U.S. patent application number 09/928410 was filed with the patent office on 2002-01-31 for substituted o6-benzyl-8-aza-guanines.
This patent application is currently assigned to The Government of the United States of America, Department of Health and Human Services. Invention is credited to Chae, Mi-Young, Dolan, M. Eileen, Moschel, Robert C., Pegg, Anthony E..
Application Number | 20020013299 09/928410 |
Document ID | / |
Family ID | 23088285 |
Filed Date | 2002-01-31 |
United States Patent
Application |
20020013299 |
Kind Code |
A1 |
Moschel, Robert C. ; et
al. |
January 31, 2002 |
Substituted O6-benzyl-8-aza-guanines
Abstract
The present invention provides AGT inactivating compounds such
as substituted O.sup.6-benzylguanines of the formula 1 7- or
9-substituted 8-aza-O.sup.6-benzylguanines, 7,8-disubstituted
O.sup.6-benzylguanines, 7,9-disubstituted O.sup.6-benzylguanines,
4(6)-substituted 2-amino-5-nitro-6 (4) -benzyloxypyrimidines, and 4
(6) -substituted 2-amino-5-nitroso-6(4)-benzyloxypyrimidines, as
well as pharmaceutical compositions comprising such compounds along
with a pharmaceutically acceptable carrier. The present invention
further provides a method of enhancing the chemotherapeutic
treatment of tumor cells in a mammal with an antineoplastic
alkylating agent, which causes cytotoxic lesions at the
O.sup.6-position of guanine, by administering to a mammal an
effective amount of one of the aforesaid compounds,
2,4-diamino-6-benzyloxy-s-triazine, 5-substituted
2,4-diamino-6-benzyloxy- pyrimidines, or
8-aza-O.sup.6-benzylguanine, and administering to the mammal an
effective amount of an antineoplastic alkylating agent which causes
cytotoxic lesions at the Deposition of guanine.
Inventors: |
Moschel, Robert C.;
(Frederick, MD) ; Pegg, Anthony E.; (Hershey,
PA) ; Dolan, M. Eileen; (Oak Park, IL) ; Chae,
Mi-Young; (Anyang-Shi, KR) |
Correspondence
Address: |
LEYDIG VOIT & MAYER, LTD
700 THIRTEENTH ST. NW
SUITE 300
WASHINGTON
DC
20005-3960
US
|
Assignee: |
The Government of the United States
of America, Department of Health and Human Services
Rockville
MD
|
Family ID: |
23088285 |
Appl. No.: |
09/928410 |
Filed: |
August 14, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09928410 |
Aug 14, 2001 |
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09333047 |
Jun 15, 1999 |
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09333047 |
Jun 15, 1999 |
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08849223 |
Apr 28, 1997 |
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5958932 |
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08849223 |
Apr 28, 1997 |
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PCT/US95/09702 |
Jul 31, 1995 |
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PCT/US95/09702 |
Jul 31, 1995 |
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08283953 |
Aug 1, 1994 |
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5525606 |
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Current U.S.
Class: |
514/150 ;
544/267; 544/276 |
Current CPC
Class: |
C07D 239/48 20130101;
C07D 239/50 20130101; C07D 473/06 20130101; A61P 35/00 20180101;
A61P 43/00 20180101; C07D 487/04 20130101; Y10S 514/922 20130101;
C07D 473/40 20130101; C07D 239/47 20130101; C07D 473/18 20130101;
C07D 251/52 20130101 |
Class at
Publication: |
514/150 ;
514/262; 514/263; 544/267; 544/276 |
International
Class: |
A61K 031/655; A61K
031/52; A61K 031/522; C07D 473/18; C07D 473/04 |
Claims
What is claimed is:
1. A compound of the formula 10wherein R.sub.1 is a substituent
selected from the group consisting of amino, hydroxy,
C.sub.1-C.sub.4 alkylamino, C.sub.1-C.sub.4 dialkylamino, and
C.sub.1-C.sub.4 acylamino, R.sub.2 is a substituent selected from
the group consisting of hydrogen, C.sub.1-C.sub.4 alkyl,
C.sub.1-C.sub.4 aminoalkyl, C.sub.1-C.sub.4 hydroxyalkyl,
C.sub.1-C.sub.4 alkylamino C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4
dialkylamino alkyl, C.sub.1-C.sub.4 cyanoalkyl, C.sub.1-C.sub.4
carbamoylalkyl, C.sub.1-C.sub.4 pivaloylalkyl, C.sub.1-C.sub.5
alkylcarbonyloxy C.sub.1C.sub.4 alkyl, C.sub.1-C.sub.4
carboalkoxyalkyl, ribose, 2'-deoxyribose, the conjugate acid form
of a C.sub.1-C.sub.4 carboxyalkyl, and the carboxylate anion of a
C.sub.1-C.sub.4 carboxyalkyl as the sodium salt, and R.sub.3 is a
substituent selected from the group consisting of halo,
C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 hydroxyalkyl, thiol,
C.sub.1-C.sub.4 alkylthio, trifluoromethylthio, C.sub.1-C.sub.4
thioacyl, hydroxy, C.sub.1-C.sub.4 alkoxy, trifluoromethoxy,
methane-sulfonyloxy, trifluoromethanesulfonylox- y, C.sub.1-C.sub.4
acyloxy, amino, C.sub.1-C.sub.4 aminoalkyl, C.sub.1-C.sub.4
alkylamino, C.sub.1-C.sub.4 dialkylamino, trifluoromethylamino,
ditrifluoromethylamino, aminomethanesulfonyl, amino C.sub.1-C.sub.4
alkylcarbonyl, aminotrifluoromethylcarbonyl, formylamino, nitro,
nitroso, C.sub.1-C.sub.4 alkyldiazo, C.sub.5-C.sub.6 aryldiazo,
trifluoromethyl, C.sub.1-C.sub.4 haloalkyl, C.sub.1-C.sub.4
cyanoalkyl, cyano, C.sub.1-C.sub.4 alkyloxycarbonyl,
C.sub.1-C.sub.4 alkylcarbonyl, phenyl, phenylcarbonyl, formyl,
C.sub.1-C.sub.4 alkoxymethyl, phenoxymethyl, C.sub.2-C.sub.4 vinyl,
C.sub.2-C.sub.4 ethynyl, and SO.sub.nR' wherein n is 0, 1, 2, or 3
and R' is hydrogen, C.sub.1-C.sub.4 alkyl, amino, or phenyl.
2. The compound of claim 1, wherein R.sub.1 is selected from the
group consisting of amino, hydroxy, C.sub.1-C.sub.4 alkylamino,
C.sub.1-C.sub.4 dialkylamino, and C.sub.1-C.sub.4
alkylcarbonylamino, R.sub.2 is selected from the group consisting
of hydrogen, C.sub.1-C.sub.4 alkyl, and C.sub.1-C.sub.6
alkylcarbonyloxy C.sub.1-C.sub.4 alkyl, and R.sub.3 is selected
from the group consisting of amino, halo, C.sub.1-C.sub.4 alkyl,
hydroxy, and trifluoromethyl.
3. The compound of claim 2, wherein R.sub.1 is selected from the
group consisting of amino, hydroxy, methylamino, dimethylamino, and
acetylamino, R.sub.2 is selected from the group consisting of
hydrogen, methyl, and pivaloyloxy methyl, and R.sub.3 is selected
from the group consisting of amino, bromo, methyl, hydroxy, and
trifluoromethyl.
4. The compound of claim 3, wherein said compound is selected from
the group consisting of 8 amino-O.sup.6-benzylguanine,
8-methyl-O.sup.6-benzylguanine, 8-hydroxy-O.sup.6-benzylguanine,
8-bromo-O.sup.6-benzylguanine,
8-trifluoromethyl-O.sup.6-benzylguanine, O.sup.6-benzylxanthine,
O.sup.6-benzyluric acid, N.sup.2-acetyl-O.sup.6-b-
enzyl-8-oxoguanine, O.sup.6-benzyl-N.sup.2-methylguanine,
O.sup.6-benzyl-N.sup.2,N.sup.2-dimethylguanine,
O.sup.6-benzyl-8-trifluor- omethyl-9-methylguanine,
O.sup.6-benzyl-8-bromo-9-methylguanine, and
O.sup.6-benzyl-8-bromo-9-(pivaloyloxymethyl)-guanine.
5. A compound of the formula 11wherein R.sub.1 is NO.sub.2 or NO,
and R.sub.2 is a substituent selected from the group consisting of
hydrogen, halo, C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4
hydroxyalkyl, thiol, C.sub.1-C.sub.4 alkylthio,
trifluoromethylthio, C.sub.1-C.sub.4 thioacyl, hydroxy,
C.sub.l-C.sub.4 alkoxy, trifluoromethoxy, methanesulfonyloxy,
trifluoromethanesulfonyloxy, C.sub.1-C.sub.4 acyloxy,
C.sub.1-C.sub.4 aminoalkyl, C.sub.1-C.sub.4 alkylamino,
C.sub.1-C.sub.4 dialkylamino, trifluoromethylamino,
ditrifluoromethylamino, aminomethanesulfonyl, amino C.sub.1-C.sub.4
alkylcarbonyl, amino trifluoromethylcarbonyl, formylamino, nitro,
nitroso, C.sub.1-C.sub.4 alkyldiazo, C.sub.5-C.sub.6 aryldiazo,
trifluoromethyl, C.sub.1-C.sub.4 haloalkyl, C.sub.1-C.sub.4
cyanoalkyl, cyano, C.sub.1-C.sub.4 alkyloxycarbonyl,
C.sub.1-C.sub.4 alkylcarbonyl, phenyl, phenylcarbonyl, formyl,
C.sub.1-C.sub.4 alkoxymethyl, phenoxymethyl, C.sub.2-C.sub.4 vinyl,
C.sub.2-C.sub.4 ethynyl, and SO.sub.nR' wherein n is 0, 1, 2, or 3
and R' is hydrogen, C.sub.1-C.sub.4 alkyl, amino, or phenyl.
6. The compound of claim 5, wherein R.sub.1 is NO.sub.2, and
R.sub.2 is selected from the group consisting of hydrogen and
C.sub.1-C.sub.4 alkyl.
7. The compound of claim 6, wherein said compound is selected from
the group consisting of 2-amino-4-benzyloxy-5-nitropyrimidine and
2-amino-4-benzyloxy-6-methyl-5-nitropyrimidine.
8. A compound of the formula 12wherein R is selected from the group
consisting of C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.6
alkylcarbonyloxy C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4
alkyloxycarbonyl C.sub.1-C.sub.4 alkyl, carboxy C.sub.1-C.sub.4
alkyl, cyano C.sub.1-C.sub.4 alkyl, aminocarbonyl C.sub.1-C.sub.4
alkyl, hydroxy C.sub.1-C.sub.4 alkyl, and C.sub.1-C.sub.4 alkyloxy
C.sub.1-C.sub.4 alkyl.
9. The compound of claim 8, wherein R is selected from the group
consisting of C.sub.1-C.sub.4 alkyl and C.sub.1-C.sub.6
alkylcarbonyloxy C.sub.1-C.sub.4 alkyl.
10. The compound of claim 9, wherein said compound is selected from
the group consisting of 8-aza-O.sup.6-benzyl-9-methylguanine and
8-aza-O.sup.6-benzyl-9-(pivaloyloxymethyl) guanine.
11. A compound of the formula 13wherein said R is selected from the
group consisting of C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.6
alkylcarbonyloxy C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4
alkyloxycarbonyl C.sub.1-C.sub.4 alkyl, carboxy C.sub.1-C.sub.4
alkyl, cyano C.sub.1-C.sub.4 alkyl, aminocarbonyl C.sub.1-C.sub.4
alkyl, hydroxy C.sub.1-C.sub.4 alkyl, and C.sub.1-C.sub.4 alkyloxy
C.sub.1-C.sub.4 alkyl.
12. The compound of claim 11, wherein said R is C.sub.1-C.sub.6
alkylcarbonyloxy C.sub.1-C.sub.4 alkyl.
13. The compound of claim 12, wherein said compound is
8-aza-O.sup.6-benzyl-7-(pivaloyloxymethyl) guanine.
14. A compound of the formula 14wherein R.sub.1 is selected from
the group consisting of hydrogen, halo, C.sub.1-C.sub.4 alkyl, halo
C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.6 alkylcarbonyloxy
C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 alkyloxycarbonyl
C.sub.1-C.sub.4 alkyl, carboxy C.sub.1-C.sub.4 alkyl, cyano
C.sub.1-C.sub.4 alkyl, aminocarbonyl C.sub.1-C.sub.4 alkyl, hydroxy
C.sub.1-C.sub.4 alkyl, and C.sub.1-C.sub.4 alkyloxy C.sub.1-C.sub.4
alkyl, and R.sub.2 is selected from the group consisting of
C.sub.1-C.sub.4 alkyl, halo C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.6
alkylcarbonyloxy C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4
alkyloxycarbonyl C.sub.1-C.sub.4 alkyl, carboxy C.sub.1-C.sub.4
alkyl, cyano C.sub.1-C.sub.4 alkyl, aminocarbonyl C.sub.1-C.sub.4
alkyl, hydroxy C.sub.1-C.sub.4 alkyl, and C.sub.1-C.sub.4 alkyloxy
C.sub.1-C.sub.4 alkyl, with the proviso that when R.sub.1 is
hydrogen, R.sub.2 is selected from the group consisting of halo
C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 alkyloxy C.sub.1-C.sub.4
alkyl, C.sub.1-C.sub.6 alkylcarbonyloxy C.sub.1-C.sub.4 alkyl,
C.sub.3-C.sub.4 alkyloxycarbonyl C.sub.1-C.sub.4 alkyl, carboxy
C.sub.2-C.sub.4 alkyl, cyano C.sub.2-C.sub.4 alkyl, aminocarbonyl
C.sub.2-C.sub.4 alkyl, and hydroxy C.sub.1-C.sub.3 alkyl.
15. The compound of claim 14, wherein said compound is selected
from the group consisting of
O.sup.6-benzyl-8-bromo-7-(pivaloyloxymethyl) guanine and
O.sup.6-benzyl-7-(pivaloyloxymethyl) guanine.
16. A pharmaceutical composition comprising a pharmaceutically
acceptable-carrier and a compound of the formula 15wherein said
pharmaceutically acceptable carrier comprises polyethylene
glycol.
17. A pharmaceutical composition comprising a pharmaceutically
acceptable carrier and a compound of the formula 16wherein R is a
substituent selected from the group consisting of hydrogen, halo,
C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 hydroxyalkyl, thiol,
C.sub.1-C.sub.4 alkylthio.sub.1 trifluoromethylthio,
C.sub.1-C.sub.4 thioacyl, hydroxy, C.sub.1-C.sub.4 alkoxy,
trifluoromethoxy, methanesulfonyloxy, trifluoromethylfonyloxy,
C.sub.1-C.sub.4 acyloxy, amino, C.sub.1-C.sub.4 aminoalkyl,
C.sub.1-C.sub.4 alkylamino, C.sub.1-C.sub.4 dialkylamino,
trifluoromethylamino, ditrifluoromethylamino, aminomethanesulfonyl,
amino C.sub.1-C.sub.4 alkylcarbonyl, ;aminotrifluoromethylcarbonyl,
formylamino, nitro, nitroso, C.sub.1-C.sub.4 alkyldiazo,
C.sub.5-C.sub.6 aryldiazo, trifluoromethyl, C.sub.1-C.sub.4
haloalkyl, cyanomethyl, C.sub.1-C.sub.4 cyanoalkyl, cyano,
C.sub.1-C.sub.4 alkyloxycarbonyl, C.sub.1-C.sub.4 alkylcarbonyl,
phenyl, phenylcarbonyl C.sub.1-C.sub.4 acyl, formyl,
C.sub.1-C.sub.4 alkoxymethyl, phenoxymethyl, C.sub.2-C.sub.4 vinyl,
C.sub.2-C.sub.4 ethynyl, and SO.sub.nR.sub.1 wherein n is 0, 1, 2,
or 3 and R.sub.1 is hydrogen, C.sub.1-C.sub.4 alkyl, amino, or
phenyl, and wherein said pharmaceutically acceptable carrier
comprises polyethylene glycol.
18. A pharmaceutical composition comprising a pharmaceutically
acceptable carrier and a compound of the formula 17wherein said
pharmaceutically acceptable carrier comprises polyethylene
glycol.
19. A method of enhancing the chemotherapeutic treatment of tumor
cells in a mammal with an antineoplastic alkylating agent which
causes cytotoxic lesions at the O.sup.6-position of guanine, which
method comprises: administering to a mammal an effective amount of
a compound of the formula 18and administering to said mammal an
effective amount of an antineoplastic alkylating agent which causes
cytotoxic lesions at the O.sup.6-position of guanine.
20. A method of enhancing the chemotherapeutic treatment of tumor
cells in a mammal with an antineoplastic alkylating agent that
causes cytotoxic lesions at the O.sup.6-position of guanine, which
method comprises: administering to a mammal an effective amount of
a compound of the formula 19wherein R is a substituent selected
from the group consisting of hydrogen, halo, C.sub.1-C.sub.4 alkyl,
C.sub.1-C.sub.4 hydroxyalkyl, thiol, C.sub.1-C.sub.4 alkylthio,
trifluoromethylthio, C.sub.1-C.sub.4 thioacyl, hydroxy,
C.sub.1-C.sub.4 alkoxy, trifluoromethoxy, methanesulfonyloxy,
trifluoromethanesulfonyloxy, C.sub.1-C.sub.4 acyloxy, amino,
C.sub.1-C.sub.4 aminoalkyl, C.sub.1-C.sub.4 alkylamino,
C.sub.1-C.sub.4 dialkylamino, trifluoromethylamino,
ditrifluoromethylamino, aminomethanesulfonyl, amino C.sub.1-C.sub.4
alkylcarbonyl, aminotrifluoromethylcarbonyl, formylamino, nitro,
nitroso, C.sub.1-C.sub.4 alkyldiazo, C.sub.5-C.sub.6 aryldiazo,
trifluoromethyl, C.sub.1-C.sub.4 haloalkyl, cyanomethyl,
C.sub.1-C.sub.4 cyanoalkyl, cyano, C.sub.1-C.sub.4
alkyloxycarbonyl, C.sub.1-C.sub.4 alkylcarbonyl, phenyl,
phenylcarbonyl, C.sub.1-C.sub.4 acyl, formyl, C.sub.1-C.sub.4
alkoxymethyl, phenoxymethyl, C.sub.2-C.sub.4 vinyl, C.sub.2-C.sub.4
ethynyl, and SO.sub.nR.sub.1 wherein n is 0, 1, 2, or 3 and R.sub.1
is hydrogen, C.sub.1-C.sub.4 alkyl, amino, or phenyl, and
administering to said mammal an effective amount of an
antineoplastic alkylating agent which causes cytotoxic lesions at
the O.sup.6-position of guanine.
