crude product was purified
on silica gel using diethyl ether/petrol ether (1/1) as eluant to
give the title compound (295 mg) as a white powder.
[0414] .sup.1H NMR (DMSO-d.sub.6) .delta. ppm: 1.5 (s, 3H,
CH.sub.3), 4.8-5.0 (m, 3H, H4', 2H5'), 5.85 (d, 1H, J=5.5 Hz, H3'),
7.15 (s, 1H, H1'), 7.38-8.08 (m, 15H), 9.15 (s, 1H, H8), 9.90 (s,
1H, H3)
Step C:
4-amino-1-(2-C-methyl-.beta.-D-ribofuranosyl)imidazo[4,5-d]pyridaz-
ine
[0415] The compound from Step B (590 mg, 0.96 mmol) was added to a
solution of ammonia in methanol and stirred in a steel bomb at
150.degree. C. for 6 hours. The reaction mixture was evaporated to
dryness to remove methanol. The crude product was purified on
silica gel reverse-phase (C18) using water as eluant to give the
title compound (35 mg) as a white powder.
[0416] .sup.1H NMR (DMSO-d.sub.6) .delta. ppm: 0.70 (s, 3H,
CH.sub.3), 3.64-3.98 (m, 4H, H3', H4', 2H5'), 5.23-5.44 (m, 3H,
3OH), 5.98 (s, 1H, H1'), 6.63 (br, 2H, NH.sub.2), 8.68 (s, 1H, H8),
9.05 (s, 1H, H3)
[0417] .sup.13C NMR (DMSO-d.sub.6) .delta. ppm: 155, 143, 132, 131,
129, 93, 83, 79, 72, 20.
[0418] Mass spectrum: m/z (FAB>0) 282 (M+H).sup.+, (FAB<0)
280 (M-H).sup.-
Example 8
Synthesis of the
4-substituted-1-(2-C-methyl-.beta.-D-ribofuranosyl)imidaz-
o[4,5d]pyridazine
[0419]
1 43 W Step A products OH NaOMe, MeOH 100.degree. C., 24 h 44 Cl
NH.sub.3/MeOH 20.degree. C., 48 h 45
1-(2-C-methyl-.beta.-D-ribofuranosyl)imidazo[4,5-d]pyridazin-4-one
[0420] .sup.1H NMR (DMSO-d.sub.6) .delta. ppm: 1.17 (s, 3H,
CH.sub.3), 3.44-3.59 (m, 1H), 3.68-3.78 (m, 1H), 3.86-3.94 (m, 1H),
4.11-4.21 (m, 1H), 4.8-5.4 (m, 3H, 3OH), 6.05 (s, 1H, H1'), 8.35
(s, 1H, H8), 8.37 (s, 1H, H3), 12.67 (br, 1H, NH).
[0421] Mass spectrum: m/z (FAB>0) 283 (2M+H).sup.+, 281
(M+H).sup.+
4-chloro-1-(2-C-methyl-.beta.-D-ribofuranosyl)imidazo[4,5-d]pyridazine
[0422] .sup.1H NMR (DMSO-d.sub.6) .delta. ppm: 0.72 (s, 3H,
CH.sub.3), 3.69-4.06 (m, 4H, H3', H4', 2H5'), 5.34-5.51 (m, 3H,
3OH), 6.19 (s, 1H, H1'), 9.18 (s, 1H, H8), 9.87 (s, 1H, H3)
[0423] Mass spectrum: m/z (FAB>0) 301 (M+H).sup.+, (FAB<0)
299 (M-H).sup.-
Example 9
Synthesis of the 4,7-diamino-imidazo[4,5-d]pyridazine nucleosides
Derivatives
[0424] 46
[0425] Step A:
[0426] To a suspension of 4,5-dicyanoimidazole (1 eq.) [for
preparation see Journal of Organic Chemistry, 1976, Vol 41, 713] in
dry DMF (0.2 M) was added the protected .beta.-D-ribofuranose
derivatives (1 eq.) at 20.degree. C. DBU (3 eq.) was added at 0 C
and the solution was stirred for 20 mn at 0.degree. C. TMSOTf (4
eq.) was added at 0.degree. C. and the mixture was heated at
60.degree. C. for 1 hour. The reaction mixture was poured into an
aqueous solution of sodium hydrogenocarbonate and extracted by
dichloromethane. The organic layer was evaporated to dryness to
give a yellow powder. The crude product was purified on silica gel
using diethyl ether/petrol ether as eluant to give the title
compound (see the following table 1).
