U.S. patent application number 10/045292 was filed with the patent office on 2003-05-08 for modified nucleosides for the treatment of viral infections and abnormal cellular proliferation.
Invention is credited to Stuyver, Lieven, Watanabe, Kyoichi.
Application Number | 20030087873 10/045292 |
Document ID | / |
Family ID | 26934330 |
Filed Date | 2003-05-08 |
United States Patent
Application |
20030087873 |
Kind Code |
A1 |
Stuyver, Lieven ; et
al. |
May 8, 2003 |
Modified nucleosides for the treatment of viral infections and
abnormal cellular proliferation
Abstract
The disclosed invention is a composition for and a method of
treating a Flaviviridae (including BVDV and HCV), Orthomyxoviridae
(including Influenza A and B) or Paramyxoviridae (including RSV)
infection, or conditions related to abnormal cellular
proliferation, in a host, including animals, and especially humans,
using a nucleoside of general formula (I)-(XXIII) or its
pharmaceutically acceptable salt or prodrug. This invention also
provides an effective process to quantify the viral load, and in
particular BVDV, HCV or West Nile Virus load, in a host, using
real-time polymerase chain reaction ("RT-PCR"). Additionally, the
invention discloses probe molecules that can fluoresce
proportionally to the amount of virus present in a sample.
Inventors: |
Stuyver, Lieven;
(Snellville, GA) ; Watanabe, Kyoichi; (Stone
Mountain, GA) |
Correspondence
Address: |
KING & SPALDING
191 PEACHTREE STREET, N.E.
ATLANTA
GA
30303-1763
US
|
Family ID: |
26934330 |
Appl. No.: |
10/045292 |
Filed: |
October 18, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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60241488 |
Oct 18, 2000 |
|
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60282156 |
Apr 6, 2001 |
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Current U.S.
Class: |
514/45 ;
514/261.1; 514/263.23; 514/269; 514/303; 514/49; 514/81;
514/86 |
Current CPC
Class: |
A61P 17/12 20180101;
C07H 19/048 20130101; C07H 19/10 20130101; C07H 21/04 20130101;
A61P 17/00 20180101; A61P 35/00 20180101; A61P 1/16 20180101; A61P
13/12 20180101; A61P 35/02 20180101; C07H 19/16 20130101; A61K
31/7064 20130101; A61P 17/06 20180101; A61P 19/02 20180101; A61P
43/00 20180101; A61P 31/00 20180101; A61P 31/16 20180101; A61P
29/00 20180101; C07H 19/20 20130101; Y02A 50/30 20180101; A61P 3/12
20180101; A61P 37/02 20180101; A61P 9/10 20180101; A61P 31/12
20180101; A61P 27/02 20180101; A61P 37/06 20180101; C12Q 1/689
20130101; C12Q 1/6895 20130101; C12Q 1/6809 20130101; A61K 31/7076
20130101; C07H 19/06 20130101; A61P 31/14 20180101; C12Q 1/6809
20130101; C12Q 2563/161 20130101 |
Class at
Publication: |
514/45 ; 514/49;
514/81; 514/86; 514/261.1; 514/263.23; 514/269; 514/303 |
International
Class: |
A61K 031/7076; A61K
031/675; A61K 031/519; A61K 031/513 |
Claims
We claim:
1. A method for the treatment or prophylaxis of host exhibiting a
Flaviviridae, Orthomyxoviridae or Paramyxoviridae viral infection
or abnormal cellular proliferation comprising administering an
effective amount of a compound of the general formula (I) or (II):
110or its .beta.-L enantiomer or its pharmaceutically acceptable
salt thereof, wherein: each D is hydrogen, alkyl, acyl,
monophosphate, diphosphate, triphosphate, monophosphate ester,
diphosphate ester, triphosphate ester, phospholipid or amino acid;
each W.sup.1 and W.sup.2 is independently CH or N; each X.sup.1 and
X.sup.2 is independently hydrogen, halogen (F, Cl, Br or I),
NH.sub.2, NHR.sup.4, NR.sup.4R.sup.4',NHOR.sup.4,NR.sup.4NR.sup-
.4'R.sup.4", OH, O.sup.4, SH or SR.sup.4; each Y.sup.1 is O, S or
Se; each Z is CH.sub.2 or NH; each R.sup.1 and R.sup.1' is
independently hydrogen, lower alkyl, lower alkenyl, lower alkynyl,
aryl, alkylaryl, halogen (F, Cl, Br or I), NH.sub.2, NHR.sup.5,
NR.sup.5R.sup.5', NHOR.sup.5, NR.sup.5NHR.sup.5',
NR.sup.5NR.sup.5'R.sup.5", OH, OR.sup.5, SH, SR.sup.5, NO.sub.2,
NO, CH.sub.2OH, CH.sub.2R.sup.5', CO.sub.2H, CO.sub.2R.sup.5,
CONH.sub.2, CONHR.sup.5, CON.sup.5R.sup.5' or CN; each R.sup.2 and
R.sup.2' independently is hydrogen or halogen (F, Cl, Br or I), OH,
SH, OCH.sub.3, SCH.sub.3, NH.sub.2, NHCH.sub.3, CH.dbd.CH.sub.2,
CN, CH.sub.2NH.sub.2, CH.sub.2O H, CO.sub.2H. each R.sup.3 and
R.sup.3 independently is hydrogen or halogen (F, Cl, Br or I), OH,
SH, OCH.sub.3, SCH.sub.3, NH.sub.2, NHCH.sub.3, CH.sub.3,
C.sub.2H.sub.5, CH.dbd.CH.sub.2, CN, CH.sub.2NH.sub.2, CH.sub.2O H,
CO.sub.2H. each R.sup.4, R.sup.4', R.sup.4", R.sup.5, R.sup.5' and
R.sup.5" independently is hydrogen, lower alkyl, lower alkenyl,
aryl, or arylalkyl such as unsubstituted or substituted phenyl or
benzyl; such that for the nucleoside of the general formula (I) or
(II) at least one of R.sup.2 and R.sup.2 is hydrogen and at least
one of R.sup.3 and R.sup.3' is hydrogen.
2. The method of claim 1, wherein the .beta.-D nucleoside of the
formula (I-a) is selected from one of the following:
27 X.sup.1 Y.sup.1 R.sup.1 R.sup.1' R.sup.2 R.sup.2' R.sup.3
R.sup.3' NH.sub.2 O H H OH H H OH NH.sub.2 O H H OH H H I NH.sub.2
O H H OH H H Cl NH.sub.2 O H H OH H H Br NH.sub.2 O H H OH H H
S--CN NH.sub.2 O H H OH H H N.sub.3 NH.sub.2 O H H H Cl H OH
NH.sub.2 O H H H Br H OH NH.sub.2 O H H H OH Br H NH.sub.2 O H H H
OH H H NH.sub.2 O H H H OH O--Ms H NH.sub.2 O H H H OH O--Ts H
NH.sub.2 O H H O--Ms H H OH NH.sub.2 O H H Cl H H OH NH.sub.2 O D D
OH H H OH NH.sub.2 O F H OH H H OH NH.sub.2 O F H H OH H OH
NH.sub.2 O F H H OH H H NH.sub.2 O F H H OH Cl H NH.sub.2 O F H H
OH Br H NH.sub.2 O F H H Cl H OH NH.sub.2 O F H H OH O--Ts H
NH.sub.2 O F H H OH O--Ms H NH.sub.2 O Cl H H OH O--Ms H NH.sub.2 O
Br H H OH O--Ms H NH.sub.2 O Br H H OH O--Ts H NH.sub.2 O Br H H OH
Cl H NH.sub.2 O Br H H OH H OH NH.sub.2 O Br H OH H H OH NH.sub.2 O
I H H OH O--Ms H NH.sub.2 O I H H OH Br H NH.sub.2 O I H H OH O--Ts
H NH.sub.2 O I H H Cl H OH NH.sub.2 O I H Br H H OH NH.sub.2 O OH H
OH H H OH NH.sub.2 O NH.sub.2 H H OH H OH NH.sub.2 O CH.sub.3 H H
OH Cl H NH.sub.2 NH H H OH H H OH NH.sub.2 S H H H Se- H H phenyl
NH-(2-Ph-Et) O H H OH H H OH NH--COCH.sub.3 O H H OH H H OH
NH--NH.sub.2 O H H OH H H OH NH--NH.sub.2 O F H OH H H OH
NH--NH.sub.2 O CH.sub.3 H H OH H OH NH--OH O H H H OH H OH NH--OH O
F H H OH H OH NH--OH O Br H H OH H OH NH--OH O I H H OH H OH NH--OH
O H H OH H H OH OH O OH H OH H H OH OH O NH.sub.2 H H OH H OH OH O
F H OH H H OH OH O F H H O--Ts H OH OH O F H H O--Ms H O--Ms OH O F
H H OH H OH OH O F H H OH H O--Ts OH O F H H H H OH O--Et O H H H
O--Bz H O--Bz S--CH.sub.3 O H H H F H OH SH O H H H OH H OH SH O F
H H OH H OH N.sub.3 O H H H H H H NH-(2-Ph-Et) O H H H OH H OH OH O
OH H H OH H OH OH O H H H OH H H
or its .beta.-L-enantiomer or its pharmaceutically acceptable salt
thereof.
3. The method of claim 1, wherein the .beta.-D nucleoside of the
formula (I-b) is selected from one of the following:
28 X.sup.1 X.sup.2 W.sup.1 R.sup.2 R.sup.2' R.sup.3 R.sup.3' OH
NH.sub.2 N H OH H OH OH NH.sub.2 CH F H H OH NH-cyclohexyl H CH H H
H H NH.sub.2 H CH H OH H F NH.sub.2 H CH H H H H NH.sub.2 NH.sub.2
N H OH H OH NH.sub.2 NH.sub.2 CH H OH H OH Cl H CH F H H H Cl I CH
H O--Ac H O--Ac Cl H CH H OH H OH NH.sub.2 H CH H OH H H Cl H CH H
OH H H
or its .beta.-L-enantiomer or its pharmaceutically acceptable salt
thereof.
4. The method of claim 1, wherein the .beta.-D nucleoside of the
formula (II-a) is selected from one of the following:
29 X.sup.1 Y.sup.1 R.sup.1 R.sup.1' R.sup.2 R.sup.3
NH-Bz-(m-NO.sub.2) O F H H H NH-Bz-(o-NO.sub.2) O F H H H NH.sub.2
O F H F H
or its .beta.-L-enantiomer or its pharmaceutically acceptable salt
thereof.
5. The method of claim 1, wherein the .beta.-D nucleoside of the
formula (II-b) is selected from one of the following:
30 X.sup.1 X.sup.2 W.sup.1 R.sup.2 R.sup.3 Cl H CH F H OH H CH H H
NH.sub.2 F CH H H NH.sub.2 F CH F H NH.sub.2 H CH H H OH NH.sub.2
CH H H OH H CH H H
or its .beta.-L-enantiomer or its pharmaceutically acceptable salt
thereof.
6. A method for the treatment or prophylaxis of host exhibiting a
Flaviviridae, Orthomyxoviridae or Paramyxoviridae viral infection
or abnormal cellular proliferation comprising administering an
effective amount of a compound of the general formula (V) or (VII):
111or its .beta.-L enantiomer or its pharmaceutically acceptable
salt thereof, wherein: each D, W.sup.1, W.sup.2, X.sup.1, X.sup.2,
Y.sup.1, Z, R.sup.1, R.sup.1', R.sup.1', R.sup.2, R.sup.3and
R.sup.3' is the same as defined previously; such that for the
nucleoside of the general formula (V) or (VI), at least one of
R.sup.2 and R.sup.2' is hydrogen and at least one of R.sup.3 and
R.sup.3' is hydrogen.
7. The method of claim 6, wherein the .beta.-D nucleoside of the
formula (V-a) is selected from one of the following:
31 X.sup.1 Y.sup.1 R.sup.1 R.sup.1' R.sup.2 R.sup.2' R.sup.3
R.sup.3' NH.sub.2 O F H H OH H OH OH H CH.sub.3 H H H H H OH O H H
H H H H NH.sub.2 O H H H OH H OH NH.sub.2 O H H H H H H OH O F H H
OH H OH NH.sub.2 O I H H H H H NH.sub.2 O I H H OH H OH NH.sub.2 O
Cl H H OH H OH
or its .beta.-L-enantiomer or its pharmaceutically acceptable salt
thereof.
8. The method of claim 6, wherein the .beta.-D nucleoside of the
formula (VII-a) is selected from one of the following:
32 X.sup.1 Y.sup.1 R.sup.1 R.sup.1' R.sup.2 R.sup.2' R.sup.3
R.sup.3' NH.sub.2 O H H H OH H OH NH.sub.2 O F H H OH H OH NH--OH O
H H H OH H OH
or its .beta.-L-enantiomer or its pharmaceutically acceptable salt
thereof.
9. The method of claim 6, wherein the .beta.-D nucleoside of the
formula (VII-b) is selected from the following:
33 X.sup.1 X.sup.2 W.sup.1 R.sup.2 R.sup.2' R.sup.3 R.sup.3'
NH.sub.2 H CH H OH H OH
or its .beta.-L-enantiomer or its pharmaceutically acceptable salt
thereof.
10. A method for the treatment or prophylaxis of host exhibiting a
Flaviviridae, Orthomyxoviridae or Paramyxoviridae viral infection
or abnormal cellular proliferation comprising administering an
effective amount of a compound of the general formula (XI): 112or
its .beta.-L enantiomer or its pharmaceutically acceptable salt
thereof, wherein: each D, W.sup.1, W.sup.2, X.sup.1, X.sup.2,
Y.sup.1, Z, R.sup.1, R.sup.1', R.sup.2, R.sup.2', R.sup.3 and
R.sup.3' is the same as defined previously; each Z.sup.1 and
Z.sup.2 independently is O, S, NR.sup.6 or Se; each R.sup.6 is
hydrogen, lower alkyl or lower acyl.
11. The method of claim 10, wherein the 5-D nucleoside of the
formula (XI-a) is selected from one of the following:
34 X.sup.1 Y.sup.1 Z.sup.1 Z.sup.2 R.sup.1 R.sup.1 NH.sub.2 O O O H
H NH.sub.2 O O S F H NH.sub.2 O O O F H
or its .beta.-L-enantiomer or its pharmaceutically acceptable salt
thereof.
12. The method of claim 10, wherein the E-D nucleoside of the
formula (XI-b) is selected from one of the following:
35 X.sup.1 X.sup.2 W.sup.1 Z.sup.1 Z.sup.2 Cl H CH O S Cl NH.sub.2
CH O S NH.sub.2 F CH O S OH H CH O O
or its .beta.-L-enantiomer or its pharmaceutically acceptable salt
thereof.
13. A method for the treatment or prophylaxis of host exhibiting a
Flaviviridae, Orthomyxoviridae or Paramyxoviridae viral infection
or abnormal cellular proliferation comprising administering an
effective amount of a compound of the general formula (XIII): 113or
its .beta.-L enantiomer or its pharmaceutically acceptable salt
thereof, wherein: each D, R.sup.1, R.sup.1', R.sup.2, R.sup.2',
R.sup.3 and R.sup.3' is the same as defined previously; each
Y.sup.2 is O, S, NH or NR; each Y.sup.3 is O, S, NH or NR.sup.8;
each X.sup.3 is OR.sup.9 or SR.sup.9; and each R.sup.7, R.sup.8 and
R.sup.9 is hydrogen, lower alkyl of C.sub.1-C.sub.6, arylalkyl or
aryl; such that for the nucleoside of the general formula (XIII-d),
at least one of R.sup.2 and R.sup.2' is hydrogen and at least one
of R.sup.3 and R.sup.3' is hydrogen.
14. The method of claim 13, wherein the .beta.-D nucleoside of the
formula (XIII-a) is selected from one of the following:
36 Y.sup.2 Y.sup.3 R.sup.1 R.sup.1' R.sup.2 R.sup.2' R.sup.3
R.sup.3' O O F H H OH H OH
or its .beta.-L-enantiomer or its pharmaceutically acceptable salt
thereof.
15. The method of claim 13, wherein the .beta.-D nucleoside of the
formula (XIII-c) is selected from one of the following:
37 Y.sup.2 Y.sup.3 R.sup.1 R.sup.1' R.sup.3 R.sup.3' O O F H H OH O
O F H H O--Ms NH O H H H O--Ms NH O H H H O--Ac NH O H H H OH NH O
F H H OH NH O F H H O--Ac
or its .beta.-L-enantiomer or its pharmaceutically acceptable salt
thereof.
16. The method of claim 13, wherein the .beta.-D nucleoside of the
formula (XIII-d) is selected from the following:
38 Y.sup.2 X.sup.3 R.sup.1 R.sup.1' R.sup.2 R.sup.2' R.sup.3
R.sup.3' O O--CH.sub.3 H H H O--Ac H O--Ac
or its .beta.-L-enantiomer or its pharmaceutically acceptable salt
thereof.
17. A method for the treatment or prophylaxis of host exhibiting a
Flaviviridae, Orthomyxoviridae or Paramyxoviridae viral infection
or abnormal cellular proliferation comprising administering an
effective amount of a compound of the general formula (XIV): 114or
its .beta.-L enantiomer or its pharmaceutically acceptable salt
thereof, wherein: each D, X.sup.1, Y.sup.1, Z.sup.1, R.sup.1,
R.sup.2, R.sup.2', R.sup.3 and R.sup.3' is the same as defined
previously; each L.sup.1 is hydrogen, Cl or Br; each L.sup.2 is OH,
OCH.sub.3, OC.sub.2H.sub.5, OC.sub.3H.sub.7, OCF.sub.3, OAc or OBz;
each Z.sup.3 can be O or CH.sub.2.
18. The method of claim 17, wherein the .beta.-D nucleoside of the
formula (XIV) is selected from one of the following:
39 X.sup.1 Y.sup.1 R.sup.1 R.sup.1' R.sup.2 R.sup.2' R.sup.3
R.sup.3' L.sup.1 L.sup.2 NH.sub.2 O NH--OH OH OH H H OH H OH OH O O
F H OH H OH Cl O--CH.sub.3 OH O O H H OH H OH Br O--CH.sub.3 OH O O
F H OH H OH Br O--COCH.sub.3 OH O O F H OH H OH Br O--CH.sub.3 OH O
O F H OH H OH Br O--Et OH O O Cl H OH H OH Br O--CH.sub.3
or its .beta.-L-enantiomer or its pharmaceutically acceptable salt
thereof.
19. A method for the treatment or prophylaxis of host exhibiting a
Flaviviridae, Orthomyxoviridae or Paramyxoviridae viral infection
or abnormal cellular proliferation comprising administering an
effective amount of a compound of the general formula (XV): 115or
its .beta.-L enantiomer or its pharmaceutically acceptable salt
thereof, wherein: each D, W.sup.1, W.sup.2, X.sup.1, Y.sup.1,
Z.sup.3, R.sup.1, R.sup.1', R.sup.2, R.sup.2', R.sup.3 and R.sup.3'
is the same as defined previously.
20. The method of claim 19, wherein the E-D nucleoside of the
formula (XV-a) is defined as the following:
40 Y.sup.1 Z.sup.3 R.sup.1 R.sup.1' R.sup.2 R.sup.2' R.sup.3
R.sup.3' O O H H H OH H OH
its .beta.-L-enantiomer or its pharmaceutically acceptable salt
thereof.
21. The method of claim 19, wherein the .beta.-D nucleoside of the
formula (XV-b) is defined as the following:
41 X.sup.1 W.sup.1 Z.sup.3 R.sup.2 R.sup.2' R.sup.3 R.sup.3'
NH.sub.2 CH O H OH H OH
its .beta.-L-enantiomer or its pharmaceutically acceptable salt
thereof.
22. A method for the treatment or prophylaxis of host exhibiting a
Flaviviridae, Orthomyxoviridae or Paramyxoviridae viral infection
or abnormal cellular proliferation comprising administering an
effective amount of a compound of the general formula (XVI): 116or
its .beta.-L enantiomer or its pharmaceuticaily acceptable salt
thereof, wherein: each D, W.sup.1, X.sup.1, X.sup.2, Y.sup.1, Z,
R.sup.1, R.sup.2, R.sup.2', R.sup.3 and R.sup.3' is the same as
defined previously; each W.sup.3 is independently N, CH or
CR.sup.1; each W.sup.4 and W.sup.5 is independently N, CH, CX.sup.1
or CR.sup.1; and each Z.sup.4and Z.sup.5 is independently NH or
C(.dbd.Y.sup.1); such that if Z.sup.4 and Z.sup.5 are covalently
bound, then Z.sup.4 is not C(.dbd.Y.sup.1) when Z.sup.5 is
C(.dbd.Y.sup.1); and there are no more than three
ring-nitrogens.
23. The method of claim 22, wherein the .beta.-D nucleoside of the
formula (XVI-a) is selected as one of the following:
42 W.sup.3 Z.sup.4 W.sup.5 W.sup.4 Z.sup.5 R.sup.2 R.sup.2' R.sup.3
R.sup.3' CH NCH.sub.3 C--OH N C.dbd.O H OH H O--Ts CH NH
C--NH.sub.2 N C.dbd.O H OH H OH CH NH C--NHAc N C.dbd.O H OH H OH
CH NH C--OH N C.dbd.O H OH H OH CH NCH.sub.3 C--NH.sub.2 N C.dbd.O
H OH H OH CH NH C--NHBz N C.dbd.O H OH H OH CH C.dbd.O C--NH.sub.2
C--SH NH H OH H OH CH NH C--OH N C.dbd.O H Cl H OH CH NH
C--NH.sub.2 N C.dbd.O H Br H OH
its .beta.-L-enantiomer or its pharmaceutically acceptable salt
thereof.
24. The method of claim 22, wherein the .beta.-D nucleoside of the
formula (XVI-c) is defined as the following:
43 W.sup.3 Z.sup.4 Z.sup.5 W.sup.4 R.sup.2 R.sup.2' R.sup.3
R.sup.3' CH N--CH.sub.3 C.dbd.O N H OH H O--Ac
its .beta.-L-enantiomer or its pharmaceutically acceptable salt
thereof.
25. The method of claim 22, wherein the .beta.-D nucleoside of the
formula (XVI-d) is defined as the following:
44 W.sup.3 Z.sup.4 Z.sup.5 W.sup.4 R.sup.3 R.sup.3' CH N C.dbd.NH N
H OH
its .beta.-L-enantiomer or its pharmaceutically acceptable salt
thereof.
26. The method of claim 22, wherein the .beta.-D nucleoside of the
formula (XVI-f) is defined as the following:
45 X.sup.1 X.sup.2 W.sup.1 R.sup.2 R.sup.2' R.sup.3 R.sup.3'
NH.sub.2 H N H OH H OH
its .beta.-L-enantiomer or its pharmaceutically acceptable salt
thereof.
27. A method for the treatment or prophylaxis of host exhibiting a
Flaviviridae, Orthomyxoviridae or Paramyxoviridae viral infection
or abnormal cellular proliferation comprising administering an
effective amount of a compound of the general formula (XVII): 117or
its .beta.-L enantiomer or its pharmaceutically acceptable salt
thereof, wherein: each D, W.sup.1, W.sup.2, X.sup.1, X.sup.2,
Y.sup.1, Z.sup.3, R.sup.1, R.sup.1', R.sup.2, R.sup.24 , R.sup.3
and R.sup.3' is the same as defined previously; each X.sup.4 and
X.sup.5 is independently hydrogen, halogen (F, Cl, Br or I),
N.sub.3, NH.sub.2, NHR.sup.8, NR.sup.8RR OH, OR.sup.8, SH or SR;
and each R.sup.8 and R.sup.8' is independently hydrogen, lower
alkyl, lower alkenyl, aryl or arylalkyl, such as an unsubstituted
or substituted phenyl or benzyl; such that for the nucleoside of
the general formula (XVII-a) or (XVII-b), X.sup.4 is not OH or
OR.sup.8.
28. The method of claim 27, wherein the .beta.-D nucleoside of the
formula (XVII-d) is defined as the following:
46 X.sup.1 X.sup.2 W.sup.1 X.sup.3 X.sup.4 NH.sub.2 F CH H OH
its .beta.-L-enantiomer or its pharmaceutically acceptable salt
thereof.
29. A method for the treatment or prophylaxis of host exhibiting a
Flaviviridae, Orthomyxoviridae or Paramyxoviridae viral infection
or abnormal cellular proliferation comprising administering an
effective amount of a compound of the general formula (XVIII):
118or its .beta.-L enantiomer or its pharmaceutically acceptable
salt thereof, wherein: each D, W.sup.1, W.sup.2, X.sup.1, X.sup.2,
Y.sup.1, R.sup.1 R.sup.1', R.sup.2, R.sup.2', R.sup.3 and R.sup.3'
is the same as defined previously;
30. A method for the treatment or prophylaxis of host exhibiting a
Flaviviridae, Orthomyxoviridae or Paramyxoviridae viral infection
or abnormal cellular proliferation comprising administering an
effective amount of a compound of the general formula (XIX): 119or
its .beta.-L enantiomer or its pharmaceutically acceptable salt
thereof, wherein: each D, R.sup.1, R.sup.4 and R.sup.4' is the same
as defined previously; each R.sup.9 is hydrogen, halogen (F, Cl, Br
or I) or each P.sup.1 is hydrogen, lower alkyl, lower alkenyl,
aryl, arylalkyl (such as an unsubstituted or substituted phenyl or
benzyl), OH, OR.sup.4, NH.sub.2, NUR.sup.4 or NR.sup.4R.sup.4'; and
each P.sup.2 and P.sup.3 is independently hydrogen, alkyl, acyl,
-Ms, -Ts, monophosphate, diphosphate, triphosphate, mono-phosphate
ester, diphosphate ester, triphosphate ester, phospholipid or amino
acid.
31. A method for the treatment or prophylaxis of host exhibiting a
Flaviviridae, Orthomyxoviridae or Paramyxoviridae viral infection
or abnormal cellular proliferation comprising administering an
effective amount of a compound of the general formula: 120or its
.beta.-L enantiomer or its pharmaceutically acceptable salt
thereof, wherein: each D and P.sup.2 is the same as defined
previously.
32. A method for the treatment or prophylaxis of host exhibiting a
Flaviviridae, Orthomyxoviridae or Paramyxoviridae viral infection
or abnormal cellular proliferation comprising administering an
effective amount of a compound of the general formula (XX): 121its
.beta.-L enantiomer or its pharmaceutically acceptable salt
thereof, wherein: each D, P.sup.1, P.sup.2, P.sup.3, R.sup.1,
R.sup.4, R.sup.4' and R.sup.9 is the same as defined
previously.
33. A method for the treatment or prophylaxis of host exhibiting a
Flaviviridae, Orthomyxoviridae or Paramyxoviridae viral infection
or abnormal cellular proliferation comprising administering an
effective amount of a compound of the general formula (XXI): 122its
.beta.-L enantiomer or its pharmaceutically acceptable salt
thereof, wherein: each D, P.sup.1, P.sup.2, P.sup.3, R.sup.1,
R.sup.4 and R.sup.4' is the same as defined previously.
34. A method for the treatment or prophylaxis of host exhibiting a
Flaviviridae, Orthomyxoviridae or Paramyxoviridae viral infection
or abnormal cellular proliferation comprising administering an
effective amount of a compound of the general formula: 123its
.beta.-L enantiomer or its pharmaceutically acceptable salt
thereof, wherein: each D, P.sup.2 and P.sup.3 is the same as
defined previously.
35. A method for the treatment or prophylaxis of host exhibiting a
Flaviviridae, Orthomyxoviridae or Paramyxoviridae viral infection
or abnormal cellular proliferation comprising administering an
effective amount of a compound of the general formula (XXII):
124its .beta.-L enantiomer or its pharmaceutically acceptable salt
thereof, wherein: each D, P.sup.1 and R.sup.1 is the same as
defined previously.
36. A method for the treatment or prophylaxis of host exhibiting a
Flaviviridae, Orthomyxoviridae or Paramyxoviridae viral infection
or abnormal cellular proliferation comprising administering an
effective amount of a compound of the general formula: 125its
.beta.-L enantiomer or its pharmaceutically acceptable salt
thereof, wherein: D is the same as defined previously.
37. A method for the treatment or prophylaxis of host exhibiting a
Flaviviridae, Orthomyxoviridae or Paramyxoviridae viral infection
or abnormal cellular proliferation comprising administering an
effective amount of a compound of the general formula (XXIII):
126its .beta.-L enantiomer or its pharmaceutically acceptable salt
thereof, wherein: each D, P.sup.1, P.sup.2, P.sup.3, R.sup.1,
R.sup.4 and R.sup.4' is the same as defined previously.
38. A method for the treatment or prophylaxis of host exhibiting a
Flaviviridae, Orthomyxoviridae or Paramyxoviridae viral infection
or abnormal cellular proliferation comprising administrating an
effective amount of a compound of the general formula: 127its
.beta.-L enantiomer or its pharmaceutically acceptable salt
thereof, wherein: each D, P.sup.2, and P.sup.3, is the same as
defined previously.
39. A method for the treatment or prophylaxis of host exhibiting a
Flaviviridae, Orthornyxoviridae or Paramyxoviridae viral infection
or abnormal cellular proliferation comprising administering an
effective amount of a compound of the general formula: 128or its
pharmaceutically acceptable salt thereof.
40. A method for the treatment or prophylaxis of host exhibiting a
Flaviviridae, Orthomyxoviridae or Paramyxoviridae viral infection
or abnormal cellular proliferation comprising administering an
effective amount of a compound of the general formula: 129or its
pharmaceutically acceptable salt thereof.
41. A method for the treatment or prophylaxis of host exhibiting a
Flaviviridae, Orthomyxoviridae or Paramyxoviridae viral infection
or abnormal cellular proliferation comprising administering an
effective amount of a compound of the general formula: 130or its
pharmaceutically acceptable salt thereof.
42. A method for the treatment or prophylaxis of host exhibiting a
Flaviviridae, Orthomyxoviridae or Paramyxoviridae viral infection
or abnormal cellular proliferation comprising administering an
effective amount of a compound of the general formula (I) or (II):
131or its pharmaceutically acceptable salt thereof.
43. A method for the treatment or prophylaxis of host exhibiting a
Flaviviridae, Orthomyxoviridae or Paramyxoviridae viral infection
or abnormal cellular proliferation comprising administering an
effective amount of a compound of the general formula: 132or its
pharmaceutically acceptable salt thereof.
44. A method for the treatment or prophylaxis of a hepatitis C
virus infection in a host comprising administering an effective
treatment amount of a compound according to any one of claims
1-29.
45. A method for the treatment or prophylaxis of a hepatitis C
virus infection in a host comprising administering an effective
treatment amount of a .beta.-D nucleoside of the formula (XIX):
133its .beta.-L enantiomer or its pharmaceutically acceptable salt
thereof, wherein: each D, R.sup.1, R.sup.4 and R.sup.4' is the same
as defined previously; each R.sup.9 is hydrogen, halogen (F, Cl, Br
or I) or OP.sup.3; each P.sup.1 is hydrogen, lower alkyl, lower
alkenyl, aryl, arylalkyl (such as an unsubstituted or substituted
phenyl or benzyl), OH, OR.sup.4, NH.sub.2, NHR.sup.4 or
NR.sup.4R.sup.4'; and each P.sup.2 and P.sup.3 is independently
hydrogen, alkyl, acyl, -Ms, -Ts, monophosphate, diphosphate,
triphosphate, mono-phosphate ester, diphosphate ester, triphosphate
ester, phospholipid or amino acid; optionally in a pharmaceutically
acceptable carrier.
46. A method for the treatment or prophylaxis of a hepatitis C
virus infection in a host comprising administering an effective
treatment amount of a E-D nucleoside of the formula: 134its
.beta.-L enantiomer or its pharmaceutically acceptable salt
thereof, wherein: each D and P.sup.2 is the same as defined
previously; optionally in a pharmaceutically acceptable
carrier.
47. A method for the treatment or prophylaxis of a hepatitis C
virus infection in a host comprising administering an effective
treatment amount of a 1-D nucleoside of the formula (XX): 135its
.beta.-L enantiomer or its pharmaceutically acceptable salt
thereof, wherein: each D, P.sup.1, P.sup.2, P.sup.3, R.sup.1,
R.sup.4, R.sup.4' and R.sup.9 is the same as defined previously;
optionally in a pharmaceutically acceptable carrier.
48. A method for the treatment or prophylaxis of a hepatitis C
virus infection in a host comprising administering an effective
treatment amount of a .beta.-D nucleoside of the formula (XXI):
136its 1-L enantiomer or its pharmaceutically acceptable salt
thereof, wherein: each D, P.sup.1, P.sup.2, P.sup.3, R.sup.1,
R.sup.4 and R.sup.4' is the same as defined previously; optionally
in a pharmaceutically acceptable camrer.
49. A method for the treatment or prophylaxis of a hepatitis C
virus infection in a host comprising administering an effective
treatment amount of a .beta.-D nucleoside of the formula: 137its
.beta.-L enantiomer or its pharmaceutically acceptable salt
thereof, wherein: each D, P.sup.2 and P.sup.3 is the same as
defined previously; optionally in a pharmaceutically acceptable
carrier.
50. A method for the treatment or prophylaxis of a hepatitis C
virus infection in a host comprising administering an effective
treatment amount of a .beta.-D nucleoside of the formula (XXII):
138its .beta.-L enantiomer or its pharmaceutically acceptable salt
thereof, wherein: each D, P.sup.1 and R.sup.1 is the same as
defined previously; optionally in a pharmaceutically acceptable
carrier.
51. A method for the treatment or prophylaxis of a hepatitis C
virus infection in a host comprising administering an effective
treatment amount of a .beta.-D nucleoside of the formula: 139its
.beta.-L enantiomer or its pharmaceutically acceptable salt
thereof, wherein: D is the same as defined previously; optionally
in a pharmaceutically acceptable carrier.
52. A method for the treatment or prophylaxis of a hepatitis C
virus infection in a host comprising administering an effective
treatment amount of a .beta.-D nucleoside of the formula (XXIII):
140its .beta.-L enantiomer or its pharmaceutically acceptable salt
thereof, wherein: each D, P.sup.1, P.sup.2, P.sup.3, R.sup.1,
R.sup.4 and R.sup.4' is the same as defined previously; optionally
in a pharmaceutically acceptable carrier.
53. A method for the treatment or prophylaxis of a hepatitis C
virus infection in a host comprising administering an effective
treatment amount of a .beta.-D nucleoside of the formula (XXIII) is
the following: 141its .beta.-L enantiomer or its pharmaceutically
acceptable salt thereof, wherein: each D, P.sup.2 and P.sup.3 is
the same as defined previously; optionally in a pharmaceutically
acceptable carrier.
54. A method for the treatment or prophylaxis of a hepatitis C
virus infection in a host comprising administering an effective
treatment amount of a nucleoside of the formula: 142or its
pharmaceutically acceptable salt thereof; optionally in a
pharmaceutically acceptable carrier.
55. A method for the treatment or prophylaxis of a hepatitis C
virus infection in a host comprising administering an effective
treatment amount of a nucleoside of the formula: 143or its
pharmaceutically acceptable salt thereof; optionally in a
pharmaceutically acceptable carrier.
56. A method for the treatment or prophylaxis of a hepatitis C
virus infection in a host comprising administering an effective
treatment amount of a nucleoside of the formula: 144or its
pharmaceutically acceptable salt thereof; optionally in a
pharmaceutically acceptable carrier.
57. A method for the treatment or prophylaxis of a hepatitis C
virus infection in a host comprising administering an effective
treatment amount of a nucleoside of the formula: 145or its
pharmaceutically acceptable salt thereof; optionally in a
pharmaceutically acceptable carrier.
58. A method for the treatment or prophylaxis of a hepatitis C
virus infection in a host comprising administering an effective
treatment amount of a nucleoside of the formula: 146or its
pharmaceutically acceptable salt thereof; optionally in a
pharmaceutically acceptable carrier.
Description
[0001] This application claims priority to U.S. provisional
application No. 60/241,488, filed Oct. 18, 2000 and U.S.
provisional application No. 60/282,156, filed on Apr. 6, 2001.
FIELD OF THE INVENTION
[0002] The present invention includes compounds and methods for the
treatment of Flaviviridae, Orthomyxoviridae, Paramyxoviridae
infections and abnormal cellular proliferation.
BACKGROUND OF THE INVENTION
[0003] Flavirididae
[0004] The Flaviviridae is a group of positive single-stranded RNA
viruses with a genome size from 9-15 kb. They are enveloped viruses
of approximately 40-50 nm. An overview of the Flaviviridae taxonomy
is available from the International Committee for Taxonomy of
Viruses. The Flaviviridae consists of three genera.
[0005] 1. Flaviviruses. This genus includes the Dengue virus group
(Dengue virus, Dengue virus type 1, Dengue virus type 2, Dengue
virus type 3, Dengue virus type 4), the Japanese encephalitis virus
group (Alfuy Virus, Japanese encephalitis virus, Kookaburra virus,
Koutango virus, Kunjin virus, Murray Valley encephalitis virus, St.
Louis encephalitis virus, Stratford virus, Usutu virus, West Nile
Virus), the Modoc virus group, the Rio Bravo virus group (Apoi
virus, Rio Brovo virus, Saboya virus), the Ntaya virus group, the
Tick-Borne encephalitis group (tick bom encephalitis virus), the
Tyuleniy virus group, Uganda S virus group and the Yellow Fever
virus group. Apart from these major groups, there are some
additional Flaviviruses that are unclassified.
[0006] 2. Hepaciviruses. This genus contains only one species, the
Hepatitis C virus (HCV), which is composed of many clades, types
and subtypes.
[0007] 3. Pestiviruses. This genus includes Bovine Viral Diarrhea
Virus-2 (BVDV-2), Pestivirus type 1 (including BVDV), Pestivirus
type 2 (including Hog Cholera Virus) and Pestivirus type 3
(including Border Disease Virus).
[0008] One of the most important Flaviviridae infections in humans
is caused by the hepatitis C virus (HCV). This is the second major
cause of viral hepatitis, with an estimated 170 million carriers
world-wide (World Health Organization; Hepatitis C: global
prevalence, Weekly Epidemiological Record, 1997, 72, 341), 3.9
million of whom reside in the United States (Centers for Disease
Control; unpublished data, http://www.cdc.gov/ncidod/diseases/
hepatitis/heptab3.htm).
[0009] The genomic organization of the Flaviviridae share many
common features. The hepatitis C virus (HCV) genome is often used
as a model. HCV is a small, enveloped virus with a positive
single-stranded RNA genome of 9.6 kb within the nucleocapsid. The
genome contains a single open reading frame (ORF) encoding a
polyprotein of just over 3,000 amino acids, which is cleaved to
generate the mature structural and nonstructural viral proteins.
The ORF is flanked by 5' and 3' non-translated regions (NTRs) of a
few hundred nucleotides in length, which are important for RNA
translation and replication. The translated polyprotein contains
the structural core (C) and envelope proteins (E1, E2, p7) at the
N-terminus, followed by the nonstructural proteins (NS2, NS3, NS4A,
NS4B, NS5A, NS5B). The mature structural proteins are generated via
cleavage by the host signal peptidase (see: Hijikata, M. et al.
Proc. Nat. Acad. Sci., USA, 1991, 88, 5547; Hussy, P. et al.
Virology, 1996, 224, 93; Lin, C. et al. J. Virol., 1994, 68, 5063;
Mizushima, H. et al. J. Virol., 1994, 68, 2731; Mizushima, H. et
al. J. Virol., 1994, 68, 6215; Santolini, E. et al. J. Virol.,
1994, 68, 3631; Selby, M. J. et al. Virology, 1994, 204, 114; and
Grakoui, A. et al. Proc. Nat. Acad. Sci., USA, 1993, 90, 10538).
The junction between NS2 and NS3 is autocatalytically cleaved by
the NS2/NS3 protease (see: Hijikata, M. et al. J. Virol., 1993, 67,
4665 and Bartenschlager, R. et al. J. Virol., 1994, 68, 5045),
while the remaining four junctions are cleaved by the N-terminal
serine protease domain of NS3 complexed with NS4A. (see: Failla, C.
et al. J. Virol., 1994, 68, 3753; Lin, C. et al. J. Virol., 1994,
68, 8147; Tanji, Y. et al. J. Virol., 1995, 69, 1575 and Tai, C. L.
et al. J. Virol., 1996, 70, 8477) The NS3 protein also contains the
NTP-dependent helicase activity which unwinds duplex RNA during
replication. The NS5B protein possesses RNA-dependent RNA
polymerase (RDRP) activity (see: Behrens, S. E. et al. EMBO J.,
1996, 15, 12; Lohmann, V. et al. J. Virol., 1997, 71, 8416-8428 and
Lohmann, V. et al. Virology, 1998, 249, 108), which is essential
for viral replication. (Ferrari, E. et al. J. Virol., 1999, 73,
1649) It is emphasized here that, unlike HBV or HIV, no DNA is
involved in the replication of HCV. Recently in vitro experiments
using NS5B, substrate specificity for HCV-RDRP was studied using
guanosine 5'-monophosphate (GMP), 5'-diphosphate (GDP),
5'-triphosphate (GTP) and the 5'-triphosphate of 2'-deoxy and
2',3'-dideoxy guanosine (dGTP and ddGTP, respectively). The authors
claimed that HCV-RDRP has a strict specificity for ribonucleoside
5'-triphosphates and requires the 2'- and 3'-OH groups. (Lohmann;
Virology, 108) Their experiments suggest that the presence of 2'-
and 3'-substituents would be the prerequisite for nucleoside
5'-triphosphates to interact with HCV-RDRP and to act as substrates
or inhibitors.
[0010] Examples of antiviral agents that have been identified as
active against the hepatitis C flavivirus include:
[0011] 1. Interferon and ribavirin (Battaglia, A. M. et al. Ann.
Pharmacother. 2000, 34, 487; Berenguer, M. et al. Antivir. Ther.
1998, 3 (Suppl. 3), 125);
[0012] 2. Substrate-based NS3 protease inhibitors (Attwood et al.
PCT WO 98/22496, 1998; Attwood et al. Antiviral Chemistry and
Chemotherapy 1999, 10, 259, Attwood et al. German Patent
Publication DE 19914474; Tung et al. PCT WO 98/17679), including
alphaketoamides and hydrazinoureas, and inhibitors that terminate
in an electrophile such as a boronic acid or phosphonate
(Llinas-Brunet et. al. PCT WO 99/07734);
[0013] 3. Non-substrate-based inhibitors such as
2,4,6-trihydroxy-3-nitro-- benzamide derivatives (Sudo K. et al.,
Biochemical and Biophysical Research Commnunications, 1997, 238,
643 and 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;
[0014] 4. Thiazolidine derivatives which show relevant inhibition
in a reverse-phase HPLC assay with an NS3/4A fusion protein and
NS5A/5B substrate (Sudo K. et al. Antiviral Research 1996, 32, 9),
especially compound RD-1-6250, possessing a fused cinnamoyl moiety
substituted with a long alkyl chain, RD4 6205 and RD4 6193;
[0015] 5. Thiazolidines and benzanilides identified in Kakiuchi N.
et al. J. EBS Letters 421, 217 and Takeshita N. et al. Analytical
Biochemistry 1997, 247, 242;
[0016] 6. A phenanthrenequinone possessing activity against HCV
protease in a SDS-PAGE and autoradiography assay isolated from the
fermentation culture broth of Streptomyces sp., Sch 68631 (Chu M.
et al. Tetrahedron Letters 1996, 37, 7229), and Sch 351633,
isolated from the fungus Penicillium griscofuluum, which
demonstrates activity in a scintillation proximity assay (Chu M. et
al., Bioorganic and Medicinal Chemistry Letters 9, 1949);
[0017] 7. Selective NS3 inhibitors based on the macromolecule elgin
c, isolated from leech (Qasim M. A. et al. Biochemistry 1997,
36,1598);
[0018] 8. HCV helicase inhibitors (Diana G. D. et al., U.S. Pat.
No. 5,633,358 and Diana G. D. et al. PCT WO 97/36554);
[0019] 9. HCV polymerase inhibitors such as nucleotide analogues,
gliotoxin (Ferrari R. et al. Journal of Virology 1999, 73, 1649),
and the natural product cerulenin (Lohmann V. et al. Virology 1998,
249, 108);
[0020] 10. Antisense phosphorothioate oligodeoxynucleotides
(S--ODN) complementary to at least a portion of a sequence of the
HCV (Anderson et al. U.S. Pat. No. 6,174,868), and in particular
the sequence stretches in the 5' non-coding region (NCR) (Alt M. et
al. Hepatology 1995, 22, 707), or nucleotides 326-348 comprising
the 3' end of the NCR and nucleotides 371-388 located in the core
coding region of the HCV RNA (Alt M. et al. Archives of Virology
1997, 142, 589 and Galderisi U. et al., Journal of Cellular
Physiology 1999, 81:2151);
[0021] 11. Inhibitors of IRES-dependent translation (Ikeda N et al.
Japanese Patent Pub. JP-08268890; Kai Y. et al. Japanese Patent
Publication JP-10101591);
[0022] 12. Nuclease-resistant ribozymes (Maccjak D. J. et al.,
Hepatology 1999, 30, abstract 995);
[0023] 13. Amantadine, such as rimantadine (Smith, Abstract from
Annual Meeting of the American Gastoenterological Association and
AASLD, 1996);
[0024] 14. Quinolones, such as ofloxacin, ciprofloxacin and
levofloxacin (AASLD Abstracts, Hepatology, October 1994, Program
Issue, 20 (4), pt.2, abstract no. 293);
[0025] 15. Nucleoside analogs (Ismaili et al. WO 01/60315; Storer
WO 01/32153), including 2'-deoxy-L-nucleosides (Watanabe et al. WO
01/34618), and
1-(.beta.-L-ribofuranosyl)-1,2,4-triazole-3-carboxamide
(levovirinTM) (Tam WO 01/46212); and
[0026] 16. Other miscellaneous compounds including
1-amino-alkylcyclohexan- es (Gold et al. U.S. Pat. No. 6,034,134),
alkyl lipids (Chojkier et al. U.S. Pat. No. 5,922,757), vitamin E
and other antioxidants (Chojkier et al. U.S. Pat. No. 5,922,757),
squalene, bile acids (Ozeki et al. U.S. Pat. No. 5,846,964),
N-(phosphonoacetyl)-L-aspartic acid, (Diana et al. U.S. Pat. No.
5,830,905), benzenedicarboxamides (Diana et al. U.S. Pat. No.
5,633,388), polyadenylic acid derivatives (Wang et al. U.S. Pat.
No. 5,496,546), 2',3'-dideoxyinosine (Yarchoan et al. U.S. Pat. No.
5,026,687), benzimidazoles (Colacino et al. U.S. Pat. No.
5,891,874), glucamines (Mueller et al. WO 01/08672),
substituted-1,5-imino-D-glucitol compounds (Mueller et al. WO
00/47198).
[0027] Orthomyxoviridae
[0028] The Orthomyxoviridae is a group of segmented negative
single-stranded RNA viruses with a genome size from 10-13.6 kb.
They are enveloped viruses of approximately 80-120 nm. An overview
of the Orthomyxoviridae taxonomy is available from the
International Committee for Taxonomy of Viruses. The
Orthomyxoviridae consists of three genera, which can be
distinguished on the basis of antigenic differences between their
nucleocapsid (NP) and matrix proteins (M).
[0029] 1. Influenzavirus A, B. This genus contains influenza A and
B viruses each of which contain eight distinct RNA segments.
Influenza B viruses show little variability in their surface
glycoproteins and only infect humans. On the other hand, influenza
A viruses have great variability in their surface glycoproteins of
influenza A viruses, and they can be divided into subtypes based on
the antigenic nature of their hemagglutinin (HA) and neuroamidase
(NA) glycoproteins and infect humans as well as swine, horses,
seals, fowl, ducks and many other species of birds.
[0030] 2. Influenzavirus C. This genus contains only one species,
influenza C, which contains only seven distinct RNA segments.
Influenza C only has a single multifunctional glycoprotein and
infects mainly humans, but has also been isolated from swine in
China.
[0031] 3. Influenzavirus D. This genus contains influenza D, which
is solely tick-borne viruses that are structurally and genetically
similar to influenza A, B and C.
[0032] One of the most important Orthomyxoviridae infections in
humans is caused by the influenza A virus. These viruses are highly
contagious and cause acute respiratory illness that has plagued
society in epidemic proportions since ancient times. One of the
earliest recordings of an influenza A epidemic can be traced to
Hippocrates in 412 BC. These epidemics are rather frequent and are
often fatal to the elderly, however these epidemics are quite
unpredictable. These viruses are unique respiratory tract viruses,
in that they undergo significant antigenic variation. Both
hemagglutinin (HA) and neuroamidase (NA) glycoproteins are capable
of antigenic drifts and shifts. There are fourteen known
hemagglutinin (H1-H14) glycoproteins and nine known neuroamidase
(N1-N9) glycoproteins. For example, since the first human influenza
virus was isolated in 1933, major antigenic shifts have occurred.
In 1957, the H2N2 subtype (Asian influenza) replaced the H1N1
subtype (Spanish influenza). Currently, the primary subtypes of
influenza are H1N1, which reappeared in 1977 and H3N2, which
reappeared in 1968.
[0033] The vast majority of research on influenza virus gene
expression and RNA replication has been carried out with the
influenza A virus. The most striking feature of the influenza A
virion is a layer of about 500 spikes radiating outward (10 to 14
nm) from the lipid envelope. These spikes are of two types:
rod-shaped spikes of HA and mushroom-shaped spikes of NA. The ratio
of HA and NA varies, but is usually 4-5 to 1. Each gene segment
encodes its own proteins, with the exception of the seventh and
eighth, which encodes M.sub.1 and M.sub.2, and NS.sub.1 and
NS.sub.2 respectively. The first 12 nucleotides at the 3'-end and
the first 13 nucleotides at the 5'-end of each vRNA segment are
conserved in all eight RNA segments. The first gene to have its
nucleotide sequence determined was HA. Since then, all 14 known HA
antigenic subtpes and many variants within the subtypes have been
determined.
[0034] In infected cells, the vRNAs are both transcribed into mRNAs
and replicated. The synthesis of mRNA is distinct, in that the RNA
is primed by 5' capped fragments derived from newly synthesized
host-cell RNA polymerase II transcripts. The mRNA chain elongates
until a stretch of uridine residues is reached 15-22 nucleotides
before the 5'-ends of the vRNAs where transcription ends and
polyadenylate is added to the mRNAs. For replication to occur, an
alternative type of transcription is required that results in the
production of full-length copies of the vRNAs. The full-length
transcripts are initiated without a primer and are not terminated
at the poly(A) site used during mRNA synthesis. The second step in
replication is the copying of the template RNAs into vRNAs. This
synthesis also occurs without a primer, since the vRNAs contain
5'-triphosphorylated ends. All three types of virus-specific
RNAs--mRNA, template RNA and vRNA--are synthesized in the
nucleus.
[0035] Examples of antiviral agents that have been identified as
active against the influenza A virus include:
[0036] 1. Actinomycin D (Barry, R. D. et al. "Participation of
deoxyribonucleic acid in the multiplication of influenza virus"
Nature, 1962, 194, 1139-1140);
[0037] 2. Amantadine (Van Voris, L. P. et al. "Antivirals for the
chemoprophylaxis and treatment of influenza" Semin Respir Infect,
1992, 7, 61-70);
[0038] 3,4-Amino- or
4-guanidino-2-deoxy-2,3-didehydro-D-N-acetylneuroamin- ic acid
-4-amino- or 4-guanidino-Neu 5 Ac2en (von Itzstein, M. et al.
"Rational design of potent sialidase-based inhibitors of influenza
virus replication" Nature, 1993, 363, 418-423);
[0039] 4. Ribavirin (Van Voris, L. P. et al. "Antivirals for the
chemoprophylaxis and treatment of influenza" Semin Respir Infect,
1992, 7, 61-70);
[0040] 5. Interferon (Came, P. E. et al. "Antiviral activity of an
interferon-inducing synthetic polymer" Proc Soc Exp Biol Med, 1969,
131, 443-446; Gerone, P. J. et al. "Inhibition of respiratory virus
infections of mice with aeresols of synthetic double-stranded
ribonucleic acid" Infect Immun, 1971, 3, 323-327; Takano, K. et al.
