U.S. patent application number 14/788507 was filed with the patent office on 2016-02-04 for kinase inhibitors, prodrug forms thereof and their use in therapy.
The applicant listed for this patent is Auckland Uniservices Limited. Invention is credited to Robert Forbes Anderson, Amir Ashoorzadeh, Kendall Marie Carlin, William Alexander Denny, Huai-Ling Hsu, Stephen Michael Frazer Jamieson, Ho Huat Lee, Guo-Liang Lu, Andrej Maroz, Alexandra Marie Mowday, Adam Vorn Patterson, Jeffrey Bruce Smaill, William Robert Wilson.
Application Number | 20160031877 14/788507 |
Document ID | / |
Family ID | 43649498 |
Filed Date | 2016-02-04 |
United States Patent
Application |
20160031877 |
Kind Code |
A1 |
Smaill; Jeffrey Bruce ; et
al. |
February 4, 2016 |
KINASE INHIBITORS, PRODRUG FORMS THEREOF AND THEIR USE IN
THERAPY
Abstract
The invention provides kinase inhibitors of Formula I:
##STR00001## wherein either: (1) R.sub.1 is H, and (a) R.sub.2 is
(3-chlorobenzyl)oxy- and R.sub.3 is chloro; (b) R.sub.2 and
R.sub.3, together with the carbon atoms to which they are attached,
form 1-(3-fluorobenzyl)-1H-pyrazole; (c) R.sub.2 is
2-pyridinylmethoxy and R.sub.3 is chloro; (d) R.sub.2 and R.sub.3
are both chloro; (e) R.sub.2 is chloro and R.sub.3 is bromo; (f)
R.sub.2 and R.sub.3 are both bromo; (g) R.sub.2 is fluoro and
R.sub.3 is ethynyl; (h) R.sub.2 is chloro and R.sub.3 is ethynyl;
(i) R.sub.2 is bromo and R.sub.3 is ethynyl; (j) other than when
R.sub.2 is in the 3-position in combination with R.sub.3 in the
4-position, R.sub.2 is bromo and R.sub.3 is fluoro; (k) R.sub.2 is
2-pyridinylmethoxy and R.sub.3 is fluoro; or (l) R.sub.2 is
2-pyridinylmethoxy and R.sub.3 is bromo; or (2) at least one of
R.sub.1, R.sub.2 and R.sub.3 is selected from benzyloxy,
3-chlorobenzyloxy and 2-pyridinylmethoxy and when at least one of
R.sub.1, R.sub.2 and R.sub.3 is not benzyloxy, 3-chlorobenzyloxy or
2-pyridinylmethoxy, each of the others is independently selected
from H, halogen, and C.sub.2-C.sub.4 alkynyl, with the proviso that
when one of R.sub.1, R.sub.2 and R.sub.3 is benzyloxy or
2-pyridinylmethoxy, the other two of R.sub.1, R.sub.2 and R.sub.3
are not H; or (3) two of R.sub.1, R.sub.2 and R.sub.3, together
with the carbon atoms to which they are attached, form
1-(3-fluorobenzyl)-1H-pyrazole; and the other is selected from H,
halogen and C.sub.2-C.sub.4 alkynyl. Also provided are reductive
prodrugs, comprising a kinase inhibitor as defined above and a
reductive trigger linked directly or indirectly to a nitrogen of
the kinase inhibitor. Further provided are pharmaceutical
compositions, comprising the kinase inhibitors or the prodrugs, and
the use of such compositions in therapy, in particular for treating
cancer.
Inventors: |
Smaill; Jeffrey Bruce;
(Auckland, NZ) ; Patterson; Adam Vorn; (Auckland,
NZ) ; Lu; Guo-Liang; (Auckland, NZ) ; Lee; Ho
Huat; (Auckland, NZ) ; Ashoorzadeh; Amir;
(Auckland, NZ) ; Anderson; Robert Forbes;
(Auckland, NZ) ; Wilson; William Robert; (Waiuku,
NZ) ; Denny; William Alexander; (Manukau, NZ)
; Hsu; Huai-Ling; (Auckland, NZ) ; Maroz;
Andrej; (Auckland, NZ) ; Jamieson; Stephen Michael
Frazer; (Auckland, NZ) ; Mowday; Alexandra Marie;
(Auckland, NZ) ; Carlin; Kendall Marie; (Auckland,
NZ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Auckland Uniservices Limited |
Auckland |
|
NZ |
|
|
Family ID: |
43649498 |
Appl. No.: |
14/788507 |
Filed: |
June 30, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
13390398 |
Apr 24, 2012 |
9101632 |
|
|
PCT/NZ2010/000174 |
Sep 2, 2010 |
|
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14788507 |
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Current U.S.
Class: |
514/264.11 ;
544/279 |
Current CPC
Class: |
A61P 19/10 20180101;
A61K 31/519 20130101; A61P 35/00 20180101; A61P 29/00 20180101;
C07D 471/04 20130101; A61P 43/00 20180101 |
International
Class: |
C07D 471/04 20060101
C07D471/04 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 2, 2009 |
NZ |
579458 |
Claims
1.-36. (canceled)
37. A method of treating cancer which includes the step of
administering a therapeutically effective amount of a kinase
inhibitor of Formula I: ##STR00042## wherein either: (1) R1 is H,
and R2 is (3-chlorobenzyl)oxy- and R3 is chloro; R2 and R3,
together with the carbon atoms to which they are attached, form
1-(3-fluorobenzyl)-1H-pyrazole; R2 is 2-pyridinylmethoxy and R3 is
chloro; R2 and R3 are both chloro; R2 is chloro and R3 is bromo; R2
and R3 are both bromo; R2 is fluoro and R3 is ethynyl; R2 is chloro
and R3 is ethynyl; R2 is bromo and R3 is ethynyl; other than when
R2 is in the 3-position in combination with R3 in the 4-position,
R2 is bromo and R3 is fluoro; R2 is 2-pyridinylmethoxy and R3 is
fluoro; or R2 is 2-pyridinylmethoxy and R3 is bromo: or at least
one of R1, R2 and R3 is selected from benzyloxy, 3-chlorobenzyloxy
and 2-pyridinylmethoxy and when at least one of R1, R2 and R3 is
not benzyloxy, 3-chlorobenzyloxy or 2-pyridinylmethoxy, each of the
others is independently selected from H, halogen, and C2-C4
alkynyl, with the proviso that when one of R1, R2 and R3 is
benzyloxy or 2-pyridinylmethoxy, the other two of R1, R2 and R3 are
not H: or two of R1, R2 and R3, together with the carbon atoms to
which they are attached, form 1-(3-fluorobenzyl)-1H-pyrazole, and
the other is selected from H, halogen and C2-C4 alkynyl; or a
pharmaceutically acceptable salt or solvate thereof, or a
pharmaceutically acceptable salt or solvate thereof, to a patient
in need of such therapy.
38. A method of treating cancer comprising the step of
administering a therapeutically effective amount of a compound of
Formula III: ##STR00043## wherein X is any negatively charged
counterion, R.sub.1, R.sub.2 and R.sub.3 are as defined for Formula
I in claim 1, R.sub.4 is selected from H, methyl, ethyl,
trifluoromethyl, --CN, --CONH.sub.2 and propyn-1-yl, and R.sub.5 is
C.sub.1-C.sub.6 alkyl; to a patient in need of such therapy.
39. A method of inhibiting kinase activity within a subject,
comprising the step of administering an inhibitory amount of a
kinase inhibitor of Formula I: ##STR00044## wherein either: (1) R1
is H, and R2 is (3-chlorobenzyl)oxy- and R3 is chloro; R2 and R3,
together with the carbon atoms to which they are attached, form
1-(3-fluorobenzyl)-1H-pyrazole; R2 is 2-pyridinylmethoxy and R3 is
chloro; R2 and R3 are both chloro; R2 is chloro and R3 is bromo; R2
and R3 are both bromo; R2 is fluoro and R3 is ethynyl; R2 is chloro
and R3 is ethynyl; R2 is bromo and R3 is ethynyl; other than when
R2 is in the 3-position in combination with R3 in the 4-position,
R2 is bromo and R3 is fluoro; R2 is 2-pyridinylmethoxy and R3 is
fluoro: or R2 is 2-pyridinylmethoxy and R3 is bromo; or at least
one of R1, R2 and R3 is selected from benzyloxy, 3-chlorobenzyloxy
and 2-pyridinylmethoxy and when at least one of R1, R2 and R3 is
not benzyloxy, 3-chlorobenzyloxy or 2-pyridinylmethoxy, each of the
others is independently selected from H, halogen, and C2-C4
alkynyl, with the proviso that when one of R1, R2 and R3 is
benzyloxy or 2-pyridinylmethoxy, the other two of R1, R2 and R3 are
not H; or two of R1, R2 and R3, together with the carbon atoms to
which they are attached, form 1-(3-fluorobenzyl)-1H-pyrazole, and
the other is selected from H, halogen and C2-C4 alkynyl; or a
pharmaceutically acceptable salt or solvate thereof, to a subject
in need of such treatment.
40. A method of inhibiting kinase activity within a subject,
comprising the step of administering an inhibitory amount of a
compound of Formula III: ##STR00045## wherein X is any negatively
charged counterion, R.sub.1, R.sub.2 and R.sub.3 are as defined for
Formula I in claim 1, R4 is selected from H, methyl, ethyl,
trifluoromethyl, --CN, --CONH.sub.2 and propyn-1-yl, and R.sub.5 is
C.sub.1-C.sub.6 alkyl; to a subject in need of such treatment.
41. The method of claim 37 or 39, wherein the compound has Formula
IIIA: ##STR00046## wherein X is any negatively charged counterion,
R.sub.1, R.sub.2 and R.sub.3 are as defined for Formula I in claim
1 and R.sub.4 is selected from H, methyl, ethyl, trifluoromethyl,
--CN, --CONH.sub.2 and propyn-1-yl.
42. The method of claim 37 or 39, wherein the compound, wherein the
compound has Formula IIIB ##STR00047## wherein X is any negatively
charged counterion, R.sub.1, R.sub.2 and R.sub.3 are as defined for
Formula IA in claim 5 and R.sub.4 is selected from H, methyl,
ethyl, trifluoromethyl, --CN, --CONH.sub.2 and propyn-1-yl.
43. The method of claim 37 or 39, wherein the compound is selected
from the group consisting of:
(2E)-4-[(4-{3-chloro-4-[(3-chlorobenzyl)oxy]anilino}pyrido[3,4-d]pyrimidi-
n-6-yl)amino]-N,N-dimethyl-N-[(1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-4-
-oxo-2-buten-1-ammonium bromide (12),
(2E)-4-[(4-{[1-(3-fluorobenzyl)-1H-indazol-5-yl]amino}pyrido[3,4-d]pyrimi-
din-6-yl)amino]-N,N-dimethyl-N-[(1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-
-4-oxo-2-buten-1-ammonium bromide (13),
(2E)-N-[(1,2-dimethyl-4-nitro-1H-imidazol-5-yl)methyl]-4-[(4-{[1-(3-fluor-
obenzyl)-1H-indazol-5-yl]amino}pyrido[3,4-d]pyrimidin-6-yl)amino]-N,N-dime-
thyl-4-oxo-2-buten-1-ammonium bromide (14),
(2E)-4-({4-[3-chloro-4-(2-pyridinylmethoxy)anilino]pyrido[3,4-d]pyrimidin-
-6-yl}amino)-N,N-dimethyl-N-[(1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-4--
oxo-2-buten-1-ammonium bromide (15),
(2E)-4-{[4-(3,4-dichloroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N,N-di-
methyl-N-[(1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-4-oxo-2-buten-1-ammon-
ium bromide (16),
(2E)-4-{[4-(3-bromo-4-chloroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N,-
N-dimethyl-N-[(1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-4-oxo-2-buten-1-a-
mmonium bromide (17),
(2E)-4-{[4-(4-bromo-3-chloroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N,-
N-dimethyl-N-[(1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-4-oxo-2-buten-1-a-
mmonium bromide (18),
(2E)-4-{[4-(3,4-dibromoanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N,N-dim-
ethyl-N-[(1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-4-oxo-2-buten-1-ammoni-
um bromide (19),
(2E)-4-{[4-(3-ethynyl-4-fluoroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}--
N,N-dimethyl-N-[(1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-4-oxo-2-buten-1-
-ammonium bromide (20),
(2E)-4-{[4-(4-chloro-3-ethynylanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}--
N,N-dimethyl-N-[(1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-4-oxo-2-buten-1-
-ammonium bromide (21),
(2E)-4-[(4-{3-chloro-4-[(3-chlorobenzyl)oxy]anilino}pyrido[3,4-d]pyrimidi-
n-6-yl)amino]-N-[(1,2-dimethyl-4-nitro-1H-imidazol-5-yl)methyl]-N,N-dimeth-
yl-4-oxo-2-buten-1-ammonium bromide (22),
(2E)-4-[(4-{3-chloro-4-[(3-chlorobenzyl)oxy]anilino}pyrido[3,4-d]pyrimidi-
n-6-yl)amino]-N-[(2-ethyl-1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-N,N-di-
methyl-4-oxo-2-buten-1-ammonium bromide (23),
(2E)-4-[(4-{3-chloro-4-[(3-chlorobenzyl)oxy]anilino}pyrido[3,4-d]pyrimidi-
n-6-yl)amino]-N,N-dimethyl-N-{[1-methyl-4-nitro-2-(trifluoromethyl)-1H-imi-
dazol-5-yl]methyl}-4-oxo-2-buten-1-ammonium bromide (24),
(2E)-4-[(4-{3-chloro-4-[(3-chlorobenzyl)oxy]anilino}pyrido[3,4-d]pyrimidi-
n-6-yl)amino]-N-[(2-cyano-1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-N,N-di-
methyl-4-oxo-2-buten-1-ammonium bromide (25),
(2E)-N-{[2-(aminocarbonyl)-1-methyl-4-nitro-1H-imidazol-5-yl]methyl}-4-[(-
4-{3-chloro-4-[(3-chlorobenzyl)oxy]anilino}pyrido[3,4-d]pyrimidin-6-yl)ami-
no]-N,N-dimethyl-4-oxo-2-buten-1-ammonium bromide (26),
(2E)-4-[(4-{3-chloro-4-[(3-chlorobenzyl)oxy]anilino}pyrido[3,4-d]pyrimidi-
n-6-yl)amino]-N,N-dimethyl-N-{[1-methyl-4-nitro-2-(1-propynyl)-1H-imidazol-
-5-yl]methyl}-4-oxo-2-buten-1-ammonium bromide (27),
(2E)-N-[(2-ethyl-1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-4-[(4-{[1-(3-f-
luorobenzyl)-1H-indazol-5-yl]amino}pyrido[3,4-d]pyrimidin-6-yl)amino]-N,N--
dimethyl-4-oxo-2-buten-1-ammonium bromide (28),
(2E)-4-[(4-{[1-(3-fluorobenzyl)-1H-indazol-5-yl]amino}pyrido[3,4-d]pyrimi-
din-6-yl)amino]-N,N-dimethyl-N-{[1-methyl-4-nitro-2-(trifluoromethyl)-1H-i-
midazol-5-yl]methyl}-4-oxo-2-buten-1-ammonium bromide (29),
(2E)-N-[(2-cyano-1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-4-[(4-({[1-(3--
fluorobenzyl)-1H-indazol-5-yl]amino}pyrido[3,4-d]pyrimidin-6-yl)amino]-N,N-
-dimethyl-4-oxo-2-buten-1-ammonium bromide (30),
(2E)-N-{[2-(aminocarbonyl)-1-methyl-4-nitro-1H-imidazol-5-yl]methyl})-4-[-
(4-{[1-(3-fluorobenzyl)-1H-indazol-5-yl]amino}pyrido[3,4-d]pyrimidin-6-yl)-
amino]-N,N-dimethyl-4-oxo-2-buten-1-ammonium bromide (31),
(2E)-4-[(4-{[1-(3-fluorobenzyl)-1H-indazol-5-yl]amino}pyrido[3,4-d]pyrimi-
din-6-yl)amino]-N,N-dimethyl-N-{[1-methyl-4-nitro-2-(1-propynyl)-1H-imidaz-
ol-5-yl]methyl}-4-oxo-2-buten-1-ammonium bromide (32),
(2E)-4-({4-[3-chloro-4-(2-pyridinylmethoxy)anilino]pyrido[3,4-d]pyrimidin-
-6-yl}amino)-N-[(1,2-dimethyl-4-nitro-1H-imidazol-5-yl)methyl]-N,N-dimethy-
l-4-oxo-2-buten-1-ammonium bromide (33),
(2E)-4-({4-[3-chloro-4-(2-pyridinylmethoxy)anilino]pyrido[3,4-d]pyrimidin-
-6-yl}amino)-N-[(2-ethyl-1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-N,N-dim-
ethyl-4-oxo-2-buten-1-ammonium bromide (34),
(2E)-4-({4-[3-chloro-4-(2-pyridinylmethoxy)anilino]pyrido[3,4-d]pyrimidin-
-6-yl}amino)-N,N-dimethyl-N-{[1-methyl-4-nitro-2-(trifluoromethyl)-1H-imid-
azol-5-yl]methyl}-4-oxo-2-buten-1-ammonium bromide (35),
(2E)-4-({4-[3-chloro-4-(2-pyridinylmethoxy)anilino]pyrido[3,4-d]pyrimidin-
-6-yl}amino)-N-[(2-cyano-1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-N,N-dim-
ethyl-4-oxo-2-buten-1-ammonium bromide (36),
(2E)-N-{[2-(aminocarbonyl)-1-methyl-4-nitro-1H-imidazol-5-yl]methyl}-4-({-
4-[3-chloro-4-(2-pyridinylmethoxy)anilino]pyrido[3,4-d]pyrimidin-6-yl}amin-
o)-N,N-dimethyl-4-oxo-2-buten-1-ammonium bromide (37),
(2E)-4-({4-[3-chloro-4-(2-pyridinylmethoxy)anilino]pyrido[3,4-d]pyrimidin-