[0427] Step B:
[0428] The compound from Step A (1 eq.) was stirred with hydrazine
monohydrate (20 eq.) and acetic acid (1.4 eq.) at 75.degree. C. for
several hours (see the following table 1). The reaction mixture was
poured into water. The aqueous layer was washed by dichloromethane
and evaporated under pressure. The residue was purified on
reverse-phase column to give the title compound (see the following
table 1).
2TABLE 1 R products from Step A yield (Step A) experiments (Step B)
products from Step B yield (Step A) H 47 63% 75.degree. C. for 1.5
h 48 58% (white powder) CH.sub.3 49 62% 75.degree. C. for 20 h. 50
15% (white powder)
4,7-diamino-1-.beta.-D-ribofuranosylimidazo[4,5-d]pyridazine
[0429] .sup.1H NMR (DMSO-d.sub.6) .delta. ppm: 3.58-4.32 (m, 5H,
H2', H3', H4', 2H5'), 5.10-5.90 (br, 7H, 2NH.sub.2, 3OH), 6.11 (s,
1H, H1'), 8.50 (s, 1H, H8)
[0430] .sup.13C NMR (DMSO-d.sub.6) .delta. ppm: 151, 144, 142, 132,
122, 89, 86, 75, 70, 61.
[0431] Mass spectrum: m/z (FAB>0) 283 (M+H).sup.+, (FAB<0)
281 (M-H).sup.-
4,7-diamino-1-(2-C-methyl-.beta.-D-ribofuranosyl)imidazo[4,5-d]pyridazine
[0432] .sup.1H NMR (DMSO-d.sub.6) .delta. ppm: 0.75 (s, 3H,
CH.sub.3), 3.67-3.76 (m, 1H), 3.84-3.94 (m, 3H), 5.32 (m, 3H, 3OH),
5.43 (br, 1H, NH.sub.2), 5.71 (br, 1H, NH.sub.2), 6.21 (s,1H, H1'),
8.78 (s, 1H, H8)
[0433] .sup.13C NMR (DMSO-d.sub.6) .delta. ppm: 151, 144, 142, 132,
123, 92, 83, 78, 71, 59, 20.
[0434] Mass spectrum: m/z (FAB>0) 593 (2M+H).sup.+, 297
(M+H).sup.+, (FAB<0) 295 (M-H).sup.-
Example 10
Synthesis of 4,7-disubstituted-imidazo[4,5-d]pyridazine
nucleosides
[0435] 51
Step A: Typical Procedure for the Preparation of the protected
4,7-dichloroimidazo[4,5-d]pyridazine nucleosides
[0436] The 4,7-dichloroimidazo[4,5-d]pyridazine [for preparation
see Journal of Heterocyclic Chemistry, 1968, Vol 5, 13] (1 eq.) was
heated at reflux in hexamethyldisilazane for 12 hours. The mixture
was evaporated to dryness to give a solid which was dissolved in
1,2-dichloroethane. The protected .beta.-D-ribofuranose derivatives
(1.1 eq.) and stannic chloride (1.4 eq.) were added at 20.degree.
C. and the solution was stirred for 3 hours. The reaction mixture
was poured into an aqueous solution of sodium hydrogenocarbonate,
filtrated through a pad of celite and washed by dichloromethane.
The organic layer was evaporated to dryness. The crude product was
purified on silica gel using dichloromethane/acetone (40/1) as
eluant to give the title compound (see the following table 2).