"Passive interferon protection in mouse influenza" J Infect Dis,
1991, 164, 969-972);
[0041] 6. Inactivated influenza A and B virus vaccines ("Clinical
studies on influenza vaccine--1978" Rev Infect Dis, 1983, 5,
721-764; Galasso, G. T. et al. "Clinical studies on influenza
vaccine--1976" J Infect Dis, 1977, 136 (suppl), S341-S746;
Jennings, R. et al. "Responses of volunteers to inactivated
influenza virus vaccines" J Hyg, 1981, 86, 1-16; Kilbourne, E. D.
"Inactivated influenza vaccine" In: Plothin S A, Mortimer EA, eds.
Vaccines Philadelphia: Saunders, 1988, 420-434; Meyer, H. M., Jr.
et al. "Review of existion vaccines for influenza" Am J Clin
Pathol, 1978, 70, 146-152; "Mortality and Morbidity Weekly Report.
Prevention and control of Influenza: Part I, Vaccines.
Recommendations of the Advisory Committee on Immunication Practices
(ACIP)" MMWR, 1993, 42 (RR-6), 1-14; Palache, A. M. et al.
"Antibody response after influenza immunization with various
vaccine doses: A double-blind, placebo-controlled, multi-centre,
dose-response study in elderly nursing-home residents and young
volunteers" Vaccine, 1993, 11,3-9; Potter, C. W. "Inactivated
influenza virus vaccine" In: Beare AS, ed. Basic and applied
influeza research, Boca Raton, Fla.: CRC Press, 1982, 119-158).
[0042] Paramyxoviridae
[0043] The Paramyxoviridae is a group of negative single-stranded
RNA viruses with a genome size from 16-20 kb. They are enveloped
viruses of approximately 150-300 nm. An overview of the
Paramyxoviridae taxonomy is available from the International
Committee for Taxonomy of Viruses. The Paramyxoviridae consists of
two subfamilies.
[0044] 1. Paramyxovirinae. This subfamily contains three
genera:
[0045] a) Paramyxovirus. This genus is represented by Sendai virus
and including human parainfluenza viruses 1 and 3;
[0046] b) Rubulavirus. This genus is represented by the mumps
virus, simian virus 5, Newcastle disease virus and the human
parainfluenza viruses 2 and 4;
[0047] c) Morbillivirus. This genus is represented by the measles
virus; and
[0048] 2. Pneumovirinae. This subfamily encode a larger number of
mRNAs than the other sub-family (ten, compared with six or seven)
and contains only one genera:
[0049] a) Pneumovirus. This genus is best represented by the
respiratory syncytial virus (RSV), but also includes bovine (BRSV),
ovine RSV (ORSC), caprine RSV (CRSV), pneumonia virus of mice (PVM)
and turkey rhinotracheitis virus (TRTV).
[0050] One of the most important Pneumovirinae infections in humans
is caused by the respiratory syncytial virus (RSV). RSV is the most
important cause of viral lower respiratory tract disease in infants
and children worldwide. In most areas, RSV outranks all other
microbial pathogens as a cause of pneumonia and bronchiolitis in
infants under one year of age. It has also been found that RSV
infection is an important agent of disease in immunosuppressed
adults and in the elderly. Additionally, BRSV has been shown to be
an economically important disease in cattle.
[0051] The 3'-end of genomic RSV RNA consists of a 44-nucleotide
extragenic leader region that is presumed to contain the major
viral promoter. The leader region is followed by the ten viral
genes, which is followed by a 155-nucleotide extragenic trailer
region. Eighty eight percent of the genomic RNA is accounted for by
the ORFs for the ten major proteins. Each gene begins with a
conserved nine-nucleotide gene-start signal. For each gene,
transcription begins at the first nucleotide of the signal. Each
gene terminates with a semi-conserved 12 to 13 nucleotide gene-end
signal that directs transcriptional termination and
polyadenylation. The first nine genes are non-overlapping and are
separated by intergenic regions that range in size from 1 to 52
nucleotides. The intergenic regions do not contain any conserved
sequence motifs or any obvious features of secondary structure. The
last two RSV genes overlap by 68 nucleotides. Thus, one of the
gene-start signals is located inside of, rather than after the
other gene.
[0052] Examples of antiviral agents that have been identified as
active against RSV include:
[0053] 1. Ribavirin (Hruska, J. F. et al. "In vivo inhibition of
respiratory syncytial virus by ribavirin" Antimicrob Agents
Chemother, 1982, 21, 125-130); and
[0054] 2. Purified human intravenous IgG--IVIG (Prince, G. A. et
al. "Effectiveness of topically administered neutralizing
antibodies in experimental immunotherapy of respiratory syncytial
virus infection in cotton rats" J Virol, 1987, 61, 1851-1954;
Prince, G. A. et al. "Immunoprophylaxis and immunotherapy of
respiratory syncytial virus infection in cotton rats" Infect Immun,
1982, 42, 81-87).
[0055] Abnormal Cellular Proliferation
[0056] Cellular differentiation, growth, function and death are
regulated by a complex network of mechanisms at the molecular level
in a multicellular organism. In the healthy animal or human, these
mechanisms allow the cell to carry out its designed function and
then die at a programmed rate.
[0057] Abnormal cellular proliferation, notably hyperproliferation,
can occur as a result of a wide variety of factors, including
genetic mutation, infection, exposure to toxins, autoimmune
disorders, and benign or malignant tumor induction.
[0058] There are a number of skin disorders associated with
cellular hyperproliferation. Psoriasis, for example, is a benign
disease of human skin generally characterized by plaques covered by
thickened scales. The disease is caused by increased proliferation
of epidermal cells of unknown cause. In normal skin the time
required for a cell to move from the basal layer to the upper
granular layer is about five weeks. In psoriasis, this time is only
6 to 9 days, partially due to an increase in the number of
proliferating cells and an increase in the proportion of cells
which are dividing (G. Grove, Int. J. Dermatol. 18:111, 1979).
Approximately 2% of the population in the United States have
psoriasis, occurring in about 3% of Caucasian Americans, in about
1% of African Americans, and rarely in native Americans. Chronic
eczema is also associated with significant hyperproliferation of
the epidermis. Other diseases caused by hyperproliferation of skin
cells include atopic dermatitis, lichen planus, warts, pemphigus
vulgaris, actinic keratosis, basal cell carcinoma and squamous cell
carcinoma.
[0059] Other hyperproliferative cell disorders include blood vessel
proliferation disorders, fibrotic disorders, autoimmune disorders,
graft-versus-host rejection, tumors and cancers.
[0060] Blood vessel proliferative disorders include angiogenic and
vasculogenic disorders. Proliferation of smooth muscle cells in the
course of development of plaques in vascular tissue cause, for
example, restenosis, retinopathies and atherosclerosis. The
advanced lesions of atherosclerosis result from an excessive
inflammatory-proliferative response to an insult to the endothelium
and smooth muscle of the artery wall (Ross, R. Nature, 1993,
362:801-809). Both cell migration and cell proliferation play a
role in the formation of atherosclerotic lesions.
[0061] Fibrotic disorders are often due to the abnormal formation
of an extracellular matrix. Examples of fibrotic disorders include
hepatic cirrhosis and mesangial proliferative cell disorders.
Hepatic cirrhosis is characterized by the increase in extracellular
matrix constituents resulting in the formation of a hepatic scar.
Hepatic cirrhosis can cause diseases such as cirrhosis of the
liver. An increased extracellular matrix resulting in a hepatic
scar can also be caused by viral infection such as hepatitis.
Lipocytes appear to play a major role in hepatic cirrhosis.
[0062] Mesangial disorders are brought about by abnormal
proliferation of mesangial cells. Mesangial hyperproliferative cell
disorders include various human renal diseases, such as
glomerulonephritis, diabetic nephropathy, malignant
nephrosclerosis, thrombotic micro-angiopathy syndromes, transplant
rejection, and glomerulopathies.
[0063] Another disease with a proliferative component is rheumatoid
arthritis. Rheumatoid arthritis is generally considered an
autoimmune disease that is thought to be associated with activity
of autoreactive T cells (See, e.g., Harris, E. D., Jr., The New
England Journal of Medicine, 1990, 322: 1277-1289), and to be
caused by autoantibodies produced against collagen and IgE.
[0064] Other disorders that can include an abnormal cellular
proliferative component include Behcet's syndrome, acute
respiratory distress syndrome (ARDS), ischemic heart disease,
post-dialysis syndrome, leukemia, acquired immune deficiency
syndrome, vasculitis, lipid histiocytosis, septic shock and
inflammation in general.
[0065] A tumor, also called a neoplasm, is a new growth of tissue
in which the multiplication of cells is uncontrolled and
progressive. A benign tumor is one that lacks the properties of
invasion and metastasis and is usually surrounded by a fibrous
capsule. A malignant tumor (i.e., cancer) is one that is capable of
both invasion and metastasis. Malignant tumors also show a greater
degree of anaplasia (i.e., loss of differentiation of cells and of
their orientation to one another and to their axial framework) than
benign tumors.
[0066] Approximately 1.2 million Americans are diagnosed with
cancer each year, 8,000 of which are children. In addition, 500,000
Americans die from cancer each year in the United States alone.
Prostate and lung cancers are the leading causes of death in men
while breast and lung cancer are the leading causes of death in
women. It is estimated that cancer-related costs account for about
10 percent of the total amount spent on disease treatment in the
United States (CNN.Cancer.Factshttp://w-
ww.cnn.com/HEALTH/9511/conquer_cancer/facts/index.html, page 2 of
2, Jul. 18, 1999).
[0067] Proliferative disorders are currently treated by a variety
of classes of compounds including alkylating agents,
antimetabolites, natural products, enzymes, biological response
modifiers, miscellaneous agents, radiophannaceuticals (for example,
Y-90 tagged to hormones or antibodies), hormones and antagonists,
such as those listed below.
[0068] Alkylating Agents
[0069] Nitrogen Mustards: Mechlorethamine (Hodgkin's disease,
non-Hodgkin's lymphomas), Cyclophosphamide, Ifosfamide (acute and
chronic lymphocytic leukemias, Hodgkin's disease, non-Hodgkin's
lymphomas, multiple myeloma, neuroblastoma, breast, ovary, lung,
Wilms' tumor, cervix, testis, soft-tissue sarcomas), Melphalan
(L-sarcolysin) (multiple myeloma, breast, ovary), Chlorambucil
(chronic lymphoctic leukemia, primary macroglobulinemia, Hodgkin's
disease, non-Hodgkin's lymphomas).
[0070] Ethylenimines and Methylmelamines: Hexamethylmelamine
(ovary), Thiotepa (bladder, breast, ovary).
[0071] Alkyl Sulfonates: Busulfan (chronic granuloytic
leukemia).
[0072] Nitrosoureas: Carmustine (BCNU) (Hodgkin's disease,
non-Hodgkin's lymphomas, primary brain tumors, multiple myeloma,
malignant melanoma), Lomustine (CCNU) (Hodgkin's disease,
non-Hodgkin's lymphomas, primary brain tumors, small-cell lung),
Semustine (methyl-CCNU) (primary brain tumors, stomach, colon),
Streptozocin (STR) (malignant pancreatic insulinoma, malignant
carcinoin).
[0073] Triazenes: Dacarbazine (DTIC;
dimethyltriazenoimidazole-carboxamide- ) (malignant melanoma,
Hodgkin's disease, soft-tissue sarcomas).
[0074] Antimetabolites
[0075] Folic Acid Analogs: Methotrexate (amethopterin) (acute
lymphocytic leukemia, choriocarcinoma, mycosis fungoides, breast,
head and neck, lung, osteogenic sarcoma).
[0076] Pyrimidine Analogs: Fluorouracil (5-fluorouracil; 5-FU)
Floxuridine (fluorodeoxyuridine; FUdR) (breast, colon, stomach,
pancreas, ovary, head and neck, urinary bladder, premalignant skin
lesions) (topical), Cytarabine (cytosine arabinoside) (acute
granulocytic and acute lymphocytic leukemias).
[0077] Purine Analogs and Related Inhibitors: Mercaptopurine
(6-mercaptopurine; 6-MP) (acute lymphocytic, acute granulocytic and
chronic granulocytic leukemia), Thioguanine (6-thioguanine: TG)
(acute granulocytic, acute lymphocytic and chronic granulocytic
leukemia), Pentostatin (2'-deoxycyoformycin) (hairy cell leukemia,
mycosis flingoides, chronic lymphocytic leukemia).
[0078] Vinca Alkaloids: Vinblastine (VLB) (Hodgkin's disease,
non-Hodgkin's lymphomas, breast, testis), Vincristine (acute
lymphocytic leukemia, neuroblastoma, Wilms' tumor,
rhabdomyosarcoma, Hodgkin's disease, non-Hodgkin's lymphomas,
small-cell lung).
[0079] Epipodophylotoxins: Etoposide (testis, small-cell lung and
other lung, breast, Hodgkin's disease, non-Hodgkin's lymphomas,
acute granulocytic leukemia, Kaposi's sarcoma), Teniposide (testis,
small-cell lung and other lung, breast, Hodgkin's disease,
non-Hodgkin's lymphomas, acute granulocytic leukemia, Kaposi's
sarcoma).
[0080] Natural Products
[0081] Antibiotics: Dactinomycin (actinonmycin D) (choriocarcinoma,
Wilms' tumor rhabdomyosarcoma, testis, Kaposi's sarcoma),
Daunorubicin (daunomycin; rubidomycin) (acute granulocytic and
acute lymphocytic leukemias), Doxorubicin (soft tissue, osteogenic,
and other sarcomas; Hodgkin's disease, non-Hodgkin's lymphomas,
acute leukemias, breast, genitourinary thyroid, lung, stomach,
neuroblastoma), Bleomycin (testis, head and neck, skin and
esophagus lung, and genitourinary tract, Hodgkin's disease,
non-Hodgkin's lymphomas), Plicamycin (mithramycin) (testis,
malignant hypercalcema), Mitomycin (mitomycin C) (stomach, cervix,
colon, breast, pancreas, bladder, head and neck).
[0082] Enzymes: L-Asparaginase (acute lymphocytic leukemia).
[0083] Biological Response Modifiers: Interferon-alfa (hairy cell
leukemia, Kaposi's sarcoma, melanoma, carcinoid, renal cell, ovary,
bladder, non Hodgkin's lymphomas, mycosis fungoides, multiple
myeloma, chronic granulocytic leukemia).
[0084] Miscellaneous Agents
[0085] Platinum Coordination Complexes: Cisplatin (cis-DDP)
Carboplatin (testis, ovary, bladder, head and neck, lung, thyroid,
cervix, endometrium, neuroblastoma, osteogenic sarcoma).
[0086] Anthracenedione: Mixtozantrone (acute granulocytic leukemia,
breast).
[0087] Substituted Urea: Hydroxyurea (chronic granulocytic
leukemia, polycythemia vera, essential thrombocytosis, malignant
melanoma).
[0088] Methylhydrazine Derivative: Procarbazine (N-methylhydrazine,
M1H) (Hodgkin's disease).
[0089] Adrenocortical Suppressant: Mitotane (o,p'-DDD) (adrenal
cortex), Amino-glutethimide (breast).
[0090] Adrenorticosteriods: Prednisone (acute and chronic
lymphocytic leukemias, non-Hodgkin's lymphomas, Hodgkin's disease,
breast).
[0091] Progestins: Hydroxprogesterone caproate, Medroxyprogesterone
acetate, Megestrol acetate (endometrium, breast). Anti-angiogenesis
Agents Angiostatin, Endostatin.
[0092] Hormones and Antagonists
[0093] Estrogens: Diethylstibestrol Ethinyl estradiol (breast,
prostate)
[0094] Antiestrogen: Tamoxifen (breast).
[0095] Androgens: Testosterone propionate Fluxomyesterone
(breast).
[0096] Antiandrogen: Flutamide (prostate).
[0097] Gonadotropin-Releasing Hormone Analog: Leuprolide
(prostate).
[0098] Toxicity associated with therapy for abnormally
proliferating cells, including cancer, is due in part to a lack of
selectivity of the drug for diseased versus normal cells. To
overcome this limitation, therapeutic strategies that increase the
specificity and thus reduce the toxicity of drugs for the treatment
of proliferative disorders are being explored. One such strategy
that is being aggressively pursued is drug targeting.
[0099] In view of the severity of these diseases and their
pervasiveness in animals, including humans, it is an object of the
present invention to provide a compound, method and composition for
the treatment of a host, including animals and especially humans,
infected with any of the viruses described above, including
flavivirus or pestivirus, influenza virus or Respiratory Syncytial
Virus ("RSV").
[0100] It is another object of the present invention to provide a
method and composition for the treatment of a host, including
animals and especially humans, with abnormal cellular
proliferation.
[0101] It is a further object to provide a method and composition
for the treatment of a host, including animals and especially
humans, infected with hepatitis C or BVDV.
[0102] It is a further object to provide a method and composition
for the treatment of a host, including animals and especially
humans, infected with influenza.
[0103] It is a further object to provide a method and composition
for the treatment of a host, including animals and especially
humans, infected with RSV.
[0104] It is a further object to provide a method and composition
for the treatment of a host, including animals and especially
humans, with a tumor, including a malignant tumor.
[0105] It is yet another object of the present invention to provide
a more effective process to quantify viral load, and in particular
of BVDV or HCV load, in a host, including animals, especially
humans.
SUMMARY OF THE INVENTION
[0106] The present invention provides a .beta.-D or .beta.-L
nucleoside of formula (I)-(XXIII) or its pharmaceutically
acceptable salt or prodrug for the treatment of a host infected
with a virus belonging to the Flaviviridae, Orthomyxoviridae and
Paramyxoviridae family. Alternatively, the .beta.-D or .beta.-L
nucleoside (I)-(XXIII) or its pharmaceutically acceptable salt or
prodrug can be used for the treatment of abnormal cellular
proliferation.
[0107] Specifically, the invention also includes methods for
treating or preventing the following:
[0108] (a) a Flaviviridae infection, including all members of the
Hepacivirus genus (HCV), Pestivirus genus (BVDV, CSFV, BDV), or
Flavivirus genus (Dengue virus, Japanese encephalitis virus group
(including West Nile Virus), and Yellow Fever virus);
[0109] (b) an Orthomyxoviridae infection, including all members of
the Influenza A, B genus, in particular influenza A and all
relevant subtypes--including H1N1 and H3N2--and Influenza B;
[0110] (c) a Paramyxoviridae infection including Respiratory
Syncytial Virus (RSV) infection; and
[0111] (d) abnormal cellular proliferation, including malignant
tumors.
[0112] In one embodiment, the anti-virally or anti-proliferatively
effective nucleoside is a .beta.-D nucleoside of the general
formula (I) or (II): 1
[0113] or its pharmaceutically acceptable salt or prodrug thereof,
wherein:
[0114] each D is hydrogen, alkyl, acyl, monophosphate, diphosphate,
triphosphate, monophosphate ester, diphosphate ester, triphosphate
ester, phospholipid or amino acid;
[0115] each W.sup.1 and W.sup.2 is independently CH or N;
[0116] each X.sup.1 and X.sup.2 is independently hydrogen, halogen
(F, Cl, Br or I), NH.sub.2, NHR.sup.4, NR.sup.4R.sup.4',
NHOR.sup.4, NR.sup.4N.sup.4'R.sup.4", OH, OR.sup.4, SH or
SR.sup.4;
[0117] each Y.sup.1 is O, S or Se;
[0118] each Z is CH.sub.2 or NH;
[0119] each R.sup.1 and R.sup.1' is independently hydrogen, lower
alkyl, lower alkenyl, lower alkynyl, aryl, alkylaryl, halogen (F,
Cl, Br or I), NH.sub.2, NHR.sup.5, NR.sup.5R.sup.5', NHOR.sup.5,
NR.sup.5NHR.sup.5', NR.sup.5NR.sup.5'R.sup.5", OH, OR.sup.5, SH,
SR.sup.5, NO.sub.2, NO, CH.sub.2OH, CH.sub.2OR.sup.5, CO.sub.2H,
CO.sub.2R.sup.5, CONH.sub.2, CONHR.sup.5, CONR.sup.5R.sup.5' or
CN;
[0120] each R.sup.2 and R.sup.2' independently is hydrogen or
halogen (F, Cl, Br or I), OH, SH, OCH.sub.3, SCH.sub.3, NH.sub.2,
NHCH.sub.3, CH.dbd.CH.sub.2, CN, CH.sub.2NH.sub.2, CH.sub.2OH,
CO.sub.2H.
[0121] each R.sup.3 and R.sup.3' independently is hydrogen or
halogen (F, Cl, Br or I), OH, SH, OCH.sub.3, SCH.sub.3, NH.sub.2,
NHCH.sub.3, CH.sub.3, C.sub.2H.sub.5, CH.dbd.CH.sub.2, CN,
CH.sub.2NH.sub.2, CH.sub.2OH, CO.sub.2H.
[0122] each R.sup.4, R.sup.4', R.sup.4", R.sup.5, R.sup.5' and
R.sup.5" independently is hydrogen, lower alkyl, lower alkenyl,
aryl, or arylalkyl such as unsubstituted or substituted phenyl or
benzyl;
[0123] such that for each nucleoside of the general formula (1) or
(II), at least one of R.sup.2 and R.sup.2' is hydrogen and at least
one of R.sup.3 and R.sup.3' is hydrogen.
[0124] In another embodiment of the invention, anti-virally or
anti-proliferatively effective nucleoside is a .beta.-L nucleoside
of the general formula (III) or (IV): 2
[0125] or its pharmaceutically acceptable salt or prodrug thereof,
wherein:
[0126] each D, W.sup.1, W.sup.2 X.sup.1, X.sup.2, Y.sup.1, Z,
R.sup.1, R.sup.1', R.sup.2, R.sup.2', R.sup.3 and R.sup.3' is the
same as defined previously;
[0127] such that for each nucleoside of the general formula (III)
or (IV), at least one of R.sup.2 and R.sup.2' is hydrogen and at
least one of R.sup.3 and R.sup.3' is hydrogen.
[0128] In one embodiment of the invention, the anti-virally or
anti-proliferatively effective nucleoside is a .beta.-D-carba-sugar
nucleoside of the general formula (V) to (VII): 3
[0129] or its pharmaceutically acceptable salt or prodrug thereof,
wherein:
[0130] D, W.sup.1, W.sup.2, X.sup.1, X.sup.2, Y.sup.1, Z, R.sup.1,
R.sup.1', R.sup.2, R.sup.2', R.sup.3 and R.sup.3' is the same as
defined previously;
[0131] such that for each nucleoside of the general formula (V) or
(VI), at least one of R.sup.2 and R.sup.2' is hydrogen and at least
one of R.sup.3 and R.sup.3' is hydrogen.
[0132] In one embodiment, anti-virally or anti-proliferatively
effective nucleoside is a .beta.-L-carba-sugar nucleoside of the
general formula (VIII) to (X): 4
[0133] or its pharmaceutically acceptable salt or prodrug thereof,
wherein:
[0134] each D, W.sup.1, W.sup.2, X.sup.1, X.sup.2, Y.sup.1, Z,
R.sup.1, R.sup.1', R.sup.2, R.sup.2', R.sup.3 and R.sup.3' is the
same as defined previously;
[0135] such that for each nucleoside of the general formula (VIII)
or (IX), at least one of R.sup.2 and R.sup.2 is hydrogen and at
least one of R.sup.3 and R.sup.3' is hydrogen.
[0136] In further embodiment of the invention, the anti-virally or
anti-proliferatively effective .beta.-D or .beta.-L-nucleoside is
of the general formula (XI) or (XII), respectively: 5
[0137] or its pharmaceutically acceptable salt or prodrug thereof,
wherein:
[0138] each D, W.sup.1, W.sup.2, X.sup.1, X.sup.2, Y.sup.1, Z,
R.sup.1, R.sup.1', R.sup.2, R.sup.2', R.sup.3 and R.sup.3' is the
same as defined previously;
[0139] each Z.sup.1 and Z.sup.2 independently is O, S, CH.sub.2,
NR.sup.6 or Se;
[0140] each R.sup.6 is hydrogen, lower alkyl or lower acyl.
[0141] In a further embodiment of this invention, the anti-virally
or anti-proliferatively effective ED or .beta.-L-nucleoside, though
preferably .beta.-D, is of the general formula (XIII): 6
[0142] or its pharmaceutically acceptable salt or prodrug thereof,
wherein:
[0143] each D, R.sup.1, R.sup.1', R.sup.2, R.sup.2', R.sup.3 and
R.sup.3' is the same as defined previously;
[0144] each Y.sup.2 is O, S, NH or NR.sup.7;
[0145] each Y.sup.3 is O, S, NH or NR.sup.8;
[0146] each X.sup.3 is OR.sup.9 or SR.sup.9; and
[0147] each R.sup.7, R.sup.8 and R.sup.9 is hydrogen, lower alkyl
of C.sub.1-C.sub.6, arylalkyl or aryl;
[0148] such that for each nucleoside of the general formula
(XIII-d), at least one of R.sup.2 and R.sup.2' is hydrogen and at
least one of R.sup.3 and R.sup.3' is hydrogen.
[0149] In another embodiment, the anti-virally or
anti-proliferatively effective compound is a .beta.-D or
.beta.-L-nucleoside, though preferably .beta.-D, resulting from the
addition of a small molecule, such as alkyl hypochlorite, alkyl
hypobromite, hypobromous acid or acyl halide to an appropriate
pyrimidine nucleoside, forming a nucleoside of the formula (XIV):
7
[0150] or its pharmaceutically acceptable salt or prodrug thereof,
wherein:
[0151] each D, X.sup.1, Y.sup.1, Z, R.sup.1, R.sup.2, R.sup.2',
R.sup.3 and R.sup.3' is the same as defined previously;
[0152] each L.sup.1 is hydrogen, Cl or Br;
[0153] each L.sup.2 is OH, OCH.sub.3, OC.sub.2H.sub.5,
OC.sub.3H.sub.7, OCF.sub.3, OAc or OBz;
[0154] each Z.sup.3 can be O or CH.sub.2.
[0155] In another embodiment, the anti-virally or
anti-proliferatively effective nucleoside is a dimeric nucleoside
(each nucleoside being in either the .beta.-D or .beta.-L
configuration) of general formula (XV), in which the two
nucleosides are linked through a disulfide bond: 8
[0156] or its pharmaceutically acceptable salt or prodrug thereof,
wherein:
[0157] each D, W.sup.1, W.sup.2, X.sup.1, Y.sup.1, Z.sup.3,
R.sup.1, R.sup.1', R.sup.2, R.sup.2', R.sup.3 and R.sup.3' is the
same as defined previously.
[0158] In one embodiment, the anti-virally or anti-proliferatively
effective nucleoside is a .beta.-or .beta.-L C-nucleoside of the
general formula (XVI): 9
[0159] or its pharmaceutically acceptable salt or prodrug thereof,
wherein:
[0160] each D, W.sup.1, X.sup.1, X.sup.2, Y.sup.1, Z, R.sup.1,
R.sup.2, R.sup.2', R.sup.3 and R.sup.3' is the same as defined
previously;
[0161] each W.sup.3 is independently N, CH or CR.sup.1;
[0162] each W.sup.4 and W.sup.5 is independently N, CH, CX.sup.1 or
CR.sup.1'; and
[0163] each Z.sup.4 and Z.sup.5 is independently NH or
C(.dbd.Y.sup.1);
[0164] such that if Z.sup.4 and Z.sup.5 are covalently bound, then
Z.sup.4 is not C(.dbd.Y.sup.1) when Z.sup.5 is C(.dbd.Y.sup.1);
and
[0165] there are no more than three ring nitrogens.
[0166] In one embodiment, the anti-virally or anti-proliferatively
effective nucleoside is a .beta.-D or .beta.-L-branched-chain sugar
nucleoside of the general formula (XVII): 10
[0167] or its pharmaceutically acceptable salt or prodrug thereof,
wherein:
[0168] each D, W.sup.1, W.sup.2, X.sup.1, X.sup.2, Z.sup.3,
R.sup.1, R.sup.2, R.sup.2', R.sup.3 and R.sup.3' is the same as
defined previously;
[0169] each X.sup.4 and X.sup.5 is independently hydrogen, halogen
(F, Cl, Br or I), N.sub.3, NH.sub.2, NHR.sup.8, NR.sup.8R.sup.8',
OH, OR.sup.8, SH or SR.sup.8; and
[0170] each R.sup.8 and R.sup.8' is independently hydrogen, lower
alkyl, lower alkenyl, aryl or arylalkyl,
[0171] such as an unsubstituted or substituted phenyl or benzyl;
such that for each nucleoside of the general formula (XVII-a) or
(XVII-b), X.sup.4 is not OH or OR.sup.8.
[0172] In one embodiment, the anti-virally or anti-proliferatively
effective nucleoside is a .alpha.-D or .alpha.-L-nucleoside of the
general formula (XVIII): 11
[0173] or its pharmaceutically acceptable salt or prodrug thereof,
wherein:
[0174] each D, W.sup.1, W.sup.2, X.sup.1, X.sup.2, Y.sup.1,
R.sup.1, R.sup.1', R.sup.2, R.sup.2" R.sup.3 and R.sup.3' is the
same as defined previously;
[0175] In a sub-embodiment of the present invention, the
anti-virally or anti-proliferatively effective E-D or .beta.-L
nucleoside is of the formula (XIX): 12
[0176] or its pharmaceutically acceptable salt or prodrug thereof,
wherein:
[0177] each D, R.sup.1, R.sup.4 and R.sup.4' is the same as defined
previously;
[0178] each R.sup.9 is hydrogen, halogen (F, Cl, Br or I) or
OP.sup.3;
[0179] each P.sup.1 is hydrogen, lower alkyl, lower alkenyl, aryl,
arylalkyl (such as an unsubstituted or substituted phenyl or
benzyl), OH, OR.sup.4, NH.sub.2, NHR.sup.4 or NR.sup.4R.sup.4;
and
[0180] each P.sup.2 and P.sup.3 is independently hydrogen, alkyl,
acyl, -Ms, -Ts, monophosphate, diphosphate, triphosphate,
mono-phosphate ester, diphosphate ester, triphosphate ester,
phospholipid or amino acid, though preferably hydrogen.
[0181] In a particular sub-embodiment of the present invention, the
anti-virally or anti-proliferatively effective .beta.-D or .beta.-L
nucleoside of the formula (XIX) is the following: 13
[0182] or its pharmaceutically acceptable salt or prodrug thereof,
wherein:
[0183] each D and P.sup.2 is the same as defined previously. In a
preferred embodiment, D and P.sup.2 are independently hydrogen.
[0184] In another sub-embodiment of the present invention, the
anti-virally or anti-proliferatively effective .beta.-D or .beta.-L
nucleoside is of the formula (XX): 14
[0185] or its pharmaceutically acceptable salt or prodrug thereof,
wherein:
[0186] each D, P.sup.1, P.sup.2, P.sup.3, R.sup.1, R.sup.4,
R.sup.4' and R.sup.9 is the same as defined previously.
[0187] In another sub-embodiment of the present invention, the
anti-virally or anti-proliferatively effective .beta.-D or .beta.-L
nucleoside is of the formula (XXI): 15
[0188] or its pharmaceutically acceptable salt or prodrug thereof,
wherein:
[0189] each D, P.sup.1, P.sup.2, P.sup.3, R.sup.1, R.sup.4 and
R.sup.4' is the same as defined previously.
[0190] In a particular sub-embodiment of the present invention, the
anti-virally or anti-proliferatively effective .beta.-D or .beta.-L
nucleoside of the formula (XXI) is the following: 16
[0191] or its pharmaceutically acceptable salt or prodrug thereof,
wherein:
[0192] each D, P.sup.2 and P.sup.3 is the same as defined
previously. In a preferred embodiment, D, P.sup.2 and P.sup.3 are
independently hydrogen.
[0193] In another sub-embodiment of the present invention, the
anti-virally or anti-proliferatively effective .beta.-D or .beta.-L
nucleoside is of the formula (XXII): 17
[0194] or its pharmaceutically acceptable salt or prodrug thereof,
wherein:
[0195] each D, P.sup.1 and R.sup.1 is the same as defined
previously. In a preferred embodiment, D and P.sup.2 are
independently hydrogen.
[0196] In a particular sub-embodiment of the present invention, the
anti-virally or anti-proliferatively effective .beta.-D or .beta.-L
nucleoside, though preferably .beta.-L, of the formula (XXII) is
the following: 18
[0197] or its pharmaceutically acceptable salt or prodrug thereof,
wherein:
[0198] D is the same as defined previously, and preferably H.
[0199] In another sub-embodiment of the present invention, the
anti-virally or anti-proliferatively effective .beta.-D or .beta.-L
nucleoside is of the formula (XXIII): 19
[0200] or its pharmaceutically acceptable salt or prodrug thereof,
wherein:
[0201] each D, P.sup.1, P.sup.2, P.sup.3, R.sup.1, R.sup.4 and
R.sup.4' is the same as defined previously.
[0202] In a particular sub-embodiment of the present invention, the
anti-virally or anti-proliferatively effective .beta.-D or .beta.-L
nucleoside of the formula (XXIII) is the following: 20
[0203] or its pharmaceutically acceptable salt or prodrug thereof,
wherein:
[0204] each D, P.sup.2 and P.sup.3 is the same as defined
previously. In a preferred embodiment, D, P.sup.2 and P.sup.3 are
independently hydrogen.
[0205] In one embodiment, the nucleoside has an EC.sub.50
(effective concentration to achieve 50% viral inhibition) when
tested in an appropriate cell-based assay, of less than 15
micromolar, and more particularly, less than 10 or 5 micromolar. In
a preferred embodiment, the nucleoside is enantiomerically
enriched.
[0206] The present invention also includes at least the following
features:
[0207] (a) use of a .beta.-D nucleoside or .beta.-L nucleoside of
formula (I)-(XXIII), as described herein, or its pharmaceutically
acceptable salt or prodrug thereof in a medical therapy, i.e. as an
antiviral or antitumor/anticancer agent, for example for the
treatment or prophylaxis of a Flaviviridae infections, including
hepatitis C infection;
[0208] (b) use of a .beta.-D nucleoside or .beta.-L nucleoside of
formula (I)-(XXIII), as described herein, or its pharmaceutically
acceptable salt or prodrug thereof in the manufacture of a
medicament for treatment of a Flaviviridae infection, including
hepatitis C infection;
[0209] (c) a pharmaceutical composition that include an antivirally
effective amount of a .beta.-D nucleoside or .beta.-L nucleoside of
formula (I)-(XXIII), as described herein, or its pharmaceutically
acceptable salt or prodrug thereof together with a pharmaceutically
acceptable carrier or diluent according to the present
invention;
[0210] (d) a pharmaceutical composition with a .beta.-D nucleoside
or .beta.-L nucleoside of formula (I)-(XXIII), as described herein,
or its pharmaceutically acceptable salt or prodrug thereof in
combination with one or more other antivirally effective agents;
and
[0211] (e) process for the preparation of .beta.-D nucleoside or
.beta.-L nucleoside of formula (I)-(XXIII), as described herein,
and their pharmaceutically acceptable salts and prodrugs
thereof.
[0212] The activity and toxicity of the compounds described herein
can be evaluated according to any known procedure. An efficient
process to quantify the viral load in a host using real-time
polymerase chain reaction ("RT-PCR") is provided below. The process
involves the use of a quenched fluorescent probe molecule, which
can be hybridized to the target viral DNA or RNA. Upon
exonucleolytic degradation, a detectable fluorescent signal can be
monitored. Using this technique, the RT-PCR amplified DNA or RNA
can be detected in real time by monitoring the presence of
fluorescence signals.
[0213] This specification demonstrates:
[0214] (a) a process to quantitate viral load in real time using
RT-PCR, as described herein;
[0215] (b) a process to quantitate viral load of a Flaviviridae in
a host, including BVDV and HCV, in a host in real time using the
RT-PCR, as described herein;
[0216] (c) a process to quantitate viral load of BVDV in a MDBK
cell line or a host sample in real time using the RT-PCR, as
described herein;
[0217] (d) a probe molecule designed to fluoresce upon
exonucleolytic degradation and to be complementary to the BVDV NADL
NS5B region, as described herein; and
[0218] (e) a probe molecule with a sequence of
5'-6-fam-AAATCCTCCTAACAAGCG- GGTTCCAGG-tamara-3' (Sequence ID No 1)
and primers with a sequence of sense: 5'-AGCCTTCAGTTTCTTGCTGATGT-3'
(Sequence ID No 2) and antisense: 5'-TGTTGCGAAAGCACCAACAG-3'
(Sequence ID No 3);
[0219] (f) a process to quantitate viral load of HCV in a host
derived sample or a cell line in real time using the RT-PCR, as
described herein;
[0220] (g) a probe molecule designed to fluoresce upon
exonucleolytic degradation and to be complementary to the HCV
5'-uncoding region, as described herein; and
[0221] (h) a probe molecule designed to fluoresce upon
exonucleolytic degradation and to be complementary to the HCV
coding region, as described herein; and
[0222] (i) a probe molecule designed to fluoresce upon
exonucleolytic degradation and to be complementary to the HCV
3'-uncoding region, as described herein; and
[0223] (j) a probe molecule with a sequence of
5'-6-fam-CCTCCAGGACCCCCCCTC- CC-tamara-3' (Sequence ID No 4) and
primers with a sequence of sense: 5'-AGCCATGGCGTTAGTA(T/C)GAGTGT-3'
(Sequence ID No 5) and antisense: 5'-TTCCGCAGACCACTATGG-3'
(Sequence ID No 6).
BRIEF DESCRIPTION OF THE FIGURES
[0224] FIG. 1 is an illustration of the increase in plaque forming
units with increasing concentration of bovine viral diarrhea virus
("BVDV") in cell culture as described in Example 51. FIG. 1
establishes that the method of Example 51 provides reliable
quantification of BVDV over a four log PFU/mL of virus.
[0225] FIG. 2 is an illustration of the BVDV replication cycle in
MDBK cells to determine the optimal harvesting time (in hours post
infection versus the log of plaque forming units ("PFU"), i.e. 22
hours after infection, which roughly corresponds to approximately
one replication cycle, where the amount of virus produced is equal
to the amount of virus inoculated into the cell, as described in
Example 52.
[0226] FIG. 3 is a bar chart graph showing the ability of certain
test compounds to inhibit the number of plaque forming units, as
described in Example 40 against BVDV.
[0227] FIG. 4 is a line graph illustrating that the prevention of
cytotoxicity of a "carba-sugar" nucleoside in CEM cells (human
T-cell lymphoma) and in SUDHL-1 cells (human anaplastic T-cell
lymphoma cell line) can be accomplished by co-administration of
natural nucleosides, namely cytidine and uridine.
[0228] FIG. 5 provides the structure of various non-limiting
examples of nucleosides of the present invention, as well as the
known nucleoside, ribavirin, which is used as a comparative example
in the text.
DETAILED DESCRIPTIlON OF THE INVENTION
[0229] The present invention provides a nucleoside of the general
formula (I)-(XXIII) or its pharmaceutically acceptable salt or
prodrug for the treatment of a host infected with a virus belonging
to the Flaviviridae, the Orthomyxoviridae, or the Paramyxoviridae
family. Alternatively, the nucleoside of the general formula (I)
(XXIII) or its pharmaceutically acceptable salt or prodrug can be
used for the treatment of abnormal cellular proliferation.
[0230] In one embodiment, a method for the treatment or prophylaxis
of an antiviral or antiproliferative agent, for example for the
treatment or prophylaxis of a viral infections, including
Flaviviridae infections, including hepatitis C infection, influenza
virus infection, including influenza A (such as H1N1 and H3N2) and
influenza B and RSV, as well as abnormal cellular proliferation
that includes the administration of an anti-virally or
anti-proliferatively effective amount of a nucleoside of the
present invention, or its pharmaceutically acceptable salt or
prodrug thereof is provided.
[0231] In another embodiment, a method for the treatment or
prophylaxis of an antiviral or antiproliferative agent, for example
for the treatment or prophylaxis of a Flaviviridae infection that
includes the administration of an antivirally amount of a
nucleoside of the present invention, or its pharmaceutically
acceptable salt or prodrug thereof in the manufacture of a
medicament for treatment is provided.
[0232] In another embodiment, a method for the treatment or
prophylaxis of an antiviral or antiproliferative agent, for example
for the treatment or prophylaxis of an Influenza virus infection
that includes the administration of an antivirally effective amount
of a nucleoside of the present invention, or its pharmaceutically
acceptable salt or prodrug thereof in the manufacture of a
medicament for treatment is provided.
[0233] In another embodiment, a method for the treatment or
prophylaxis of an antiviral or antiproliferative agent, for example
for the treatment or prophylaxis of a RSV infection that includes
the administration of an antivirally effective amount of the
present invention, or its pharmaceutically acceptable salt or
prodrug thereof in the manufacture of a medicament for treatment is
provided.
[0234] In another embodiment, a method for the treatment or
prophylaxis of an antiviral or antiproliferative agent, for example
for the treatment or prophylaxis of a disease characterized by
abnormal cellular proliferation that includes the administration of
an anti-proliferatively effective amount of a nucleoside of the
present invention.
[0235] In another embodiment, the invention is the use of one of
the compounds described herein in the manufacture of a medicament
for the treatment of a viral infection or abnormal cellular
proliferation, as provided herein.
[0236] In another embodiment, the invention is the use of one of
the compounds described herein in the treatment of a host
exhibiting a viral infection or abnormal cellular proliferation, as
provided herein.
[0237] In another embodiment, a pharmaceutical composition that
includes an antivirally or anti-proliferatively effective amount of
a nucleoside of the present invention, or its pharmaceutically
acceptable salt or prodrug thereof together with a pharmaceutically
acceptable carrier or diluent according to the present invention is
provided.
[0238] In another embodiment, a pharmaceutical composition with a
nucleoside of the present invention, or its pharmaceutically
acceptable salt or prodrug thereof in combination with one or more
other antivirally or anti-proliferatively effective agents is
provided.
[0239] In another embodiment, a process for the preparation of the
nucleosides of the present invention, and its pharmaceutically
acceptable salt and prodrug thereof is provided.
[0240] In an additional embodiment, a method of treating a mammal
having a virus-associated disorder which comprises administering to
the mammal a pharmaceutically effective amount of a nucleoside of
the present invention, or their pharmaceutically acceptable salts
or prodrugs thereof, is provided.
[0241] In an additional embodiment, a method of treating a mammal
having disorder associated with abnormal cellular proliferation,
which comprises administering to the mammal a pharmaceutically
effective amount of a nucleoside of the present invention, or their
pharmaceutically acceptable salts or prodruigs thereof, is
provided.
[0242] In particular, the invention includes the described
compounds in methods for treating or preventing, or uses for the
treatment or prophylaxis of, or uses in the manufacture of a
medicament for following:
[0243] (a) a Flaviviridae infection, including all members of the
Hepacivirus genus (HCV), Pestivirus genus (BVDV, CSFV, BDV), or
Flavivirus genus (Dengue virus, Japanese encephalitis virus group
(including West Nile Virus), and Yellow Fever virus);
[0244] (b) an Orthomyxoviridae infection, including all members of
the Influenza A, B genus, in particular influenza A and all
relevant subtypes--including H1N1 and H3N2-and Influenza B;
[0245] (c) a Paramyxoviridae infection, including Respiratory
Syncytial Virus (RSV) infection; and
[0246] (d) abnormal cellular proliferation, including malignant
tumors.
[0247] I. Compounds of the Invention
[0248] In one embodiment, the anti-virally or anti-proliferatively
effective nucleoside is a .beta.-D nucleoside of the general
formula (I) or (II): 21
[0249] or its pharmaceutically acceptable salt or prodrug thereof,
wherein:
[0250] each D is hydrogen, alkyl, acyl, monophosphate, diphosphate,
triphosphate, monophosphate ester, diphosphate ester, triphosphate
ester, phospholipid or amino acid, though preferably hydrogen;
[0251] each W.sup.1 and w.sup.2 is independently CH or N;
[0252] each X.sup.1 and X.sup.2 is independently hydrogen, halogen
(F, Cl, Br or I), NH.sub.2, NHR.sup.4, NR.sup.4 R.sup.4',
NHOR.sup.4, NR.sup.4NR.sup.4'R.sup.4", OH, OR.sup.4, SH or
SR.sup.4;
[0253] each Y.sup.1 is O, S or Se;
[0254] each Z is CH.sub.2 or NH;
[0255] each R.sup.1 and R.sup.1' is independently hydrogen, lower
alkyl, lower alkenyl, lower alkynyl, aryl, alkylaryl, halogen (F,
Cl, Br or I), NH.sub.2, NHR.sup.5, NR.sup.5R.sup.5, NHOR.sup.5,
NR.sup.5NHR.sup.5', NR.sup.5NR.sup.5'R.sup.5", OH, OR.sup.5, SH,
SR.sup.5, NO.sub.2, NO, CH.sub.2O H, CH.sub.2OR.sup.5, CO.sub.2H,
CO.sub.2R.sup.5, CONH.sub.2, CONHR.sup.5, CONR.sup.5R.sup.5' or
CN;
[0256] each R.sup.2 and R.sup.2' independently is hydrogen or
halogen (F, Cl, Br or I), OH, SH, OCH.sub.3, SCH.sub.3, NH.sub.2,
NHCH.sub.3, CH.dbd.CH.sub.2, CN, CH.sub.2NH.sub.2, CH.sub.2OH,
CO.sub.2H.
[0257] each R.sup.3 and R.sup.3' independently is hydrogen or
halogen (F, Cl, Br or I), OH, SH, OCH.sub.3, SCH.sub.3, NH.sub.2,
NHCH.sub.3, CH.sub.3, C.sub.2H.sub.5, CH.dbd.CH.sub.2, CN,
CH.sub.2NH.sub.2, CH.sub.2O H, CO.sub.2H.
[0258] each R.sup.4, R.sup.4', R.sup.4", R.sup.5, R.sup.5' and
R.sup.5" independently is hydrogen, lower alkyl, lower alkenyl,
aryl, or arylalkyl such as unsubstituted or substituted phenyl or
benzyl;
[0259] such that for each nucleoside of the general formula (I) or
(II), at least one of R.sup.2 and R.sup.2' is hydrogen and at least
one of R.sup.3 and R.sup.3' is hydrogen.
[0260] In another embodiment of the invention, anti-virally or
anti-proliferatively effective nucleoside is a .beta.-L nucleoside
of the general formula (III) or (IV): 22
[0261] or its pharmaceutically acceptable salt or prodrug thereof,
wherein:
[0262] each D, W.sup.1, W.sup.2, X.sup.1, X.sup.2, Y.sup.1, Z,
R.sup.1, R.sup.1', R.sup.2, R.sup.2', R.sup.3 and R.sup.3' is the
same as defined previously;
[0263] such that for each nucleoside of the general formula (III)
or (IV), at least one of R.sup.2 and R.sup.2' is hydrogen and at
least one of R.sup.3 and R.sup.3' is hydrogen.
[0264] In one embodiment of the invention, the anti-virally or
anti-proliferatively effective nucleoside is a .beta.-D-carba-sugar
nucleoside of the general formula (V) to (VII): 23
[0265] or its pharmaceutically acceptable salt or prodrug thereof,
wherein:
[0266] D, W.sup.1, W.sup.2, X.sup.1, X.sup.2, Y.sup.1, Z, R.sup.1,
R.sup.1', R.sup.2, R.sup.2', R.sup.3 and R.sup.3' is the same as
defined previously;
[0267] such that for each nucleoside of the general formula (V) or
(VI), at least one of R.sup.2 and R.sup.2' is hydrogen and at least
one of R.sup.3 and R.sup.3' is hydrogen.
[0268] In one embodiment, anti-virally or anti-proliferatively
effective nucleoside is a L-carba-sugar nucleoside of the general
formula (VIII) to (X): 24
[0269] or its pharmaceutically acceptable salt or prodrug thereof,
wherein:
[0270] each D, W.sup.1, W.sup.2, X.sup.1, X.sup.2, Y.sup.1, Z,
R.sup.1, R.sup.1', R.sup.2, R.sup.2', R.sup.3 and R.sup.3' is the
same as defined previously;
[0271] such that for each nucleoside of the general formula (VIII)
or (IX), at least one of R.sup.2 and R.sup.2' is hydrogen and at
least one of R.sup.3 and R.sup.3' is hydrogen.
[0272] In further embodiment of the invention, the anti-virally or
anti-proliferatively effective .beta.-D or .beta.-L-nucleoside is
of the general formula (XI) or (XII), respectively: 25
[0273] or its pharmaceutically acceptable salt or prodrug thereof,
wherein:
[0274] each D, W.sup.1, W.sup.2, X.sup.1, X.sup.2, Y.sup.1, Z,
R.sup.1, R.sup.1', R.sup.2, R.sup.2', R.sup.3 and R.sup.3' is the
same as defined previously;
[0275] each Z.sup.1 and Z.sup.2 independently is O, S, NR.sup.6 or
Se;
[0276] each R.sup.6 is hydrogen, lower alkyl or lower acyl.
[0277] In a further embodiment of this invention, the anti-virally
or anti-proliferatively effective .beta.-D or .beta.-L-nucleoside,
though preferably .beta.-D, is of the general formula (XIII):
26
[0278] or its pharmaceutically acceptable salt or prodrug thereof,
wherein:
[0279] each D, R.sup.1, R.sup.1', R.sup.2, R.sup.2', R.sup.3 and
R.sup.3' is the same as defined previously;
[0280] each Y.sup.2 is O, S, NH or NR.sup.7;
[0281] each Y.sup.3 is O, S, NH or NR.sup.8;
[0282] each X.sup.3 is OR.sup.9 or SR.sup.9; and
[0283] each R.sup.7, R.sup.8 and R.sup.9 is hydrogen, lower alkyl
of C.sub.1-C.sub.6, arylalkyl or aryl;
[0284] such that for each nucleoside of the general formula
(XIII-d), at least one of R.sup.2 and R.sup.2' is hydrogen and at
least one of R.sup.3 and R.sup.3 is hydrogen.
[0285] In another embodiment, the anti-virally or
anti-proliferatively effective is a .beta.-D or
.beta.-L-nucleoside, though preferably .beta.-D, resulting from the
addition of a small molecule, such as alkyl hypochlorite, alkyl
hypobromite, hypobromous acid or acyl halide to an appropriate
pyrimidine nucleoside, forming a nucleoside of the formula (XIV):
27
[0286] or its pharmaceutically acceptable salt or prodrug thereof,
wherein:
[0287] each D, X.sup.1, Y.sup.1, Z.sup.1, R.sup.2, R.sup.2',
R.sup.3 and R.sup.3' is the same as defined previously;
[0288] each L.sup.1 is hydrogen, Cl or Br;
[0289] each Z.sup.3 can be O or CH.sub.2.
[0290] In another embodiment, the anti-virally or
anti-proliferatively effective nucleoside is a dimeric nucleoside
(each nucleoside being in either the .beta.-D or .beta.-L
configuration) of general formula (XV), in which the two
nucleosides are linked through a disulfide bond: 28
[0291] or its pharmaceutically acceptable salt or prodrug thereof,
wherein:
[0292] each D, W.sup.1, W.sup.2, X.sup.1, Y.sup.1, Z.sup.3,
R.sup.1, R.sup.1', R.sup.2, R.sup.2', R.sup.3 and R.sup.3' is the
same as defined previously.
[0293] In one embodiment, the anti-virally or anti-proliferatively
effective nucleoside is a .beta.-D or .beta.-L C-nucleoside of the
general formula (XVI): 29
[0294] or its pharmaceutically acceptable salt or prodrug thereof,
wherein:
[0295] each D, W.sup.1, X.sup.1, X.sup.2, Y.sup.1, Z, R.sup.1,
R.sup.2, R.sup.2', R.sup.3 and R.sup.3' is the same as defined
previously;
[0296] each W.sup.3 is independently N, CH or CR.sup.1;
[0297] each W.sup.4and W.sup.5 is independently N, CH, CX.sup.1 or
CR.sup.1'; and
[0298] each Z.sup.4 and Z.sup.5 is independently NH or
C(.dbd.Y.sup.1);
[0299] such that if Z.sup.4 and Z.sup.5 are covalently bound, then
Z.sup.4 is not C(.dbd.Y.sup.1) when Z.sup.5 is C(.dbd.Y.sup.1);
and
[0300] there are no more than three ring-nitrogens.