-6-yl}amino)-N,N-dimethyl-N-{[1-methyl-4-nitro-2-(1-propynyl)-1H-imidazol--
5-yl]methyl}-4-oxo-2-buten-1-ammonium bromide (38),
(2E)-4-{[4-(3,4-dichloroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N-[(1,-
2-dimethyl-4-nitro-1H-imidazol-5-yl)methyl]-N,N-dimethyl-4-oxo-2-buten-1-a-
mmonium bromide (39),
(2E)-4-{[4-(3,4-dichloroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N-[(2--
ethyl-1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-N,N-dimethyl-4-oxo-2-buten-
-1-ammonium bromide (40),
(2E)-4-{[4-(3,4-dichloroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N,N-di-
methyl-N-{[1-methyl-4-nitro-2-(trifluoromethyl)-1H-imidazol-5-yl]methyl}-4-
-oxo-2-buten-1-ammonium bromide (41),
(2E)-N-[(2-cyano-1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-4-{[4-(3,4-dic-
hloroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N,N-dimethyl-4-oxo-2-buten-
-1-ammonium bromide (42),
(2E)-N-{[2-(aminocarbonyl)-1-methyl-4-nitro-1H-imidazol-5-yl]methyl}-4-{[-
4-(3,4-dichloroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N,N-dimethyl-4-o-
xo-2-buten-1-ammonium bromide (43),
(2E)-4-{[4-(3,4-dichloroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N,N-di-
methyl-N-{[1-methyl-4-nitro-2-(1-propynyl)-1H-imidazol-5-yl]methyl}-4-oxo--
2-buten-1-ammonium bromide (44),
(2E)-4-{[4-(3-bromo-4-chloroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N--
[(1,2-dimethyl-4-nitro-1H-imidazol-5-yl)methyl]-N,N-dimethyl-4-oxo-2-buten-
-1-ammonium bromide (45),
(2E)-4-{[4-(3-bromo-4-chloroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino)}-N-
-[(2-ethyl-1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-N,N-dimethyl-4-oxo-2--
buten-1-ammonium bromide (46),
(2E)-4-{[4-(3-bromo-4-chloroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N,-
N-dimethyl-N-{[1-methyl-4-nitro-2-(trifluoromethyl)-1H-imidazol-5-yl]methy-
l}-4-oxo-2-buten-1-ammonium bromide (47),
(2E)-4-{[4-(3-bromo-4-chloroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N--
[(2-cyano-1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-N,N-dimethyl-4-oxo-2-b-
uten-1-ammonium bromide (48),
(2E)-N-{[2-(aminocarbonyl)-1-methyl-4-nitro-1H-imidazol-5-yl]methyl}-4-{[-
4-(3-bromo-4-chloroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N,N-dimethyl-
-4-oxo-2-buten-1-ammonium bromide (49),
(2E)-4-{[4-(3-bromo-4-chloroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N,-
N-dimethyl-N-{[1-methyl-4-nitro-2-(1-propynyl)-1H-imidazol-5-yl]methyl}-4--
oxo-2-buten-1-ammonium bromide (50),
(2E)-4-{[4-(4-bromo-3-chloroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N--
[(1,2-dimethyl-4-nitro-1H-imidazol-5-yl)methyl]-N,N-dimethyl-4-oxo-2-buten-
-1-ammonium bromide (51),
(2E)-4-{[4-(4-bromo-3-chloroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N--
[(2-ethyl-1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-N,N-dimethyl-4-oxo-2-b-
uten-1-ammonium bromide (52),
(2E)-4-{[4-(4-bromo-3-chloroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N,-
N-dimethyl-N-{[1-methyl-4-nitro-2-(trifluoromethyl)-1H-imidazol-5-yl]methy-
l}-4-oxo-2-buten-1-ammonium bromide (53),
(2K)-4-{[4-(4-bromo-3-chloroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N--
[(2-cyano-1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-N,N-dimethyl-4-oxo-2-b-
uten-1-ammonium bromide (54),
(2E)-N-{[2-(aminocarbonyl)-1-methyl-4-nitro-1H-imidazol-5-yl]methyl}-4-{[-
4-(4-bromo-3-chloroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N,N-dimethyl-
-4-oxo-2-buten-1-ammonium bromide (55),
(2E)-4-{[4-(4-bromo-3-chloroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N,-
N-dimethyl-N-{[1-methyl-4-nitro-2-(1-propynyl)-1H-imidazol-5-yl]methyl)}-4-
-oxo-2-buten-1-ammonium bromide (56),
(2E)-4-{[4-(3,4-dibromoanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N-[(1,2-
-dimethyl-4-nitro-1H-imidazol-5-yl)methyl]-N,N-dimethyl-4-oxo-2-buten-1-am-
monium bromide (57),
(2E)-4-{[4-(3,4-dibromoanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N-[(2-e-
thyl-1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-N,N-dimethyl-4-oxo-2-buten--
1-ammonium bromide (58),
(2E)-4-{[4-(3,4-dibromoanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N,N-dim-
ethyl-N-{[1-methyl-4-nitro-2-(trifluoromethyl)-1H-imidazol-5-yl]methyl}-4--
oxo-2-buten-1-ammonium bromide (59),
(2E)-N-[(2-cyano-1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-4-{[4-(3,4-dib-
romoanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N,N-dimethyl-4-oxo-2-buten--
1-ammonium bromide (60),
(2E)-N-{[2-(aminocarbonyl)-1-methyl-4-nitro-1H-imidazol-5-yl]methyl}-4-{[-
4-(3,4-dibromoanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N,N-dimethyl-4-ox-
o-2-buten-1-ammonium bromide (61),
(2E)-4-{[4-(3,4-dibromoanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N,N-dim-
ethyl-N-{[1-methyl-4-nitro-2-(1-propynyl)-1H-imidazol-5-yl]methyl}-4-oxo-2-
-buten-1-ammonium bromide (62),
(2E)-N-[(1,2-dimethyl-4-nitro-1H-imidazol-5-yl)methyl]-4-{[4-(3-ethynyl-4-
-fluoroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N,N-dimethyl-4-oxo-2-but-
en-1-ammonium bromide (63),
(2E)-N-[(2-ethyl-1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-4-{[4-(3-ethyn-
yl-4-fluoroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N,N-dimethyl-4-oxo-2-
-buten-1-ammonium bromide (64),
(2E)-4-{[4-(3-ethynyl-4-fluoroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}--
N,N-dimethyl-N-{[1-methyl-4-nitro-2-(trifluoromethyl)-1H-imidazol-5-yl]met-
hyl}-4-oxo-2-buten-1-ammonium bromide (65),
(2E)-N-[(2-cyano-1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-4-{[4-(3-ethyn-
yl-4-fluoroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N,N-dimethyl-4-oxo-2-
-buten-1-ammonium bromide (66),
(2E)-N-{[2-(aminocarbonyl)-1-methyl-4-nitro-1H-imidazol-5-yl]methyl}-4-{[-
4-(3-ethynyl-4-fluoroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N,N-dimeth-
yl-4-oxo-2-buten-1-ammonium bromide (67),
(2E)-4-{[4-(3-ethynyl-4-fluoroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}--
N,N-dimethyl-N-{[1-methyl-4-nitro-2-(1-propynyl)-1H-imidazol-5-yl]methyl}--
4-oxo-2-buten-1-ammonium bromide (68),
(2E)-4-{[4-(4-chloro-3-ethynylanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}--
N-[(1,2-dimethyl-4-nitro-1H-imidazol-5-yl)methyl]-N,N-dimethyl-4-oxo-2-but-
en-1-ammonium bromide (69),
(2E)-4-{[4-(4-chloro-3-ethynylanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}--
N-[(2-ethyl-1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-N,N-dimethyl-4-oxo-2-
-buten-1-ammonium bromide (70),
(2E)-4-{[4-(4-chloro-3-ethynylanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}--
N,N-dimethyl-N-{[1-methyl-4-nitro-2-(trifluoromethyl)-1H-imidazol-5-yl]met-
hyl}-4-oxo-2-buten-1-ammonium bromide (71),
(2E)-4-{[4-(4-chloro-3-ethynylanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}--
N-[(2-cyano-1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-N,N-dimethyl-4-oxo-2-
-buten-1-ammonium bromide (72),
(2E)-N-{[2-(aminocarbonyl)-1-methyl-4-nitro-1H-imidazol-5-yl]methyl}-4-{[-
4-(4-chloro-3-ethynylanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N,N-dimeth-
yl-4-oxo-2-buten-1-ammonium bromide (73),
(2E)-4-{[4-(4-chloro-3-ethynylanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}--
N,N-dimethyl-N-{[1-methyl-4-nitro-2-(1-propynyl)-1H-imidazol-5-yl]methyl}--
4-oxo-2-buten-1-ammonium bromide (74),
(2E)-4-{[4-(4-bromo-3-ethynylanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N-
,N-dimethyl-N-[(1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-4-oxo-2-buten-1--
ammonium bromide (75),
(2E)-4-{[4-(4-bromo-3-ethynylanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N-
-[(1,2-dimethyl-4-nitro-1H-imidazol-5-yl)methyl]-N,N-dimethyl-4-oxo-2-bute-
n-1-ammonium bromide (76),
(2E)-4-{[4-(4-bromo-3-ethynylanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N-
-[(2-ethyl-1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-N,N-dimethyl-4-oxo-2--
buten-1-ammonium bromide (77),
(2E)-4-{[4-(4-bromo-3-ethynylanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N-
,N-dimethyl-N-{([1-methyl-4-nitro-2-(trifluoromethyl)-1H-imidazol-5-yl]met-
hyl}-4-oxo-2-buten-1-ammonium bromide (78),
(2E)-4-{[4-(4-bromo-3-ethynylanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N-
-[(2-cyano-1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-N,N-dimethyl-4-oxo-2--
buten-1-ammonium bromide (79),
(2E)-N-{[2-(aminocarbonyl)-1-methyl-4-nitro-1H-imidazol-5-yl]methyl}-4-{[-
4-(4-bromo-3-ethynylanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N,N-dimethy-
l-4-oxo-2-buten-1-ammonium bromide (80),
(2E)-4-{[4-(4-bromo-3-ethynylanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N-
,N-dimethyl-N-{[1-methyl-4-nitro-2-(1-propynyl)-1H-imidazol-5-yl]methyl}-4-
-oxo-2-buten-1-ammonium bromide (81),
(2E)-4-{[4-(4-bromo-3-fluoroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N,-
N-dimethyl-N-[(1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-4-oxo-2-buten-1-a-
mmonium bromide (82),
(2E)-4-{[4-(4-bromo-3-fluoroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N--
[(1,2-dimethyl-4-nitro-1H-imidazol-5-yl)methyl]-N,N-dimethyl-4-oxo-2-buten-
-1-ammonium bromide (83),
(2E)-4-{[4-(4-bromo-3-fluoroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N--
[(2-ethyl-1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-N,N-dimethyl-4-oxo-2-b-
uten-1-ammonium bromide (84),
(2E)-4-{[4-(4-bromo-3-fluoroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N,-
N-dimethyl-N-{[1-methyl-4-nitro-2-(trifluoromethyl)-1H-imidazol-5-yl]methy-
l}-4-oxo-2-buten-1-ammonium bromide (85),
(2E)-4-{[4-(4-bromo-3-fluoroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N--
[(2-cyano-1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-N,N-dimethyl-4-oxo-2-b-
uten-1-ammonium bromide (86),
(2E)-N-{[2-(aminocarbonyl)-1-methyl-4-nitro-1H-imidazol-5-yl]methyl}-4-{[-
4-(4-bromo-3-fluoroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N,N-dimethyl-
-4-oxo-2-buten-1-ammonium bromide (87),
(2E)-4-{[4-(4-bromo-3-fluoroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N,-
N-dimethyl-N-{[1-methyl-4-nitro-2-(1-propynyl)-1H-imidazol-5-yl]methyl}-4--
oxo-2-buten-1-ammonium bromide (88),
(2E)-4-({4-[3-fluoro-4-(2-pyridinylmethoxy)anilino]pyrido[3,4-d]pyrimidin-
-6-yl}amino)-N,N-dimethyl-N-[(1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-4--
oxo-2-buten-1-ammonium bromide (91),
(2E)-N-[(1,2-dimethyl-4-nitro-1H-imidazol-5-yl)methyl]-4-({4-[3-fluoro-4--
(2-pyridinylmethoxy)anilino]pyrido[3,4-d]pyrimidin-6-yl}amino)-N,N-dimethy-
l-4-oxo-2-buten-1-ammonium bromide (92),
(2E)-N-[(2-ethyl-1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-4-({4-[3-fluor-
o-4-(2-pyridinylmethoxy)anilino]pyrido[3,4-d]pyrimidin-6-yl}amino)-N,N-dim-
ethyl-4-oxo-2-buten-1-ammonium bromide (93),
(2E)-4-({4-[3-fluoro-4-(2-pyridinylmethoxy)anilino]pyrido[3,4-d]pyrimidin-
-6-yl}amino)-N,N-dimethyl-N-{[1-methyl-4-nitro-2-(trifluoromethyl)-1H-imid-
azol-5-yl]methyl}-4-oxo-2-buten-1-ammonium bromide (94),
(2E)-N-[(2-cyano-1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-4-({4-[3-fluor-
o-4-(2-pyridinylmethoxy)anilino]pyrido[3,4-d]pyrimidin-6-yl}amino)-N,N-dim-
ethyl-4-oxo-2-buten-1-ammonium bromide (95),
(2E)-N-{[2-(aminocarbonyl)-1-methyl-4-nitro-1H-imidazol-5-yl]methyl}-4-({-
4-[3-fluoro-4-(2-pyridinylmethoxy)anilino]pyrido[3,4-d]pyrimidin-6-yl}amin-
o)-N,N-dimethyl-4-oxo-2-buten-1-ammonium bromide (96),
(2E)-4-({4-[3-fluoro-4-(2-pyridinylmethoxy)anilino]pyrido[3,4-d]pyrimidin-
-6-yl}amino)-N,N-dimethyl-N-{[1-methyl-4-nitro-2-(1
I-propynyl)-1H-imidazol-5-yl]methyl}-4-oxo-2-buten-1-ammonium
bromide (97),
(2E)-4-({4-[3-bromo-4-(2-pyridinylmethoxy)anilino]pyrido[3,4-d]pyri-
midin-6-yl}amino)-N,N-dimethyl-N-[(1-methyl-4-nitro-1H-imidazol-5-yl)methy-
l]-4-oxo-2-buten-1-ammonium bromide (98),
(2E)-4-({4-[3-bromo-4-(2-pyridinylmethoxy)anilino]pyrido[3,4-d]pyrimidin--
6-yl}amino)-N-[(1,2-dimethyl-4-nitro-1H-imidazol-5-yl)methyl]-N,N-dimethyl-
-4-oxo-2-buten-1-ammonium bromide (99),
(2E)-4-({4-[3-bromo-4-(2-pyridinylmethoxy)anilino]pyrido[3,4-d]pyrimidin--
6-yl}amino)-N-[(2-ethyl-1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-N,N-dime-
thyl-4-oxo-2-buten-1-ammonium bromide (100),
(2E)-4-({4-[3-bromo-4-(2-pyridinylmethoxy)anilino]pyrido[3,4-d]pyrimidin--
6-yl}amino)-N,N-dimethyl-N-{[1-methyl-4-nitro-2-(trifluoromethyl)-1H-imida-
zol-5-yl]methyl}-4-oxo-2-buten-1-ammonium bromide (101),
(2E)-4-({4-[3-bromo-4-(2-pyridinylmethoxy)anilino]pyrido[3,4-d]pyrimidin--
6-yl}amino)-N-[(2-cyano-1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-N,N-dime-
thyl-4-oxo-2-buten-1-ammonium bromide (102),
(2E)-N-{[2-(aminocarbonyl)-1-methyl-4-nitro-1H-imidazol-5-yl]methyl}-4-({-
4-[3-bromo-4-(2-pyridinylmethoxy)anilino]pyrido[3,4-d]pyrimidin-6-yl}amino-
)-N,N-dimethyl-4-oxo-2-buten-1-ammonium bromide (103) and
(2E)-4-({4-[3-bromo-4-(2-pyridinylmethoxy)anilino]pyrido[3,4-d]pyrimidin--
6-yl}amino)-N,N-dimethyl-N-{[1-methyl-4-nitro-2-(1-propynyl)-1H-imidazol-5-
-yl]methyl}-4-oxo-2-buten-1-ammonium bromide (104); and
pharmaceutically acceptable solvates thereof.
44. The compound according to claim 42, which is
(2E)-4-{[4-(3-bromo-4-chloroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N,-
N-dimethyl-N-[(1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-4-oxo-2-buten-1-a-
mmonium bromide (17), or a pharmaceutically acceptable solvate
thereof.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application is continuation of U.S. patent application
Ser. No. 13/390,398, filed on Apr. 24, 2012, which is a U.S.
national phase of International Application No. PCT/NZ2010/000174
filed Sep. 2, 2010 which designated the U.S. and claims priority to
New Zealand Application No. 579458, filed Sep. 2, 2009, the entire
contents of each of which are hereby incorporated by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to kinase inhibitors, to
kinase inhibitors in prodrug form, compositions and medicaments
containing them, and processes for the preparation and use of such
inhibitors, compositions and medicaments.