21. A method of enhancing the chemotherapeutic treatment of tumor
cells in a mammal with an antineoplastic alkylating agent that
causes cytotoxic lesions at the O.sup.6-position of guanine, which
method comprises: administering to a mammal an effective amount of
a compound of the formula 20and administering to said mammal an
effective amount of an antineoplastic alkylating agent which causes
cytotoxic lesions at the O.sup.6-position of guanine.
22. A compound of the formula 21wherein R.sub.1 is a substituent
selected from the group consisting of hydroxy, C.sub.1-C.sub.4
alkylamino, and C.sub.1-C.sub.4 dialkylamino, R.sub.2 is a
substituent selected from the group consisting of hydrogen,
C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 aminoalkyl, C.sub.1-C.sub.4
hydroxyalkyl, C.sub.1-C.sub.4 alkylamino C.sub.1-C.sub.4 alkyl,
C.sub.1-C.sub.4 dialkylamino alkyl, C.sub.1-C.sub.4 cyanoalkyl,
C.sub.1-C.sub.4 carbamoylalkyl, C.sub.1-C.sub.4 pivaloylalkyl,
C.sub.1-C.sub.6 alkylcarbonyloxy C.sub.1-C.sub.4alkyl,
C.sub.1-C.sub.4 carboalkoxyalkyl, ribose, 2'-deoxyribose, the
conjugate acid form of a C.sub.1-C.sub.4 carboxyalkyl, and the
carboxylate anion of a C.sub.1-C.sub.4 carboxyalkyl as the sodium
salt, and R.sub.3 is a substituent selected from the group
consisting of hydrogen, halo, C.sub.1-C.sub.4 alkyl,
C.sub.1-C.sub.4 hydroxyalkyl, thiol, C.sub.1-C.sub.4 alkylthio,
trifluoromethylthio, C.sub.1-C.sub.4 thioacyl, hydroxy,
C.sub.1-C.sub.4 alkoxy, trifluoromethoxy, methanesulfonyloxy,
trifluoromethanesulfonyloxy, C.sub.1-C.sub.4 acyloxy, amino,
C.sub.1-C.sub.4 aminoalkyl, C.sub.1-C.sub.4 alkylamino,
C.sub.1-C.sub.4 dialkylamino, trifluoromethylamino,
ditrifluoromethylamino, aminomethanesulfonyl, amino C.sub.1-C.sub.4
alkylcarbonyl, aminotrifluoromethylcarbonyl, formylamino, nitro,
nitroso, C.sub.1-C.sub.4alkyldiazo, C.sub.5-C.sub.6 aryldiazo,
trifluoromethyl, C.sub.1-C.sub.4 haloalkyl, C.sub.1-C.sub.4
cyanoalkyl, Cyano, C.sub.1-C.sub.4 alkyloxycarbonyl,
C.sub.1-C.sub.4 alkylcarbonyl, -phenyl, phenylcarbonyl, formyl,
C.sub.1-C.sub.4 alkoxymethyl, phenoxymethyl, C.sub.2-C.sub.4 vinyl,
C.sub.2-C.sub.4 ethynyl, and SO.sub.nR' wherein a is 0, 1, 2, or 3
and R' is hydrogen, C.sub.1-C.sub.4 alkyl, amino, or phenyl, with
the proviso that R.sub.1 is not methylamino when R.sub.2 is ribose
or 2'-deoxyribose and R.sub.3 is hydrogen.
23. A compound of the formula 22wherein R.sub.1 is a substituent
selected from the group consisting of amino, hydroxy,
C.sub.1-C.sub.4 alkylamino, C.sub.1-C.sub.4 acylamino, and
C.sub.1-C.sub.4-dialkylamino, R.sub.2 is a substituent selected
from the group consisting of C.sub.1-C.sub.4 aminoalkyl,
C.sub.1-C.sub.4 alkylamino C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4
dialkylamino alkyl, C.sub.1-C.sub.4 carbamoylalkyl, C.sub.1-C.sub.4
pivaloylalkyl, C.sub.1-C.sub.6 alkylcarbonyloxy C.sub.1-C.sub.4
alkyl, C.sub.1-C.sub.4 carboalkoxyalkyl, ribose, and
2'-deoxyribose, and R.sub.3 is a substituent selected from the
group consisting of hydrogen, halo, C.sub.1-C.sub.4 alkyl,
C.sub.1-C.sub.4 hydroxyalkyl, thiol, C.sub.1-C.sub.4 alkylthio,
trifluoromethylthio, C.sub.1-C.sub.4 thioacyl, hydroxy,
C.sub.1-C.sub.4 alkoxy, trifluoromethoxy, methanesulfonyloxy,
trifluoromethanesulfonyloxy, C.sub.1-C.sub.4 acyloxy, amino,
C.sub.1-C.sub.4 aminoalkyl, C.sub.1-C.sub.4 alkylamino,
C.sub.1-C.sub.4 dialkylamino, trifluoromethylamino,
ditrifluoromethylamino, aminomethanesulfonyl, amino C.sub.1-C.sub.4
alkylcarbonyl, aminotrifluoromethylcarbonyl, formylamino, nitro,
nitroso, C.sub.1-C.sub.4 alkyldiazo, C.sub.5-C.sub.6 aryldiazo,
trifluorom.ethyl, C.sub.1-C.sub.4 haloalkyl, C.sub.1-C.sub.4
cyanoalkyl, cyano, C.sub.1-C.sub.4 alkyloxycarbonyl,
C.sub.1-C.sub.4 alkylcarbonyl, phenyl, phenylcarbonyl, formyl,
C.sub.1-C.sub.4 alkoxymethyl, phenoxymethyl, C.sub.2-C.sub.4 vinyl,
C.sub.2-C.sub.4 ethynyl, and SO.sub.nR' wherein n is 0, 1, 2, or 3
and R' is hydrogen, C.sub.1-C.sub.4 alkyl, amino, or phenyl, with
the proviso that R.sub.1 is not methylamino when R.sub.2 is ribose
or 2'-deoxyribose and R.sub.3 is hydrogen.
24. A pharmaceutical composition comprising a pharmaceutically
acceptable carrier and at least one compound of any of claims 1-15
or 22-23.
25. The pharmaceutical composition of claim 24, wherein said
pharmaceutically acceptable carrier comprises polyethylene
glycol.
26. A method of enhancing the chemotherapeutic treatment of tumor
cells in a mammal with an antineoplastic alkylating agent that
causes cytotoxic lesions at the O.sup.6-position of guanine, which
method comprises: administering to a mammal an effective amount of
a compound of any of claims 1-15 or 22-23, and administering to
said mammal an effective amount of an antineoplastic alkylating
agent which causes cytotoxic lesions at the O.sup.6-position of
guanine.
Description
TECHNICAL FIELD OF THE INVENTION
[0001] The present invention relates to substituted
O.sup.6-benzylguanines, O.sup.6-benzyl-8-azaguanines, and
6(4)-benzyloxypyrimidines, pharmaceutical compositions comprising
such compounds, and-methods of using such compounds. The subject
compounds are particularly useful in inactivating the human DNA
repair protein O.sup.6-alkylguanine-DNA alkyltransferase.
BACKGROUND OF THE INVENTION
[0002] The inactivation of the human DNA repair protein
O.sup.6-alkylguanine-DNA alkyltransferase (AGT) by
O.sup.6-benzylguanine leads to a dramatic enhancement in the
cytotoxic response of human tumor cells and tumor xenografts to
chemotherapeutic drugs whose mechanism of action involves
modification of DNA guanine residues at the O.sup.6-position (Dolan
et al., Proc. Natl. Acad. Sci. U.S.A., 87, 5368-5372 (1990); Dolan
et al., Cancer Res., 51, 3367-3372 (1991); Dolan et al., Cancer
Commun., 2, 371-377 (1990); Mitchell et al., Cancer Res., 52,
1171-1175 (1992); Friedman et al., J. Natl. Cancer Inst., 84,
1926-1931 (1992); Felker et al., Cancer Chem. Pharmacol., 32,
471-476 (1993); Dolan et al., Cancer Chem. Pharmacol., 32, 221-225
(1993); Dolan et al., Biochem. Pharmacol., 46, 285-290 (1993)). The
AGT inactivating activity of a large number of
O.sup.6-benzylguanine analogs have been compared with the aim of
obtaining information about the types of substituent groups and the
sites at which they could be attached to O.sup.6-benzylguanine
without significantly lowering its AGT-inactivating activity
(Moschel et al., J. Med. Chem., 35, 4486-4491 (1992); Chae et al.,
J. Med. Chem., 37, 342-347 (1994)). While these studies led to the
production of a variety of analogs that were as potent or somewhat
less potent than O.sup.6-benzylguanine, none of the analogs were
better than O.sup.6-benzylguanine.
[0003] Thus, there remains a need for additional compounds which
are capable of enhancing the chemotherapeutic treatment of tumor
cells in a mammal with an antineoplastic alkylating agent which
causes cytotoxic lesions at the O.sup.6-position of guanine. The
present invention provides such compounds and associated
pharmaceutical compositions and treatment methods. These and other
objects and advantages of the present invention, as well as
additional inventive features, will be apparent from the
description of the invention provided herein.
BRIEF SUMMARY OF THE INVENTION
[0004] The present invention provides 7- and 8-substituted
O.sup.6-benzylguanine derivatives, 7,8-disubstituted
O.sup.6-benzylguanine derivatives, 7,9-disubstituted benzylguanine
derivatives, 8-aza-O.sup.6-benzylguanine derivatives, and
4(6)-substituted 2-amino-5-nitro-6(4)-benzyloxypyrimidine and
2-amino-5-nitroso-6(4)-benzyloxypyrimidine derivatives which have
been found to be effective AGT inactivators, as well as
pharmaceutical compositions comprising such derivatives along with
a pharmaceutically acceptable carrier. The present invention
further provides a method of enhancing the chemotherapeutic
treatment of tumor cells in a mammal with an antineoplastic
alkylating agent which causes cytotoxic lesions at the
O.sup.6-position of guanine, by administering to a mammal an
effective amount of one of the aforesaid derivatives,
2,4-diamino-6-benzyloxy-s-tri- azine, 5-substituted
2,4-diamino-6-benzyloxypyrimidines, or 8-aza-O.sup.6-benzylguanine,
and administering to the mammal an effective amount of an
antineoplastic alkylating agent which causes cytotoxic lesions at
the O.sup.6-position of guanine.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0005] The present invention provides a compound of the formula I
2
[0006] wherein R.sub.1 is a substituent selected from the group
consisting of amino, hydroxy, C.sub.1-C.sub.4 alkylamino,
C.sub.1-C.sub.4 dialkylamino, and C.sub.1-C.sub.4 acylamino
(although, as explained in further detail below, other substituents
can be placed at this 2-position), R.sub.2 is a substituent
selected from the group consisting of hydrogen, C.sub.1-C.sub.4
alkyl, C.sub.1-C.sub.4 aminoalkyl, C.sub.1-C.sub.4 hydroxyalkyl,
C.sub.1-C.sub.4 alkylaminoalkyl, C.sub.1-C.sub.4 dialkylaminoalkyl,
C.sub.1-C.sub.4 cyanoalkyl, C.sub.1-C.sub.4 carbamoylalkyl,
C.sub.1-C.sub.4 pivaloylalkyl, C.sub.1-C.sub.6 alkylcarbonyloxy
C.sub.1-C.sub.4 alkyl, carbo C.sub.1-C.sub.4 alkoxyalkyl, ribose,
2'-deoxyribose, the conjugate acid form of a C.sub.1-C.sub.4
carboxyalkyl, and the carboxylate anion of a C.sub.1-C.sub.4
carboxyalkyl as the sodium salt (although, as explained in further
detail below, other substituents can be placed at this
N.sup.9-position), and R.sub.3 is a substituent selected from the
group consisting of hydrogen, halo, C.sub.1-C.sub.4 alkyl,
C.sub.1-C.sub.4 hydroxyalkyl, thiol, C.sub.1-C.sub.4 alkylthio,
trifluoromethylthio, C.sub.1-C.sub.4 thioacyl, hydroxy,
C.sub.1-C.sub.4 alkoxy, trifluoromethoxy, methanesulfonyloxy,
trifluoromethanesulfonyloxy, C.sub.1-C.sub.4 acyloxy, amino,
C.sub.1-C.sub.4 aminoalkyl, C.sub.1-C.sub.4 alkylamino,
C.sub.1-C.sub.4 dialkylamino, trifluoromethylamino,
ditrifluoromethylamino, aminomethanesulfonyl, C.sub.1-C.sub.4
aminoacyl, aminotrifluoromethylcarbonyl, formylamino, nitro,
nitroso, C.sub.1-C.sub.4 alkyldiazo, C.sub.5-C.sub.6 aryldiazo,
trifluoromethyl, C.sub.1-C.sub.4 haloalkyl, halomethyl,
C.sub.1-C.sub.4 cyanoalkyl, cyanomethyl, cyano, C.sub.1-C.sub.4
alkyloxycarbonyl, C.sub.1-C.sub.4 alkylcarbonyl, phenyl,
phenylcarbonyl, formyl, C.sub.1-C.sub.4 alkoxymethyl,
phenoxymethyl, C.sub.2-C.sub.4 vinyl, C.sub.2-C.sub.4 ethynyl, and
SO.sub.nR' wherein n is 0, 1, 2, or 3 and R' is hydrogen,
C.sub.1-C.sub.4 alkyl, amino, or phenyl, with the proviso that
R.sub.1 is not amino when both R.sub.2 and R.sub.3 are hydrogen,
and with the proviso that R.sub.1 is not amino or methylamino when
R.sub.2 is ribose or 2'-deoxyribose and R.sub.3 is hydrogen. It is
to be understood that the substituents are defined herein such that
the group farthest from the point of attachment of the substituent
is named first. By way of illustration, C.sub.1-C.sub.6
alkylcarbonyloxy C.sub.1-C.sub.4 alkyl includes
pivaloyloxymethyl.
[0007] Suitable compounds of the above formula include those
compounds wherein R.sub.1 is selected from the group consisting of
amino, hydroxy, C.sub.1-C.sub.4 alkylamino, C.sub.1-C.sub.4
dialkylamino, and C.sub.1-C.sub.4 alkylcarbonylamino, R.sub.2 is
selected from the group consisting of hydrogen, C.sub.1-C.sub.4
alkyl, and C.sub.1-C.sub.6 alkylcarbonyloxy C.sub.1-C.sub.4 alkyl,
and R.sub.3 is selected from the group consisting of amino, halo,
C.sub.1-C.sub.4 alkyl, hydroxy, and trifluoromethyl. Other suitable
compounds include those wherein R.sub.1 is selected from the group
consisting of amino, hydroxy, methylamino, dimethylamino, and
acetylamino, R.sub.2 is selected from the group consisting of
hydrogen, methyl, and pivaloyloxymethyl, and R.sub.3 is selected
from the group consisting of amino, bromo, methyl, hydroxy, and.
trifluoromethyl. Examples of suitable compounds include
8-amino-O.sup.6-benzylguanine, 8-methyl-O.sup.6-benzylguanine,
8-hydroxy-O.sup.6-benzylguanine, 8-bromo-O.sup.6-benzylguanine,
8-trifluoromethyl-O.sup.6-benzylguanine, O.sup.6-benzylxanthine,
O.sup.6-benzyluric acid,
N.sup.2-acetyl-O.sup.6-benzyl-8-oxoguanine,
O.sup.6-benzyl-N.sup.2-methylguanine,
O.sup.6-benzyl-N.sup.2,N.sup.2-dime- thylguanine,
6-benzyl-8-trifluoromethyl-9-methylguanine,
O.sup.6-benzyl-8-bromo-9-methylguanine, and
O.sup.6-benzyl-8-bromo-9-(piv- aloyloxymethyl)guanine.
[0008] The present invention also provides a compound of the
formula II 3
[0009] wherein R.sub.1 is NO.sub.2 or NO, and R.sub.2 is a
substituent selected from the group consisting of hydrogen, halo,
C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 hydroxyalkyl, thiol,
C.sub.1-C.sub.4 alkylthio, trifluoromethylthio, C.sub.1-C.sub.4
thioacyl, hydroxy, C.sub.1-C.sub.4 alkyloxy, trifluoromethoxy,
methanesulfonyloxy, trifluoromethanesulfonylo- xy, C.sub.1-C.sub.4
acyloxy, C.sub.1-C.sub.4 aminoalkyl, C.sub.1-C.sub.4 alkylamino,
C.sub.1-C.sub.4 dialkylamino, trifluoromethylamino,
ditrifluoromethylamino, aminomethanesulfonyl, amino C.sub.1-C.sub.4
alkylcarbonyl, aminotrifluoromethylcarbonyl, formylamino, nitro,
nitroso, C.sub.1-C.sub.4 alkyldiazo, C.sub.5-C.sub.6 aryldiazo,
trifluoromethyl, C.sub.1-C.sub.4 haloalkyl, cyanomethyl,
C.sub.1-C.sub.4 cyanoalkyl, cyano, C.sub.1-C.sub.4
alkyloxycarbonyl, C.sub.1-C.sub.4 alkylcarbonyl, phenyl,
phenylcarbonyl, formyl, C.sub.1-C.sub.4 alkoxymethyl,
phenoxymethyl, C.sub.2-C.sub.4 vinyl, C.sub.2-C.sub.4 ethynyl, and
SO.sub.nR' wherein n is 0, 1, 2, or 3 and R' is hydrogen,
C.sub.1-C.sub.4 alkyl, amino, or phenyl. Suitable compounds include
those compounds wherein R.sub.1 is NO.sub.2 and R.sub.2 is hydrogen
or a C.sub.1-C.sub.4 alkyl. Examples of suitable compounds include
2-amino-4-benzyloxy-5-nitro- pyrimidine and
2-amino-4-benzyloxy-6-methyl-5-nitropyrimidine.