[0301] In one embodiment, the anti-virally or anti-proliferatively
effective nucleoside is a .beta.-D or .beta.-L-branched-chain sugar
nucleoside of the general formula (XVII): 30
[0302] or its pharmaceutically acceptable salt or prodrug thereof,
wherein:
[0303] each D, W.sup.1, W.sup.2, X.sup.1, X.sup.1, Y.sup.1,
Z.sup.3, R.sup.1, R.sup.1', R.sup.2, R.sup.2', R.sup.3 and R.sup.3'
is the same as defined previously;
[0304] each X.sup.4 and X.sup.5 is independently hydrogen, halogen
(F, Cl, Br or I), N.sub.3, NH.sub.2, NHR.sup.8, NR.sup.8R.sup.8',
OH, OR SH or SR.sup.8; and
[0305] each R.sup.8 and R.sup.8' is independently hydrogen, lower
alkyl, lower alkenyl, aryl or arylalkyl, such as an unsubstituted
or substituted phenyl or benzyl;
[0306] such that for each nucleoside of the general formula
(XVII-a) or (XVII-b), X.sup.4 is not OH or OR.sup.8.
[0307] In one embodiment, the anti-virally or anti-proliferatively
effective nucleoside is a .alpha.-D or .alpha.-L-nucleoside of the
general formula (XVIII): 31
[0308] or its pharmaceutically acceptable salt or prodrug thereof,
wherein:
[0309] each D, W.sup.1, W.sup.2, X.sup.1, X.sup.2, Y.sup.1,
R.sup.1, R.sup.', R.sup.2, R.sup.2', R.sup.3 and R.sup.3' is the
same as defined previously;
[0310] In a sub-embodiment of the present invention, the
anti-virally or anti-proliferatively effective .beta.-D or .beta.-L
nucleoside is of the formula (XIX): 32
[0311] or its pharmaceutically acceptable salt or prodrug thereof,
wherein:
[0312] each D, R.sup.1, R.sup.4 and R.sup.4' is the same as defined
previously;
[0313] each R.sup.9 is hydrogen, halogen (F, Cl, Br or I) or
OP.sup.3;
[0314] each P.sup.1 is hydrogen, lower alkyl, lower alkenyl, aryl,
arylalkyl (such as an unsubstituted or substituted phenyl or
benzyl), OH, OR.sup.4, NH.sub.2, NHR.sup.4 or NR.sup.4R.sup.4';
and
[0315] each P.sup.2 and P.sup.3 is independently hydrogen, alkyl,
acyl, -Ms, -Ts, monophosphate, diphosphate, triphosphate,
mono-phosphate ester, diphosphate ester, triphosphate ester,
phospholipid or amino acid, though preferably hydrogen.
[0316] In a particular sub-embodiment of the present invention, the
anti-virally or anti-proliferatively effective .beta.-D or .beta.-L
nucleoside of the formula (XIX) is the following: 33
[0317] or its pharmaceutically acceptable salt or prodrug thereof,
wherein:
[0318] each D and P.sup.2 is the same as defined previously. In a
preferred embodiment, D and P.sup.2 are independently hydrogen.
[0319] In another sub-embodiment of the present invention, the
anti-virally or anti-proliferatively effective .beta.-D or .beta.-L
nucleoside is of the formula (XX): 34
[0320] or its pharmaceutically acceptable salt or prodrug thereof,
wherein:
[0321] each D, P.sup.1, P.sup.2, P.sup.3, R.sup.1, R.sup.4,
R.sup.4' and R.sup.9 is the same as defined previously.
[0322] In another sub-embodiment of the present invention, the
anti-virally or anti-proliferatively effective .beta.-D or .beta.-L
nucleoside is of the formula (XXI): 35
[0323] or its pharmaceutically acceptable salt or prodrug thereof,
wherein:
[0324] each D, P.sup.1, P.sup.2, P.sup.3, R.sup.1, R.sup.4 and
R.sup.4' is the same as defined previously.
[0325] In a particular sub-embodiment of the present invention, the
anti-virally or anti-proliferatively effective E-D or .beta.-L
nucleoside of the formula (XXI) is the following: 36
[0326] or its pharmaceutically acceptable salt or prodrug thereof,
wherein:
[0327] each D, P.sup.2 and P.sup.3 is the same as defined
previously. In a preferred embodiment, D, P.sup.2 and P.sup.3 are
independently hydrogen.
[0328] In another embodiment, N-hydroxycytosine is used as the base
attached to any of the sugar or carba-sugar moieties described in
this application, as if each were fully described a separate
specific embodiment.
[0329] In another sub-embodiment of the present invention, the
anti-virally or anti-proliferatively effective .beta.-D or .beta.-L
nucleoside is of the formula (XXII): 37
[0330] or its pharmaceutically acceptable salt or prodrug thereof,
wherein:
[0331] each D, P.sup.1 and R.sup.1 is the same as defined
previously. In a preferred embodiment, D and P.sup.2 are
independently hydrogen.
[0332] In a particular sub-embodiment of the present invention, the
anti-virally or anti-proliferatively effective .beta.-D or .beta.-L
nucleoside, though preferably .beta.-L, of the formula (XXII) is
the following: 38
[0333] or its pharmaceutically acceptable salt or prodrug thereof,
wherein:
[0334] D is the same as defined previously, and preferably H.
[0335] In another sub-embodiment of the present invention, the
anti-virally or anti-proliferatively effective .beta.-D or .beta.-L
nucleoside is of the formula (XXIII): 39
[0336] or its pharmaceutically acceptable salt or prodrug thereof,
wherein:
[0337] each D, P.sup.1, P.sup.2, P.sup.3, R.sup.1, R.sup.4 and
R.sup.4' is the same as defined previously.
[0338] In a particular sub-embodiment of the present invention, the
anti-virally or anti-proliferatively effective .beta.-D or .beta.-L
nucleoside of the formula (XXIII) is the following: 40
[0339] or its pharmaceutically acceptable salt or prodrug thereof,
wherein:
[0340] each D, P.sup.2 and P.sup.3 is the same as defined
previously. In a preferred embodiment, D, P.sup.2 and P.sup.3 are
independently hydrogen.
[0341] In a preferred embodiment, the .beta.-D and .beta.-L
nucleosides of general formula (I-a) and (III-a) are represented by
the non-limiting examples provided in Table 1.
1TABLE 1 41 42 ID X.sup.1 Y.sup.1 R.sup.1 R.sup.1' R.sup.2 R.sup.2'
R.sup.3 R.sup.3' AA NH.sub.2 O H H OH H H OH AB NH.sub.2 O H H OH H
H I AC NH.sub.2 O H H OH H H Cl AD NH.sub.2 O H H OH H H Br AE
NH.sub.2 O H H OH H H S--CN AF NH.sub.2 O H H OH H H N.sub.3 AG
NH.sub.2 O H H H Cl H OH AH NH.sub.2 O H H H Br H OH AI NH.sub.2 O
H H H OH Br H AJ NH.sub.2 O H H H OH H H AK NH.sub.2 O H H H OH
O--Ms H AL NH.sub.2 O H H H OH O--Ts H AM NH.sub.2 O H H O--Ms H H
OH AN NH.sub.2 O H H Cl H H OH AO NH.sub.2 O D D OH H H OH AP
NH.sub.2 O F H OH H H OH AQ NH.sub.2 O F H H OH H OH AR NH.sub.2 O
F H H OH H H AS NH.sub.2 O F H H OH Cl H AT NH.sub.2 O F H H OH Br
H AU NH.sub.2 O F H H Cl H OH AV NH.sub.2 O F H H OH O--Ts H AW
NH.sub.2 O F H H OH O--Ms H AX NH.sub.2 O Cl H H OH O--Ms H AY
NH.sub.2 O Br H H OH O--Ms H AZ NH.sub.2 O Br H H OH O--Ts H BA
NH.sub.2 O Br H H OH Cl H BB NH.sub.2 O Br H H OH H OH BC NH.sub.2
O Br H OH H H OH BD NH.sub.2 O I H H OH O--Ms H BE NH.sub.2 O I H H
OH Br H BF NH.sub.2 O I H H OH O--Ts H BG NH.sub.2 O I H H Cl H OH
BH NH.sub.2 O I H Br H H OH BI NH.sub.2 O OH H OH H H OH BJ
NH.sub.2 O NH.sub.2 H H OH H OH BK NH.sub.2 O CH.sub.3 H H OH Cl H
BL NH.sub.2 NH H H OH H H OH BM NH.sub.2 S H H H Se-phenyl H H BN
NH-(2-Ph--Et) O H H OH H H OH BO NH--COCH.sub.3 O H H OH H H OH BP
NH--NH.sub.2 O H H OH H H OH BQ NH--NH.sub.2 O F H OH H H OH BR
NH--NH.sub.2 O CH.sub.3 H H OH H OH BS NH--OH O H H H OH H OH BT
NH--OH O F H H OH H OH BU NH--OH O Br H H OH H OH BV NH--OH O I H H
OH H OH BW NH--OH O H H OH H H OH BX OH O OH H OH H H OH BY OH O
NH.sub.2 H H OH H OH BZ OH O F H OH H H OH CA OH O F H H O--Ts H OH
CB OH O F H H O--Ms H O--Ms CC OH O F H H OH H OH CD OH O F H H OH
H O--Ts CE OH O F H H H H OH CF O--Et O H H H O--Bz H O--Bz CG
S--CH.sub.3 O H H H F H OH CH SH O H H H OH H OH CI SH O F H H OH H
OH CJ N.sub.3 O H H H H H H CK NH-(2-Ph--Et) O H H H OH H OH CL OH
O OH H H OH H OH CM OH O H H H OH H H
[0342] In a preferred embodiment, the .beta.-D and .beta.-L
nucleosides of general formula (I-b) and (III-b) are represented by
the non-limiting examples provided in Table 2.
2TABLE 2 43 44 ID X.sup.1 X.sup.2 W.sup.1 R.sup.2 R.sup.2' R.sup.3
R.sup.3' DA OH NH.sub.2 N H OH H OH DB OH NH.sub.2 CH F H H OH DC
NH-cyclohexyl H CH H H H H DD NH.sub.2 H CH H OH H F DE NH.sub.2 H
CH H H H H DF NH.sub.2 NH.sub.2 N H OH H OH DG NH.sub.2 NH.sub.2 CH
H OH H OH DH Cl H CH F H H H DI Cl I CH H O--Ac H O--Ac DJ Cl H CH
H OH H OH DK NH.sub.2 H CH H OH H H DL Cl H CH H OH H H
[0343] In a preferred embodiment, the .beta.-D and .beta.-L
nucleosides of general formula (II-a) and (IV-a) are represented by
the non-limiting examples provided in Table 3.
3TABLE 3 45 46 ID X.sup.1 Y.sup.1 R.sup.1 R.sup.1' R.sup.2 R.sup.3
EA NH--Bz-(m-NO.sub.2) O F H H H EB NH--Bz-(o-NO.sub.2) O F H H H
EC NH.sub.2 O F H F H
[0344] In a preferred embodiment, the .beta.-D and .beta.-L
nucleosides of general formula (II-b) and (IV-b) are represented by
the non-limiting examples provided in Table 4.
4TABLE 4 47 48 ID X.sup.1 X.sup.2 W.sup.1 R.sup.2 R.sup.3 FA Cl H
CH F H FB OH H CH H H FC NH.sub.2 F CH H H FD NH.sub.2 F CH F H FE
NH.sub.2 H CH H H FF OH NH.sub.2 CH H H FG OH H CH H H
[0345] In a preferred embodiment, the .beta.-D and .beta.-L
nucleosides of general formula (V-a) and (VIII-a) are represented
by the non-limiting examples provided in Table 5.
5TABLE 5 49 50 ID X.sup.1 Y.sup.1 R.sup.1 R.sup.1' R.sup.2 R.sup.2'
R.sup.3 R.sup.3' GA NH.sub.2 O F H H OH H OH GB OH H CH.sub.3 H H H
H H GC OH O H H H H H H GD NH.sub.2 O H H H OH H OH GE NH.sub.2 O H
H H H H H GF OH O F H H OH H OH GG NH.sub.2 O I H H H H H GH
NH.sub.2 O I H H OH H OH GI NH.sub.2 O Cl H H OH H OH
[0346] In a preferred embodiment, the .beta.-D and .beta.-L
nucleosides of general formula (VII-a) and (X-a) are represented by
the non-limiting examples provided in Table 6.
6TABLE 6 51 52 ID X.sup.1 Y.sup.1 R.sup.1 R.sup.1' R.sup.2 R.sup.2'
R.sup.3 R.sup.3' HA NH.sub.2 O H H H OH H OH HB NH.sub.2 O F H H OH
H OH HC NH--OH O H H H OH H OH
[0347] In a preferred embodiment, the .beta.-D and .beta.-L
nucleosides of general formula (VII-b) and (X-b) are represented by
the non-limiting examples provided in Table 7.
7TABLE 7 53 54 ID X.sup.1 X.sup.2 W.sup.1 R.sup.2 R.sup.2' R.sup.3
R.sup.3' IA NH.sub.2 H CH H OH H OH
[0348] In a preferred embodiment, the .beta.-D or .beta.-L
nucleosides of general formula (XI-a) or (XIII-a) are represented
by the non-limiting examples provided in Table 8.
8TABLE 8 55 56 ID X.sup.1 Y.sup.1 Z.sup.1 Z.sup.2 R.sup.1 R.sup.1'
JA NH.sub.2 O O O H H JB NH.sub.2 O O S F H JC NH.sub.2 O O O F
H
[0349] In a preferred embodiment, the .beta.-L nucleosides of
general formula (XII-b) are represented by the non-limiting
examples provided in Table 9.
9TABLE 9 57 58 ID X.sup.1 X.sup.2 W.sup.1 Z.sup.1 Z.sup.2 KA Cl H
CH O S KB Cl NH.sub.2 CH O S KC NH.sub.2 F CH O S KD OH H CH O
O
[0350] In a preferred embodiment, the .beta.-D nucleosides of
general formula (XIII-a) are represented by the non-limiting
examples provided in Table 10.
10TABLE 10 59 ID Y.sup.2 Y.sup.3 R.sup.1 R.sup.1' R.sup.2 R.sup.2'
R.sup.3 R.sup.3' LA O O F H H OH H OH
[0351] In a preferred embodiment, the .beta.-D nucleosides of
general formula (XIII-c) are represented by the non-limiting
examples provided in Table 11.
11TABLE 11 [XIII-c] 60 ID Y.sup.2 Y.sup.3 R.sup.1 R.sup.1' R.sup.3
R.sup.3' MA O O F H H OH MB O O F H H O--Ms MC NH O H H H O--Ms MD
NH O H H H O--Ac ME NH O H H H OH MF NH O F H H OH MG NH O F H H
O--Ac
[0352] In a preferred embodiment, the .beta.-D nucleosides of
general formula (XIII-d) are represented by the non-limiting
examples provided in Table 12.
12TABLE 12 [XIII-d] 61 ID Y.sup.2 X.sup.3 R.sup.1 R.sup.1' R.sup.2
R.sup.2' R.sup.3 R.sup.3' NA O O--CH.sub.3 H H H O--Ac H O--Ac
[0353] In a preferred embodiment, the .beta.-D nucleosides of
general formula (XIV) are represented by the non-limiting examples
provided in Table 13.
13TABLE 13 [XIV] 62 ID X.sup.1 Y.sup.1 R.sup.1 R.sup.1' R.sup.2
R.sup.2' R.sup.3 R.sup.3' L.sup.1 L.sup.2 OA NH.sub.2 O NH--OH OH
OH H H OH H OH OB OH O O F H OH H OH Cl O--CH.sub.3 OC OH O O H H
OH H OH Br O--CH.sub.3 OD OH O O F H OH H OH Br O--COCH.sub.3 OE OH
O O F H OH H OH Br O--CH.sub.3 OF OH O O F H OH H OH Br O--Et OG OH
O O Cl H OH H OH Br O--CH.sub.3
[0354] In a preferred embodiment, the nucleosides of general
formula (XV-a) are represented by the non-limiting examples
provided in Table 14.
14TABLE 14 [XV-a] 63 ID Y.sup.1 Z.sup.3 R.sup.1 R.sup.1' R.sup.2
R.sup.2' R.sup.3 R.sup.3' PA O O H H H OH H OH
[0355] In a preferred embodiment, the nucleosides of general
formula (XV-b) are represented by the non-limiting examples
provided in Table 15.
15TABLE 15 [XV-b] 64 ID X.sup.1 W.sup.1 Z.sup.3 R.sup.2 R.sup.2'
R.sup.3 R.sup.3' QA NH.sub.2 CH O H OH H OH
[0356] In a preferred embodiment, the nucleosides of general
formula (XVI-a) are represented by the non-limiting examples
provided in Table 16.
16TABLE 16 [XVI-a] 65 ID W.sup.3 Z.sup.4 W.sup.5 W.sup.4 Z.sup.5
R.sup.2 R.sup.2' R.sup.3 R.sup.3' RA CH NCH.sub.3 C--OH N C.dbd.O H
OH H O--Ts RB CH NH C--NH.sub.2 N C.dbd.O H OH H OH RC CH NH
C--NHAc N C.dbd.O H OH H OH RD CH NH C--OH N C.dbd.O H OH H OH RE
CH NCH.sub.3 C--NH.sub.2 N C.dbd.O H OH H OH RF CH NH C--NHBz N
C.dbd.O H OH H OH RG CH C.dbd.O C--NH.sub.2 C--SH NH H OH H OH RH
CH NH C--OH N C.dbd.O H Cl H OH RI CH NH C--NH.sub.2 N C.dbd.O H Br
H OH
[0357] In a preferred embodiment, the nucleosides of general
formula (XVI-c) are represented by the non-limiting examples
provided in Table 17.
17TABLE 17 [XVI-c] 66 ID W.sup.3 Z.sup.4 Z.sup.5 W.sup.4 R.sup.2
R.sup.2' R.sup.3 R.sup.3' SA CH N--CH.sub.3 C.dbd.O N H OH H
O--Ac
[0358] In a preferred embodiment, the nucleosides of general
formula (XVI-d) are represented by the non-limiting examples
provided in Table 18.
18TABLE 18 [XVI-d] 67 ID W.sup.3 Z.sup.4 Z.sup.5 W.sup.4 R.sup.3
R.sup.3' TA CH N C.dbd.NH N H OH
[0359] In a preferred embodiment, the nucleosides of general
formula (XVI-f) are represented by the non-limiting examples
provided in Table 19.
19TABLE 19 [XVI-f] 68 ID X.sup.1 X.sup.2 W.sup.1 R.sup.2 R.sup.2'
R.sup.3 R.sup.3' UA NH.sub.2 H N H OH H OH
[0360] In a preferred embodiment, the nucleosides of general
formula (XVII-d) are represented by the non-limiting examples
provided in Table 20.
20TABLE 20 [XVII-d] 69 ID X.sup.1 X.sup.2 W.sup.1 X.sup.4 X.sup.5
VA NH.sub.2 F CH H OH
[0361] In one embodiment, the nucleoside has an EC.sub.50
(effective concentration to achieve 50% viral inhibition) when
tested in an appropriate cell-based assay, of less than 15
micromolar, and more particularly, less than 10 or 5 micromolar. In
a preferred embodiment, the nucleoside is enantiomerically
enriched.
[0362] II. Stereoisomerism and Polymorphism
[0363] Compounds of the present invention having a chiral center
may exist in and be isolated in optically active and racemic forms.
Some compounds may exhibit polymorphism. The present invention
encompasses racemic, optically-active, polymorphic, or
stereoisomeric form, or mixtures thereof, of a compound of the
invention, which possess the useful properties described herein.
The optically active forms can be prepared by, for example,
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 or by enzymatic resolution.
[0364] As shown below, a nucleoside contains at least two critical
chiral carbon atoms (*). In general, the substituents on the chiral
carbons [the specified purine or pyrimidine base (referred to as
the C1 substituent when using the sugar ring intermediate
numbering) and CH.sub.2O H (referred to as the C4 substituent)] of
the nucleoside can be either cis (on the same side) or trans (on
opposite sides) with respect to the sugar ring system. Both the cis
and trans racemates consist of a pair of optical isomers. Hence,
each compound has four individual stereoisomers. The four
stereoisomers are represented by the following configurations (when
orienting the sugar moiety in a horizontal plane such that the
-O--moiety is in back): (1) cis, with both groups "up", which is
referred to as .beta.-D; (2) the mirror image, i.e., cis, with both
groups "down", which is the mirror image is referred to as
.beta.-L; (3) trans with the C4 substituent "up" and the C1
substituent "down" (referred to as .alpha.-D); and (4) trans with
the C4 substituent "down" and the C1 substituent "up" (referred to
as .alpha.-L). The two cis enantiomers together are referred to as
a racemic mixture of .beta.-enantiomers, and the two trans
enantiomers are referred to as a racemic mixture of
.alpha.-enantiomers. 70
[0365] The four possible stereoisomers of the claimed compounds are
illustrated below. 71
[0366] III. Definitions
[0367] The term "alkyl," as used herein, unless otherwise
specified, refers to a saturated straight, branched, or cyclic,
primary, secondary, or tertiary hydrocarbon, including but not
limited to those of C.sub.1 to C.sub.16, and specifically includes
methyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, isobutyl,
t-butyl, pentyl, cyclopentyl, isopentyl, neopentyl, hexyl,
isohexyl, cyclohexyl, cyclohexylmethyl, 3-methylpentyl,
2,2-dimethylbutyl, and 2,3-dimethylbutyl. The alkyl group can be
optionally substituted with one or more moieties selected from the
group consisting of alkyl, halo, haloalkyl, hydroxyl, carboxyl,
acyl, acyloxy, amino, amido, carboxyl derivatives, alkylamino,
dialkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, azido,
thiol, imine, sulfonic acid, sulfate, sulfonyl, sulfanyl, sulfinyl,
sulfamonyl, ester, carboxylic acid, amide, phosphonyl, phosphinyl,
phosphoryl, phosphine, thioester, thioether, acid halide,
anhydride, oxime, hydrozine, carbamate, phosphonic acid, phosphate,
phosphonate, or any other viable functional group that does not
inhibit the pharmacological activity of this compound, either
unprotected, or protected as necessary, as known to those skilled
in the art, for example, as taught in Greene, et al., Protective
Groups in Organic Synthesis, John Wiley and Sons, Second Edition,
1991, hereby incorporated by reference.
[0368] The term "lower alkyl," as used herein, and unless otherwise
specified, refers to a C.sub.1 to C.sub.4 saturated straight,
branched, or if appropriate, a cyclic (for example, cyclopropyl)
alkyl group, including both substituted and unsubstituted
forms.
[0369] The term "alkylene" or "alkenyl" refers to a saturated
hydrocarbyldiyl radical of straight or branched configuration,
including but not limited to those that have from one to ten carbon
atoms. Included within the scope of this term are methylene,
1,2-ethane-diyl, 1,1-ethane-diyl, 1,3-propane-diyl,
1,2-propane-diyl, 1,3-butane-diyl, 1,4-butane-diyl and the like.
The alkylene group or other divalent moiety disclosed herein can be
optionally substituted with one or more moieties selected from the
group consisting of alkyl, halo, haloalkyl, hydroxyl, carboxyl,
acyl, acyloxy, amino, amido, carboxyl derivatives, alkylamino,
azido, dialkylamino, arylamino, alkoxy, aryloxy, nitro, cyano,
sulfonic acid, thiol, imine, sulfonyl, sulfanyl, sulfinyl,
sulfamonyl, ester, carboxylic acid, amide, phosphonyl, phosphinyl,
phosphoryl, phosphine, thioester, thioether, acid halide,
anhydride, oxime, hydrozine, carbamate, phosphonic acid,
phosphonate, or any other viable functional group that does not
inhibit the pharmacological activity of this compound, either
unprotected, or protected as necessary, as known to those skilled
in the art, for example, as taught in Greene, et al., Protective
Groups in Organic Synthesis, John Wiley and Sons, Second Edition,
1991, hereby incorporated by reference.
[0370] 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 bromo, chloro,
fluoro, iodo, hydroxyl, azido, amino, alkylamino, arylamino,
alkoxy, aryloxy, nitro, cyano, sulfonic acid, sulfate, phosphonic
acid, phosphate, or phosphonate, either unprotected, or protected
as necessary, as known to those skilled in the art, for example, as
taught in Greene, et al., Protective Groups in Organic Synthesis,
John Wiley and Sons, Second Edition, 1991.
[0371] The term "aralkyl," as used herein, and unless otherwise
specified, refers to an aryl group as defined above linked to the
molecule through an alkyl group as defined above. The term
"alkaryl" or "alkylaryl" as used herein, and unless otherwise
specified, refers to an alkyl group as defined above linked to the
molecule through an aryl group as defined above. In each of these
groups, the alkyl group can be optionally substituted as describe
above and the aryl group can be optionally substituted with one or
more moieties selected from the group consisting of alkyl, halo,
haloalkyl, hydroxyl, carboxyl, acyl, acyloxy, amino, amido, azido,
carboxyl derivatives, alkylamino, dialkylamino, arylamino, alkoxy,
aryloxy, nitro, cyano, sulfonic acid, thiol, imine, sulfonyl,
sulfanyl, sulfinyl, sulfamonyl, ester, carboxylic acid, amide,
phosphonyl, phosphinyl, phosphoryl, phosphine, thioester,
thioether, acid halide, anhydride, oxime, hydrozine, carbamate,
phosphonic acid, phosphonate, or any other viable functional group
that does not inhibit the pharmacological activity of this
compound, either unprotected, or protected as necessary, as known
to those skilled in the art, for example, as taught in Greene, et
al., Protective Groups in Organic Synthesis, John Wiley and Sons,
Second Edition, 1991, hereby incorporated by reference.
Specifically included within the scope of the term aryl are phenyl;
naphthyl; phenylmethyl; phenylethyl; 3,4,5-trihydroxyphenyl;
3,4,5-trimethoxyphenyl; 3,4,5-triethoxy-phenyl; 4-chlorophenyl;
4-methylphenyl; 3,5-di-tertiarybutyl-4-hydroxyphenyl;
4-fluorophenyl; 4-chloro-1-naphthyl; 2-methyl-1-naphthylmethyl;
2-naphthylmethyl; 4-chlorophenylmethyl; 4-t-butylphenyl;
4-t-butylphenylmethyl and the like.
[0372] The term "alkylamino" or "arylamino" refers to an amino
group that has one or two alkyl or aryl substituents,
respectively.
[0373] The term "halogen," as used herein, includes fluorine,
chlorine, bromine and iodine.
[0374] The term "enantiomerically enriched" is used throughout the
specification to describe a nucleoside which includes at least
about 95%, preferably at least 96%, more preferably at least 97%,
even more preferably, at least 98%, and even more preferably at
least about 99% or more of a single enantiomer of that nucleoside.
When a nucleoside of a particular configuration (D or L) is
referred to in this specification, it is presumed that the
nucleoside is an enantiomerically enriched nucleoside, unless
otherwise stated.
[0375] The term "host," as used herein, refers to a unicellular or
multicellular organism in which the virus can replicate, including
cell lines and animals, and preferably a human. Alternatively, the
host can be carrying a part of the viral genome, whose replication
or 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 viral genome and animals,
in particular, primates (including chimpanzees) and humans.
Relative to abnormal cellular proliferation, the term "host" refers
to unicellular or multicellular organism in which abnormal cellular
proliferation can be mimicked. The term host specifically refers to
cells that abnormally proliferate, either from natural or unnatural
causes (for example, from genetic mutation or genetic engineering,
respectively), 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 bovine viral diarrhea virus in cattle,
hog cholera virus in pigs, and border disease virus in sheep).
[0376] The term "pharmaceutically acceptable salt or prodrug" is
used throughout the specification to describe any pharmaceutically
acceptable form (such as an ester, phosphate ester, salt of an
ester or a related group) of a compound which, upon administration
to a patient, provides the active compound. Pharmaceutically
acceptable salts include those derived from pharmaceutically
acceptable inorganic or organic bases and acids. Suitable salts
include those derived from alkali metals such as potassium and
sodium, alkaline earth metals such as calcium and magnesium, among
numerous other acids well known in the pharmaceutical art.
Pharmaceutically acceptable prodrugs refer to a compound that is
metabolized, for example hydrolyzed or oxidized, in the host to
form the compound of the present invention. Typical examples of
prodrugs include compounds that have biologically labile protecting
groups on a functional moiety of the active compound. Prodrugs
include compounds that can be oxidized, reduced, aminated,
deaminated, hydroxylated, dehydroxylated, hydrolyzed, dehydrolyzed,
alkylated, dealkylated, acylated, deacylated, phosphorylated,
dephosphorylated to produce the active compound.
[0377] IV. Pharmaceutically Acceptable Salts and Prodrugs
[0378] In cases where compounds are sufficiently basic or acidic to
form stable nontoxic acid or base salts, administration of the
compound as a pharmaceutically acceptable salt may be appropriate.
Pharmaceutically acceptable salts include those derived from
pharmaceutically acceptable inorganic or organic bases and acids.
Suitable salts include those derived from alkali metals such as
potassium and sodium, alkaline earth metals such as calcium and
magnesium, among numerous other acids well known in the
pharmaceutical art. In particular, examples of pharmaceutically
acceptable salts are organic acid addition salts formed with acids,
which form a physiological acceptable anion, for example, tosylate,
methanesulfonate, acetate, citrate, malonate, tartarate, succinate,
benzoate, ascorbate, a-ketoglutarate, and a-glycerophosphate.
Suitable inorganic salts may also be formed, including, sulfate,
nitrate, bicarbonate, and carbonate salts.
[0379] 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.
[0380] Any of the nucleosides described herein can be administered
as a nucleotide prodrug to increase the activity, bioavailability,
stability or otherwise alter the properties of the nucleoside. A
number of nucleotide prodrug ligands are known. In general,
alkylation, acylation or other lipophilic modification of the mono,
di or triphosphate of the nucleoside will increase the stability of
the nucleotide. Examples of substituent groups that can replace one
or more hydrogens on the phosphate moiety are alkyl, aryl,
steroids, carbohydrates, including sugars, 1,2-diacylglycerol and
alcohols. Many are described in R. Jones and N. Bischofberger,
Antiviral Research, 27 (1995) 1-17. Any of these can be used in
combination with the disclosed nucleosides to achieve a desired
effect.
[0381] The active nucleoside can also be provided as a
5'-phosphoether lipid or a 5'-ether lipid, as disclosed in the
following references, which are incorporated by reference herein:
Kucera, L. S., N. Iyer, E. Leake, A. Raben, Modest E. K., D. L. W.,
and C. Piantadosi. 1990. "Novel membrane-interactive ether lipid
analogs that inhibit infectious HIV-1 production and induce
defective virus formation." AIDS Res. Hum. Retro Viruses.
6:491-501; Piantadosi, C., J. Marasco C. J., S. L. Morris-Natschke,
K. L. Meyer, F. Gumus, J. R. Surles, K. S. Ishaq, L. S. Kucera, N.
Iyer, C. A. Wallen, S. Piantadosi, and E. J. Modest. 1991.
"Synthesis and evaluation of novel ether lipid nucleoside
conjugates for anti-HIV activity." J. Med. Chem. 34:1408.1414;
Hosteller, K. Y., D. D. Richman, D. A. Carson, L. M. Stuhmiller, G.
M. T. van Wijk, and H. van den Bosch. 1992. "Greatly enhanced
inhibition of human immunodeficiency virus type I replication in
CEM and HT4-6C cells by 3'-deoxythymidine diphosphate
dimyristoylglycerol, a lipid prodrug of 3.beta.-deoxythymidine."
Antimicrob. Agents Chemother. 36:2025.2029; Hosetler, K. Y., L. M.
Stuhmiller, H. B. Lenting, H. van den Bosch, and D. D. Richman,
1990. "Synthesis and antiretroviral activity of phospholipid
analogs of azidothymidine and other antiviral nucleosides." J.
Biol. Chem. 265:61127.
[0382] Nonlimiting examples of U.S. patents that disclose suitable
lipophilic substituents that can be covalently incorporated into
the nucleoside, preferably at the 5'-OH position of the nucleoside
or lipophilic preparations, include U.S. Pat. Nos. 5,149,794 (Sep.
22, 1992, Yatvin et al.); 5,194,654 (Mar. 16, 1993, Hostetler et
al., 5,223,263 (Jun. 29, 1993, Hostetler et al.); 5,256,641 (Oct.
26, 1993, Yatvin et al.); 5,411,947 (May 2, 1995, Hostetler et
al.); 5,463,092 (Oct. 31, 1995, Hostetler et al.); 5,543,389 (Aug.
6, 1996, Yatvin et al.); 5,543,390 (Aug. 6, 1996, Yatvin et al.);
5,543,391 (Aug. 6, 1996, Yatvin et al.); and 5,554,728 (Sep. 10,
1996; Basava et al.), all of which are incorporated herein by
reference. Foreign patent applications that disclose lipophilic
substituents that can be attached to the nucleosides of the present
invention, or lipophilic preparations, include WO 89/02733, WO
90/00555, WO 91/16920, WO 91/18914, WO 93/00910, WO 94/26273, WO
96/15132, EP 0 350 287, EP 93917054.4, and WO 91/19721.
[0383] V. Pharmaceutical Compositions
[0384] Pharmaceutical compositions based upon a .beta.-D or
.beta.-L compound of formula (I)-(XXIII) or its pharmaceutically
acceptable salt or prodrug can be prepared in a therapeutically
effective amount for treating a Flaviviridae, Orthomyxoviridae or
Paramyxoviridae viral infection or abnormal cellular proliferation,
optionally in combination with a pharmaceutically acceptable
additive, carrier or excipient. The therapeutically effective
amount may vary with the infection or condition to be treated, its
severity, the treatment regimen to be employed, the
pharmacokinetics of the agent used, as well as the patient
treated.
[0385] In one aspect according to the present invention, the
compound according to the present invention is formulated
preferably in admixture with a pharmaceutically acceptable carrier.
In general, it is preferable to administer the pharmaceutical
composition in orally administrable form, but formulations may be
administered via parenteral, intravenous, intramuscular,
transdermal, buccal, subcutaneous, suppository or other route.
Intravenous and intramuscular formulations are preferably
administered in sterile saline. One of ordinary skill in the art
may modify the formulation within the teachings of the
specification to provide numerous formulations for a particular
route of administration without rendering the compositions of the
present invention unstable or compromising its therapeutic
activity. In particular, a modification of a desired compound to
render it more soluble in water or other vehicle, for example, may
be easily accomplished by routine modification (salt formulation,
esterification, etc.).
[0386] In certain pharmaceutical dosage forms, the prodrug form of
the compound, especially including acylated (acetylated or other)
and ether derivatives, phosphate esters and various salt forms of
the present compounds, is preferred. One of ordinary skill in the
art will recognize how to readily modify the present compound to a
prodrug form to facilitate delivery of active compound to a
targeted site within the host organism or patient. The artisan also
will take advantage of favorable pharmacokinetic parameters of the
prodrug form, where applicable, in delivering the desired compound
to a targeted site within the host organism or patient to maximize
the intended effect of the compound in the treatment of
Flaviviridae (including HCV), Orthomyxoviridae (including Influenza
A and B), Paramyxoviridae (including RSV) infections or conditions
related to abnormal cellular proliferation.
[0387] The amount of compound included within therapeutically
active formulations, according to the present invention, is an
effective amount for treating the infection or condition, in
preferred embodiments, a Flaviviridae (including HCV),
Orthomyxoviridae (including Influenza A and B), Paramyxoviridae
(including RSV) infection or a condition related to abnormal
cellular proliferation. In general, a therapeutically effective
amount of the present compound in pharmaceutical dosage form
usually ranges from about 0.1 mg/kg to about 100 mg/kg or more,
depending upon the compound used, the condition or infection
treated and the route of administration. For purposes of the
present invention, a prophylactically or preventively effective
amount of the compositions, according to the present invention,
falls within the same concentration range as set forth above for
therapeutically effective amount and is usually the same as a
therapeutically effective amount.
[0388] Administration of the active compound may range from
continuous (intravenous drip) to several oral administrations per
day (for example, Q.I.D., B.I.D., etc.) and may include oral,
topical, parenteral, intramuscular, intravenous, subcutaneous,
transdermal (which may include a penetration enhancement agent),
buccal and suppository administration, among other routes of
administration. Enteric-coated oral tablets may also be used to
enhance bioavailability and stability of the compounds from an oral
route of administration. The most effective dosage form will depend
upon the pharmacokinetics of the particular agent chosen, as well
as the severity, of disease in the patient. Oral dosage forms are
particularly preferred, because of ease of administration and
prospective favorable patient compliance.
[0389] To prepare the pharmaceutical compositions according to the
present invention, a therapeutically effective amount of one or
more of the compounds according to the present invention is
preferably mixed with a pharmaceutically acceptable carrier
according to conventional pharmaceutical compounding techniques to
produce a dose. A carrier may take a wide variety of forms
depending on the form of preparation desired for administration,
e.g., oral or parenteral. In preparing pharmaceutical compositions
in oral dosage form, any of the usual pharmaceutical media may be
used. Thus, for liquid oral preparations such as suspensions,
elixirs and solutions, suitable carriers and additives including
water, glycols, oils, alcohols, flavoring agents, preservatives,
coloring agents and the like may be used. For solid oral
preparations such as powders, tablets, capsules, and for solid
preparations such as suppositories, suitable carriers and additives
including starches, sugar carriers, such as dextrose, mannitol,
lactose and related carriers, diluents, granulating agents,
lubricants, binders, disintegrating agents and the like may be
used. If desired, the tablets or capsules may be enteric-coated for
sustained release by standard techniques. The use of these dosage
forms may significantly impact the bioavailability of the compounds
in the patient.
[0390] For parenteral formulations, the carrier will usually
comprise sterile water or aqueous sodium chloride solution, though
other ingredients, including those that aid dispersion, also may be
included. Where sterile water is to be used and maintained as
sterile, the compositions and carriers must also be sterilized.
Injectable suspensions may also be prepared, in which case
appropriate liquid carriers, suspending agents and the like may be
employed.
[0391] Liposomal suspensions (including liposomes targeted to viral
antigens) may also be prepared by conventional methods to produce
pharmaceutically acceptable carriers. This may be appropriate for
the delivery of free nucleosides, acyl nucleosides or phosphate
ester prodrug forms of the nucleoside compounds according to the
present invention.
[0392] In particularly preferred embodiments according to the
present invention, the compounds and compositions are used to
treat, prevent or delay the onset of Flaviviridae (including HCV),
Orthomyxoviridae (including Influenza A and B), Paramyxoviridae
(including RSV) infections or conditions related to abnormal
cellular proliferation. Preferably, to treat, prevent or delay the
onset of the infection or condition, the compositions will be
administered in oral dosage form in amounts ranging from about 250
micrograms up to about 1 gram or more at least once a day,
preferably, or up to four times a day. The present compounds are
preferably administered orally, but may be administered
parenterally, topically or in suppository form.
[0393] The compounds according to the present invention, because of
their low toxicity to host cells in certain instances, may be
advantageously employed prophylactically to prevent Flaviviridae
(including HCV), Orthomyxoviridae (including Influenza A and B),
Paramyxoviridae (including RSV) infections or conditions related to
abnormal cellular proliferation or to prevent the occurrence of
clinical symptoms associated with the viral infection or condition.
Thus, the present invention also encompasses methods for the
prophylactic treatment of viral infection, and in particular
Flaviviridae (including HCV), Orthomyxoviridae (including Influenza
A and B), Paramyxoviridae (including RSV) infections or of a
condition related to abnormal cellular proliferation. In this
aspect, according to the present invention, the present
compositions are used to prevent or delay the onset of a
Flaviviridae (including HCV), Orthomyxoviridae (including Influenza
A and B), Parainyxoviridae (including RSV) infection or a condition
related to abnormal cellular proliferation. This prophylactic
method comprises administration to a patient in need of such
treatment, or who is at risk for the development of the virus or
condition, an amount of a compound according to the present
invention effective for alleviating, preventing or delaying the
onset of the viral infection or condition. In the prophylactic
treatment according to the present invention, it is preferred that
the antiviral or antiproliferative compound utilized should be low
in toxicity and preferably non-toxic to the patient. It is
particularly preferred in this aspect of the present invention that
the compound that is used should be maximally effective against the
virus or condition and should exhibit a minimum of toxicity to the
patient. In the case of Flaviviridae (including HCV),
Orthomyxoviridae (including Influenza A and B), Paramyxoviridae
(including RSV) infections or conditions related to abnormal
cellular proliferation, compounds according to the present
invention, which may be used to treat these disease states, may be
administered within the same dosage range for therapeutic treatment
(i.e., about 250 micrograms up to 1 gram or more from one to four
times per day for an oral dosage form) as a prophylactic agent to
prevent the proliferation of a Flaviviridae (including HCV),
Orthomyxoviridae (including Influenza A and B), Paramyxoviridae
(including RSV) infections or conditions related to abnormal
cellular proliferation, or alternatively, to prolong the onset of a
Flaviviridae (including HCV), Orthomyxoviridae (including Influenza
A and B), Paramyxoviridae (including RSV) infections or conditions
related to abnormal cellular proliferation, which manifests itself
in clinical symptoms.
[0394] In addition, compounds according to the present invention
can be administered in combination or alternation with one or more
antiviral, anti-HBV, anti-HCV or anti-herpetic agent or interferon,
anti-cancer or antibacterial agents, including other compounds of
the present invention. Certain compounds according to the present
invention may be effective for enhancing the biological activity of
certain agents according to the present invention by reducing the
metabolism, catabolism or inactivation of other compounds and as
such, are co-administered for this intended effect.
[0395] This invention is further illustrated in the following
sections. The Experimental Details section and Examples contained
therein are set forth to aid in an understanding of the invention.
This section is not intended to, and should not be interpreted to,
limit in any way the invention set forth in the claims that follow
thereafter.
[0396] VI. Therapies for the Treatment of Flaviviridae
Infection
[0397] It has been recognized that 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 the viral replication cycle, and
most typically in the case of HCV, the RNA-dependent-RNA
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 stresses on the virus.
[0398] Examples of agents that have been identified as active
against the hepatitis C virus, and thus can be used in combination
or alternation with one or more nucleosides of general formula
(I)-(XXIII) include:
[0399] (a) interferon and ribavirin (Battaglia, A. M. et al. Ann.
Pharmacother. 2000, 34, 487; Berenguer, M. et al. Antivir. Ther.
1998, 3 (Suppl. 3), 125);
[0400] (b) Substrate-based NS3 protease inhibitors (Attwood et al.
PCT WO 98/22496, 1998; Attwood et al. Antiviral Chemistry and
Chemotherapy 1999, 10, 259,; Attwood et al. German Patent
Publication DE 19914474; Tung et al. PCT WO 98/17679), including
alphaketoamides and hydrazinoureas, and inhibitors that terminate
in an electrophile such as a boronic acid or phosphonate
(Llinas-Brunet et. al. PCT WO 99/07734);
[0401] (c) Non-substrate-based inhibitors such as
2,4,6-trihydroxy-3-nitro- -benzamide derivatives (Sudo K. et al.,
Biochemical and Biophysical Research Communications, 1997, 238, 643
and 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
apara-phenoxyphenyl group;
[0402] (d) Thiazolidine derivatives which show relevant inhibition
in a reverse-phase HPLC assay with an NS3/4A fusion protein anti
NS5A/5B substrate (Sudo K. et al. Antiviral Research 1996, 32, 9),
especially compound RD-1-6250, possessing a fused cinnamoyl moiety
substituted with a long alkyl chain, RD4 6205 and RD4 6193;
[0403] (e) Thiazolidines and benzanilides identified in Kakiuchi N.
et al. J. EBS Letters 421, 217 and Takeshita N. et al. Analytical
Biochemistry 1997, 247, 242;
[0404] (f) A phenanthrenequinone possessing activity against HCV
protease in a SDS-PAGE and autoradiography assay isolated from the
fermentation culture broth of Streptomyces sp., Sch 68631 (Chu M.
et al. Tetrahedron Letters 1996, 37, 7229), and Sch 351633,
isolated from the fungus Penicillium griscofuluum, which
demonstrates activity in a scintillation proximity assay (Chu M. et
al., Bioorganic and Medicinal Chemistry Letters 9, 1949);
[0405] (g) Selective NS3 inhibitors based on the macromolecule
elgin c, isolated from leech (Qasim M. A. et al. Biochemistry 1997,
36,1598);
[0406] (h) HCV helicase inhibitors (Diana G. D. et al, U.S. Pat.
No. 5,633,358 and Diana G.
[0407] D. et al. PCT WO 97/36554);
[0408] (i) HCV polymerase inhibitors such as nucleotide analogues,
gliotoxin (Ferrari R. et al. Journal of Virology 1999, 73, 1649),
and the natural product cerulenin (Lohmann V. et al. Virology 1998,
249, 108);
[0409] (j) Antisense phosphorothioate oligodeoxynucleotides
(S--ODN) complementary to at least a portion of a sequence of the
HCV (Anderson et al. U.S. Pat. No. 6,174,868), and in particular
the sequence stretches in the 5' non-coding region (NCR) (Alt M. et
al. Hepatology 1995, 22, 707), or nucleotides 326-348 comprising
the 3' end of the NCR and nucleotides 371-388 located in the core
coding region of the HCV RNA (Alt M. et al. Archives of Virology
1997, 142, 589 and Galderisi U. et al., Journal of Cellular
Physiology 1999, 81:2151);
[0410] (k) Inhibitors of IRES-dependent translation (Ikeda N et al.
Japanese Patent Pub. JP-08268890; Kai Y. et al. Japanese Patent
Publication JP-10101591);
[0411] (l) Nuclease-resistant ribozymes (Maccjak D. J. et al.,
Hepatology 1999, 30, abstract 995);
[0412] (m) Amantadine, such as rimantadine (Smith, Abstract from
Annual Meeting of the American Gastoenterological Association and
AASLD, 1996);
[0413] (n) Quinolones, such as ofloxacin, ciprofloxacin and
levofloxacin (AASLD Abstracts, Hepatology, October 1994, Program
Issue, 20 (4), pt.2, abstract no. 293);
[0414] (o) Nucleoside analogs (Ismaili et al. WO 01/60315; Storer
WO 01/32153), including 2'-deoxy-L-nucleosides (Watanabe et al. WO
01/34618), and
1-(.beta.-L-ribofuranosyl)-1,2,4-triazole-3-carboxamide
(levovirin.TM.) (Tam WO 01/46212); and
[0415] (p) Other miscellaneous compounds including
I-amino-alkylcyclohexan- es (Gold et al. U.S. Pat. No. 6,034,134),
alkyl lipids (Chojkier et al. U.S. Pat. No. 5,922,757), vitamin E
and other antioxidants (Chojkier et al. U.S. Pat. No. 5,922,757),
squalene, bile acids (Ozeki et al. U.S. Pat. No. 5,846,964),
N-(phosphonoacetyl)-L-aspartic acid, (Diana et al. U.S. Pat. No.
5,830,905), benzenedicarboxamides (Diana et al. U.S. Pat. No.
5,633,388), polyadenylic acid derivatives (Wang et al. U.S. Pat.
No. 5,496,546), 2',3'-dideoxyinosine (Yarchoan et al U.S. Pat. No.
5,026,687), benzimidazoles (Colacino et al. U.S. Pat. No.
5,891,874), glucamines (Mueller et al. WO 01/08672),
substituted-1,5-imino-D-glucitol compounds (Mueller et al. WO
00/47198).
[0416] VII. Therapies for the Treatment of Orthomyxoviridae
Infection
[0417] It has been recognized that drug-resistant variants of
influenza 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 the viral replication cycle,
resulting in antigenic shifts or drifts. It has been demonstrated
that the efficacy of a drug against influenza infection can be
prolonged, augmented, or restored by administering the compound in
combination or alternation with a second, and perhaps third,
antiviral compound that induces a different mutation from that
caused by the principle drug. Alternatively, the pharmacokinetics,
biodistribution or other parameter of the drug can be altered by
such combination or alternation therapy. In general, combination
therapy is typically preferred over alternation therapy because it
induces multiple simultaneous stresses on the virus.
[0418] Examples of agents that have been identified as active
against the influenza virus, and thus can be used in combination or
alternation with one or more nucleosides of general
formula(I)-(XXIII) include:
[0419] (a) actinomycin D (Barry, R. D. et al. "Participation of
deoxyribonucleic acid in the multiplication of influenza virus"
Nature, 1962, 194, 1139-1140);
[0420] (b) amantadine (Van Voris, L. P. et al. "Antivirals for the
chemoprophylaxis and treatment of influenza" Semin Respir Infect,
1992, 7, 61-70);
[0421] (c) 4-amino- or
4-guanidino-2-deoxy-2,3-didehydro-D-N-acetylneuroam- inic
acid-4-amino- or 4-guanidino-Neu 5 Ac2en (von Itzstein, M. et al.
"Rational design of potent sialidase-based inhibitors of influenza
virus replication" Nature, 1993, 363, 418-423);
[0422] (d) ribavirin (Van Voris, L. P. et al. "Antivirals for the
chemoprophylaxis and treatment of influenza" Semin Respir Infect,
1992, 7, 61-70);
[0423] (e) interferon (Came, P. E. et al. "Antiviral activity of an
interferon-inducing synthetic polymer" Proc Soc Exp Biol Med, 1969,
131, 443-446; Gerone, P. J. et al. "Inhibition of respiratory virus
infections of mice with aeresols of synthetic double-stranded
ribonucleic acid" Infect Immun, 1971, 3, 323-327; Takano, K. et al.
"Passive interferon protection in mouse influenza" J Infect Dis,
1991, 164, 969-972);
[0424] (f) inactivated influenza A and B virus vaccines ("Clinical
studies on influenza vaccine -1978" Rev Infect Dis, 1983, 5,
721-764; Galasso, G. T. et al. "Clinical studies on influenza
vaccine-1976" J Infect Dis, 1977, 136 (suppl), S341-S746; Jennings,
R. et al. "Responses of volunteers to inactivated influenza virus
vaccines" J Hyg, 1981, 86, 1-16; Kilbourne, E. D. "Inactivated
influenza vaccine" In: Plothin S A, Mortimer E A, eds. Vaccines
Philadelphia: Saunders, 1988, 420-434; Meyer, H. M., Jr. et al.
"Review of existion vaccines for influenza" Am J Clin Pathol, 1978,
70, 146-152; "Mortality and Morbidity Weekly Report. Prevention and
control of Influenza: Part I, Vaccines. Recommendations of the
Advisory Committee on Immunication Practices (ACIP)" MMWR, 1993, 42
(RR-6), 1-14; Palache, A. M. et al. "Antibody response after
influenza immunization with various vaccine doses: A double-blind,
placebo-controlled, multi-centre, dose-response study in elderly
nursing-home residents and young volunteers" Vaccine, 1993, 11,3-9;
Potter, C. W. "Inactivated influenza virus vaccine" In: Beare A S,
ed. Basic and applied influeza research, Boca Raton, Fla.: CRC
Press, 1982, 119-158).
[0425] VIII. Therapies for the Treatment of Paramyxoviridae
Infection
[0426] It has been recognized that drug-resistant variants of RSV
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 the viral replication cycle. It has been
demonstrated that the efficacy of a drug against RSV infection can
be prolonged, augmented, or restored by administering the compound
in combination or alternation with a second, and perhaps third,
antiviral compound that induces a different mutation from that
caused by the principle drug. Alternatively, the pharmacokinetics,
biodistribution or other parameter of the drug can be altered by
such combination or alternation therapy. In general, combination
therapy is typically preferred over alternation therapy because it
induces multiple simultaneous stresses on the virus.
[0427] Examples of agents that have been identified as active
against RSV, and thus can be used in combination or alternation
with one or more nucleosides of general formula (I)-(XXIII)
include:
[0428] (a) ribavirin (Hruska, J. F. et al. "In vivo inhibition of
respiratory syncytial virus by ribavirin" Antimicrob Agents
Chemother, 1982, 21, 125-130); and
[0429] (b) purified human intravenous IgG-IVIG (Prince, G. A. et
al. "Effectiveness of topically administered neutralizing
antibodies in experimental immunotherapy of respiratory syncytial
virus infection in cotton rats" J Virol, 1987, 61, 1851-1954;
Prince, G. A. et al. "Immunoprophylaxis and immunotherapy of
respiratory syncytial virus infection in cotton rats" Infect Immun,
1982, 42, 81-87).
[0430] IX. Therapies for the Treatment of Abnormal Cellular
Proliferation
[0431] Examples of agents that have been identified as active
against abnormal cellular proliferation, and thus can be used in
combination or alternation with one or more nucleosides of general
formula (I)-(XXIII) include:
[0432] A. Alkylating Agents
[0433] Nitrogen Mustards: Mechlorethamine (Hodgkin's disease,
non-Hodgkin's lymphomas), Cyclophosphamide, Ifosfamide (acute and
chronic lymphocytic leukemias, Hodgkin's disease, non-Hodgkin's
lymphomas, multiple myeloma, neuroblastoma, breast, ovary, lung,
Wilms' tumor, cervix, testis, soft-tissue sarcomas), Melphalan
(L-sarcolysin) (multiple myeloma, breast, ovary), Chlorambucil
(chronic lymphoctic leukemia, primary macroglobulinemia, Hodgkin's
disease, non-Hodgkin's lymphomas).