BACKGROUND OF THE INVENTION
[0003] Kinases represent a large family of enzymes that catalyse
the phosphorylation of proteins, lipids and metabolites and play a
central role in the regulation of a wide variety of cellular
processes. Abnormal kinase activity has been related to a wide
range of disorders, including cancers. This has led to the
development of kinase inhibitors as therapeutic agents.
[0004] This invention generally relates to compounds having
activity as kinase inhibitors, including their prodrug forms, as
well as to the application of such compounds in therapy.
SUMMARY OF THE INVENTION
[0005] In a first aspect, the invention provides a kinase inhibitor
of Formula I:
##STR00002##
wherein either: [0006] (1) R.sub.1 is H, and [0007] (a) R.sub.2 is
(3-chlorobenzyl)oxy- and R.sub.3 is chloro; [0008] (b) R.sub.2 and
R.sub.3, together with the carbon atoms to which they are attached,
form 1-(3-fluorobenzyl)-1H-pyrazole; [0009] (c) R.sub.2 is
2-pyridinylmethoxy and R.sub.3 is chloro; [0010] (d) R.sub.2 and
R.sub.3 are both chloro; [0011] (e) R.sub.2 is chloro and R.sub.3
is bromo; [0012] (f) R.sub.2 and R.sub.3 are both bromo; [0013] (g)
R.sub.2 is fluoro and R.sub.3 is ethynyl; [0014] (h) R.sub.2 is
chloro and R.sub.3 is ethynyl; [0015] (i) R.sub.2 is bromo and
R.sub.3 is ethynyl; [0016] (j) other than when R.sub.2 is in the
3-position in combination with R.sub.3 in the 4-position, R.sub.2
is bromo and R.sub.3 is fluoro; [0017] (k) R.sub.2 is
2-pyridinylmethoxy and R.sub.3 is fluoro; or [0018] (l) R.sub.2 is
2-pyridinylmethoxy and R.sub.3 is bromo; or [0019] (2) at least one
of R.sub.1, R.sub.2 and R.sub.3 is selected from benzyloxy,
3-chlorobenzyloxy and 2-pyridinylmethoxy and when at least one of
R.sub.1, R.sub.2 and R.sub.3 is not benzyloxy, 3-chlorobenzyloxy or
2-pyridinylmethoxy, each of the others is independently selected
from H, halogen, and C.sub.2-C.sub.4 alkynyl, with the proviso that
when one of R.sub.1, R.sub.2 and R.sub.3 is benzyloxy or
2-pyridinylmethoxy, the other two of R.sub.1, R.sub.2 and R.sub.3
are not H; or [0020] (3) two of R.sub.1, R.sub.2 and R.sub.3,
together with the carbon atoms to which they are attached, form
1-(3-fluorobenzyl)-1H-pyrazole, and the other is selected from H,
halogen and C.sub.2-C.sub.4 alkynyl; [0021] or a pharmaceutically
acceptable salt or solvate thereof.
[0022] In certain embodiments, the invention provides kinase
inhibitors of the Formula IA
##STR00003##
[0023] wherein R.sub.1 is H, and either [0024] (a) R.sub.2 is
(3-chlorobenzyl)oxy- and R.sub.3 is chloro; [0025] (b) R.sub.2 and
R.sub.3, together with the carbon atoms to which they are attached,
form 1-(3-fluorobenzyl)-1H-pyrazole; [0026] (c) R.sub.2 is
2-pyridinylmethoxy and R.sub.3 is chloro; [0027] (d) R.sub.2 and
R.sub.3 are both chloro; [0028] (e) R.sub.2 is chloro and R.sub.3
is bromo; [0029] (f) R.sub.2 is bromo and R.sub.3 is chloro [0030]
(g) R.sub.2 and R.sub.3 are both bromo; [0031] (h) R.sub.2 is
fluoro and R.sub.3 is ethynyl; [0032] (i) R.sub.2 is chloro and
R.sub.3 is ethynyl; [0033] (j) R.sub.2 is bromo and R.sub.3 is
ethynyl; [0034] (k) R.sub.2 is bromo and R.sub.3 is fluoro; [0035]
(l) R.sub.2 is 2-pyridinylmethoxy and R.sub.3 is fluoro; or [0036]
(m) R.sub.2 is 2-pyridinylmethoxy and R.sub.3 is bromo;
[0037] and pharmaceutically acceptable salts and solvates
thereof.
[0038] Preferably, the kinase inhibitor is selected from the
following: [0039]
(2E)-N-(4-{3-chloro-4-[(3-chlorobenzyl)oxy]anilino}pyrido[3,4-d]py-
rimidin-6-yl)-4-(dimethylamino)-2-butenamide (1), [0040]
(2E)-4-(dimethylamino)-N-(4-{[1-(3-fluorobenzyl)-1H-indazol-5-yl]amino}py-
rido[3,4-d]pyrimidin-6-yl)-2-butenamide (2), [0041]
(2E)-N-{4-[3-chloro-4-(2-pyridinylmethoxy)anilino]pyrido[3,4-]pyrimidin-6-
-yl}-4-(dimethylamino)-2-butenamide (3), [0042]
(2E)-N-[4-(3,4-dichloroanilino)pyrido[3,4-d]pyrimidin-6-yl]-4-(dimethylam-
ino)-2-butenamide (4), [0043]
(2E)-N-[4-(3-bromo-4-chloroanilino)pyrido[3,4-d]pyrimidin-6-yl]-4-(dimeth-
ylamino)-2-butenamide (5), [0044]
(2E)-N-[4-(4-bromo-3-chloroanilino)pyrido[3,4-d]pyrimidin-6-yl]-4-(dimeth-
ylamino)-2-butenamide (6), [0045]
(2E)-N-[4-(3,4-dibromoanilino)pyrido[3,4-d]pyrimidin-6-yl]-4-(dimethylami-
no)-2-butenamide (7), [0046]
(2E)-4-(dimethylamino)-N-[4-(3-ethynyl-4-fluoroanilino)pyrido[3,4-d]pyrim-
idin-6-yl]-2-butenamide (8), [0047]
(2E)-N-[4-(4-chloro-3-ethynylanilino)pyrido[3,4-d]pyrimidin-6-yl]-4-(dime-
thylamino)-2-butenamide (9), [0048]
(2E)-N-[4-(4-bromo-3-ethynylanilino)pyrido[3,4-d]pyrimidin-6-yl]-4-(dimet-
hylamino)-2-butenamide (10), [0049]
(2E)-N-[4-(4-bromo-3-fluoroanilino)pyrido[3,4-d]pyrimidin-6-yl]-4-(dimeth-
ylamino)-2-butenamide (11), [0050]
(2E)-4-(dimethylamino)-N-{4-[3-fluoro-4-(2-pyridinylmethoxy)anilino]pyrid-
o[3,4-d]pyrimidin-6-yl}-2-butenamide (89) and [0051]
(2E)-N-{4-[3-bromo-4-(2-pyridinylmethoxy)anilino]pyrido[3,4-d]pyrimidin-6-
-yl}-4-(dimethylamino)-2-butenamide (90); and pharmaceutically
acceptable salts and hydrates thereof.
[0052] The structures of the compounds in the list above are
below:
##STR00004## ##STR00005## ##STR00006##
[0053] In another aspect, the invention provides the use of a
kinase inhibitor of Formula I as defined above, or a salt or
solvate thereof, in the preparation of a medicament.
[0054] In one preferred embodiment, the medicament is a prodrug and
the compound of Formula I or the salt or solvate thereof is linked
to a reductive trigger.
[0055] In yet a further aspect, the invention provides a reductive
prodrug comprising a kinase inhibitor of Formula I as defined
above, or a salt or solvate thereof, and a reductive trigger linked
directly or indirectly to a nitrogen of the kinase inhibitor.
[0056] In certain embodiments, the reductive trigger is of Formula
II:
##STR00007##
wherein * is a point of attachment to a nitrogen of said kinase
inhibitor, and where in Formula II R.sub.5 is selected from
C.sub.1-C.sub.6 alkyl and R.sub.4 is selected from H, methyl,
ethyl, trifluoromethyl, --CN, --CONH.sub.2 and propyn-1-yl.
[0057] In certain embodiments, the reductive trigger is selected
from the group consisting of Formulae IIa to IIg:
##STR00008##
[0058] In certain embodiments, R.sub.5 is selected from methyl,
ethyl and propyl, preferably methyl.
[0059] In certain preferred embodiments, the reductive trigger is
of Formula IIa, wherein R.sub.5 is selected from methyl, ethyl and
propyl.
[0060] Most preferably, the reductive trigger is of Formula IIa,
wherein R.sub.5 is selected from methyl.
[0061] In one embodiment, the prodrug is a compound of Formula
III:
##STR00009##
wherein X is any negatively charged counterion, R.sub.1, R.sub.2
and R.sub.3 are as defined for Formula I, R.sub.4 is selected from
H, methyl, ethyl, trifluoromethyl, --CN, --CONH.sub.2 and
propyn-1-yl, and R.sub.5 is C.sub.1-C.sub.6 alkyl.
[0062] In certain embodiments, the prodrug is a compound of Formula
IIIA
##STR00010##
wherein X is any negatively charged counterion, R.sub.1, R.sub.2
and R.sub.3 are as defined for Formula I and R.sub.4 is selected
from H, methyl, ethyl, trifluoromethyl, --CN, --CONH.sub.2 and
propyn-1-yl.
[0063] In yet another embodiment, the prodrug is a compound of
Formula IIIB
##STR00011##
wherein X is any negatively charged counterion, R.sub.1, R.sub.2
and R.sub.3 are as defined for Formula IA and R.sub.4 is selected
from H, methyl, ethyl, trifluoromethyl, --CN, --CONH.sub.2 and
propyn-1-yl.
[0064] In preferred embodiments, X is selected from halide
(fluoride, chloride, bromide, iodide), methanesulfonate,
trifluoromethanesulfonate, acetate, trifluoroacetate, tosylate,
lactate, citrate and formate.
[0065] More preferably, X is halide, with bromide being most
preferred.
[0066] Preferably, the compound of Formula III is selected from:
[0067]
(2E)-4-[(4-{3-chloro-4-[(3-chlorobenzyl)oxy]anilino}pyrido[3,4-d]pyrimidi-
n-6-yl)amino]-N,N-dimethyl-N-[(1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-4-
-oxo-2-buten-1-ammonium bromide (12), [0068]
(2E)-4-[(4-{[1-(3-fluorobenzyl)-1H-indazol-5-yl]amino)}pyrido[3,4-d]pyrim-
idin-6-yl)amino]-N,N-dimethyl-N-[(1-methyl-4-nitro-1H-imidazol-5-yl)methyl-
]-4-oxo-2-buten-1-ammonium bromide (13), [0069]
(2E)-N-[(1,2-dimethyl-4-nitro-1H-imidazol-5-yl)methyl]-4-[(4-{[1-(3-fluor-
obenzyl)-1H-indazol-5-yl]amino)}pyrido[3,4-d]pyrimidin-6-yl)amino]-N,N-dim-
ethyl-4-oxo-2-buten-1-ammonium bromide (14), [0070]
(2E)-4-(({4-[3-chloro-4-(2-pyridinylmethoxy)anilino]pyrido[3,4-d]pyrimidi-
n-6-yl)}amino)-N,N-dimethyl-N-[(1-methyl-4-nitro-1H-imidazol-5-yl)methyl]--
4-oxo-2-buten-1-ammonium bromide (15), [0071]
(2E)-4-{[4-(3,4-dichloroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N,N-di-
methyl-N-[(1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-4-oxo-2-buten-1-ammon-
ium bromide (16), [0072]
(2E)-4-{[4-(3-bromo-4-chloroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N,-
N-dimethyl-N-[(1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-4-oxo-2-buten-1-a-
mmonium bromide (17), [0073]
(2E)-4-{[4-(4-bromo-3-chloroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N,-
N-dimethyl-N-[(1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-4-oxo-2-buten-1-a-
mmonium bromide (18), [0074]
(2E)-4-{[4-(3,4-dibromoanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N,N-dim-
ethyl-N-[(1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-4-oxo-2-buten-1-ammoni-
um bromide (19), [0075]
(2E)-4-{[4-(3-ethynyl-4-fluoroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino)}-
-N,N-dimethyl-N-[(1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-4-oxo-2-buten--
1-ammonium bromide (20), [0076]
(2E)-4-{[4-(4-chloro-3-ethynylanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}--
N,N-dimethyl-N-[(1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-4-oxo-2-buten-1-
-ammonium bromide (21), [0077]
(2E)-4-[(4-{3-chloro-4-[(3-chlorobenzyl)oxy]anilino}pyrido[3,4-d]pyrimidi-
n-6-yl)amino]-N-[(1,2-dimethyl-4-nitro-1H-imidazol-5-yl)methyl]-N,N-dimeth-
yl-4-oxo-2-buten-1-ammonium bromide (22), [0078]
(2E)-4-[(4-{3-chloro-4-[(3-chlorobenzyl)oxy]anilino}pyrido[3,4-d]pyrimidi-
n-6-yl)amino]-N-[(2-ethyl-1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-N,N-di-
miethyl-4-oxo-2-buten-1-ammonium bromide (23), [0079]
(2E)-4-[(4-{3-chloro-4-[(3-chlorobenzyl)oxy]anilino}pyrido[3,4-d]pyrimidi-
n-6-yl)amino]-N,N-dimiethyl-N-{[1-methyl-4-nitro-2-(trifluoromethyl)-1H-im-
idazol-5-yl]methyl}-4-oxo-2-buten-1-ammonium bromide (24), [0080]
(2E)-4-[(4-{3-chloro-4-[(3-chlorobenzyl)oxy]anilino}pyrido[3,4-d]pyrimidi-
n-6-yl)amino]-N-[(2-cyano-1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-N,N-di-
methyl-4-oxo-2-buten-1-ammonium bromide (25), [0081]
(2E)-N-{[2-(aminocarbonyl)-1-methyl-4-nitro-1H-imidazol-5-yl]methyl}-4-[(-
4-{3-chloro-4-[(3-chlorobenzyl)oxy]anilino}pyrido[3,4-d]pyrimidin-6-yl)ami-
no]-N,N-dimethyl-4-oxo-2-buten-1-ammonium bromide (26), [0082]
(2E)-4-[(4-{3-chloro-4-[(3-chlorobenzyl)oxy]anilino}pyrido[3,4-d]pyrimidi-
n-6-yl)amino]-N,N-dimiethyl-N-{[1-methyl-4-nitro-2-(1-propynyl)-1H-imidazo-
l-5-yl]methyl}-4-oxo-2-buten-1-ammonium bromide (27), [0083]
(2E)-N-[(2-ethyl-1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-4-[(4-{[1-(3-f-
luorobenzyl)-1H-indazol-5-yl]amino}pyrido[3,4-d]pyrimidin-6-yl)amino]-N,N--
dimethyl-4-oxo-2-buten-1-ammonium bromide (28), [0084]
(2E)-4-[(4-{[1-(3-fluorobenzyl)-1H-indazol-5-yl]amino}pyrido[3,4-d]pyrimi-
din-6-yl)amino]-N,N-dimiethyl-N-{[1-methyl-4-nitro-2-(trifluoromethyl)-1H--
imidazol-5-yl]methyl}-4-oxo-2-buten-1-ammonium bromide (29), [0085]
(2E)-N-[(2-cyano-1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-4-[(4-{[1-(3-f-
luorobenzyl)-1H-indazol-5-yl]amino}pyrido[3,4-d]pyrimidin-6-yl)amino]-N,N--
dimethyl-4-oxo-2-buten-1-ammonium bromide (30), [0086]
(2E)-N-{[2-(aminocarbonyl)-1-methyl-4-nitro-1H-imidazol-5-yl]methyl}-4-[(-
4-{[1-(3-fluorobenzyl)-1H-indazol-5-yl]amino}pyrido[3,4-d]pyrimidin-6-yl)a-
mino]-N,N-dimiethyl-4-oxo-2-buten-1-ammonium bromide (31), [0087]
(2E)-4-[(4-{[1-(3-fluorobenzyl)-1H-indazol-5-yl]amino}pyrido[3,4-d]pyrimi-
din-6-yl)amino]-N,N-dimiethyl-N-{[1-methyl-4-nitro-2-(1-propynyl)-1H-imida-
zol-5-yl]methyl}-4-oxo-2-buten-1-ammonium bromide (32), [0088]
(2E)-4-({4-[3-chloro-4-(2-pyridinylmethoxy)anilino]pyrido[3,4-d]pyrimidin-
-6-yl}amino)-N-[(1,2-dimethyl-4-nitro-1H-imidazol-5-yl)methyl]-N,N-dimethy-
l-4-oxo-2-buten-1-ammonium bromide (33), [0089]
(2E)-4-({4-[3-chloro-4-(2-pyridinylmethoxy)anilino]pyrido[3,4-d]pyrimidin-
-6-yl}amino)-N-[(2-ethyl-1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-N,N-dim-
iethyl-4-oxo-2-buten-1-ammonium bromide (34), [0090]
(2E)-4-({4-[3-chloro-4-(2-pyridinylmethoxy)anilino]pyrido[3,4-d]pyrimidin-
-6-yl}amino)-N,N-dimiethyl-N-{[1-methyl-4-nitro-2-(trifluoromethyl)-1H-imi-
dazol-5-yl]methyl}-4-oxo-2-buten-1-ammonium bromide (35), [0091]
(2E)-4-({4-[3-chloro-4-(2-pyridinylmethoxy)anilino]pyrido[3,4-d]pyrimidin-
-6-yl}amino)-N-[(2-cyano-1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-N,N-dim-
iethyl-4-oxo-2-buten-1-ammonium bromide (36), [0092]
(2E)-N-{[2-(aminocarbonyl)-1-methyl-4-nitro-1H-imidazol-5-yl]methyl}-4-({-
4-[3-chloro-4-(2-pyridinylmethoxy)anilino]pyrido[3,4-d]pyrimidin-6-yl}amin-
o)-N,N-dimethyl-4-oxo-2-buten-1-ammonium bromide (37), [0093]
(2E)-4-({4-[3-chloro-4-(2-pyridinylmethoxy)anilino]pyrido[3,4-d]pyrimidin-
-6-yl}amino)-N,N-dimiethyl-N-{[1-methyl-4-nitro-2-(1-propynyl)-1H-imidazol-