[0010] The present invention further provides a compound of the
formula III 4
[0011] wherein R is selected from the group consisting of
C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 alkyloxycarbonyl
C.sub.1-C.sub.4 alkyl, carboxy C.sub.1-C.sub.4 alkyl, cyano
C.sub.1-C.sub.4 alkyl, aminocarbonyl C.sub.1-C.sub.4 alkyl, hydroxy
C.sub.1-C.sub.4 alkyl, and C.sub.1-C.sub.4 alkyloxy C.sub.1-C.sub.4
alkyl. Suitable compounds of the above formula include those
wherein R is selected from the group consisting of C.sub.1-C.sub.4
alkyl and C.sub.1-C.sub.6 alkylcarbonyloxy C.sub.1-C.sub.4 alkyl.
Examples of suitable compounds include
8-aza-O.sup.6-benzyl-9-methylguanine and
8-aza-O.sup.6-benzyl-9-(pivaloyl- oxymrethyl) guanine.
[0012] The present invention further provides a compound of the
formula IV 5
[0013] wherein R is selected from the group consisting of
C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.6 alkylcarbonyloxy
C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 alkyloxycarbonyl
C.sub.1-C.sub.4 alkyl, carboxy C.sub.1-C.sub.4 alkyl, cyano
C.sub.1-C.sub.4 alkyl, aminocarbonyl C.sub.1-C.sub.4 alkyl, hydroxy
C.sub.1-C.sub.4 alkyl, and C.sub.1-C.sub.4 alkyloxy C.sub.1-C.sub.4
alkyl. Suitable compounds include those wherein said R is
C.sub.1-C.sub.6 alkylcarbonyloxy C.sub.1-C.sub.4 alkyl. An example
of a suitable compound is 8-aza-O.sup.6-benzyl-7-(pivaloyloxymeth-
yl)guanine.
[0014] The present invention further provides a compound of the
formula V 6
[0015] wherein R.sub.1 is selected from the group consisting of
hydrogen, halo, C.sub.1-C.sub.4 alkyl, halo C.sub.1-C.sub.4 alkyl,
C.sub.1-C.sub.6 alkylcarbonyloxy C.sub.1-C.sub.4 alkyl,
C.sub.1-C.sub.4 alkyloxycarbonyl C.sub.1-C.sub.4 alkyl, carboxy
C.sub.1-C.sub.4 alkyl, cyano C.sub.1-C.sub.4 alkyl, aminocarbonyl
C.sub.1-C.sub.4 alkyl, hydroxy C.sub.1-C.sub.4 alkyl, and
C.sub.1-C.sub.4 alkyloxy C.sub.1-C.sub.4 alkyl, and R.sub.2 is
selected from the group consisting of C.sub.1-C.sub.4 alkyl, halo
C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.6 alkylcarbonyloxy
C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 alkyloxycarbony
C.sub.1-C.sub.4 alkyl, carboxy C.sub.1-C.sub.4 alkyl, cyano
C.sub.1-C.sub.4 alkyl, aminocarbonyl C.sub.1-C.sub.4 alkyl, hydroxy
C.sub.1-C.sub.4 is alkyl, and C.sub.1-C.sub.4 alkyloxy
C.sub.1-C.sub.4 alkyl, with the proviso that when R.sub.1 is
hydrogen, R.sub.2 is selected from the group consisting of halo
C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 alkyloxy C.sub.1-C.sub.4
alkyl, C.sub.1-C.sub.6 alkylcarbonyloxy C.sub.1-C.sub.4 alkyl,
C.sub.3-C.sub.4 alkyloxycarbonyl C.sub.1-C.sub.4 alkyl, carboxy
C.sub.2-C.sub.4 alkyl, cyano C.sub.2-C.sub.4 alkyl, aminocarbonyl
C.sub.2-C.sub.4 alkyl, and hydroxy C.sub.1-C.sub.3 alkyl. Suitable
compounds include those wherein R.sub.1 is hydrogen or halo, and
R.sub.2 is C.sub.1-C.sub.6 alkylcarbonyloxy C.sub.1-C.sub.4 alkyl.
Examples of suitable compounds include
O.sup.6-benzyl-8-bromo-7-(pivaloyloxymethyl)guanine and
O.sup.6-benzyl-7-(pivaloyloxymethyl)guanine.
[0016] The present invention additionally provides treatment
methods, which are generally administered via pharmaceutical
compositions comprising one or more of the O.sup.6-substituted
compounds of the present invention. In particular, the present
invention provides a method of enhancing the chemotherapeutic
treatment of tumor cells in a mammal with an antineoplastic
alkylating agent that causes cytotoxic lesions at the
O.sup.6-position of guanine, which method comprises administering
to a mammal an effective amount of one or more of the
aforedescribed present inventive compounds of formulas I-V, and
administering to the mammal an effective amount of an
antineoplastic alkylating agent that causes cytotoxic lesions at
the O.sup.6-position of guanine. The present invention also
includes the method of enhancing the chemotherapeutic treatment of
tumor cells in a mammal with an antineoplastic alkylating agent
that causes cytotoxic lesions at the O.sup.6-position of guanine,
which method comprises (i) administering to a mammal an effective
amount of
[0017] (a) 8-aza-O.sup.6-benzylguanine 7
[0018] (b) a compound of the formula VI 8
[0019] wherein R is a substituent selected from the group
consisting of hydrogen, halo, C.sub.1-C.sub.4 alkyl,
C.sub.1-C.sub.4 hydroxyalkyl, thiol, C.sub.1-C.sub.4 alkylthio,
trifluoromethylthio, C.sub.1-C.sub.4 thioacyl; hydroxy,
C.sub.1-C.sub.4 alkoxy, trifluoromethoxy, methanesulfonyloxy,
trifluoromethanesulfonyloxy, C.sub.1-C.sub.4 acyloxy, amino,
C.sub.1-C.sub.4 aminoalkyl, C.sub.1-C.sub.4 alkylamino,
C.sub.1-C.sub.4 dialkylamino, trifluoromethylamino,
ditrifluoromethylamino, aminomethanesulfonyl, amino C.sub.1-C.sub.4
alkylcarbonyl, aminotrifluoromethylcarbonyl, formylamino, nitro,
nitroso, C.sub.1-C.sub.4 alkyldiazo, C.sub.5-C.sub.6 aryldiazo,
trifluoromethyl, C.sub.1-C.sub.4 haloalkyl, halomethyl,
cyanomethyl, C.sub.1-C.sub.4 cyanoalkyl, cyano, C.sub.1-C.sub.4
alkyloxycarbonyl, C.sub.1-C.sub.4 alkylcarbonyl, phenyl,
phenylcarbonyl, formyl, C.sub.1-C.sub.4 alkoxymethyl,
phenoxymethyl, C.sub.2-C.sub.4 vinyl, C.sub.2-C.sub.4 ethynyl, and
SO.sub.nR' wherein n is 0, 1, 2, or 3 and R' is hydrogen,
C.sub.1-C.sub.4 alkyl, amino, or phenyl, or
[0020] (c) 2,4-diamino-O.sup.6-benzyl-s-triazine, and (ii)
administering to the mammal an effective amount of an
antineoplastic alkylating agent which causes cytotoxic lesions at
the O.sup.6-position of guanine.
[0021] Several O.sup.6-substituted compounds were tested for their
ability to inactivate the human DNA repair protein,
O.sup.6-alkylguanine-DNA alkyltransferase (AGT, alkyltransferase).
Two classes of compounds were identified as being significantly
better than O.sup.6-benzylguanine (the prototype
low-molecular-weight irradiator) in inactivating AGT in human HT29
colon tumor cell extracts. These were 8-substituted
O.sup.6-benzylguanines bearing electron-withdrawing groups at the
8-position and 5-substituted 2,4-diamino-6-benzyloxypyrimidines
bearing electron-withdrawing groups at the 5-position. The latter
derivatives were also more effective than O.sup.6-benzylguanine in
inactivating AGT in intact HT29 colon tumor cells. Both types of
compounds were as effective or more effective than
O.sup.6-benzylguanine in enhancing cell killing by
1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) of colon, breast and
prostate cancer cells grown in culture. Provided 8-substituted
O.sup.6-benzylguanine derivatives bearing electron-withdrawing
substituents at the 8-position and 5-substituted
2,4-diamino-6-benzyloxyp- yrimidines bearing electron-withdrawing
substituents at the 5-position do not exhibit undesirable toxicity,
they should be superior to O.sup.6-benzylguanine as
chemotherapeutic adjuvants for enhancing the effectiveness of
antitumor drugs whose mechanism of action involves modification of
the O.sup.6-position of DNA guanine residues. The specific
compounds surveyed-for AGT inactivating activity are illustrated
below. 9
[0022] Preparations of the 8-substituted O.sup.6-benzylguanine
derivatives 8-amino-O.sup.6-benzylguanine (1a) and
O.sup.6-benzyl-8-methylguanine (1b) were accomplished by treating
2,8-diamino-6-chloropurine and 2-amino-6-chloro-8-methylpurine,
respectively, with sodium benzyloxide in benzyl alcohol.
O.sup.6-Benzyl-8-oxoguanine (O.sup.6-benzyl-7,8-dihydro-8-
-oxoguanine, 1c) was prepared by reacting 1,1'-carbonyldiimidazole
with 2,4,5-triamino-6-benzyloxypyrimidine (Pfleiderer et al., Chem.
Ber., 94, 12-18 (1961)). For convenience, the compound is
illustrated -in the 8-hydroxy tautomeric form although it most
probably exists in solution in the 8-keto form with a hydrogen
attached to the 7-nitrogen atom. O.sup.6-Benzyl-8-bromoguanine (1d)
was prepared by brominatlon of O.sup.6-benzylguanine.
O.sup.6-Benzyl-8-trifluoromethylguanine (1e) was prepared by
reacting 2-amino-6-chloro-8-trifluoro-methylpurine with sodium
benzyloxide in benzyl alcohol. 8-Aza-O.sup.6-benzylguanine (2) was
prepared through ridrous acid treatment of 2,4,
5-triamina-6-benzyloxypyr- imidine. Compound 2 had been prepared
previously by another route (Shealy et al., J. Org. Chem., 27,
4518-4523 (1962)).
[0023] With respect to the pyrimidine derivatives (3a-f),
4-amino-6-benzyloxy-5-nitropyrimidine (3a) was prepared by treating
4-amino-6-chloro-5-nitropyrimidine (Boon et al., J. Chem. Soc.,
96-102 (1951)) with sodium benzyloxide in benzyl alcohol.
Derivatives 3b-d were prepared by the method of Pfleiderer et al.
(Chem. Ber., 94, 12-18 (1961)).
2,4-Diamino-6-benzyloxy-5-nitropyrimidine (3e) and
2,4-diamino-6-benzyloxy-5-bromopyrimidine (3f) were prepared
previously by Kosary et al. (Acta Pharm. Hung., 49, 241-247
(1989)).
[0024] The purines, O.sup.6-benzylxanthine (4a) and
O.sup.6-benzyluric acid (4b) were prepared by nitrous acid
domination of O.sup.6-benzylguanine and
O.sup.6-benzyl-8-oxoguanine, respectively.
N.sup.2-Acetyl-O.sup.6-benzyl-8-oxoguanine
(N.sup.2-acetyl-O.sup.6-benzyl- -7,8-dihydro-8-oxoguanine) (4d) was
prepared through acetylation of O.sup.6-benzyl-8-oxoguanine (1c).
O.sup.6-Benzyl-2-fluorohypoxanthine (4c) was prepared previously by
Robins and Robins (J. Org. Chem., 34, 2160-2163 (1969)). This
material was treated with methylamine and dimethylamine to produce
O.sup.6-benzyl-N.sup.2-methylguanine (4e) and
O.sup.6-benzyl-N.sup.2,N.sup.2-dimethylguanine (4f),
respectively.
[0025] Compounds 5a (2-amino-4-benzyloxy-5-nitropyrimidine) and 5b
(2-amino-4-benzyloxy-6-methyl-5-nitropyrimidine) were prepared by
treating 2-amino-4-chloro-5-nitropyrimidine and
2-amino-4-chloro-6-methyl- -5-nitropyrimidine (Boon et al., J.
Chem. Soc., 96-102 (1951)), respectively, with sodium benzyloxide
in benzyl alcohol. Compound 6 (2,4-diamino-6-benzyloxy-s-triazine)
was prepared previously under similar conditions (Wakabayashi et
al., Nippon Dojo-Hiryyogaku Zasshi, 41, 193-200 (1970)).
O.sup.6-Benzyl-8-trifluoromethyl-9-methylguanine (7) was prepared
by treating the anion of 1e with methyl iodide in
N,N-dimethylformamide.
[0026] Compound 8a was prepared by methylating the sodium salt of
8-aza-O.sup.6-benzylguanine using methyl iodide as the methylating
agent. Compounds 8b and 9 were prepared by the reaction of the
sodium salt of 8-aza-O.sup.6-benzylguanine and chloromethyl
pivalate. Compound 10a was prepared by direct bromination of
O.sup.6-benzyl-9-methylguanine. Compounds 10b and 11 were prepared
by the reaction of the sodium salt of O.sup.6-benzyl-8-bromoguanine
and chloromethyl pivalate. Compound 12 was prepared by the reaction
of the sodium salt of O.sup.6-benzylguanine and chloromethyl
pivalate.
[0027] The ability of these compounds to inactivate the AGT protein
in HT29 human colon tumor cell extracts and in intact HT29 cells is
summarized in Tables 1 and 2. The data represent the dose of
compound required to produce 50% inactivation in cell-free extracts
upon incubation for 30 min or in cells upon incubation for 4
hr.
1TABLE 1 AGT-Inactivating Activity of 6-Benzyloxypurine,
6(4)-Benzyloxypyrimidine, and 6-Benzyloxy-s-triazine Derivatives
ED.sub.50 (mM).sup.a In HT29 In cell-free HT29 Compound extract
cells 2,4-diamino-6-benzyloxy-5- 0.06 0.02 nitrosopyrimidine (3d)
2,4-diamino-6-benzyloxy-5-nitropyrimidine 0.06 0.02 (3e)
8-aza-O.sup.6-benzylguanine (2) 0.07 0.06
O.sup.6-benzyl-8-bromoguanine (1d) 0.08 0.06 O.sup.6-benzylguanine
0.2 0.05 O.sup.6-benzyl-8-methyl-guanine (1b) 0.3 0.1
O.sup.6-benzyl-8-oxoguanine (1c) 0.3 0.15 O.sup.6-benzyl-8-trifluo-
romethylguanine (1e) 0.4 0.25 2,4,5-triamino-6-benzyloxypyrimidine
(3c) 0.4 0.3 2-amino-4-benzyloxy-6-methyl-5- 0.4 0.06
nitropyrimidine (5b) 2-amino-4-benzyloxy-5-nitropyrimidine (5a) 0.4
0.05 8-amino-O.sup.6-benzylguanine (1a) 0.7 2
2,4-diamino-6-benzyloxy-5-bromopyrimidine 2 0.8 (3f)
2,4-diamino-6-benzyloxy-s-triazine (6) 4 1.0
2,4-diamino-6-benzyloxypyrimidine (3b) 15 5 O.sup.6-benzyluric acid
(4b) 25 45 4-amino-6-benzyloxy-5-nitropyrimidine (3a) 28 8
O.sup.6-benzyl-2-fluorohypoxanthine (4c) 48 12
O.sup.6-benzylxanthine (4a) 60 35 N.sup.2-acetyl-O.sup.6-benzyl-8--
oxoguanine (4d) 65 11 O.sup.6-benzyl-N.sup.2-methylguanine (4e) 160
60 O.sup.6-benzyl-N.sup.2,N.sup.2-dimethylguanine (4f) 200 110
.sup.aThe effective dose required to produce 50% inactivation in
cell-free extracts upon incubation for 30 min or in cells upon
incubation for 4 hr. The values for O.sup.6-benzylguanine are from
Moschel et al., J. Med. Chem., 35, 4486-4491 (1992) .
[0028] Within these series of compounds,
O.sup.6-benzyl-N.sup.2-methyl-and
O.sup.6-benzyl-N.sup.2,N.sup.2-dimethylguanine (4e and 4f) were the
least active agents exhibiting so values for inactivation of AGT in
HT29 cell extracts of 160 and 200 mM, respectively. For comparison,
the ED.sub.50 value exhibited by O.sup.6-benzylguanine was 0.2 mM
(Table 1). The other 2- and/or 8-substituted 6-benzyloxypurines,
N.sup.2-acetyl-O.sup.6-benzyl- -8-oxoguarine (4d),
O.sup.6-benzylxanthine (4a), O.sup.6-benzyl-2-fluorohy- poxanthine
(4c) and O.sup.6-benzyluric acid (4b), together with the
substituted pyrimidines 4-amino-6-benzyloxy-5-nitropyrimidine (3a)
and 2,4-diamino-6-benzyloxypyrimidine (3b), comprised a group of
increasingly more active AGT inactivating agents exhibiting
intermediate ED.sub.50 values in the range of 65 to 15 mM.
2,4-Diamino-6-benzyloxy-s-triazine (6) and
2,4-diamino-6-benzyloxy-5-bromopyrimidine (3f) were considerably
more active than 3b indicating that electron-withdrawing groups at
the 5-position of a 2,4-diamino-6-benzyloxypyrimidine derivative
are positive contributors to efficient AGC inactivation. This is
further emphasized by the very high activity exhibited by
2,4-diamino-6-benzyloxy-5-nitrasopyri- midine (3d) and
2,4-diamino-6-benzyloxy-5-nitropyrimidine (3e), which contain
strongly electron-withdrawing nitroso and nitro substituents,
respectively. These two derivatives are the most active AGT
inactivators tested to date. The observation that
2-amino-4-benzyloxy-5-nitropyrimidin- e (5a) is much more active
than 3a indicates that a 2-amino group is critical for high
activity for a 6(4)-benzyloxy-5-nitropyrimidine derivative. An
additional alkyl group at the 4(6)-position (e.g., as in 5b) does
not enhance activity significantly over that for 5a although an
amino group at the 4(6)-position significantly enhances activity.
Thus, AGT inactivating activity increases substantially over the
series 5a=5b<3d=3e. With these considerations in mind the
activity of 2,4,5-triamino-6-benzyloxypyrimidine (3c) seems
exceptional and the reasons for its relatively high activity are
unclear at present. It is also significant that pyrimidines 5a and
5b are quite active in cells, which is not totally predicted by
their corresponding activity in HT29cell extracts.