Step B: Typical Procedure for the Preparation of the
4,7-dichloroimidazo[4,5-d]pyridazine nucleosides
[0437] The compound from Step A (1 eq.) was stirred with sodium
methoxide (0.1 eq.) in methanol for several hours. The reaction
mixture was evaporated under pressure. Water was added to the
residue. The aqueous layer was washed by ethyl acetate and was
evaporated under pressure. The residue was purified on
reverse-phase column to give the title compound (see the following
table 2).
Step C: Typical Procedure for the Preparation of the
imidazo[4,5-d]pyridazine nucleosides
[0438] A mixture of the compound from Step A (1 eq.), palladium on
charcoal (10%), sodium acetate (4.2 eq.) in acetyl acetate was
stirred under hydrogen until the compound from Step A was consumed.
The reaction mixture was evaporated under pressure and was purified
on silica gel to give the title protected compound which was
stirred with sodium methoxide (3.3 eq.) in methanol. The reaction
mixture was evaporated under pressure. Water was added to the
residue. The aqueous layer was washed by ethyl acetate and was
evaporated under pressure. The residue was purified on
reverse-phase column to give the title compound (see the following
table 2).
Step D: Typical Procedure for the Preparation of the
chloro-methoxy-imidazo[4,5-d]pyridazine nucleosides
[0439] The compound from Step A (1 eq.) was stirred with sodium
methoxide (3.3 eq.) in methanol 0.3M at 20.degree. C. for several
hours. The reaction mixture was evaporated under pressure. Water
was added to the residue. The aqueous layer was washed by ethyl
acetate and was evaporated under pressure. The residue was purified
on reverse-phase column to give a compound whose regioselectivity
was not given (see the following table 2).
Step E: Typical Procedure for the Preparation of the
methoxy-imidazo[4,5-d]pyridazine nucleosides
[0440] A mixture of the compound from Step D (1 eq.), palladium on
charcoal (10%), sodium acetate (4.2 eq.) in water/ethanol (1/1) was
stirred under hydrogen until the compound from Step A was consumed.
The reaction mixture was evaporated under pressure and was purified
on reverse-phase column to give the title compound whose
regioselectivity was not given (see the following table 2).
Step F: Typical Procedure for the Preparation of the Protected
4,7-diazidoimidazo[4,5-d]pyridazine nucleosides
[0441] The compound from Step A (1 eq.) was treated at 50.degree.
C. with sodium azide (1.5 eq.) in DMF. Water was added to the
mixture. The aqueous layer was extracted by ethyl acetate. The
organic layer was evaporated under pressure. The crude product was
purified on silica gel using diethyl ether/petrol ether (7/3) as
eluant to give the title compound (see the following table 2).
Step G: Typical Procedure for the Preparation of the
azido-methoxy-imidazo[4,5-d]pyridazine nucleosides
[0442] The compound from Step F (1 eq.) was stirred at 50.degree.
C. with sodium methoxide (1 eq.) in methanol. The reaction mixture
was evaporated under pressure. Water was added to the residue. The
aqueous layer was washed by ethyl acetate and was evaporated under
pressure. The residue was purified on reverse-phase column using
water/acetonitrile as eluant to give the title compound whose
regioselectivity was not given (see the following table 2).
Step H: Typical Procedure for the Preparation of the
amino-azido-imidazo[4,5-d]pyridazine nucleosides
[0443] A mixture of the compound from Step F (1 eq.), palladium on
charcoal (10%), sodium acetate (4.2 eq.) in ethyl acetate was
stirred under hydrogen until the compound from Step F was consumed.
The reaction mixture was filtrated over celite and was evaporated
under pressure The crude product was purified on silica gel to give
the title protected compound whose regioselectivity was not given
(see the following table 1). This compound was stirred with sodium
methoxide (3 eq.) in methanol. The reaction mixture was evaporated
under pressure. Water was added to the residue. The aqueous layer
was washed by ethyl acetate and was evaporated under pressure. The
residue was purified on reverse-phase column to give the title
compound whose regioselectivity was not given (see the following
table 2).