[0434] Ethylenimines and Methylmelamines: Hexamethylmelamine
(ovary), Thiotepa (bladder, breast, ovary).
[0435] Alkyl Sulfonates: Busulfan (chronic granuloytic
leukemia).
[0436] Nitrosoureas: Carmustine (BCNU) (Hodgkin's disease,
non-Hodgkin's lymphomas, primary brain tumors, multiple myeloma,
malignant melanoma), Lomustine (CCNU) (Hodgkin's disease,
non-Hodgkin's lymphomas, primary brain tumors, small-cell lung),
Semustine (methyl-CCNU) (primary brain tumors, stomach, colon),
Streptozocin (STR) (malignant pancreatic insulinoma, malignant
carcinoin).
[0437] Triazenes: Dacarbazine (DTIC;
dimethyltriazenoimidazole-carboxamide- ) (malignant melanoma,
Hodgkin's disease, soft-tissue sarcomas).
[0438] B. Antimetabolites
[0439] Folic Acid Analogs: Methotrexate (amethopterin) (acute
lymphocytic leukemia, choriocarcinoma, mycosis fungoides, breast,
head and neck, lung, osteogenic sarcoma).
[0440] Pyrimidine Analogs: Fluorouracil (5-fluorouracil; 5-FU)
Floxuridine (fluorodeoxyuridine; FUdR) (breast, colon, stomach,
pancreas, ovary, head and neck, urinary bladder, premalignant skin
lesions) (topical), Cytarabine (cytosine arabinoside) (acute
granulocytic and acute lymphocytic leukemias).
[0441] Purine Analogs and Related Inhibitors: Mercaptopurine
(6-mercaptopurine; 6-MP) (acute lymphocytic, acute granulocytic and
chronic granulocytic leukemia), Thioguanine (6-thioguanine: TG)
(acute granulocytic, acute lymphocytic and chronic granulocytic
leukemia), Pentostatin (2'-deoxycyoformycin) (hairy cell leukemia,
mycosis fungoides, chronic lymphocytic leukemia).
[0442] Vinca Alkaloids: Vinblastine (VLB) (Hodgkin's disease,
non-Hodgkin's lymphomas, breast, testis), Vincristine (acute
lymphocytic leukemia, neuroblastoma, Wilms' tumor,
rhabdomyosarcoma, Hodgkin's disease, non-Hodgkin's lymphomas,
small-cell lung).
[0443] Epipodophylotoxins: Etoposide (testis, small-cell lung and
other lung, breast, Hodgkin's disease, non-Hodgkin's lymphomas,
acute granulocytic leukemia, Kaposi's sarcoma), Teniposide (testis,
small-cell lung and other lung, breast, Hodgkin's disease,
non-Hodgkin's lymphomas, acute granulocytic leukemia, Kaposi's
sarcoma).
[0444] C. Natural Products
[0445] Antibiotics: Dactinomycin (actinonmycin D) (choriocarcinoma,
Wilms' tumor rhabdomyosarcoma, testis, Kaposi's sarcoma),
Daunorubicin (daunomycin; rubidomycin) (acute granulocytic and
acute lymphocytic leukemias), Doxorubicin (soft tissue, osteogenic,
and other sarcomas; Hodgkin's disease, non-Hodgkin's lymphomas,
acute leukemias, breast, genitourinary thyroid, lung, stomach,
neuroblastoma), Bleomycin (testis, head and neck, skin and
esophagus lung, and genitourinary tract, Hodgkin's disease,
non-Hodgkin's lymphomas), Plicamycin (mithramycin) (testis,
malignant hypercalcema), Mitomycin (mitomycin C) (stomach, cervix,
colon, breast, pancreas, bladder, head and neck).
[0446] Enzymes: L-Asparaginase (acute lymphocytic leukemia).
[0447] Biological Response Modifiers: Interferon-alfa (hairy cell
leukemia, Kaposi's sarcoma, melanoma, carcinoid, renal cell, ovary,
bladder, non Hodgkin's lymphomas, mycosis fungoides, multiple
myeloma, chronic granulocytic leukemia).
[0448] D. Miscellaneous Agents
[0449] Platinum Coordination Complexes: Cisplatin (cis-DDP)
Carboplatin (testis, ovary, bladder, head and neck, lung, thyroid,
cervix, endometrium, neuroblastoma, osteogenic sarcoma).
[0450] Anthracenedione: Mixtozantrone (acute granulocytic leukemia,
breast).
[0451] Substituted Urea: Hydroxyurea (chronic granulocytic
leukemia, polycythemia vera, essential thrombocytosis, malignant
melanoma).
[0452] Methylhydrazine Derivative: Procarbazine (N-methylhydrazine,
M1H) (Hodgkin's disease).
[0453] Adrenocortical Suppressant: Mitotane (o,p'-DDD) (adrenal
cortex), Aminoglutethimide (breast).
[0454] Adrenorticosteriods: Prednisone (acute and chronic
lymphocytic leukemias, non-Hodgkin's lymphomas, Hodgkin's disease,
breast).
[0455] Progestins: Hydroxprogesterone caproate, Medroxyprogesterone
acetate, Megestrol acetate (endometrium, breast).
[0456] E. Antioangiogenesis Agents
[0457] Angiostatin, Endostatin.
[0458] F. Hormones and Antagonists
[0459] Estrogens: Diethylstibestrol Ethinyl estradiol (breast,
prostate)
[0460] Antiestrogen: Tamoxifen (breast).
[0461] Androgens: Testosterone propionate Fluxomyesterone
(breast).
[0462] Antiandrogen: Flutamide (prostate).
[0463] Gonadotropin-Releasing Hormone Analog: Leuprolide
(prostate).
[0464] X. Synthetic Protocol
[0465] Compounds of formula (I)-(XXIII) can be synthesized by any
means known in the art. In particular, the compounds can be made
via three distinct routes: (a) from a pre-formed nucleoside, (b)
condensation of a modified sugar or unmodified ribose with purine
or pyrimidine, and (c) combination of the two routes. Since the
3-deoxy-D-erythropentofuranose structure is found in the nucleoside
antibiotic, cordycepin, a number of total syntheses of this
antibiotic have been reported during 1960s (see: Lee, W. W. et al.
J. Am. Chem. Soc., 1961, 83, 1906; Walton, E. et al. J. Am. Chem.
Soc., 1964, 86, 2952; Suhadolnik, R. J. et al. Carbohydr. Res.,
1968, 61, 545; Ikehara, M. et al. Chem. Pharm. Bull., 1967, 15, 94;
Kaneko, M. et al. Chem. Pharm. Bull. 1972, 20, 63). In a preferred
embodiment of the invention, preparation of 3'-deoxy nucleosides
from preformed nucleosides are performed in the following ways;
[0466] A. Compounds of Types Ia-c and IIIa-c.
[0467] (i) Synthesis from Pre-Formed Nucleosides:
[0468] From the teachings of Marumoto, R. et al. Chem. Pharm. Bull.
1974, 22, 128 where N4-acetylcytidine is treated with acetyl
bromide to give
2',5'-di-O-acetyl-3'-bromo-3'-deoxy-.beta.-D-xylofuranosyl-cytosine
(2, R=Ac), N.sup.4-protected-cytidine nucleosides can be
derivatized to form pyrimidine nucleosides (I-a) as shown in Scheme
1. 72
[0469] An N.sup.4-protected-D-cytidine nucleoside 1 can be treated
with an acid halide, such as acetyl bromide, to give the
corresponding 3'-halo-xylo-nucleoside 2. Deacetylation of 2 to 3,
followed reductive dehalogenation affords the desired
3'-deoxycytidine derivatives 4. Treatment of 2 with an acid,
preferably boiling aqueous acetic acid, gives the corresponding
protected uracil nucleoside 5, which can be readily converted into
free 3'-bromo-xylo nucleoside 6a, from which 3'-deoxyuridine
derivatives 6b can be obtained by reductive debromination. In a
similar manner, starting from N.sup.4-protected-L-cyt- idine, the
L-enantiomer (III-a) of 4 and 6 can be synthesized.
[0470] In an alternate embodiment for the preparation of
nucleosides I-a, 2', 5'-di-O-tritylation of a ribonucleoside gives
7 (R.sup.2' R.sup.5'=Tr) which is converted into the corresponding
3'-O-mesylates 8 (Scheme 2). Treatment of 8 with diluted potassium
or sodium hydroxide gives the corresponding xylo derivative 10 via
anhydronucleoside 9, which, after de-O-tritylation, affords 12.
Mesylation of 10, followed by de-O-tritylation yields the
3'-O-mesyl xylo-nucleoside. Upon treatment of 8 with lithium
bromide or sodium iodide, the corresponding 3'-deoxy-3'-halogeno
derivative 11 is formed via 9, which, after de-O-tritylation,
followed by hydrogenolysis, is converted into the desired
3'-deoxyuridine derivative 6b. In a similar manner, starting from
an L-ribonucleoside, the L-nucleoside (III-a) counterparts of 4 and
6 are synthesized. 73
[0471] An example for the preparation of type I-b compound, purine
nucleoside, is the synthesis of 3'-deoxypurine nucleosides (Scheme
3). Ribonucleoside 13 is treated with 2-methoxyisobutyryl halide
(X=Cl or Br) to give a mixture of 3'-halogeno-xylo-furanosyl and 2
'-halogeno-arabinofuranosyl derivatives (14 and 15).
Hydrogenolysis, followed by chromatographic separation affords the
corresponding 3'-deoxynucleoside 17 along with the
2'-deoxynucleoside 16. Saponification of 17 gives the desired
3'-deoxynucleoside 20. Treatment of the reaction mixture of 14 and
15 with a base gives the single epoxide 18 in quantitative yield,
which, upon treatment with ammonium or sodium iodide affords
exclusively the 3'-xylo-iodide 19. Hydrogenolysis of 19 affords 20.
Reduction of 18 with a reducing agent such as Raney nickel, lithium
aluminum hydride or sodium borohydride also yields 20.
[0472] In a similar manner, starting from a purine
L-ribonucleoside, the L-nucleoside counterpart of 20, which belongs
to III-b, can be synthesized. 74
[0473] For the synthesis of a compound of formula I-c, the starting
material is a 5-nitropyrimidine or pyridine nucleoside (Scheme 4).
Treatment of 5-nitrouridine (21, vide supra) with azide ion in a
solvent such as alcohol or dimethylformamide at a temperature range
of from 20.degree. C. to 100.degree. C., preferably from 25.degree.
C. to 80.degree. C. Nucleophilic attack of azide ion at C-6 of 21
results in the formation of aci-nitro salt 22 which cyclizes to 23.
Neutralization of 23 furnishes the bicyclic nucleoside 24. 75
[0474] (ii) Synthesis by Condensation of an Appropriate Sugar with
Base.
[0475] The appropriate sugar derivatives must be prepared for
condensation with the selected base. Though there are several
methods for the synthesis of 3-deoxy-D-erythropentofuranose
(3-deoxy-D-ribofuranose) derivatives (see: Lee, W. W. et al. J. Am.
Chem. Soc., 1961, 83, 1906; Walton, E. et al. J. Am. Chem. Soc.,
1964, 86, 2952; Lin, T. -S. et al. J. Med. Chem., 1991, 34, 693;
Ozols, A. M. et al. Synthesis, 1980, 557), new methods were
developed for the present invention as shown in Scheme 5. 76
[0476] 1,2-O-Isopropylidene-5-O-methoxycarbonyl-cc-D-xylo-furanose
(25) is converted into the corresponding 3-thiocarbonyl
derivative26, followed by free radical deoxygenation using
trialkyltin hydride in the presence of a radical initiator, such as
2,2'-azobisisobutyronitrile. The deoxygenated product 27 is
acylated with a mixture of acetic acid, acetic anhydride and
sulfuric acid to give 28, which then is condensed with a silylated
base using Vorbruggen's procedure (see: Niedballa, U. et al. J.
Org. Chem., 1976, 41, 2084; Vorbruggen, H. et al. Chem. Ber., 1981,
114, 1234; Kazinierczuk, Z. et al. J. Am. Chem. Soc., 1984, 106,
6379) to obtain the pyrimidine nucleoside 29 (Type I-a) or a
related purine nucleoside (Type I-b). The 5-OH group can be
alternatively protected with other acyl groups, such as benzoyls,
p-nitrobenzoyls, p-chlorobenzoyls or p-methoxybenzoyls as well as
other silyl groups, such as t-butyldimethylsilyl or t-butyldiphenyl
groups. Similarly, L-xylose can be converted into the L-sugar
counterpart of 25, which can be further derivatized to attain the
L-nucleoside of 30.
[0477] Alternatively, as shown in Scheme 6,
1,2-O-isopropylidene-5-O-(t-bu-
tyldiphenylsilyl)-.alpha.-D-xylofuranose (31) can be sulfonylated
with mesyl chloride, tosyl chloride or tresyl chloride in pyridine
to obtain32. After methanolysis of 32, the methyl xyloside 33 can
be treated with a base, such as sodium methoxide in methanol, to
afford the ribo-epoxide 34. Opening of the epoxide 34 with lithium
aluminum hydride stereoselectively produces 3-deoxy sugar 36.
Treatment of 34 with lithium bromide or sodium iodide in acetone or
2-butanone gives 3-halogeno-3-deoxy xyloside 35. Reductive
dehalogenation of 35 affords 36. Removal of the 5'-silyl protecting
group with a fluoride ion source, such as tri-n-butylammonium
fluoride in tetrahydrofuran or triethylammonium hydrogen fluoride
gives 37. Acylation of 37 with acetic anhydride and acetic acid in
the presence of sulfuric acid gives
tri-O-acetyl-3-deoxy-D-ribofuranose 38. Also, fluoride treatment
converts 33 into 39, which, upon acetylation, affords 40. These
acetylated sugars 38 and 40 can be condensed with
pertrimethylsilylated pyrimidine or purine bases using
Vorbrueggen's procedure to give the 3'-modified nucleoside. The
t-butyldiphenylsilyl protecting group can be replaced by
t-butyldimethylsilyl group. 77
[0478] (iii) Post Synthetic Modifications (1-6)
[0479] (a) Modification at C-4 of Pyrimidine Nucleosides (I-a and
III-a)
[0480] After condensation of 28 or 38 with uracil or 5-substituted
uracil, the protected 3'-deoxyuridine derivative (29,
R.sup.5'.dbd.CH.sub.3OCO, R.sup.2'.dbd.Ac or
R.sup.5'.dbd.R.sup.2'.dbd.Ac) is treated with phosphorus
pentasulfide in pyridine or Lawesson's reagent in toluene to give
4-thiouracil nucleoside 41, which, upon treatment with ammonia, is
converted into 3'-deoxycytidine (43, R.sub.1.dbd.R.sub.2.dbd.H).
Alternatively, methylation of 41 with methyliodide or
dimethylsulfate in base gives the 4-S-methyl derivative 42.
Displacement of the 4-S-methylgroup of 42 with various nucleophiles
affords the corresponding N.sup.4-substituted 3'-deoxycytidines 43.
Also, 29 can be converted into the 4-(triazol-2-yl) derivative 44,
which can be reacted with ammonia or various amines to give 43.
Alternatively, treatment of 44 with various alcohols or phenols
affords the corresponding 4-O-substituted-3'-deoxyuri- dines.
78
[0481] Alternatively, a uracil nucleoside, such as a
sugar-protected uridine 45 (R.dbd.H) i converted into the
4-(methylimidazolium) 46 (Scheme 8) or
4-O-(2,4,6-triisopropylbenzenesulfonyl) intermediate 47 and then
treated with a nucleophile, such as hydroxylamine, to give the
corresponding C-4 modified nucleoside, such as N4-hydroxy-X
cytidine (48, R.dbd.H). 79
[0482] In similar manners starting from the L-nucleoside
counterparts, the corresponding III-a nucleosides are prepared.
[0483] (b) Modification at C-5 of Pyrimidine Nucleosides (I-a and
III-a)
[0484] (i) Halogenation (Scheme 9)
[0485] 3'-Deoxyuridine (6, R.dbd.H) can be fluorinated with
fluorinating agents, some non-limiting examples include fluorine in
acetic acid, selectfluor in an inert solvent or solvents such as
tetrahydrofuran or cesium fluoroxisulfate in alcohol (see: Stovber,
S. et al. J. Chem. Soc. Chem. Commun., 1983, 563), to give
5-fluoro-3'-deoxyuridine (49). The 5-chloro, 5-bromo and
5-iodouridine derivatives (50-52) are obtained using the
appropriate N-halogenosuccinimde. Treatment of 6 with bromine in
water or iodine in acetic acid in the presence of an oxidizing
agent such as nitric acid affords the 5-bromo- or 5-iodo-uracil
nucleoside, respectively. The cytosine derivative 43 (R.dbd.H) also
can be converted into the corresponding 5-halogeno derivative
(44-56). 5-Fluoro-3'-deoxycytidine (53, R.dbd.H) is prepared by
condensing 28 or 38 with 5-fluorocytosine, followed by
saponification. 80
[0486] In similar manners starting from the L-nucleoside
counterparts, the corresponding III-a nucleosides are prepared.
Scheme 10 depicts the conversion of the brominated compound 51 into
5-hydroxy-3'-deoxyuridine (63) by treatment with sodium bicarbonate
solution. Alkylation of 55 with an alkyl iodide with base affords
62. Prolonged reaction of 51 with an alkali metal cyanide gives the
5-cyano-uracil derivative 57, which can be hydrated to
5-carboxamide 58 and 5-carboxylic acid 59. Conversion of 59 into an
alkyl ester 60, followed by reduction with sodium borohydride
yields the 5-hydroxymethyl derivative 61. Compound 60 alternatively
can be treated with dihydropyran and a catalytic amount of acid,
such as hydrochloric, sulfuric or p-toluenesulfonic acid, to yield
the 2',5'-di-O-protected nucleoside 64. Sodium borohydride
reduction of 64 affords 65. Due to allylic nature of 65, treatment
with mesyl chloride or tosyl chloride gives the
5-chloromethyl-uracil derivative 66. Alkoxide treatment of 66,
followed by deprotection gives the corresponding
5-alkoxymethyl-3'-deoxyuridine (69). Similarly, reaction of various
amines with 66 affords 67, which, upon mild acid hydrolysis, is
converted into 68. Reaction with 66 and thiourea gives
mercaptomethyl derivative (70, R.dbd.H), while treatment with
sodium mercaptide gives thioalkyl derivative 70 (R=alkyl), which
can be oxidized with hydrogen peroxide to the corresponding sulfone
(71).In similar manners starting from the L-nucleoside
counterparts, the corresponding III-a nucleosides are prepared.
81
[0487] (ii) Nitration (Scheme 11)
[0488] Treatment of uridine 6 with nitronium tetrafluoroborate in
sulfolane (see: Huang, G. -F. et al. J. Org. Chem., 1977, 42, 3821;
Huang, G. -F. et al. J. Carbohyd. Nucleosides Nucleotides, 1978, 5,
317) affords the corresponding 5-nitro derivative 72. Catalytic
hydrogenation of the nitro-nucleoside 72 gives the corresponding
5-amino derivative 73. Diazotization of 73 with nitrous acid gives
the 5-diazo-3'-deoxyuridine (74), which, upon hydrolysis, can be
converted into the 1,2,3-triazole 75. Similar conversions of
5-aminouridine into ribosilyltriazole have been reported (see:
Roberts, M. et al. J. Am. Chem. Soc., 1952, 74, 668; Thurber, T. C.
et al. J. Am. Chem. Soc., 1973, 95, 3081; J. Org. Chem., 1976, 41,
1041). Reaction of 72 with sodium azide in dimethylformamide
affords the triazolopyrimidine (8-azapurine) nucleoside 76.
[0489] In similar manners starting from the L-nucleoside
counterparts, the corresponding III-a nucleosides are prepared.
82
[0490] A similar sequence of reactions is shown in Scheme 12,
starting from 3'-deoxycytidine 4 gives 5-nitro-3'-deoxycytidine
(77), followed by 5-amino-3'-deoxycytidine (78). However, treatment
of 78 with nitrous acid results in the formation of another
8-azapurine nucleoside 79. The same sequence of reactions can be
applied to the corresponding L-nucleosides III-a. 83
[0491] (iii) Hydroxymethylation
[0492] Treatment of 6 (R.dbd.H,
R.sup.5'.dbd.R.sup.3'.dbd.R.sup.3"H) with formaldehyde in base such
as aqueous potassium hydroxide or sodium hydroxide gives
5-hydroxymethyl-3'-deoxyuridine (80) as shown in Scheme 13, which
is converted into 5-ethoxymethyl-3'-deoxyuridine (81,
X.dbd.OCH.sub.2CH.sub.3) by treatment with ethanolic hydrogen
chloride. Compound 80 (R.sup.5'=R.sup.3'.dbd.TBDPS) can also be
prepared from the thymine derivative 6 (R.dbd.CH.sub.3,
R.sup.5.dbd.R.sup.3.dbd.TBDPS) by photochemical bromination to 81
(X.dbd.Br), followed by hydrolysis (Matulic-Adamic, J. et al. Chem.
Pharm. Bull., 1988, 36, 1554). Compound 80 is converted into
5-chloromethyl derivative (81, X.dbd.Cl) by action of hydrochloric
acid or 5-fluoromethyl derivative (81, X.dbd.F) by treatment with
diethylaminosulfur trifluoride (DAST). Oxidation of 80
(R.sup.5'.dbd.R.sup.2.dbd.TBDPS, R.sup.3".dbd.H) with manganese
dioxide affords the 5-formyl derivative 82, which is a good
substrate for various reactions including Wittig, Wittig-Horner,
Grignard or Reformatsky reaction. For example, treatment of 82 with
ethoxymethylene triphenylphosphorane [EtOC(.dbd.O)CH.dbd.PPh.sub.3]
gives 5-(2-ethoxycarbonyl)ethylene-3'-deoxyuridine derivative (83),
which can be converted into 5-ethylene-, 5-(2-chloroethylene)- or
5-(2-bromoethylene)-3'-deoxyuridine derivative (85) by way of the
5-(ethylene-2-carboxylic acid) derivative 84. 5-Difluoromethyl
derivative 86 can be obtained by treatment of 82 with DAST. These
synthetic pathways are shown in Scheme 13.
[0493] The same sequence of reactions can be applied to the
corresponding L-nucleosides III-a. 84
[0494] (iv) Metallation
[0495] In aqueous buffer, 6 or 4 can be treated with mercuric
acetate, followed by sodium chloride, to give the corresponding
5-chloromercuri derivative 87 or 91, respectively (Scheme 14), in
quantitative yield. Reaction of 87 or 91 with iodine in ethanol
gives the 5-iodo derivative 52 or 56, respectively. Compound 52 can
be converted to 5-ethynyl derivatives 88 by reaction with 1-alkynes
and bis(triphenylphosphine)pall- adium chloride
(Ph.sub.3P).sub.2PdCl.sub.2 in the presence of cuprous iodide and
triethylamine. Treatment with trifluoroiodomethane and powdered
copper, on the other hand, converts 52 into
5-trifluoromethyl-3'-deoxyuridine 89. Treatment of 87 with lithium
paladium chloride (Li.sub.2PdCl.sub.4) and alyl chloride affords
5-allyl-3'-deoxyuridine (90). Methyl acrylate reacts with 87 or 91
in the presence of Li.sub.2PdCl.sub.2 to give
5-(E)-(2-methoxy-carbonyl)vinyl-3'- -deoxyuridine (83) or -cytidine
(92), respectively. Saponification of 83 to 84, followed by
N-halogenosuccinimide yields 5-(E)-halogenovinyluracil nucleoside
85 (X.dbd.Cl, Br or J). Thermal decarboxylation of 84 gives
5-vinyluracil derivative 85 (X=H). Compound 85 (X.dbd.H) c an also
be prepared by treatment of 52 with vinyl acetate in the presence
of palladium acetate-triphenylphosphine complex. Similarly, 91 can
be converted into the corresponding acrylate derivative 92, which,
after hydrolysis to 93, is reacted with N-halogenosuccinimide to
give 5-(E)-(2-halogenovinyl)-3'-deoxycytidines (94). It should be
noted that catalytic hydrogenation of 5-vinyl derivatives gives the
corresponding 5ethyl-pyrimidine nucleosides. Hydration of
5-ethynyl-3'-deoxyuridine (88, R.dbd.H) with diluted sulfuric acid
gives 5-acetyl-3'-deoxyuridine in high yield.
[0496] In a similar manner but starting from the L-nucleoside
counterparts, the corresponding III-a nucleosides are prepared.
85
[0497] (c) Modification at C-6 of Pyrimidine Nucleosides (I-a and
III-a)
[0498] Treatment of 5-bromo-3'-deoxyuridine (51, Scheme 15) with
sodium or potassium cyanide in dimethylformamide at room
temperature affords 6-cyano-3'-deoxyuridine (95) in high yield.
Further treatment at elevated temperature converts 95 into the
5-cyano isomer 59. Hydrolysis of 95 furnishes 3'-deoxyorotidine 96.
Methanolysis of 95 gives the methyl ester 97, which, upon
amminolysis, is converted into 98, wherein R' is lower alkyl of
from C.sub.1 to C.sub.6 or benzyl or phenyl group. Reduction of 97
with sodium borohydride affords 6-hydroxymethyl derivative 99,
which is converted into 6-chloromethyluracil nucleoside 100 by
action of hydrochloric acid. Reaction with various amines, 100 is
converted into the corresponding 6-aminomethyl-3'-deoxyuridine
(101). A similar sequence of reactions starting from
3'-deoxycytidine (55) gives 3'-deoxycytidin-6-yl-carboxylic acid
(103) or its methyl ester 104 via the 6-cyano intermediate 102.
Various 6-carbox-amidocytosine nucleosides 105 can be obtained by
treatment of 104 with the corresponding amines. Borohydride
reduction of 104 affords 6-hydroxymethyl derivative 104 which can
be converted into 6-chloromethyl-3'-deoxycytidine 107 by action of
hydrochloric acid. Compound 107 can be converted into the
corresponding 6-aminomethyl-3'-deoxcytidine (108) by reaction with
various amines. The same sequence of reactions can be applied to
the corresponding L-nucleosides III-a. 86
[0499] Further derivatization is shown in Scheme 16. Lithiation of
uracil and cytosine nucleosides occurs at C-6.(see: Tanaka, H. et
al. Tetrahedron Lett., 1979, 4755; Sergueeva, Z. A. et al.
Nucleosides Nucleotides Nucleic Acids, 2000, 19, 275) Thus,
treatment of fully trimethylsilylated 3'-deoxycytidine (109,
R.sup.3'.dbd.R.sup.3".dbd.H) with n-butyllithium at -45.degree. C.,
followed by treatment with methyl iodide or carbon dioxide, gives
6-methyl-3'-deoxycytidine (110) or 3'-deoxycytidine-6-carboxylic
acid (103), respectively. In a similar manner but starting from the
L-nucleoside counterparts, the corresponding III-a nucleosides are
prepared. 87
[0500] Treatment of
2',5'-di-O-(tetrahydropyran-2-yl)-3'-deoxyuridine (6,
R.sup.2'.dbd.R.sup.5'.dbd.THP, R.sup.3'.dbd.R.sup.3".dbd.H) with
lithium diusopropylamide in tetrahydrofuran at -78.degree. C. and
subsequent reaction with alkyl halide result in the formation of
6-alkyl-3'-deoxyuridines (111). Oxidation of 111 (n=0) with
selenium dioxide gives 3'-deoxyuridine-6-carboxaldehyde (112),
which, upon treatment with nitromethane in the presence of base
gives the nitroalkene 113. Compound 112 reacts with various Wittig
reagents to give the corresponding olefins 114-117. Also, Grignard
treatment of 112 gives 6-hydroxyalkyl derivatives 121. Oxidation of
121 affords the corresponding 6-acyl derivatives 120 (R=alkyl). On
the other hand lithiated 6 (R.sup.5'.dbd.R.sup.2'.dbd.THP,
R.sup.3'.dbd.R.sup.3"'H) with benzaldehyde produces 6-hydroxybenzyl
derivative 119 which is converted into 6-benzoyl-3'-deoxyuridine
(120, R.dbd.Ph) by mild oxidation. Also, reaction of lithiated 6
with diphenyldisulfide affords 6-phenylthio-3'-deoxyuridine 118, as
shown in Scheme 17.
[0501] In a similar manner but starting from the L-nucleoside
counterparts, the corresponding III-a nucleosides are prepared.
88
[0502] (d) Modification at C-6 of Purine Nucleosides (I-b and
III-b)
[0503] Compound 28 or 38 is converted into halogenase 122 (Scheme
18) by treatment with hydrogen chloride or hydrogen bromide in
acetic acid or hydrogen bromide in dichloromethane and condensed
with 6-chloropurine by the sodio procedure in acetonitrile affords
3'-deoxynucleoside 123. Aqueous sodium or potassium hydroxide
treatment of 123 gives 3'-deoxyinosine (124). Treatment of 123 with
sodium methoxide in methanol affords 6-O-methyl-3'-deoxyinosine
(125). Mild saponification, followed by catalytic 110
hydrogenolysis of 123 results in the production of
3'-deoxynebularine (126). Thiourea reacts with 123 to give
6-thiopurine nucleoside 127, which is S-alkylated to 128. Compounds
123, 127 and 128 readily react with various amines, hydroxylamine,
hydrazine and aminoalcohols to give 3'-deoxyadenosine analogues
129-133. Treatment of 123 with sodium azide gives 6-azidopurine
nucleoside 134.
[0504] The same sequence of reactions can be applied to the
corresponding L-nucleosides III-b. 89
[0505] These compounds can also be synthesized by nitrous acid
treatment of 6-hydrazidopurine nucleoside 130. Reduction of 129,
130 or 134 gives 3'-deoxyadenosine (i.e., cordycepin). Compound 125
or cordycepin are expected to be converted in vivo into 124 by
action of adenosine deaminase. 6-Unsubstituted purine nucleoside
126 may be oxidized in vivo to 124.
[0506] Condensation of 122 with 2-substituted-6-chloropurine gives
the 2-substituted analogue of 123. The 6-chloro functionality can
be converted into various functional groups by nucleophilic
substitution reactions. Thus, 2-amino-6-chloropurine is converted
into 135 (Scheme 19), which can be converted various 2-aminopurine
nucleosides (136-147). It should be noted that the
2,6-diamino-(141) and 2-amino-purine (138) nucleosides are
potential precursors for 3'-deoxy-guanosine (136). In a similar
manner but starting from the L-nucleoside counterparts, the
corresponding 111-b nucleosides are prepared. 90
[0507] In a similar manner, 2-oxo-, 2-methoxy-, 2-thio-,
2-alkylmercapto-, 2-methyl-, 2-methyl-amino- or
2-dimethylamino-purine nucleosides (148-154) are synthesized
(Scheme 20). Also, in a similar manner but using the corresponding
L-nucleosides, compounds of III-b type are prepared. 91
[0508] (e) Modification at C-2 of Purine Nucleosides (I-b and
III-b).)
[0509] The 2-amino group of 135-147 can be modified to obtain 155
(Scheme 21) by acylation with various alkanoyl or aroyl halides.
Then, 155 can further be derivatized into the corresponding
2-alkylamino or 2-arylamino derivative 156 by reduction with a
borane-amine complex (Sergueeva, Z. A. et al. Nucleosides
Nucleotides Nucleic Acids, 2000, 19, 275). Alternatively, the
2-amino group of compound 135 can be substituted by undergoing a
Schiemann reaction, diazotizing in the presence of fluoroborate,
followed by thermal decomposition, to give 2-fluoro-6-chloropurine
nucleoside 157. Furthermore, the 6-chloro substituent of these
nucleosides can be displaced with various nucleophilic reagents as
described above. It should be noted that the presence of 2-fluoro
substituent protects the 6-amino group from adenosine deaminase
attack. 92
[0510] (f) Modification at C-8 of Purine Nucleosides (I-b)
[0511] It should be noted that modification of the 8-position of
purine nucleosides is important as the substitution at this
position alters the preferred conformation of the nucleosides to be
syn.
[0512] Cordycepin (158, R.sup.3'.dbd.R.sup.3".dbd.H),
3'-deoxyinosine (124, R.sup.3.dbd.R.sup.3".dbd.H) and
3'-deoxyguanosine (136, R.sup.3'=R.sup.3"=H) can be brominated at
the C-8 position by treatment with bromine in acetic acid in the
presence of sodium acetate to 159-161 (Scheme 22). The C-8 bromine
substituent in 159-161can be replaced with sulfur by the action of
thiourea to obtain 162-164, which can be alkylated or aralkylated
with alkyl or aralkyl halide in a polar solvent, such as water,
alcohol or dimethylformamide, in the presence of base, such as
sodium or potassium carbonate, to give 165-167. The methylmercapto
derivative 165-167 (R=methyl) can be oxidized to the corresponding
sulfone 168-170. Upon treatment of these sulfones with various
amines, the corresponding 8-amino derivatives 171-173 are obtained.
Many of the 8-amino derivatives can be obtained directly from
159-161 by treatment with amines. Also, 159 can be converted into
the 8-oxo derivative 174 by treatment with sodium acetate in acetic
anhydride, followed by hydrolysis. O-Alkylation of 174 with
triethyloxonium fluoroborate gives 8-ethoxycordycepin 175. 93
[0513] 8-Alkyl derivatives 176 (Scheme 23) are prepared from 123
(R.sup.5'=R.sup.2'=THP) by treatment with lithium diisopropylamide
in tetrahydrofuran below -70.degree. C., followed by alkyl halide
treatment. This method was successfully used in other
ribonucleosides (Tanaka, H. et al. Chem. Pharm. Bull., 1983, 31,
787) but never been applied to 3'-deoxynucleosides. When carbon
dioxide is used instead of alkyl halide, purine nucleo side
8-carboxylic acid 177 is obtained. Esterification to 178, followed
by ammonolysis gives amide 181, which is dehydrated to
8-cyanopurine nucleoside 182. Reduction of 178 with
borane-dimethylsulfide affords the alcohol 179. Mild oxidation with
dimethylsulfoxide and oxalic chloride affords aldehyde 180.
Compounds 179 and 180 are versatile intermediates for various
modifications. 94
[0514] B. Compounds of Types IIa-c and IVa-c.
[0515] (i) Synthesis from Pre-Formed Nucleosides:
[0516] Several methods are available to introduce a
2',3'-unsaturation into a preformed nucleosides. An example is
shown in Scheme 24.
[0517] Selective O-silylation of nucleoside 7, preferably with
t-butyldimethylsilyl halide or t-butyldiphenylsilyl halide, in
base, preferably in pyridine at from 0.degree. C. to 80.degree. C.,
preferably at room temperature, followed by sulfonylation,
preferably with mesyl chloride or tosyl chloride in base,
preferably in pyridine at from 0.degree. C. to 80.degree. C.,
preferably at room temperature, gives 8 in high yield, which can be
readily converted into the lyxo-epoxide 183 by treatment with base.
Reaction of 183 with halide ion, preferably iodide ion, such as
treatment with sodium iodide in acetone or methylethylketone gives
exclusively the trans-iodohydrin 184, X =1). Mesylation of 184
gives in high yield of the olifin 186 via 185. Compound 185 can be
isolated in poor yield after short reaction time. De-O-silyation of
186 with fluoride, such as tetrabutyl ammonium fluoride affords the
desired olefin 187, type II-a nucleoside, in high yield. 95
[0518] Starting from 2'-deoxy nucleosides, e.g., 188 (Scheme 25),
the type II-a olefinic sugar nucleoside also can be prepared.
Sulfonylation of 188, preferably with mesyl chloride in pyridine at
temperature range from -10.degree. C. to 80.degree. C., preferably
at room temperature, gives the di-O-mesylate 189, which, upon
treatment with aqueous base such as sodium hydroxide solution gives
3',5'-anhydrosugar nucleoside 190. The latter nucleoside can be
readily converted into the desired 187 in high yield by treatment
with strong, anhydrous base, such as with potassium tert-butoxide
in dimethylsulfoxide at temperature range of from -10.degree. C. to
80.degree. C., preferably at room temperature for 10 minutes to
overnight, preferably 1.5 to 3 hours. 96
[0519] An example for preparation of 2'-substituted olefinic sugar
nucleoside of type II-a is given in Scheme 26.
1-(2'-Deoxy-2'-fluoro-.bet- a.-D-arabinofuranosyl)thymine (191) is
selectively protected, preferably with trityl chloride or
t-butyldimethylsilyl chloride or t-butyldiphenylsilyl chloride, in
pyridine to give 192. Sulfonylation of 192, preferably with mesyl
chloride in pyridine, gives the mesylate 193, which, upon treatment
with non-nucleophilic base, such as DBU or DBN in anhydrous inert
solvent, such as methylene chloride, affords 2,3'-anhydro
nucleoside 194. This compound is readily converted into
2'-fluoro-olefinic sugar nucleoside 195 upon treatment with
potassium t-butoxide in dimethylsulfoxide. De-protection of 195
gives the desired 2'-fluorinated II-a type nucleoside 196.
5'-O--Silyl protection gives better overall yield than trityl
protection. 97
[0520] All these reactions can be applied to the corresponding
pyrimidine L-nucleosides for the preparation of IV-a type
nucleosides.
[0521] Nucleosides of type I-b can be prepared readily from 197
(Scheme 27). Selective protection of 197 at the 5'-position, e.g.,
with t-butyldimethylsilyl or t-butyldiphenylsilyl group affords
198. Sulfonylation with tosyl halide or mesyl halide in base such
as in pyridine affords the protected olefinic nucleoside 199.
De-O-silylation of 199 with fluoride, such as tetrabutyl ammonium
fluoride affords the desired olefin 200, type I-b nucleoside, in
high yield.
[0522] Alternatively, treatment of 15 (see Scheme 3) with chromous
acetate gives, after deprotection with base 200 in good yield.
98
[0523] By the same procedure but using purine L-nucleosides, the
corresponding olefinic sugar L-nucleosides of type IV-b can be
obtained.
[0524] (ii) Synthesis by Condensation of Base and Unsaturated Sugar
Derivative
[0525] Commercially available 4-hydroxymethyl-2-pentenone (201,
Scheme 28) is silylated, preferably with t-butyldimethylsilyl
halide in base, preferably in pyridine, to give 202, which is
reduced with borohydride to 203. After acetylation, the product 204
is condensed with silylated base, e.g., 5-substituted uracil. A
complicated mixture is 7ff obtained in which the anomeric
nucleosides (205) are the major components. After chromatographic
separation of the anomers 206 and 207, followed by desilylation of
each anomer affords the P-nucleoside 208 (type II-a) and
a-nucleoside 209 (type XVIII-c), respectively. 99
[0526] Another example is shown in Scheme 29. 2-Fluoro-lactone 212
can be prepared by Wittig condensation of aldehyde 210 with
Ph.sub.3P.dbd.CFCO.sub.2Et. Silyl protection and DIBAH reduction,
followed by acetylation of the product affords 213. Condensation of
213 with silylated purine, such as 6-chloropurine, in the presence
of Lewis acid, such as trimethylsilyl triflate or tin
tetrachloride, in an inert solvent, such as methylene or ethylene
chloride gives anomeric mixture 214. These anomers are separated on
a silica gel column. After desilylation of each component, the
corresponding .beta.-nucleoside 215 (type II-b) and (x-nucleoside
216 (type XVIII-d) can be obtained. 100
[0527] C. Synthesis of Carba-Sugar Nucleosides (V-X)
[0528] Only carba-nucleosides so far found in nature are adenine
nucleosides, i.e., aristeromycin and neplanocins, and they are
either extremely expensive or commercially not available. Thus,
these types of nucleosides are chemically synthesized from scratch.
The carba-sugar derivative is prepared first and then the
heterocyclic aglycon is It constructed on the sugar to prepare
carba-sugar nucleosides or in the case of purine nucleoside, the
base is directly condensed with the carba-sugar.
[0529] Scheme 30 illustrates the synthesis of
5-fluoro-garba-cytidine (227, Type V-a). The carba-sugar
interrediate 219 can be synthesized by any means known in the art.
It is disclosed by Ali et al. (Tetrahedron Letters, 1990, 31, 1509)
that D-ribonolactone 217 is converted into the pentanone
intermediate 218. The ketone 218 can then be reduced by any known
reducing agent, preferably sodium borohydride in methanol at
0.degree. C. for 1 hour to afford alcohol 219. Sulfonylation of
219, preferably with mesyl chloride in methylene chloride in the
presence of triethylamine at 0.degree. C. for 2 hours gives 220,
which is then treated with sodium azide in DMF at 140.degree. C.
ovemight to give 221. The azide 221 can readily be reduced with any
known reducing agent, e.g., Ph.sub.3P (Staudinger procedure) or
catalytic hydrogenolysis, preferably over palladium on carbon. The
resulting amine 222 is subjected to Warrener-Shaw reaction with
.beta.-methoxyacryloylisocyanate in DMF, followed by ammonium
hydroxide treatment to form protected carba-uridine 224 via the
linear intermediate 223. Protected 5-fluoro-carba-uridine (225) can
be obtained by fluorination of 224 with any fluorinating agent.
Preferably, the fluorinating agent is fluorine in acetic acid.
After quenching the reaction with base, preferably triethylamine.
Conversion of uracil nucleoside 225 into protected
carba-5-fluorocytidine (226) can be achieved in a similar manner as
described with Scheme 7. The protecting groups of 226 are removed
with acid, preferably with trifluoroacetic acid/water (2:1 v/v) at
50.degree. C. for 3 hours, to give 227.
[0530] Sulfonylation of 219 with triflyl chloride in methylene
chloride in the presence of triethylamine gives triflate, which,
upon reaction with purine base, such as adenine, and sodium hydride
in an inert solvent, such as acetonitrile or DMF directly affords
the corresponding purine nucleoside (V-b type).
[0531] By using the same procedure but starting from
L-ribonolactone, the corresponding L-nucleosides counterparts (type
VIII nucleosides) can be obtained. 101
[0532] Alternatively, commercially available
(1R)-(-)-azabicyclo[2.2.1]hep- t-5-en-3-one (228, Scheme 31) is
converted into 2,3-dihydroxy-lactam 229 by osmium tetroxide
oxidation. After methanolysis of 229 with methanolic hydrogen
chloride, the product 230 is treated with 2,2-dimethoxypropane in
acetone or l,l-dimethoxycyclohexane in cyclohexanol to give a
ketal, e.g., 231, which is reduced to 232 with sodium borohydride.
The aminoalcohol 232 is converted into
2',3'-O-cyclohexylidene-carba-uridine by reaction with
.beta.-methoxyacryloylisocyanate, followed by ammonia treatment.
Acid treatment, preferably with trifluoroacetic acid in methanol,
gives carba-uridine (233). carba-5-Fluorocytidine (227) can be
obtained readily from 233 by the well-known means in the art.
102
[0533] In a similar sequence of reactions but starting from the
other optical isomer, (IR)-(+)-azabicyclo[2.2.1]hept-5-en-3-one,
the corresponding L-nucleoside analogue (type VIII) can be
obtained.
[0534] Nucleoside of type VI is prepared from nucleoside of type V.
An example is shown in Scheme 32. Aristeromycin (234) or any
carba-ribonucleoside is converted into the corresponding
N-[(dimethylamino)methylene]-5'-O-trityl derivative 235 by
treatment with dimethylformamide dimethylacetal in DMF, followed by
tritylation. Reaction of 235 with thiocarbonyldiimidazole gives
2',3'-O-thiocarbonate 236, which, upon radical reduction with
tri-n-butyltin hydride in the presence of
2,2'-azobis(2-methylpropionitrile) affords olefin 237 along with
3'-deoxy- and 2'-deoxy-aristeromycine derivatives 238 and 239,
respectively. These products can be readily separated on a silica
gel column. Each of these produces the corresponding free
nucleoside, 240, 241 and 242, respectively, upon acid treatment.
This procedure is particularly suited for preparation of small
amounts of several nucleosides in short time for screening.
[0535] By the same procedure but using type VIII nucleosides
instead of type V, the corresponding L-nucleosides of type IX can
be obtained. 103
[0536] Stereoselective conversion of type V to type VI is also
possible as shown in Scheme 33. 5-Fluoro-carba-uridine (233) is
converted into the 5'-O-trityl-2',3'-di-O-mesylderivative 243.
Aqueous base treatment of 243 affords lyxo epoxide 245 via
2,2'anhydro nucleoside intermediate 244. Epoxide ring-opening with
sodium iodide in acetone or butanone gives trans iodohydrin 246,
which, upon mesylation affords the olefin 248 via 247.
De-O-tritylation of 247 furnishes 249. Instead of 5'-O-trityl
protection, silyl protection with t-butyldimethylsilyl or
t-butyldiphenylsilyl protection can also be used. Also, instead of
mesylation, other sulfonylation using an agent, such as tosyl
chloride, trifyl chloride or triflyl anhydride can be used.
[0537] By using the same procedure but using type VIII nucleosides
instead of type V, the corresponding L-nucleosides of type IX can
be obtained. 104
[0538] Also, nucleosides of type VI-b can be synthesized starting
from 2-cyclopenten-1-one (250, Scheme 34). Michael addition of
t-butoxymethyllithiumcuprate [(t-BuOCH.sub.2).sub.2CuLi] to 250
yields the adduct 251. Phenylselenation of 251 according to Wilson
et al. (Synthesis, 1995, 1465) mainly occurs trans to
t-butoxymethyl group to give 252. DIBAH reduction reduces the
carbonyl group to hydroxyl group in a stereoselective manner to
give 253. Sulfonylation, preferably with triflyl chloride or
triflic anhydride in base, to 254, followed by condensation with
sodio-purine, produced, e.g., adenine and NaH, in an inert solvent
such as acetonitrile affords 255 in a stereoselective manner.
Oxidation of the selenide 255 with hydrogen peroxide in pyridine
smoothly converts 255 into the olefin 256. Mild acid treatment of
256 gives free nucleoside 240.
[0539] Alternatively, acetylation of 253, followed by condensation
with silylated pyrimidine, such as
tris(trimethylsilyl)-5-fluorocytosine in the presence of
trimethylsilyl trifluoromethylsulfonate gives high yield of the
corresponding pyrimidine nucleoside, from which VI-a type
nucleoside can readily prepared by oxidation and acid removal of
t-butyl group of the product.
[0540] By using the same procedure but using type VIII nucleosides
instead of type V, the corresponding L-nucleosides of type IX can
be obtained. 105
[0541] Furthermore, racemic carba analogues of 2',3'-unsaturated
nucleosides can be prepared by the procedure of Shi et al. (J. Med.
Chem., 1999, 42, 859) who achieved multi-step preparation of
racemic cis-3,4-epoxy-cyclopentanemethanol 257 (Scheme 35) from
ethyl cyclopentene-4-carboxylate. Opening of the epoxide with
diphenyldiselenide affords 258, which, after acetylation followed
by peroxide treatment, gives diacetate 259. Treatment of 259 with
sodiopyrimidine, prepared by reaction of uracil or cytosine
derivative with NaH in dimethylsulfoxide, in the presence of
Pd(PPh.sub.3).sub.4 in an inert solvent, e.g., tetrahydrofuran,
gives 260 in 10-70% yield after deacetylation of the product.
106
[0542] Scheme 36 shows the synthesis of 3,4-unsaturated carba
nucleoside of type VII Wolfe et al (J. Org. Chem., 1990, 55, 4712)
prepared 261 from D-ribonolactone. Quenching the Michael addition
of t-butoxyrnethyl group to (261, Scheme 36) with sulfinyl
chloride, followed by heating the product with calcium carbonate
gives cyclopentenone 262. Reduction of 262 with DTBAH followed by
sulfonylation affords 263. Condensation of 263 (preferably
R.dbd.CF.sub.3) with purine base with NaH as described earlier
gives purine nucleoside VII-b, e.g., neplanocin A (264). Treatment
of 263 (preferably R.dbd.Me) with NaN.sub.3 gives 265 which can be
readily converted into various pyrimidine nucleosides (VII-a)
including 266 by the procedure already described with Scheme
30.
[0543] Starting from L-ribonolactone, the corresponding
L-nucleoside counterparts (X-a and X-b) can be readily prepared.
107
[0544] D. Synthesis of Nucleosides of Types XI and XII.
[0545] There are several methods are available for the synthesis of
these types of nucleosides, Some nucleosides used in the present
invention are prepared mainly in the following manner.
1-Mentylester of 2,2-dimethoxyacetic acid (267, Scheme 37) is
condensed with thioglycolic acid to give a diastereomeric mixture
268, which can readily be separated on a silica gel column.
Reduction of 268 with NaBH.sub.4 in ethanol, followed by
acetylation affords 269, which is condensed with silylated base in
the presence of tin tetrachloride. Mainly the desired protected
.beta.-nucleoside is obtained and is purified by chromatography.
De-O-acetylation affords the corresponding unprotected nucleoside
270. Also, 270 is obtained starting from 2,2-dimethoxyethyl ester
of N-t-Boc-L-proline. This compound is treated with 3 equivalents
of thioglycolic acid in methylene chloride in the presence of
MgSO.sub.4 and CAS to give 271 as a diastereomeric mixture, which
is separated chromatographically. Reduction of each diastereomer of
271 with Li(t-BuO).sub.3AIH in tetrahydrofuran and subsequent
acetylation affords 272, which is condensed with silylated base,
followed by deprotection of the product to give 270. 108
[0546] Nucleosides of type XIII used in this invention are prepared
by using means known in the art. In a preferred embodiment, XIII-a
type nucleosides are prepared in one or two-step synthesis reported
(Nucleic Acid Chem., 1978, 1, 272 and 343) by activating the 5'-OH
by sulfonylation followed by base treatment or direct treatment of
unprotected nucleosides with Ph3P and diethyl diazocarboxylate.
[0547] Preparation of nucleosides of type XIV used in the present
invention are synthesized by methods somewhat analogous to those
utilized for the synthesis of the corresponding
5-fluorodeoxyuridine adducts by Duschinsky et al. (J. Med. Chem.,
1967, 10, 47). Some examples are shown in Scheme 38 using
5-fluorouridine (273). Any pyrimidine nucleoside containing a
strongly electron-withdrawing substituent at C-5 undergoes similar
adduct formation. Treatment of 273 with bromine in methanol gives
adduct 274 which can be reduced to 275 by catalytic hydrogenation.
Treatment in water gives the bromohydrin 277 while action of
bromine in acetic acid in the presence of acetic anhydride affords
276. The corresponding other adducts can be prepared by using other
hypohalites, e.g., hypochlorite gives 278. Each of these adducts
are diastereomeric mixture and are used for screening as such.
109
[0548] E. Nucleosides of Type XV-XVIII.
[0549] Nucleosides used in this invention are prepared by oxidation
of 4-thiouridine and 6-thiolnosine derivatives according to the
well-known means in the art. Type XVI compounds are C-nucleosides.
XVI-a nucleosides are synthesized from .psi.-uridine by methods
known in the art (Watanabe, "The Chemistry of C--Nucleosides",
Townsend, L. 4 B., Ed., In "Chemistry of Nucleosides and
Nucleotides", Plenum, Publ., New York, Vol., 3, 421, 1994), or
condensation of an aromatic compound to protected ribonolactone,
followed by manupulation of the products (e.g., Kabat et al., J.
Med. Chem., 1987, 30, 924). Nucleosides XVI-b and XVI-c are
prepared according to a modified procedure developed by Pankiewicz
et al., (Carbohydr. Res., 1984, 127, 227; Nucleosides Nucleotides,
1991, 10, 1333). The purine-type XVI-d C-nucleosides are
synthesized according to the method reported by Chu et al., (J.
Heterocycl. Chem., 1980, 17, 1435). Nucleosides of type XVII used
in this invention are synthesized either by cross-aldol reaction of
4'-formyl nucleosides with formaldehyde or condensation of
preformed sugar with a base. Preparation of some of the type XVIII
nucleosides have already discussed earlier.
[0550] The following working examples provide a further
understanding of the method of the present invention. These
examples are of illustrative purposes, and are not meant to limit
the scope of the invention. Equivalent, similar or suitable
solvents, reagents or reaction conditions may be substituted for
those particular solvents, reagents or reaction conditions
described without departing from the general scope of the
method.