[0029] All the O.sup.6-benzylguanine analogs 1a-d were much more
active than the purines in the series 4a-f and the activity
differences among 1a-d also reflect enhancements due to
introduction of electron withdrawing groups. Thus, activity
increased in the series 8-amino-O.sup.6-benzylguanine (1a)
<O.sup.6-benzyl-8-axoguanine (1c)
<O.sup.6-benzyl-8-methylguanine (1b)
<O.sup.6-benzyl-8-bromoguanine (1d)
<8-aza-O.sup.6-benzylguanine (2). Indeed, derivatives 1d and 2
were essentially as active as pyrimidines 3d and 3e in cell-free
extracts although 1d and 2 were somewhat less active in cells than
expected from their activity in cell-free extracts.
[0030] The compounds listed in Table 2 also had AGT-inactivating
activity in cell free extracts and in cells. The activity of 7, 8a,
and 10a in cells is significantly higher than their activity in
cell-free extracts. Thus the ratio of ED50 values in cell-free
extracts/intact cells is 1.6, 1.6, 1.1, respectively, for
derivatives id,. 1e, and 2 (Table 1). This ratio increases to 7.2,
6.3, and 6.3, respectively, for the corresponding methylated
derivatives 10a, 7, and 8a. It is believed that the higher activity
of the methylated derivatives in the cells is due to the fact that
these compounds do not possess readily dissociable hydrogens in the
imidazole portion of the purine ring system and therefore they can
readily enter the cells as neutral molecules.
2TABLE 2 AGT-Inactivating Activity of 7, 8- and 8, 9- Disubstituted
O.sup.6-Benzylguanine Derivatives and Related Compounds ED.sub.50
value (mM).sup.a In HT29 In cell-free HT29 Compound extract cells
O.sup.6-benzyl-8-trifluoromethyl-9-methylguanine 2.5 0.4 (7)
8-aza-O.sup.6-benzyl-9-methylguanine (8a) 0.5 0.08
8-aza-O.sup.6-benzyl-9- 0.28.sup.b 0.23 (pivaloyloxymethyl)guanine
(8b) 8-aza-O.sup.6-benzyl-7- 0.11.sup.c 0.16
(pivaloyloxymethyl)guanine (9) O.sup.6-benzyl-8-bromo-9-methylguan-
ine (10a) 1.9 0.25 O.sup.6-benzyl-8-bromo-9- 0.08.sup.d 0.05
(pivaloyloxymethyl)guanine (10b) O.sup.6-benzyl-7-(pivaloyloxymeth-
yl)guanine (12) 9.sup.e 0.3
O.sup.6-benzyl-9-(pivaloyloxymethyl)gua- nine 3.1.sup.f,g 0.3
O.sup.6-benzyl-9-methylguanine 2.6.sup.g 0.4 .sup.aThe dose
required to produce 50% inactivation in cell-free extracts upon
incubation for 30 min. or in cells upon incubation for 4 hr.
.sup.bED.sub.50 = 4 with purified human AGT. .sup.cED.sub.50 = 2
with purified human AGT. .sup.dED.sub.50 > 100 with purified
human AGT. .sup.eED.sub.50 >> 100 with purified human AGT.
.sup.fED.sub.50 = 95 with purified human AGT. .sup.gData from Chae
et al., J. Med. Chem., 37, 342-347 (1994) .
[0031] The ability of increasing concentrations of 1a-d, 2, and
3c-e to enhance the killing of human HT29 colon cancer cells,
DU-145 prostate cancer cells, and MCF-71 breast cancer cells by
BCNU (40 mM) is shown in Tables 3, 4, and 5, respectively. The data
reflect the number of cell colonies that result following exposure
to AGT inactivator alone or AGT inactivator 2 hr before exposure to
BCNU as described in Dolan et al. (Proc. Natl. Acad. Sci., U.S.A.,
87, 5368-5372 (1990)). Data for O.sup.6-benzylguanine are included
for comparison. As indicated, at 10 mM concentrations, all the
8-substituted purines with the exception of la were as effective as
O.sup.6-benzylguanine in enhancing the cytotoxicity of BCNU (40
mM); such treatment killed essentially all the tumor cells.
Treatment of the cells with the modified 8-substituted
O.sup.6-benzylguanine alone or BCNU alone had no significant effect
on cell colony number. The comparatively low activity of la in all
but the breast cancer cells may reflect its poor transport into
other tumor cell types or its rapid metabolic conversion to an
ineffective AGT inactivator. Its ineffective enhancement of BCNU
cytotoxicity parallels its relatively poor AGT inactivating ability
in colon tumor cells (Table 1).
[0032] For the pyrimidines tested,
2,4,5-triamino-6-benzyloxypyrimidine (3c) was as effective as the
8-substituted O.sup.6-bezylguanine derivatives and
O.sup.6-benzylguanine itself in enhancing BCNU toxicity although
the nitroso- and nitropyrimldine derivatives (3d and 3e) were
similarly effective at a 4-fold lower dose.
3TABLE 3 Killing of HT-29 Colon Cancer Cells by BCNU Combined with
AGT Inactivators Colony Inactivator Formation Concentration BCNU
per 1000 Inactivator (mM) (mM) cells None None 435 .+-. 63 None 40
442 .+-. 34 O.sup.6-benzylguanine 10 None 431 .+-. 33 10 40 13 .+-.
6 2.5 40 38 .+-. 15 1 40 277 .+-. 25 8-aza-O.sup.6-benzylguanine 10
None 537 .+-. 48 (2) 10 40 2 .+-. 1 1 40 423 .+-. 42
O.sup.6-benzyl-8-bromoguanine 10 None 401 .+-. 22 (1d) 10 40 1 .+-.
0 1 40 299 .+-. 30 O.sup.6-benzyl-8-oxoguanine 10 None 401 .+-. 22
(1c) 10 40 <1 1 40 221 .+-. 15 O.sup.6-benzyl-8- 10 None 513
.+-. 76 methylguanine (1b) 10 40 <1 1 40 230 .+-. 51
O.sup.6-benzyl-8-aminoguanine 10 None 504 .+-. 30 (1a) 10 40 430
.+-. 41 1 40 475 .+-. 26 2,4,5-triamino-6- 10 None 453 .+-. 59
benzyloxypyrimidine (3c) 10 40 3 .+-. 1 1 40 487 .+-. 32
2,4-diamino-6-benzyloxy- 2.5 None 528 .+-. 64 5-nitrosopyrimidine
(3d) 2.5 40 <1 1 40 19 .+-. 4 2,4-diamino-6-benzyloxy- 2.5 None
438 .+-. 25 5-nitropyrimidine (3e) 2.5 40 <1 1 40 45 .+-. 4
[0033]
4TABLE 4 Killing of DU-145 Prostate Cancer Cells by BCNU Combined
with AGT Inactivators Colony Inactivator Formation Concentration
BCNU per 1000 Inactivator (mM) (mM) cells None None 453 .+-. 81
None 40 394 .+-. 76 O.sup.6-benzylguanine 10 None 462 .+-. 68 10 40
28 .+-. 5 1 40 299 .+-. 18 8-aza-O.sup.6-benzylguanine 10 None 452
.+-. 72 (2) 10 40 28 .+-. 5 1 40 248 .+-. 21 O.sup.6-benzyl-8- 10
None 493 .+-. 90 bromoguanine (1d) 10 40 16 .+-. 3 1 40 267 .+-. 39
O.sup.6-benzyl-8-oxoguanine 10 None 379 .+-. 34 (1c) 10 40 34 .+-.
3 1 40 329 .+-. 43 O.sup.6-benzyl-8- 10 None 357 .+-. 43
methylguanine (1b) 10 40 50 .+-. 7 1 40 306 .+-. 157
O.sup.6-benzyl-8- 10 None 380 .+-. 36 aminoguanine (1a) 10 40 435
.+-. 70 1 40 295 .+-. 45 2,4,5-triamino-6- 10 None 429 .+-. 101
benzyloxypyrimidine 10 40 57 .+-. 7 (3c) 1 40 378 .+-. 60
2,4-diamino-6- 2.5 None 403 .+-. 35 benzyloxy-5- 2.5 40 7 .+-. 3
nitrosopyrimidine 1 40 25 .+-. 4 (3d) 0.25 40 192 .+-. 17
2,4-diamino-6- 2.5 None 407 .+-. 80 benzyloxy-5- 2.5 40 9 .+-. 2
nitropyrimidine (3e) 1 40 59 .+-. 6 0.25 40 129 .+-. 26
[0034]
5TABLE 5 Killing of MCF-71 Breast Cancer Cells by BCNU Combined
with AGT Inactivators Colony Inactivator Formation Concentration
BCNU per 1000 Inactivator (mM) (mM) cells None None 426 .+-. 78
None 40 364 .+-. 60 O.sup.6-benzylguanine 10 None 455 .+-. 63 10 40
4 .+-. 2 2.5 40 12 .+-. 6 8-aza-O.sup.6-benzylguanine 10 None 483
.+-. 27 (2) 10 40 2 .+-. 1 O.sup.6-benzyl-8-bromoguanine 10 None
380 .+-. 109 (1d) 10 40 3 .+-. 1 2.5 40 4 .+-. 3
O.sup.6-benzyl-8-oxoguani- ne 10 None 522 .+-. 78 (1c) 10 40 4 .+-.
2 O.sup.6-benzyl-8-methylguanine 10 None 376 .+-. 76 (1b) 10 40 2
.+-. 1 O.sup.6-benzyl-8-aminoguanine 10 None 432 .+-. 36 (1a) 10 40
95 .+-. 8 2,4,5-triamino-6- 10 None 448 .+-. 55 benzyloxypyrimidine
(3c) 10 40 12 .+-. 4 2,4-diamino-6-benzyloxy- 2.5 None 447 .+-. 87
5-nitrosopyrimidine (3d) 2.5 40 2 .+-. 1 2,4-diamino-6-benzyloxy-
2.5 None 314 .+-. 49 5-nitropyrimidine (3e) 2.5 40 2 .+-. 1
[0035] Although the human alkyltransferase is very sensitive to
inactivation by O.sup.6-benzylguanine and the various compounds
described above, a number of mutants have been generated that are
resistant to O.sup.6-benzylguanine (Crone and Pegg, Cancer Res.,
53, 4750-4753 (1993)). This resistance is probably caused by a
reduction in the space surrounding the active site of the
alkyltransferase, which limits the access to O.sup.6-benzylguanine;
These mutants are produced by single base changes in the
alkyltransferase DNA-coding sequence causing changes in one or two
amino acids in the alkyltransferase (Crone and Pegg, Cancer Res.,
53, 4750-4753 (1993)). Thus, as indicated in Table 6, changing the
proline residue at position 140 to alanine (protein 2140A) or the
glycine residue at position 156 to an alanine (protein G156A)
causes a 20-fold and a 240-fold increase in resistance to
O.sup.6-benzylguanine, respectively. The alkyltransferase
containing an arginine in place of a praline at residue 138
together with an arginine in place of a praline at residue 140
(protein P138A/P140A) is 88-fold more resistant to inactivation by
O.sup.6-benzylguanine. It is possible that such resistant mutants
will arise or be selected for in tumors under the selective
pressure generated by treatment with O.sup.6-benzylguanine plus an
alkylating agent. More potent inhibitors and/or those of a smaller
size that are better able to fit into the space of the active site
of the mutant alkyltransferase can be used to advantage to overcome
this resistance.
6TABLE 6 Inhibition of Mutant Alkyltransferase Proteins by
O.sup.6-Benzylguanine or 2,4-Diamino-6-benzyloxy-5-ni-
trosopyrimidine ED.sub.50 value (mM).sup.a 2,4-diamino-6-
benzyloxy-5-nitroso- Protein O.sup.6-benzylguanine pyrimidine
Control 0.25 0.05 P140A 5 0.1 P138A/P140A 22 0.3 G156A 60 1
.sup.aThe concentration needed to inactivate 50% of the activity in
30 minutes.
[0036] As shown in Table 6,
2,4-diamino-6-benzyloxy-5-nitrasopyrimidine (3d) was 50 to 60 times
better at inactivating the mutant alkyltransferase than
O.sup.6-benzylguanine. Doses of
2,4-diamino-6-benzyloxy-5-nitrasopyrimidine leading to
intracellular concentrations greater than 5 mM will therefore be
effective at inactivating such resistant alkyltransferase.
Concentrations greater than 200 mM of O.sup.6-benzylguanine would
be needed to get such inactivation, and these are much more than
can be achieved with this compound in current formulations.
However, 8-substituted O.sup.6-benzylguanine derivatives that are
significantly more potent than O.sup.6-benzylguanine may be useful
in inactivating mutant alkyltransferase provided their required
intracellular concentrations can be achieved. These data for mutant
alkyltransferase inactivation and the data presented earlier
indicate that pyrimidine derivatives bearing electron-withdrawing
groups at the 5-position as well as substituted
O.sup.6-benzylguanine derivatives bearing electron-withdrawing
groups at the 8-position are superior to O.sup.6-benzylguanine for
use as adjuvants in chemotherapy with agents whose mechanism of
action, like that of BCNU, involves modification of the
O.sup.6-position of DNA guanine residues.
[0037] Other 8-substituted O.sup.6-benzylguanine derivatives
bearing electron-withdrawing 8-substituents (e.g., NO.sub.2) are
readily available. For example, O.sup.6-benzyl-8-nitroguanine could
be prepared by treatment of 8-nitroguanine (Jones and Robins, J.
Am. Chem. Soc., 82, 3773-3779 (1960)) with phosphorus oxychloride
to produce 2-amino-6-chloro-8-nitropurine which when treated with
sodium benzyloxide in benzyl alcohol would produce the desired
O.sup.6-benzyl-8-nitroguanine- .
[0038] Additional 2,4-diamino-6-benzyloxypyrimidine derivatives
bearing electron-withdrawing groups other than halogen or nitro
groups (e.g., formyl or cyano groups) could also be readily
prepared. 2,4-Diamino-5-formyl-6-hydroxypyrimidine, a known
compound (Delia and Otteman, Heterocycles, 20, 1805-1809 (1983)),
can be treated with phosphorus oxychloride to produce a
2,4-diamino-6-chloro-5-formylpyrimidi- ne intermediate, which on
treatment with sodium benzyloxide in benzyl alcohol produces
2,4-diamino-6-benzyloxy-5-formylpyrimidine. Treatment of the formyl
pyrimidine with hydroxylamine affords 2,4-diamino-6-benzyloxy--
5-cyanopyrimidine. The preparation of a large number of
5-substituted 6(4)-benzyloxypyrimidines or 8-substituted
O.sup.6-benzylguanine derivatives is possible for those skilled in
the art of synthesis of heterocyclic aromatic compounds (D. J.
Brown, "The Pyrimidines," in The Chemistry of Hetezogyclic
Compounds, Vol. 16, A. Weissberger, Ed., Wiley Interscience, New
York, 1962; D. J. Brown, "The Pyrimidines," Supplement I, in The
Chemistry of Heterocyclic Compounds, Vol. 16, A. Weissberger and E.
C. Taylor,. Eds., Wiley Interscience, New York, 1970; J. H. Lister,
"Fused Pyrimidines Part II Purines," in The Chemistry of
Heterocyclic Compounds, Vol. 24 Part II, A. Weissberger and E. C.
Taylor, Eds., Wiley Interscience, New York, 1971).
[0039] Because many 9-substituted O.sup.6-benzylguanine derivatives
exhibit excellent AGT inactivation properties (Moschel et al., J.
Med. Chear., 35, 4486-4491 (1992); Chae et al., J. Med. Chem., 37,
342-347 (1994)), 8,9-disubstituted analogs are expected to be
similarly active. These can be readily prepared by reacting the
anion of 8-substituted O.sup.6-benzylguanines (e.g., 1a-e) or the
anion of 8-aza-O.sup.6-benzylguanine (2) with any of the range of
compounds already described (Moschel et al., J. Med. Chem., 35,
4486-4491 (1992); Chae et al., J. Med. Chem., 37, 342-347 (1994))
to produce a mixture of isomeric 7,8- and 8,9-disubstituted
O.sup.6-benzylguanine derivatives. The desired 8,9-disubstituted
derivative can be isolated and purified by silica gel column
chromatography as already described (Moschel et al., J. Med. Chem.,
35, 4486-4491 (1992); Chae et al., J. Med. Chem., 37, 342-347
(1994)). Compound 7 was prepared by treating the anion of compound
1e with methyl iodide in N,N-dimethylformamide. Compounds 8-12 were
prepared using similar procedures.
[0040] The O.sup.6-substituted compounds of the present invention
can be administered in any suitable manner to a mammal for the
purpose of enhancing the chemotherapeutic treatment of a particular
cancer. Although more than one route can be used to administer a
particular compound, a particular route can provide a more
immediate and more effective reaction than another route.
Accordingly, the described methods provided herein are merely
exemplary and are in no way limiting.
[0041] Generally, the O.sup.6-substituted compounds of the present
invention as described above will be administered in a
pharmaceutical composition to an individual afflicted with a
cancer. Those undergoing or about to undergo chemotherapy can be
treated with the O.sup.6-substituted compounds separately or in
conjunction with other treatments, as appropriate. In therapeutic
applications, compositions are administered to a patient in an
amount sufficient to elicit an effective depression of AGT activity
thereby potentiating the cytotoxicity of the aforedescribed
chemotherapeutic treatment. An amount adequate to accomplish this
is defined as a "therapeutically effective dose," which is also an
"AGT inactivating effective amount." Amounts effective for a
therapeutic or prophylactic use will depend on, e.g., the stage and
severity of the disease being treated, the age, weight, and general
state of health of the patient, and the judgment of the prescribing
physician. The size of the dose will also be determined by the
O.sup.6-substituted compound selected, method of administration,
timing and frequency of administration as well as the existence,
nature, and extent of any adverse side-effects that might accompany
the administration of a particular O.sup.6-substituted compound and
the desired physiological effect. It will be appreciated by one of
skill in the art that various disease states may require prolonged
treatment involving multiple administrations, perhaps using a
series of different AGT inactivators and/or chemotherapeutic agents
in each or various rounds of administration.