3TABLE 2 experi- ments products yield Step A 52 60% (white powder)
Step A 53 34% (white powder) Step B 54 (white powder) Step C 55 71%
(white powder) Step D 56 55% (white powder) Step E 57 55% (white
powder) Step F 58 50% (yellow powder) Step F 59 50% (yellow powder)
Step G 60 36% (beige powder) Step H 61 98% (white powder) Step H 62
49% (white powder)
4,7-dichloro-1-(2,3,5-tri-O-Benzoyl-.beta.-D-ribofuranosyl)imidazo[4,5-d]p-
yridazine
[0444] .sup.1H NMR (DMSO-d.sub.6) .delta. ppm: 4.8-5.0 (m, 3H, H4',
2H5'), 6.05 (s, 1H, H3'), 6.25 (s, 1H, H2'), 7.1 (d, 1H, J=4 Hz,
H1'), 7.4-8.0 (m, 15H), 9.25 (s, 1H, H8).
[0445] Mass spectrum: m/z (FAB>0) 633 (M+H).sup.+
4,7-dichloro-1-(2-C-methyl-2,3,5-tri-O-Benzoyl-.beta.-D-ribofuranosyl)imid-
azo[4,5-d]pyridazine
[0446] .sup.1H NMR (DMSO-d.sub.6) .delta. ppm: 1.65 (s, 3H,
CH.sub.3), 4.9-5.0 (m, 3H, H4', 2H5'), 5.8 (s, 1H, H3'), 7.35-8.05
(m, 16H including H1'), 9.3 (s, 1H, H8).
4,7-dichloro-1-(2-C-methyl-.beta.-D-ribofuranosyl)imidazo[4,5-d]pyridazine
[0447] .sup.1H NMR (DMSO-d.sub.6) .delta. ppm: 0.84 (s, 3H,
CH.sub.3), 3.77 (m, 1H), 3.88-4.04 (m, 3H), 5.30-5.60 (m, 3H, OH),
6.5 (s, 1H, H1'), 9.44 (s, 1H, H8).
1-(2-C-methyl-.beta.-D-ribofuranosyl)imidazo[4,5-d]pyridazine
[0448] .sup.1H NMR (DMSO-d.sub.6) .delta. ppm: 0.80 (s, 3H,
CH.sub.3), 3.75 (m, 1H), 3.80-4.00 (m, 3H), 5.40 (br, 3H, OH), 6.2
(s, 1H, H1'), 9.0 (s, 1H), 9.65 (s, 1H), 9.85 (s, 1H).
7-chloro-4-methoxy-1-.beta.-D-ribofuranosylimidazo[4,5-d]pyridazine
or
4-chloro-7-methoxy-1-.beta.-D-ribofuranosylimidazo[4,5-d]pyridazine
[0449] .sup.1H NMR (DMSO-d.sub.6) .delta. ppm: 3.6-4.5 (m, 8H,
2H5', H4', H3', H2', OCH.sub.3), 5.40 (m, 3H, OH), 6.2 (s, 1H,
H1'), 9.0 (s, 1H, H8).
[0450] 675 (2M+H).sup.+, Mass spectrum: m/z (FAB>0) 317
(M+H).sup.+, (FAB<0) 315 (M-H).sup.-
4-methoxy-1-.beta.-D-ribofuranosylimidazo[4,5-d]pyridazine or
7-methoxy-1-.beta.-D-ribofuranosylimidazo[4,5-d]pyridazine
[0451] .sup.1H NMR (DMSO-d.sub.6) .delta. ppm: 3.54-3.79 (m, 2H),
3.95 (m, 1H), 4.15 (m, 1H, H3'), 4.2 (s, 3H, OCH.sub.3), 4.4 (m,
1H, H2'), 5.05-5.70 (m, 3H, OH), 6.2 (d, 1H, J=4.8 Hz, H1'), 8.95
(s, 1H), 9.3 (s, 1H).
[0452] .sup.13C NMR (DMSO-d.sub.6) .delta. ppm: 154, 145, 143, 142,
121, 90, 85, 75, 70, 61, 55.