-5-yl]methyl}-4-oxo-2-buten-1-ammonium bromide (38), [0094]
(2E)-4-{[4-(3,4-dichloroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N-[(1,-
2-dimethyl-4-nitro-1H-imidazol-5-yl)methyl]-N,N-dimiethyl-4-oxo-2-buten-1--
ammonium bromide (39), [0095]
(2E)-4-{[4-(3,4-dichloroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N-[(2--
ethyl-1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-N,N-dimethyl-4-oxo-2-buten-
-1-ammonium bromide (40), [0096]
(2E)-4-{[4-(3,4-dichloroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N,N-di-
methyl-N-{[1-methyl-4-nitro-2-(trifluoromethyl)-1H-imidazol-5-yl]methyl}-4-
-oxo-2-buten-1-ammonium bromide (41), [0097]
(2E)-N-[(2-cyano-1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-4-{[4-(3,4-dic-
hloroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N,N-dimethyl-4-oxo-2-buten-
-1-ammonium bromide (42), [0098]
(2E)-N-{[2-(aminocarbonyl)-1-methyl-4-nitro-1H-imidazol-5-yl]methyl}-4-{[-
4-(3,4-dichloroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N,N-dimiethyl-4--
oxo-2-buten-1-ammonium bromide (43), [0099]
(2E)-4-{[4-(3,4-dichloroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N,N-di-
methyl-N-{[1-methyl-4-nitro-2-(1-propynyl)-1H-imidazol-5-yl]methyl}-4-oxo--
2-buten-1-ammonium bromide (44), [0100]
(2E)-4-{[4-(3-bromo-4-chloroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N--
[(1,2-dimethyl-4-nitro-1H-imidazol-5-yl)methyl]-N,N-dimethyl-4-oxo-2-buten-
-1-ammonium bromide (45), [0101]
(2E)-4-{[4-(3-bromo-4-chloroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N--
[(2-ethyl-1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-N,N-dimethyl-4-oxo-2-b-
uten-1-ammonium bromide (46), [0102]
(2E)-4-{[4-(3-bromo-4-chloroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N,-
N-dimethyl-N-{[1-methyl-4-nitro-2-(trifluoromethyl)-1H-imidazol-5-yl]methy-
l}-4-oxo-2-buten-1-ammonium bromide (47), [0103]
(2E)-4-{[4-(3-bromo-4-chloroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N--
[(2-cyano-1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-N,N-dimethyl-4-oxo-2-b-
uten-1-ammonium bromide (48), [0104]
(2E)-N-{[2-(aminocarbonyl)-1-methyl-4-nitro-1H-imidazol-5-yl]methyl}-4-{[-
4-(3-bromo-4-chloroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N,N-dimethyl-
-4-oxo-2-buten-1-ammonium bromide (49), [0105]
(2E)-4-{[4-(3-bromo-4-chloroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N,-
N-dimiethyl-N-{[1-methyl-4-nitro-2-(1-propynyl)-1H-imidazol-5-yl]methyl}-4-
-oxo-2-buten-1-ammonium bromide (50), [0106]
(2E)-4-{[4-(4-bromo-3-chloroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N--
[(1,2-dimethyl-4-nitro-1H-imidazol-5-yl)methyl]-N,N-dimethyl-4-oxo-2-buten-
-1-ammonium bromide (51), [0107]
(2E)-4-{[4-(4-bromo-3-chloroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N--
[(2-ethyl-1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-N,N-dimiethyl-4-oxo-2--
buten-1-ammonium bromide (52), [0108]
(2E)-4-{[4-(4-bromo-3-chloroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N,-
N-dimethyl-N-{[1-methyl-4-nitro-2-(trifluoromethyl)-1H-imidazol-5-yl]methy-
l}-4-oxo-2-buten-1-ammonium bromide (53), [0109]
(2E)-4-{[4-(4-bromo-3-chloroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N--
[(2-cyano-1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-N,N-dimiethyl-4-oxo-2--
buten-1-ammonium bromide (54), [0110]
(2E)-N-{[2-(aminocarbonyl)-1-methyl-4-nitro-1H-imidazol-5-yl]methyl}-4-{[-
4-(4-bromo-3-chloroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N,N-dimethyl-
-4-oxo-2-buten-1-ammonium bromide (55), [0111]
(2E)-4-{[4-(4-bromo-3-chloroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N,-
N-dimiethyl-N-{[1-methyl-4-nitro-2-(1-propynyl)-1H-imidazol-5-yl]methyl}-4-
-oxo-2-buten-1-ammonium bromide (56), [0112]
(2E)-4-{[4-(3,4-dibromoanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N-[(1,2-
-dimethyl-4-nitro-1H-imidazol-5-yl)methyl]-N,N-dimethyl-4-oxo-2-buten-1-am-
monium bromide (57), [0113]
(2E)-4-{[4-(3,4-dibromoanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N-[(2-e-
thyl-1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-N,N-dimethyl-4-oxo-2-buten--
1-ammonium bromide (58), [0114]
(2E)-4-{[4-(3,4-dibromoanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N,N-dim-
iethyl-N-{[1-methyl-4-nitro-2-(trifluoromethyl)-1H-imidazol-5-yl]methyl}-4-
-oxo-2-buten-1-ammonium bromide (59), [0115]
(2E)-N-[(2-cyano-1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-4-{[4-(3,4-dib-
romoanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N,N-dimethyl-4-oxo-2-buten--
1-ammonium bromide (60), [0116]
(2E)-N-{[2-(aminocarbonyl)-1-methyl-4-nitro-1H-imidazol-5-yl]methyl}-4-{[-
4-(3,4-dibromoanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N,N-dimethyl-4-ox-
o-2-buten-1-ammonium bromide (61), [0117]
(2E)-4-{[4-(3,4-dibromoanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N,N-dim-
ethyl-N-{[1-methyl-4-nitro-2-(1-propynyl)-1H-imidazol-5-yl]methyl}-4-oxo-2-
-buten-1-ammonium bromide (62), [0118]
(2E)-N-[(1,2-dimethyl-4-nitro-1H-imidazol-5-yl)methyl]-4-{[4-(3-ethynyl-4-
-fluoroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N,N-dimethyl-4-oxo-2-but-
en-1-ammonium bromide (63), [0119]
(2E)-N-[(2-ethyl-1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-4-{[4-(3-ethyn-
yl-4-fluoroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N,N-dimiethyl-4-oxo--
2-buten-1-ammonium bromide (64), [0120]
(2E)-4-{[4-(3-ethynyl-4-fluoroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}--
N,N-dimiethyl-N-{[1-methyl-4-nitro-2-(trifluoromethyl)-1H-imidazol-5-yl]me-
thyl}-4-oxo-2-buten-1-ammonium bromide (65), [0121]
(2E)-N-[(2-cyano-1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-4-{[4-(3-ethyn-
yl-4-fluoroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N,N-dimiethyl-4-oxo--
2-buten-1-ammonium bromide (66), [0122]
(2E)-N-{[2-(aminocarbonyl)-1-methyl-4-nitro-1H-imidazol-5-yl]methyl}-4-{[-
4-(3-ethynyl-4-fluoroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N,N-dimiet-
hyl-4-oxo-2-buten-1-ammonium bromide (67), [0123]
(2E)-4-{[4-(3-ethynyl-4-fluoroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}--
N,N-dimiethyl-N-{[1-methyl-4-nitro-2-(1-propynyl)-1H-imidazol-5-yl]methyl}-
-4-oxo-2-buten-1-ammonium bromide (68), [0124]
(2E)-4-{[4-(4-chloro-3-ethynylanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}--
N-[(1,2-dimethyl-4-nitro-1H-imidazol-5-yl)methyl]-N,N-dimethyl-4-oxo-2-but-
en-1-ammonium bromide (69), [0125]
(2E)-4-{[4-(4-chloro-3-ethynylanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}--
N-[(2-ethyl-1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-N,N-dimiethyl-4-oxo--
2-buten-1-ammonium bromide (70), [0126]
(2E)-4-{[4-(4-chloro-3-ethynylanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}--
N,N-dimiethyl-N-{[1-methyl-4-nitro-2-(trifluoromethyl)-1H-imidazol-5-yl]me-
thyl}-4-oxo-2-buten-1-ammonium bromide (71), [0127]
(2E)-4-{[4-(4-chloro-3-ethynylanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}--
N-[(2-cyano-1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-N,N-dimiethyl-4-oxo--
2-buten-1-ammonium bromide (72), [0128]
(2E)-N-{[2-(aminocarbonyl)-1-methyl-4-nitro-1H-imidazol-5-yl]methyl}-4-{[-
4-(4-chloro-3-ethynylanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N,N-dimiet-
hyl-4-oxo-2-buten-1-ammonium bromide (73), [0129]
(2E)-4-{[4-(4-chloro-3-ethynylanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}--
N,N-dimiethyl-N-{[1-methyl-4-nitro-2-(1-propynyl)-1H-imidazol-5-yl]methyl}-
-4-oxo-2-buten-1-ammonium bromide (74), [0130]
(2E)-4-{[4-(4-bromo-3-ethynylanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N-
,N-dimiethyl-N-[(1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-4-oxo-2-buten-1-
-ammonium bromide (75), [0131]
(2E)-4-{[4-(4-bromo-3-ethynylanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N-
-[(1,2-dimethyl-4-nitro-1H-imidazol-5-yl)methyl]-N,N-dimethyl-4-oxo-2-bute-
n-1-ammonium bromide (76), [0132]
(2E)-4-{[4-(4-bromo-3-ethynylanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N-
-[(2-ethyl-1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-N,N-dimethyl-4-oxo-2--
buten-1-ammonium bromide (77), [0133]
(2E)-4-{[4-(4-bromo-3-ethynylanilino)pyrido[3,4-d]pyrimidin-6-yl]amino)}--
N,N-dimethyl-N-{[1-methyl-4-nitro-2-(trifluoromethyl)-1H-imidazol-5-yl]met-
hyl}-4-oxo-2-buten-1-ammonium bromide (78), [0134]
(2E)-4-{[4-(4-bromo-3-ethynylanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N-
-[(2-cyano-1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-N,N-dimethyl-4-oxo-2--
buten-1-ammonium bromide (79), [0135]
(2E)-N-{[2-(aminocarbonyl)-1-methyl-4-nitro-1H-imidazol-5-yl]methyl}-4-{[-
4-(4-bromo-3-ethynylanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N,N-dimethy-
l-4-oxo-2-buten-1-ammonium bromide (80), [0136]
(2E)-4-{[4-(4-bromo-3-ethynylanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N-
,N-dimethyl-N-{[1-methyl-4-nitro-2-(1-propynyl)-1H-imidazol-5-yl]methyl)}--
4-oxo-2-buten-1-ammonium bromide (81), [0137]
(2E)-4-{[4-(4-bromo-3-fluoroanilino)pyrido[3,4-]pyrimidin-6-yl]amino)}-N,-
N-dimethyl-N-[(1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-4-oxo-2-buten-1-a-
mmonium bromide (82), [0138]
(2E)-4-{[4-(4-bromo-3-fluoroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N--
[(1,2-dimethyl-4-nitro-1H-imidazol-5-yl)methyl]-N,N-dimethyl-4-oxo-2-buten-
-1-ammonium bromide (83), [0139]
(2E)-4-{[4-(4-bromo-3-fluoroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N--
[(2-ethyl-1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-N,N-dimethyl-4-oxo-2-b-
uten-1-ammonium bromide (84), [0140]
(2E)-4-{[4-(4-bromo-3-fluoroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N,-
N-dimethyl-N-{[1-methyl-4-nitro-2-(trifluoromethyl)-1H-imidazol-5-yl]methy-
l}-4-oxo-2-buten-1-ammonium bromide (85), [0141]
(2E)-4-{[4-(4-bromo-3-fluoroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N--
[(2-cyano-1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-N,N-dimethyl-4-oxo-2-b-
uten-1-ammonium bromide (86), [0142]
(2E)-N-{[2-(aminocarbonyl)-1-methyl-4-nitro-1H-imidazol-5-yl]methyl}-4-{[-
4-(4-bromo-3-fluoroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N,N-dimethyl-
-4-oxo-2-buten-1-ammonium bromide (87), [0143]
(2E)-4-{[4-(4-bromo-3-fluoroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N,-
N-dimethyl-N-{[1-methyl-4-nitro-2-(I-propynyl)-1H-imidazol-5-yl]methyl}-4--
oxo-2-buten-1-ammonium bromide (88), [0144]
(2E)-4-({4-[3-fluoro-4-(2-pyridinylmethoxy)anilino]pyrido[3,4-d]pyrimidin-
-6-yl}amino)-N,N-dimethyl-N-[(1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-4--
oxo-2-buten-1-ammonium bromide (91), [0145]
(2E)-N-[(1,2-dimethyl-4-nitro-1H-imidazol-5-yl)methyl]-4-({4-[3-fluoro-4--
(2-pyridinylmethoxy)anilino]pyrido[3,4-d]pyrimidin-6-yl}amino)-N,N-dimethy-
l-4-oxo-2-buten-1-ammonium bromide (92),
[0146]
(2E)-N-[(2-ethyl-1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-4-({4-[-
3-fluoro-4-(2-pyridinylmethoxy)anilino]pyrido[3,4-d]pyrimidin-6-yl}amino)--
N,N-dimethyl-4-oxo-2-buten-1-ammonium bromide (93), [0147]
(2E)-4-({4-[3-fluoro-4-(2-pyridinylmethoxy)anilino]pyrido[3,4-d]pyrimidin-
-6-yl}amino)-N,N-dimiethyl-N-{[1-methyl-4-nitro-2-(trifluoromethyl)-1H-imi-
dazol-5-yl]methyl}-4-oxo-2-buten-1-ammonium bromide (94), [0148]
(2E)-N-[(2-cyano-1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-4-({4-[3-fluor-
o-4-(2-pyridinylmethoxy)anilino]pyrido[3,4-d]pyrimidin-6-yl}amino)-N,N-dim-
ethyl-4-oxo-2-buten-1-ammonium bromide (95), [0149]
(2E)-N-{[2-(aminocarbonyl)-1-methyl-4-nitro-1H-imidazol-5-yl]methyl}-4-({-
4-[3-fluoro-4-(2-pyridinylmethoxy)anilino]pyrido[3,4-d]pyrimidin-6-yl}amin-
o)-N,N-dimethyl-4-oxo-2-buten-1-ammonium bromide (96), [0150]
(2E)-4-({4-[3-fluoro-4-(2-pyridinylmethoxy)anilino]pyrido[3,4-d]pyrimidin-
-6-yl}amino)-N,N-dimiethyl-N-{[1-methyl-4-nitro-2-(1-propynyl)-1H-imidazol-
-5-yl]methyl}-4-oxo-2-buten-1-ammonium bromide (97), [0151]
(2E)-4-({4-[3-bromo-4-(2-pyridinylmethoxy)anilino]pyrido[3,4-d]pyrimidin--
6-yl}amino)-N,N-dimiethyl-N-[(1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-4--
oxo-2-buten-1-ammonium bromide (98), [0152]
(2E)-4-({4-[3-bromo-4-(2-pyridinylmethoxy)anilino]pyrido[3,4-d]pyrimidin--
6-yl}amino)-N-[(1,2-dimethyl-4-nitro-1H-imidazol-5-yl)methyl]-N,N-dimethyl-
-4-oxo-2-buten-1-ammonium bromide (99), [0153]
(2E)-4-({4-[3-bromo-4-(2-pyridinylmethoxy)anilino]pyrido[3,4-d]pyrimidin--
6-yl}amino)-N-[(2-ethyl-1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-N,N-dimi-
ethyl-4-oxo-2-buten-1-ammonium bromide (100), [0154]
(2E)-4-({4-[3-bromo-4-(2-pyridinylmethoxy)anilino]pyrido[3,4-d]pyrimidin--
6-yl}amino)-N,N-dimethyl-N-{[1-methyl-4-nitro-2-(trifluoromethyl)-1H-imida-
zol-5-yl]methyl}-4-oxo-2-buten-1-ammonium bromide (101), [0155]
(2E)-4-({4-[3-bromo-4-(2-pyridinylmethoxy)anilino]pyrido[3,4-d]pyrimidin--
6-yl}amino)-N-[(2-cyano-1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-N,N-dime-
thyl-4-oxo-2-buten-1-ammonium bromide (102), [0156]
(2E)-N-{[2-(aminocarbonyl)-1-methyl-4-nitro-1H-imidazol-5-yl]methyl}-4-({-
4-[3-bromo-4-(2-pyridinylmethoxy)anilino]pyrido[3,4-d]pyrimidin-6-yl}amino-
)-N,N-dimethyl-4-oxo-2-buten-1-ammonium bromide (103) and [0157]
(2E)-4-({4-[3-bromo-4-(2-pyridinylmethoxy)anilino]pyrido[3,4-d]pyrimidin--
6-yl}amino)-N,N-dimiethyl-N-{[1-methyl-4-nitro-2-(1-propynyl)-1H-imidazol--
5-yl]methyl}-4-oxo-2-buten-1-ammonium bromide (104).