[0042] Suitable chemotherapeutic agents usefully administered in
coordination with the O.sup.6-substituted compounds of the present
invention include alkylating agents, such as chloroethylating and
methylating agents. Such agents may be administered using
conventional techniques such as those described in Wasserman et
al., Cancer, 36, pp. 1258-1268 (1975), and Physicians' Desk
Reference, 48th ed., Edward R. Barnhart publisher (.1994). For
example, 1,3-bis(2-chloroethyl)-1-nitroso- urea (carmustine or
BCNU, Bristol-Myers, Evansville, Ind.) may be administered
intravenously at a dosage of from about 150 to 200 mg/m.sup.2 every
six weeks. Another alkylating agent,
1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (lomustine or CCNU,
Bristol-Myers), may be administered orally at a dosage of about 130
mg/m.sup.2 every six weeks. Other alkylating agents may be
administered in appropriate dosages via appropriate routes of
administration known to skilled medical practitioners.
[0043] Suitable doses and dosage regimens can be determined by
conventional range-finding techniques known to those of ordinary
skill in the art. Generally, treatment is initiated with smaller
dosages that are less than the optimum dose of the compound.
Thereafter, the dosage is increased by small increments until the
optimum effect under the circumstances is reached. The present
inventive method typically will involve the administration of about
0.1 mg to about 50 mg of one or more of the compounds described
above per kg body weight of the individual. For a 70 kg patient,
dosages of from about 10 mg to about 200 mg of O.sup.6-substituted
compound would be more commonly used, possibly followed by further
lesser dosages from about 1 mg to about 1 mg of O.sup.6-substituted
compound over weeks to months, depending on a patient's
physiological response, as determined by measuring cancer-specific
antigens or other measurable parameters related to the tumor load
of a patient.
[0044] It must be kept in mind that the compounds and compositions
of the present invention generally are employed in serious disease
states, that is, life-threatening or potentially life-threatening
situations. In such cases, in view of the minimization of
extraneous substances and the relative nontoxic nature of the
O.sup.6-substituted compounds, it is possible and may be felt
desirable by the treating physician to administer substantial
excesses of these O.sup.6-substituted compounds.
[0045] Single or multiple administrations of the compounds can be
carried out with dose levels and pattern being selected by the
treating physician. In any event, the pharmaceutical formulations
should provide a quantity of AGT-inactivating compounds of the
invention sufficient to effectively enhance the cytotoxic impact of
the chemotherapy.
[0046] The pharmaceutical compositions for therapeutic treatment
are intended for parental, topical, oral or local administration
and generally comprise a pharmaceutically acceptable carrier and an
amount of the active ingredient sufficient to reduce, and
preferably prevent, the activity of the AGT protein. The carrier
may be any of those conventionally used and is limited only by
chemico-physical considerations, such as solubility and lack of
reactivity with the compound, and by the route of
administration.
[0047] Examples of pharmaceutically-acceptable acid addition salts
for use in the present inventive pharmaceutical composition include
those derived from mineral acids, such as hydrochloric,
hydrobromic, phosphoric, metaphosphoric, nitric and sulfuric acids,
and organic acids, such as tartaric, acetic, citric, malic, lactic,
fumaric, benzoic, glycolic, gluconic, succinic, p-toluenesulphonic
acids, and arylsulphanic, for example.
[0048] The pharmaceutically acceptable excipients described herein,
for example, vehicles, adjuvants, carriers or diluents, are
well-known to those who are skilled in the art and are readily
available to the public. It is preferred that the pharmaceutically
acceptable carrier be one that is chemically inert to the active
compounds and one that has no detrimental side effects or toxicity
under the conditions of use. Such pharmaceutically acceptable
excipients preferably include saline (e.g., 0.9% saline), Cremophor
EL (which is a derivative of castor oil and ethylene oxide
available from Sigma Chemical Co., St. Louis, Mo.) (e.g., 5%
Cremophor EL/5% ethanol/90% saline, 10% Cremophor EL/90% saline, or
50% Cremophor EL/50% ethanol), propylene glycol (e.g., 40%
propylene glycol/10% ethanol/50% water), polyethylene glycol (e.g.,
40% PEG 400/60% saline), and alcohol (e.g., 40% t-butanol/60%
water). The most preferred pharmaceutical excipient for use in
conjunction with the present invention is polyethylene glycol, such
as PEG 400, and particularly a composition comprising 40% PEG 400
and 60% water or saline.
[0049] The choice of excipient will be determined in part by the
particular O.sup.6-substituted compound chosen, as well as by the
particular method used to administer the comparison. Accordingly,
there is a wide variety of suitable formulations of the
pharmaceutical composition of the present invention.
[0050] The following formulations for oral, aerosol, parenteral,
subcutaneous, intravenous, intraarterial, intramuscular,
interperitoneal, rectal, and vaginal administration are merely
exemplary and are in no way limiting.
[0051] The pharmaceutical compositions can be administered
parenterally, e.g., intravenously, intraarterially, subcutaneously,
intradermally, or intramuscularly. Thus, the invention provides
compositions for parenteral administration that comprise a solution
of the O.sup.6-substituted compound dissolved or suspended in an
acceptable carrier suitable for parenteral administration,
including aqueous and non-aqueous, isotonic sterile injection
solutions.
[0052] Overall, the requirements for effective pharmaceutical
carriers for parenteral compositions are well known to those of
ordinary skill in the art. See Pharmaceutics and Pharmacy Practice,
J. B. Lippincott Company, Philadelphia, Pa., Banker and Chalmers,
eds., pages 238-250 (1982), and ASHP Handbook on Injectable Drugs,
Toissel, 4th ed., pages 622-630 (1986). Such solutions can contain
anti-oxidants, buffers, bacteriostats, and solutes that render the
formulation isotonic with the blood of the intended recipient, and
aqueous and non-aqueous sterile suspensions that can include
suspending agents, solubilizers, thickening agents, stabilizers,
and preservatives. The compound may be administered in a
physiologically acceptable diluent in a pharmaceutical carrier,
such as a sterile liquid or mixture of liquids, including water,
saline, aqueous dextrose and related sugar solutions, an alcohol,
such as ethanol, isopropanol, or hexadecyl alcohol, glycols, such
as propylene glycol or polyethylene glycol, dimethylsulfoxide,
glycerol ketals, such as 2,2-dimethyl-1,3-dioxolane-4-methanol,
ethers, such as poly(ethyleneglycol) 400, an oil, a fatty acid, a
fatty acid ester or glyceride, or an acetylated fatty acid
glyceride with or without the addition of a pharmaceutically
acceptable surfactant, such as a soap or a detergent, suspending
agent, such as pectin, carbomers, methylcellulose,
hydroxypropylmethylcellulose, or carboxymethylcellulose, or
emulsifying agents and other pharmaceutical adjuvants.
[0053] Oils useful in parenteral formulations include petroleum,
animal, vegetable, or synthetic oils. Specific examples of oils
useful in such formulations include peanut, soybean, sesame,
cottonseed, corn, olive, petrolatum, and mineral. Suitable fatty
acids for use in parenteral formulations include oleic acid,
stearic acid, and isostearic acid. Ethyl oleate and isopropyl
myristate are examples of suitable fatty acid esters.
[0054] Suitable soaps for use in parenteral formulations include
fatty alkali metal, ammonium, and triethanolamine salts, and
suitable detergents include (a) cationic detergents such as, for
example, dimethyl dialkyl ammonium halides, and alkyl pyridinium
halides, (b) anionic detergents such as, for example, alkyl, aryl,
and olefin sulfonates, alkyl, olefin, ether, and monoglyceride
sulfates, and sulfosuccinates, (c) nonionic detergents such as, for
example, fatty amine oxides, fatty acid alkanolamides, and
polyoxyethylenepolypropylene copolymers, (d) amphoteric detergents
such as, for example, alkyl-b-aminopropionates, and
2-alkyl-imidazoline quaternary ammonium salts, and (e) mixtures
thereof.
[0055] The parenteral formulations typically will contain from
about 0.5% to about 25% by weight of the active ingredient in
solution. Preservatives and buffers may be used. In order to
minimize or eliminate irritation at the site of injection, such
compositions may contain one or more nonionic surfactants having a
hydrophile-lipophile balance (HLB) of from about 12 to about 17.
The quantity of surfactant in such formulations will typically
range from about 5% to about 15% by weight. Suitable surfactants
include polyethylene sorbitan fatty acid esters, such as sorbitan
monooleate and the high molecular weight adducts of ethylene oxide
with a hydrophobic base, formed by the condensation of propylene
oxide with propylene glycol. The parenteral formulations can be
presented in unit-dose or multi-dose sealed containers, such as
ampules and vials, and can be stored in a freeze-dried
(lyophilized) condition requiring only the addition of the sterile
liquid excipient, for example, water, for injections, immediately
prior to use. Extemporaneous injection solutions and suspensions
can be prepared from sterile powders, granules, and tablets of the
kind previously described.
[0056] Topical formulations, including those that are useful for
transdermal drug release, are well-known to those of skill in the
art and are suitable in the context of the present invention for
application to skin.
[0057] Formulations suitable for oral administration require extra
considerations considering the peptidyl and/or carbohydrate nature
of some of the O.sup.6-substituted compounds of the present
invention and the likely breakdown thereof if such compounds are
administered orally without protecting them from the digestive
secretions of the gastrointestinal tract. Such a formulation can
consist of (a) liquid solutions, such as an effective amount of the
compound dissolved in diluents, such as water, saline, or orange
juice; (b) capsules, sachets, tablets, lozenges, and troches, each
containing a predetermined amount of the active ingredient, as
solids or granules; (c) powders; (d) suspensions in an appropriate
liquid; and (e) suitable emulsions. Liquid formulations may include
diluents, such as water and alcohols, for example, ethanol, benzyl
alcohol, and the polyethylene alcohols, either with or without the
addition of a pharmaceutically acceptable surfactant, suspending
agent, or emulsifying agent. Capsule forms can be of the ordinary
hard- or soft-shelled gelatin type containing, for example,
surfactants, lubricants, and inert fillers, such as lactose,
sucrose, calcium phosphate, and corn starch. Tablet forms can
include one or more of lactose, sucrose, mannitol, corn starch,
potato starch, alginic acid, microcrystalline cellulose, acacia,
gelatin, guar gum, colloidal silicon dioxide, croscarmellose
sodium, talc, magnesium stearate, calcium stearate, zinc stearate,
stearic acid, and other excipients, colorants, diluents, buffering
agents, disintegrating agents, moistening agents, preservatives,
flavoring agents, and pharmacologically compatible excipients.
Lozenge forms can comprise the active ingredient in a flavor,
usually sucrose and acacia or tragacanth, as well as pastilles
comprising the active ingredient in an inert base, such as gelatin
and glycerin, or sucrose and acacia, emulsions, gels, and the like
containing, in addition to the active ingredient, such excipients
as are known in the art.
[0058] The O.sup.6-substituted compounds of the present invention,
alone or in combination with other suitable components, can be made
into aerosol formulations to be administered via inhalation. The
compounds are preferably supplied in finely divided form along with
a surfactant and propellant. Typical percentages of active compound
are 0.01-20% by weight, preferably 1%-10%. The surfactant must, of
course, be nontoxic, and preferably soluble in the propellant.
Representative of such surfactants are the esters or partial esters
of fatty acids containing from 6 to 22 carbon atoms, such as
caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic,
olesteric and oleic acids with an aliphatic polyhydric alcohol or
its cyclic anhydride. Mixed esters, such as mixed or natural
glycerides may be employed. The surfactant may constitute 0.1%-20%
by weight of the composition, preferably 0.25-5%. The balance of
the composition is ordinarily propellant. A carrier can also be
included as desired, e.g., lecithin for intranasal delivery. These
aerosol formulations can be placed into acceptable pressurized
propellants, such as dichlorodifluoromethane, propane, nitrogen,
and the like. They also may be formulated as pharmaceuticals for
non-pressured preparations, such as in a nebulizer or an atomizer.
Such spray formulations may be used to spray mucosa.
[0059] Additionally, the compounds and polymers useful in the
present inventive methods may be made into suppositories by mixing
with a variety of bases, such as emulsifying bases or water-soluble
bases. Formulations suitable for vaginal administration may be
presented as pessaries, tampons, creams, gels, pastes, foams, or
spray formulas containing, ain addition to the active ingredient,
such carriers as are known in the art to be appropriate.
[0060] The concentration of the O.sup.6-substituted compounds of
the present invention in the pharmaceutical formulations can vary
widely, i.e., from less than about 1%, usually at or at least about
10%, to as much as 20% to 50% or more by weight, and will be
selected primarily by fluid volumes, viscosities, etc., in
accordance with the particular mode of administration selected.
[0061] Thus, a typical pharmaceutical composition for intravenous
infusion could be made up to contain 250 ml of sterile Ringer's
solution, and 100 mg of the O.sup.6-substituted compound. Actual
methods for preparing parenterally administrable compounds will be
known or apparent to those skilled in the art and are described in
more detail in, for example, Remington's Pharmaceutical Science
(17th ed., Mack Publishing Company, Easton, Pa., 1985).
[0062] It will be appreciated by one of ordinary skill in the art
that, in addition to the aforedescribed pharmaceutical
compositions, the O.sup.6-substituted compounds of the present
inventive method may be formulated as inclusion complexes, such as
cyclodextrin inclusion complexes, or liposomes. Liposomes serve to
target the compounds to a particular tissue, such as lymphoid
tissue or cancerous hepatic cells. Liposomes can also be used to
increase the half-life of the O.sup.6-substituted compound.
Liposomes useful in the present invention include emulsions, foams,
micelles, insoluble monolayers, liquid crystals, phospholipid
dispersions, lamellar layers and the like. In these preparations,
the O.sup.6-substituted compound to be delivered is incorporated as
part of a liposome, alone or in conjunction with a suitable
chemotherapeutic agent. Thus, liposomes filled with a desired
O.sup.6-substituted compound of the invention can be directed to
the site of a specific tissue type, hepatic cells, for example,
where the liposomes then deliver the selected
chemotherapeutic-enhancement compositions. Liposomes for use in the
invention are formed from standard vesicle-forminq lipids, which
generally include neutral and negatively charged phospholipids and
a sterol, such as cholesterol. The selection of lipids is generally
guided by consideration of, for example, liposome size and
stability of the liposomes in the blood stream. A variety of
methods are available for preparing liposomes, as described in, for
example, Szoka et al., Ann. Rev. Biophys. Bioeng., 9, 467 (1980),
and U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.
For targeting to the cells of a particular tissue type, a ligand to
be incorporated into the liposome can include, for example,
antibodies or fragments thereof specific for cell surface
determinants of the targeted tissue type. A liposome suspension
containing an O.sup.6-substituted compound may be administered
intravenously, locally, topically, etc. in a dose that varies
according to the mode of administration, the O.sup.6-substituted
compound being delivered, the stage of disease being treated,
etc.
[0063] While the efficacy of the O.sup.6-substituted compounds of
the present invention has been demonstrated with respect to
particular types of cancerous cells, e.g., colon, prostate, and
breast cancer cells, the present invention has applicability to the
treatment of any type of cancer capable of being treated with an
antineoplastic alkylating agent which causes cytotoxic lesions at
the O.sup.6-position of guanine. Such cancers include, for example,
colon tumors, prostrate tumors, brain tumorsL lymphomas, leukemias,
breast tumors, ovarian tumors, lung tumors, Wilms' tumor,
rhabdomyosarcoma, multiplemyeloma, stomach tumors, soft-tissue
sarcomas, Hodgkin's disease, and non-Hodgkin's lymphomas.
[0064] Similarly, in view of the mode of action of the
O.sup.6-substituted compounds of the present invention, such
compounds can be used in conjunction with any type of
antineoplastic alkylating agent which causes cytotoxic lesions at
the O.sup.6-position of guanine. Such antineoplastic alkylating
agents include, for example, chloroethylating agents (e.g.
chloroethylnitrosoureas and chloroethyltriazines) and
monofunctional alkylating agents such as Streptozotocin,
Procarbazine, Dacarbazine, and Temozolomide.
[0065] Among the chloroethylating agents, the most frequently used
chemotherapeutic drugs are
1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU, lomustLne),
1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU, carmustine),
1-(2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitrosourea (MeCCNU,
semustine), and 1-(2-chloroethyl)-3-(4-amino-2-methyl-S-pyrimidi-
nyl)methyl-1-nitrosourea (ACNU). These agents have been used
clinically against tumors of the central nervous system, multiple
myeloma, melanoma, lymphoma, gastrointestinal tumors, and other
solid tumors (Colvin and Chabner, Alkylating Agents. In: Cancer
Chemotherapy: Principles and Practice, Chabner and Collins, eds.,
Lippincott, Philadelphia, pp. 276-313 (1990); McCormick and
McElhinney, Eur. J. Cancer, 26, 207-221 (1990)). Chloroethylating
agents currently under development with fewer side effects are
1-(2-chloroethyl)-3-(2-hydroxyethyl)-1-nitrosourea (HECNU),
2-chloroethyl-methylsulfonylmethanesulfonate (Clomesone), and
1-[N-(2-chloroethyl)-N-nitrosoureido]ethylphosphonic acid diethyl
ester (Fotemustine) (Colvin and Chabner, Alkylating Agents. In:
Cancer Chemotherapy: Principles and Practice, Chabner and Collins,
eds., Lippincott, Philadelphia, pp. 276-313 (1990); McCormick and
McElhinney, Eur. J. Cancer, 26, 207-221 (1990)). Methylating
chemotherapeutic agents include Streptozotocin
(2-deoxy-2-(3-methyl-3-nitrosoureido)-D-glucopyran- ose),
Procarbazine
(N-(1-methylethyl)-4-[(2-methylhydrazino)methyl]benzami- de),
Dacarbazine or DTIC (5-(3,3-dimethyl-1-triazenyl)
-1H-imidazole-4-carboxamide), and Temozolomide
(8-carbamoyl-3-methylimida-
zo[5,1-d]-1,2,3,5-tetrazine-4-(3H)-one). Temozolomide is active
against malignant melanomas, brain tumors, and mycosis fungoides.
Streptozotocin is effective against pancreatic tumors. Procarbazine
is used to treat Hodgkin's disease and brain tumors, and DTIC is
used in treatment of melanoma and lymphomas (Colvin and Cabner,
Alkylating Agents. In: Cancer Chemotherapy: Principles and
Practice, Chabner and Collins, eds., Lippincott, Philadelphia, pp.
276-313 (1990); Longo, Semin. Concol., 17, 716-735 (1990)).