[0453] Mass spectrum: m/z (FAB>0) 283 (M+H).sup.+, (FAB<0)
281 (M-H).sup.-
4,7-diazido-1-(2,3,5-tri-O-Benzoyl-.beta.-D-ribofuranosyl)imidazo[4,5-d]py-
ridazine
[0454] .sup.1H NMR (DMSO-d.sub.6) .delta. ppm: 4.81-5.1 (m, 3H,
H4', 2H5'), 6.24-6.49 (m, 2H, H2', H3'), 7.2 (d, 1H, J=5 Hz, H1'),
7.4-8.0 (m, 15H), 9.12 (s, 1H, H8).
[0455] Mass spectrum: m/z (FAB>0) 647 (M+H).sup.+
4,7-diazido-1-(2-C-methyl-2,3,5-tri-O-Benzoyl-.beta.-D-ribofuranosyl)imida-
zo[4,5-d]pyridazine
[0456] .sup.1H NMR (DMSO-d.sub.6) .delta. ppm: 1.6 (s, 3H,
CH.sub.3), 4.96 (m, 3H, H4', 2H5'), 6.02 (m, 1H, H3'), 7.24 (s, 1H,
H1'), 7.40-7.52 (m, 6H), 7.60-7.71 (m, 3H), 7.93-8.1 (m, 6H), 9/10
(s, 1H, H8).
[0457] Mass spectrum: m/z (FAB>0) 661 (M+H).sup.+
4-azido-7-methoxy-(2-C-methyl-.beta.-D-ribofuranosyl)imidazo[4,5-d]pyridaz-
ine or
7-azido-4-methoxy-1-(2-C-methyl-.beta.-D-ribofuranosyl)imidazo[4,5--
d]pyridazine
[0458] .sup.1H NMR (DMSO-d.sub.6) .delta. ppm: 0.75 (s, 3H,
CH.sub.3), 3.70-3.98 (m, 2H), 4.05 (m, 2H), 4.2 (s, 3H, OCH.sub.3),
5.32-5.61 (br, 3H, OH), 6.36 (d, 1H, J=5.7 Hz, H1'), 9.18 (s, 1H,
H8),
[0459] .sup.13C NMR (DMSO-d.sub.6) 8 ppm: 156, 143, 136, 129, 123,
94, 83, 79, 71, 59, 57, 20.
[0460] Mass spectrum: m/z (FAB>0) 675 (2M+H).sup.+, 338
(M+H).sup.+, (FAB<0) 673 (2M-H).sup.-, 336 (M-H)
4-amino-7-azido-1-(2-C-methyl-2,3,5-tri-O-Benzoyl-.beta.-D-ribofuranosyl)i-
midazo[4,5-d]pyridazine or
7-amino-4-azido-1-(2-C-methyl-2,3,5-tri-O-Benzo-
yl-.beta.-D-ribofuranosyl)imidazo[4,5-d]pyridazine
[0461] .sup.1H NMR (DMSO-d.sub.6) .delta. ppm: 01.64 (s, 3H,
CH.sub.3), 4.95 (m, 3H), 6.06 (m, 1H, H3'), 7.18 (s, 1H, H1'),
7.40-7.52 (m, 6H), 7.63-7.74 (m, 5H, including NH.sub.2), 7.91-8.04
(m, 6H), 8.96 (s, 1H, H8),
[0462] Mass spectrum: m/z (FAB>0) 1269 (2M+H).sup.+, 635
(M+H).sup.+, (FAB<0) 633 (M-H).sup.-
4-amino-7-azido-(2-C-methyl-.beta.-D-ribofuranosyl)imidazo[4,5-d]pyridazin-
e or
7-amino-4-azido-1-(2-C-methyl-.beta.-D-ribofuranosyl)imidazo[4,5-d]py-
ridazine
[0463] .sup.1H NMR (DMSO-d.sub.6) .delta. ppm: 0.75 (s, 3H,
CH.sub.3), 3.65-4.15 (m, 4H), 5.30-5.55 (br, 3H, 3.times.OH), 6.27
(s, 1H, H1'), 7.63 (br, 2H, NH.sub.2), 9.03 (s, 1H, H8),
[0464] Mass spectrum: m/z (FAB>0) 323 (M+H).sup.+, (FAB<0)
321 (M-H)
Example 11
Synthesis of 4-amino-6-substituted-imidazo[4,5-d]-.nu.-triazine
nucleosides
[0465] 63
Step A:
4-amino-6-bromo-7-(.beta.-D-ribofuranosyl)imidazo[4,5-d]-.nu.-tria-
zine
[0466] The 2-azaadenosine [for preparation see Patent WO 01/16149.