[0158] The structures of the prodrug compounds in the list above
are below:
##STR00012## ##STR00013## ##STR00014## ##STR00015## ##STR00016##
##STR00017## ##STR00018## ##STR00019## ##STR00020## ##STR00021##
##STR00022## ##STR00023##
[0159] In yet a further aspect, the invention provides a compound
of Formula I or salt or solvate thereof as defined above, a
reductive prodrug as defined above, or a compound of Formula III or
salt or solvate thereof as defined above, for use in therapy.
[0160] In still another aspect, the invention provides
pharmaceutical compositions comprising a compound of Formula I or
salt or solvate thereof as defined above, a reductive prodrug as
defined above, or a compound of Formula III or salt or solvate
thereof as defined above, in combination with one or more
pharmaceutically acceptable excipients or diluents.
[0161] In yet a further aspect, the invention provides a method of
therapy which includes the step of administering a therapeutically
effective amount of a compound of Formula I or salt or solvate
thereof as defined above, a reductive prodrug as defined above, or
a compound of Formula III or salt or solvate thereof as defined
above, to a patient in need of such therapy.
[0162] Preferably, the method of therapy is the treatment of
cancer.
[0163] In yet a further aspect, the invention provides a method of
inhibiting kinase activity within a subject which involves the step
of administering to said subject an inhibitory amount of a compound
of Formula I or salt or solvate thereof as defined above, a
reductive prodrug as defined above, or a compound of Formula III or
salt or solvate thereof as defined above.
[0164] Preferably, said inhibiting of kinase activity is for a
therapeutic purpose, including an anti-cancer purpose.
[0165] In another aspect, the present invention provides a method
for the production of an anti-cancer effect in a warm-blooded
animal such as a human, wherein the method comprises administering
to the animal an effective amount of a compound of Formula I or
salt or solvate thereof as defined above, a reductive prodrug as
defined above, or a compound of Formula III or salt or solvate
thereof as defined above.
[0166] In a further aspect, the present invention provides a method
for the production of an anti-cancer effect in a cell, wherein the
method comprises contacting the cell with an effective amount of a
compound of Formula I or salt or solvate thereof as defined above,
a reductive prodrug as defined above, or a compound of Formula III
or salt or solvate thereof as defined above.
[0167] In a further aspect, the present invention provides a method
for the treatment of a cancer in a warm-blooded animal such as a
human, which comprises administering to the animal an effective
amount of a compound of Formula I or salt or solvate thereof as
defined above, a reductive prodrug as defined above, or a compound
of Formula III or salt or solvate thereof as defined above.
[0168] While the invention is broadly as defined above, it is not
limited thereto and also includes embodiments of which the
following description provides examples.
BRIEF DESCRIPTION OF THE DRAWINGS
[0169] The invention will now be described in more detail, with
reference to the accompanying Figures, in which:
[0170] FIG. 1 shows median H1975 tumour growth after q3dx4
treatment with kinase inhibitors 1, 2, 3 (n=3);
[0171] FIG. 2 shows median H1975 tumour growth after q3dx4
treatment with kinase inhibitors 4, 5 (n=3);
[0172] FIG. 3 shows median H1975 tumour growth after q3dx4
treatment with kinase inhibitors 7, 8 (n=3-4);
[0173] FIG. 4 shows median A431 tumour growth after q3dx4 treatment
with kinase inhibitors 5, 6, 9 (n=3);
[0174] FIG. 5 shows median H1975 tumour growth after q3dx4
treatment with prodrugs 12, 13, 15 (n=3);
[0175] FIG. 6 shows median H1975 tumour growth after q3dx4
treatment with prodrugs 16, 17 (n=3);
[0176] FIG. 7 shows median H1975 tumour growth after q3dx4
treatment with prodrugs 19, 20 (n=3-4);
[0177] FIG. 8 shows median A431 tumour growth after q3dx4 treatment
with prodrugs 17, 18, 21 (n=3);
[0178] FIG. 9 shows median H1975 tumour growth after q3dx4
treatment with prodrug 15 and its cognate kinase inhibitor 3
(n=3);
[0179] FIG. 10 shows median (A) and mean (B) H1975 tumour growth
after q3dx4 treatment with prodrug 17 and its cognate kinase
inhibitor 5 (n=3);
[0180] FIG. 11 shows median H1975 tumour growth after treatment
with prodrug 17 at q3dx4, q5dx4 and q7dx4 dosing schedules
(n=3);
[0181] FIG. 12 shows inhibition of erbB1 (EGFR) autophosphorylation
and p44/42 MAPK (Erk 1/2) phosphorylation in intact A431 cells
following 1 hour treatment with a range of concentrations of
compound 3;
[0182] FIG. 13 shows inhibition of erbB1 (EGFR) autophosphorylation
and p44/42 MAPK (Erk 1/2) phosphorylation in intact A431 cells
following 1 hour treatment with a range of concentrations of
compound 5;
[0183] FIG. 14 shows inhibition of erbB1 (EGFR) autophosphorylation
and p44/42 MAPK (Erk 1/2) phosphorylation in intact A431 cells
following 1 hour treatment with a range of concentrations of
compound 6; and
[0184] FIG. 15 shows inhibition of erbB1 (EGFR) autophosphorylation
and p44/42 MAPK (Erk 1/2) phosphorylation in intact A431 cells
following 1 hour treatment with a range of concentrations of
prodrug 15.
[0185] FIG. 16 is a graphical plot of band densitometry analysis of
relative (.beta.-actin corrected) inhibition of erbB1 (EGFR)
autophosphorylation in intact A431 cells following 1 hour treatment
with a range of concentrations of prodrug 15 and compound 3.
DESCRIPTION OF THE INVENTION
Definitions
[0186] As used herein, the terms "alkyl" and "alkynyl", unless
otherwise specified, include both straight chain and branched chain
groups, and unsubstituted and substituted groups. The optional
substituents may include, without limitation, halogen,
C.sub.1-C.sub.6 alkoxy, CN, OH, NH.sub.2, NO.sub.2,
NH(C.sub.1-C.sub.6 alkyl), N(C.sub.1-C.sub.6 alkyl).sub.2,
CONH.sub.2, CO(C.sub.1-C.sub.6 alkyl), SO.sub.2NH.sub.2 and
SO.sub.2(C.sub.1-C.sub.6 alkyl).
[0187] "Anti-cancer effects" include, but are not limited to,
anti-tumour effects, the response rate, the time to disease
progression and the survival rate. "Anti-tumour" effects include
but are not limited to inhibition of tumour growth, tumour growth
delay, regression of tumour, shrinkage of tumour, increased time to
regrowth of tumour on cessation of treatment and slowing of disease
progression.
[0188] "Effective amount" means an amount of a compound that, when
administered to a subject for treating a cancer, is sufficient to
effect such treatment for the cancer. The "effective amount" will
vary depending on the cancer to be treated, the compound to be
administered, the severity of the cancer treated, the age and
relative health of the subject, the route and form of
administration, whether the treatment is monotherapy or combination
therapy, the judgement of the attending clinician, and other
factors.
[0189] "Pharmaceutically acceptable" means that which is useful in
preparing a pharmaceutical composition that is generally safe,
non-toxic, and neither biologically nor otherwise undesirable and
includes that which is acceptable for veterinary as well as human
pharmaceutical use.
[0190] "Pharmaceutically acceptable salts" of a compound means
salts that are pharmaceutically acceptable, as defined herein, and
that possess the desired pharmacological activity of the parent
compound. Such salts include:
[0191] acid addition salts formed with inorganic acids such as
hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,
phosphoric acid and the like; or formed with organic acids such as
acetic acid, methanesulfonic acid, maleic acid, tartaric acid,
citric acid and the like; or
[0192] salts formed when an acidic proton present in the parent
compound either is replaced by a metal ion, e.g. an alkali metal
ion, an alkaline earth ion, or an aluminium ion; or coordinates
with an organic or inorganic base. Acceptable organic bases include
ethanolamine, diethanolamine, N-methylglucamine, triethanolamine
and the like. Acceptable inorganic bases include aluminium
hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate
and sodium hydroxide.
[0193] "Warm blooded animal" means any member of the mammalia class
including, but not limited to humans, non-human primates such as
chimpanzees and other apes and monkey species, farm animals such as
cattle, horses, sheep, goats, and swine; domestic animals such as
rabbits, dogs and cats; laboratory animals including rodents, such
as rats, mice and guinea pigs, and the like.
Compounds of the Invention
[0194] As defined above, in broad terms the invention relates to
compounds useful as kinase inhibitors. Such compounds have
application in therapy.
[0195] In one embodiment, the compounds of the invention are
4-anilinopyrido[3,4-d]pyrimidine irreversible erbB1, 2, 4 kinase
inhibitors of Formula I.
[0196] In another embodiment, the compounds comprise a kinase
inhibitor of Formula I and a reductive trigger. The reductive
trigger fragments when reduced. Preferably fragmentation of the
trigger occurs at the one-electron reduction level and is
effectively suppressed by the presence of oxygen, thus providing
selective activation in hypoxic environments. This suppression by
oxygen may occur through reoxidation of the one-electron radical by
oxygen, or by oxidation of reducing intermediates required for
prodrug reduction. The latter would include, for example,
scavenging by oxygen of radiation-induced reducing radicals such as
the aquated electron, or oxidation of reducing intermediates in the
catalytic cycle of reductase enzymes. The reduction equivalents
required to reduce the trigger may be provided by enzymes,
radiation-induced radicals, or chemical reducing agents.
[0197] In preferred forms, the trigger is a reductively-activated
aromatic nitroheterocycle or aromatic nitrocarbocycle trigger and
is linked directly to a quaternisable nitrogen of a compound of
Formula I such that a quaternary nitrogen is formed. It is however
particularly preferred that the trigger has the structure of
Formula II above.
[0198] In such embodiments, the compounds act as prodrugs, with
reduction of the trigger releasing the kinase inhibitor. It is
presently most preferred that the prodrug forms of the compounds of
the invention are of Formula III as defined above.
Therapeutic Applications of Compounds of the Invention
[0199] The compounds of the invention have application in any
therapeutic approach in which inhibition of the activity of a
kinase is desirable. The invention therefore relates to methods for
treating and preventing diseases, for example, hyper-proliferative,
inflammatory and angiogenesis disorders and osteoporosis in mammals
by administering a compound of this invention or a pharmaceutical
composition comprising one or more compounds of this invention.
[0200] The invention particularly relates to a method of treating
or preventing cancer and other hyperproliferative disorders by
administering a compound of this invention or a pharmaceutical
composition comprising one or more compounds of this invention,
whether alone as a monotherapy or in combination with a second
anti-proliferative agent.
[0201] Optional anti-proliferative agents which can also be
adminstered include but are not limited to compounds listed on the
cancer chemotherapy drug regimens in the 14.sup.th Edition of the
Merck Index (2006), which is hereby incorporated by reference, such
as asparaginase, bleomycin, carboplatin, carmustine, chlorambucil,
cisplatin, colaspase, cyclophosphamide, cytarabine, dacarbazine,
dactinomycin, daunorubicin, doxorubicin (adriamycine), epirubicin,
etoposide, 5-fluorouracil, hexamethylmelamine, hydroxyurea,
ifosfamide, irinotecan, leucovorin, lomustine, mechlorethamine,
6-mercaptopurine, mesna, methotrexate, mitomycin C, mitoxantrone,
prednisolone, prednisone, procarbazine, raloxifen, streptozocin,
tamoxifen, thioguanine, topotecan, vinblastine, vincristine, and
vindesine.
[0202] Additional anti-proliferative agents include other molecular
targeted agents which modulate parallel pathways such as MEK 1/2
inhibitors, AKT inhibitors and mTOR inhibitors, monoclonal
antibodies, oxaliplatin, gemcitabine, gefinitib, taxotere, ara A,
ara C, herceptin, BCNU, CCNU, DTIC, and actinomycin D. Still
further anti-proliferative agents include but are not limited to
those compounds acknowledged to be used in the treatment of
neoplastic diseases in Goodman and Gilman's The Pharmacological
Basis of Therapeutics (Eleventh Edition), editor Molinoff et al.,
publ. by McGraw-Hill, pages 1225-1287 (2006), which is hereby
incorporated by reference, such as aminoglutethimide,
L-asparaginase, azathioprine, 5-azacytidine cladribine, busulfan,
diethylstilbestrol, 2',2'-difluorodeoxycytidine, docetaxel,
erythrohydroxynonyladenine, ethinyl estradiol,
5-fluorodeoxyuridine, 5-fluorodeoxyuridine monophosphate,
fludarabine phosphate, fluoxymesterone, flutamide,
hydroxyprogesterone caproate, idarubicin, interferon,
medroxyprogesterone acetate, megestrol acetate, melphalan,
mitotane, paclitaxel, pentostatin, N-phosphonoacetyl-L-aspartate
(PALA), plicamycin, semustine, tenipdside, testosterone propionate,
thiotepa, trimethylmelamine, uridine, and vinorelbine.
[0203] Cancer and hyperproliferative disorders as used herein
include but are not limited to solid tumors, such as cancers of the
breast, respiratory tract, brain, reproductive organs, digestive
tract, urinary tract, eye, liver, skin, head and neck, thyroid,
parathyroid and their distant metastases. Those disorders also
include lymphomas, sarcomas, and leukemias.
[0204] Examples of breast cancer include, but are not limited to
invasive ductal carcinoma, invasive lobular carcinoma, ductal
carcinoma in situ, and lobular carcinoma in situ.
[0205] Examples of cancers of the respiratory tract include, but
are not limited to small-cell and non-small-cell lung carcinoma, as
well as bronchial adenoma and pleuropulmonary blastoma.
[0206] Examples of brain cancers include, but are not limited to
brain stem and hypophthalmic glioma, cerebellar and cerebral
astrocytoma, medulloblastoma, ependymoma, as well as
neuroectodermal and pineal tumor.
[0207] Tumors of the male reproductive organs include, but are not
limited to prostate and testicular cancer.
[0208] Tumors of the female reproductive organs include, but are
not limited to endometrial, cervical, ovarian, vaginal, and vulvar
cancer, as well as sarcoma of the uterus.
[0209] Tumors of the digestive tract include, but are not limited
to anal, colon, colorectal, esophageal, gallblader, gastric,
pancreatic, rectal, small-intestine, and salivary gland
cancers.
[0210] Tumors of the urinary tract include, but are not limited to
bladder, penile, kidney, renal pelvis, ureter, and urethral
cancers.
[0211] Eye cancers include, but are not limited to intraocular
melanoma and retinoblastoma.
[0212] Examples of liver cancers include, but are not limited to
hepatocellular carcinoma (liver cell carcinomas with or without
fibrolamellar variant), cholangiocarcinoma (intrahepatic bile duct
carcinoma), and mixed hepatocellular cholangiocarcinoma.
[0213] Skin cancers include, but are not limited to squamous cell
carcinoma, Kaposi's sarcoma, malignant melanoma, Merkel cell skin
cancer, and non-melanoma skin cancer.
[0214] Head-and-neck cancers include, but are not limited to
laryngeal/hypopharyngeal/nasopharyngeal/oropharyngeal cancer, and
lip and oral cavity cancer. Lymphomas include, but are not limited
to AIDS-related lymphoma, non-Hodgkin's lymphoma, cutaneous T-cell
lymphoma, Hodgkin's disease, and lymphoma of the central nervous
system.
[0215] Sarcomas include, but are not limited to sarcoma of the soft
tissue, osteosarcoma, malignant fibrous histiocytoma,
lymphosarcoma, and rhabdomyosarcoma. Leukemias include, but are not
limited to acute myeloid leukemia, acute lymphoblastic leukemia,
chronic lymphocytic leukemia, chronic myelogenous leukemia, and
hairy cell leukemia.
[0216] These disorders have been well characterized in man, but
also exist with a similar etiology in other warm-blooded animals,
and can be treated by pharmaceutical compositions of the present
invention.
[0217] Conditions within a human or other warm-blooded animal which
can be treated by administering a compound of this invention
include tumor growth, retinopathy, including diabetic retinopathy,
ischemic retinal-vein occlusion, retinopathy of prematurity and age
related macular degeneration; psoriasis, or bullous disorder
associated with subepidermal blister formation, including bullous
pemphigoid, erythema multiforme, or dermatitis herpetiformis,
rheumatoid arthritis, osteoarthritis, septic arthritis, tumor
metastasis, periodontal disease, cornal ulceration, proteinuria,
coronary thrombosis from atherosclerotic plaque, aneurismal aortic,
dystrophobic epidermolysis bullosa, degenerative cartilage loss
following traumatic joint injury, osteopenias mediated by MMP
activity, tempero mandibular joint disease or demyelating disease
of the nervous system.
[0218] It will be appreciated by those skilled in the art that a
particular method of therapy will employ a selected route of
administration which will in turn depend on a variety of factors,
all of which are considered routinely when administering
therapeutics. It will be further appreciated by one skilled in the
art that the optimal course of treatment, i.e., the mode of
treatment and the daily number of doses of a compound of this
invention given for a defined number of days, can be ascertained by
those skilled in the art using conventional treatment tests.
[0219] Therapeutic dosages will likely be in the range of 1 mg to
3000 mg per day. The specific dose level selected for any
particular patient will depend upon a variety of factors, including
the activity of the specific compound employed, the age, body
weight, general health, sex, diet, time of administration, route of
administration, and rate of excretion, drug combination and the
severity of the condition undergoing therapy.