EXAMPLES
[0551] Melting points were determined in open capillary tubes on an
Electrothermal digit melting point apparatus and are uncorrected.
The UV absorption spectra were recorded on an Uvikon 931 (KONTRON)
spectrophotometer in ethanol. .sup.1H-NMR spectra were run at room
temperature with a Varian Unity Plus 400 spectrometer. Chemical
shifts are given in ppm downfield from internal tetramethylsilane
as reference. Deuterium exchange, decoupling experiments or 2D-COSY
were performed in order to confirm proton assignments. Signal
multiplicities are represented by s (singlet), d (doublet), dd
(doublet of doublets), t (triplet), q (quadruplet), br (broad), m
(multiplet). All J-values are in Hz. FAB mass spectra were recorded
in the positive-(FAB>0) or negative-(FAB<0) ion mode on a
JEOL DX 300 mass spectrometer The matrix was 3-nitrobenzyl alcohol
(NBA) or a mixture (50:50, v/v) of glycerol and thioglycerol (GT).
Specific rotations were measured on a Perkin-Elmer 241
spectropolarimeter (path length 1 cm) and are given in units of
10.sup.-1deg cm.sup.2 g.sup.-1. Elemental analyses were performed
by Atlantic Microlab Inc. (Norcross, GA). Analyses indicated by the
symbols of the elements or functions were within .+-.0.4% of
theoretical values. Thin layer chromatography was performed on
Whatman PK5F silica gel plates, visualization of products being
accomplished by UV absorbency followed by charring with 10%
ethanolic sulfuric acid and heating. Column chromatography was
carried out on Silica Gel (Fisher, S733-1) at atmospheric
pressure.
Example 1
[0552]
1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-.beta.-D-xylofuranosyl)-N.sup.4--
acetylcytosine (2, R.dbd.H).
[0553] To a suspension of N.sup.4-acetylcytidine (5.7 g, 0.02 mol)
in acetonitrile (300 mL) is added acetyl bromide (15 mL, 0.2 mol)
over 30 minutes under reflux. The mixture is refluxed for 4 hours,
and then concentrated in vacuo to dryness. The residue is dissolved
in methylene chloride (150 mL) and washed with water (150 mL). The
organic layer is dried (Na.sub.2SO.sub.4), evaporated, and the
residue crystallized from ethanol to give
I-(2,5-Di-O-acetyl-3-bromo-3-deoxy-.beta.-D-xylofuranosyl-
)-N.sup.4-acetylcytosine (2, R.dbd.H, 3.4 g, 40%), mp
179-180.degree. C. .sup.1H NMR (CDCl.sub.3) .delta.: 10.2 (bs, 1H,
NHAC), 8.1 (d, 1H, H-6, 35,6=7.5 Hz), 7.5 (d, 1H, H-5,
J.sub.5,6=7.5 Hz), 6.0 (d, 1H, H-1', J.sub.1',2'<1 Hz), 5.5 (d,
1H, H-2', J.sub.1',2'<1, J.sub.2',3'=0 Hz), 4.2-4.7 (m, 4H,
H-3',4',5',5"), 2.0-2.4 (3s, 9H, 3Ac).
[0554] In a similar manner but using the corresponding N-acylated
cytidine, the following nucleosides and their L-counterparts are
prepared:
[0555]
1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-.beta.-D-xylofuranosyl)-N.sup.4--
acetyl-5-fluorocytosine,
[0556]
1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-.beta.-D-xylofuranosyl)-N.sup.4--
acetyl-5-chlororocytosine,
[0557]
1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-.beta.-D-xylofuranosyl)-N.sup.4--
acetyl-5-bromocytosine,
[0558]
1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-.beta.-D-xylofuranosyl)-N.sup.4--
acetyl-5-iodocytosine,
[0559]
1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-.beta.-D-xylofuranosyl)-N.sup.4--
acetyl-5-ethylcytosine,
[0560]
1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-.beta.-D-xylofuranosyl)-N.sup.4--
acetyl-5-n-propylcytosine,
[0561]
1-(2,5-Di-O-acetyl-3-bromo-31-deoxy-.beta.-D-xylofuranosyl)-N.sup.4-
-acetyl-5-i-propylcytosine,
[0562]
1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-.beta.-D-xylofuranosyl)-N.sup.4--
acetyl-5-vinylcytosine,
[0563]
1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-.beta.-D-xylofuranosyl)-N.sup.4--
acetyl-5-(2-chlorovinyl)cytosine,
[0564]
1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-.beta.-D-xylofuranosyl)-N.sup.4--
acetyl-5-(2-bromovinyl)cytosine,
[0565]
1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-.beta.-D-xylofuranosyl)-N.sup.4--
acetyl-5-(2-iodovinyl)cytosine,
[0566]
1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-.beta.-D-xylofuranosyl)-N4-acety-
l-5-(2-methoxylcarbonyl-vinyl)-cytosine,
[0567]
1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-.beta.-D-xylofuranosyl)-N.sup.4--
acetyl-5-(2-hydroxycarbonyl-vinyl)-cytosine,
[0568]
1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-.beta.-D-xylofuranosyl)-N.sup.4--
acetyl-5-phenylcytosine,
[0569]
1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-.beta.-D-xylofuranosyl)-N.sup.4--
acetyl-5-benzylcytosine,
[0570]
1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-.beta.-D-xyloftiranosyl)-N.sup.4-
-benzoylcytosine,
[0571]
1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-.beta.-D-xylofuranosyl)-N.sup.4--
benzoyl-5-fluorocytosine,
[0572]
1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-.beta.-D-xylofiiranosyl)-N.sup.4-
-benzoy1-5-cromocytosine,
[0573]
1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-.beta.-D-xylofuranosyl)-N.sup.4--
benzoyl-5-chlrorocytosine,
[0574]
1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-.beta.-D-xylofuranosyl)-N.sup.4--
benzoyl-5-bromocytosine,
[0575]
1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-.beta.-D-xylofuranosyl)-N.sup.4--
benzoyl-5-methylcytosine,
[0576]
1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-.beta.-D-xylofuranosyl)-N.sup.4--
benzoyl-5-ethylcytosine,
[0577]
1-(2,5-Di-O-aceyl-3-bromo-3-deoxy-.beta.-D-xylofuranosyl)-N.sup.4-b-
enzoyl-5-ethylcytosine,
[0578]
1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-.beta.-D-xylofuranosyl)-N.sup.4--
benzoyl-5-n-propylcytosine,
[0579]
1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-.beta.-D-xylofuranosyl)-N.sup.4--
benzoyl-5--i-poylcytosine,
[0580]
1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-.beta.-D-xylofuranosyl)-N.sup.4--
benzoyl-5-(2-chlorovinyl)cytosine,
[0581]
1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-.beta.-D-xylofuranosyl)-N.sup.4--
benzoyl-5-(2-bromovinyl)cytosine,
[0582]
1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-.beta.-D-xylofuranosyl)-N.sup.4--
benzoyl-5-(2-iodovinyl)cytosine,
[0583]
1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-.beta.-D-xylofuranosyl)-N.sup.4--
benzoyl-5-(2-methoxylcarbonyl-vinyl)cytosine,
[0584]
1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-.beta.-D-xylofuranosyl)-N.sup.4--
benzoyl-5-(2-hydroxycarbonyl-vinyl)cytosine,
[0585]
1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-.beta.-D-xylofuranosyl)-N.sup.4--
benzoyl-5-phenylcytosine,
[0586]
1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-.beta.-D-xylofuranosyl)-N.sup.4--
benzoyl-5-benzylcytosine,
[0587]
1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-.beta.-D-xylofuranosyl)-N.sup.4--
anisoylcytosine,
[0588]
1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-.beta.-D-xylofuranosyl)-N.sup.4--
anisoyl-5-chluorocytosine,
[0589]
1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-.beta.-D-xylofuranosyl)-N.sup.4--
anisoyl-5-chlrorocytosine,
[0590]
1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-.beta.-D-xylofuranosyl)-N.sup.4--
anisoyl-5-bromocytosine,
[0591]
1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-.beta.-D-xylofuranosyl)-N.sup.4--
anisoyl-5-methycytosine,
[0592]
1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-.beta.-D-xylofuranosyl)-N.sup.4--
anisoyl-5-methylcytosine,
[0593]
1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-.beta.-D-xylofuranosyl)-N.sup.4a-
nisoyl-5-nproylcytosine,
[0594]
1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-.beta.-D-xylofuranosyl)-N.sup.4a-
nisoyl-5-i-propylcytosine,
[0595]
1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-.beta.-D-xylofuranosyl)-N.sup.4--
anisoyl-5-vipoylcytosine,
[0596]
1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-.beta.-D-xylofuranosyl)-N.sup.4--
anisoyl-5-minylcytosine,
[0597]
1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-.beta.-D-xylofuranosyl)-N.sup.4--
anisoyl-5-(2-chlorovinyl)cytosine,
[0598]
1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-.beta.-D-xylofuranosyl)-N.sup.4--
anisoyl-5-(2-cbromovinyl)cytosine,
[0599]
1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-.beta.-D-xylofuranosyl)-N.sup.4--
anisoyl-5-(2-iodovinyl)cytosine,
[0600]
1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-.beta.-D-xylofuranosyl)-N.sup.4--
anisoyl-5-(2methoxylcarbon incoin yl)-cytosine,
[0601]
1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-.beta.-D-xylofuranosyl)-N.sup.4--
anisoyl-5-(2-mehydroxycarbonyl-vinyl)-cytosine,
[0602]
1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-.beta.-D-xylofuranosyl)-N.sup.4--
anisoyl-5-phenylcytosine, and
[0603]
1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-.beta.-D-xylofuranosyl)-N.sup.4--
anisoyl-5-benzylcytosine.
Example 2
[0604]
1-(2,5-Di-O-acetyl-3-deoxy-.beta.-erythropentofiuranosyl)-N.sup.4-a-
cetylcytosine.
[0605]
1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-.beta.-D-xylofuranosyl)-N.sup.4--
acetylcytosine (2.15 g, 5 mmol) in 50% aqueous methanol (100 mL) is
hydrogenated in a Parr apparatus in the presence of powdered
calcium carbonate (1 g) and Pd-BaSO.sub.4 catalyst (0.5 g) at the
initial pressure of 45 psi. The catalyst is removed by filtration,
and the filtrate is concentrated in vacuo. The residue is
crystallized from ethanol to give
1-(2,5-di-O-acetyl-3-deoxy-P3-D-erythropentofuranosyl)-N.-
sup.4-acetylcytosine (3, R.dbd.H, 1.06 g, 60%), mp 174-177.degree.
C. .sup.1H NMR (CDCl.sub.3) .delta.: 10.30 (bs, 1H, NHAc), 8.05 (d,
1H, H-6, J5,6=7.5 Hz), 7.43 (d, 1H, H-5, J5,6=7.5 Hz), 5.90 (d, 1H,
H-1', J1',2'=1.0 Hz), 5.46 (m, 1H, H-2'), 4.30-4.80 (3H, nm,
H-4',5',5"), 2.10, 2.27 (2s, 9H, 3Ac), 1.60-2.00 (m, 2H,
H-3',3").
[0606] In a similar manner but using the corresponding
3'-bromo-xylo nucleosides, the following nucleosides and their
L-counterparts are prepared:
[0607]
1-(2,5-Di-O-acetyl-3-deoxy-.beta.-D-erythropentofuranosyl)-N.sup.4--
benzoylcytosine,
[0608]
1-(2,5-Di-O-acetyl-3-deoxy-.beta.-D-erythropentofuranosyl)-N.sup.4--
benzoyl-5-methylcytosine,
[0609]
1-(2,5-Di-O-acetyl-3-deoxy-.beta.D-erythropentofuranosyl)-N.sup.4-b-
enzoyl-5-ethylcytosine,
[0610]
1-(2,5-Di-O-acetyl-3-deoxy-.beta.-D-erythiropentofuranosyl)-N.sup.4-
-benzoyl-5-n-propylcytosine,
[0611]
1-(2,5-Di-O-acetyl-3-deoxy-.beta.-D-erythropentofuranosyl)-N.sup.4--
benzoyl-5-i-propylcytosine,
[0612]
1-(2,5-Di-O-acetyl-3-deoxy-.beta.-D-erythropentofuranosyl)-N.sup.4--
benzoyl-5-phenylcytosine,
[0613]
1(2,5-Di-O-acetyl-3-deoxy-.beta.-D-erythropentofuranosyl)-N.sup.4-b-
enzoyl-5-benzylcytosine,
[0614]
1-(2,5-Di-O-acetyl-3-deoxy-.beta.-D-erythropentofuranosyl)-N.sup.4--
anisoylcytosine,
[0615]
1-(2,5-Di-O-acetyl-3-deoxy-.beta.-D-erythropentofuranosyl)-N.sup.4--
anisoyl-5-methylcytosine,
[0616]
1-(2,5-Di-O-acetyl-3-deoxy-.beta.-D-erythropentofuranosyl)-N.sup.4--
anisoyl-5-ethylcylosine,
[0617]
1-(2,5-Di-O-acetyl-3-deoxy-.beta.-D-erythropentofuranosyl)-N.sup.4--
anisoyl-5-n-propylcytosine,
[0618]
1-(2,5-Di-O-acetyl-3-deoxy-.beta.-D-erythropentofuranosyl)-N.sup.4--
anisoyl-5-i-propylcytosine,
[0619]
1-(2,5-Di-O-acetyl-3-deoxy-.beta.-D-erythropentofuranosyl)-N.sup.4--
anisoyl-5-phenylcytosine and
[0620] 1-(2,5-Di-O-acetyl-3-deoxy-.beta.-D-erythropentofuranosyl)-N
4_anisoyl-5-benzylcytosine.
Example 3
[0621] 1-(3-Bromo-3-deoxy-.beta.-D-xylofuranosyl)cytosine (3,
R.dbd.H).
[0622]
1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-.beta.-D-xylofuranosyl)-N.sup.4--
acetylcytosine (4.31 g, 0.01 mol) is treated with saturated
methanolic ammonia (100 mL) at 0.degree. C. for 30 minutes, and
then concentrated in vacuo below 35.degree. C. The residue is
crystallized from methanol to give
1-(3-bromo-3-deoxy-.beta.-D-xylofuranosyl)cytosine (3, R.dbd.H).
The UV and .sup.1H NMR (D.sub.2O) are consistent with the
xylo-structure.
[0623] In a similar manner but using the corresponding N-acylated
cytidines, the following nucleosides and their L-counterparts are
prepared:
[0624]
1-(3-Bromo-3-deoxy-.beta.-D-xylofuranosyl)-5-fluorocytosine,
[0625]
1-(3-Bromo-3-deoxy-.beta.-D-xylofuranosyl)-5-chlororocytosine,
[0626]
1-(3-Bromo-3-deoxy-.beta.-D-xylofuranosyl)-5-bromocytosine,
[0627]
1-(3-Bromo-3-deoxy-.beta.-D-xylofuranosyl)-5-iodocytosine,
[0628]
1-(3-Bromo-3-deoxy-.beta.-D-xylofuranosyl)-5-methylcytosine,
[0629]
1-(3-Bromo-3-deoxy-.beta.-D-xylofuranosyl)-5-ethylcytosine,
[0630]
1-(3-Bromo-3-deoxy-.beta.-D-xylofuranosyl)-5-n-propylcytosine,
[0631]
1-(3-Bromo-3-deoxy-.beta.-D-xylofuranosyl)-5-i-propylcytosine,
[0632]
1-(3-Bromo-3-deoxy-.beta.-D-xylofuranosyl)-5-vinylcytosine,
[0633]
1-(3-Bromo-3-deoxy-.beta.-D-xylofuranosyl)-5-(2-chorovinyl)cytosine-
,
[0634]
1-(3-Bromo-3-deoxy-.beta.-D-xylofuranosyl)-5-(2-bromovinyl)cytosine-
,
[0635]
1-(3-Bromo-3-deoxy-.beta.-D-xylofuranosyl)-5-(2-bodovinyl)cytosine,
[0636]
1-(3-Bromo-3-deoxy-.beta.-D-xylofuranosyl)-5-(2-aininocarbonylvinyl-
)cytosine,
[0637]
1-(3-Bromo-3-deoxy-.beta.-D-xylofuranosyl)-5-(2-hydroxycarbonylviny-
l)cytosine,
[0638] 1-(3-Bromo-3-deoxy-.beta.-D-xylofuranosyl)-5-phenylcytosine
and
[0639]
1-(3-Bromo-3-deoxy-.beta.-D-xylofuranosyl)-5-benzylcytosine.
Example 4
[0640] 3'-Deoxyctidiine (4, R.dbd.H).
[0641]
1-(2,5-Di-O-acetyl-3-deoxy-.beta.-D-erythropentofuranosyl)-N.sup.4--
acetylcytosine (3, R H, 700 mg, 2 mmol) is dissolved in methanolic
ammonia (20 mL, saturated at 0.degree. C.) and the solution is kept
overnight at room temperature. The solvent is removed by
evaporation in vacuo, and the residue is dissolved in ethanol (20
mL), and then the pH of the solution is adjusted to 3 with 2N
sulfuric acid. The precipitates are collected and crystallized from
water-ethanol to give 3'-deoxycytidine (4) as hemisulfate (408 mg,
74%). Mp 202-203.degree. C. (decomp). .sup.1H NMR (D.sub.2O)
.delta.: 8.23 (d, 1H, H-6, J.sub.5,6=8.0 Hz), 6.27 (d, 1H, H-5,
J.sub.5,6=80 Hz), 5.84 (d, 1H, H-1', J.sub.1',2'=1.0 Hz), 4.6 (m,
1H, H-2'), 3.9 (m, 3H, H-4',5',5"), 1.95-2.15 (m, 2H, H-2',2").
[0642] In a similar manner but using the corresponding acylated
3'-deoxynucloesides, the following nucleosides and their
L-counterparts are prepared: 3'-deoxy-5-methylcytidine,
3'-deoxy-5-ethylcytidine, 3'-deoxy-5-n-propylcytidine,
3'-deoxy-5-i-propylcytidine, 3'-deoxy-5-phenylcytidine, and
3'-deoxy-5-benzylcytidine.
Example 5
[0643] 2',5'-Di-O-acetyl-3'-deoxyuridine.
[0644] 2',5'-Di-O-acetyl-3-deoxy-N.sup.4-acetylcytidine (1.06 g, 3
mol) is dissolved in 70% acetic acid, and the solution is gently
refluxed overnight. After concentration of the mixture in vacuo,
the residue is crystallized from ethanol to give
2',5'-di-O-acetyl-3'-deoxyuridine (660 mg, 96%). .sup.1H NMR
spectrum shows that it contains two acetyl groups, two methylene
groups and two olefinic protons.
[0645] In a similar manner but using the corresponding
3'-deoxycytidines (4), the following
2',5'-di-O-acetyl-3'-deoxyuridines and their L-counterparts are
prepared: 2',5'-Di-O-acetyl-3-deoxy-5-methyluridine,
2',5'-di-O-acetyl-3-deoxy-5-ethyluridine,
2',5'-di-O-acetyl-3-deoxy-5-n-p- ropyluridine,
2',5'-di-O-acetyl-3-deoxy-5-i-propyluridine,
2',5'-di-O-acetyl-3-deoxy-5-phenylunrdine and
2',5'-di-O-acetyl-3-deoxy-5- -benzyluridine.
[0646] In a similar manner but using the corresponding 3'-deoxy
cytosine nucleosides (2), the following uracil nucleosides and
their L-counterparts are prepared:
[0647] 2,5'-Di-O-acetyl-3-deoxy5-fluorouridine,
[0648] 2', 5'-Di-O-acetyl-3-deoxy-5-chlorouridine,
[0649] 2', 5'-Di-O-acetyl-3-deoxy-5-bromouridine,
[0650] 2',5'-Di-O-acetyl-3-deoxy-5-iodouridine,
[0651] 2', 5'-Di-O-acetyl-3-deoxy-5-methyluridine,
[0652] 2', 5'-Di-O-acetyl-3-deoxy-5-ethyluridine,
[0653] 2', 5'-Di-O-acetyl-3-deoxy-5-n-propyluridine,
[0654] 2',5'-Di-O-acetyl-3-deoxy-5-i-propyluridine,
[0655] 2',5'-Di-O-acetyl-3-deoxy-5-vinyluridine,
[0656] 2', 5'-Di-O-acetyl-3-deoxy-5-(2-chlorovinyl)uridine,
[0657] 2', 5'-Di-O-acetyl-3-deoxy-5-(2-bromovinyl)uridine,
[0658] 2',5'-Di-O-acetyl-3-deoxy-5-(2-iodovinyl)uridine,
[0659] 2',
5'-Di-O-acetyl-3-deoxy-5-(2-methoxylcarbonylvinyl)uridine,
[0660] 2',
5'-Di-O-acetyl-3-deoxy-5-(2-hydroxycarbonylvinyl)uridine,
[0661] 2',5'-Di-O-acetyl-3-deoxy-5-phenyluridine and
[0662] 2',5'-Di-O-acetyl-3-deoxy-5-benzyluridine.
Example 6
[0663]
1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-.beta.-D-xylofuranosyl)uracil
(5, R.dbd.H).
[0664]
1-(2,5-Di-O-acetyl-3-bromo-3-deoxy-.beta.-D-xylofuranosyl)-N4-acety-
lcytosine (2, R.dbd.H, R'.dbd.CH.sub.3) (4.31 g, 0.01 mol) is
dissolved in 70% acetic acid, and the solution is gently refluxed
for 4 hours. After concentration of the mixture in vacuo, the
residue is crystallized from ethanol to give
2',5'-di-O-acetyl-3'-bromo-3'-deoxyuridine (5, 2.80 g, 91%).
.sup.1H NMR spectrum shows that it contains two acetyl groups, two
methylene groups and two olefinic protons.
[0665] In a similar manner but using the corresponding
2',5'-di-O-acetyl-3'-bromo-3'-deoxy-N.sup.4-acylcytidines (2), the
following 1,5-di-O-acetyl-3'-bromo-3'-deoxyuridines and their
L-counterparts are prepared:
[0666]
1-(2,5-Di-O-acetyl-3-deoxy-.beta.-D-xylofuranosyl)-5-fluorouracil,
[0667]
1-(2,5-Di-O-acetyl-3-deoxy-.beta.-D-xylofuranosyl)-5-chlororouracil-
,
[0668]
1-(2,5-Di-O-acetyl-3-deoxy-.beta.-D-xylofuranosyl)-5-bromouracil,
[0669]
1-(2,5-Di-O-acetyl-3-deoxy-.beta.-D-xylofuranosyl)-5-iodouracil,
[0670]
1-(2,5-Di-O-acetyl-3-deoxy-.beta.-D-xylofuranosyl)-5-methyluracil,
[0671]
1-(2,5-Di-O-acetyl-3-deoxy-.beta.-D-xylofuranosyl)-5-ethyluracil,
[0672]
1-(2,5-Di-O-acetyl-3-deoxy-.beta.-D-xylofuranosyl)-5-n-propyluracil-
,
[0673]
1-(2,5-Di-O-acetyl-3-deoxy-.beta.-D-xylofuranosyl)-5-i-propyluracil-
,
[0674]
1-(2,5-Di-O-acetyl-3-deoxy-.beta.-D-xylofuranosyl)-5-vinyluracil,
[0675]
1-(2,5-Di-O-acetyl3-deoxy-.beta.-D-xylofuranosyl)-5-(2-chlorovinyl)-
uracil,
[0676]
1-(2,5-Di-O-acetyl-3-deoxy-.beta.-D-xylofuranosyl)-5-(2-bromovinyl)-
uracil,
[0677]
1-(2,5-Di-O-acetyl-3-deoxy-.beta.-D-xylofuranosyl)-5-(2-iodovinyl)u-
racil,
[0678]
1-(2,5-Di-O-acetyl-3-deoxy-.beta.-D-xylofuranosyl)-5-(2-methoxylcar-
bonylvinyl)uracil,
[0679]
1-(2,5-Di-O-acetyl-3-deoxy-.beta.D-xylofuranosyl)-5-(2-hydroxycarbo-
nylvinyl)uracil,
[0680]
1-(2,5-Di-O-acetyl-3-deoxy-.beta.-D-xylofuranosyl)-5-phenyluracil
and
[0681]
1-(2,5-Di-O-acetyl-3-deoxy-.beta.-D-xylofuranosyl)-5-benzyluracil.
Example 7
[0682] 3'-Deoxyuridine (6b, R.dbd.H).
[0683] 2',5'-Di-O-acetyl-3'-deoxyuridine (1.06 g, 3 mol) is
dissolved in methanolic ammonia (10 mL, saturated at 0.degree. C.)
overnight. After concentration of the mixture in vacuo, the residue
is crystallized from ethanol to give 3'-deoxyuridine (6b, 660 mg,
96%).
[0684] In a similar manner but using the corresponding acylated
3'-deoxy-uracil nucleosides (6b) or their L-counterparts, the
following nucleosides are prepared: 3-Deoxy-5-methyluridine,
3-deoxy-5-ethyluridine, 3-deoxy-5-n-propyluridine,
3-deoxy-5-i-propyluridine, 3-deoxy-5-phenyluridine, and
3-deoxy-5-benzyluridine.
Example 8
[0685] 1-(3-Bromo-3-deoxy-fD-xylofuranosyl)uracil (6a,
R.dbd.H).
[0686]
1-(2',5'-Di-O-acetyl-3'-bromo-3'-deoxy-.beta.-D-xylofuranosyl)uraci-
l (5, R.dbd.H) is dissolved in methanolic ammonia (10 mL, saturated
at 0.degree. C.). After 1 hour at 0.degree. C., the mixture is
concentrated in vacuo, and the residue is crystallized from ethanol
to give 3'-bromo-3'-deoxyuridine (6a, 660 mg, 96%). The UV and
.sup.1H NMR are consistent with the structure.
[0687] In a similar manner but using the corresponding acylated
3'-bromo-xylosyluracils, the following nucleosides and their
L-counterparts are prepared:
[0688]
1-(3-Bromo-3-deoxy-.beta.-D-xylofuranosyl)-5-fluorouracil,
[0689]
1-(3-Bromo-3-deoxy-.beta.-D-xylofuranosyl)-5-chlororouracil,
[0690]
1-(3-Bromo-3-deoxy-.beta.-D-xylofuranosyl)-5-bromouracil,
[0691] 1-(3-Bromo-3-deoxy-.beta.-D-xylofuranosyl)-5-iodouracil,
[0692]
1-(3-Bromo-3-deoxy-.beta.-D-xylofuranosyl)-5-methyluracil,
[0693]
1-(3-Bromo-3-deoxy-.beta.-D-xylofuranosyl)-5-ethyluracil,
[0694]
1-(3-Bromo-3-deoxy-.beta.-D-xylofuranosyl)-5-n-propyluracil,
[0695]
1-(3-Bromo-3-deoxy-.beta.-D-xylofuranosyl)-5-i-propyluracil,
[0696]
1-(3-Bromo-3-deoxy-.beta.-D-xylofuranosyl)-5-vinyluracil,
[0697]
1-(3-Bromo-3-deoxy-.beta.-D-xylofuranosyl)-5-(2-chlorovinyl)uracil,
[0698]
1-(3-Bromo-3-deoxy-.beta.-D-xylofuranosyl)-5-(2-bromovinyl)uracil,
[0699]
1-(3-Bromo-3deoxy-.beta.-D-xylofuranosyl)-5-(2-iodovinyl)uracil,
[0700]
1-(3-Bromo-3-deoxy-.beta.-D-xylofuranosyl)-5-(2-aminocarbonylvinyl)-
uracil,
[0701]
1-(3-Bromo-3-deoxy-.beta.-D-xylofuranosyl)-5-(2-hydroxycarbonylviny-
l)uracil,
[0702] 1-(3-Bromo-3-deoxy-.beta.-D-xylofuranosyl)-5-phenyluracil
and
[0703]
1-(3-Bromo-3-deoxy-.beta.-D-xylofuranosyl)-5-benzyluracil.
Example 9
[0704] 2',5-Di-O-triphenylmethyluridine (7, R.dbd.H).
[0705] A mixture of uridine (24.4 g, 0.1 mol) and
triphenylchloromethane (83.5 g, 0.3 mol) in anhydrous pyridine (250
mL) is stirred overnight at room temperature, and then is refluxed
for 4 hours. After cooling to room temperature, the mixture is
poured into water with vigorous stirring. The water is removed by
decantation, and the gummy residue is treated with water, stirred
and the water decanted. This process is repeated several times,
after which the residue is treated with hot water (500 mL), stirred
and the water decanted. This process is repeated twice. The residue
is dissolved in methylene chloride, dried (Na.sub.2SO.sub.4), and
concentrated in vacuo. The residue is dissolved in minimum amount
of benzene, and the solution diluted with ethyl ether to turbidity,
and the mixture left overnight at 15.degree. C. The precipitates
are collected and recrystallized from benzene-ethyl ether to give 7
(R H) (22.8 g, 31%), mp 224-225.degree. C. The combined fitrates
are concentrated, and the residue dissolved in methylene chloride
and chromatographed over a silica gel column using methylene
chloride-ethanol (99:1 v/v), (98:2 v/v) and (97:3 v/v). Compound 7
is eluted first (10 g, 14%), followed by
3',5'-di-O-triphenylmethyluridine (3 1.0 g, 42.5%).
[0706] In a similar manner but using the corresponding nucleosides,
the following 2',5'-di-O-protected and 3',5'-di-O-protected
nucleosides and their L-counterparts are prepared:
[0707] 2', 5'-Di-O-triphenylmethyl-5-fluorouridine,
[0708] 2', 5'-Di-O-triphenylmethyl-5-chlorouridine,
[0709] 2', 5'-Di-O-triphenylmethyl-5-bromouridine,
[0710] 2',5'-Di-O-triphenylmethyl-5-iodouridine,
[0711] 2', 5'-Di-O-triphenylmethyl-5-methyluridine,
[0712] 2',5'-Di-O-triphenylmethyl-5-ethyluridine,
[0713] 2',5'-Di-O-triphenylmethyl-5-n-propyluridine,
[0714] 2', 5'-Di-O-triphenylmethyl-5-i-propyluridine,
[0715] 2',5'-Di-O-triphenylmethyl-5-vinyluridine,
[0716] 2',5'-Di-O-triphenylmethyl-5-ethynyluridine,
[0717] 2',5'-Di-O-triphenylmethyl-5-(2-chlorovinyl)uridine,
[0718] 2',5'-Di-O-triphenylmethyl-5-(2-bromovinyl)uridine,
[0719] 2',5'-Di-O-triphenylmethyl-5-(2-iodovinyl)uridine,
[0720]
2',5'-Di-O-triphenylmethyl-5-(2-methoxylcarbonylvinyl)uridine,
[0721]
2',5'-Di-O-triphenylmethyl-5-(2-hydroxycarbonylvinyl)uridine,
[0722] 2',5'-Di-O-triphenylmethyl-5-phenyluridine,
[0723] 2',5'-Di-O-triphenylmethyl-5-benzyluridine,
[0724] 3',5'-Di-O-triphenylmethyl-5-fluorouridine,
[0725] 3',5'-Di-O-triphenylmethyl-5-chlorouridine,
[0726] 3', 5'-Di-O-triphenylmethyl-5-bromouridine,
[0727] 3',5'-Di-O-triphenylmethyl-5-bodouridine,
[0728] 3',5'-Di-O-triphenylmethyl-5-methyluridine,
[0729] 3',5'-Di-O-triphenylmethyl-5-ethyluridine,
[0730] 3',5'-Di-O-triphenylmethyl-5-n-propyluridine,
[0731] 3',5'-Di-O-triphenylmethyl-5-i-propyluridine,
[0732] 3',5'-Di-O-triphenylmethyl-5-vinyluridine,
[0733] 3', 5'-Di-O-triphenylmethyl-5-ethynyluridine,
[0734] 3',5'-Di-O-triphenylmethyl-5-(2-chlorovinyl)uridine,
[0735] 3',5'-Di-O-triphenylmethyl-5-(2-bromovinyl)uridine,
[0736] 3',5'-Di-O-triphenylmethyl-5-(2-iodovinyl)uridine,
[0737]
3',5'-Di-O-triphenylmethyl-5-(2-methoxylcarbonylvinyl)uridine,
[0738]
3',5'-Di-O-triphenylmethyl-5-(2-hydroxycarbonylvinyl)uridine,
[0739] 3',5'-Di-O-triphenylmethyl-5-phenyluridine and
[0740] 3',5'-Di-O-triphenylmethyl-5-benzyluridine.
Example 10
[0741] 3'-O-Mesyl-2,5'-di-O-triphenylmethyluridine (8,
R.dbd.H).
[0742] To a cooled solution of 2',5'-di-O-triphenylmethyluridine
(7, R.dbd.H, 7.28 g, Immol) in pyridine (100 mL) is added drop wise
mesyl chloride (1 mL), and the reaction is kept overnight at
4.degree. C. The reaction is quenched by addition of ethanol (5
mL). After 2 hours of stirring at room temperature, the mixture is
concentrated in vacuo. The residue is triturated with ethanol (250
mL), and the solid collected, and recrystallized from ethanol to
give 8 (R.dbd.H) (7.45 g, 92%), mp 225-226.degree. C.
[0743] In a similar manner but using the corresponding nucleosides,
the following 2',5'-di-O-triphenylmethylated and
3',5'-di-O-triphenylmethylat- ed nucleosides and their
L-counterparts are prepared:
[0744] 3'-O-Mesyl-2',5'-di-O-triphenylmethyl-5-fluorouridine,
[0745] 3'-O-Mesyl-2',5'-di-O-triphenylmethyl-5-chlorouridine,
[0746] 3'-O-Mesyl-2',5'-di-O-triphenylmethyl-5-bromouridine,
[0747] 3'-O-Mesyl-2',5'-di-O-triphenylmethyl-5-iodouridine,
[0748] 3'-O-Mesyl-2',5'-di-O-triphenylmethyl-5-methyluridine,
[0749] 3'-O-Mesyl-2',5'-di-O-triphenylmethyl-5-ethyluridine,
[0750] 3'-O-Mesyl-2',5'-di-O-triphenylmethyl-5-n-propylunrdine,
[0751] 3'-O-Mesyl-2',5'-di-O-triphenylmethyl-5-i-propyluridine,
[0752] 3'-O-Mesyl-2',5'-di-O-triphenylmethyl-5-vinyluridine,
[0753] 3'-O-Mesyl-2',5'-di-O-triphenylmethyl-5-ethynyluridine,
[0754]
3'-O-Mesyl-2',5'-di-O-triphenylmethyl-5-(2-chlorovinyl)uridine,
[0755]
3'-O-Mesyl-2',5'-di-O-triphenylmethyl-5-(2-bromovinyl)uridine,
[0756]
3'-O-Mesyl-2',5'-di-O-triphenylmethyl-5-(2-iodovinyl)uridine,
[0757]
3'-O-Mesyl-2',5'-di-O-triphenylmethyl-5-(2-methoxylcarbonylvinyl)ur-
idine,
[0758]
3'-O-Mesyl-2',5'-di-O-triphenylmethyl-5-(2-hydroxycarbonylvinyl)uri-
dine,
[0759] 3'-O-Mesyl-2',5'-di-O-triphenylmethyl-5-phenyluridine,
[0760] 3'-O-Mesyl-2',S '-di-O-triphenylmethyl-5-benzyluridine,
[0761] 2'-O-Mesyl-3',5'-di-O-triphenylmethyl-5-fluorouridine,
[0762] 2'-O-Mesyl-3',5'-di-O-triphenylmethyl-5-chlorouridine,
[0763] 2'-O-Mesyl-3',5'-di-O-triphenylmethyl-5-bromouridine,
[0764] 2'-O-Mesyl-3',5'-di-O-triphenylmethyl-5-iodouridine,
[0765] 2'-O-Mesyl-3',5'-di-O-triphenylmethyl-5-methyluridine,
[0766] 2'-O-Mesyl-3',5'-di-O-triphenylmethyl-5-ethyluridine,
[0767] 2'-O-Mesyl-3',5'-di-O-triphenylmethyl-5-n-propyluridine,
[0768] 2'-O-Mesyl-3',5'-di-O-triphenylmethyl-5-i-propyluidine,
[0769] 2'-O-Mesyl3',5'-di-O-triphenylmethyl-5-vinyluridine,
[0770] 2'-O-Mesyl-3',5'-di-O-triphenylmethyl-5-ethynyluridine,
[0771]
2'-O-Mesyl-3',5'-di-O-triphenylmethyl-5-(2-chlorovinyl)uridine,
[0772] 2'-O-Mesyl-3',5'-di-O-triphenylmethyl-5-(2-bromovinyl)ue,
dine,
[0773]
2'-O-Mesyl-3',5'-di-triphenylmethyl-5-(2-iodovinyl)uridine,
[0774]
2'-O-Mesyl-3',5'-di-O-triphenylmethyl-5-(2-methoxylcarbonylvinyl)ur-
idine,
[0775] 2'-O-Mesyl-3',5'-di-O-triphenylmethyl-5-(2-hydroxycaibonyl
vinyl)uridine,
[0776] 2'-O-Mesyl-3',5'-di-O-triphenylmethyl-5-phenyluridine
and
[0777] 2'-O-Mesyl-3',5'-di-O-triphenylmethyl-5-benzyluridine.
Example 11
[0778]
2,3'-Anhydro-1-(2,5-di-O-triphenylmethyl-.beta.-.sub.D-xylofuranosy-
l)uracil (9, R.dbd.H X'.dbd.OH).
[0779] A mixture of 3'-O-mesyl-2',5'-di-O-triphenylmethyluridine
(806 mg, 1 mmol), sodium benzoate (2 g) in dimethylformamide (40
mL) is heated at 130-140.degree. C. overnight. The mixture is
cooled to room temperature, and poured onto IL of water with
stirring. The precipitates are collected by decantation and
triturated with ethanol (100 mL) to give
3'-anhydro-1-(2,5-di-O-triphenylmethyl-.beta.-D-xylofuranosyl)uracil
(9, R H, X'.dbd.OH), (500 mg, 75%), mp 237.degree. C.
[0780] In a similar manner but using the corresponding
5-substituted 3'-O-mesyl-2',5'-di-O-triphenylmethyluridines (8),
the following 2,3'-anhydro-di-O-triphenylmethylated nucleosides and
their L-counterparts are prepared:
2,3'-Anhydro-1-(2,5-di-O-triphenylmethyl-.be-
ta.-D-xylofuranosyl)-5-fluorouracil.
[0781]
2,3'-Anhydro-1-(2,5-di-O-triphenylmethyl-.beta.-D-xylofuranosyl)-5--
chlorouridine,
[0782]
2,3'-Anhydro-1-(2,5-di-O-triphenylmethyl-.beta.-D-xylofuranosyl)-5--
bromouridine,
[0783]
2,3'-Anhydro-1-(2,5-di-O-triphenylmethyl-.beta.-D-xylofuranosyl)-5--
iodouridine,
[0784]
2,3'-Anhydro-1-(2,5-di-O-triphenylmethyl-.beta.-D-xylofuranosyl)-5--
methyluridine,
[0785]
2,3'-Anhydro-1-(2,5-di-O-triphenylmethyl-.beta.-D-xylofuranosyl)-5--
ethyluridine,
[0786]
2,3'-Anhydro-1-(2,5-di-triphenylmethyl-.beta.-D-xylofuranosyl)-5-n--
propyluridine,
[0787]
2,3'-Anhydro-1-(2,5-di-O-triphenylmethyl-.beta.-D-xylofuranosyl)-5--
i-propyluridine,
[0788]
2,3'-Anhydro-1-(2,5-di-O-triphenylmethyl-.beta.-D-xylofuranosyl)-5--
vinyluridine,
[0789]
2,3'-Anhydro-1-(2,5-di-O-triphenylmethyl-.beta.-D-xylofuranosyl)-5--
ethynyluridine,
[0790] 2,3'-Anhydro-1-s
[0791]
2,3'-Anhydro-1-(2,5-di-O-triphenylmethyl-.beta.-D-xylofuranosyl)-5--
(2-bromovinyl)uridine,
[0792]
2,3'-Anhydro-1-(2,5-di-O-triphenylmethyl-.beta.-D-xylofuranosyl)-5--
(2-iodovinyl)uridine,
[0793]
2,3'-Anhydro-1-(2,5-di-O-triphenylmethyl-.beta.-D-xylofuranosyl)-5--
(2-methoxylcarbonylvinyl)-uridine,
[0794]
2,3'-Anhydro-1-(2,5-di-O-triphenylmethyl-.beta.-D-xylofuranosyl)-5--
(2-hydroxycarbonylvinyl)-uridine,
[0795]
2,3'-Anhydro-1-(2,5-di-O-triphenylmethyl-.beta.-D-xylofuranosyl)-5--
phenyluridine and
[0796]
2,3'-Anhydro-1-(2,5-di-O-triphenylmethyl-.beta.-D-xylofuranosyl)-5--
benzyluridine.
[0797] In a similar manner but using the corresponding
5-substituted 2'-O-mesyl-3',5'-di-O-triphenylmethyluridines, the
following 2,2'-anhydro-3',5'-di-O-triphenylmethylated nucleosides
and their L-counterparts are prepared:
[0798]
2,2'-Anhydro-1-(3,5-di-O-triphenylmethyl-.beta.-D-arabinofuranosyl)-
-5-fluorouracil,
[0799] 2,2'-Anhydro-1-(3
,5-di-O-triphenylmethyl-.beta.-D-arabinofuranosyl-
)-5-chlorouridine,
[0800] 2,2'-Anhydro-1-(3
,5-di-O-triphenylmethyl-.beta.-D-arabinofuranosyl-
)-5-bromouridine,
[0801] 2,2'-Anhydro-1-(3
,5-di-O-triphenylmethyl-.beta.-D-arabinofuranosyl-
)-5-iodouridine,
[0802]
2,2'-Anhydro-1-(3,5-di-O-triphenylmethyl-.beta.-D-arabinofuranosyl)-
-5-methyluridine,
[0803]
2,2'-Anhydro-1-(3,5-di-O-triphenylmethyl-.beta.-D-arabinofuranosyl)-
-5-ethyluridine,
[0804] 2,2'-Anhydro-1-(3
,5-di-O-triphenylmethyl-.beta.-D-arabinofuranosyl-
)-5-n-propyluridine,
[0805]
2,2'-Anhydro-1-(3,5-di-O-triphenylmethyl-.beta.-D-arabinofuranosyl)-
-5-i-propyluridine,
[0806]
2,2'-Anhydro-1-(3,5-di-O-triphenylmethyl-.beta.-D-arabinofuranosyl)-
-5-vinyluridine,
[0807]
2,2'-Anhydro-1-(3,5-di-O-triphenylmethyl-.beta.-D-arabinofuranosyl)-
-5-ethynyluridine,
[0808] 2,2'-Anhydro-1-(3
,5-di-O-triphenylmethyl-.beta.-D-arabinofuranosyl-
)-5-(2-chlorovinyl)uridine,
[0809] 2,2'-Anhydro-1-(3
,5-di-O-triphenylmethyl-.beta.-D-arabinofuranosyl-
)-5-(2-bromovinyl)uridine,
[0810]
2,2'-Anhydro-1-(3,5-di-O-triphenylmethyl-.beta.-D-arabinofuranosyl)-
-5-(2-iodoviny)uridine,
[0811]
2,2'-Anhydro-1-(3,5-di-O-triphenylmethyl-.beta.-D-arabinofuranosyl)-
-5-(2-methoxylcarbonylvinyl)-uridine,
[0812] 2,2'-Anhydro-1-(3,5-di-O-triphenylmethyl-.beta.-D-arabino
furanosyl)-5-(2-hydroxycarbonylvinyl)-uridine,
[0813]
2,2'-Anhydro-1-(3,5-di-O-triphenylmethyl-.beta.-D-arabinofuranosyl)-
-5-phenyluridine and
[0814]
2,2'-Anhydro-1-(3,5-di-O-triphenylmethyl-.beta.-D-arabinofuranosyl)-
-5-benzyluridine.
Example 12
[0815] 3'-Deoxy-3'-iodo-2',5'-di-O-triphenylmethyluridine (11,
R.dbd.H, X.dbd.I, X'.dbd.OH).
[0816] A mixture of 3'-O-mesyl-2',5'-di-O-triphenylmethyluridine
(8, 1.61 g, 2 mmol), sodium iodide (3 g, 20 mmol) in
1,2-dimethoxyethane (40 mL) is heated at reflux overnight. The
solvent is removed by evaporation in vacuo, the residue is
dissolved in methylene chloride. The solution is washed
successively with 5% sodium thiosulfate and water, dried over
sodium sulfate, and concentrated to dryness in vacuo. The residue
is chromatographed over a silica gel column using methylene
chloride-ethyl ether (3:1 v/v) as the eluent to give 703 mg (42%)
of 3'-deoxy-3'-iodo-2',5'-di-O-triphenylmethyluridine (11, R.dbd.H,
X.dbd.I, X'.dbd.OH).
[0817] In a similar manner but using the corresponding
5-substituted 3'-O-mesyl-2',5'-di-O-triphenylmethyluridines (8),
the following 3'-iodo derivatives are and their L-counterparts
prepared:
[0818]
3'-Deoxy-3'-iodo-2',5'-di-O-triphenylmethyl-5-fluorouridine,
[0819]
3'-Deoxy-3'-iodo-2',5'-di-O-triphenylmethyl-5-chlorouridine,
[0820]
3'-Deoxy-3'-iodo-2',5'-di-O-triphenylmethyl-5-bromouridine,
[0821]
3'-Deoxy-3'-iodo-2',5'-di-O-triphenylmethyl-5-iodoun'dine,
[0822]
3'-Deoxy-3'-iodo-2',5'-di-O-triphenylmethyl-5-methyluridine,
[0823]
3'-Deoxy-3'-iodo-2',5'-di-O-triphenylmethyl-5-ethyluridine,
[0824]
3'-Deoxy-3'-iodo-2',5'-di-O-triphenylmethyl-5-n-propyluridine,
[0825]
3'-Deoxy-3'-iodo-2',5'-di-O-triphenylmethyl-5-i-propyluridine,
[0826]
3'-Deoxy-3'-iodo-2',5'-di-O-triphenylmethyl-5-vinyluridine,
[0827]
3'-Deoxy-3'-iodo-2',5'-di-O-triphenylmethyl-5-ethynyluridine,
[0828]
3'-Deoxy-3'-iodo-2',5'-di-O-triphenylmethyl-5-(2-chlorovinyl)uridin-
e,
[0829]
3'-Deoxy-3'-iodo-2',5'-di-O-triphenylmethyl-5-(2-bromovinyl)uridine-
,
[0830]
3'-Deoxy-3'-iodo-2',5'-di-O-triphenylmethyl-5-(2-iodovinyl)uridine,
[0831]
3'-Deoxy-3'-iodo-2',5'-di-O-triphenylmethyl5-(2-methoxylcarbonylvin-
yl)uridine,
[0832]
3'-Deoxy-3'-iodo-2',5'-di-O-triphenylmethyl-5-(2-hydroxycarbonylvin-
yl)ur idine,
[0833] 3'-Deoxy-3'-iodo-2',5'-di-O-triphenylmethyl-5-phenyluridine
and
[0834]
3'-Deoxy-3'-iodo-2',5'-di-O-triphenylmethyl-5-benzyluridine.
[0835] In a similar manner but using the corresponding
5-substituted 2'-O-mesyl-3',5'-di O-triphenylmethyluridines, the
following 2'-iodo derivatives and their L-counterparts are
prepared:
[0836]
2'-Deoxy-2'-iodo-3',5'-di-O-triphenylmethyl-5-fluorouridine,
[0837]
2'-Deoxy-2'-iodo-3',5'-di-O-triphenylmethyl-5-chyorourbdine,
[0838]
2'-Deoxy-2'-iodo-3',5'-di-O-triphenylmethyl-5-bromouridine,
[0839]
2'-Deoxy-2'-iodo-3',5'-di-O-triphenylmethyl-5-iodouridine,
[0840]
2'-Deoxy-2'-iodo-3',5'-di-O-triphenylmethyl-5-methyluridine,
[0841]
2'-Deoxy-2'-iodo-3',5'-di-O-triphenylmethyl-5-ethyluridine,
[0842]
2'-Deoxy-2'-iodo-3',5'-di-triphenylmethyl-5-n-propyluridine,
[0843]
2'-Deoxy-2'-iodo-3',5'-di-O-triphenylmethyl-5-i-propyluridine,
[0844]
2'-Deoxy-2'-iodo-3',5'-di-O-triphenylmethyl-5-vinyluridine,
[0845]
2'-Deoxy-2'-iodo-3',5'-di-O-triphenylmethyl-5-ethynyluridine,
[0846]
2'-Deoxy-2'-iodo-3',5'-di-O-triphenylmethyl-5-(2-chlorovinyl)uridin-
e,
[0847]
2'-Deoxy-2'-iodo-3',5'-di-O-triphenylmethyl-5-(2-bromovinyl)uridine-
,
[0848]
2'-Deoxy-2'-iodo-3',5'-di-O-triphenylmethyl-5-(2-iodovinyl)uridine,
[0849]
2'-Deoxy-2'-iodo-3',5'-di-O-triphenylmethyl-5-(2-methoxylcarbonylvi-
nyl)uridine,
[0850]
2'-Deoxy-2'-iodo-3',5'-di-O-triphenylmethyl-5-(2-hydroxycarbonylvin-
yl)uridine,
[0851] 2'-Deoxy-2'-iodo-3',5'-di-O-triphenylmethyl-5-phenyluridine
and
[0852]
2'-Deoxy-2'-iodo-3',5'-di-O-triphenylmethyl-5-benzyluridine.
Example 13
[0853] 3'-Iodo-3'-deoxyuridine.
[0854] 3'-Deoxy-3'-iodo-2',5'-di-O-triphenylmethyluridine (840 mg,
1 mmol) (11, R.dbd.H, X.dbd.I, X'.dbd.OH) is dissolved in a 10:1
mixture of methylene chloride and trifluoroacetic acid (20 mL), and
the mixture is kept at room temperature. The solvent is removed in
vacuo, and the residue is triturated with ethyl ether (15
mL.times.2). The ether-insoluble residue is crystallized from
methanol ether to give 3'-iodo-3'-deoxyuridine (312 mg, 88.1%).
[0855] In a similar manner but using the corresponding
5-substituted 3'-deoxy-3'-iodo-2',5'-di-O-triphenylmethyluridines,
the following 3'-iodouridine derivatives and their L-counterparts
are prepared: 3'-Deoxy-3'-iodo-5-iluorouridine,
3'-deoxy-3'-iodo-5-chlorouridine, 3'-deoxy-3'-iodo-5-bromo-uridine,
3'-deoxy-3'-iodo-5-iodouridine, 3'-deoxy-3'-iodo-S-methyl-urndine,
3'-deoxy-3'-iodo-5-ethyluridine,
3'-deoxy-3'-iodo-5-n-propyluridine,
3'-deoxy-3'-iodo-5-i-propyl-uridine,
3'-deoxy-3'-iodo-5-vinyluridine, 3'-deoxy-3'-iodo-5-ethynyluri
dine, 3'-deoxy-3'-iodo-5-(2-chloro-vinyl)-uridine,
3'-deoxy-3'-iodo-5-(2-bromov- inyl) uridine,
3'-deoxy-3'-iodo-5-(2-iodovinyl)uridine,
3'deoxy-3'-iodo-5-(2-methoxylcarbonyl-vinyl)uridine,
3'-deoxy-3'-iodo-5-(2-hydroxy-carbonyl-vinyl)-uridine,
3'-deoxy-3'-iodo-5-phenyluridine, and
3'-deoxy-3'-iodo-5-benzyl-uridine.
[0856] In a similar manner but using the corresponding
5-substituted 2'-deoxy-2'-iodo-3',5'-di-O-triphenylmethyluridines,
the following 2'-iodouridine derivatives and their L-counterparts
are prepared: 2'-deoxy-2'-iodo-5-fluorouridine,
2'-deoxy-2'-iodo-5-chlorouridine, 2'-deoxy-2'-iodo-5-bromo-uridine,
2'-deoxy-2'-iodo-5-iodouridine, 2'-deoxy-2'-iodo-5-methyl-uridine,
2'-deoxy-2'-iodo-5-ethyluridine,
2'-deoxy-2'-iodo-5-n-propyluridine,
2'-deoxy-2'-iodo-5-i-propyl-uridine,
2'-deoxy-2'-iodo-5-vinyluridine, 2'-deoxy-2'-iodo-5-ethynyluridine,
2'deoxy-2'-iodo-5-(2-chlorovinyl)-uridine,
2'-deoxy-2'-iodo-5-(2-bromovin- yl)uridine,
2'-deoxy-2'-iodo-5-(2-iodovinyl)uridine,
2'-deoxy-2'-iodo-5-(2-methoxylcarbonylvinyl)uridine,
2'-deoxy-2'-iodo-5-(2-hydroxycarbonyl-vinyl)-uridine,
2'-deoxy-2'-iodo-5-phenyluridine, and
2'-deoxy-2'-iodo-5-benzyluridine.