2001] (70 mg, 0.26 mmol) was added to a solution of sodium acetate
0.5M (1.4 mL). The solution was heated until the 2-azaadenosine was
solubilized. A solution of bromine (100 .mu.L of Br.sub.2 in 10 mL
of water) (6.3 mL, 1.22 mmol) was added and the mixture was stirred
at 20.degree. C. for 3 days. A second portion of the bromine's
solution (6.3 mL, 1.22 mmol) was added and the mixture was stirred
at 20.degree. C. for 3 hours. The reaction mixture was evaporated
to dryness. The crude product was purified on silica gel
reverse-phase (C18) using water/acetonitrile (9/1) as eluant to
give the title compound as a yellow powder.
[0467] .sup.1H NMR (DMSO-d.sub.6) .delta. ppm: 3.55 (m, 1H,
H.sub.5'), 3.71 (m, 1H, H.sub.5'), 4.01 (m, 1H, H.sub.4'), 4.31 (m,
1H, H.sub.3'), 5.17 (m, 1H, H.sub.2'), 5.19 (m, 1H, OH), 5.36 (m,
1H, OH), 5.58 (m, 1H, OH), 5.93 (d, 1H, J=6.47 Hz, H1'), 8.08 (br,
2H, NH.sub.2).
[0468] Mass spectrum: m/z (FAB>0) 349 (M+2H).sup.+, m/z
(FAB<0) 345 (M-2H).sup.-.
Step B:
4-amino-6-methyl-7-(.beta.-D-ribofuranosyl)imidazo[4,5-d]-.nu.-tri-
azine
[0469] The compound from Step A (112 mg, 0.3 mmol) was heated at
reflux in hexamethyldisilazane (15 mL) for 16 hours. The mixture
was evaporated to dryness to give a syrup which was dissolved in
dry THF (12 mL). PPh.sub.3 (10 mg; 0.04 mmol), PdCl.sub.2 (3.5 mg;
0.02 mmol) and AlMe.sub.3 (100 .mu.l; 0.94 mmol) were added. The
mixture was reflux for 5 hours. The mixture was evaporated to
dryness. The crude product was dissolved in methanol (30 mL) in the
presence of ammonium chloride. The mixture was evaporated to
dryness and the residue was purified on silica gel reverse-phase
(C18) using water/acetonitrile (from 9/1 to 6/4) as eluant to give
the title compound (35 mg) as a yellow powder.
[0470] .sup.1H NMR (DMSO-d.sub.6) .delta. ppm: 2.67 (s, 3H,
CH.sub.3), 3.60 (m, 1H, H.sub.5'), 3.72 (m, 1H, H.sub.5'), 4.03 (m,
1H, H.sub.4'), 4.24 (m, 1H, H.sub.3'), 4.93 (m, 1H, H.sub.2'), 5.48
(m, 3H, OH), 5.92 (d, 1H, J=6.82 Hz, H.sub.1'), 7.78 (br, 2H,
NH.sub.2).
[0471] Mass spectrum: m/z (FAB>0) (FAB>0) 283 (M+H).sup.+,
m/z (FAB<0) 281 (M-H).sup.-.