Pharmaceutical Compositions of Compounds of the Invention
[0220] The invention also includes pharmaceutical compositions
including a compound of this invention, and a pharmaceutically
acceptable carrier.
[0221] The compounds may be administered orally, topically,
parenterally, by inhalation or spray or rectally in dosage unit
formulations. The term `administration by injection` includes
intravenous, intramuscular, subcutaneous and parenteral injections,
as well as use of infusion techniques. One or more compounds may be
present in association with one or more non-toxic pharmaceutically
acceptable carriers and if desired other active ingredients.
[0222] Compositions intended for oral use may be prepared according
to any suitable method known to the art for the manufacture of
pharmaceutical compositions. Such compositions may contain one or
more agents selected from the group consisting of diluents,
sweetening agents, flavoring agents, coloring agents and preserving
agents in order to provide palatable preparations. Tablets contain
the active ingredient in admixture with non-toxic pharmaceutically
acceptable excipients which are suitable for the manufacture of
tablets. These excipients may be, for example, inert diluents, such
as calcium carbonate, sodium carbonate, lactose, calcium phosphate
or sodium phosphate; granulating and disintegrating agents, for
example, corn starch, or alginic acid; and binding agents, for
example magnesium stearate, stearic acid or talc. The tablets may
be uncoated or they may be coated by known techniques to delay
disintegration and adsorption in the gastrointestinal tract and
thereby provide a sustained action over a longer period. For
example, a time delay material such as glyceryl monostearate or
glyceryl distearate may be employed. These compounds may also be
prepared in solid, rapidly released form.
[0223] Formulations for oral use may also be presented as hard
gelatin capsules wherein the active ingredient is mixed with an
inert solid diluent, for example, calcium carbonate, calcium
phosphate or kaolin, or as soft gelatin capsules wherein the active
ingredient is mixed with water or an oil medium, for example peanut
oil, liquid paraffin or olive oil.
[0224] Aqueous suspensions contain the active materials in
admixture with excipients suitable for the manufacture of aqueous
suspensions. Such excipients are suspending agents, for example
sodium carboxymethylcellulose, methylcellulose, hydroxypropyl
methylcellulose, sodium alginate, polyvinylpyrrolidone, gum
tragacanth and gum acacia; dispersing or wetting agents may be a
naturally occurring phosphatide, for example, lecithin, or
condensation products or an alkylene oxide with fatty acids, for
example polyoxyethylene stearate, or condensation products of
ethylene oxide with long chain aliphatic alcohols, for example
heptadecaethylene oxycetanol, or condensation products of ethylene
oxide with partial esters derived from fatty acids and hexitol such
as polyoxyethylene sorbitol monooleate, or condensation products of
ethylene oxide with partial esters derived from fatty acids and
hexitol anhydrides, for example polyethylene sorbitan monooleate.
The aqueous suspensions may also contain one or more preservatives,
for example ethyl, or n-propyl p-hydroxybenzoate, one or more
coloring agents, one or more flavoring agents, and one or more
sweetening agents, such as sucrose or saccharin.
[0225] Dispersible powders and granules suitable for preparation of
an aqueous suspension by the addition of water provide the active
ingredient in admixture with a dispersing or wetting agent,
suspending agent and one or more preservatives. Suitable dispersing
or wetting agents and suspending agents are exemplified by those
already mentioned above. Additional excipients, for example,
sweetening, flavoring and coloring agents, may also be present.
[0226] The compounds may also be in the form of non-aqueous liquid
formulations, e.g., oily suspensions which may be formulated by
suspending the active ingredients in a vegetable oil, for example
arachis oil, olive oil, sesame oil or peanut oil, or in a mineral
oil such as liquid paraffin. The oily suspensions may contain a
thickening agent, for example beeswax, hard paraffin or cetyl
alcohol. Sweetening agents such as those set forth above, and
flavoring agents may be added to provide palatable oral
preparations. These compositions may be preserved by the addition
of an anti-oxidant such as ascorbic acid.
[0227] Pharmaceutical compositions of the invention may also be in
the form of oil-in-water emulsions. The oily phase may be a
vegetable oil, for example olive oil or arachis oil, or a mineral
oil, for example liquid paraffin or mixtures of these. Suitable
emulsifying agents may be naturally-occurring gums, for example gum
acacia or gum tragacanth, naturally-occurring phosphatides, for
example soy bean, lecithin, and esters or partial esters derived
from fatty acids and hexitol anhydrides, for example sorbitan
monooleate, and condensation products of the said partial esters
with ethylene oxide, for example polyoxyethylene sorbitan
monooleate. The emulsions may also contain sweetening and flavoring
agents.
[0228] Syrups and elixirs may be formulated with sweetening agents,
for example glycerol, propylene glycol, sorbitol or sucrose. Such
formulations may also contain a demulcent, a preservative and
flavoring and coloring agents.
[0229] The compounds may also be administered in the form of
suppositories for rectal administration of the drug. These
compositions can be prepared by mixing the drug with a suitable
non-irritating excipient which is solid at ordinary temperatures
but liquid at the rectal temperature and will therefore melt in the
rectum to release the drug. Such materials include cocoa butter and
polyethylene glycols.
Preparation of Quaternary Nitrogen Salt Prodrugs of the
Invention
[0230] The prodrug compounds of the present invention comprise an
effector moiety linked to a reductive trigger, which is preferably
a nitroheterocyclic reductive trigger of Formula II, more
preferably of Formula IIa, as defined above.
[0231] The effector moiety is a 4-anilinopyrido[3,4-d]pyrimidine
irreversible erbB1, 2, 4 kinase inhibitor of Formula I.
[0232] The 4-anilinopyrido[3,4-d]pyrimidine irreversible erbB1, 2,
4 kinase inhibitors of Formula I possess an amide Michael acceptor
in the 6-position, where the Michael acceptor features a double
bond that is substituted at the beta carbon with a methylene group
that terminates with a tertiary dimethylamine group, as shown
below.
##STR00024##
[0233] It will be appreciated that the remainder of the effector
moiety not shown in Formula IVa has a bicyclic aromatic ring
structure as defined in Formula I.
[0234] The preferred prodrug compounds are of Formula III as
defined above. In general terms, the preferred prodrug compounds of
Formula III may be prepared by quaternising an aliphatic tertiary
dimethylamine effector moiety with a nitroheterocyclic reductive
trigger moiety. Such methods are described in more detail
below.
Preparation of 4-anilinopyrido[3,4-d]pyrimidine Irreversible erbB1,
2, 4 Kinase Inhibitors
[0235] Effector compounds related to those of the Formula I as
shown above, where the central inhibitor scaffold is a
4-anilinopyrido[3,4-d]pyrimidine and the 6-position is substituted
with an amide Michael acceptor may be prepared according to methods
described in the art when the amide Michael acceptor, for example,
is unsubstituted (Smaill et al. J Med Chem, 1999, 42, 1803-1815),
substituted at the N-position, alpha-position or beta-position with
a range of groups (Smaill et al. J Med Chem, 2001, 44, 429-440)
excluding dimethylaminomethylene as described above and when the
Michael acceptor contains a triple bond substituted at the
beta-position (Klutchko et al. J Med Chem, 2006, 49, 1475-1485;
U.S. Pat. No. 6,602,863). Compounds of Formula I of the present
invention may be prepared using analogous methods.
[0236] Scheme 1 below illustrates the preparation of
4-anilinopyrido[3,4-d]pyrimidine effector compounds of the
invention, from the known intermediate
6-fluoropyrido[3,4-d]pyrimidinone (200) (Rewcastle et al, J Chem
Soc, Perkins Trans 1, 1996, 2221-2226) using the methods of
Rewcastle et al, J Chem Soc, Perkins Trans 1, 1996, 2221-2226 and
Soyka et al, US 2005/0085495 A1.
##STR00025##
[0237] Scheme 2 below illustrates the preparation of specific
effector compounds of the invention. Thionyl chloride mediated
conversion of 6-fluoropyrido[3,4-d]pyrimidinone (200) to
4-chloro-6-fluoropyrido[3,4-d]pyrimidine, followed by reaction with
the appropriate aniline provided the
4-anilino-6-fluoropyrido[3,4-d]pyrimidines (201-209). Fluorine
displacement with 4-methoxybenzyl amine then gave the benzylamines
(210-218), which were reacted with trifluoroacetic acid (TFA) to
provide the amines (219-227). CDI-promoted amide coupling with
2-(diethoxyphosphoryl)acetic acid gave the phosphonates (228-236).
Horner-Wadsworth-Emmons coupling of these with the aldehyde derived
from in situ hydrolysis of 2,2-diethoxy-N,N-dimethylethanamine then
provided the exemplary effector compounds 1-9.
##STR00026##
Preparation of Prodrugs
[0238] The prodrug compounds of the invention may, in general
terms, be prepared by reacting an aliphatic tertiary amine-bearing
kinase inhibitor of Formula I with an appropriate nitroheterocyclic
or nitrocarbocylic .alpha.-methyl halide/mesylate/tosylate, in a
suitable solvent and for a suitable length of time (for example in
N-methyl-2-pyrrolidinone for about 15 hours), to produce a
quaternary nitrogen salt comprising the nitroheterocyclic or
nitrocarbocyclic reductive trigger moiety linked directly or
indirectly to a nitrogen of the kinase inhibitor.
[0239] Preferred reductive trigger moieties suitable for use in the
prodrugs of the invention are of Formula II shown below:
##STR00027##
where R.sub.4 and R.sub.5 are as defined above.
[0240] Particularly preferred reductive trigger moieties are of
Formulae IIa-IIg, shown below:
##STR00028##
[0241] The .alpha.-methyl halides of Formula IIa may be prepared as
described previously (bromide; Stribbling et al, PCT International
patent publication WO 2008/039087) (chloride; Tercel et al, J Med
Chem, 2001, 44, 3511-3522).
[0242] Scheme 3 below illustrates two alternate methods to the
.alpha.-methyl bromide 239, from commercially available starting
materials.
##STR00029##
[0243] Scheme 4 below illustrates a route to the .alpha.-methyl
bromide 244, from commercially available starting materials.
##STR00030##
[0244] Scheme 5 below illustrates a route to the .alpha.-methyl
bromide 250, from 1,5-dimethyl-4-nitro-1H-imidazole (238) (Scheme
3).
##STR00031##
[0245] Scheme 6 below illustrates a route to the .alpha.-methyl
bromide 261 from the commercially available oxazole (251).
##STR00032##
[0246] Scheme 7 below illustrates two alternate routes to the
.alpha.-methyl bromide 264, from .alpha.-methyl bromide 246 (Scheme
5) and 2-bromo-1,5-dimethyl-4-nitro-1H-imidazole (245) (Scheme 5),
respectively. Scheme 7 below also illustrates a route to the
.alpha.-methyl bromide 266, from .alpha.-methyl bromide 264.
##STR00033##
[0247] Scheme 8 below illustrates a route to the .alpha.-methyl
bromide 270, from (2-bromo-1-methyl-4-nitro-1H-imidazol-5-yl)methyl
acetate (247) (Scheme 5).
##STR00034##
[0248] Scheme 9 below illustrates a route to quaternary nitrogen
salt compounds of Formula III by reacting an aliphatic tertiary
amine-bearing kinase inhibitor of Formula I with an appropriate
nitroheterocyclic .alpha.-methyl halide/mesylate/tosylate (Formula
VIII), in a suitable solvent and for a suitable length of time (for
example in N-methyl-2-pyrrolidinone for about 15 hours).
##STR00035##
[0249] Scheme 10 below illustrates the preparation of a number of
prodrug compounds of Formula III according to the invention. The
4-anilinopyrido[3,4-d]pyrimidine effector compounds 1-9 (Scheme 2)
were reacted with the .alpha.-methyl bromide 239 (Scheme 3) in
N-methyl-2-pyrrolidinone (NMP) at room temperature for
approximately 15 hours, before the addition of acetonitrile, to
provide the quaternary ammonium salts (12, 13, 15-21) as a fine
precipitate that was collected by filtration and washed with
acetonitrile, ethyl acetate and hexane.
##STR00036##
[0250] Scheme 11 below illustrates the preparation of a prodrug
compound according to the invention.
##STR00037##
[0251] Schemes 12 to 14 below illustrate the preparation of alkyl
trigger bromides in which R.sub.5 is C.sub.1 to C.sub.6 alkyl.
##STR00038##
##STR00039## ##STR00040##
##STR00041##
[0252] Without further elaboration, it is believed that one skilled
in the art can, using the preceding description, utilize the
present invention to its fullest extent. The following examples
are, therefore, to be construed as merely illustrative and not
limitative of the remainder of the disclosure in any way
whatsoever.
Experimental
1. Synthesis
1.1 Chemical Synthesis
[0253] Combustion analyses were performed by the Microchemical
Laboratory, University of Otago, Dunedin, NZ. Melting points were
determined using either an Electrothermal Model 9200 and are as
read. .sup.1H NMR spectra were measured either on a Bruker
Avance-400 spectrometer and are referenced to Me.sub.4Si. High
resolution mass spectra were recorded on a Varian VG-70SE
spectrometer at nominal 5000 resolution. Mass spectrometry was
performed on a ThermoFinnigan MSQ single quadrupole mass
spectrometer. Mass detection was performed with an APCI source,
using simultaneous positive and negative ion acquisition. Unless
otherwise indicated, compounds were purified by flash column
chromatography on Silica gel 60 support (Scharlau, 230-400 mesh
ASTM), using the indicated eluants.
1.1.1 the Synthesis of Kinase Inhibitor Effectors
1.1.1.1 Preparation of
(2E)-N-(4-{3-chloro-4-[(3-chlorobenzyl)oxy]anilino)}pyrido[3,4-d]pyrimidi-
n-6-yl)-4-(dimethylamino)-2-butenamide (1) (Scheme 2)
[0254] A heterogeneous mixture of
6-fluoropyrido[3,4-d]pyrimidin-4(3H)-one (200) (1.0 g, 6.06 mmol),
thionyl chloride (20 mL) and a catalytic amount of DMF (2 drops)
was stirred under reflux for 40 min to give a homogeneous mixture.
It was evaporated under reduced pressure at 40.degree. C. (bath
temperature) to give a light brown solid. To this solid was added a
mixture of 3-chloro-4-(3-chlorobenzyloxy)aniline (1.79 g, 6.67
mmol) and dry DMA (10 mL). The residue of
3-chloro-4-(3-chlorobenzyloxy)aniline was washed down with more DMA
(2.times.2 mL). The reaction mixture was stirred at room
temperature for 1 h 30 min. It was partitioned in between ethyl
acetate (400 mL) and water (400 mL). The ethyl acetate layer was
separated and washed further with water (2.times.200 mL), dried
(MgSO.sub.4) and evaporated to give a crude product of
N-(3-chloro-4-(3-chlorobenzyloxy)phenyl)-6-fluoropyrido[3,4-d]pyrimidin-4-
-amine (201). Chromatography on silica gel
(dichloromethane/MeOH=25:1) gave pure 201 (2.52 g, 100%) as a
yellow/orange solid, mp 196-198.degree. C.; .sup.1H NMR .delta.
[(CD.sub.3).sub.2SO]10.03 (s, 1H), 8.94 (s, 1H), 8.69 (s, 1H), 8.21
(s, 1H), 8.07 (d, J=2.5 Hz, 1H), 7.75 (dd, J=9.0, 2.5 Hz, 1H),
7.57-7.54 (m, 1H), 7.49-7.39 (m, 3H), 7.31 (d, J=9.0 Hz, 1H). Anal.
Calcd for C.sub.20H.sub.3Cl.sub.2FN.sub.4O: C, 57.85; H, 3.16; N,
13.49. Found C, 57.60; H, 3.45; N, 13.32.
[0255] A mixture of compound 201 (2.60 g, 6.27 mmol) and
4-methoxybenzylamine (8.24 mL, 62.7 mmol) in dry DMSO (15 mL) was
stirred under a nitrogen atmosphere at 71-72.degree. C. (bath
temperature) for 91 h. It was partitioned in between ethyl acetate
(500 mL) and water (300 mL). The ethyl acetate layer was separated
and washed further with water (3.times.300 mL); dried (MgSO.sub.4)
and evaporated to give a crude product of
N.sup.4-(3-chloro-4-(3-chlorobenzyloxy)phenyl)-N.sup.6-(4-methoxybenzyl)p-
yrido[3,4-d]pyrimidine-4,6-diamine (210). Chromatography on silica
gel (ethyl acetate/petroleum ether=1:1) gave pure 210 (2.02 g, 61%)
as a yellow/orange solid, mp 120-122.degree. C.; .sup.1H NMR
.delta. [(CD.sub.3).sub.2SO]9.59 (s, 1H), 8.72 (s, 1H), 8.33 (s,
1H), 7.99 (d, J=2.4 Hz, 1H), 7.57-7.53 (m, 1H), 7.48-7.38 (m, 3H),
7.36-7.19 (m, 4H), 7.15 (br s, 1H), 6.91-6.85 (m, 2H), 5.25 (s,
1H), 4.48 (d, J=6.3 Hz, 2H), 3.71 (s, 3H). Anal. Calcd for
C.sub.28H.sub.23Cl.sub.2N.sub.5O.sub.2: C, 63.16; H, 4.35; N,
13.15. Found C, 62.96; H, 4.63; N, 13.12.
[0256] To a stirred heterogeneous mixture of compound 210 (1.46 g,
2.74 mmol) and dry DCM (27 mL) was added trifluoroacetic acid (2.1
mL, 27.4 mmol), followed by anisole (0.60 mL, 5.48 mmol), and the
mixture was stirred further at room temperature for 91 h. It was
poured into petroleum ether (300 mL) and stirred at room
temperature for ca. 30 min. Petroleum ether layer was decanted and
discarded. The process was repeated with more petroleum ether (300
mL). The residue left behind was dissolved in mixed solvents of
acetone-water=1:1 (ca. 100 mL) and stirred with 5M NH.sub.3 (100
mL) at room temperature for 1 h. The solid was filtered and washed
successively with acetone-water=1:4 (5.times.20 mL), petroleum
ether-ethyl acetate=3:1 (5.times.20 mL), and dried to give
N.sup.4-(3-chloro-4-(3-chlorobenzyloxy)phenyl)pyrido[3,4-d]pyrimidine-4,6-
-diamine (219) (1.10 g, 97%), mp 251-254.degree. C. (decomp);
.sup.1H NMR .delta. [(CD.sub.3).sub.2SO]9.70 (s, 1H), 8.68 (s, 1H),
8.34 (s, 1H), 8.04 (br s, 1H), 7.73 (br d, J=8.0 Hz, 1H), 7.57-7.53
(m, 1H), 7.48-7.39 (m, 3H), 7.26 (d, J=9.0 Hz, 1H), 7.12 (br s,
1H), 7.65 (br s, 2H), 5.24 (s, 2H). Anal. Calcd for
C.sub.20H.sub.15Cl.sub.2N.sub.5O: C, 58.27; H, 3.67; N, 16.99.