Example 14
[0857] 9-(2-O-Acetyl-3-bromo-3-deoxy-,8-D-xylofuranosyl)adenitne
(14, R.dbd.H, X'.dbd.Br, Y.dbd.NH.sub.2, Z=H).
[0858] Compound 14 (R=2,5,5-trimethyl-1,3-dioxolan-4-on-2-yl,
X.dbd.Br, Y.dbd.NH.sub.2, Z=H, 500 mg, I mmol) is dissolved in
methanolic hydrogen chloride prepared by addition of 3 drops of
acetyl chloride in 10 mL of methanol. After 30 minutes at room
temperature, 3 mL of saturated sodium bicarbonate solution is
added, and the mixture concentrated in vacuo to dryness. The
residue is triturated with ethanol until supernatant does not show
significant UV absorption at 260 nm, The ethanol extracts are
concentrated, and the residue is crystallized from methanol to give
the desired 14 (R.dbd.H, X.dbd.Br, Y.dbd.NH.sub.2, Z=H), 325 g
(87%). .sup.1H NMR (D.sub.6-DMSO) .delta.: 8.16, 8.32 (2s, H-2 and
H-8), 6. 10 (d, 1H, H-1', J.sub.1',2'=3.9 Hz), 5.91 (dd, 1H, H-2',
J.sub.1',2'=3.9, J.sub.2',3'=4.1 Hz), 5.85 (dd, 1H, H-3',
J.sub.2',3'=4.1, J.sub.3',4'=5.1 Hz), 4.38 (dt, 1H, H-4',
J.sub.3',4'=5.1, J.sub.4',5'=J.sub.4',5=5.0 Hz), 3.79 (dd, 2H,
H-5', 5"), 2.09 (s, 3H, Ac).
[0859] In a similar manner but using the corresponding purine
nucleosides, the following 2'-O-acetyl-3'-bromo-3'-deoxy-D-xylo
nucleosides (14) and their L-counterparts are prepared:
[0860]
9-(2-O-Acetyl-3-bromo-3-deoxy-.beta.-D-xylofuranosyl)guanine,
[0861]
9-(2-O-Acetyl-3-bromo-3-deoxy-.beta.-D-xylofuranosyl)-6-chloropurin-
e,
[0862]
9-(2O-Acetyl-3-bromo-3-deoxy-.beta.-D-xylofuranosyl)-2,6-dichloropu-
rine,
[0863]
9-(2-O-Acetyl-3-bromo-3-deoxy-.beta.-D-xylofuranosyl)-2-amino-6-chl-
oropurine,
[0864]
9-(2-O-Acetyl-3-bromo-3-deoxy-.beta.-D-xylofuranosyl)-6-methylthiop-
urine and
[0865]
9-(2-O-Acetyl-3-bromo-3-deoxy-.beta.-D-xylofuranosyl)-6-methoxypuri-
ne.
Example 15
[0866] 9-[2-O-Acetyl-3-bromo-3-deoxy-5-O-(2,5,
5-trimethyl-1,3-dioxolan-4--
on-2-yl)-.beta.-D-xylofuranosyl]adenine (14,
R=2,5,5-trimethyl-1,3-dioxala- n-2-one-2-yl, X.dbd.Br,
Y.dbd.NH.sub.2, Z.dbd.H).
[0867] A mixture of adenosine (13, Y.dbd.NH.sub.2, Z.dbd.H, 10 g,
0.037 mol) and .alpha.-acetoxy-isobutyryl bromide (24 g, 0.117 mol)
in acetonitrile (120 mL) is stirred at room temperature for 45
minutes. The solvent is removed in vacuo, and the residue is
dissolved in ethyl acetate, washed with sodium bicarbonate solution
and water, dried over sodium sulfate, and concentrated in vacuo.
The residue is crystallized from methanol to give 6.5 g (35%) of 14
(X.dbd.Br, Y.dbd.NH.sub.2, Z.dbd.H), mp 169-170.degree. C. .sup.1H
NMR (D.sub.6-DMSO) .delta.: 8.17, 8.26 (2s, 1H each, H-2 and H-6),
6.16 (d, 1H, H-1', J.sub.1',2'=3.5 Hz), 5.94 (dd, 1H, H-2',
J.sub.1',2'=3.5 Hz, J.sub.2',3'=3.0 Hz), 4.92 (dd, 1H, H-3',
J.sub.2',3'=3.0 Hz, J.sub.3',4'=4.8 Hz), 4.54 (m, 1H, H-4'), 3.94
(m, 2H, H-5',5"), 2.10 (s, 3H, Ac), 1.73, 1.58, 1.47 (3s, 3H each,
CH.sub.3 groups on 5'). The mother liquor of crystallization of 14
contains a mixture of 2'-bromo-2'-deoxy-D-arabinosyl isomer 15, as
judged by .sup.1H NMR.
[0868] In a similar manner but using the corresponding purine
nucleosides, the following 3'-bromo-3'-deoxy derivatives (14) and
their L-counterparts are prepared:
[0869]
9-[2-O-Acetyl-3-bromo-3-deoxy-5-O-(2,5,5-trimethyl-1,3-dioxolan-4-o-
n-2-yl)-.beta.-D-xylofuranosyl]-guanine,
[0870]
9-[2-O-Acetyl-3-bromo-3-deoxy-5-O-(2,5,5-trimethyl-1,3-dioxolan-4-o-
n-2-yl)-.beta.-D-xylofuranosyl]-6-chloropurine,
[0871]
9-[2-O-Acetyl-3-bromo-3-deoxy-5-O-(2,5,5-trimethyl-1,3-dioxolan-4-o-
n-2-yl)-.beta.-D-xylofuranosyl]-2,6-dichloropurine,
[0872]
9-[2-O-Acetyl-3-bromo-3-deoxy-5-O-(2,5,5-trimethyl-1,3-dioxolan-4-o-
n-2-yl)-.beta.-D-xylofuranosyl]-2-amino-6-chloropurine,
[0873]
9-[2-O-Acetyl-3-bromo-3-deoxy-5-O-(2,5,5-trimethyl-1,3-dioxolan-4-o-
n-2-yl)-.beta.-D-xylofuranosyl]-6-methylthiopurine,
[0874]
9-[2-O-Acetyl-3-bromo-3-deoxy-5-O-(2,5,5-trimethyl-1,3-dioxolan-4-o-
n-2-yl)-.beta.-D-xylofuranosyl]-6-methoxypurine,
[0875] 9-[3-O-Acetyl-2-bromo-2-deoxy-5-O-(2,5
,5-trimethyl-1,3-dioxolan-4--
on-2-yl)-.beta.-D-arabino-furanosyl]guanine,
[0876]
9-[3-O-Acetyl-2-bromo-2-deoxy-5-O-(2,5,5-trimethyl-1,3-dioxolan-4-o-
n-2-yl)-.beta.-D-arabino-furanosyl]-6-chloropurine,
[0877]
9-[3-O-Acetyl-2-bromo-2-deoxy-5-O-(2,5,5-trimethyl-1,3-dioxolan-4-o-
n-2-yl)-.beta.-D-arabino-furanosyl]-2,6-dichloropurine,
[0878]
9-[3-O-Acetyl-2-bromo-2-deoxy-5-O-(2,5,5-trimethyl-1,3-dioxolan-4-o-
n-2-yl)-.beta.-D-arabino-furanosyl]-2-amino-6-chloropurine,
[0879]
9-[3-O-Acetyl-2-bromo-2-deoxy-5-O-(2,5,5-trimethyl-1,3-dioxolan-4-o-
n-2-yl)-.beta.-D-arabino-furanosyl]-6-methylthiopurine and
[0880] (i)
9-[3-O-Acetyl-2-bromo-2-deoxy-5-O-(2,5,5-trimethyl-1,3-dioxolan-
-4-on-2-yl)-.beta.-D-arabino-furanosyl]-6-methoxypurine.
Example 16
[0881] 2', 3'-Anhydroadenosine (18, Y.dbd.NH.sub.2, Z.dbd.H).
[0882]
9-[2-O-Acetyl-3-bromo-3-deoxy-5-O-(2,5,5-trimethyl-1,3-dioxolan-4-o-
n-2-yl)-.beta.-D-xylo-furanosyl]adenine 14 (5.0 g, 0.01 mol) is
treated with 1M sodium methoxide in methanol (20 mL) for 1 hour at
room temperature. The mixture is neutralized with glacial acetic
acid, and is kept refrigerator overnight. Crystalline 18 deposited
is collected by filtration, 2.1 g (84%). .sup.1H NMR spectrum of
this sample is identical with the one prepared by an alternative
procedure by Mendez, E. et al. J. Virol. 1998, 72, 4737.
[0883] In a similar manner but using the corresponding purine
nucleosides, the following 2',3'-anhydro-.beta.-ribo derivatives
(18) and their L-counterparts are prepared: 2',3'-anhydroguanosine,
9-(2,3-anhydro-.beta.-D-ribofuranosyl]-6-methylmercaptopurine, and
9-(2,3-anhydro-.beta.-D-ribo-furanosyl]-2-amino-6-methoxypurine.
Example 17
[0884] 9-(3-Deoxy-3-iodo-.beta.-D-xylofuranosyl)adenine (19,
X.dbd.I, Y.dbd.NH.sub.2, Z.dbd.H).
[0885] A mixture of 18 (Y.dbd.NH.sub.2, Z.dbd.H, 1 g, 4 mmol),
sodium iodide (1.5 g, 10 mmol), sodium acetate (100 mg) and acetic
acid (5 mL) in butanone (30 mL) is gently refluxed for 3 hours.
Evaporation of the solvent in vacuo, and trituration of the residue
with water afford 19 (X.dbd.I, Y.dbd.NH.sub.2, Z.dbd.H), 1.2 g
(80%). .sup.1H NMR (D.sub.6-DMSO) .delta.: 8.24, 8.34 (2s, 1H each,
H-2 and H-8), 5.90 (d, 1H, H-1', J.sub.1',2'=4.7 Hz), 4.96 (dd, 1H,
H-2', J.sub.1',2'=4.7, J.sub.2',3'=4.9 Hz), 4.60 (dd, 1H, H-3',
J.sub.2',3'=4.9, J.sub.3',4'=4.7 Hz), 4.80 (d, 2H, H-5',5"), 4.40
(m, 1H, H-4').
[0886] In a similar manner but using the corresponding
2',3'-anhydro-D-ribo purine nucleosides (14), the following
3'-deoxy-3'-iodo-D-xylo nucleosides and their L-counterparts are
prepared:
[0887] 9-(3-Deoxy-3-iodo-.beta.-D-xylofuranosyl)guanine,
[0888]
9-(3-Deoxy-3-iodo-.beta.-D-xylofuranosyl)-6-methylmercaptopurine,
[0889]
9-(3-Deoxy-3-iodo-.beta.-D-xylofuranosyl)-6-methoxypurine,
[0890]
9-(3-Deoxy-3-iodo-.beta.-D-xylofuranosyl)-2-amino-6-methylmercaptop-
urine and
[0891]
9-(3-Deoxy-3-iodo-.beta.-D-xylofuranosyl)-2-amino-6-methoxypurine.
Example 18
[0892] 3'-Deoxyadenosine (20, Y.dbd.NH.sub.2, Z.dbd.H).
[0893] A solution of 19 (Y.dbd.NH.sub.2, Z.dbd.H, 380 mg, 1 mmol)
in methanol (75 mL) is shaken in an atmosphere of hydrogen in the
presence of 5% Pd/BaSO.sub.4 catalyst (100 mg) and triethylamine (1
mL) at the initial pressure of 3 atm overnight. After removal of
the catalyst, the solvent is evaporated in vacuo, and the residue
is crystallized from methanol to give 3'-deoxyadenosine 20
(Y.dbd.NTH.sub.2, Z.dbd.H), 200 mg (80%). The .sup.1H NMR spectrum
of this sample is identical with that of cordycepin.
[0894] In a similar manner but using the corresponding
3'-iodo-D-xylo purine nucleosides (19), the following
3'-deoxy-nucleosides and their L-counterparts are prepared:
9-(3-Deoxy-.beta.-D-erythropentofuranosyl)gu- anine,
9-(3-deoxy-.beta.-D-erythropentofuranosyl) purine,
9-(3-deoxy-.beta.-D-erythropentofuranosyl)-6-methoxypurine,
9-(3-deoxy-.beta.-D-erythropento-furanosyl)-2-amino-purine and
9-(3-deoxy-.beta.-D-erythropentofuranosyl)-2-amino-6-methoxypurine.
Example 19
[0895] 3-(.beta.-D-Ribofuranosyl)-8-azaxanthine (24, X.dbd.OH,
Y.dbd.N).
[0896] To a solution of 5-nitrouridine (300 mg) in DMF (60 mL) is
added sodium azide (100 mg), and the mixture is stirred overnight
at room temperature. The solvent is removed in vacuo, and the
residue is dissolved in minimal amount of hot water and the pH
adjusted to 3-4 with diluted hydrochloric acid. The precipitates
are recrystallized from water, mp 164-166.degree. C. (dec). anal
Calcd for C.sub.9H.sub.11N.sub.5O.sub.6H.sub.2O : C, 35.64; H,
4.29; N, 23.1. Found: C, 35.96; H, 4.01; N, 23.43.
Example 20
[0897]
1,2-O-Isopropylidene-5-O-methoxycarbonyl-3-O-phenoxythiocarbonyl-.a-
lpha.-D-xylofuranose (26, R.dbd.Ph).
[0898] To a solution of
1,2-O-isopropylidene-5-O-methoxycarbonyl-cc-D-xylo- furanose (25,
25.0g, 0.1 mol) and 4-dimethylaminopyridine (25 g, 0.2 mol) in dry
pyridine (250 mL) is added drop wise a solution of phenyl
chlorothionoformate (50 g, 0.3 mol) in acetonitrile (100 mL), and
the reaction mixture is stirred at 50-60.degree. C. for 24 hours.
The solution is concentrated in vacuo, and the residue is
partitioned between methylene chloride and water. The organic layer
is washed successively with water, 0.1N sodium hydroxide, water,
0.1N hydrochloric acid and water, and dried over sodium sulfate,
and concentrated in vacuo to give 26 (R.dbd.Ph) as a syrup in
quantitative yield (38.2 g). This syrup is used directly in the
next step.
Example 21
[0899]
3-Deoxy-1,2-O-isopropylidene-5-O-methoxycarbonyl-.beta.-D-erythrope-
ntofuranose (27).
[0900] A solution of tri-n-butyltin hydride (58 g, 0.2 mol) in
toluene (300 mL) is added over a period of 3 hours to a refluxing
solution of compound 26 (R.dbd.Ph) above (19.2 g, 50 mmol) and
2,2'-azobisisobutyronitrile (2.5 g, 15 mmol) in toluene (400 mL).
The mixture is concentrated in vacuo, and the residue is dissolved
in acetonitrile (300 mL), and the solution is extracted with
petroleum ether (4.times.100 mL) to remove tri-n-butyltin
derivatives. The acetonitrile layer is concentrated. The thin layer
chromatography of the residue shows one major spot and .sup.1H NMR
spectrum indicates the presence of three methyl groups and no
aromatic protons but contamination of a small amount of butyltin
derivatives. Without further purification, this product is used in
the next step.
Example 22
[0901]
1,2-Di-O-acetyl-3-deoxy-5-O-methoxycarbonyl-D-erythropentofuranose
(28).
[0902] To a stirred solution of 23 (2.32 g, 0.01 mol) in a mixture
of acetic acid (60 mL) and acetic anhydride (6 mL) is added drop
wise concentrated sulfuric acid (3 mL) with ice-cooling at such a
rate that the temperature is maintained at 15-25.degree. C. After
standing overnight at room temperature, ice (250 g) is added to the
solution, and then the mixture is extracted with methylene chloride
(3.times.50 mL). The combined extracts are washed with saturated
sodium bicarbonate solution (3.times.30 mL), dried over sodium
sulfate, and concentrated in vacuo to give 28 (2.8 g, 100%) as an
anomeric mixture. This compound is sufficiently pure to be used in
the next step without further purification.
Example 23
[0903]
1-(2-O-acetyl-3-deoxy-5-O-methoxycarbonyl-pBD-erythropentofuranosyl-
)-5-fluorouracil (29, X.dbd.OH, Z.dbd.F).
[0904] A mixture of 5-fluorouracil (2.6 g, 0.02 mol), ammonium
sulfate (ca. 30 mg) in hexamethyldisilazane (15 mL) is refluxed
until a clear solution is obtained. The solvent is removed in
vacuo, and the residue is dissolved in 1,2-dichloroethane (20 mL),
and 1,2-di-O-acetyl-3-deoxy-5-O--
methoxycarbonyl-D-erythropentofuranose (28, 5.5 g, 0.02 mol) in
1,2-dichloroethane (20 mL) is added. To the solution is added tin
tetrachloride (5.2 g, 0.02 mol), and the mixture is stirred
overnight at room temperature, then is heated for 3 hours at
40-50.degree. C. for 3 hours. Saturated sodium bicarbonate solution
(40 mL) is added and stirred until carbon dioxide evolution ceases.
The mixture is filtered through a Celite pad. The organic layer is
separated, washed carefully with saturated sodium bicarbonate
solution (20 mL.times.2) and water (20 mL.times.2), dried over
sodium sulfate, and concentrated to dryness in vacuo. The residue
is crystallized from ethanol to give 29 (4.3 g, 62%).
[0905] In a similar manner but using the corresponding pyrimidine
bases, the following 2',5'-protected 3'-deoxy-nucleosides and their
L-counterparts are prepared:
[0906]
1-(2-Acetyl-3-deoxy-5-O-methoxycarbonyl-.beta.-D-erythropentofurano-
syl)-5-chlorouracil,
[0907]
1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-,p-D-erythropentofuranosy-
l)-5-bromouracil,
[0908]
1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-.beta.-D-erythropentofara-
nosyl)-5-iodouracil,
[0909]
1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-.beta.-D-erythropentofura-
nosyl)-5-cyanouracil,
[0910]
1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-.beta.-D-erythropentofura-
nosyl)-5-ethoxycarbonyl-uracil,
[0911]
1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-.beta.-D-erythropentofura-
nosyl)-5-aminocarbonyl-uracil,
[0912]
1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-.beta.-D-erylhropentofura-
nosyl)-5-acetyluracil,
[0913]
1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-.beta.-D-erythropentofura-
nosyl)-5-methyluracil,
[0914]
1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-.beta.-D-erythropentofura-
nosyl)-5-ethyluracil,
[0915]
1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-.beta.-D-erythropentofura-
nosyl)-5-n-propyluracil,
[0916]
1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-.beta.-D-erythropentofura-
nosyl)-5-i-propyluracil,
[0917]
1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-.beta.-D-erythropentofura-
nosyl)-5-vinyluracil,
[0918]
1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-.beta.-D-erythropentofura-
nosyl)-5-allyluracil,
[0919]
1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-.beta.-D-erythropentofura-
nosyl)-5-ethynyluracil,
[0920]
1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-.beta.-D-erythropentofura-
nosyl)-5-(2-chlorovinyl)-uracil,
[0921]
1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-.beta.-D-erythropentofura-
nosyl)-5-(2-bromovinyl)-uracil,
[0922]
1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-.beta.-D-erythropentofura-
nosyl)-5-(2-iodovinyl)-uracil,
[0923]
1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-.beta.-D-erythropentofura-
nosyl)-5-(2-methoxylcarbonyl-vinyl)uracil,
[0924]
1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-.beta.-D-erythropentofura-
nosyl)-5-(2-hydroxycarbonyl-vinyl)uracil,
1-(2-O-Acetyl-3-deoxy-5-O-methox-
ycarbonyl-.beta.-D-erythropentofuranosyl)-5-phenyluracil,
[0925]
1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-3-D-erythropentofuranosyl-
)-5-benzyluracil,
1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-.beta.-D-eryth-
ropentofuranosyl)-5-fluorocytosine,
[0926]
1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-.beta.-D-erythropentofura-
nosyl)-5-chlorocytosine,
[0927]
1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-.beta.-D-erythropentofura-
nosyl)-5-bromocytosine,
[0928]
1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-.beta.-D-erythropentofura-
nosyl)-5-iodocytosine,
[0929]
1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-.beta.-D-erythropentofura-
nosyl)-5-ethylcyanocytosine,
[0930]
1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-.beta.-D-erythropentofura-
nosyl)-5aiethoxycarbonyl-cytosine,
[0931]
1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-.beta.-D-erythropentofura-
nosyl)-5-aminocarbonyl-cye osine,
[0932]
1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-.beta.-D-erythropentofura-
nosyl)-5-acetylcytosine,
[0933]
1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-.beta.-D-erythropentofura-
nosyl)-5-methylcytosine,
[0934]
1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-.beta.-D-erythropentofura-
nosyl)-5-ethylcytosine,
[0935]
1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-.beta.-D-erythropentofura-
nosyl)-5-n-propylcytosine,
[0936]
1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-.beta.-D-erythropentofura-
nosyl)-5-i-propyleytosine,
[0937]
1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-.beta.-D-erythropentofura-
nosyl)-5-vinyicytosine,
[0938]
1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-.beta.-D-erythropentofura-
nosyl)-5-allylcytosine,
[0939]
1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-.beta.-D-erythropentofura-
nosyl)-5-ethynylcytosine,
[0940]
1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-.beta.-D-erythropentofura-
nosyl)-5-(2-chlorovinyl)-cytosine,
[0941]
1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-.beta.-D-erythropentofura-
nosyl)-5-(2-bromovinyl)-cytosine,
[0942]
1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-.beta.-D-erythropentofura-
nosyl)-5-(2-iodovinyl)-cytosine,
[0943]
1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-.beta.-D-erythropentofura-
nosyl)-5-(2-methoxyl-carbonylvinyl)cytosine,
[0944]
1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-.beta.-D-erythropentofura-
nosyl)-5-(2-hydroxycarbonylvinyl)cytosine,
[0945]
1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-.beta.-D-erythropentofura-
nosyl)-5-phenylcytosine and
[0946]
1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-.beta.-D-erythropentofura-
nosyl)-5-benzylcytosine.
[0947] In a similar manner but using the corresponding pyrimidine
and purine bases, the following 2',5'-di-O-acetyl
3'-deoxy-nucleosides and their L-counterparts are prepared:
[0948]
1-(2,5-Di-O-acetyl-3-deoxy-.beta.-D-erythropentofuranosy)-5-chlorou-
racil,
[0949]
1-(2,5-Di-O-acetyl-3-deoxy-.beta.-D-erythropentofuranosyl)-5-bromou-
racil,
[0950]
1-(2,5-Di-O-acetyl-3-deoxy-.beta.-D-erythropentofuranosyl)-5-iodour-
acil,
[0951]
1-(2,5-Di-O-acetyl-3-deoxy-.beta.-D-erythropentofuranosyl)-5-cyanou-
racil,
[0952]
1-(2,5-Di-O-acetyl-3-deoxy-.beta.-D-erythropentofuranosyl)-5-ethoxy-
carbonyluracil,
[0953]
1-(2,5-Di-O-acetyl-3-deoxy-.beta.-D-erythropentofuranosyl)-5-ethoca-
rbonyluracil,
[0954]
1-(2,5-Di-O-acetyl-3-deoxy-.beta.-D-erythropentofuranosyl)-5-acetyl-
uraci l ,
[0955]
1-(2,5-Di-O-acetyl-3-deoxy-.beta.-D-erythropentofuranosyl)-5-methyl-
uracil,
[0956]
1-(2,5-Di-O-acetyl-3-deoxy-.beta.-D-erythropentofuranosyl)-5-ethylu-
racil,
[0957]
1-(2,5-Di-O-acetyl-3-deoxy-.beta.-D-erythropentofuranosyl)-5-n-prop-
yluracil,
[0958]
1-(2,5-Di-O-acetyl-3-deoxy-.beta.-D-erythropentofuranosyl)-5-i-prop-
yluracil,
[0959]
1-(2,5-Di-O-acetyl-3-deoxy-.beta.-D-erythropentofuranosyl)-5-vinylu-
racil,
[0960]
1-(2,5-Di-O-acetyl-3-deoxy-.beta.-D-erythropentofuranosyl)-5-allylu-
racil,
[0961]
1-(2,5-Di-O-acetyl-3-deoxy-.beta.-D-erythropentofuranosyl)-5-ethyny-
luracil,
[0962]
1-(2,5-Di-O-acetyl-3-deoxy-.beta.-D-erythropentofuranosyl)-5-(2-chl-
orovinyl)uracil,
[0963]
1-(2,5-Di-O-acetyl-3-deoxy-.beta.-D-erythropentofuranosyl)-5-(2-bro-
movinyl)uracil,
[0964]
1-(2,5-Di-O-acetyl-3-deoxy-.beta.-D-erythropentofuranosyl)-5-(2-iod-
ovinyl)uracil,
[0965]
1-(2,5-Di-O-acetyl-3-deoxy-.beta.-D-erythropentofaranosyl)-5-(2-met-
hoxylcarbonylvinyl)uracil,
[0966]
1-(2,5-Di-O-acetyl-3-deoxy-.beta.-D-crythropentofuranosyl)-5-(2-hyd-
roxycarbonylvinyl)uracil,
[0967]
1-(2,5-Di-O-acetyl-3-deoxy-.beta.-D-erythropentofuranosyl)-5-phenyl-
uracil,
[0968]
1-(2,5-Di-O-acetyl-3-deoxy-.beta.-D-erythropentofuranosyl)-5-benzyl-
uracil,
[0969]
1-(2,5-Di-O-acetyl-3-deoxy-.beta.-D-erythropentofuranosyl)-5-fluoro-
cytosine,
[0970]
1-(2,5-Di-O-acetyl-3-deoxy-.beta.-D-erythropentofuranosyl)-5-chloro-
cytosine,
[0971]
1-(2,5-Di-O-acetyl-3-deoxy-.beta.-D-erythropentofuranosyl)-5-bromoc-
ytosine,
[0972]
1-(2,5-Di-O-acetyl-3-deoxy-.beta.-D-erythropentofuranosyl)-5-iodocy-
tosine,
[0973]
1-(2,5-Di-O-acetyl-3-deoxy-.beta.-D-erythropentofuranosyl)-5-cyanoc-
ytosine,
[0974]
1-(2,5-Di-O-acetyl-3-deoxy-.beta.-D-erythropentofuranosyl)-5-ethoxy-
carbonylcytosine,
[0975]
1-(2,5-Di-O-acetyl-3-deoxy-.beta.D-erythropentofuranosyl)-5-aminoca-
rbonylcytosine,
[0976]
1-(2,5-Di-O-acetyl-3-deoxy-.beta.-D-erythropentofuranosyl)-5-acetyl-
cytosine,
[0977]
1-(2,5-Di-O-acetyl-3-deoxy-.beta.-D-erytfropentofiranosyl)-5-methyl-
cytosine,
[0978]
1-(2,5-Di-O-acetyl-3-deoxy-.beta.-D-erythropentofuranosyl)-5-ethylc-
yt osine,
[0979]
1-(2,5-Di-O-acetyl-3-deoxy-.beta.-D-erythropentofuranosyl)-5-n-prop-
ylcytosine,
[0980]
1-(2,5-Di-O-acetyl-3-deoxy-.beta.-D-erythropentofuranosyl)-5-i-prop-
ylcytosine,
[0981]
1-(2,5-Di-O-acetyl-3-deoxy-.beta.-D-erythropentofuranosyl)-5-vinylc-
ytosine,
[0982]
1-(2,5-Di-O-acetyl-3-deoxy-.beta.-D-erythropentofuranosyl)-5-allylc-
ytosine,
[0983]
1-(2,5-Di-O-acetyL3-deoxy-.beta.-D-erythropentofuranosyl)-5-ethynyl-
cytosine,
[0984]
1-(2,5-Di-O-acetyl-3-deoxy-.beta.-D-erythropentofuranosyl)-5-(2-cmh-
orovinyl)cytosine,
[0985]
1-(2,5-Di-O-acetyl-3-deoxy-.beta.-D-erythropentofuranosyl)-5-(2-bro-
movinyl)cytosine,
[0986]
1-(2,5-Di-O-acetyl-3-deoxy-.beta.-D-erythropentofuranosyl)-5-(2idvi-
nylcytosine,
[0987]
1-(2,5-Di-O-acetyl-3-deoxy-.beta.-D-erythropentofuranosyl)-5-(2meth-
oxylcarbonylvinyl)cytosine,
[0988]
1-(2,5Di-O-acetyl-3-deoxy-.beta.-D-erythropentofuranosyl)-5-(2-hydr-
oxycarbonylvinyl)cyto sine,
[0989] 1
(2,5-Di-O-acetyl-3-deoxy-.beta.-D-erythropentofuranosyl)-5-phenyl-
cytosine,
[0990]
1-(2,5-Di-O-acetyl-3-deoxy-.beta.-D-erythropentofuranosyl)-5-benzyl-
cytosine,
[0991]
1-(2,5-Di-O-acetyl-3-deoxy-.beta.-D-erythropentofuranosyl)-N6-benz:-
oyladenine,
[0992]
1-(2,5-Di-O-acetyl-3deoxy-.beta.-D-erythropentofuranosyl)-6-chlorop-
urine,
[0993]
1-(2,5-Di-O-acetyl-3-deoxy-.beta.-D-erythropentofuranosyl)-2,6-dich-
loroputine,
[0994]
1-(2,5-Di-O-acetyl-3-deoxy-.beta.-D-erythropentofuranosyl)-6-methox-
ypurine and
[0995]
1(2,5-Di-O-acetyl-3-deoxy-.beta.-D-erythropentofuranosyl)-6-methylm-
ercaptopurine.
Example 24
[0996]
1-(2-O-acetyl-3-deoxy-5-O-methoxycarbonyl-.beta.-D-erythropentofura-
nosyl)-6-chloropurine (30, X.dbd.Cl, Y.dbd.H).
[0997] A mixture of 6-chloropurine (3.1 g, 0.02 mol), ammonium
sulfate (ca. 30 mg) in hexamethyldisilazane (25 mL) is refluxed
until a clear solution is obtained. The solvent is removed in
vacuo, and the residue is dissolved in 1,2-dichloroethane (30 mL),
and 1,2-di-O-acetyl-3-deoxy-5-O--
methoxycarbonyl-D-erythropentofuranose (28, 5.5 g, 0.02 mol) in
1,2-dichloroethane (20 mL) is added. To the solution is added tin
tetrachloride (5.2 g, 0.02 mol), and the mixture is stirred
overnight at room temperature, then is heated for 3 hours at
40-50.degree. C. for 3 hours. Saturated sodium bicarbonate solution
(50 mL) is added and stirred until carbon dioxide evolution ceases.
The mixture is filtered through a Celite pad. The organic layer is
separated, washed carefully with saturated sodium bicarbonate
solution (30 mL.times.2) and water (30 mL.times.2), dried over
sodium sulfate, and concentrated to dryness in vacuo. The residue
is crystallized from ethanol to give 30 (4.3 g, 62%).
[0998] In a similar manner but using the corresponding purine
bases, the following 2',5'-protected 3'-deoxy-nucleosides and their
L-counterparts are prepared:
[0999]
1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-.beta.-D-erythropentofura-
nosyl)-N.sup.6 benzoyladenine,
[1000]
1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-.beta.-D-erythropentofura-
nosyl)-6-chloropurine,
[1001]
1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-.beta.-D-erythropentofura-
nosyl)-2,6-dichloropurine,
[1002]
1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-.beta.-D-erythropentofura-
nosyl)-2-acetamido-6-chloropurine,
[1003]
1-(2-Acetyl-3-deoxy-5-O-methoxycarbonyl-.beta.-D-erythropentofurano-
syl)-2-acetamido-6-methoxypurine,
[1004]
1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-.beta.-D-erythropentofura-
nosyl)-6-methoxypurine and
[1005]
1-(2-O-Acetyl-3-deoxy-5-O-methoxycarbonyl-.beta.-D-erythropentofura-
nosyl)-6-methylmercapto-purine.
Example 25
[1006]
1,2-O-Isopropylidene-5-O-t-butyldiphenylsilyl-.alpha.-D-xylofuranos-
e (31).
[1007] A mixture of 1,2-O-isopropylidene-a-D-xylofuranose (38.0 g,
0.2 mol), t-butyl-diphenylchlorosilane (70 g, 0.25 mol) and
imidazole (21.5 g, 0.4 mol) in N,N-dimethylformamide (50 mL) is
stirred at room temperature for 1 hour. The solvent is removed in
vacuo, and the residue is dissolved in ethyl acetate (1 L), and
extracted with water (300 mL.times.2) and brine (300 mL), dried
over sodium sulfate, and concentrated to dryness in vacuo to give
crude 31 (86 g, 100%), which is used directly in the next step
without further purification.
Example 26
[1008]
1,2-O-Isopropylidene-3-O-mesyl-5-O-t-butyldiphenylsilyl-.beta.-D-xy-
lofuranose (32, R.dbd.Ms).
[1009] Mesyl chloride (17 g, 0.15 mol) is added drop wise to a
solution of crude 31 (43 g, 0.1 mol) in pyridine (100 mL), and the
mixture is kept standing overnight at room temperature. Crashed ice
(1 L) is added to the mixture, and the product is extracted with
methylene chloride (300 mL.times.3). The extracts are combined,
washed with water (300 mL.times.2) and brine (300 mL), dried over
sodium sulfate, and concentrated in vacuo to dryness. Traces of
pyridine are removed by repeated azeotropic distillation with
toluene. The residue is dissolved in methylene chloride (500 mL)
and washed with O.IN hydrochloric acid (250 mL.times.2) and water,
dried over sodium sulfate, and concentrated to dryness to give
crude 32 (R Ms), 50.1 g (99%). The .sup.1H NMR spectrum of this
material is sufficiently pure to be used directly in the next
step.
Example 27
[1010] Methyl 3-O-mesyl-5-O-t-butyldiphenylsilyl-D-xylofuranoside
(33, R.dbd.Ms).
[1011] A solution of crude 32 (50 g, 0.1 mol) in 1% anhydrous
methanolic hydrogen chloride (IL) is kept overnight at room
temperature, and then evaporated in vacuo to a syrup which is
partitioned between water (100 mL) and methylene chloride (150 mL).
The organic layer is separated, washed with water (100 mL), dried
over sodium sulfate, and concentrated in vacuo, giving crude 33, a
syrup, weighing 48 g (100%). This material is not further purified
but used directly in the next step.
Example 28
[1012] Methyl 2,3-anhydro-5-O-t-butyldiphenylsilyl-D-fibofuranoside
(34).
[1013] Crude 33 (48 g, 0.1 mol) is dissolved in methylene chloride
(100 mL) and treated with 2M methanolic sodium methoxide (60 mL),
and refluxed for 2 hours. Insoluble salt is removed by filtration,
and the filtrate is concentrated in vacuo to dryness. The residue
is dissolved in methylene chloride (150 mL), washed with water (100
mL.times.2), dried over sodium sulfate, and concentrated to dryness
to give crude 30 (38 g, 100%), which can be used directly in the
next step without purification.
Example 29
[1014] Methyl
3-deoxy-3-iodo-5-O-t-butyldiphenylsilyl-D-ribofuranoside (35,
X.dbd.I).
[1015] A mixture of 34 (38 g, 0.1 mol), sodium iodide (60 g, 0.4
mol), sodium acetate (0.6 g) and acetic acid (70 mL) in acetone
(500 mL) is heated under reflux for 8 hours. The acetone is removed
in vacuo, and the residue is partitioned between methylene chloride
(500 mL) and water (250 mL). The organic layer is separated, washed
with 250 mL each of water, 0.1 M sodium thiosulfate solution, water
and dried over sodium sulfate. After removal of the solvent in
vacuo, the residue is crystallized from ethanol to afford 31 g
(60.5%) of 35 (X.dbd.I).
Example 30
[1016] Methyl
3-deoxy-5-O-t-butyldiphenylsilyl-D-erythropentofuranoside (37, from
35).
[1017] Compound 35 (X.dbd.I, 25.6 g, 0.05 mol) is hydrogenated in
ethyl acetate (250 mL) with 5% palladium on charcoal (2 g). After
the consumption of hydrogen ceased, the mixture is filtered, and
the filtrate is washed with water (150 mL.times.2), dried over
sodium sulfate, and concentrated to dryness to give crude 37 (19 g,
quantitative yield) which is sufficiently pure to be used directly
in the next step.
Example 31
[1018] Methyl
3-deoxy-5-O-t-butyldiphenylsilyl-D-erythropentofuranoside (36, from
34).
[1019] A suspension of lithium aluminum hydride (8.4 g, 0.2 mol) in
dry ethyl ether (220 mL) is stirred under nitrogen atmosphere and
cooled in an ice bath. To this suspension is added drop wise a
solution of 34 (19 g, 0.05 mol) in dry tetrahydrofuran (250 mL) at
such a rate that the temperature remains below 25.degree. C. After
2 hours, another l g of lithium aluminum hydride is charged, and
the mixture is stirred overnight at room temperature. The stirred
mixture is cooled in an ice bath, and isopropanol (100 mL) is added
drop wise, followed by acetone (50 mL). The mixture is concentrated
in vacuo, and the residue is partitioned between ethyl ether (250
mL) and water (150 mL). Insoluble materials are filtered through
Celite pad which is washed with ether. The ether layer is
separated, washed successively with 0.2N hydrochloric acid (150
mL.times.2) and water (150 mL.times.2), dried over sodium sulfate,
and then concentrated to dryness to give crude 36 (16.5 g,
87%).
Example 32
[1020] Methyl 3-deoxy-D-erythropentofuranoside (38).
[1021] To a solution of crude 36 (13 g, 0.03 mol) in
tetrahydrofuran (320 mL) is added drop wise IM solution of
triethylammonium hydrogen fluoride (100 mL), and the mixture is
stirred for 24 hours. The mixture is concentrated in vacuo, and the
residue is dissolved in water (200 mL). Powdered calcium carbonate
(20 g) is added, and the mixture is stirred overnight at room
temperature, and then filtered. The filtrate is concentrated in
vacuo to a syrup which is dissolved in chloroform (200 mL),
filtered, and evaporated in vacuo to afford crude 38 (4.5 g,
100%).
Example 33
[1022] 1,2,5-Tri-O-acetyl-3-deoxy-D-erythropentofuranose (38).
[1023] To a vigorously stirred mixture of crude methyl
3-deoxy-D-erythropentofuranoside 37 (4.5 g, 0.03 mol) and acetic
acid (80 mL) is added acetic anhydride (40 mL), followed by
sulfuric acid (4 mL), and the reaction mixture is stirred overnight
at room temperature. The mixture is partitioned between methylene
chloride (150 mL) and ice-water (400 mL). The water layer is
extracted with methylene chloride (100 mL.times.2). The combined
organic layers are washed twice with equal volumes of a saturated
solution of sodium bicarbonate, once with water, dried over sodium
sulfate, and concentrated to dryness in vacuo. Traces of acetic
acid are removed by several azeotropic distillations with toluene
to give crude 38 (5.1 g, 66%). The .sup.1H NMR spectrum shows that
the major constituent of this product contains 3 acetyl groups and
is the .beta.-anomer.
Example 34
[1024] 1-(3-Deoxy-.beta.-D-erythropentofuranosyl)-5-fluorouraceil
(3'-deoxy-5-fluorouridine,6b, X.dbd.OH, R.dbd.F).
[1025] A mixture of an acetyl derivative of 39 (X.dbd.OH, Z.dbd.F,
3.3 g, 0.01 mol) and triethylamine (3 mL) in methanol (100 mL) is
stirred overnight at room temperature. The mixture is concentrated
in vacuo to dryness, and the residue is crystallized from ethanol
to give 3'-deoxy-5-fluorouridine (2.0 g, 83%), mp 169-171.degree.
C. .sup.1H NMR (D.sub.6-DMSO) .delta.: 11.7 (bs, 1H, N.sup.3-H,
exchangeable), 8.44 (d, 1H, H-6, J.sub.6,F=7.1 Hz),5.7 (d, 1H,
2'-OH, exchangeable), 5.5 (narrow m, 1H, H-i'), 5.3 (t, 1H, 5'-OH,
exchangeable), 4.1-4.5 (m, 2H, H-2' and H-4'), 3.5-3.9 (m, 2H,
H-5',5"), 1.6-2.2 (m, 2H, H-3',3").
[1026] In a similar manner but using the corresponding
2',5'-di-O-acetyl pyrimidine and purine nucleoside, the following
3'-deoxy-nucleosides and their L-counterparts are prepared:
[1027]
1-(3-Deoxy-.beta.-D-erythropentofuranosyl)-5-chlorouracil,
[1028]
1-(3-Deoxy-.beta.-D-erythropentofuranosyl)-5-bromouracil,
[1029] 1-(3-Deoxy--D-erythropentofuranosyl)-5-iodouracil,
[1030]
1-(3-Deoxy-.beta.-D-erythropentofuranosyl)-5-cyanouracil,
[1031]
1-(3-Deoxy-.beta.-D-erythropentofuranosyl)-5-ethoxycarbonyluracil,
[1032]
1-(3-Deoxy-.beta.-D-erythropentofuranosyl)-5-aminocarbonyluracil,
[1033]
1-(3-Deoxy-.beta.-D-erythropentofuranosyl)-5-acetyluracil,
[1034]
1-(3-Deoxy-.beta.-D-erythropentofuranosyl)-5-methyluracil,
[1035]
1-(3-Deoxy-.beta.-D-erythropentofuranosyl)-5-ethyluracil,
[1036]
1-(3-Deoxy-.beta.-D-erythropentofuranosyl)-5-n-propyluracil,
[1037]
1-(3-Deoxy-.beta.-D-erythropentofuranosyl)-5-i-propyluracil,
[1038]
1-(3-Deoxy-.beta.-D-erythropentofuranosyl)-5-vinyluracil,
[1039]
1-(3-Deoxy-.beta.-D-erythropentofuranosyl)-5-anlyluracil,
[1040]
1-(3-Deoxy-.beta.-D-erythropentofuranosyl)-5-ethynyluracil,
[1041]
1-(3-Deoxy-.beta.-D-erythropentofuranosyl)-5-(2-chlorovinyl)uracil,
[1042]
1-(3-Deoxy-.beta.-D-erythropentofuranosyl)-5-(2-bromovinyl)uracil,
[1043]
1-(3-Deoxy-.beta.-D-erythropentofuranosyl)-5-(2-iodovinyl)uracil,
[1044]
1-(3-Deoxy-.beta.-D-erythropentofuranosyl)-5-(2-methoxylcarbonylvin-
yl)uracil,
[1045]
1-(3-Deoxy-.beta.-D-erythropentofuranosyl)-5-(2-hydroxycarbonylviny-
l)uracil,
[1046]
1-(3-Deoxy-.beta.-D-erythropentofuranosyl)-5-phenyluracil,
[1047]
1-(3-Deoxy-.beta.-D-erythropentofuranosyl)-5-benzyluracil,
[1048] 1-(3-Deoxy-.beta.-D-erythropentofuranosyl)cytosine,
[1049]
1-(3-Deoxy-.beta.-D-erythropentofuranosyl)-5-fluorocytosine,
[1050]
1-(3-Deoxy-.beta.-D-erythropentofuranosyl)-5-chlorocytosine,
[1051]
1-(3-Deoxy-.beta.-D-erythropentofuranosyl)-5-bromocytosine,
[1052]
1-(3-Deoxy-.beta.-D-erythropentofuranosyl)-5-iodocytosine,
[1053]
1-(3-Deoxy-.beta.-D-erythropentofuranosyl)-5-cyanocytosine,
[1054]
1-(3-Deoxy-.beta.-D-erythropentofuranosyl)-5-ethoxycarbonylcytosine-
,
[1055]
1-(3-Deoxy-.beta.-D-erythropentofuranosyl)-5-aminocarbonylcytosine,
[1056]
1-(3-Deoxy-.beta.-D-erythropentofuranosyl)-5-acetylcytosine,
[1057]
1-(3-Deoxy-.beta.-D-erythropentofuranosyl)-5-methylcytosine,
[1058]
1-(3-Deoxy-.beta.-D-erythropentofuranosyl)-5-ethylcytosine,
[1059]
1-(3-Deoxy-.beta.-D-erythropentofuranosyl)-5-n-propylcytosine,
[1060]
1-(3-Deoxy-.beta.-D-erythropentofuranosyl)-5-i-propylcytosine,
[1061]
1-(3-Deoxy-.beta.-D-erythropentofuranosyl)-5-vinylcytosine,
[1062] 1-(3-Deoxy-.beta.-erythropentofuranosyl)-5-allycytosine,
[1063]
1-(3-Deoxy-.beta.-D-erythropentofuranosyl)-5-ethynylcytosine,
[1064]
1-(3-Deoxy-.beta.-D-erythropentofuranosyl)-5-(2-chlorovinyl)cytosin-
e,
[1065]
1-(3-Deoxy-.beta.-D-erythropentofuranosyl)-5-(2-bromovinyl)cytosine-
,
[1066]
1-(3Deoxy--D-erythropentofuranosyl)-5-(2-iodovinyl)cytosine,
[1067]
1-(3-Deoxy-.beta.-D-erythropentofuranosyl)-5-(2-methoxylcarbonylvin-
yl)cytosine,
[1068]
1-(3-Deoxy-.beta.-D-erythropentofuranosyl)-5-(2-hydroxycarbonyl
vinyl)cytosine,
[1069]
1-(3-Deoxy-.beta.-D-erythropentofuranosyl)-5-phenylcytosine,
[1070]
1-(3-Deoxy-.beta.-D-erythropentofuranosyl)-5-benzylcytosine,
[1071]
1-(3-Deoxy-.beta.-D-erythropentofuranosyl)-2-chloroadenine,
[1072]
1-(3-Deoxy-.beta.-D-erythropentofuranosyl)-6-chloropurine,
[1073]
1-(3-Deoxy-.beta.-D-erythropentofuranosyl)-2,6-dichloropurine,
[1074]
1-(3-Deoxy-.beta.-D-erythropentofuranosyl)-2-acetamido-6-chloropuri-
ne,
[1075]
1-(3-Deoxy-.beta.-D-erythropentofuranosyl)-2-acetamido-6-methoxypur-
ine,
[1076] 1-(3-Deoxy-.beta.-D-erythropentofuranosyl)-6-methoxypurine
and
[1077]
1-(3-Deoxy-.beta.-D-erythropentofuranosyl)-6-methylmercaptopurine.
Example 35
[1078]
1-(2,5-Di-O-acetyl-3-O-mesyl-.beta.-D-xylofuranosyl)-5-fluorouracil-
.
[1079] A mixture of 5-fluorouracil (0.02 mol), ammonium sulfate
(ca. 30 mg) in hexamethyldisilazane (15 mL) is refluxed until a
clear solution is obtained. The solvent is removed in vacuo, and
the residue is dissolved in 1,2-dichloroethane (20 mL), and
1,2,5-tri-O-acetyl-3-O-mesyl-D-xylofur- anose (5.5 g, 0.02 mol) in
1,2-dichloroethane (20 mL) is added. To the solution is added tin
tetrachloride (5.2 g, 0.02 mol), and the mixture is stirred
overnight at room temperature, then is heated for 3 hours at
40-50.degree. C. for 3 hours. Saturated sodium bicarbonate solution
(40 mL) is added and stirred until carbon dioxide evolution ceases.
The mixture is filtered through a Celite pad. The organic layer is
separated, washed carefully with saturated sodium bicarbonate
solution (20 mL.times.2) and water (20 mL.times.2), dried over
sodium sulfate, and concentrated to dryness in vacuo. The residue
is crystallized from ethanol to give the title product (62%). The
.sup.1H NMR spectrum of this sample is compatible with the
structure indicated.