Example 12
Synthesis of imidazo[4,5d]-triazin-4-one nucleosides
[0472] 64
Step A:
7-(.beta.-D-Ribofuranosyl)imidazo[4,5-d]-.nu.-triazin-4-one
[0473] The AICAR [for preparation see Synthesis, 2003, No 17, 2639]
(1 g, 3.87 mmol) was added to a solution of chlorhydrique acid 6N
(25 mL) at -30.degree. C. A solution of sodium nitrite 3M (4 ml,
11.62 mmol) was added and the mixture was stirred at -30.degree.
for 2 hours. A pre-cooled (-30.degree. C.) solution of ethanol (25
mL) was added. A solution of ammonia (28%) was added at -20.degree.
C. to pH=7. The reaction mixture was evaporated to dryness. The
crude product was purified on silica gel reverse-phase (C18) using
water as eluant to give the title compound (0.81 gr) as a white
powder.
[0474] .sup.1H NMR (DMSO-d.sub.6) .delta. ppm: 3.58 (d, 1H, J=11.85
Hz, H.sub.5'), 3.70 (d, 1H, J=11.85 Hz, H.sub.5'), 4.00 (dd, 1H,
J=3.92 Hz, 4.02 Hz, H4'), 4.18 (dd, 1H, J=4.27 Hz, 4.78 Hz,
H.sub.3'), 4.54 (dd, 1H, J=4.86 Hz, 5.19 Hz, H.sub.2'), 5.18 (br,
1H, OH), 5.35 (br, 1H, OH), 5.73 (br, 1H, OH), 6.08 (d, 1H, J=5.11
Hz, H.sub.1'), 8.65 (s, 1H, H.sub.8).
Step B:
7-(2,3,5-Tri-O-actyl-.beta.-D-ribofuranosyl)imidazo[4,5-d]-.nu.-tr-
iazin-4-one
[0475] The compound from Step A (1.68 gr, 6.24 mmol) was stirred in
pyridine (20 mL). The anhydride acetic (2.3 ml , 25 mmol) was added
and the mixture was stirred at 20.degree. C. for 16 hours. The
mixture was evaporated to dryness to give a syrup which was
dissolved in water. The aqueous layer was extracted by acetyl
acetate. The organic layer was evaporated to dryness to give the
title compound (1.5 gr) as a brown foam.
[0476] .sup.1H NMR (DMSO-d.sub.6) .delta. ppm: 2.04 (s, 3H,
COCH.sub.3), 2.09 (s, 3H, COCH.sub.3), 2.10 (s, 3H, COCH.sub.3),
4.39 (m, 3H, 2.times.H.sub.5' et H.sub.4'), 5.53 (dd, 1H, J=4.39
Hz, 5.4 Hz, H.sub.3'), 5.80 (t, 1H, J=5.4 Hz, H.sub.2'), 6.26 (d,
1H, J=5,4 Hz, H1'), 8.15 (s, 1H, H.sub.8).
Example 13
Alternative Methods for Ribofuranosyl-Purine Analogues
Synthesis
I. Preparation of
4-methylamino-7-(.beta.-D-ribofuranosyl)imidazo[4,5-d]-.-
nu.-triazine
[0477] The
4-methylamino-7-(.beta.-D-ribofuranosyl)imidazo[4,5-d]-.nu.-tri-
azine Va may be prepared according the following synthesis, where
the starting material used is the AICAR I. The AICAR may be
prepared according to the published synthesis of Y. Yamamoto and N.
Kohyama, Synthesis, 2003, 17:2639-2646.