Found C, 58.33; H, 3.57; N, 17.2.
[0257] To a stirred mixture of CDI (0.53 g, 3.25 mmol) and dry THF
(2 mL), at room temperature and under a nitrogen atmosphere, was
added a solution of 2-(diethoxyphosphoryl)acetic acid (0.64 g, 3.25
mmol) in THF (1 mL). After addition the reaction mixture was
stirred further at 40.degree. C. (bath) for 15 min (whence
evolution of gases ceased). A solution of compound 219 (1.03 g,
2.50 mmol) in a mixed solvents of dry THF (2.5 mL) and DMA (2.5 mL)
was added and stirred further at 40.degree. C. The reaction was
monitored by TLC (dichloromethane-MeOH=20:1) and found only ca. 60%
reaction occurred after 50 min. Thus, another batch of reagent was
prepared from CDI [0.53 g, 3.25 mmol; dry THF (2 mL)] and
2-(diethoxyphosphoryl)acetic acid [0.64 g, 3.25 mmol); THF (1 mL)]
and added to the reaction. It was stirred further for 3 h at
40.degree. C. The reaction mixture was poured into water (300 mL)
and stirred with petroleum ether (400 mL) at room temperature for
12 h. Petroleum ether layer was decanted. More petroleum ether (200
mL) was added and stirred for 15 min. The solid was collected by
suction filtration; washed with water (5.times.20 mL) and dried to
give diethyl
2-(4-(3-chloro-4-(3-chlorobenzyloxy)phenylamino)pyrido[3,4-d]pyrimidin-6--
ylamino)-2-oxoethylphosphonate (228) (1.37 g, 93%); mp
106-109.degree. C.; .sup.1H NMR .delta. (CDCl.sub.3) 9.41 (s, 1H),
9.02 (s, 1H), 8.70 (s, 1H), 8.54 (s, 1H), 7.91 (d, J=2.6 Hz, 1H),
7.66 (s, 1H), 7.54 (dd, J=8.8, 2.6 Hz, 1H), 7.48 (s, 1H), 7.40-7.29
(m, 3H), 6.99 (d, J=8.9 Hz, 1H), 5.15 (s, 2H), 4.30-4.18 (m, 4H),
3.15 (d, J=21.1 Hz, 2H), 1.39 (t, J=7.0 Hz, 6H). .delta.
[(CD.sub.3).sub.2SO]: 10.82 (s, 1H), 10.25 (s, 1H), 8.99 (poorly
resolved d, J=0.6 Hz, 1H), 8.41 (s, 1H), 8.58 (s, 1H), 7.95 (d,
J=2.6 Hz, 1H), 7.71 (dd, J=9.0, 2.6 Hz, 1H), 7.56 (br s, 1H),
7.50-7.38 (m, 3H), 7.27 (d, J=9.0 Hz, 1H), 5.26 (s, 2H), 4.15-4.04
(m, 4H), 3.34 (d, partially obscured by water peak, 2H), 1.26 (t,
J=7.0 Hz, 6H). Anal. Calcd for
C.sub.26H.sub.26Cl.sub.2N.sub.5O.sub.5P.0.5H.sub.2O: C, 52.10; H,
4.54; N, 11.68%. Found C, 52.09; H, 4.60; N, 11.70%.
[0258] To a stirred mixture of 2,2-diethoxy-N,N-dimethylethanamine
(1.16 g, 7.20 mmol) and water (1.2 mL) at room temperature and
under a nitrogen atmosphere was added an aq. 37% HCl (1.21 mL, 14.4
mmol). After addition the mixture was stirred at 40.degree. C.
(bath) for 24 h. It was cooled to 0.degree. C. (bath). This called
solution A. KOH (1.03 g, 18.4 mmol) was dissolved in water (5.5 mL)
at room temperature under a nitrogen atmosphere. It was cooled to
0.degree. C. (bath). This called solution B. To a stirred
heterogeneous mixture of compound 228 (1.70 g, 2.88 mmol) and THF
(5.5 mL) at room temperature and under a nitrogen atmosphere was
added minimum amount of DMA (3 mL) to give a homogeneous solution.
LiCl (122 mg, 2.88 mmol) was added and stirred at 0.degree. C.
(bath) for 15 min. The cold solution B was added and stirred at
0.degree. C. for 2 min. Then the cold solution A was added and the
final reaction mixture was continued to stir at 0.degree. C. under
a nitrogen atmosphere. The reaction was monitored by TLC
(DCM-MeOH=20:1). After 25 min more KOH (s) (0.34 g, 6.13 mmol) was
added and stirred further at 0.degree. C. for another 35 min. It
was poured into water (200 mL). Petroleum ether (200 mL) was added
and stirred at room temperature for 15 min. Petroleum ether layer
was decanted. More petroleum ether (200 mL) was added and stirred
for 15 min. The solid was collected by suction filtration; washed
with water (4.times.20 mL); dried under reduced pressure over
silica gel/KOH to give
(2E)-N-(4-{3-chloro-4-[(3-chlorobenzyl)oxy]anilino)}pyrido[3,4-d]-
pyrimidin-6-yl)-4-(dimethylamino)-2-butenamide (1) (1.28 g, 85%) as
a pale yellow solid, mp 195-198; HPLC: 96.8% pure; .sup.1H NMR
.delta. [(CD.sub.3).sub.2SO]10.88 (s, 1H), 10.19 (s, 1H), 8.99 (s,
1H), 8.97 (s, 1H), 8.58 (s, 1H), 7.99 (d, J=2.6 Hz, 1H), 7.73 (dd,
J=9.0, 2.6 Hz, 1H), 7.56 (br s, 1H), 7.49-7.38 (m, 3H), 7.27 (d,
J=9.0 Hz, 1H), 6.87 (dt, J=15.4, 6.0 Hz, 1H), 6.51 (br d, J=15.4
Hz, 1H), 5.26 (s, 2H), 3.09 (br d, J=6.0 Hz, 2H), 2.19 (s, 6H).
Anal. Calcd for C.sub.26H.sub.24Cl.sub.2N.sub.6O.sub.2.0.3H.sub.2O:
C, 59.05; H, 4.69; N, 15.89. Found C, 58.96; H, 4.62; N, 15.73.
1.1.1.2 Preparation of
(2E)-4-(dimethylamino)-N-(4-{[1-(3-fluorobenzyl)-1H-indazol-5-yl]amino}py-
rido[3,4-d]pyrimidin-6-yl)-2-butenamide (2) (Scheme 2)
[0259] A heterogeneous mixture of
6-fluoropyrido[3,4-d]pyrimidin-4(3H)-one (200) (1.65 g, 10.0 mmol),
thionyl chloride (30 mL) and a catalytic amount of DMF (2 drops)
was stirred under reflux for 5 h to give a homogeneous mixture. It
was evaporated under reduced pressure at 45.degree. C. (bath
temperature) to give a light brown solid. To this solid was added a
solution of 1-(3-fluorobenzyl)-1H-indazol-5-amine (PCT Int. Appl.,
2005058245, 30 Jun. 2005) (2.65 g, 11.0 mmol) in dry DMA (15 mL).
The residue of 1-(3-fluorobenzyl)-1H-indazol-5-amine was washed
down with more DMA (2.times.2 mL). The reaction mixture was stirred
at room temperature for 65 h. It was poured into water (200 mL).
The pH was adjusted to ca. 9 using an aqueous solution of
Na.sub.2CO.sub.3 at room temperature. Petroleum ether (200 mL) was
added and stirred at room temperature for 2 h. The petroleum ether
layer was decanted. It was repeated once more with petroleum ether
(200 mL). The solid was collected by filtration and washed with
water (5.times.25 mL). It was suspended in acetone (80 mL) and
stirred at room temperature for 20 min. Water (160 mL) was added
and stirred further for 1.5 h. The solid was filtered, dried and
finally purified by a silica column (MeOH/dichloromethane: gradient
from 0-10%) to give
6-fluoro-N-(1-(3-fluorobenzyl)-1H-indazol-5-yl)pyrido[3,4-d]pyrimidin-4-a-
mine (202) (3.11 g, 80%) as a pale brown solid, mp 218-221.degree.
C.; .sup.1H NMR .delta. [(CD.sub.3).sub.2SO]10.15 (s, 1H), 8.92 (s,
1H), 8.64 (s, 1H), 8.30 (d, J=1.3 Hz, 1H), 8.28 (br s, 1H), 8.19
(poorly resolved d, J=0.7 Hz, 1H), 7.77 (d, J=9.0 Hz, 1H), 7.72
(dd, J=9.0, 1.8 Hz, 1H), 7.42-7.32 (m, 1H), 7.15-7.01 (m, 3H), 5.71
(s, 2H). Anal. Calcd for C.sub.21H.sub.14F.sub.2N.sub.6: C, 64.94;
H, 3.63; N, 21.64. Found C, 65.21; H, 3.71; N, 21.63.
[0260] A mixture of compound 202 (3.00 g, 7.73 mmol) and
4-methoxybenzylamine (10.2 mL, 77.3 mmol) in dry DMSO (18 mL) was
stirred under a nitrogen atmosphere at 68-70.degree. C. (bath
temperature) for 165 h. The solution was then cooled and petroleum
ether (200 mL) was added. It was stirred at room temperature for 15
min. The layers were allowed to separate and the petroleum ether
layer was decanted. This procedure was repeated with more petroleum
ether (200 ml). Water (200 mL) was added and the mixture was
stirred at room temperature for 45 min. The oil was deposited.
Thus, the product was extracted into ethyl acetate; dried
(MgSO.sub.4) and solvent removed to give a brown oil. It was
purified by a silica column (EtOAc/petroleum ether: gradient from
30% to pure EtOAc) to give pure
N.sup.4-(1-(3-fluorobenzyl)-1H-indazol-5-yl)-N.sup.6-(4-methoxybenzyl)pyr-
ido[3,4-d]pyrimidine-4,6-diamine (211) (2.38 g, 61%) as a
yellow/orange solid, mp 196-198.degree. C.; .sup.1H NMR .delta.
[(CD.sub.3).sub.2SO]9.75 (s, 1H), 8.72 (s, 1H), 8.29 (s, 1H), 8.22
(poorly resolved d, J=1.3 Hz, 1H), 8.15 (d, J=0.7 Hz, 1H), 7.73 (br
d, J=9.0 Hz, 1H), 7.68 (dd, J=9.0, 1.8 Hz, 1H), 7.40-7.31 (m, 3H),
7.24-7.17 (m, 2H), 7.13-7.01 (m, 3H), 6.92-6.84 (m, 2H), 5.70 (s,
2H) 4.49 (d, J=6.3 Hz, 2H), 3.71 (s, 3H). Anal. Calcd for
C.sub.29H.sub.24FN.sub.7O.1.5H2O: C, 65.40; H, 5.11; N, 18.41%.
Found C, 65.45; H, 5.07; N, 18.58%.
[0261] To a stirred heterogeneous mixture of compound 211 (2.27 g,
4.48 mmol) and DCM (45 ml) was added trifluoroacetic acid (3.45 ml,
44.8 mmol), followed by anisole (0.99 ml, 8.96 mmol), and the
mixture was stirred further at room temperature for 5 days. It was
poured into petroleum ether (500 ml) and stirred at room
temperature for ca. 20 min. Petroleum ether layer was decanted and
discarded. The process was repeated with more petroleum ether (300
ml). To the solid left behind was added 5M NH.sub.3 (80 ml) at
0.degree. C. and stirred at room temperature for 15 min. The solid
was collected and washed successively with water (6.times.10 ml),
petroleum ether-ethyl acetate=3:1 (3.times.20 ml), and dried to
give
N.sup.4-(1-(3-fluorobenzyl)-H-indazol-5-yl)pyrido[3,4-d]pyrimidine-4,6-di-
amine (220) (1.68 g, 97%), mp 241-244.degree. C.; .sup.1H NMR
.delta. [(CD.sub.3).sub.2SO]9.72 (s, 1H), 8.67 (s, 1H), 8.30 (s,
1H), 8.27 (br s, 1H), 8.15 (s, 1H), 7.75-7.68 (m, 2H), 7.41-7.33
(m, 1H), 7.18 (s, 1H), 7.14-7.01 (m, 3H), 6.20 (s, 2H), 5.69 (s,
2H). Anal. Calcd for C.sub.21H.sub.16FN.sub.7.1.5H.sub.2O: C,
61.16; H, 4.64; N, 23.77. Found C, 61.23; H, 4.71; N, 23.87.
[0262] To a stirred mixture of CDI (1.41 g, 8.73 mmol) and dry THF
(6 mL), at room temperature and under a nitrogen atmosphere, was
added a solution of 2-(diethoxyphosphoryl)acetic acid (1.56 g, 7.94
mmol) in THF (4 mL). After addition the reaction mixture was
stirred further at 40.degree. C. (bath) for 15 min (whence
evolution of gases ceased). A solution of compound 220 (1.53 g,
3.97 mmol) in a mixed solvents of dry THF (4 mL) and DMA (5 mL) was
added and stirred further at 40.degree. C. The reaction was
monitored by TLC (dichloromethane-MeOH=15:1). After stirred
overnight (16 h), it was found only ca. 70% reaction occurred.
Thus, another batch of reagent was prepared from CDI [0.42 g, 2.62
mmol; DCM (3 mL)] and 2-(diethoxyphosphoryl)acetic acid [0.47 g,
2.38 mmol); DCM (2 mL)] and added to the reaction. It was stirred
further for 4 h at 40.degree. C. The reaction mixture was poured
into water (200 mL) and stirred with petroleum ether (400 mL) at
room temperature for 20 min. Petroleum ether layer was decanted. It
was repeated once more with petroleum ether (200 mL). The sticky
solid was collected by suction filtration; washed with water
(5.times.30 mL). It was dissolved in acetone (50 mL) and stirred
with water (100 mL) and petroleum ether (200 mL) at room
temperature for 68 h. The solid was collected, washed with
acetone/water=1:10 (5.times.30 mL) and dried to give diethyl
2-(4-(1-(3-fluorobenzyl)-1H-indazol-5-ylamino)pyrido[3,4-d]pyrimidin-6-yl-
amino)-2-oxoethylphosphonate (229) (1.95 g, 87%); mp
111-114.degree. C.; .sup.1H NMR .delta. [(CD.sub.3).sub.2SO]: 10.81
(s, 1H), 10.36 (s, 1H), 8.98 (s, 1H), 8.88 (s, 1H), 8.52 (s, 1H),
8.16 (d, J=0.7 Hz, 1H), 8.13 (d, J=1.3 Hz, 1H), 7.73 (d, J=9.0 Hz,
1H), 7.68 (dd, J=9.0, 1.8 Hz, 1H), 7.42-7.32 (m, 1H), 7.15-7.00 (m,
3H), 5.70 (s, 2H), 4.17-4.03 (m, 4H), 3.32 (d, partially obscured
by water peak, 2H), 1.26 (t, J=7.0 Hz, 6H). Anal. Calcd for
C.sub.27H.sub.27FN.sub.7O.sub.4P.1.6H.sub.2O: C, 54.75; H, 5.14; N,
16.55%. Found C, 54.60; H, 5.21; N, 16.60%.
[0263] To a stirred mixture of 2,2-diethoxy-N,N-dimethylethanamine
(1.32 g, 8.20 mmol) and water (1.4 mL) at room temperature and
under a nitrogen atmosphere was added an aq. 37% HCl (1.38 mL, 16.4
mmol). After addition the mixture was stirred at 45.degree. C.
(bath) for 25 h. It was cooled to 0.degree. C. (bath). This called
solution A. KOH (2.36 g, 42.0 mmol) was dissolved in water (7 mL)
at room temperature under a nitrogen atmosphere. It was cooled to
0.degree. C. (bath). This called solution B. To a stirred
heterogeneous mixture of compound 229 (1.85 g, 3.28 mmol) and THF
(7 mL) at room temperature and under a nitrogen atmosphere was
added minimum amount of DMA (4 mL) to give a homogeneous solution.
LiCl (139 mg, 3.28 mmol) was added and stirred at 0.degree. C.