[1080] In a similar manner but using the corresponding pyrimidine
and purine bases, the following 2',5'-di-O-acetyl 3'-substituted
xylo-nucleosides and their L-counterparts are prepared:
[1081]
1-(2,5-Di-O-acetyl-3-O-mesyl-.beta.-D-xylofuranosyl)-5-chlorouracil-
,
[1082]
1-(2,5-Di-O-acetyl-3-O-mesyl-.beta.-D-xylofuranosyl)-5-bromouracil,
[1083]
1-(2,5-Di-O-acetyl-3-O-mesyl-.beta.-D-xylofuranosyl)-5-iodouracil,
[1084]
1-(2,5-Di-O-acetyl-3-O-mesyl-.beta.-D-xylofuranosyl)-5-cyanouracil,
[1085]
1-(2,5-Di-O-acetyl-3-O-mesyl-.beta.-D-xylofuranosyl)-5-ethoxycarbon-
yluracil,
[1086]
1-(2,5-Di-O-acetyl-3-O-mesyl-.beta.-D-xylofuranosyl)-5-aminocarbony-
luracil,
[1087]
1-(2,5-Di-O-acetyl-3-O-mesyl-.beta.-D-xylofuranosyl)-5-acetyluracil-
,
[1088]
1-(2,5-Di-O-acetyl-3-O-mesyl-.beta.-D-xylofuranosyl)-5-methyluracil-
,
[1089]
1-(2,5-Di-O-acetyl-3-O-mesyl-.beta.-D-xylofuranosyl)-5-ethyluracil,
[1090]
1-(2,5-Di-O-acetyl-3-O-mesyl-.beta.-D-xylofuranosyl)-5-n-propylurac-
il,
[1091]
1-(2,5-Di-O-acetyl-3-O-mesyl-.beta.-D-xylofuranosyl)-5-i-propylurac-
il,
[1092]
1-(2,5-Di-O-acetyl-3-O-mesyl-.beta.-D-xylofuranosyl)-5-vinyluracil,
[1093]
1-(2,5-Di-O-acetyl-3-O-mesyl-.beta.-D-xylofuranosyl)-5-allyluraeil,
[1094]
1-(2,5-Di-O-acetyl-3-O-mesyl-.beta.-D-xylofuranosyl)-5-ethynyluraci-
l,
[1095]
1-(2,5-Di-O-acetyl-3-O-mesyl-.beta.-D-xylofuranosyl)-5-(2-chlorovin-
yl)uracil,
[1096]
1-(2,5-Di-O-acetyl-3-O-mesyl-.beta.-D-xylofuranosyl)-5-(2-bromoviny-
l)uracil,
[1097]
1-(2,5-Di-O-acetyl-3-O-mesyl-.beta.-D-xylofuranosyl)-5-(2-iodovinyl-
)uracil,
[1098]
1-(2,5-Di-O-acetyl-3-O-mesyl-.beta.-D-xylofuranosyl)-5-(2-methoxylc-
arbonylvinyl)uracil,
[1099]
1-(2,5-Di-O-acetyl-3-O-mesyl-.beta.-D-xylofuranosyl)-5-(2-hydroxyca-
rbonylvinyl)uracll,
[1100]
1-(2,5-Di-O-acetyl-3-O-mesyl-.beta.-D-xylofuranosyl)-5-phenyluracil-
,
[1101]
1-(2,5-Di-O-acetyl-3-O-mesyl-.beta.-D-xylofuranosyl)-5-benzyluracil-
,
[1102]
1-(2,5-Di-O-acetyl-3-O-mesyl-.beta.-D-xylofuranosy)-5-fluorocytosin-
e,
[1103]
1-(2,5-Di-O-acetyl-3-O-mesyl-.beta.-D-xylofuranosyl)-5-chlorocytosi-
ne,
[1104]
1-(2,5-Di-O-acetyl-3-O-mesyl-.beta.-D-xylofuranosyl)-5-bromocytosin-
e,
[1105]
1-(2,5-Di-O-acetyl-3-O-mesyl-.beta.-D-xylofuranosyl)-5-iodocytosine-
,
[1106]
1-(2,5-Di-O-acetyl-3-O-mesyl-.beta.-D-xylofuranosyl)-5-cyanocytosin-
e,
[1107]
1-(2,5-Di-O-acetyl-3-O-mesyl-.beta.-D-xylofuranosyl)-5-ethoxycarbon-
ylcytosine,
[1108]
1-(2,5-Di-O-acetyl-3-O-mesyl-.beta.-D-xylofuranosyl)-5-ethnoxcarbon-
ylcytosine,
[1109] 1-(2,5-Di-O-acetyl-3-O-mesyl-.beta.-D-xylofuranosyl)-5-amet
ylcytosine,
[1110]
1-(2,5-Di-O-acetyl-3-O-mesyl-.beta.-D-xylofuranosyl)-5-methylcytosi-
ne,
[1111]
1-(2,5-Di-O-acetyl-3-O-mesyl-.beta.-D-xylofuranosyl)-5-npoyleytosin-
e,
[1112]
1-(2,5-Di-O-acetyl-3-O-mesyl-.beta.-D-xylofuranosyl)-5-i-propylcyto-
sine,
[1113]
1-(2,5-Di-O-acetyl-3-O-mesyl-.beta.-D-xylofuranosyl)-5-ethylcytosin-
e,
[1114]
1-(2,5-Di-O-acetyl-3-O-mesyl-.beta.-D-xylofuranosyl)-5-n-propylcyto-
sine,
[1115]
1-(2,5-Di-O-acetyl-3-O-mesyl-.beta.-D-xylofuranosyl)-5-ethynylcytos-
ine,
[1116]
1-(2,5-Di-O-acetyl-3-O-mesyl-.beta.-D-xylofuranosyl)-5-vinylcytosin-
e,
[1117]
1-(2,5-Di-O-acetyl-3-O-mesyl-.beta.-D-xylofuranosyl)-5-(2-chlorovin-
yl)cytosine,
[1118]
1-(2,5-Di-O-acetyl-3-O-mesyl-.beta.-D-xylofuranosyl)-5-(2-bromoviny-
l)cytosine,
[1119]
1-(2,5-Di-O-acetyl-3-O-mesyl-.beta.-D-xylofuranosyl)-5-(2-methoxylc-
arbonylvinyl)cytosine,
[1120]
1-(2,5-Di-O-acetyl-3-O-mesyl-.beta.-D-xylofuranosyl)-5-(2-hydroxyca-
rbonylvinyl)cytosine,
[1121]
1-(2,5-Di-O-acetyl-3-O-mesyl-.beta.-D-xylofuranosyl)-5-phenylcytosi-
ne,
[1122]
1-(2,5-Di-O-acetyl-3-O-mesyl-.beta.-D-xylofuranosyl)-5-benzylcytosi-
ne,
[1123]
1-(2,5-Di-O-acetyl-3-O-mesyl-.beta.-D-xylofuranosyl)-N.sup.6-benzoy-
ladenine,
[1124]
1-(2,5-Di-O-acetyl-3-O-mesyl-.beta.-D-xylofuranosyl)-6-chloropurine-
,
[1125]
1-(2,5-Di-O-acetyl-3-O-mesyl-.beta.-D-xylofuranosyl)-2,6-dichloropu-
rine,
[1126]
1-(2,5-Di-O-acetyl-3-O-mesyl-.beta.-D-xylofuranosyl)-2-acetamido-6--
chloropurine,
[1127]
1-(2,5-Di-O-acetyl-3-O-mesyl-.beta.-D-xylofuranosyl)-2-acetamido-6--
methoxypurine,
[1128]
1-(2,5-Di-O-acetyl-3-O-mesyl-.beta.-D-xylofuranosyl)-6-methoxypurin-
e,
[1129]
1-(2,5-Di-O-acetyl-3-O-mesyl-.beta.-D-xylofuranosyl)-6-methylmercap-
topurine,
[1130]
1-(2,5-Di-O-acetyl-3-O-tosyl-.beta.-D-xylofuranosyl)-5-chlorouracil-
,
[1131]
1-(2,5-Di-O-acetyl-3-O-tosyl-.beta.-D-xylofuranosyl)-5-bromouracil,
[1132]
1-(2,5-Di-O-acetyl-3-O-tosyl-.beta.-D-xylofuranosyl)-5-iodouracil,
[1133]
1-(2,5-Di-O-acetyl-3-O-tosyl-.beta.-D-xylofuranosyl)-5-cyanouracil,
[1134]
1-(2,5-Di-O-acetyl-3-O-tosyl-.beta.-D-xylofuranosyl)-5-ethoxycarbon-
yluracil,
[1135]
1-(2,5-Di-O-acetyl-3-O-tosyl-.beta.-D-xylofuranosyl)-5-aminocarbony-
luracil,
[1136]
1-(2,5-Di-O-acetyl-3-O-tosyl-.beta.-D-xylofuranosyl)-5-acetyluracil-
,
[1137]
1-(2,5-Di-O-acetyl-3-O-tosyl-.beta.-D-xylofuranosyl)-5-methyluracil-
,
[1138]
1-(2,5-Di-O-acetyl-3-O-tosyl-.beta.-D-xylofuranosyl)-5-ethyluracil,
[1139]
1-(2,5-Di-O-acetyl-3-O-tosyl-.beta.-D-xylofuranosyl)-5-n-propylurac-
il,
[1140]
1-(2,5-Di-O-acetyl-3-O-tosyl-.beta.-D-xylofuranosyl)-5-i-propylurac-
il,
[1141]
1-(2,5-Di-O-acetyl-3-O-tosyl-.beta.-D-xylofuranosyl)-5-vinyluracil,
[1142]
1-(2,5-Di-O-acetyl-3-O-tosyl-.beta.-D-xylofuranosyl)-5-allyluracil,
[1143]
1-(2,5-Di-O-acetyl-3-O-tosyl-.beta.-D-xylofuranosyl)-5-ethynyluraci-
l,
[1144]
1-(2,5-Di-O-acetyl-3-O-tosyl-.beta.-D-xylofuranosyl)-5-(2-chlorovin-
yl)uracil,
[1145]
1-(2,5-Di-O-acetyl-3-O-tosyl-.beta.-D-xylofuranosyl)-5-(2-bromoviny-
l)uracil,
[1146]
1-(2,5-Di-O-acetyl-3-O-tosyl-.beta.-D-xylofuranosyl)-5-(2-iodovinyl-
)uracil,
[1147]
1-(2,5-Di-O-acetyl-3-O-tosyl-.beta.-D-xylofuranosyl)-5-(2-methoxylc-
arbonylvinyl)uracil,
[1148]
1-(2,5-Di-O-acetyl-3-O-tosyl-.beta.-D-xylofuranosyl)-5-(2-hydroxyca-
rbonylvinyl)uracil,
[1149]
1-(2,5-Di-O-acetyl-3-O-tosyl-.beta.-D-xylofuranosyl)-5-phenyluracil-
,
[1150]
1-(2,5-Di-O-acetyl-3-O-tosyl-.beta.-D-xylofuranosyl)-5-benzyluracil-
,
[1151]
1-(2,5-Di-O-acetyl-3-O-tosyl-.beta.-D-xylofuranosyl)-5-fluorocytosi-
ne,
[1152]
1-(2,5-Di-O-acetyl-3-O-tosyl-.beta.-D-xylofuranosyl)-5-chlorocytosi-
ne,
[1153]
1-(2,5-Di-O-acetyl-3-O-tosyl-.beta.-D-xylofuranosyl)-5-bromocytosin-
e,
[1154]
1-(2,5-Di-O-acetyl-3-O-tosyl-.beta.-D-xylofuranosyl)-5-iodocytosine-
,
[1155]
1-(2,5-Di-O-acetyl-3-O-tosyl-.beta.-D-xylofuranosyl)-5-cyanocytosin-
e,
[1156]
1-(2,5-Di-O-acetyl-3-tosyl-.beta.-D-xylofuranosyl)-5-ethoxycarbonyl-
cytosine,
[1157]
1-(2,5-Di-O-acetyl-3-tosyl-.beta.-D-xylofuranosyl)-5-aminocarbonylc-
ytosine,
[1158]
1-(2,5-Di-O-acetyl-3-O-tosyl-.beta.-D-xylofuranosyl)-5-acetylcytosi-
ne,
[1159]
1-(2,5-Di-O-acetyl-3-O-tosyl-.beta.-D-xylofuranosyl)-5-methylcytosi-
ne,
[1160]
1-(2,5-Di-O-acetyl-3-O-tosyl-.beta.-D-xylofuranosyl)-5-ethylcytosin-
e,
[1161]
1-(2,5-Di-O-acetyl-3-O-tosyl-.beta.-D-xylofuranosyl)-5-n-propylcyto-
sine,
[1162]
1-(2,5-Di-O-acetyl-3-O-tosyl-.beta.-D-xylofuranosyl)-5-i-propylcyto-
sine,
[1163]
1-(2,5-Di-O-acetyl-3-O-tosyl-.beta.-D-xylofuranosyl)-5-vinylcytosin-
e,
[1164]
1-(2,5-Di-O-acetyl-3-O-tosyl-.beta.-D-xylofuranosyl)-5-allylcytosin-
e,
[1165]
1-(2,5-Di-O-acetyl-3-O-tosyl-.beta.-D-xylofuranosyl)-5-ethynylcytos-
ine,
[1166]
1-(2,5-Di-O-acetyl-3-O-tosyl-.beta.-D-xylofuranosyl)-5-(2-chlorovin-
yl)cytosine,
[1167]
1-(2,5-Di-O-acetyl-3-O-tosyl-.beta.-D-xylofuranosyl)-5-(2-bromoviny-
l)cytosine,
[1168]
1-(2,5-Di-O-acetyl-3-O-tosyl-.beta.-D-xylofuranosyl)-5-(2-iodovinyl-
)cytosine,
[1169]
1-(2,5-Di-O-acetyl-3-O-tosyl-.beta.-D-xylofuranosyl)-5-(2-methoxylc-
arbonylvinyl)cytosine,
[1170]
1-(2,5-Di-O-acetyl-3-O-tosyl-.beta.-D-xylofuranosyl)-5-(2-hydroxyca-
rbonylvinyl)cytosine,
[1171]
1-(2,5-Di-O-acetyl-3-O-tosyl-.beta.-D-xylofuranosyl)-5-phenylcytosi-
ne,
[1172]
1-(2,5-Di-O-acetyl-3-O-tosyl-.beta.-D-xylofuranosyl)-5-benzylcytosi-
ne,
[1173] 1-(2,5-Di-O-acetyl-3-O-tosyl-.beta.-D-xylofuranosyl)-N
-benzoyladenine,
[1174]
1-(2,5-Di-O-acetyl-3-O-tosyl-.beta.-D-xylofuranosyl)-6-chloropurine-
,
[1175]
1-(2,5-Di-O-acetyl-3-O-tosyl-.beta.-D-xylofuranosyl)-2,6-dichloropu-
rine,
[1176]
1-(2,5-Di-O-acetyl-3-O-tosyl-.beta.-D-xylofuranosyl)-2-acetamido-6--
chloropurine,
[1177]
1-(2,5-Di-O-acetyl-3-O-tosyl-.beta.-D-xylopentofuranosyl)-2-acetami-
do-6-methoxypurine,
[1178]
1-(2,5-Di-O-acetyl-3-O-tosyl-.beta.-D-xylopentofuranosyl)-6-methoxy-
purine and
[1179]
1-(2,5-Di-O-acetyl-3-O-tosyl-.beta.-D-xylofuranosy.sup.1)-6-methylm-
ercaptopurine.
Example 36
[1180] 1-(2,3, 5-Tri-O-acetyl-.beta.-D-xylofuranosyl)thymine.
[1181] A mixture of thymine (0.02 mol), ammonium sulfate (ca. 30
mg) in hexamethyldisilazane (15 mL) is refluxed until a clear
solution is obtained. The solvent is removed in vacuo, and the
residue is dissolved in 1,2-dichloroethane (20 mL), and
1,2,3,5-tri-O-acetyl-D-xylofuranose (5.5 g, 0.02 mol) in
1,2-dichloroethane (20 mL) is added. To the solution is added tin
tetrachloride (5.2 g, 0.02 mol), and the mixture is stirred
overnight at room temperature, then is heated for 3 hours at
40-50.degree. C. for 3 hours. Saturated sodium bicarbonate solution
(40 mL) is added and stirred until carbon dioxide evolution ceases.
The mixture is filtered through a Celite pad. The organic layer is
separated, washed carefully with saturated sodium bicarbonate
solution (20 mL.times.2) and water (20 mL.times.2), dried over
sodium sulfate, and concentrated to dryness in vacuo. The residue
is crystallized from ethanol to give product (4.3 g, 62%). The
.sup.1H NMR spectrum of this sample is compatible with the
structure indicated.
[1182] In a similar manner but using the corresponding pyrimidine
and purine bases, the following 2',5'-di-O-acetyl 3'-substituted
xylo-nucleosides and their L-counterparts are prepared:
[1183]
1-(2,3,5-Tri-O-acetyl-.beta.-D-xylofuranosyl)-5-fluorouracil,
[1184]
1-(2,3,5-Tri-O-acetyl-.beta.-D-xylofuranosyl)-5-chlorouracil,
[1185]
1-(2,3,5-Tri-O-acetyl-.beta.-D-xylofuranosyl)-5-bromouracil,
[1186]
1-(2,3,5-Tri-O-acetyl-.beta.-D-xylofuranosyl)-5-iodouracil,
[1187] 1-(2,3
,5-Tri-O-acetyl-.beta.-D-xylofuranosyl)-5-cyanouracil,
[1188]
1-(2,3,5-Tri-O-acetyl-.beta.-D-xylofuranosyl)-5-ethoxycarbonyluraci-
l,
[1189]
1-(2,3,5-Tri-O-acetyl-.beta.-D-xylofuranosyl)-5-aminocarbonyluracil-
,
[1190] 1-(2,3
,5-Tri-O-acetyl-.beta.-D-xylofuranosyl)-5-acetyluracil,
[1191]
2-(2,3,5-Tri-O-acetyl-.beta.-D-xylofuranosyl)-5-methyluracil,
[1192] 1-(2,3
,5-Tri-O-acetyl-.beta.-D-xylofuranosyl)-5-ethyluracil,
[1193]
1-(2,3,5-Tri-O-acetyl-.beta.-D-xylofuranosyl)-5-n-propyluracil,
[1194]
1-(2,3,5-Tri-O-acetyl-.beta.-D-xylofuranosyl)-5-i-propyluracil,
[1195] 1-(2,3
,5-Tri-O-acetyl-.beta.-D-xylofuranosyl)-5-vinyluracil,
[1196] 1-(2,3
,5-Tri-O-acetyl-.beta.-D-xylofuranosyl)-5-allyluracil,
[1197]
1-(2,3,5-Tri-O-acetyl-.beta.-D-xylofuranosyl)-5-ethynyluracil,
[1198] 1-(2,3
,5-Tri-O-acetyl-.beta.-D-xylofuranosyl)-5-(2-chlorovinyl)ura-
cil,
[1199] 1-(2,3
,5-Tri-O-acetyl-.beta.-D-xylofuranosyl)-5-(2-bromovinyl)urac-
il,
[1200]
1-(2,3,5-Tri-O-acetyl-.beta.-D-xylofuranosyl)-5-(2-iodovinyl)uracil-
,
[1201] 1-(2,3
,5-Tri-O-acetyl-.beta.-D-xylofuranosyl)-5-(2-methoxylcarbony-
lvinyl)uracil,
[1202]
1-(2,3,5-Tri-O-acetyl-.beta.-D-xylofuranosyl)-5-(2-hydroxycarbonylv-
inyl)uracil,
[1203] 1-(2,3
,5-Tri-O-acetyl-.beta.-D-xylofuranosyl)-5-phenyluracil,
[1204] 1-(2,3
,5-Tri-O-acetyl-.beta.-D-xylofuranosyl)-5-benzyluracil,
[1205]
1-(2,3,5-Tri-O-acetyl-.beta.-D-xylofuranosyl)-5-fluorocytosine,
[1206] 1-(2,3
,5-Tri-O-acetyl-.beta.-D-xylofuranosyl)-5-chlorocytosine,
[1207]
1-(2,3,5-Tri-O-acetyl-.beta.-D-xylofuranosyl)-5-bromocytosine,
[1208]
1-(2,3,5-Tri-O-acetyl-.beta.-D-xylofuranosyl)-5-iodocytosine,
[1209] 1-(2,3
,5-Tri-O-acetyl-.beta.-D-xylofuranosyl)-5-cyanocytosine,
[1210]
1-(2,3,5-Tri-O-acetyl-.beta.-D-xylofuranosyl)-5-ethoxycarbonylcytos-
ine,
[1211]
1-(2,3,5-Tri-O-acetyl-.beta.-D-xylofuranosyl)-5-aminocarbonylcytosi-
ne,
[1212]
1-(2,3,5-Tri-O-acetyl-.beta.-D-xylofuranosyl)-5-acetylcytosine,
[1213]
1.beta.-(2,3,5-Tri-O-acetyl-.beta.-D-xylofuranosyl)-5-methycytosine-
,
[1214]
1-(2,3,5-Tri-O-acetyl-.beta.-D-xylofuranosyl)-5-ethylcytosine,
[1215]
1-(2,3,5-Tri-O-acetyl-.beta.-D-xylofuranosyl)-5-n-propylcytosine,
[1216]
1-(2,3,5-Tri-O-acetyl-.beta.-D-xylofuranosyl)-5-i-propylcytosine,
[1217]
1-(2,3,5-Tri-O-acetyl-.beta.-D-xylofuranosyl)-5-vinylcytosine,
[1218]
1-(2,3,5-Tri-O-acetyl-.beta.-D-xylofuranosyl)-5-allylcytosine,
[1219]
1-(2,3,5-Tri-O-acetyl-.beta.-D-xylofuranosyl)-5-ethynylcytosine,
[1220]
1-(2,3,5-Tri-O-acetyl-.beta.-D-xylofuranosyl)-5-(2-chlorovinyl)cyto-
sine,
[1221]
1-(2,3,5-Tri-O-acetyl-.beta.-D-xylofuranosyl)-5-(2-bromovinyl)cytos-
ine,
[1222] 1-(2,3 ,5-Tri-O-acetyl-.beta.-D-xylofuranosyl)-5-1-(2,3
,5-Tri-O-acetyl-.beta.-D-xylofuranosyl)-5-(2-methoxylcarbonylvinyl)cytosi-
ne,
[1223] 1-(2,3
,5-Tri-O-acetyl-.beta.-D-xylofuranosyl)-5-phenylcytosine,
[1224]
1-(2,3,5-Tri-O-acetyl-.beta.-D-xylofuranosyl)-N.sup.6-benzoyladenin-
e,
[1225] 1-(2,3
,5-Tri-O-acetyl-.beta.-D-xylofuranosyl)-6-chloropurine,
[1226]
1-(2,3,5-Tri-O-acetyl-.beta.-D-xylofuranosyl)-2,6-dichloropurine,
[1227]
1-(2,3,5-Tri-O-acetyl-.beta.-D-xylofuranosyl)-2-acetamido-6-chlorop-
urine,
[1228] 1-(2,3
,5-Tri-O-acetyl-.beta.-D-xylopentofuranosyl)-2-acetamido-6-m-
ethoxypurine,
[1229] 1-(2,3
,5-Tri-O-acetyl-.beta.-D-xylopentofuranosyl)-6-methoxypurine
and
[1230]
1-(2,3,5-Tri-O-acetyl-.beta.-D-xylofuranosyl)-6-methylmercaptopurin-
e.
Example 37
[1231]
1-(3-Deoxy-3-O-mesyl-.beta.-D-xylofuranosyl)-5-fluorouracil.
[1232] A mixture of
1-(2,5-Di-O-acetyl-3-O-mesyl-.beta.-D-xylofuranosyl)-5-
-fluorouracil (4.24 g, 0.01 mol) in methanolic ammonia (100 mL) is
stirred for 30 minutes at 0.degree. C., and is concentrated in
vacuo to dryness, and the residue is crystallized from ethanol to
give 1-(3-deoxy-3-O-mesyl-.beta.-D-xylofuranosyl)-5-fluorouracil
(2.82 g, 83%). .sup.1H NMR (D.sub.6-DMSO) showed that there is no
acetyl group but one mesyl group in the molecule.
[1233] In a similar manner but using the corresponding
2',5'-di-O-acetyl pyrimidine and purine nucleosides, the following
3'-O-mesyl-nucleosides and their L-counterparts are prepared:
[1234] 1-(3-5-Mesyl-.beta.-D-xylofuranosyl)-5-chlorouracil,
[1235] 1-(3-O-Mesyl-.beta.-D-xylofuranosyl)-5-bromouracil,
[1236] 1-(3-O-Mesyl-.beta.-D-xylofuranosyl)-5-iodouracil,
[1237] 1-(3-O-Mesyl-.beta.-D-xylofuranosyl)-5-cyanouracil,
[1238]
1-(3-O-Mesyl-.beta.-D-xylofuranosyl)-5-ethoxycarbonyluracil,
[1239]
2-(3-O-Mesyl-.beta.-D-xylofuranosyl)-5-aminocarbonyluracil,
[1240] 1-(3-O-Mesyl-.beta.-D-xylofuranosyl)-5-acetyluracil,
[1241] 1-(3-O-Mesyl-.beta.-D-xylofuranosyl)-5-methyluracil,
[1242] 1-(3-O-Mesyl-.beta.-D-xylofuranosyl)-5-ethyluracil,
[1243] 1-(3-O-Mesyl-.beta.-D-xylofuranosyl)-5-n-propyluracil,
[1244] 1-(3-O-Mesyl-.beta.-D-xylofuranosyl)-5-i-propyluracil,
[1245] 1-(3-O-Mesyl-.beta.-D-xylofuranosyl)-5-vinyluracil,
[1246] 1-(3-O-Mesyl-.beta.-D-xylofuranosyl)-5-allyluracil,
[1247] 1-(3-O-Mesyl-.beta.-D-xylofuranosyl)-5-ethynyluracil,
[1248]
1-(3-O-Mesyl-.beta.-D-xylofuranosyl)-5-(2-chlorovinyl)uracil,
[1249]
1-(3-O-Mesyl-.beta.-D-xylofuranosyl)-5-(2-bromovinyl)uracil,
[1250]
1-(3-O-Mesyl-.beta.-D-xylofuranosyl)-5-(2-iodovinyl)uracil,
[1251]
1-(3-O-Mesyl-.beta.-D-xylofuranosyl)-5-(2-methoxylcarbonylvinyl)ura-
cil,
[1252]
1-(3-O-Mesyl-.beta.-D-xylofuranosyl)-5-(2-hydroxycarbonylvinyl)urac-
il,
[1253] 1-(3-O-Mesyl-.beta.-D-xylofuranosyl)-5-phenyluracil,
[1254] 1-(3-O-Mesyl-.beta.-D-xylofuranosyl)-5-benzyluracil,
[1255] 1-(3-O-Mesyl-.beta.-D-xylofuranosyl)cytosine,
[1256] 1-(3-O-Mesyl-.beta.-D-xylofuranosyl)-5-fluorocytosine,
[1257] 1-(3-O-Mesyl-.beta.-D-xylofuranosyl)-5-chlorocytosine,
[1258] 1-(3-O-Mesyl-.beta.-D-xylofaranosyl)-5-bromocytosine,
[1259] 1-(3-O-Mesyl-.beta.-D-xylofuranosyl)-5-iodocytosine,
[1260] 1-(3-O-Mesyl-.beta.-D-xylofuranosyl)-5-cyanocytosine,
[1261]
1-(3-O-Mesyl-.beta.-D-xylofuranosyl)-5-ethoxycarbonylcytosine,
[1262]
1-(3-O-Mesyl-.beta.-D-xylofuranosyl)-5-aminocarbonylcytosine,
[1263] 1-(3-O-Mesyl-.beta.-D-xylofuranosyl)-5-acetylcytosine,
[1264] 1-(3-O-Mesyl-.beta.-D-xylofuranosyl)-5-methylcytosine,
[1265] 1-(3-O-Mesyl-.beta.-D-xylofuranosyl)-5-ethylcytosine,
[1266] 1q-(3-Mesyl-.beta.-D-xylofuranosyl)-5-n-propylcytosine,
[1267] 1-(3-O-Mesyl-.beta.-D-xylofuranosyl)-5-i-propylcytosine,
[1268] 1-(3-O-Mesyl-.beta.-D-xylofuranosyl)-5-vinylcytosine,
[1269] 1-(3-O-Mesyl-.beta.-D-xylofuranosyl)-5-allylcytosine,
[1270] 1-(3-O-Mesyl-.beta.-D-xylofuranosyl)-5-ethynylcytosine,
[1271] 1-(3-O-Mesyl-.beta.-D-xylofuranosyl)-5-(2-chlorovinyl)cyto
sine,
[1272]
1-(3-O-Mesyl-.beta.-D-xylofuranosyl)-5-(2-bromovinyl)cytosine,
[1273]
1-(3-O-Mesyl-.beta.-D-xylofuranosyl)-5-(2-iodovinyl)cytosine,
[1274]
1-(3-O-Mesyl-3-D-xylofuranosyl)-5-(2-methoxylearbonylviny)cytosine,
[1275]
1-(3-O-Mesyl-.beta.-D-xylofuranosyl)-5-(2-hydroxycarbonylvinyl)cyto-
sine,
[1276] 1-(3-O-Mesyl-.beta.-D-xylofuranosyl)-5-phenylcytosine,
[1277] 1-(3-O-Mesyl-.beta.-D-xylofuranosyl)-5-benzylcytosine,
[1278] 1-(3-O-Mesyl-.beta.-D-xylofuranosyl)-2-chloroadenine,
[1279] 1-(3-O-Mesyl-.beta.-D-xylofuranosyl)-6-chloropurine,
[1280] 1-(3-O-Mesyl-.beta.-D-xylofuranosyl)-2,6-dichloropurine,
[1281]
1-(3-O-Mesyl-.beta.-D-xylofuranosyl)-2-acetamido-6-chloropurine,
[1282]
1-(3-O-Mesyl-.beta.-D-xylofuranosyl)-2-acetamido-6-methoxypurine,
[1283] 1-(3-O-Mesyl-.beta.-D-xylofuranosyl)-6-methoxypurine and
[1284] 1-(3-O-Mesyl-o-D-xylofuranosyl)-6-methylmercaptopurine.
Example 38
[1285] 1-.beta.-D-Xylofuranosyl)-5-fluorouracil.
[1286] A mixture of
1-(2,3,5-tri-O-acetyl-.beta.-D-xylofuranosyl)-5-fluoro- uracil
(3.88 g, 0.01 mol) and triethylamine (3 mL) in methanol (100 mL) is
stirred overnight at room temperature. The mixture is concentrated
in vacuo to dryness, and the residue is crystallized from ethanol
to give I-(.beta.-D-xylo-furanosyl)-5-fluorouracil (2.0 g, 76%).
The UV and .sup.1H NMR (Me.sub.2SO-d6) spectra of this sample are
consistent with the product structure.
[1287] In a similar manner but using the corresponding
2',5'-di-O-acetyl pyrimidine and purine bases, the following
xylo-nucleosides and their L-counterparts are prepared:
[1288] 1-(.beta.-D-Xylofuranosyl)-5-chlorouracil,
[1289] 1-(.beta.-D-Xylofuranosyl)-5-bromouracil,
[1290] 1-(3-D-Xylofuranosyl)-5-iodouracil,
[1291] 1-(.beta.-D-Xylofuranosyl)-5-cyanouracil,
[1292] 1-(.beta.-D-Xylofuranosyl)-5-ethoxycarbonyluracil,
[1293] 1-(.beta.-D-Xylofuranosyl)-5-aminocarbonyluracil,
[1294] 1-(.beta.-D-Xylofuranosyl)-5-acetyluracil,
[1295] 1-(.beta.-D-Xylofuranosyl)-5-methyluracil,
[1296] 1-(.beta.-D-Xylofuranosyl)-5-ethyluracil,
[1297] 1-(.beta.-D-Xylofuranosyl)-5-n-propyluracil,
[1298] 1-(.beta.-D-Xylofuranosyl)-5-i-propyluracil,
[1299] 1-(.beta.-D-Xylofuranosyl)-5-vinyluracil,
[1300] 1-(.beta.-D-Xylofuranosyl)-5-allyluracil,
[1301] 1-(.beta.-D-Xylofuranosyl)-5-ethynyluracil,
[1302] 1-(.beta.-D-Xylofuranosyl)-5-(2-chlorovinyl)uracil,
[1303] 1-(.beta.-D-Xylofuranosyl)-5-(2-bromovinyl)uracil,
[1304] 1-(.beta.-D-Xylofuranosyl)-5-(2-iodovinyl)uracil,
[1305]
1-(.beta.-D-Xylofuranosyl)-5-(2-methoxylcarbonylvinyl)uracil,
[1306]
1-(.beta.-D-Xylofuranosyl)-5-(2-hydroxycarbonylvinyl)uracil,
[1307] 1-(.beta.-D-Xylofuranosyl)-5-phenyluracil,
[1308] 1-(.beta.-D-Xylofuranosyl)-5-benzyluracil,
[1309] 1-(.beta.-D-Xylofuranosyl)cytosine,
[1310] 1-(.beta.-D-Xylofuranosyl)-5-fluorocytosine,
[1311] 1-(.beta.-D-Xylofuranosyl)-5-chlorocytosine,
[1312] 1-(.beta.-D-Xylofuranosyl)-5-bromocytosine,
[1313] 1-(.beta.-D-Xylofuranosyl)-5-iodocytosine,
[1314] 1-(.beta.-D-Xylofuranosyl)-5-cyanocytosine,
1-(.beta.-D-Xylofuranos- yl)-5-ethoxycarbonylcytosine,
[1315] 1-(.beta.-D-Xylofuranosyl)-5-aminocarbonylcytosine,
[1316] 1-(.beta.-D-Xylofuranosyl)-5-acetylcytosine,
[1317] 1-(.beta.-D-Xylofuranosyl)-5-methylcytosine,
[1318] 1-(.beta.-D-Xylofuranosyl)-5-ethylcytosine,
[1319] 1-(.beta.-D-Xylofuranosyl)-5-n-propylcytosine,
[1320] 1-(.beta.-D-Xylofuranosyl)-5-i-propylcytosine,
[1321] 1-(.beta.-D-Xylofuranosyl)-5-viny lcytosine,
[1322] 1-(.beta.-D-Xylofuranosyl)-5-allylcytosine,
[1323] 1-(.beta.-D-Xylofuranosyl)-5-ethynylcytosine,
[1324] 1-(.beta.-D-Xylofuranosyl)-5-(2-chlorovinyl)cytosine,
[1325] 1-(.beta.-D-Xylofuranosyl)-5-(2-bromovinyl)cytosine,
[1326] 1-(.beta.-D-Xylofuranosyl)-5-(2-iodovinyl)cytosine,
[1327] 1-(.beta.-D-Xylofuranosyl)-5-(2-methoxylcarbonyl
vinyl)cytosine,
[1328]
3-(.beta.-D-Xylofuranosyl)-5-(2-hydroxycarbonylvinyl)cytosine,
[1329] 1-(.beta.-D-Xylofuranosyl)-5-phenylcytosine,
[1330] 1-(.beta.-D-Xylofuranosyl)-5-benzylcytosine,
[1331] 1-(.beta.-D-Xylofuranosyl)-2-chloroadenine,
[1332] 1-(.beta.-D-Xylofuranosyl)-6-chloropurine,
[1333] 1-(.beta.-D-Xylofuranosyl)-2,6-dichloropurine,
[1334] 1-(.beta.-D-Xylofuranosyl)-2-acetamido-6-chloropurine,
[1335] 1-(.beta.-D-Xylofuranosyl)-2-acetamido-6-methoxypurine,
[1336] 1-(.beta.-D-Xylofuranosyl)-6-methoxypurine and
[1337] 1-(.beta.-D-Xylofuranosyl)-6-methylmercaptopurine.
Example 39
[1338]
2',3'-O-Isopropylidene-5'-O-triphenylmethyl-N4-hydroxycytidine.
[1339] To a stirred solution of
2',3'-O-isopropylidene-5-O-triphenylmethyl- uridine (1 g) in 50 mL
of anhydrous acetonitrile and triethylamine (0.76 g) are added
2,4,6-triisopropylbenzenesulfonyl chloride (1.15 g) and DMAP (232
mg) at 0.degree. C., and the reaction mixture is stirred for 1 day
at room temperature. Hydroxylamine hydrochloride (263 mg) is then
added, and the mixture is further stirred for 1 day at room
temperature. The reaction is quenched by addition of water, and the
product is extracted with chloroform (200 mL). The organic layer is
washed with brine, dried over MgSO.sub.4, and concentrated in
vacuo. The residue is purified by silica gel column chromatography
(5% MeOH in CHCl.sub.3) to give
2',3'-O-isopropylidene-5'-O-trityl-N.sup.4-hydroxy-cytidine (723
mg, 70%) as a white solid. Mp: 99-101.degree. C. .sup.1H NMR
(CDCl.sub.3) .delta. 1.34 (s, 3H), 1.56 (s, 3H), 3.40-3.73 (m, 2H),
4.26 (br s, 1H), 4.79-4.81 (m, 2H), 5.34 (d, J=8.12 Hz, 1H), 5.88
(br s, 1H), 6.88 m(d, J=8.12 Hz, 1H), 7.22-7.41 (m, 15H).
[1340] In a similar manner but using the corresponding
5-substituted uracil nucleosides, the following
N.sup.4-hydroxy-2',3'-O-isopropylidene--
5'-O-triphenylmethylcytidine derivatives are synthesized:
[1341]
2',3'-O-Isopropylidene-5'-O-triphenylmethyl-5-fluoro-N.sup.4-hydrox-
ycytidine,
[1342]
2',3'-O-Isopropylidene-5'-O-triphenylmethyl-5-chloro-N.sup.4-hydrox-
ycytidine,
[1343]
2',3'-O-Isopropylidene-5'-O-triphenylmethyl-5-bromo-N.sup.4-hydroxy-
cytidine,
[1344]
2',3'-O-Isopropylidene-5'-O-triphenylmethyl-5-iodo-N.sup.4-hydroxyc-
ytidine,
[1345]
2',3'-O-Isopropylidene-5'-O-triphenylmethyl-5-methyl-N.sup.4-hydrox-
ycytidine,
[1346]
2',3'-O-Isopropylidene-5'-O-triphenylmethyl-5-ethyl-N.sup.4-hydroxy-
cytidine,
[1347] 2',
3'-O-Isopropylidene-5'-O-triphenylmethyl-5-n-propyl-N.sup.4-hyd-
roxycytidine,
[1348]
2',3'-O-Isopropylidene-5'-O-triphenylmethyl-5-i-propyl-N.sup.4-hydr-
oxycytidine,
[1349]
2',3'-O-Isopropylidene-5'-O-triphenylmethyl-5-vinyl-N.sup.4-hydroxy-
cytidine,
[1350] 2
2',3'-O-Isopropylidene-5'-O-triphenylmethyl-5-ethynyl-N.sup.4-hyd-
roxycytidine,
[1351] 2',
3'-O-Isopropylidene-5'-O-triphenylmethyl-5-(2-chlorovinyl)-N.su-
p.4-hydroxycytidine,
[1352]
2',3'-O-Isopropylidene-5'-O-triphenylmethyl-5-(2-bromovinyl)-N.sup.-
4-hydroxycytidine,
[1353]
2',3'-O-Isopropylidene-5'-O-triphenylmethyl-5-(2-iodovinyl)-N.sup.4-
-hydroxycytidine,
[1354]
2',3'-O-Isopropylidene-5'-O-triphenylmethyl-5-(2-methoxycarbonylvin-
yl)-N.sup.4-hydroxycytidine,
[1355]
2',3'-O-Isopropylidene-5'-O-triphenylmethyl-5-(2-hydroxycarbonylvin-
yl)-N.sup.4-hydroxycytidine,
[1356]
2',3'-O-Isopropylidene-5'-O-triphenylmethyl-5-phenyl-N.sup.4-hydrox-
ycytidine and
[1357]
2',3'-O-Isopropylidene-5'-O-triphenylmethyl-5-benzyl-N.sup.4-hydrox-
ycytidine.
[1358] In a similar manner but using the corresponding
5-substituted 2',5'-di-O-acetyl-3'-deoxyuridines, the following
N4-hydroxy-2',5'-di-O-a- cetyl-3'-deoxycytidine derivatives are
synthesized:
[1359]
1-(2,5-Di-O-acetyl-3-deoxy-.beta.-D-erythropentofuranosyl)-N.sup.4--
hydroxycytosine,
[1360]
1-(2,5-Di-O-acetyl-3-deoxy-.beta.-D-erythropentofuranosyl)-5-fluoro-
-N.sup.4-hydroxycytosine,
[1361]
1-(2,5-Di-O-acetyl-3-deoxy-.beta.-D-erythropentofuranosyl)-5-chloro-
-N.sup.4-hydroxycytosine,
[1362]
1-(2,5-Di-O-acetyl-3-deoxy-.beta.-D-erythropentofuranosyl)-5-cyano--
N4-hydroxycytosine,
[1363]
1-(2,5-Di-O-acetyl-3-deoxy-.beta.-D-erythropentofuranosyl)-5-ethoxy-
carbonyl-N.sup.4-hydroxycytosine,
[1364]
1-(2,5-Di-O-acetyl-3-deoxy-.beta.-D-erythropentofuranosyl)-5-aminoc-
arbonyl-N.sup.4-hydroxycytosine,
[1365]
1-(2,5-Di-O-acetyl-3-deoxy-.beta.-D-erythropentofuranosyl)-5-acetyl-
-N.sup.4-hydroxycytosine,
[1366]
1-(2,5-Di-O-acetyl-3-deoxy-.beta.-D-erythropentofuranosyl)-5-methyl-
-N.sup.4-hydroxycytosine,
[1367]
1-(2,5-Di-O-acetyl-3-deoxy-.beta.-D-erythropentofuranosyl)-5-n-prop-
yl-N.sup.4-hydroxycytosine,
[1368]
1-(2,5-Di-O-acetyl-3-deoxy-.beta.-D-erythropentofuranosyl)-5-n-prop-
yl-N.sup.4-hydroxycytosine,
[1369]
1-(2,5-Di-O-acetyl-3-deoxy-.beta.-D-erythropentofuranosyl)-5-vinyl--
N.sup.4-hydroxycytosine,
[1370]
1-(2,5-Di-O-acetyl-3-deoxy-.beta.-D-erythropentofuranosyl)-5-allyl--
N.sup.4-hydroxycytosine,
[1371]
5-(2,5-Di-O-acetyl-3-deoxy-.beta.-D-erythropentofuranosyl)-5-allyl--
N.sup.4-hydroxycytosine,
[1372]
1-(2,5-Di-O-acetyl-3-deoxy-.beta.-D-erythropentofuranosyl)-5-ethyov-
inyl)-N.sup.4-hydroxycytosine
[1373]
1-(2,5-Di-O-acetyl-3-deoxy-.beta.-D-erythropentofuranosyl)-5-(2-bro-
movinyl)-N.sup.4-hydroxycytosine,
[1374]
1-(2,5-Di-O-acetyl-3-deoxy-.beta.-D-erythropentofuranosyl)-5-(2-iod-
ovinyl)-N.sup.4-hydroxycytosine,
[1375]
1-(2,5-Di-O-acetyl-3-deoxy-.beta.-D-erythropentofuranosyl)-5-(2-met-
hoxylcarbonylvinyl)-N.sup.4-hydroxycytosine,
[1376]
1-(2,5-Di-O-acetyl-3-deoxy-.beta.-D-erythropentofuranosyl)-5-(2-hyd-
roxycarbonylvinyl)-N.sup.4-hydroxycytosine,
[1377]
1-(2,5-Di-O-acetyl-3-deoxy-.beta.-D-erythropentofuranosyl)-5-phenyl-
-N.sup.4-hydroxycytosine and
[1378]
1-(2,5-Di-O-acetyl-3-deoxy-.beta.-D-erythropentofuranosyl)-5-benzyl-
-N4-hydroxycytosine,
[1379] In a similar manner but using the corresponding
5-substituted 3',5'-di-O-acetyl-2'-deoxyuridines, the following
N.sup.4-hydroxy-3',5'-d-
i-O-acetyl-N.sup.4-hydroxy-2'-deoxycytidine derivatives are
synthesized:
[1380] 3',5'-Di-O-acetyl-2'-deoxy-N.sup.4-hydroxycytidine,
[1381]
3',5'-Di-O-acetyl-2'-deoxy-5-fluoro-N.sup.4-hydroxycytidine,
[1382] 3',5'-D
1-O-acetyl-2'-deoxy-5-chloro-N.sup.4-hydroxycytidine,
[1383]
3',5'-Di-O-acetyl-2'-deoxy-5-bromo-N.sup.4-hydroxycytidine,
[1384]
3',5'-Di-O-acetyl-2'-deoxy-5-iodo-N.sup.4-hydroxycytosine,
[1385]
3',5'-Di-O-acetyl-2'-deoxy-5-cyano-N.sup.4-hydroxycytidine,
[1386]
3',5'-Di-O-acetyl-2'-deoxy-5-ethoxycarbonyl-N.sup.4-hydroxycytidine-
,
[1387]
3',5'-Di-O-acetyl-2'-deoxy-5-aminocarbonyl-N.sup.4-hydroxycytidine,
[1388]
3',5'-Di-O-acetyl-2'-deoxy-5-acetyl-N.sup.4-hydroxycytidine,
[1389]
3',5'-Di-O-acetyl-2'-deoxy-5-methyl-N.sup.4-hydroxycytidine,
[1390]
3',5'-Di-O-acetyl-2'-deoxy-5-ethyl-N.sup.4-hydroxycytosine,
[1391] 3',
5'-Di-O-acetyl-2'-deoxy-5-n-propyl-N.sup.4-hydroxycytidine,
[1392] 3',
5'-Di-O-acetyl-2'-deoxy-5-i-propyl-N.sup.4-hydroxycytidine,
[1393] 3',
5'-Di-O-acetyl-2'-deoxy-5-vinyl-N.sup.4-hydroxycytidine,
[1394]
3',5'-Di-O-acetyl-2'-deoxy-5-allyl-N.sup.4-hydroxycytidine,
[1395] 3',
5'-Di-O-acetyl-2'-deoxy-5-ethynyl-N.sup.4-hydroxycytidine,
[1396]
3',5'-Di-O-acetyl-2'-deoxy-5-(2-chlorovinyl)-N.sup.4-hydroxycytidin-
e,
[1397]
3',5'-Di-O-acetyl-2'-deoxy-5-(2-bromovinyl)-N.sup.4-hydroxycytidine-
,
[1398]
3',5'-Di-O-acetyl-2'-deoxy-5-(2-iodovinyl)-N.sup.4-hydroxycytidine,
[1399]
3',5'-Di-O-acetyl-2'-deoxy-5-(2-methoxylcarbonylvinyl)-N.sup.4-hydr-
oxycytidine,
[1400]
3',5'-Di-O-acetyl-2'-deoxy-5-(2-hydroxycarbonylvinyl)-N.sup.4-hydro-
xycytosine,
[1401] 3',5'-Di-O-acetyl-2'-deoxy-5-phenyl-N.sup.4-hydroxycytidine
and
3',5'-Di-O-acetyl-2'-deoxy-5-benzyl-N.sup.4-hydroxycytidine.
Example 40
[1402] N.sup.4-Hydroxycytidine.
[1403] 2',3'-O-Isopropylidene-5'-O-trityl-N.sup.4-hydroxycytidine
(500 mg, 0.92 mmol) is dissolved in 50 mL of a mixture of
trifluoroacetic acid and water (2:1, v/v), and the solution is
stirred for 3 h at 50.degree. C. After cooling to room temperature,
the solvent is removed by evaporation and coevaporated with ethanol
(3.times.20 mL). The residue is purified by silica gel column
chromatography (20% MeOH in CHCl.sub.3) to give
N.sup.4-hydroxycytidine (215 mg) as a white solid which is
recrystallized from hot ethanol; mp. 173-176.degree. C. .sup.1H NMR
(DMSO-d.sub.6) .delta. 3.66-3.71 (m, 2H), 3.93 (br s, 1H),
4.08-4.15 (m, 2H), 5.17-5.23 (m, 2H, D.sub.2O exchangeable), 5.43
(d, J=6.00 Hz, 1H, D.sub.2O exchangeable), 5.73 (d, J=8.16 Hz, 1H),
5.90 (d, J=8.12 Hz, 1H), 7.28 (d, J=8.40 Hz, 1H), 9.65 (s, 1H,
D.sub.2O exchangeable), 10.15 (s, 1H, D.sub.20 exchangeable). Anal.
Calcd for C9H13N3O6: C, 41.70; H, 5.05; N, 16.21. Found: C, 41.85;
H, 5.14; N, 16.34.
[1404] In a similar manner but using the corresponding
5-substituted
2',3'-O-isopropylidene-5-O-triphenylmethyl-N.sup.4-hydroxycytidine
nucleosides, the following N.sup.4-hydroxy-5-substituted cytidine
are synthesized:
[1405] 5-Fluoro-N.sup.4-hydroxycytidine,
[1406] 5-Chloro-N.sup.4-hydroxycytidine,
[1407] 5-Bromo-N.sup.4-hydroxycytidine,
[1408] 5-Iodo-N.sup.4-hydroxycytidine,
[1409] 5-Methyl-N.sup.4-hydroxycytidine,
[1410] 5-Ethyl-N.sup.4-hydroxycytidine,
[1411] 5-n-Propyl-N.sup.4-hydroxycytidine,
[1412] 5-i-Propyl-N.sup.4-hydroxycytidine,
[1413] 5-Vinyl-N.sup.4-hydroxycytidine,
[1414] 5-Ethynyl-N.sup.4-hydroxycytidine,
[1415] 5-(2-chlorovinyl)-N.sup.4-hydroxycytidine,
[1416] 5-(2-bromovinyl)-N.sup.4-hydroxycytidine,
[1417] 5-(2-iodovinyl)-N.sup.4-hydroxycytidine,
[1418] 5-(2-methoxycarbonylvinyl)-N.sup.4-hydroxycytidine,
[1419] 5-(2-hydroxycarbonylvinyl)-N.sup.4-hydroxycytidine,
[1420] 5-phenyl-N.sup.4-hydroxycytidine and
[1421] 5-benzyl-N.sup.4-hydroxycytidine.
[1422] In a similar manner but using methanolic ammonia instead of
trifluoroacetic acid, and the corresponding 5-substituted
1-(2,5-di-O-acetyl-3-deoxy-.beta.-D-erythro-pento-furanosyl)-N.sup.4-hydr-
oxycytosine nucleosides, the following
N.sup.4-hydroxy-5-substituted 3'-deoxycytidine are synthesized:
[1423] 5-Fluoro-3'-deoxy-N.sup.4-hydroxycytidine,
[1424] 5-Chloro-3'-deoxy-N.sup.4-hydroxycytidine,
[1425] 5-Bromo-3'-deoxy-N.sup.4-hydroxycytidine,
[1426] 5-Iodo-3'-deoxy-N.sup.4-hydroxycytidine,
[1427] 5-Methyl-3'-deoxy-N.sup.4-hydroxycytidine,
[1428] 5-Ethyl-3'-deoxy-N.sup.4-hydroxycytidine,
[1429] 5-n-Propyl-3'-deoxy-N.sup.4-hydroxycytidine,
[1430] 5-i-Propyl-3'-deoxy-N.sup.4-hydroxycytidine,
[1431] 5-Vinyl-3'-deoxy-N.sup.4-hydroxycytidine,
[1432] 5-Ethynyl-3'-deoxy-N.sup.4-hydroxycytidine,
[1433] 5-(2-chlorovinyl)-3'-deoxy-N.sup.4-hydroxycytidine,
[1434] 5-(2-bromovinyl)-3'-deoxy-N.sup.4-hydroxycytidine,
[1435] 5-(2-iodovinyl)-3'-deoxy-N.sup.4-hydroxycytidine,
[1436]
5-(2-methoxycarbonylnylyl)-3'-deoxy-N.sup.4-hydroxycytidine,
[1437]
5-(2-hydroxycarbonylvinyl)-3'-deoxy-N.sup.4-hydroxycytidine,
[1438] 5-phenyl-3'-deoxy-N.sup.4-hydroxycytidine and
[1439] 5-benzyl-3'-deoxy-N.sup.4-hydroxycytidine.
[1440] In a similar manner but using methanolic ammonia instead of
trifluoroacetic acid, and the corresponding 5-substituted
3',5'-di-O-acetyl-2'-deoxy-N4-hydroxycytosine nucleosides, the
following N.sup.4-hydroxy-5-substituted 2'-deoxycytidine are
synthesized:
[1441] 5-Fluoro-2'-deoxy-N.sup.4-hydroxycytidine,
[1442] 5-Chloro-2'-deoxy-N.sup.4-hydroxycytidine,
[1443] 5-Bromo-2'-deoxy-N.sup.4-hydroxycytidine,
[1444] 5-Iodo-2'-deoxy-N.sup.4-hydroxycytidine,
[1445] 5-Methyl-2'-deoxy-N.sup.4-hydroxycytidine,
[1446] 5-Ethyl-2'-deoxy-N.sup.4-hydroxycytidine,
[1447] 5-n-Propyl-2'-deoxy-N.sup.4-hydroxycytidine,
[1448] 5-i-Propyl-2'-deoxy-N.sup.4-hydroxycytidine,
[1449] 5(2-Vinyl2'.beta.-deoxy-N.sup.4-hydroxycytidine,
[1450] 5-E Vinyl-2'-deoxy-N4-hydroxycytidine,
[1451] 5-(2-iodovinyl)-2'-deoxy-N.sup.4-hydroxycytidine,
[1452] 5-(2-bromovinyl)-2'-deoxy-N.sup.4-hydroxycytidine,
[1453] 5-(2-iodovinyl)-2'-deoxy-N.sup.4-hydroxycytidine,
[1454]
2,5-(2-methoxycarbonylvinyl)-2''-deoxy-N.sup.4-hydroxycytidine,
[1455]
5-(2-hydroxycarbonylvinyl)-2'-deoxy-N.sup.4-hydroxycytidine,
[1456] 5-phenyl-2'-deoxy-N.sup.4-hydroxycytidine and
[1457] 5-benzyl-2'-deoxy-N.sup.4-hydroxycytidine.
Example 41
[1458]
2,3'-Anhydro-1-(2-deoxy-2-fluoro-5-O-trityl-)-D-lyxofuranosyl)thymi-
ne (194, R=Tr).
[1459] A solution of
1-(2-deoxy-2-fluoro-3-O-mesyl-5-O-triphenylmethyl-.be-
ta.-D-arabino-furanosyl)thymine (193, R=Tr, 6.0 g) and DBU (3.0 mL)
in methylene chloride (50 mL) is heated at reflux for 16 hours.
After concentration of the mixture in vacuo, the residue is
chromatographed on a silica gel column using chloroform as the
eluent to give 4.4 g of
2,3'-anhydro-1-(2'-deoxy-2'-fluoro-5-O-trityl-.beta.-D-lyxofuranosyl)thym-
ine (194, R=Tr), mp 252-255.degree. C. after recrystallization from
methanol. .sup.1H NMR (DMSO-d.sub.6); .delta. 1.80 (s, 3H, Me),
4.61 (1H, m), 5.40 (dm, 1H), 5.89 (1H, ddd), 5.96 (1H, dd, H-1'),
7.30 (15H, Tr), 7.66 (s, 1H, H-6).
Example 42
[1460]
1-(2,3-Dideoxy-2'fluoro-5'-O-trityl-.beta.D-glycero-pento-2-enofura-
nosyl)-thymine (195, R=Tr).
[1461] A suspension of 194 (646 mg) and t-BuOK (270 mg) in DMSO (10
mL) is stirred at room temperature for 2 hours and then filtered.
The filtrate is concentrated in vacuo and the residue is
chromatographed on a silica gel column (CHCl.sub.3/MeOH, 49:1 v/v)
to give 600 mg of 195, mp. 176-180.degree. C. (from EtOH). .sup.1H
NMR (DMSO-d.sub.6) .delta. 1.27 (s, 3H, Me), 3.21 (m, 2H, H-5,5"),
4.98 (m, 1H, H-4'), 6.17 (t, 1H, H-1', J1',2'=J1',F=1.5 Hz), 6.81
(m, IH, H-3'), 7.32 (m, 16H, H-6, Tr), 11.52 (s, 1H, NH
exchangeable).
Example 43
[1462]
1-(2,3-Dideoxy-2-fluoro-.beta.-D-glycero-2-enofuranosyl)thymine
(196).