[0066] The examples set forth below describe the syntheses of the
aforedescribed compounds. As regards the methods and materials set
forth in these examples, .sup.1H--NMR spectra were recorded on a
Varian VXR 500S spectrometer equipped with Sun 2/110 data stations
or a Varian XL 200 instrument interfaced to an Advanced data
system. Samples were dissolved in DMSO-d.sub.6 with Me.sub.4Si as
an internal standard. EI mass spectra were obtained on a reversed
geometry VG Micromass ZAB-2F spectrometer interfaced to a VG 2035
data system. Elemental analyses were performed by Galbraith
Laboratories, Inc., Knoxville, Tenn.
[0067] Most of the reagents and solvents were from Aldrich Chemical
Co., Inc., Milwaukee, Wis. 8-Aza-O.sup.6-benzylguanine (2) (Shealy
et al., J. Org. Chem., 27, 4518-4523 (1962)), 2,
4-diamino-6-benzyloxypyrimidine (3b) (Pfleiderer and Lohzmann,
Chem. Ber., 94, 12-18 (1961)), 2,4,5-triamino-6-benzyloxypyrimidine
(3c) (Pfleiderer and Lohrmann, Chem. Ber., 94, 12-18 (1961)),
2,4-diamino-6-benzyloxy-5-nitrasopyrimidine (3d) (Pfleiderer and
Lohrman, Chem. Ber., 94, 12-18 (1961)),
2,4-diamino-6-benzyloxy-5-nitropyrimidine (3e) (Kosary et al., Acta
Pharm. Hung. 49, 241-247 (1989)),
2,4-diamino-6-benzyloxy-5-bromapyrimidi- ne (3f) (Kosary et al.,
Acta Phazm. Hung., 49, 241-247 (1989)),
4-amino-6-benzyloxy-5-nitropyrimidine (3a) and
O.sup.6-benzyl-2-fluorohyp- oxanthine (4c) (Robins and Robins, J.
Org. Chem., 34, 2160-2163 (1969)) were prepared previously.
Alternative synthetic methods are provided below for some of these
compounds together with spectroscopic data not provided previously.
AGT inactivation studies were carried out as described in Moschel
et al., J. Med. Chem., 35, 4486-4491 (1992). Cell killing
experiments involving various AGT inactivators in combination with
BCNU were carried out as in Dolan, et al. (Proc. Natl. Acad. Sci.
U.S.A., 87; 5368-5372 (1990)). Cells were treated for 2 h with AGT
inactivator prior to exposure to BCNU.
EXAMPLE 1
[0068] 2,8-Diamino-6-chloropurine
[0069] A suspension of 8-aminoguanine (Fischer, Z. Physiol. Chem.,
60, 69 (1909); Beaman et al., in Zorbach and Tipson, Synthetic
Procedures in Nucleic Acid Chemistry, Vol. 1, pp 41-43, John Wiley
& Sons, New York, 1968) (3.0 g, 18.1 mmol) in phosphorus
oxychloride (90 mL) and N,N-diethylaniline (3 mL) was refluxed for
30 min and the excess phosphorus oxychloride was evaporated under
reduced pressure. Ice (20 g) was added slowly to the resulting
solution and the pH was adjusted to 6 with a concentrated aqueous
sodium hydroxide solution. A yellow solid formed and was collected
by filtration, washed with water, and dried to give a green solid.
Crystallization from water with charcoal treatment produced
2,8-diamino-6-chloropurine as a white solid: yield, 2.11 g (63%);
mp >275.degree. C. dec.; .sup.1H NMR d 6.09 (s, 2 H, NH.sub.2,
exchange with D.sub.2O), 6.71 (s, 2 H, NH.sub.2, exchange with
D.sub.2O); MS (EI) calcd. m/z for C.sub.5H.sub.5N.sub.6.sup.35Cl
184.0264, found 184.0266; calcd. m/z C.sub.5H.sub.5N.sub.6.sup.37Cl
186.0235, found 186.0237.
EXAMPLE 2
[0070] 8-Amino-O.sup.6-benzylguanine (1a)
[0071] 2,8-Diamino-6-chloropurine (0.9 g, 4.9 mmol) was added to
the solution of sodium (0.22 g, 10 mmol) in benzyl alcohol (9.0
mL). The solution was heated in a 130.degree. C. oil bath for 5 h,
and was poured into water (100 mL) with constant stirring for 10
min. Undissolved solid was removed by filtration and the filtrate
was neutralized with glacial acetic acid. The solution was mixed
with methanol (100 mL), and half of the aqueous methanol solution
was loaded an a 3.times.80 cm Sephadex LH-20 column eluted with
methanol/water (1:1) at 1 mL/min. Column eluent was continuously
monitored at 280 nm and fractions (10 mL) were collected. The
remainder of the reaction mixture in MeOH/H.sub.2O was
chromatographed separately under identical conditions. The desired
product eluted in fractions 100-130. Evaporation of solvent from
the pooled fractions 100-130 from both chromatographic runs
afforded analytically pure 1a: yield, 0.26 g (21%); mp
269-271.degree. C. dec.; UV (pH 1) 1.sub.max 241 nm
(e=0.699.times.10.sup.4), 300 (1.109.times.10.sup.4); (pH 6.9) 250
(sh) (0.447.times.10.sup.4), 292 (1.027.times.10.sup.4) ; (pH 13)
255 (sh) (0.355.times.10.sup.4), 295 (0.932.times.10.sup.4);
.sup.1H NMR d 5.41 (s, 2 H, ArCH.sub.2), 5.70 (S, 2 H, NH.sub.2,
exchange with D.sub.2O), 6.18 (s, 2 H, NH.sub.2, exchange with
D.sub.2O), 7.25-7.55 (m, 5 H, ArH), 11.1 (br s, 1 H, NH, exchanges
with D.sub.2O) ; MS (EI) calcd. m/z for C.sub.12H.sub.12N.sub.6O
256.1072, found 256.1059; Anal. (CH.sub.12H.sub.12N.sub.6O) C, H,
N.
EXAMPLE 3
[0072] 2-Amino-6-chloro-8-methylpurine
[0073] A suspension of 8-methylguanine (Daves et al., J. Am. Chem.
Soc., 82, 2633-2640 (1960)) (1.0 g, 6.1 mmol) in phosphorous
oxychloride (30 mL) and N,N-diethylaniline (1 mL) was refluxed for
3 h. The excess phosphorous oxychloride was evaporated under
reduced pressure. The resulting brown oil was dissolved in
ice-water and was neutralized with a concentrated aqueous NaOH
solution.
[0074] After evaporation of the solvent, the solid residue was
suspended in 70 mL of H.sub.2O. Undissolved solid was filtered off,
and the filtrate was loaded an a 3.times.60 cm Sephadex LH-20
column eluted with methanol/water (1:1) at 1 mL/min. Column eluent
was continuously monitored at 280 nm and fractions (10 mL) were
collected. Evaporation of pooled fractions 50-60 produced
2-amino-6-chloro-8-methylpurine as a crude solid. Crystallization
from ethanol/water with charcoal treatment afforded
2-amino-6-chloro-8-methylpurine as a white solid: yield, 0.57
g(51%); mp >265.degree. C. dec.; .sup.1H NMR d 2.39 (s, 3 H,
CH.sub.3), 6.62 (s, 2 H, NH.sub.2, exchange with D.sub.2O), 12.56
(s, 1 H, NH, exchanges with D.sub.2O); MS (EI) calcd. m/z for
C.sub.6H.sub.6N.sub.5.sup.35Cl 183.0312, found 183.0309; calcd. m/z
for C.sub.6H.sub.6N.sub.5.sup.37Cl 185.0283, found 185.0286.
EXAMPLE 4
[0075] O.sup.6Benzyl-8-methylguanine (1b)
[0076] Sodium (0.1 g, 4.4 mmol) was stirred in 4.1 mL of benzyl
alcohol until all sodium had reacted.
2-Amino-6-chloro-8-methylpurine (0.41 g, 2.2 mmol) was added, and
the reaction mixture was heated in a 130.degree. C. oil bath ror 5
h. After cooling to room temperature 40 mL of ether was added to
remove excess benzyL alcohol. The sticky precipitate that formed
was collected by filtration and was dissolved in water (50 mL). The
pH of the yellow solution was adjusted to 5-6 with glacial acetic
acid. The solution was mixed with methanol (50 mL) and was loaded
on a 3.times.80 cm Sephadex LH-20 column eluted with methanol/water
(1:1) at 1 mL/min. Column eluent was continuously monitored at 280
nm and fractions (10 mL) were collected. Evaporation of pooled
fractions 78-93 afforded analytically pure 1b: yield, 0.25 g (44%);
mp 214-216.degree. C.; UV (pH 1) 1.sub.max 238 nm (sh)
(e=0.648.times.10.sup.4), 290 (1.136.times.10.sup.4) ; (pH 6.9) 242
(0.758.times.10.sup.4), 284 (0.897.times.10.sup.4); (pH 13) 240
(sh) (0.495.times.10.sup.4) 286 (0.932.times.10.sup.4); .sup.1H NMR
d 2.33 (s, 3 H, CH.sub.3), 5.46 (s, 2 H, ArCH.sub.2), 6.17 (s, 2 H,
NH.sub.2, exchange with D.sub.2O),7.34-7.51 (m, 5 H, ArH), 12.18
(br s, 1 H, NH, exchanges with D.sub.2O),; MS (EI) calcd. m/z for
C.sub.13H.sub.13N.sub.5O 255.1120, found 255.1125; Anal.
(C.sub.13H.sub.13N.sub.5O. 1/4 H.sub.2O) C, H, N.
EXAMPLE 5
[0077] O.sup.6-Benzyl-8-oxoguanine (1c)
[0078] 2,4,5-Triamino-6-benzyloxypyrimidine (Pfleiderer et al.,
Chem. Ber., 94, 12-18 (1961)) (1.85 g, 8 mmol) and
1,1'-carbonyldiimidazole (1.30 g, 8 mmol) were dissolved in
anhydrous N,N-dimethvlformamide (5 mL) under argon. The solution
was stirred at room temperature overnight and was mixed with water
(200 mL) to precipitate a white solid. The solid was collected by
filtration, and dissolved in 250 mL of aqueous 2 N NaOH solution.
Undissolved material was removed by filtration, and the filtrate
was neutralized with glacial acetic acid to precipitate a white
solid. The solid was collected by filtration, was washed with
water, and was recrystallized from 50% aqueous ethanol to afford
analytically pure 1c: yield, 1.63 g (79%); mp 256-257.degree. C.
dec.; UV (pH 1) 1.sub.max 243 nm (e=0.717.times.10.sup.4), 306
(1.499.times.10.sup.4); (pH 6.9) 243 (0.915.times.10.sup.4), 290
(1.108.times.10.sup.4); (pH 13) 249 (sh) (0.443.times.10.sup.4),
293 (1.368.times.10.sup.4); 1H NMR d 5.41 (s, 2 H, ArCH.sub.2),
6.13 (s, 2 H, NH.sub.2, exchange with D.sub.2O), 7.33-7.51 (m, 5 H,
ArH), 10.46 (s, 1 H, exchanges with D.sub.2O), 11.04 (s, 1 H,
exchanges with D.sub.2O); MS (ET) Calcd. m/z for
C.sub.12H.sub.11N.sub.5O.sub.2:257.0912. Found: 257.0914. Anal.
(C.sub.12H.sub.11N.sub.5O.sub.2. 1/2 H.sub.2O) C, N, H.
EXAMPLE 6
[0079] O.sup.6-Benzyl-8-bromoguanine (1d)
[0080] Bromine (0.26 mL, 5.1 mmol) was added slowly to the solution
of O.sup.6-benzylguanine (1.205 g, 5.0 mmol) in anhydrous DMF (10
mL) under argon. The resulting deep green solution was stirred at
room temperature overnight. The solution was mixed with water (70
mL) to precipitate crude product. This product was collected by
filtration and was dissolved in 50% aqueous methanol (100 mL). The
solution was loaded on a 3.times.80 cm Sephadex LH-20 column eluted
with methanol/water (1:1) at 1 mL/min. Column eluent was
continuously monitored at 280 nm and fractions (10 mL) were
collected. The desired product eluted in fractions 110-190.
Evaporation of solvent from the pooled fractions 110-190 afforded
Id as a pale yellow solid. Crystallization from ethanol/water (1:1)
produced analytically pure lid: yield, 0.166 g (10%); mp
135-137.degree. C. dec.; UV (pH 1) 1.sub.max 236 nm (sh)
(e=0.517.times.10.sup.4), 294 (1.429.times.10.sup.4); (pH 6.9) 244
(0.666.times.10.sup.4), 287 (1.043.times.10.sup.4); (pH 13) 245
(sh) (0.544.times.10.sup.4), 289 (1.030.times.10.sup.4); .sup.1H
NMR d 5.45 (s, 2 H, ArCHz), 6.35 (s, 2, H, NH.sub.2, exchange with
D.sub.2O), 7.34-7.52 (m, 5 H, ArH), 13.08 (b s, 1 H, NH, exchanges
with D.sub.2O) ; MS (EI) calcd. m/z for
C.sub.12H.sub.10N.sub.5O.sup.79Br 319.0068, found 319.0069; calcd.
m/z for C.sub.12H.sub.10N.sub.5O.sup.81 Br 321.0048, found
321.0048; Anal. (C.sub.12H.sub.10N.sub.5OBR{fraction (3/2)}
H.sub.2O) C, H, N, Br.
EXAMPLE 7
[0081] 8-Aza-O.sup.6-benzylguanine (2)
[0082] Glacial acetic acid (1 mL) was added into the mixture of
2,4,5-triamino-6-benzyloxypyrimidine (0.231 g, 1.0 mmol) and sodium
nitrite (0.069 g, 1.0 mmol) in acetone (5 ml). The resulting
mixture was stirred at room temperature for 2 h. The solution was
poured in water (100 mL) with stirring to precipitate a crude
solid. The solid was collected by filtration and air dried.
Crystallization from ethanol/water (1:1) with charcoal treatment
produced 2 as a white solid: yield, 105 mg (43%); mp
191-192.degree. C. (192-193.degree. C.; Shealy et. al., J. Org.
Chem., 27, 4518-4523 (1962)); .sup.1H NMR d 5.56 (s, 2 H,
ArCH.sub.2), 7.00 (s, 2 H, NH.sub.2, exchange with D.sub.2O),
7.41-7.58 (m, 5 H, ArH) ; MS (EI) calcd. m/z for
C.sub.11H.sub.10N.sub.6O 242.0916, found 242.0924.
EXAMPLE 8
[0083] 4-Amino-6-benzyloxy-5-nitropyrimidine (3a)
[0084] 4-sAmino-6-chloro-5-nitropyrimidine (Boon et al., J. Chem.
Soc., 96-102 (1951)) (1.5 g, 8.6 mmol) was added to a solution of
sodium (0.23 g, 9.9 mmol) in benzyl alcohol (14 mL). The solution
was heated in a 130.degree. C. oil bath for 3.5 h, and was poured
into benzene (50 mL). A yellow solid was collected by filtration
and washed with benzene. Crystallization from benzene/ether
afforded an analytically pure sample of 3a: yield, 0.71 g (34%); mp
149-150.degree. C.; UV (pH 1) 1.sub.max 284 nm
(e=0.368.times.10.sup.4), 333 (0.488.times.10.sup.4); (pH 6.9) 284
(0.329.times.10.sup.4), 336 (0.470.times.10.sup.4); (pH 13) 290
(0.344.times.10.sup.4) 333 (0.494.times.10.sup.4); .sup.1H NMR d
5.50 (s, 2 H, ArCH.sub.2), 7.33-7.49 (m, 5 H, ArH), 8.12-8.24 (br
d, 2 H, NH.sub.a and NE.sub.b, exchange with D.sub.2O), 8.24 (s, 1
H, H-2); MS (EI) calcd. m/z for C.sub.11H.sub.10N.sub.4O.sub.3
246.0752, found 246.0751; Anal. (C.sub.11H.sub.10N.sub.4O.sub.3) C,
H, N.
EXAMPLE 9
[0085] 2,4-Diamina-6-benzyloxy-5-nitropyrimidine (3e)
[0086] 2,4-Diamino-6-chloro-5-nitropyrimidine (O'Brien et. al., J.
Med. Chem., 9, 573-575 (1966)) (1.0 g, 5.28 mmol) was added to a
solution of sodium (0.14 g, 6.08 =mol) in benzyl alcohol (9 mL).
The solution was heated in a 160.degree. C. oil bath for 3.5 h and
the solvent was evaporated under reduced pressure to provide a
yellow solid. This solid was washed with water, and air dried.
Crystallization from benzene/ether gave a pale yellow filamentous
solid: yield, 0.69 g (50%); mp 194-195.degree. C. (171.degree. C.;
Kosary et. al., Acta. Pharm. Hung., 49, 241-247 (1989)); UV (pH 1)
1.sub.max 236 nm (sh) (e=1.452.times.10.sup.4) 264
(0.522.times.10.sup.4) 321 (1.294.times.10.sup.4) ; (pH 6.9) 242
(sh) (0.965.times.10.sup.4), 337 (1.493.times.10.sup.4); (pH 13)
242 (sh) (0.952.times.10.sup.4), 338 (1.479.times.10.sup.4);
.sup.1H NMR d 5.43 (s, 2 H, ArCH.sub.2), 7.26 (br s, 2 H, NH.sub.2,
exchange with D.sub.2O), 7.33-7.51 (m, 5 H, ArH), 7.93 (br s, 2 H,
NH.sub.2, exchange with D.sub.2O); MS (EI) calcd. m/z for
C.sub.11H.sub.11N.sub.5O.sub.3 261.0861, found 261.0866; Anal.
(C.sub.11H.sub.11N.sub.5O.sub.3).