[0478] The other synthesis of
4-methylamino-7-(.beta.-D-ribofuranosyl)imid-
azo[4,5-d]-.nu.-triazine Va was described from 2-azainosine II
according to the published synthesis of L. Towsend and Co,
Nucleosides, Nucleotides & Nucleic Acids, 2000,
19(1&2):39-68. 65
II. Preparation of
4-substituted-7-(2,3-dideoxy-.beta.-D-glycero-pentofura-
nosyl)-imidazo-[4,5-d]-.nu.-triazine Derivative Compounds
[0479] The
4-substituted-7-(2,3-dideoxy-.beta.-D-glycero-pentofuranosyl)im-
idazo[4,5-d]-.nu.-triazine compounds IXa, IXb and IXc may be
prepared according the following synthesis according to the
published synthesis of R. Panzica and Co, Bioorganic &
Medicinal Chemistry, 1999, 7:2373-2379. 66
III. Preparation of
4-substituted-7-(2,3-dideoxy-.beta.-D-glycero-pent-2-e-
ne-furanosyl)-imidazo-[4,5-d]-.nu.-triazine Derivative
Compounds
[0480] The
4-substituted-7-(2,3-dideoxy-.beta.-D-glycero-pent-2-ene-furano-
syl)imidazo[4,5-d]-.nu.-triazine derivative compounds XIa, XIb and
XIc may be prepared according the following synthesis: 67
Example 14
Phosphorylation Assay of Nucleoside to Active Triphosphate
[0481] To determine the cellular metabolism of the compounds, HepG2
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 subcultured once a week. After
detachment of the adherent monolayer with a 10 minute exposure to
30 mL of trypsin-EDTA and three consecutive washes with medium,
confluent HepG2 cells 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 (PS).
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.
Example 15
Bioavailability Assay in Cynomolgus Monkeys
[0482] Within 1 week prior to the study initiation, the cynomolgus
monkey is surgically implanted with a chronic venous catheter and
sucutaneous venous access port (VAP) to facilitate lood collection
and underwent a physical examination including hematology and serum
chemistry evaluations and the body weight was recorded. Each monkey
(six total) receives approximately 250 .mu.Ci of .sup.3H activity
with each dose of active compound at a dose level of 10 mg/kg at a
dose concentration of 5 mg/mL, either via an intravenous olus (3
monkeys, IV), or via oral gavage (3 monkeys, PO). Each dosing
syringe is weighed efore 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).
Example 16
Bone Marrow Toxicity Assay
[0483] Human one 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-propoxymet- hyl)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 one
marrow progenitor cells" Biochemical Pharmacology 1992;
44:1921-1925. The culture assays for CFU-GM and FU-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 inhiition of
colony formation in the presence of drug compared to solvent
control cultures.
Example 17
Mitochondria Toxicity Assay
[0484] HepG2 cells are cultured in 12-well plates as described
above and exposed to various concentrations of drugs as taught by
Pan-Zhou X-R, Cui L, Zhou X-J, Sommadossi J-P, Darley-Usmer V M.
"Differential effects of antiretroviral nucleoside analogs on
mitochondrial function in HepG2 cells" Antimicro Agents Chemother
2000; 44:496-503. Lactic acid levels in the culture medium after 4
day drug exposure are measured using a Boehringer lactic acid assay
kit. Lactic acid levels are normalized by cell number as measured
by hemocytometer count.
Example 18
Cytotoxicity Assay
[0485] Cells are seeded at a rate of between 5.times.10.sup.3 and
5.times.10.sup.4/well into 96-well plates in growth medium
overnight at 37.degree. C. in a humidified CO.sub.2 (5%)
atmosphere. New growth medium containing serial dilutions of the
drugs is then added. After incubation 5 for 4 days, cultures are
fixed in 50% TCA and stained with sulforhodamine. The optical
density was read at 550 nm. The cytotoxic concentration was
expressed as the concentration required to reduce the cell numer by
50% (CC.sub.50). The preliminary results are tabulated in the Table
3 below.
4TABLE 3 MDK versus Human Hepatoma CC.sub.50, .mu.M Compound MDK
.beta.-D-4'-CH.sub.3-riboG >250 .beta.-D-4'-CH.sub.3-ribo-4-th-
ioU >250 .beta.-D-4'-CH.sub.3-riboC >250
.beta.-D-4'-CH.sub.3-ribo-5-fluoroU >167
.beta.-D-4'-CH.sub.3-riboT >250 .beta.-D-4'-CH.sub.3-riboA
>250
[0486] This invention has been described with reference to its
preferred embodiments. Variations and modifications of the
invention will be obvious to those skilled in the art from the
foregoing detailed description of the invention. It is intended
that all of these variations and modifications be included within
the scope of this invention.
* * * * *