[1463] A solution of 195 (600 mg) in 80% aqueous acetic acid (10
mL) is heated under reflux for 20 minutes and then concentrated to
dryness in vacuo. The residue is chromatographed on a column of
silica gel (CHCl.sub.3/MeOH, 9:1 v/v) to give 100 mg of 196, mp
154-159.degree. C. (from EtOH-H.sub.2O ). .sup.1H NMR
(DMSO-d.sub.6) .delta. 1.76 (s, 3H, Me), 3.61 (m, 2H, H-5',5"),
4.79 (m, 1H, H-4'), 5.15 (t, 1H, 5'-OH, exchangeable), 5.99 (m, 1H,
H-1'), 6.76 (m, 1H, H-3'), 7.88 (s, 1H, H-6), 11.43 (s, 1H, NH,
exchangeable).
Example 44
[1464] (1S,2S, 3R,
4R)-4-(tert-Butoxymethyl)-2,3-(isopropylidenedioxy)cycl-
opentan-1-ol (219).
[1465] To a solution of 4-(t-butoxymethyl)cyclopentane-2,3-diol
(218, 5 g) and CeCl.sub.3 7H.sub.2O (7.69 g) in methanol (80 mL) is
added NaBH.sub.4 (1.01 g) at 0.degree. C., and the mixture is
stirred for 1 hour at 0.degree. C. The reaction is quenched by
addition of cold water, and extracted with ethyl acetate
(2.times.300 mL). The combined organic extracts are washed with
brine (2.times.200 mL), dried over Na2SO4, and then concentrated in
vacuo. The residue is chromatographed on a silica gel column (30%
ethyl acetate in n-hexane) to give 219 (4.8 g, 95%) as a syrup.
.sup.1H-NMR (CDCl.sub.3) .delta. 1.13 (s, 9H, t-Bu), 1.34 (s, 3H,
Me), 1.48 (s, 3H, Me), 1.83 (m, 2H, 5a,b-H), 2.19 (m, 1H, 4-H),
2.44 (d, OH, exchangeable), 3.20 (dd, J=4.5, 8.8 Hz, 1H, 6a-H),
3.31 (dd, J=4.5, 8.8 Hz, 1H, 6b-H), 4.23 (m, 1H, 1-H), 4.44 (m, 2H,
2-H, 3-H). Anal. Calcd for C.sub.13H.sub.24O.sub.4: C, 63.91; H,
9.90. Found: C, 64.09; H, 9.87.
Example 45
[1466] (1S, 2S, 3R,
4R)-4-(tert-Butoxymethyl)-2,3-(isopropylidenedioxy)-1--
mesyloxycyclopentane (220).
[1467] To a solution of 219 (6.50 g) and triethylamine (7.3 g) in
methylene chloride (170 mL) is added mesyl chloride (4.73 g)
dropwise at 0.degree. C. After 45 minutes, water (270 mL) is added.
The aqueous layer is extracted with methylene chloride (3.times.200
mL). The organic layers are combined, washed with brine
(2.times.200 mL), dried over Na.sub.2SO.sub.4, and concentrated in
vacuo to give crude 220, which is sufficiently pure to be used
directly in the next step.
Example 46
[1468] (1R, 2S, 3R,
4R)-1-Azido-4-(tert-butoxymethyl)-2,3-(isopropylidened-
ioxy)cyclopentane (221).
[1469] A mixture of 220 obtained above and sodium azide (17.3 g) in
DMF (300 mL) is heated at 140.degree. C. overnight with stirring.
The mixture is filtered and the filtrate is concentrated in vacuo.
The residue is partitioned between ethyl acetate (150 mL) and water
(50 mL). The organic layer is dried over Na.sub.2SO.sub.4,
concentrated in vacuo, and the residue is chromatographed on a
silica gel column (1-4% gradient, ethyl acetate in n-hexane) to
give 221 (5.9 g) as an oil. .sup.1H NMR (CDCl.sub.3) .delta. 1.18
(s, 9H, t-Bu), 1.30 (s, 3H, Me), 1.46 (s, 3H, Me), 1.71 (m, 1H,
5a-H), 2.29 (m, 2H, 4-H, 5b-H), 3.29 (dd, J=6.7, 8.8 Hz, 1H, 6a-H),
3.37 (dd, J=7.0, 8.8 Hz, 1H, 6b-H), 3.96 (m, 1H, 1-H), 4.40 (dd,
J=2.3, 6.1 Hz, 1H, 3-H), 4.48 (dd, J=2.0, 6.1 Hz, 1H, 2-H). Anal.
Calcd for C.sub.13H.sub.23N.sub.3O.sub.30.13EtOAc: C, 57.95; H,
8.65, N, 14.99. Found: C, 58.25; H, 8.71; N, 14.76.
Example 47
[1470] (1R, 2S, 3R,
4R)-4-(tert-Butoxymethyl)-2,3-(isopropylidenedioxy)-1--
cyclopentylamine (222).
[1471] A suspension of 221 (4.0 g) and 10% Pd/C (1.0 g) in
anhydrous ethanol (140 mL) is shaken under 20 psi of H.sub.2 for 5
hours. The mixture is filtered, and the filtrate is concentrated in
vacuo to give crude 222 (3.6 g, quantitative), which is used
directly in the next step without further purification. .sup.1H NMR
(CDCl.sub.3) .delta. 1.18 (s, 9H, t-Bu), 1.28 (s, 3H, Me), 1.36 (m,
1H, 5a-H), 1.45 (s, 3H, Me), 1.89 (br s, 2H, NH.sub.2), 2.24-2.36
(m, 2H, 4-H, 5b-H), 3.34-3.43 (m, 3H, 1-H, 6a,b-H), 4.21 (dd,
J=2.6, 6.2 Hz, 1H, 3-H), 4.48 (dd, J=2.8, 6.2 Hz, 1H, 2-H). Anal.
Calcd for C.sub.13H.sub.26NO.sub.30.16H.sub.2O : C, 63.41; H,
10.37, N, 5.69. Found: C, 63.09; H, 10.16; N, 5.59.
Example 48
[1472]
N-{[1R,2S,3R,4R)-4-(tert-Butoxymethyl)-2,3-(isopropylidenedioxy)cyc-
lopentyl]-aminocarbonyl}-3-methoxy-2-propenamide (223).
[1473] A mixture of silver cyanate (7.60 g, dried in vacuo over
phosphorus pentoxide in the dark at 100.degree. C. for 3 hours),
.beta.-methoxyacryloyl chloride (2.64 g) in anhydrous benzene (30
mL) is heated under reflux for 30 minutes, and then is allowed to
cool to room temperature. After precipitation is settled, 22.5 mL
of the supernatant, which contains .beta.-methoxyacryloyl
isocyanate) is added during 15 minutes to a solution of 222 (3.0 g)
in dry DMF (50 mL) at -15 to -20.degree. C. under nitrogen. The
mixture is stirred for 2 hours at -15.degree. C. and then 10 more
hours at room temperature under nitrogen. After concentration in
vacuo and coevaporation with toluene (2.times.20 mL), the product
223 solidifies (4.0 g). .sup.1H NMR (CDCl.sub.3) .delta. 1.17 (s,
9H, t-Bu), 1.28 (s, 3H, Me), 1.47 (s, 3H, Me), 1.58 (m, 1H, 5'a-H),
2.28 (m, 1H, 4-H), 2.36-2.43 (m, 1H, 5'b-H), 3.33-3.42 (m, 2H,
6'a,b-H), 3.73 (s, 3H, OMe), 4.20 (m, 1H, 3'-H), 4.45 (m, 2H, 1'-H,
2'-H), 5.35 (d, J 12.3 Hz, 1H, 5-H), 7.67 (d, J 12.3 Hz, 1H, 6-H),
8.72 (br s, 1H, NH), 9.35 (br s, 1H, NH). Anal. Calcd for
C.sub.18H.sub.30N.sub.2O.sub.6': C, 58.36; H, 8.16, N, 7.56. Found:
C, 58.28; H, 8.16; N, 7.60.
Example 49
[1474] (1'R, 2 S, 3R, 4
R)-1-[4-(tert-Butoxymethyl)-2,3-isopropylidenediox- y)
cyclopentan-1-yl]uracil
(5'-tert-Butyl-2,3'-O-isopropylidene-carba-urid- ine, 224).
[1475] A solution of 223 (4.2 g) in ethanol (25 mL) and ammonium
hydroxide (30% 11 mL) is heated at 100.degree. C. in a steel bomb
for 12 hours. After removal of the solvents, the residue is
chromato graphed over a silica gel column (ethylacetate-n-hexane,
1:1 v/v) to give 224 (3.21 g) as a white foam. UV (MeOH)
k.lambda..sub.max 266.0 nmn. .sup.1H NMR (CDCl.sub.3) .delta. 1.19
(s, 9H, t-Bu), 1.30 (s, 3H, Me), 1.54 (s, 3H, Me), 1.97 (mn, 1H,
5'a-H), 2.32-2.41 (mn, 2H, 4'-H, 5'b-H), 3.43-3.50 (mn, 2H,
6'a,b-H), 4.48 (dd, J=4.1, 6.5 Hz, 1H, 3'-H), 4.65-4.75 (mn, 2H,
1'-H, 2'-H), 5.72 (d, J=8.0 Hz, 1H, 5-H), 7.35 (d, J=8.0 Hz, 1H,
6-H), 8.63 (br s, 1H, NH). Anal. Calcd for
C.sub.17H.sub.26N.sub.2O.sub.5: C, 60.34; H, 7.74, N, 8.28. Found:
C, 60.06; H, 7.70; N, 8.14.
Example 50
[1476] (1'R,2 'S,3 'R,
4'R)-1-[4-(tert-Butoxymethyl)-2,3-isopropylidenedio-
xy)cyclopentan-1-yl]-5-fluorouracil (5'-O-tert-Butyl-2 ,
3'-O-isopropylidene-carba-5-fluorouridine, 225).
[1477] A fluorine-nitrogen mixture containing 5% of fluorine is
bubbled carefully into a solution of 224 (2.50 g) in acetic acid
(600 mL) for 30 minutes at room temperature. The mixture is stirred
until no UV absorption is detected on TLC plate. The solvent is
removed in vacuo, and the residue is coevaporated with acetic acid
(20 mL) to dryness. The residue is treated with triethylamine for
1.5 hours at 50.degree. C., and then concentrated in vacuo to
dryness. The residue is purified by silica gel column
chromatography (ethylacetate-n-hexane, 1:1 v/v) to give 225 (1.31
g) as a white foam. TV (MeOH) .lambda..sub.max 271.5 nm. .sup.1H
NMR (CDCl.sub.3) .delta. 1.22 (s, 9H, t-Bu), 1.31 (s, 3H, Me), 1.55
(s, 3H, Me), 1.85 (m, 1H, 5'a-H), 2.38-2.51 (m, 2H, 4'-H, 5'b-H),
3.44-3.52 (m, 2H, 6'a,b-H), 4.47 (dd, J=3.4, 6.2 Hz, 1H, 3'-H),
4.58 (t, J=6.0 Hz, 1H, 1'-H), 4.87 (dd, J=8.9, 14.5 Hz, 1H, 2'-H),
7.61 (d, J=6.1 Hz, 1H, 6-H), 8.77 (br s, 1H, NH). Anal. Calcd for
C.sub.17H.sub.25FN.sub.2O.sub.- 5.0.25H.sub.2O : C, 56.58; H, 7.12,
N, 7.76. Found: C, 56.20; H, 7.02; N, 7.50.
Example 51
[1478] (1'R, 2'S, 3
'R,4'R)-1-[4-(tert-Butoxymethyl)-2,3-isopropylidenedio-
xy)cyclopentan-1-yl]-5-fluorocytosine (226).
(5'-O-tert-Butyl-2',3'-O-isop-
ropylidene-carba-5-fluorocytidine)
[1479] A mixture of 225 (350 mg), triethylamine (190 mg),
2,4,6-triisopropylbenzenesulfonyl chloride (590 mg) and DMAP (230
mg) in acetonitrile (50 mL) is stirred for 1 day at room
temperature. Ammonium hydroxide solution (30%, 15 mL) is added, and
the mixture is further stirred 5 hours. The reaction is quenched by
addition of chloroform (250 mL) and water (10 mL). The organic
layer is washed with brine, dried over Na.sub.2SO.sub.4, and
concentrated in vacuo. The residue is purified by silica gel column
chromatography (5% MeOH in CHCl.sub.3, v/v) to give 226 (205 mg),
mp 128-130.degree. C. UV (MeOH) .lambda..sub.max 286.5 nm. .sup.1H
NMR (CDCl.sub.3) .delta. 1.19 (s, 9H, t-Bu), 1.29 (s, 3H, Me), 1.53
(s, 3H, Me), 2.02 (dt, J=10.2, 12.8 Hz, 1H, 5'a-H), 2.32 (m, 1H,
4'-H), 2.42 (dt, J=8.0, 12.7 Hz, 1H, 5'b-H), 3.42 (dd, J=6.1, 8.7
Hz, 1H, 6'a-H), 3.52 (dd, J=4.1, 8.8 Hz, 1H, 6'b-H), 4.49 (dd,
J=5.1, 6.3 Hz, 1H, 3'-H), 4.60 (m, 1H, 1'-H), 4.79 (dd, J=5.0, 6.4
Hz, 1H, 2'-H), 7.49 (d, J 6.1 Hz, 1H, 6-H). HR-FAB MS Obsd; m/z
356.1981. Calcd for C.sub.17H.sub.26FN.sub.3O.sub.4: m/z 356.1986
(M +1).sup.+.
Example 52
[1480] (1'R,2 'S, 3 'R,
4'R)-1-[2,3-Dihydroxy-4-(hydroxymethyl)cyclopentan-
-1-yl]-5-fluorocytosine (carba-5-fluorocytidine, 227).
[1481] A solution of 226 (180 mg) in a 2:1 (v/v) mixture of
trifluoroacetic acid and water (40 mL) is stirred for 3 hours at
50.degree. C. The solvents are removed in vacuo, and the residue is
coevaporated with ethanol (2.times.30 mL), and purified on a silica
gel column (MeOH-CHCl3, 1:5 v/v) to give 227 (47.5 mg) as a foam.
WV (H.sub.2O ) .lambda..sub.max 284 nm (s 5,876, pH 7), 293.5 nm
(.epsilon. 7,440, pH 2), 284 5 nm (.epsilon. 5,883, pH 11). .sup.1H
NMR (DMSO-d.sub.6) .delta. 1.19 (m, 1H, 5'a-H), 1.92 (m, 1H,
4'a-H), 2.00 (ddd, J=8.3, 8.7, 12.5 Hz, 1H, 5'b-H), 3.42 (m, 2H,
6'ab-H), 3.70 (dd, J=2.9, 5.3 Hz, 1H, 3'b-H), 3.98 (dd, J=5.2, 9.0
Hz, 1H, 2'-H), 4.10 (d, J=4.5, 1H, OH, exchangeable), 4.51 (br s,
1H, OH, exchangeable), 4.60 (dd, J=9.0, 18.2 Hz, 1H, 1'-H), 4.73
(d, J=6.1 Hz, 1H, OH, exchangeable), 7.33 (bs, 1H, exchangeable),
7.55 (bs, 1H, exchangeable), 7.98 (d, J=7.3 Hz, 1H, 6-H). HR-FAB MS
Obsd; m/z 260.1054. Calcd for C.sub.17H.sub.26FN.sub.3O.sub.4: m/z
260.1047 (M+1).sup.+.
[1482] In a similar manner but using the corresponding
5-substituted derivatives, the following 5-substituted
carba-nucleosides are prepared: -Chloro-carba-uridine,
[1483] 5-Bromo-carba-uridine,
[1484] 5-Bodo-carba-uridine,
[1485] 5-Cyano-carba-uridine,
[1486] cara-Uridine-5-carboxylic acid,
[1487] 5-Ethoxycarbonyl-carba-uridine,
[1488] carba-Uridine-5-carboxamide,
[1489] 5-Hydroxymethyl-carba-uridine,
[1490] 5-Nitro-carba-uridine,
[1491] 5-Amino-carba-uridine
[1492] 5-Chloro-carba-cytidine,
[1493] 5-Bromo-carba-cytidine,
[1494] 5-lodo-carba-cytidine,
[1495] 5-Cyano-carba-cytidine,
[1496] cara-Cytidine-5-carboxylic acid,
[1497] 5-Ethoxycarbonyl-carba-cytidine,
[1498] carba-Cytidine-5-carboxamide,
[1499] 5-Hydroxymethyl-carba-cytidine,
[1500] 5-Nitro-carba-cytidine and
[1501] 5-Amino-carba-cytidine.
[1502] XI. Biological Methods
[1503] This invention further provides an efficient process to
quantify the viral load in a host using quantitative real-time
reverse-transcription polymerase chain reaction ("Q-RT-PCR"). The
process involves using a quenched fluorescent probe molecule that
can be hybridized to a target viral DNA or RNA. Therefore, upon
exonucleolytic degradation, a detectable fluorescent signal can be
monitored. Therefore, the RT-PCR amplified DNA or RNA can be
detected in real time by monitoring the presence of fluorescence
signals.
[1504] In a specific embodiment of the invention, the use of RT-PCR
to quantitate the viral load of a Flaviviridae virus is
provided.
[1505] In a more specific embodiment, the use of RT-PCR to
quantitate the viral load of BVDV in a MDBK cell line or a host
sample is provided.
[1506] In a further embodiment of the invention, a probe molecule
designed to fluoresce upon exonucleolytic degradation and to be
complementary to the BVDV NADL NS5B region is provided.
[1507] In a more specific embodiment of the invention, a probe
molecule with a sequence of
5',6-fam-AAATCCTCCTAACAAGCGGGTTCCAGG-tamara 3' (Sequence ID No 1)
and primers with a sequence of sense:
5'-AGCCTTCAGTTTCTTGCTGATGT-3'(Sequence ID No 2) and antisense:
5'-TGTTGCGAAAGCACCAACAG-3' (Sequence ID No 3) is provided.
[1508] In a specific embodiment of the invention, the use of RT-PCR
to quantitate viral load of HCV in a host derived sample or a cell
line in real time is provided;
[1509] In a more specific embodiment of the invention, the use of
RT-PCR, a probe molecule designed to fluoresce upon exonucleolytic
degradation and to be complementary to the HCV genome is provided
In a more specific embodiment of the invention, the use of RT-PCR,
a probe molecule designed to fluoresce upon exonucleolytic
degradation and to be complementary to the HCV 5' untranslated
region is provided In a more specific embodiment of the invention,
a probe molecule with a sequence of
5',6-fam-CCTCCAGGACCCCCCCTCCC-tamara 3' (Sequence ID No 4) and
primers with a sequence of sense: 5'-AGCCATGGCGTTAGTA(T/C)GAGTGT-3'
(Sequence ID No 5) and antisense: 5'-TTCCGCAGACCACTATGG-3'
(Sequence ID No 6) is provided.
[1510] A. RNA Isolation and Quantitative RT-PCR Analysis
[1511] An effective process to quantify the viral load in a host,
termed real-time polymerase chain reaction ("RT-PCR") is provided.
The process involves using a quenched fluorescent probe molecule
that can be hybridized to viral DNA or RNA. Therefore, upon
exonucleolytic degradation, a detectable fluorescent signal can be
monitored. Therefore, the RT-PCR amplified DNA or RNA is detected
in real time by monitoring the presence of fluorescence
signals.
[1512] As one illustration of this method, in the case of BVDV in
MDBK cells, in a first step, viral RNA is isolated from 140 .mu.L
of the cell culture supernatant by means of a commercially
available column (Viral RNA extraction kit, QiaGen, Calif.). The
viral RNA is then eluted from the column to yield a total volume of
60 .mu.L, and subsequently amplified with a quantitative RT-PCR
protocol using a suitable primer for the BVDV NADL strain. A
quenched fluorescent probe molecule is hybridized to the BVDV DNA,
which then undergoes exonucleolytic degradation resulting in a
detectable fluorescent signal. Therefore, the RT-PCR amplified DNA
was detected in real time by monitoring the presence of
fluorescence signals. The TaqMan probe molecule
(5'-6-fam-AAATCCTCCTAACAA- GCGGGTTCCAGG-tamara 3' [Sequence ID No
1] and primers (sense: 5'-AGCCTTCAGTTTCTTGCTGATGT-3' [Sequence ID
No 2]; and antisense: 5'-TGTTGCGAAAGCACCAACAG-3' [Sequence ID No
3]) were designed with the aid of the Primer Express software
(PE-Applied Biosystems) to be complementary to the BVDV NADL NS5B
region. A total of 10 .mu.L of RNA was analyzed in a 50 .mu.L
RT-PCR mixture. Reagents and conditions used in quantitative PCR
were purchased from PE-Applied Biosystems. The standard curve that
was created using the undiluted inoculum virus ranged from 6000
plaque forming units (PFU) to 0.6 PFU per RT-PCR mixture. A linear
range of over 4-logs was routinely obtained.
[1513] A comparable approach can be taken to measure the amount of
other Flaviviridae (more importantly HCV, YFV, Dengue, West Nile
Virus and others) in a clinical sample or in a tissue culture
sample. For example, the combination of HCV RNA purification with
real-time RT-PCR using the following primers
(5'-TTCCGCAGACCACTATGG-3' [Sequence ID No. 4] and
5'-AGCCATGGCGTTAGTATGAGTGT-3' [Sequence ID No. 5]) and probe
(5'-6-fam-CCTCCAGGACCCCCCCTCCC-tamara-3' [Sequence ID No. 6])
resulted in a 7-log linear range of viral load detection.
[1514] B. Cell/Viral Materials
[1515] One of the best characterized members of the Pestivirus
genus is BVDV. BVDV and HCV share at least three common features,
which are the following: (1) they both undergo IRES-mediated
translation; (2) NS4A cofactor is required by their NS3 serine
protease; and (3) they undergo similar polyprotein processing
within the non-structural region, especially at the NS5A and NS5B
junction site.
[1516] The BVDV replication system was used for the discovery of
anti-Flaviviridae compounds. The compounds described herein are
active against Pestiviruses, Hepaciviruses and/or Flaviviruses.
[1517] Maldin-Darby bovine kidney (MDBK) cells were grown and
maintained in a modified eagle medium (DMEM/F12; GibcoBRL),
supplemented with 10% heat inactivated horse serum at 37.degree. C.
in a humidified, 5% CO.sub.2, incubator.
[1518] Bovine viral diarrhea virus (BVDV), strain NADL, causes a
cytopathogenic effect (CPE) after infection of these cells.
[1519] C. Antiviral Assay
[1520] MDBK-cells, grown in DMEM/F12-10% horse serum (HS), were
isolated in standard techniques using trypsin-EDTA. Cells were
seeded in a 96-well plate at 5.times.10.sup.4 cells/well, with test
compound (20 micromolar (.mu.M) concentration) to give a total
volume of 100 microliters (.mu.L). After one hour, the media was
removed and the cells were infected at a multiplicity of infection
(MOI) of 0.02 or 0.002 in a total volume of 50 .mu.L for 45
minutes. Thereafter, the virus was removed and the cells were
washed twice with 100 .mu.L of assay media. Finally, the infected
cells were incubated in a total volume of 100 .mu.L containing the
test compound at 10, 40 or 100 .mu.M concentration. After 22 hours,
the cell supernatant was collected by removing the cellular debris
by low-speed centrifugation, and subsequently tested for the
presence of virus in a quantitative manner.
[1521] D. Cytotoxicity Testing of Anti-Flaviviridae Compounds
[1522] The cytotoxicity testing as performed here is a standard
technique. Briefly, cells are seeded in 96-well plates at various
concentrations (dependent on cell type, duration of assay),
typically at 5.times.10.sup.3 cells per well, in the presence of
increasing concentrations of the test compound (0, 1, 3, 10, 33,
and 100 .mu.M). After a three day-incubation, cell viability and
mitochondrial activity are measured by adding the MTS-dye
(Promega), followed by a 3 hours incubation. Afterwards the plates
containing the dye are read at 490 nm. Such methodologies are well
described and available from the manufacturer (Promega).
Example 53
[1523] The BVDVRT-PCR Quantification Standard Curve
[1524] The standard BVDV virus stock contained 2.times.10.sup.6
PFU/mL, as determined by routine plaque assay (Mendez, E. et al. J.
Virol. 1998, 72, 4737). Viral RNA was extracted from 140 .mu.L of
this inoculum material and eluted from a column using 60 .mu.L of
an elution buffer. This purified RNA material then was diluted
stepwise from 10.sup.-1 to 10.sup.-5. Using the real-time RT-PCR
amplification technique, 10 .mu.L of each dilution was tested. The
results of this dilution series are plotted in FIG. 1, relating PFU
to concentration of standard. From this experiment, it is clear
that this technology allows for reliable quantification over 4-logs
of virus (from 6000 to 0.6 PFU/input in amplification mix). The
lower limit of detection in this experiment is 0.6 PFU or -0.22 log
PFU. Therefore, the real-time RT-PCR quantification values of test
samples below this detection limit were considered
non-reliable.
Example 54
[1525] The B VD V Replication Cycle in MDBK Cells
[1526] In order to measure the BVDV production in MDBK cells and to
determine the optimal harvesting time over a certain period of
time, cells were seeded at 5.times.10.sup.4 cells/well and infected
either with MOI=0.02 or MOI=0.002. After infection, the inoculum
was removed and the cells were washed twice with culture medium. At
different time points, the cell supernatant was harvested; and, the
amount of virus was measured and compared to the original inoculum
and the cell wash. At least 2 wash-steps were needed to remove the
inoculum virus, as shown in FIG. 2. The amount of virus produced 22
hours after infection approximately equals the amount of virus used
to inoculate the cells. Based on these results, the time required
for one replication cycle of BVDV in MDBK cells was 22 hours. Note
that the detection level set in these experiments was based on the
lower limit of detection as determined by the standard curve.
Example 55
[1527] Evaluation of Antiviral Compounds Using RT-PCR
[1528] MDBK cells were seeded at 5.times.10.sup.4 cells/ well,
infected with BVDV with a multiplicity of infection (MOI) equal to
0.02 and grown for 22 hours in the presence of a test compound.
Cells that were not treated with a test compound were considered a
negative control, while ribavirin served as a positive control.
Viral RNA was extracted and analyzed by real time RT-PCR. A typical
experiment, shown in FIG. 3, demonstrates that the negative control
and the majority of the treated cells produced comparable amounts
of virus (between 1.5 and 2 log PFU/input), effectively showing the
test compounds as non-active. However, the cells treated with the
positive control, ribavirin (RIB) or with 5-hydroxyuridine
(.beta.-D-CL) show an almost complete absence of viral RNA. RIB and
.beta.-D-CL reduce viral production by approximately 2 log PFU, or
99%, in the 22 hour reproduction period. The exact potency of these
compounds cannot be deduced from this kind of experiment, since the
detection limit in this experiment is set at -0.22 log PFU and only
one cycle of viral replication occurs under the stated experimental
conditions.
[1529] Potencies, or the effect concentration of compounds that
inhibits virus production by 50% or 90% (EC.sub.50 or EC.sub.90
values, respectively), of anti-BVDV compounds were determined in a
similar set of experiments, but over a broad range of test compound
concentrations (0, 1, 3, 10, 33, 100 .mu.M). The EC.sub.90 value
refers to the concentration necessary to obtain a 1-log reduction
in viral production within a 22 hour period. Compounds that showed
potent antiviral activity are listed in Table 21. This table gives
the maximal viral load reduction observed at a given concentration
22 hours post infection.
21TABLE 21 BVDV viral load 22 hours post infection ID n conc.
(.mu.M) Ave. Log Reduction .beta.-D-AA 4 100 2.43 .beta.-D-AI 3 100
1.52 .beta.-D-AJ 3 100 1.34 .beta.-D-AK 4 100 1.90 .beta.-D-AL 3
100 1.55 .beta.-D-AN 2 100 1.21 .beta.-D-AO 2 100 2.24 .beta.-D-AP
3 100 1.36 .beta.-D-AQ 3 100 0.87 .beta.-D-AT 4 100 1.42
.beta.-D-BE 3 100 1.23 .beta.-D-BL 2 100 1.20 .beta.-D-BO 3 100
0.80 .beta.-D-BS 2 10 1.48 .beta.-D-CL 6 40 3.10 .beta.-D-CM 3 40
1.77 .beta.-D-DJ 1 40 1.58 .beta.-D-DK 2 100 2.17 .beta.-D-DL 2 100
1.33 .beta.-D-HA 1 100 2.87 .beta.-D-HB 2 100 2.26 .beta.-D-MD 1
100 2.16 .beta.-D-ME 4 100 2.41 .beta.-D-MF 4 100 1.41 .beta.-D-QA
1 100 1.50 .beta.-D-TA 1 100 1.30 .beta.-D-VA 1 100 4.69
.beta.-L-FC 2 100 2.39
Example 56
[1530] Alternate Cell Culture Systems for Determining Antiviral
Activities
[1531] The assay described above can be adapted to the other
members of the Flaviviridae by changing the cell system and the
viral pathogen. Methodologies to determine the efficacy of these
antiviral compounds include modifications of the standard
techniques as described by Holbrook, M R et al. Virus Res. 2000, 69
(1), 31; Markland, W et al. Antimicrob. Agents. Chemother. 2000, 44
(4), 859; Diamond, M S et al., J. Virol. 2000, 74 (17), 7814;
Jordan, I. et al. J. Infect. Dis. 2000, 182, 1214; Sreenivasan, V.
et al. J. Virol. Methods 1993, 45 (1), 1; or Baginski, S G et al.
Proc. Natl. Acad. Sci. U.S.A. 2000, 97 (14), 7981 or the real-time
RT-PCR technology. Specifically, an HCV replicon system in HuH7
cells (Lohmann, V et al. Science, 1999, 285 (5424), 110) or
modifications thereof (Rice et al. 2000, abstract Xth International
Symposium for Viral Hepatitis and Liver Disease, Atlanta, Ga.) can
be used.
Example 57
[1532] Cytotoxicity Testing of Candidate Compounds
[1533] The cytotoxicity testing as performed herein is a standard
technique. Briefly, cells are seeded in 96-well plates at various
concentrations (dependent on cell type, duration of assay),
typically at 5.times.10.sup.3 cells per well, in the presence of
increasing concentrations of the test compound (0, 1, 3, 10, 33,
and 100 .mu.M). After three (Vero cells), or four (CEM cells), or
five (PBM cells) day-incubation, cell viability and mitochondrial
activity are measured by adding the MTT-dye (Promega), followed by
a 8 hours incubation. Afterwards the plates containing the dye are
fixed by adding a stop-solution followed by another eight hour
incubation. Finally, absorbance is read at 570 nm. Such
methodologies are well described and available from the
manufacturer (Promega).
[1534] A relevant list of compounds tested in this methodology is
listed in Table 22. While the tested compounds are generally not
cytotoxic, compound .beta.-D-GA showed a selective cytotoxic effect
on CEM cells.
22TABLE 22 Cytotoxicity* of V-a and VIIIa ID PBM cells* CEM Cells*
Vero Cells* .beta.-D-GA >100 (11.3) 1.9 >57.4 .beta.-D-GF
>100 (-46.2) >100 (11.2) >100 (4.3) .beta.-L-GA >100
(-113.2) >100 (1.1) >100 (27.9) .beta.-L-GB >100 (33)
>100 (8.3) .about.171 .beta.-L-GC >100 (-53.2) >100 (-1.2)
>100 (-13.4) .beta.-L-GD >100 (-12.9) >100 (-79.7) >100
(0.8) .beta.-L-GE >100 (-59.7) >100 (0.0) >100 (10.6)
.beta.-L-GF >100 (-70.4) >100 (35.1) >100 (33.8)
.beta.-L-GG >100 (-34.6) >100 (17.3) >100 (33.6)
.beta.-L-GH >100 (-52.1) >100 (19.7) >100 (27.0)
.beta.-L-GI >100 (-47.8) >100 (18.0) >100 (31.9)
*IC.sub.50 in .mu.M (% inhibition at 100 .mu.M)
Example 58
[1535] Antiviral Testing of Candidate Compounds for Respiratory
Viruses
[1536] During the course of these experiments, compounds from
general formula (I) have ;.z 5 been tested for their antiviral
activities against a set of viruses infecting the upper respiratory
tract. The methodologies used for these purposes are well
described. The following protocols are standard operating
procedures taken from the Virology Branch, Division of Microbiology
and Infectious Diseases, NIAID, NIH.
[1537] A. Viruses and Cell-Lines Used in Primary Screen
[1538] (i) Influenza A and B
[1539] Virus strains: A/Beijing/262/95 (HIN1) (Source CDC);
A/Sydney/05/97 (H3N2) (source CDC); B/Beijing/184/93 (source:
CDC).
[1540] Cell line: Maldin Darby Canine Kidney (MDCK)
[1541] (ii) Respiratory Syncytial Virus (RSV)
[1542] Virus strain A2 (source: ATCC).
[1543] Cell Line: African Green Monkey kidney (MA-104) cells
[1544] (iii) Parainfluenza Type 3 Virus
[1545] Virus Strain: 14702 (source: isolate 5/95 Boivin, Montreal
Canada)
[1546] Cell line: African Green Monkey kidney (MA-104) cells
[1547] B. Methods for Antiviral Activity
[1548] (i) Inhibition of Viral Cytopathic Effect (CPE)
[1549] This test is run in 96-well micro-titer plates. In this CPE
inhibition test, four loglo dilutions of each test compound will be
added to 3 cups containing the cell mono-layer; within 5 min, the
virus is then added and the plate sealed, incubated at 37.degree.
C. and CPE read microscopically when untreated infected controls
develop a 3 to 4+ CPE (approximately 72 to 120 hours). A known
positive control drug is evaluated in parallel with test drug in
each test. This drug is Ribavirin for influenza, measles, RSV and
para-influenza.
[1550] (ii) Increase in Neutral Red (NR) Dye Uptake.
[1551] This test is run to validate the CPE inhibition seen in the
initial test, and utilizes the same 96-well micro-plate after CPE
has been red. Neutral red is added to the medium; cells not damaged
by virus take up greater amount of dye, which is read on a
computerized micro-plate reader. The method as described by McManus
(Appl. Environment. Microbiol. 31:35-38, 1976) is used. An
EC.sub.50 is determined from this dye uptake.
[1552] (iii) Confirmatory Test: CPE-Visual and Virus Yield
Assay
[1553] Compounds considered active by CPE inhibition and by NR dye
uptake will be retested using both CPE inhibition and effect on
reduction of virus yield. Collected eluates from the initial
testing are assayed for virus titer by serial dilution onto
mono-layers of susceptible cells. Development of CPE in these cells
is indicative for the presence of infectious virus The EC.sub.90,
which is the drug that inhibits the virus production by 1-log is
determined from these data.
[1554] Table 23 summarizes the results of part of the antiviral
testing. .beta.-D-BS has potent anti-flaviviridae activity and
potent in vitro antiviral capacities against influenza A and B, as
well as some activities against RSV. There is no activity against
Parainfluenza type 3 virus, illustrating that this compound is
exerting a specific antiviral effect against certain classes of RNA
viruses, but not all.
[1555] In addition, compound .beta.-D-CL is a potent in-vitro
anti-RSV compound with a selectivity index of 150.
23TABLE 23 Antiviral effect on respiratory viruses Initial Test,
Antiviral Screening with Respiratory Viruses by CPE Inhibition
(Visual) .beta.-D-AJ .beta.-D-BS .beta.-D-CL .beta.-D-DJ Influenza
A EC.sub.50(.mu.M) 150 1.5 >5 >500 (H1N1) SI** 2 50 0 0
Influenza A EC.sub.50(.mu.M) >500 1.5 >5 >500 (H3N2) SI**
0 50 0 0 Influenza B EC.sub.50(.mu.M) 150 0.5 >5 50 SI** 2 150 0
>10 RSV* EC.sub.50(.mu.M) >500 0.5 0.5 500 SI** 0 80 150 0
Parainfluenza EC.sub.50(.mu.M) >500 >500 90 >500 Type 3
Virus SI** 0 0 0 0 Initial Test, Antiviral Screening with
Respiratory Viruses by Neutral Red .beta.-D-AJ .beta.-D-BS
.beta.-D-CL .beta.-D-DJ Influenza A EC.sub.50(.mu.M) 150 1.2 8
>500 (H1N1) SI** >3.3 116 1.1 0 Influenza A EC.sub.50(.mu.M)
>500 4 >5 >500 (H3N2) SI** 0 20 0 0 Influenza B
EC.sub.50(.mu.M) 150 1.2 >5 110 SI** >3.3 133 0 >4.5 RSV*
EC.sub.50(.mu.M) >500 <0.5 <0.5 >500 SI** 0 >30
>170 0 Parainfluenza EC.sub.50(.mu.M) >500 40 40 500 Type 3
Virus SI** 0 1 1 >1 Confirmatory Test, Antiviral Screening with
Respiratory Viruses by Visual (EC.sub.50) .beta.-D-BS Influenza A
EC.sub.50(.mu.M) 1.3 (H1N1) SI** >246 Influenza A
EC.sub.50(.mu.M) 0.5 (H3N2) SI** >640 Influenza B
EC.sub.50(.mu.M) 0.6 SI** >533 Confirmatory Test, Antiviral
Screening with Respiratory Viruses by Yield (EC.sub.90) .beta.-D-BS
Influenza A EC.sub.50(.mu.M) 0.4 (H1N1) SI** >800 Influenza A
EC.sub.50(.mu.M) 0.32 (H3N2) SI** >1000 Influenza B
EC.sub.50(.mu.M) 0.6 SI** >533 *RSV: Respiratory Syncytial Virus
A **SI: Selectivity Index (IC.sub.50/EC.sub.90)
Example 59
[1556] Antiviral Testing of Candidate Compounds for
Flaviviridae
[1557] A. The HCV Replicon System in Huh7 Cells.
[1558] Huh7 cells harboring the HCV replicon can be cultivated in
DMEM media (high glucose, no pyruvate) containing 10% fetal bovine
serum, 1.times. non-essential Amino Acids, Pen-Strep-Glu (100
units/liter, 100 microgram/liter, and 2.92 mg/liter, respectively)
and 500 to 1000 microgram/milliliter G418. Antiviral screening
assays can be done in the same media without G418 as follows: in
order to keep cells in logarithmic growth phase, seed cells in a
96-well plate at low density, for example 1000 cells per well. Add
the test compound immediate after seeding the cells and incubate
for a period of 3 to 7 days at 37.degree. C. in an incubator. Media
is then removed, and the cells are prepared for total nucleic acid
extraction (including replicon RNA and host RNA). Replicon RNA can
then be amplified in a Q-RT-PCR protocol, and quantified
accordingly. The observed differences in quantification of replicon
RNA is one way to express the antiviral potency of the test
compound. A typical experiment demonstrates that in the negative
control and in the non-active compounds-settings a comparable
amount of replicon is produced. This can be concluded because the
measured threshold-cycle for HCV RT-PCR in both setting is close to
each other. In such experiments, one way to express the antiviral
effectiveness of a compound is to subtract the threshold RT-PCR
cycle of the test compound with the average threshold RT-PCR cycle
of the negative control. This value is called DeltaCt (.DELTA.Ct or
DCt). A .DELTA.Ct of 3.3 equals a 1-log reduction (equals
EC.sub.90) in replicon production. Compounds that result in a
reduction of HCV replicon RNA levels of greater than 2 ACt values
(75% reduction of replicon RNA) are candidate compounds for
antiviral therapy. Such candidate compounds are belonging to
structures with general formula (I)-(XXIII). Table 24 gives the
average .DELTA.Ct values (N=times tested) that can be obtained if
the target compounds are incubated in the described way for 96
hours. As a positive control, recombinant interferon alfa-2a
(Roferon-A, Hoffmann-Roche, New Jersey, USA) is taken alongside as
positive control.
[1559] However, this HCV .DELTA.Ct value does not include any
specificity parameter for the replicon encoded viral RNA-dependent
RNA polymerase. In a typical setting, a compound might reduce both
the host RNA polymerase activity and the replicon-encoded
polymerase activity. Therefore, quantification of rRNA (or any
other host RNA polymerase I product) or beta-actin mRNA (or any
other host RNA polymerase II) and comparison with RNA levels of the
no-drug control is a relative measurement of the effect of the test
compound on host RNA polymerases. Table 24 also illustrates the ACt
values for rRNA of the test compounds.
[1560] With the availability of both the HCV .DELTA.Ct data and the
rRNA .DELTA.Ct, a specificity parameter can be introduced. This
parameter is obtained by subtracting both .DELTA.Ct values from
each other. This results in Delta-DeltaCT values (.DELTA..DELTA.ct
or DDCt); a value above 0 means that there is more inhibitory
effect on the replicon encoded polymerase, a .DELTA..DELTA.ct value
below 0 means that the host rRNA levels are more affected than the
replicon levels. The antiviral activity of tested compounds,
expressed as .DELTA..DELTA.Ct values, is given in Table 24. As a
general rule, .DELTA..DELTA.Ct values above 2 are considered as
significantly different from the no-drug treatment control, and
hence, exhibits appreciable antiviral activity. However, compounds
with a .DELTA..DELTA.Ct value of less than 2, but showing limited
molecular cytotoxicty data (rRNA .DELTA.CT between 0 and 2) are
also possible active compounds.
[1561] In another typical setting, a compound might reduce the host
RNA polymerase activity, but not the host DNA polymerase activity.
Therefore, quantification of rDNA or beta-actin DNA (or any other
host DNA fragment) and comparison with DNA levels of the no-drug
control is a relative measurement of the inhibitory effect of the
test compound on cellular DNA polymerases. Table 25 illustrates the
.DELTA.Ct values for rDNA of the test compounds.
[1562] With the availability of both the HCV .DELTA.Ct data and the
rDNA .DELTA.Ct, a specificity parameter can be introduced. This
parameter is obtained by subtracting both .DELTA.Ct values from
each other. This results in .DELTA..DELTA.Ct values; a value above
0 means that there is more inhibitory effect on the replicon
encoded polymerase, a .DELTA..DELTA.Ct value below 0 means that the
host rDNA levels are more affected than the replicon levels. The
antiviral activity of tested compounds, expressed as
.DELTA..DELTA.Ct values, is given in Table 25. As a general rule,
.DELTA..DELTA.Ct values above 2 are considered as significantly
different from the no-drug treatment control, and hence, is an
interested compound for further evaluation. However, compounds with
a .DELTA..DELTA.Ct value of less than 2, but with limited molecular
cytotoxicty (rDNA ACT between 0 and 2) may be desired.
[1563] Compounds that result in the specific reduction of HCV
replicon RNA levels, but with limited reductions in cellular RNA
and/or DNA levels are candidate compounds for antiviral therapy.
Candidate compounds belonging to general formula group (I)-(XXIII)
were evaluated for their specific capacity of reducing Flaviviridae
RNA (including BVDV and HCV), and potent compounds were detected
(Tables 21, 24 and 25).
24TABLE 24 Ave. HCV RNA Ave. rRNA ID n .DELTA.Ct .DELTA.Ct Ave.
.DELTA..DELTA.Ct .beta.-D-AA 3 3.83 2.41 1.42 .beta.-D-AI 3 2.93
2.43 0.48 .beta.-D-AJ 22 2.92 1.74 1.18 .beta.-D-AK 4 3.73 2.48
1.25 .beta.-D-AL 2 3.08 2.72 0.36 .beta.-D-AN 6 3.33 2.11 1.22
.beta.-D-AO 1 4.10 2.13 1.97 .beta.-D-AP 2 3.27 3.23 0.05
.beta.-D-AQ 7 4.45 3.22 1.22 .beta.-D-AT 2 3.71 3.07 0.64
.beta.-D-BE 2 4.44 2.80 1.64 .beta.-D-BF 2 4.37 2.69 1.68
.beta.-D-BH 1 3.06 0.91 2.15 .beta.-D-BJ 2 5.06 3.62 1.44
.beta.-D-BL 1 2.28 1.93 0.35 .beta.-D-BO 1 4.52 2.95 1.57
.beta.-D-BS 40 4.89 1.05 3.83 .beta.-D-BT 5 4.83 3.59 1.24
.beta.-D-BU 4 3.46 2.18 1.06 .beta.-D-BV 3 1.88 0.65 1.22
.beta.-D-CC 6 5.04 4.82 0.21 .beta.-D-DD 1 6.60 4.99 1.61
.beta.-D-DH 3 4.13 2.91 1.21 .beta.-D-DJ 5 3.51 3.62 -0.11
.beta.-D-EB 1 3.33 1.42 1.90 .beta.-D-FA 2 3.80 3.58 1.44
.beta.-D-GA 4 6.04 2.10 3.93 .beta.-D-HA 2 5.52 3.85 1.68
.beta.-D-HB 5 2.94 1.65 1.30 .beta.-D-KB 2 3.61 2.52 1.10
.beta.-D-LA 3 3.85 4.10 0.89 .beta.-D-MD 3 3.57 1.95 1.62
.beta.-D-ME 1 2.89 1.25 1.64 .beta.-D-MF 2 3.79 2.69 1.10
.beta.-D-OE 1 4.51 4.20 0.31 .beta.-D-QA 3 2.91 3.81 -0.89
.beta.-D-RB 2 4.30 3.18 1.12 .beta.-D-TA 1 4.00 3.31 0.69
.beta.-D-UA 1 2.91 1.61 1.3 .beta.-D-VA 1 5.56 4.17 1.39
.beta.-L-FC 3 5.55 5.13 0.42 .beta.-L-JB 1 3.65 4.55 -0.90
.beta.-L-KA 1 4.10 4.84 -0.74 .beta.-L-KC 2 1.19 1.35 -0.16 IFN 4
5.21 0.69 4.52 ribavirin 2 3.13 2.35 0.78
[1564]
25TABLE 25 Ave. HCV RNA Ave. rDNA ID N .DELTA.Ct .DELTA.Ct average
.DELTA..DELTA.Ct .beta.-D-AA 3 3.83 2.53 1.88 .beta.-D-AI 1 3.76
-0.96 4.55 .beta.-D-AJ 16 2.75 0.43 2.33 .beta.-D-AK 1 3.51 2.69
0.79 .beta.-D-AL 1 3.18 2.56 0.61 .beta.-D-AN 2 3.86 2.53 1.88
.beta.-D-AO 1 4.10 1.84 2.26 .beta.-D-AP 2 3.27 2.26 1.02
.beta.-D-AQ 3 4.75 1.78 2.73 .beta.-D-AT 1 3.81 2.43 1.43
.beta.-D-BE 1 4.99 2.06 2.98 .beta.-D-BF 1 5.27 2.04 3.28
.beta.-D-BH 1 3.06 1.42 1.64 .beta.-D-BJ 1 4.34 0.81 3.53
.beta.-D-BL 1 2.28 1.62 0.65 .beta.-D-BS 14 4.81 0.38 4.45
.beta.-D-BT 2 4.44 1.17 3.39 .beta.-D-BU 4 3.46 1.10 1.16
.beta.-D-BV 3 1.88 0.31 1.65 .beta.-D-CC 3 5.84 2.17 3.66
.beta.-D-DD 1 6.60 3.30 3.30 .beta.-D-DH 1 4.14 0.89 3.25
.beta.-D-DJ 1 4.84 2.70 2.14 .beta.-D-EB 1 3.33 0.96 2.37
.beta.-D-FA 2 3.80 1.92 0.78 .beta.-L-FC 1 4.41 1.00 3.41
.beta.-D-HA 1 5.12 2.04 3.16 .beta.-D-HB 1 1.90 1.19 0.40
.beta.-D-KB 1 3.81 0.00 3.81 .beta.-L-JB 1 3.65 1.20 2.45
.beta.-L-KA 1 4.10 0.42 3.69 .beta.-L-KC 1 2.73 -0.81 3.54
.beta.-D-LA 1 3.54 1.56 1.98 .beta.-D-MD 2 3.50 1.58 1.46
.beta.-D-ME 1 2.89 1.53 1.36 .beta.-D-MF 2 3.79 2.17 1.65
.beta.-D-OE 1 4.51 -0.04 4.60 .beta.-D-QA 1 4.85 2.30 2.55
.beta.-D-RB 1 4.00 1.27 2.74 .beta.-D-TA 1 4.00 3.07 0.93
.beta.-D-UA 1 2.91 0.50 2.41
Example 60
[1565] Toxicity Profile of .beta.-D-GA
[1566] Cytotoxicity testing as performed here are standard
techniques. Briefly, cells are seeded in 96-well plates at various
concentrations (dependent on cell type, duration of assay),
typically at 5.times.10.sup.3 cells per well, in the presence of
increasing concentrations of the test compound (0, 1, 3, 10, 33,
and 100 .mu.M). Depending on the cell-type incubation with test
compound can vary in time, but is usually within the range of 3 to
5 days. Cell viability and mitochondrial activity are measured by
adding the MTT-dye (Promega), followed by eight hours of
incubation. Afterwards the plates containing the dye are fixed by
adding a stop-solution followed by another eight hour incubation.
Finally, absorbance is read at 570 nm. Such methodologies are well
described and available from the manufacturer (Promega).
[1567] While the tested compounds are generally not cytotoxic,
surprisingly enough .beta.-D-GA showed a selective cytotoxic effect
on CEM cells (Table 21). In order to explore the complete potential
of this compound, a set of human malignant T and B cells and
various tumor cell lines were incubated with .beta.-D-GA at varying
concentrations, and after the absorbance was read, an IC.sub.50
value was calculated. As a control, Ara-C, 5FU, and cycloheximide
was taken alongside (Table 26).
[1568] .beta.-D-GA has potent toxicity in human malignant T and B
cells, but not in human PBM cells and non-T or B neoplastic cells.
Compared to Ara-C and 5-FU, the anticancer activity of .beta.-D-GA
is highly selective for T and B cells.
26TABLE 26 Toxicity profile of .beta.-D-GA against various tumor
cell lines (IC.sub.50, .mu.M)* .beta.-D-GA Ara-C 5-FU Cycloheximide
PBM >100 7 13.7 2.6 Vero >100 0.8 65 2.1 CEM 2.9 0.6 90.5 0.1
SUDHL-1 0.7 3.7 .smallcircle. 0.3 SupT1 0.3 .smallcircle. 53.6 0.6
H9 1.4 .smallcircle. 14.2 1 JY 3 .smallcircle. 7.5 0.8 BL41 <1.0
.smallcircle. 24.1 0.3 LNCaP 45.7 .smallcircle. 22.1 2.4 SK-MES-1
>100 .smallcircle. 13.1 3.4 SK-MEL-28 >100 .smallcircle. 11.2
1 HEPG2 >100 .smallcircle. 40.6 3.6 MCF-7 >100 .smallcircle.
43.7 1.5 *MTT assay (incubation time of 3-5 days) PBM: Human
peripheral blood mononuclear cells Vero: African green monkey
kidney cell line CEM: Human T-cell lymphoma cell line SUDHL-1:
Human anaplastic large T-cell lymphoma cell line Supt1: Human
T-cell lymphoblast cell line H9: Human T-cell lymphoblast cell line
JY: Human T-cell lymphoblast cell line (transformed with EBV) BL41:
Human T-cell lymphoblast cell line LNCap: Human prostate
adenocarcinoma cell line SK-MES-1: Human lung squamous carcinoma
cell line SK-MEL-28: Human melnoma cell line HEPG2: Human liver
carcinoma cell line MCF-7: Human breast carcinoma cell line
[1569] The prevention of .beta.-D-GA-related cytotoxicity in CEM
cells (human T-cell lymphoma) and in the SUDHL-1 cells (human
anaplastic large T-cell lymphoma cell line) was studied by adding
natural nucleosides. This experiment was initiated by adding 50
.mu.M of natural nucleosides into the media, together with
increasing concentration of .beta.-D-GA. CEM cells were seeded at
2500 cells per well and incubated for 4 days (=fast growing cell
line with a doubling time of .about.1.3 days). SUDHL-1 cells were
seeded at 10,000 cells/well, and incubated for 3 days (=slow
growing cell line, doubling time .about.3days). The result of this
experiment is plotted in FIG. 4. This figure illustrates that
cytidine and uridine markedly prevent .beta.-D-GA toxicity in
SUDHL-1 cells and also in CEM cells (similar plot, not shown).
2'-Deoxycytidine has modest preventive activity effect. These data
allow to conclude that .beta.-D-GA is equally effective against
slower growing SUDHL-1 cells and fast growing CEM cells and that
Cytidine and uridine prevent the compound related toxicity in both
cell lines. The action of .beta.-D-GA may be related to synthesis
and functions of host RNA molecules, but not DNA.
[1570] The invention has been described with reference to various
specific and preferred embodiments and techniques. However, it
should be understood that many variations and modifications will be
obvious to those skilled in the art from the foregoing detailed
description of the invention and may be made while remaining within
the spirit and scope of the invention.
* * * * *
References