EXAMPLE 10
[0087] O.sup.6-Benzylxanthine (4a)
[0088] A suspension of O.sup.6-benzylguanine (0.83 g, 3.4 mmol) in
acetone (15 mL) was poured into a solution of sodium nitrite (5 g)
in 15 mL of H.sub.2O. Acetic acid (8 mL) was added to the
suspension with stirring. Minimum amounts of acetone were added as
necessary to dissolve any suspended solid. The resulting pale
yellow-green solution was stirred for 3 h. A pale green precipitate
that formed was collected by filtration and washed with water (200
mL). Recrystallization of the air-dried solid from ethanol/water
(1:1) afforded analytically pure 4a: yield, 0.43 g (52%); mp
145-147.degree. C. dec.; UV (pH 1) 1.sub.max 270 nm
(e=0.749.times.10.sup.4); (pH 6.9) 286 (1.143.times.10.sup.4); (pH
13) 290 (0.914.times.10.sup.4); .sup.1H NMR d 5.49 (s, 2 H,
ArCH.sub.2), 7.36-7.54 (m, 5 H, ArH), 8.02 (s, 1 H, H-8), 11.8 (br
s, 1 H, NH, exchanges with D.sub.2O), 13.2 (br s, 1 H, NH,
exchanges with D.sub.2O); MS (EI) calcd. m/z for
C.sub.12H.sub.10N.sub.4O.sub.2 242.0803, found 242.0828; Anal.
(C.sub.12H.sub.10N.sub.4O.sub.2. H.sub.2O) C, H, N.
EXAMPLE 11
[0089] O.sup.6-Benzyluric acid (4b)
[0090] Sodium nitrite (1.5 g, 43 mmol) dissolved in water (5 mL)
was added to a suspension of O.sup.6-benzyl-8-oxoguanine (1c)
(0.257 g, 1.0 mmol) in acetone (5 mL). Glacial acetic acid (3 mL)
was added to the suspension with stirring. After stirring for 3 h
at room temperature a bright yellow precipitate formed. The
suspension was mixed with water (150 mL) and undissolved solid was
filtered off. Saturated aqueous sodium carbonate solution was added
to the filtrate to adjust the pH to approximately 5. A yellow
precipitate (130 mg) was collected and washed with water. This
solid was crystallized from 50% aqueous ethanol to give an
analytically pure sample of 4b: yield, 75 mg (29%); mp
>230.degree. C.; UV (pH 1) 1.sub.max 236 nm (sh)
(e=0.972.times.10.sup.4), 299 (1.427.times.10.sup.4); (pH 6.9) 240
(sh) (0.821.times.10.sup.4), 304 (2.134.times.10.sup.4); (pH 13)
245 (sh) (0.846.times.10.sup.4), 297 (1.861.times.10.sup.4);
.sup.1H NMR d 5.43 (s; 2 H, ArCH.sub.2), 7.35-7.51 (m, 5 H, ArH),
10.76 (s, 1 H, NH, exchanges with D.sub.2O), 11.23-(s, 1 H, NH,
exchanges with D.sub.2O), 11.39 (s, 1 H, NH, exchanges with
D.sub.2O); MS (EI) calcd. m/z for C.sub.12H.sub.10N.sub.4O.sub.3
258.0752, found 258.0753; Anal.
(C.sub.12H.sub.10N.sub.4O.sub.3.{fraction (5/2)} H.sub.2O) C, H,
N.
EXAMPLE 12
[0091] Diacetyl-O.sup.6-benzyl-8-oxoguanine
[0092] Acetic anhydride (2 mL) was added to the suspension of
O.sup.6-benzyl-8-oxoguanine (1c) (0.257 g, 1.0 mmol) in dry toluene
(10 mL). The suspension was vigorously refluxed for 24 hr, and was
cooled to room temperature. After storing at 4.degree. C. for 4 hr,
the resulting precipitate was collected by filtration, washed with
benzene and air dried to give an analytically pure sample of a
diacetylated product: yield, 0.287 g (84%); mp 272-274.degree. C.
dec.; UV (100% MeOH) 1.sub.max 275 nm (e=1.313.times.10.sup.4); (pH
1) 275 (1.143.times.10.sup.4); (pH 6.9) 238 (0.995.times.10.sup.4),
276 (1.115.times.10.sup.4); (pH 13) 285 (2.138.times.10.sup.4);
.sup.1H NMR d 2.18 (s, 3 H, CH.sub.3), 2.57 (s, 3 H, CH.sub.3),
5.51 (s, 2 H, ArCH.sub.2), 7.30-7.57 (m, 5 H, ArH), 10.41 (s, 1 H,
exchanges with D.sub.2O), 12.30 (s, 1 H, exchanges with D.sub.2O);
MS (EI) Calcd. m/z for C.sub.16H.sub.15N.sub.5O.sub.4:341.1123.
Found: 341.1130. Anal. (C.sub.16H.sub.15N.sub.5O.sub.4) C, N,
H.
EXAMPLE 13
[0093] N.sup.2-Acetyl-O.sup.6-benzyl-8-oxoguanine (4d)
[0094] Diacetyl-O.sup.6-benzyl-8-oxoguanine (85 mg, 0.25 mmol) was
dissolved in methanol (10 mL) and ammonium hydroxide (28%, 5 mL)
and was allowed stand for 1 hr. The clear solution became cloudy
and a precipitate formed on standing. The precipitate was collected
by filtration, washed with water, and dried to give an analytically
pure sample of 4d: yield, 48 mg (65%); mp 335-337.degree. C. dec.;
UV (pH 1) 1.sub.max 276 nm (e=1.723.times.10.sup.4), 303 (sh)
(0.679.times.10.sup.4); (pH 6.9) 276 (1.379.times.10.sup.4); (pH
13) 284 (1.683.times.10.sup.4); .sup.1H NMR d 2.15 (s, 3 H,
CH.sub.3), 5.49 (s, 2 H, ArCH.sub.2), 7.30-7.55 (m, 5 H, ArH),
10.21 (s, 1 H, exchanges with D.sub.2O), 10.99 (s, 1 H, exchanges
with D.sub.2O), 11.60 (s, 1 H, exchanges with D.sub.2O; MS (EI)
Calcd. m/z for C.sub.14H.sub.13N.sub.5O.- sub.3:299.1018. Found:
299.1023. Anal. (C.sub.14H.sub.13N.sub.5O.sub.3) C, N, H.
EXAMPLE 14
[0095] O.sup.6-Benzyl-2-fluorchypoxanthine (4c)
[0096] O.sup.6-Benzylguanine (1.21 g, 5 mmol) was added to 100 mL
of 48% fluoboric acid at -20.degree. C. Sodium nitrite (1.23 g, 35
mmole) was dissolved in water (5 mL) and 2.5 mL of this sodium
nitrite solution was added slowly to the cold fluoboric acid
solution. The resulting mixture was stirred for 1 h at or below
-15.degree. C. Additional fluoboric acid (25 mL) was added followed
by an additional 2.5 mL of the aqueous sodium nitrite solution.
After stirring for an additional 1 h below -15.degree. C.,
fluoboric acid (25 mL) was again added and stirring was continued
for 1 h. The resulting solution was neutralized with saturated
aqueous sodium carbonate solution at -20.degree. C. and was allowed
to warm to room temperature. A white precipitate that formed was
collected by filtration and was washed with water and dried under
vacuum to afford crude 4c: yield, 0.52 g, 43%. An analytical sample
was prepared by chromatography on a Sephadex LH-20 column
(3.times.80 cm) eluted with methanol/water (1:1) at 1 mL/min. The
desired4c eluted in fractions 66-77: up 182-183.degree. C.
(184-185.degree. C.; Robins and Robins, J. Org. Chem., 34,
2160-2163 (1969)); UV (pH 1) 1.sub.max 256 nm
(e=1.117.times.10.sup.4- ); (pH 6.9) 257 (1.078.times.10.sup.4) (pH
13) 264 (1.063.times.10.sup.4); .sup.1H NMR d 5.60 (s, 2 H,
ArCH.sub.2), 7.37-7.57 (m, 5 H, ArH), 8.40 (s, 1 H, H-8), 13.60 (s,
1 H, NH, exchanges with D.sub.2O), .sup.19F NMR d 23.54 downfield
from trifluoroacetic acid standard; MS (EI) calcd. m/z for
CH.sub.19H.sub.9FN.sub.4O 244.0760, found 244.0756; Anal.
(C.sub.12H.sub.9FN.sub.4O.2/3 H.sub.2O) C, H, N.
EXAMPLE 15
[0097] O.sup.6-Benzyl-N.sup.2-methylguanine (4e)
[0098] Fluoboric acid (48%, 30 mL) was cooled to -20.degree. C. in
an dry ice-acetone bath. 06-Benzylguanine (0.362 g, 1.5 mmol) was
added with stirring. Sodium nitrite (0.369 g, 10.5 zmmol) was
dissolved in water (1 mL) and 0.5 mL of this solution was added
slowly to the cold fluoboric acid solution. The resulting solution
was stirred at or below -15.degree. C. for 1 h. More fluoboric acid
(5 mL) was then added followed by 0.5 mL of the sodium nitrite
solution. After stirring for 1 h at or below -15.degree. C.,
fluoboric acid (5 mL) was again added and stirring was continued
for an additional 1 h. Methylamine (40% in water, 60 mL) was then
added at -20.degree. C., and the resulting basic solution was
stirred at room temperature for 2 days. The solvent was evaporated
under reduced pressure to produce a white solid. The solid was
suspended in 50 mL of H.sub.2O with stirring for 10 min.
Undissolved material was collected by filtration and washed with
water. This solid was dissolved in 40 mL methanol/water (1:1) to
which was added 1.2 mL of 28% aqueous ammonia solution. The
solution was loaded on a 3.times.80 cm Sephadex LH-20 column eluted
with MeOH/H.sub.2O/NH.sub.4OH (30:70:3) at 1 mL/min. Column eluent
was continuously monitored at 280 nm and fractions (10 mL) were
collected. Evaporation of the pooled fractions 106-127 gave an
analytically pure sample of 4e: yield, 85 mg (22%); mp 189
-190.degree. C.; UV (pH 1) 1.sub.max 238 nm (sh)
(e=0.665.times.10.sup.4), 297 (0.904.times.10.sup.4); (pH 6.9) 246
(0.898.times.10.sup.4), 290 (0.676.times.10.sup.4); (pH 13) 240
(sh) (0.615.times.10.sup.4), 294 (0.674.times.10.sup.4); .sup.1H
NMR d 2.30 (d, 3 H, CH.sub.3), 5.50 (s, 2 H, ArCH), 6.75 (m, 1 H,
MeNH, exchanges with D.sub.2O), 7.31-7.53 (m, 5 H, ArH), 7.82 (s, 1
H, H-8), 12.53 (s, 1 H, NH, exchanges with D.sub.2O); MS (EI)
calcd. m/z for C.sub.13H.sub.13N.sub.5O 255.1120, found 255.1107;
Anal. (C.sub.13H.sub.13N.sub.5O.1/2 H.sub.2O) C, H, N.
EXAMPLE 16
[0099] O.sup.6-Benzyl-N.sup.2,N.sup.2-dimethylguanine (4f)
[0100] Fluoboric acid (48%, 40 mL) was cooled to -20.degree. C. in
an dry ice-acetone bath. O.sup.6-Benzylguanine (0.482 g, 2.0 mmol)
was added with stirring. Sodium nitrite (0.492 g, 14.0 mmol) was
dissolved in water (2 mL) and 1 mL of this solution was added
slowly to the cold fluoboric acid solution. The resulting solution
was stirred at or below -15.degree. C. tor 1 h. More fluoboric acid
(10 mL) was added followed by the addition of 1 mL of the sodium
nitrite solution. After stirring for 1 h at or below -15.degree.
C., additional fluoboric acid (10 mL) was added with stirring for 1
h. Dimethylamine (40% in water, 60 mL) was then added to the
solution at -20.degree. C., and the resulting mixture was allowed
to warm to room temperature. The suspension became a clear solution
and a precipitate formed within 10 min. After standing overnight at
room temperature the precipitate was collected by filtration and
was washed with water. The solid was crystallized from 50% aqueous
ethanol to give an analytically pure sample of 4f: yield, 0.25 g
(46%); mp 220-221.degree. C. dec.; UV (pH 1) 1.sub.max 248 nm (sh)
(e=0.512.times.10.sup.4), 303 (0.908.times.10.sup.4); (pH 6.9) 251
(1.152.times.10.sup.4) 299 (0.686.times.10.sup.4); (pH 13) 248 (sh)
(0.766.times.10.sup.4) 299 (0.710.times.10.sup.4); .sup.1H NMR d
3.12 (s, 6 H, CH.sub.3), 5.54 (s, 2 H, ArCH.sub.2), 7.36-7.51 (m, 5
H, ArH), 7.84 (s, 1 H, H-8), 12.56 (s, 1 H, NH, exchanges with
D.sub.2O); MS (EI) calcd. m/z for C.sub.14H.sub.15N.sub.5O
269.1276, found 269.1254; Anal. (C.sub.14H.sub.15N.sub.5O) C, H,
N.
EXAMPLE 17
[0101] 2,4-Diamino-6-benzyloxy-5-bromopyrimidine (3f)
[0102] 2, 4-Diamino-5-bromo-6-chloropyrimidine (Phillips et. al.,
J. Org. Chem., 29, 1488-1490 (1963)) (2.3 g, 10 mmol) was added to
a solution of sodium (0.29 g, 12.5 mmol) in benzyl alcohol (10 mL)
under argon. The solution was heated in a 130.degree. C. oil bath
for 3 h and the benzyl alcohol was evaporated under reduced
pressure to give a white solid. This solid was washed with water,
and air dried. Crystallization from 50% aqueous ethanol gave white
crystalline needles of 3f: yield, 2.32 g (76%); mp 165-166.degree.
C. (lit. 136.degree. C.; Kosary et. al., Acta Phazm. Hung., 49,
241-247 (1989)); UV (pH 1) 1.sub.max 236 nm
(e=0.873.times.10.sup.4), 291 (1.388.times.10.sup.4); (pH 6.9) 236
(0.850.times.10.sup.4), 277 (0.835.times.10.sup.4); (pH 13) 234
(0.869.times.10.sup.4), 277 (0.827.times.10.sup.4); .sup.1H NMR d
5.30 (s, 2 H, ArCH.sub.2), 6.15 (s, 2 H, NH.sub.2, exchange with
D.sub.2O), 6.32 (s, 2 H, NH.sub.2, exchange with D.sub.2O),
7.31-7.45 (m, 5 H, ArH); MS (El) calcd. m/z for
C.sub.11H.sub.11N.sub.4O.sup.79Br 294.0115, found 294.0127; calcd.
m/z for C.sub.11H.sub.11N.sub.4O.sup.81Br 296.0094, found 296.0083;
Anal. (C.sub.11H.sub.11N.sub.4OBr) C, H, N.
EXAMPLE 18
[0103] 2-Amino-4-chloro-5-nitropyrimidine
[0104] A suspension of 2-amino-4-hydroxy-5-nitropyrimidine (5.0 g,
32.1 mmol) in phosphorous oxychloride (100 mL) was refluxed
overnight, and the excess phosphorous oxychloride was evaporated
under reduced pressure. The residue was mixed with ice (100 g) in
an ice-bath, and the mixture was neutralized with concentrated
aqueous sodium carbonate solution. A yellow precipitate was
collected by filtration and washed with water: yield, 1.39 g (25%);
mp 191-194.degree. C. dec.; .sup.1H NMR d 8.45 (br s, 2 H,
NH.sub.2, exchange with D.sub.2O), 9.03 (s, 1 H, H-6); MS (EI)
calcd. nmz for C.sub.4H.sub.3N.sub.4O.sub.2.sup.35Cl 173.9944,
found 173.9934; calcd. m/z for
C.sub.4H.sub.3N.sub.4O.sub.2.sup.37Cl 175.9915, found 175.9916.
EXAMPLE 19
[0105] 2-Amino-4-benzyloxy-5-nitropyrimidine (5a)
[0106] 2-Amino-4-chloro-S-nitropyrimldine (0.70 g, 4.0 mmol) was
added to a solution of sodium (0.12 g, 5.2 mmol) in benzyl alcohol
(8 mL) under argon. The solution was heated in a 130.degree. C. oil
bath for 3 h, and approximately half of the benzyl alcohol was
evaporated under reduced pressure. The residue was poured into
water (50 mL) with constant stirring for 10 min. After
neutralization with glacial acetic acid, a brown precipitate formed
which was collected by filtration and washed with water. This solid
was crystallized from benzene to give 5a as a golden crystalline
solid: yield, 126 mg (13%); mp 164-167.degree. C.; UV (pH 1)
1.sub.max 262 nm (e=0.879.times.10.sup.4), 295 (sh)
(0.571.times.10.sup.4); (pH 6.9) 235 (sh) (0.448.times.10.sup.4)
273 (0.360.times.10.sup.4), 326 (1.085.times.10.sup.4); (pH 13) 273
(0.404.times.10.sup.4), 327 (1.055.times.10.sup.4); .sup.1H NMR d
5.51 (s, 2 H, ArCH.sub.2), 7.35-7.54 (m, 5 H, ArH), 8.05 (d, 2 H,
NH.sub.2, exchange with D.sub.2O), 8.92 (s, 1 H, H-6); MS (EI)
calcd. m/z for C.sub.11H.sub.10N.sub.4O.sub.3 246.0752, found
246.0758; Anal. (C.sub.11H.sub.10N.sub.4O.sub.3) C, H, N.
EXAMPLE 20
[0107] 2-Amina-4-benzyloxy-6-methyl-5-nitropyrimidine (5b)
[0108] 2-Amino-4-chloro-6-methyl-5-nitropvrimidine (Boon et al., J.
Chem. Sac., 96-102 (1951)) (1.24 g, 6.58 mmol) was added to a
solution of sodium (0.21 g, 9.13 mmol) in benzyl alcohol (14 mL)
under argon. The solution was heated in a 135.degree. C. oil bath
for 3.5 h, and was poured into water (70 mL) with constant stirring
for 10 min. After neutralization with glacial acetic acid, a yellow
precipitate formed which was collected by filtration and washed
with water. This solid was crystallized from benzene to give 5b as
a bright yellow crystalline solid: yield, 0.57 g (33%); mp
159-160.degree. C.; UV (pH 1) 1.sub.max 268 nm
(e=0.783.times.10.sup.4), 345 (sh) (0.104.times.10.sup.4); (pH 6.9)
282 (0.564.times.10.sup.4), 345 (sh) (0.338.times.10.sup.4); (pH
13) 282 (0.549.times.10.sup.4), 345 (sh) (0.332.times.10.sup.4);
.sup.1H NMR d 2.35 (s, 3 H, CH.sub.3), 5.44 (s, 2 H, ArCH.sub.2),
7.34-7.46 (m, 5 H, ArH), 7.64 (b s, 2 H, NH.sub.2, exchange with
D.sub.2O); MS (EI) calcd. m/z for C.sub.12H.sub.12N.sub.4O.sub.3
260.0908, found 260.0913; Anal. (C.sub.12H.sub.12N.sub.4O.sub.3) C,
H, N.
EXAMPLE 21
[0109] 2,4-Diamino-6-benzyloxy-s-triazine (6)
[0110] 2,4-Diamino-6-chloro-s-triazine (2.25 g, 15.0 mmol) was
added to a solution of sodium (0.43 g, 18.8 mmol) in benzyl alcohol
(30 mL) under argon. The suspension was heated in a 130.degree. C.
oil bath for 3.5 h. The excess benzyl alcohol was removed under
vacuum and the resulting solid was collected with the aid of
benzene, and washed with water (100 mL): yield, 1.83 g (56%); mp
184-185.degree. C. (lit. 186-188.degree. C.; Wakabayashi et al.,
Nippon Dojo-Hiryogaku Zasshi, 41, 193-200 (1970)); UV. (pH 1)
1.sub.max 233 nm (sh) (e=0.589.times.10.sup.4); (pH 6.9) 238 (sh)
(0.111.times.10.sup.4)i (pH 13) 240 (sh) (0.073.times.10.sup.4) ;
.sup.1H NMR d 5.25 (s; 2 H, ArCH.sub.2), 6.63 (s, 4 H, NH.sub.2,
exchange with D.sub.2O), 7.30-7.42 (m, 5 H, ArH); MS (EI) calcd.
m/z for C.sub.10H.sub.11N.sub.5O 217.0963, found 217.0955.
EXAMPLE 22
[0111] 2-Amino-6-chloro-8-trifluoromethylpurine
[0112] A suspension of 8-trifluoromethylguanine (Pfleiderer arid
Shanshal, Liebigs Ann. Chem., 726, 201-215 (1969)) (2.0 g, 9.1
mmol) in phosphorous oxychloride (20 mL) was refluxed for 3 h.
Excess phosphorous oxychloride was evaporated under reduced
pressure. The resulting residue was mixed with ice-water (100 g),
and the pH was adjusted to 3-4 with a concentrated aqueous NaOH
solution. The resulting solution was mixed with MeOH (100 mL) and
approximately half (i.e., 100 mL) or the aqueous methanol solution
was loaded on a 3.times.80 cm Sephadex LH-20 column eluted with
methanol/water (1:1) at 1 mL/min. Column eluent was continuously
monitored at 280 nm and fractions (10 mL) were collected. The
remainder of the reaction mixture in MeOH/H.sub.2O was
chromatographed separately under identical conditions. The desired
product eluted in fractions 73-85. Evaporation of solvent from the
pooled fractions 73-85 from both chromatographic nins afforded
analytically pure 2-amino-6-chloro-8-trifluoromethylpurine: yield,
0.94 g (43%); mp >225.degree. C. dec.; UV (pH 1) 1.sub.max 245
nm (e=0.501.times.10.sup.4), 314 (0.746.times.10.sup.4); (pH 6.9)
270 (0.265.times.10.sup.4), 315 (0.612.times.10.sup.4); (pH 13) 272
(0.269.times.10.sup.4) 314 (0.612.times.10.sup.4); 1H NMR d 7.19
(s, 2 H, NH.sub.2, exchange with D.sub.2O), 14.25 (br s, 1 H, NH,
exchanges with D.sub.2O); MS (EI) calcd. m/z for
C.sub.6H.sub.3N.sub.5F.sub.3.sup.37Cl 237.0029, found 237.0011;
calcd. m/z for C.sub.6H.sub.3N.sub.5F.sub.3.sup- .37Cl 239.0000,
found 238.9987; Anal. (C.sub.6H.sub.3N.sub.5F.sub.3C) C, H, N, F,
Cl.
EXAMPLE 23
[0113] O.sup.6-Benzyl-8-trifluoromethylguanine (1e)
[0114] Sodium (0.10 g,. 4.3 mmol) was stirred in 5 mL of benzyl
alcohol until all had reacted.
2-Amino-6-chloro-8-trifluoromethylpurine (0.475 g, 2.0 mmol) was
added, and the reaction mixture was heated in a 135.degree. C. oil
bath for 3.5 h. The benzyl alcohol was removed by vacuum
distillation yielding a brown oil. The oil was dissolved in water
(50 mL) and was acidified with glacial acetic acid to produce a
pale yellow precipitate. The precipitate was collected by
filtration and washed with water. The crude product was loaded on a
2.5.times.35 cm silica gel column (Davisil grade 633, 200-425 mesh,
60 A). Elution was carried out with 5% EtOH in CHCI.sub.3 to
provide analytically pure O.sup.6-benzyl-8-trifluoromethylguanine
(1e): yield, 0.42 g (67%); mp 214-216.degree. C. dec.; UV (pH 1)
1.sub.max 291 nm (e=1.229.times.10.sup.4); (pH 6.9) 244
(0.470.times.10.sup.4), 289 (1.023.times.10.sup.4); (pH 13) 247
(sh) (0.393.times.10.sup.4), 290 (0.923.times.10.sup.4); .sup.1H
NMR d 5.51 (s, 2 H, ArG.sub.2), 6.82 (s, 2 H, NH.sub.2, exchange
with D.sub.2O), 7.38-7.55 (m, 5 H, ArH), 13.75 (br s, 1 H, NH,
exchanges with D.sub.2O); MS (EI) calcd. m/z for
C.sub.13H.sub.10N.sub.5OF.sub.3 309.0837, found 309.0827; Anal.
(C.sub.13H.sub.10N.sub.5OF.sub.3) C, H, N, F.
EXAMPLE 24
[0115] O.sup.6-Benzyl-8-trifluoromethyl-9-methylguanine (7)
[0116] To O.sup.6-benzyl-8-trifluoromethylguanine (1e) (200 mg,
0.65 mmol) under argon was added 0.66 mL of a 1.0 M solution of
sodium ethoxide in ethanol. The solution was stirred for 10 min and
the ethanol was removed under vacuum. The remaining solid was
dissolved in anhydrous DMF (1.5 mL), and methyl iodide (49 .mu.L,
0.78 mmol) was added to the solution. This solution was stirred at
room temperature for 1 h, and 1.5 mL additional DMF was added. The
solution was stirred at room temperature overnight. The solvent was
evaporated under reduced pressure. The crude solid was loaded on a
2.5.times.35 cm silica gel column (Davisil grade 633, 200-425 mesh,
60 A). Elution was carried out with chloroform/hexane (3:1) to
provide analytically pure O.sup.6-benzyl-8-trifluoromethyl-9-met-
hylguanine (7): yield, 95 mg (45%); mp 86-89.degree. C.; UV (pH 1)
1.sub.max 244 nm (e=0.581.times.10.sup.4), 286
(1.274.times.10.sup.4); (pH, 6.9) 252 (0.608.times.10.sup.4),
288(1.022.times.10.sup.4); (pH 13) 252 (0.618.times.10.sup.4), 288
(1.038.times.10.sup.4); .sup.1H NMR d 3.70 (s, 3 H, CH.sub.3), 5.51
(s, 2 H, ArCH.sub.2), 6.91 (s, 2 H, NH.sub.2, exchange with
D.sub.2O), 7.38-7.54 (m, 5 H, ArH); MS (El) calcd. m/z for
C.sub.14H.sub.12N.sub.3OF.sub.3 323.0994, found 323.0978; Anal.
(C.sub.14H.sub.12N.sub.3OF.sub.3) C, H, N, F.
EXAMPLE 25
[0117] 8-Aza-O.sup.6-benzyl-9-methylguanine (8a)
[0118] 8-Aza-O.sup.6-benzylguanine (0.484 g, 2.0 mmol) was mixed.
with 4 mL of 0.5 M sodium ethoxide in ethanol and stirred for 30
min. The ethanol was evaporated under reduced pressure. The residue
was dissolved in anhydrous DMF (6 mL), and methyl iodide (0.15 mL,
2.4 mmol) was added. The clear solution became cloudy within 10
min, and the resulting mixture was stirred overnight at room
temperature. DMF was evaporated under reduced pressure to give a
brown solid. The solid was dissolved in chloroform and loaded on a
silica gel column (Davisil grade 633, 200-425 mesh, 60 A). Product
8a was eluted with chloroform; yield, 138 mg (27%); mp
178-179.degree. C.; UV (pH 1) 1.sub.max 243 nm (sh)
(e=0.556.times.10.sup.4), 284 (1.112.times.10.sup.4); (pH 6.9) 243
(0.553.times.10.sup.4), 290 (0.998.times.10.sup.4); (pH 13) 242
(0.549.times.10.sup.4), 290 (1.010.times.10.sup.4); .sup.1H NMR
3.96 (s, 3 H, CH.sub.3), 5.57 (s, 2 H, ArCH.sub.2), 7.18 (s, 2 H,
NH.sub.2, exchange with D.sub.2O) 7.38-7.57 (m, 5 H, ArH); MS (EI)
calcd m/z for C.sub.12H.sub.12N.sub.6O 256.1072, found 256.1086;
Anal. (C.sub.12H.sub.12N.sub.6O) C, H, N.
EXAMPLE 26
[0119] 8-Aza-O.sup.6-benzyl-9-(pivaloyloxymethyl) guanine (8b) and
8-Aza-O.sup.6-benzyl-7-(pivaloyloxymethyl)guanine (9)
[0120] 8-Aza-O.sup.6-benzylguanine (0.484 g, 2.0 mmol) was mixed
with 4 mL of 0.5 M sodium ethoxide in ethanol and stirred for 30
min. The ethanol was evaporated under reduced pressure. The residue
was dissolved in anhydrous DMF (6 mL), and chloromethyl pivalate
(0.3 mL, 2.1 mmol) was added. The clear solution was stirred for 8
h at room temperature. DMF was evaporated under reduced pressure to
give a brown solid. The solid was dissolved in chloroform and
loaded on a silica gel column (Davisil grade 633, 200-425 mesh, 60
A). The 9-isomer (8b) was eluted from the column with
CHCl.sub.3:hexane (4:1) while the 7-isomer (9) was subseuently
eluted with CHCl.sub.3. 8-Aza-O.sup.6-benzyl-9-(pivaloyloxyme-
thyl)guanine (8b): yield, 405 mg (57%); mp 119-120.degree. C.; UV
(pH 1) 1.sub.max 246 nm (e=0.494.times.10.sup.4), 286
(0.878.times.10.sup.4); (pH 6.9) 247 (0.472.times.10.sup.4), 288
(0.819.times.10.sup.4); (pH 13) (decomposes to
8-aza-O.sup.6-benzylguanine); .sup.1H NMR d 1.10 (s, 9 E,
C(CH.sub.3).sub.3), 5.50 (s, 2 H, ArCH.sub.2), 6.31 (s, 2H,
CH.sub.2), 7.38 (s, 2 H, NH.sub.2, exchange with D.sub.2O),
7.40-7.54 (m, 5 H, ArH); MS (EI) calcd m/z for
C.sub.17H.sub.20N.sub.6O.sub.3 356.1596, found 356.1578; Anal.
(C.sub.17H.sub.20N.sub.6O.sub.3. {fraction (1.5)}H.sub.2O) C, H, N.
8-Aza-O.sup.6-benzyl-7-(pivaloyloxymethyl)guanin- e (9): yield, 103
mg (15%); mp 153-154.degree. C.; UV (pH 1) 1.sub.max 244 nm
(e=0.820.times.10.sup.4), 294 (1.249.times.10.sup.4) ; (pH 6.9) 250
(sh) (0.296.times.10.sup.4) 313 (0.503.times.10.sup.4); (pH 13)
(decomposes to 8-aza-O.sup.6-benzylguanine); .sup.1H NMR d 1.12 (s,
9 H, C(CH.sub.3).sub.3), 5.56 (s, 2 H, ArCH.sub.2), 6.40 (s, 2 H,
CH.sub.2), 7.04 (s, 2 H, NH.sub.2, exchange with D.sub.2O),
7.4-7.58 (m, 5 H, ArH); MS (EI) calcd m/z for
C.sub.17h.sub.20.sub.N.sub.6O.sub.3 356.1596, found 356.1602; Anal.
(C.sub.17H.sub.20N.sub.6O.sub.3).
EXAMPLE 27
[0121] O.sup.6-Benzyl-8-bromo-9-methylguanine (10a)
[0122] O.sup.6-Benzyl-9-methylguanine (0.252 g, 1.0 mmol) and
sodium bicarbonate (0.084 g, 1.0 mmol) were dissolved in anhydrous
DMF (2 mL) under argon. Bromine (52 mL, 1.0 mmol) was added to the
solution and the resulting mixture was stirred overnight at room
temperature. The solvent was evaporated under reduced pressure. The
residue was dissolved in chloroform, and loaded on a silica gel
column (Davisil grade 633, 200-425 mesh, 60 A). Product 10a was
eluted with chloroform; yield, 180 mg (52%); mp 150-152.degree. C.;
UV (pH1) 1.sub.max 248 nm (e=0.753.times.10.sup.4)- , 292
(1.398.times.10.sup.4); (pH 6.9) 251 (0.919.times.10.sup.4), 287
(1.306.times.10.sup.4); (pH 13) 251 (0.906.times.10.sup.4), 287
(1.296.times.10.sup.4); .sup.1H NMR d 3.53 (s, 3 H, CH.sub.3), 5.47
(s, 2 H, ArCH;), 6.61 (s, 2 H, NH.sub.2, exchange with D.sub.2O),
7.35-7.52 (m, 5 H, ArH); MS (EI) calcd m/z for
C.sub.13H.sub.12N.sub.5O.sup.79Br 333.0225, found 333.0228; calcd
m/z for C.sub.13H.sub.12N.sub.5O.sup.81 Br 335.0205, found
335.0188; Anal. (C.sub.13H.sub.12N.sub.5OBr) C, H, N, Br:
EXAMPLE 28
[0123] O.sup.6-Benzyl-8-bromo-9-(pivaloyloxmethyl) guanine (10b)
and O.sup.6-benzyl-8-bromo-7-(pivaloyloxymthyl) guanine (11)
[0124] O.sup.6-Benzyl-8-bromoguanine (Chae et al., J. Med. Chem.,
38, 342-347 (1995)) (0.48 a, 1.5 mmol) was mixed with 1.5 mL of a
1.0 M solution of sodium ethoxide in ethanol and was stirred for 20
min. The ethanol was removed under reduced pressure and the solid
residue was dissolved in DMF (5 mL). Chloromethylpivalate (0.24 mL,
1.65 mmol) was then added and the solution was stirred overnight.
The DMF was removed under reduced pressure. The residue was
dissolved in chloroform and was loaded on a silica gel column
(Davisil grade 633, 200-425 mesh, 60A) eluted with chloroform. The
9-isomer (10b) eluted earlier than the 7-isomer with chloroform and
10b was recovered in pure for under these conditions.
O.sup.6-Benzyl-8-bromo-9-(pivaloyloxymethyl)guanine (1b): yield,
150 mg (23%); mp 217-218.degree. C. UV (pH 1) 1.sub.max 250 nm
(e=0.944.times.10.sup.4), 291 (1.166.times.10.sup.4); (pH 6.9) 266
(0.916.times.10.sup.4), 295 (0.916.times.10.sup.4); (pH 13)
decomposes to O.sup.6-benzyl-8-bromoguanine; .sup.1H NMR d 1.13 (s,
9H, C(CH.sub.3).sub.3), 5.48 (s, 2H, ArCH.sub.2), 5.93 (s, 2H,
CH.sub.2), 6.80 (s, 2H, NH.sub.2, exchange with D.sub.2O),
7.35-7.52 (m, 5H, ArH). MS (EI) calcd m/z for
C.sub.18H.sub.20N.sub.5O.sub.3.sup.79Br 433.0750, found 433.0725;
calcd m/z for C.sub.18H.sub.20N.sub.5O.sub.3.sup.81Br 435.0729,
found 435.0672; Anal. (C.sub.18H.sub.20N.sub.5O.sub.3Br) C, H, N,
Br. The recovered 7-isomer (11) was rechromatographed on a silica
gel column (Davisil grade 633, 200-425 mesh, 60 A) which was eluted
first with CHCl.sub.3/hexane (1:1) followed by CHCl.sub.3 to
recover O.sup.6-benzyl-8-bromo-7-(pivaloyloxymethyl)guanine
(11).
EXAMPLE 29
[0125] O.sup.6-Benzyl-7-(pivaloyloxmethyl) guanine (12)
[0126] O.sup.6-Benzylguanine (2.41 g, 10 mmol) was mixed with 10 mL
of a 1.0 M solution of sodium ethoxide in ethanol and was stirred
for 30 min. The ethanol was evaporated under reduced pressure. The
residue was dissolved in anhydrous DMF (30 mL), and chloromethyl-
pivalate (Aldrich) (1.5 mL, 10.4 mmol) was added. The clear
solution was stirred overnight at room temperature. DMF was
evaporated under reduced pressure to give a pale peach-colored
solid. The solid was dissolved in chloroform/ethanol (9:1) and
loaded on a silica gel column (Davisil grade 633, 200-425 mesh, 60
A). The column was eluted with chloroform/ethanol (9:1) to elute
the 9-isomer (Chae et al., J. Med. Chem., 37, 342-347 (1994))
followed by the 7-isomer. The 7-isomer (12) was further purified by
silica gel column chromatography (Davisil grade 633, 200-425 mesh,
60 A) using chloroform/ethanol (98:2) as eluent: yield, 36 mg (1%);
mp 166-168.degree. C. dec; UV (pH 1) 1. 240 nm (sh)
(e=0.656.times.10.sup.4)- , 290 (1.164.times.10.sup.4) ; (pH 6.9)
240 (sh) (0.635.times.10.sup.4) 293 (0.528.times.10.sup.4); (pH 13)
decomposes to O.sup.6-benzylguanine; .sup.1H NMR d 0.98 (s, 9 H,
C(CH.sub.3).sub.3), 5.51 (s, 2 H, ArCH.sub.2), 6.07 (s, 2 H,
CH.sub.2), 6.32 (s, 2 H, NH.sub.2, exchange with D.sub.2O),
7.36-7.58 (m, 5 H, ArH), 8.25 (s, 1 H, H-8); MS (EI) calcd m/z for
C.sub.18H.sub.21N.sub.5O.sub.3 355.1644, found 355.1626.
* * * * *