(bath) for 15 min. The cold solution B was added and stirred at
0.degree. C. for 2 min. Then the cold solution A was added and the
final reaction mixture was continued to stir at 0.degree. C. under
a nitrogen atmosphere. The reaction was monitored by TLC
(DCM-MeOH=10:1). After stirred further for 30 min. it was poured
into water (200 mL). Petroleum ether (200 mL) was added and stirred
at room temperature for 15 min. Petroleum ether layer was decanted.
More petroleum ether (200 mL) was added and stirred for 15 min. The
solid was collected by suction filtration; washed with water
(4.times.30 mL); dried under reduced pressure over silica gel/KOH
to give
(2E)-4-(dimethylamino)-N-(4-{[1-(3-fluorobenzyl)-1H-indazol-5-yl]amino}py-
rido[3,4-d]pyrimidin-6-yl)-2-butenamide (2) (1.55 g, 95%) as a pale
yellow solid, mp 240-243; HPLC: 96.7% pure; .sup.1H NMR .delta.
[(CD.sub.3).sub.2SO]10.87 (s, 1H), 10.29 (s, 1H), 9.01 (s, 1H),
8.98 (s, 1H), 8.53 (s, 1H), 8.18 (d, J=0.7 Hz, 1H), 8.16 (d, J=0.4
Hz, 1H), 7.73 (br d, J=9.0 Hz, 1H), 7.70 (dd, J=9.0, 1.7 Hz, 1H),
7.41-7.33 (m, 1H), 7.14-7.02 (m, 3H), 6.87 (dt, J=15.4, 6.0 Hz,
1H), 6.52 (br d, J=15.4 Hz, 1H), 5.70 (s, 2H), 3.09 (dd, J=6.0, 1.1
Hz, 2H), 2.19 (s, 6H). Anal. Calcd for
C.sub.27H.sub.25FN.sub.8O.1.2H.sub.2O: C, 62.59; H, 5.33; N,
21.63%. Found C, 62.59; H, 5.25; N, 21.59%.
1.1.1.3 Preparation of
(2E)-N-{4-[3-chloro-4-(2-pyridinylmethoxy)anilino]pyrido[3,4-d]pyrimidin--
6-yl}-4-(dimethylamino)-2-butenamide (3) (Scheme 2)
[0264] A heterogeneous mixture of
6-fluoropyrido[3,4-d]pyrimidin-4(3H)-one (200) (1.65 g, 10.0 mmol),
thionyl chloride (30 ml) and a catalytic amount of DMF (3 drops)
was stirred under reflux for 2 h 30 min to give a homogeneous
mixture. It was evaporated under reduced pressure at 40.degree. C.
(bath temperature) to give a light brown solid. To this solid was
added a solution of 3-chloro-4-(pyridin-2-ylmethoxy)aniline (2.58
g, 11.0 mmol) in dry DMA (15 ml). The residue of
3-chloro-4-(pyridin-2-ylmethoxy)aniline was washed down with more
DMA (2.times.5 ml). The reaction mixture was stirred at room
temperature for 18 h. It was poured into water (300 ml). The pH was
adjusted to ca. 9 using an aqueous solution of Na.sub.2CO.sub.3 at
room temperature. Petroleum ether (300 mL) was added and stirred at
room temperature for 30 min. The petroleum ether layer was
decanted. It was repeated once more with petroleum ether (300 mL).
The solid was collected by filtration and washed with water
(4.times.50 mL). It was stirred with hot (ca. 50.degree. C.)
acetone (300 mL) for 15 min. The insoluble materials was filtered
off and the filtrate was evaporated under reduced pressure to give
N-(3-chloro-4-(pyridin-2-ylmethoxy)phenyl)-6-fluoropyrido[3,4-d]pyri-
midin-4-amine (203) (3.18 g, 83%) as a yellow/orange solid, mp
216-219.degree. C.; .sup.1H NMR .delta. [(CD.sub.3).sub.2SO]10.04
(s, 1H), 8.94 (s, 1H), 8.69 (s, 1H), 8.63-8.57 (m, 1H), 8.22 (br s,
1H), 8.08 (d, J=2.6 Hz, 1H), 7.85 (td, J=7.7, 1.8 Hz, 1H), 7.75
(dd, J=9.0, 2.6 Hz, 1H), 7.59 (br d, J=7.8 Hz, 1H), 7.40-7.35 (m,
1H), 7.32 (d, J=9.0 Hz, 1H), 5.31 (s, 2H). Anal. Calcd for
C.sub.19H.sub.13ClFN.sub.5O.0.9H.sub.2O: C, 57.34; H, 3.75; N,
17.60. Found C, 57.46; H, 3.82; N, 17.63.
[0265] A mixture of compound 203 (3.07 g, 8.04 mmol) and
4-methoxybenzylamine (10.6 mL, 80.4 mmol) in dry DMSO (20 mL) was
stirred under a nitrogen atmosphere at 70.degree. C. (bath
temperature) for 118 h. The mixture was then cooled and petroleum
ether (200 mL) was added. It was stirred at room temperature for 15
min. The layers were allowed to separate and the petroleum ether
layer was decanted. This procedure was repeated with more petroleum
ether (200 ml). Water (200 mL) was added and the mixture was
stirred at room temperature overnight (22 h). It was filtered and
washed solid with water (5.times.30 mL), then with petroleum ether
(3.times.30 mL). The sticky yellow/orange solid was stirred with
acetone (150 mL) at room temperature for 20 min. Water (150 mL) was
added slowly and stirred further at room temperature for 45 min.
The fine solid was collected by suction-filtration, washed with
water/acetone (1:1) (4.times.20 mL), petroleum ether (3.times.20
mL) and dried in vacuum over silica-gel/KOH to give pure
N.sup.4-(3-chloro-4-(pyridin-2-ylmethoxy)phenyl)-N.sup.6-(4-methoxybenzyl-
)pyrido[3,4-d]pyrimidine-4,6-diamine (212) (3.08 g, 77%) as a
yellow/orange solid, mp 182-184.degree. C.; .sup.1H NMR .delta.
[(CD.sub.3).sub.2SO]9.60 (s, 1H), 8.72 (s, 1H), 8.64-8.56 (m, 1H),
8.33 (s, 1H), 8.01 (d, J=2.6 Hz, 1H), 7.88 (td, J=7.7, 1.8 Hz, 1H),
7.71 (dd, J=9.0, 2.6 Hz, 1H), 7.58 (d, J=7.7 Hz), 7.42-7.19 (m,
5H), 7.15 (s, 1H), 6.93-6.84 (m, 2H), 5.25 (s, 2H), 4.48 (d, J=6.3
Hz, 2H), 3.71 (s, 3H). Anal. Calcd for
C.sub.27H.sub.23ClN.sub.6O.sub.2.0.6acetone: C, 64.80; H, 5.02; N,
15.74%. Found C, 64.47; H, 5.00; N, 15.95%.
[0266] To a stirred heterogeneous mixture of compound 212 (2.88 g,
5.77 mmol) and DCM (60 ml) was added trifluoroacetic acid (4.38 ml,
57.7 mmol), followed by anisole (1.27 ml, 11.5 mmol), and the
mixture was stirred further at room temperature. After 72 h another
batch of trifluoroacetic acid (4.38 ml, 57.7 mmol) was added and
stirred further for 49 h. It was poured into petroleum ether (250
ml) and stirred at room temperature for ca. 20 min. Petroleum ether
layer was decanted and discarded. The process was repeated with
more petroleum ether (250 ml). The solid left behind was dissolved
in acetone (150 mL) and 5M NH.sub.3 (125 ml) added at 0.degree. C.
The mixture was stirred at room temperature for 30 min. The solid
was collected and washed successively with acetone/water (1:4)
(5.times.30 ml), petroleum ether/ethyl acetate=3:1 (5.times.30 ml),
and dried to give
N.sup.4-(3-chloro-4-(pyridin-2-ylmethoxy)phenyl)pyrido[3,4-a]pyrimidine-4-
,6-diamine (221) (1.94 g, 89%), mp 247-250.degree. C.; .sup.1H NMR
.delta. [(CD.sub.3).sub.2SO]9.64 (s, 1H), 8.68 (s, 1H), 8.62-8.56
(m, 1H), 8.33 (s, 1H), 8.06 (d, J=2.6 Hz, 1H), 7.88 (td, J=7.7, 1.8
Hz, 1H), 7.73 (dd, J=9.0, 2.6 Hz, 1H), 7.58 (br d, J=7.8 Hz, 1H),
7.40-7.34 (m, 1H), 7.26 (d, J=9.0 Hz, 1H), 7.12 (s, 1H), 6.23 (s,
2H), 5.29 (s, 2H). Anal. Calcd for
C.sub.19H.sub.15ClN.sub.6O.1.5H.sub.2O: C, 56.23; H, 4.47; N,
20.71%. Found C, 56.55; H, 4.45; N, 20.42%.
[0267] To a stirred mixture of CDI (2.25 g, 13.9 mmol) and dry THF
(9 mL), at room temperature and under a nitrogen atmosphere, was
added a solution of 2-(diethoxyphosphoryl)acetic acid (2.43 g, 12.4
mmol) in THF (7 mL). After addition the reaction mixture was
stirred further at 40.degree. C. (bath) for 15 min (whence
evolution of gases ceased). A mixture of compound 221 (1.88 g, 4.96
mmol) in a mixed solvents of dry THF (10 mL) and DMA (16 mL) was
added and stirred further at 40.degree. C. The reaction was
monitored by TLC (dichloromethane-MeOH=15:1). After stirred for 17
h, it was poured into water (300 mL) and stirred with petroleum
ether (300 mL) at room temperature for 30 min. Petroleum ether
layer was decanted. It was repeated once more with petroleum ether
(300 mL). The solid was collected by suction filtration; washed
with water (5.times.20 mL) and dried to give diethyl
2-(4-(3-chloro-4-(pyridin-2-ylmethoxy)phenylamino)pyrido[3,4-d]pyrimidin--
6-ylamino)-2-oxoethylphosphonate (230) (2.56 g, 93%); mp
113-116.degree. C.; .sup.1H NMR .delta. [(CD.sub.3).sub.2SO]: 10.82
(s, 1H), 10.25 (s, 1H), 8.99 (s, 1H), 8.84 (s, 1H), 8.63-8.56 (m,
2H), 7.96 (d, J=2.6 Hz, 1H), 7.88 (td, J=7.7, 1.8 Hz, 1H), 7.70
(dd, J=8.9, 2.6 Hz, 1H), 7.59 (br d, J=7.8 Hz, 1H), 7.41-7.34 (m,
1H), 7.28 (d, J=9.0 Hz, 3H), 4.16-4.03 (m, 4H), 3.31 (d, partially
obscured by water peak, 2H), 1.26 (t, J=7.0 Hz, 6H). Anal. Calcd
for C.sub.25H.sub.26ClN.sub.6O.sub.5P.1.2H.sub.2O: C, 51.90; H,
4.95; N, 14.53%. Found C, 51.85; H, 4.94; N, 14.52%.
[0268] To a stirred mixture of 2,2-diethoxy-N,N-dimethylethanamine
(1.76 g, 11.0 mmol) and water (1.8 mL) at room temperature and
under a nitrogen atmosphere was added an aq. 37% HCl (1.84 mL, 21.9
mmol). After addition the mixture was stirred at 50.degree. C.
(bath) for 22 h. It was cooled to 0.degree. C. (bath). This called
solution A. KOH (3.14 g, 56.1 mmol) was dissolved in water (9 mL)
at room temperature under a nitrogen atmosphere. It was cooled to
0.degree. C. (bath). This called solution B. To a stirred
heterogeneous mixture of compound 230 (2.44 g, 4.38 mmol) and THF
(9 mL) at room temperature and under a nitrogen atmosphere was
added minimum amount of DMA (6 mL) to give a homogeneous solution.
LiCl (186 mg, 4.38 mmol) was added and stirred at 0.degree. C.
(bath) for 15 min. The cold solution B was added and stirred at
0.degree. C. for 2 min. Then the cold solution A was added and the
final reaction mixture was continued to stir at 0.degree. C. under
a nitrogen atmosphere. The reaction was monitored by TLC
(DCM-MeOH=10:1). After stirred for 30 min. it was poured into water
(300 mL). Petroleum ether (400 mL) was added and stirred at room
temperature for 15 min. Petroleum ether layer was decanted. More
petroleum ether (300 mL) was added and stirred for 15 min. The
solid was collected by suction filtration; washed with water
(4.times.30 mL); dried under reduced pressure over silica gel/KOH
to give compound 3 (1.79 g, 83%) which was found only 87% pure by
HPLC. Thus, the sample was stirred with warm MeOH (200 mL) for 30
min and cooled to room temperature. The insoluble materials were
filtered off and to the filtrates was added one volume of water to
precipitate out the required product. The solid was collected,
washed with water/MeOH=1:1 several times and dried to give
(2E)-N-{4-[3-chloro-4-(2-pyridinylmethoxy)anilino]pyrido[3,4-d]pyrimidin--
6-yl}-4-(dimethylamino)-2-butenamide (3) (1.54 g, 72%) as a pale
yellow solid, mp 196-199; HPLC: 94.6% pure; .sup.1H NMR .delta.
[(CD.sub.3).sub.2SO]10.88 (s, 1H), 10.19 (s, 1H), 8.99 (s, 1H),
8.64-8.55 (m, 2H), 8.00 (d, J=2.5 Hz, 1H), 7.88 (td, J=7.7, 1.8 Hz,
1H), 7.73 (br dd, J=8.9, 2.5 Hz, 1H), 7.59 (br d, J=7.8 Hz, 1H),
7.41-7.34 (m, 1H), 7.28 (d, J=9.0 Hz, 1H), 6.87 (dt, J=15.4, 6.1
Hz, 1H), 6.51 (br d, J=15.4 Hz, 1H), 5.30 (s, 2H), 3.09 (dd, J=6.1,
1.2 Hz, 2H), 2.19 (s, 6H). Anal. Calcd for
C.sub.24H.sub.24ClN.sub.7O.sub.2.1.5H.sub.2O: C, 58.08; H, 5.26; N,
18.97%. Found C, 58.17; H, 5.36; N, 19.00%.
1.1.1.4 Preparation of
(2E)-N-[4-(3,4-dichloroanilino)pyrido[3,4-d]pyrimidin-6-yl]-4-(dimethylam-
ino)-2-butenamide (4) (Scheme 2)
[0269] A heterogeneous mixture of
6-fluoropyrido[3,4-d]pyrimidin-4(3H)-one (200) (1.50 g, 9.09 mmol),
thionyl chloride (20 ml) and a catalytic amount of DMF (2 drops)
was stirred under reflux for 1.5 h to give a homogeneous mixture.
It was evaporated under reduced pressure at 50.degree. C. (bath
temperature) to give a light brown solid. To this solid was added a
solution of 3,4-dichloroaniline (1.62 g, 10.0 mmol) in dry DMA (15
mL). The residue of 3,4-dichloroaniline was washed down with more
DMA (2.times.2 ml). The reaction mixture was stirred at room
temperature for 20 h. It was poured into water (200 mL). The pH was
adjusted to ca. 8 using an aqueous solution of Na.sub.2CO.sub.3 at
room temperature. Petroleum ether (300 mL) was added and stirred at
room temperature for 30 min. The petroleum ether layer was
decanted. It was repeated once more with petroleum ether. The solid
was collected by filtration and washed with water (5.times.25 mL).
It was dried in vacuum over silica gel/KOH overnight to give
N-(3,4-dichlorophenyl)-6-fluoropyrido[3,4-d]pyrimidin-4-amine (204)
(2.81 g, 100%) as a yellow/orange solid, mp 262-264.degree. C.;
.sup.1H NMR .delta. [(CD.sub.3).sub.2SO]10.17 (s, 1H), 8.99 (s,
1H), 8.79 (s, 1H), 8.35 (d, J=2.5 Hz, 1H), 8.26 (poorly resolved d,
J=0.7 Hz, 1H), 7.92 (dd, J=8.8, 2.5 Hz, 1H), 7.69 (d, J=8.8 Hz,
1H). Anal. Calcd for C.sub.13H.sub.7Cl.sub.2FN.sub.4: C, 50.51; H,
2.28; N, 18.12. Found C, 50.50; H, 2.27; N, 18.42.
[0270] A mixture of compound 204 (2.95 g, 9.55 mmol) and
4-methoxybenzylamine (12.6 mL, 95.5 mmol) in dry DMSO (20 mL) was
stirred under a nitrogen atmosphere at 52.degree. C. (bath
temperature) for 10 days. The solution was then cooled and
petroleum ether (200 mL) was added. It was stirred at room
temperature for 10 min. The layers were allowed to separate and the
petroleum ether layer was decanted. This procedure was repeated
with more petroleum ether (2.times.200 ml). Water (200 mL) was
added and the mixture was stirred at room temperature for 15 min.
It was filtered and washed solid with water (5.times.30 mL), then
with petroleum ether (3.times.30 mL). The sticky yellow/orange
solid was stirred with acetone (100 mL) at 35-45.degree. C.
(internal temperature) for 10 min. Water (100 mL) was added and
stirred at room temperature for 20 min. The solid was collected by
suction-filtration, washed with water/acetone (1:1) (5.times.20
mL), and dried in vacuum over silica-gel/KOH to give pure
N.sup.4-(3,4-dichlorophenyl)-N.sup.6-(4-methoxybenzyl)pyrido[3,4-d]pyrimi-
dine-4,6-diamine (213) (2.59 g, 64%) as a yellow/orange solid, mp
209-211.degree. C.; .sup.1H NMR .delta. [(CD.sub.3).sub.2SO]9.76
(s, 1H), 8.77 (s, 1H), 8.42 (s, 1H), 8.30 (poorly resolved d, J=2.1
Hz, 1H), 7.89 (dd, J=8.8, 2.1 Hz, 1H), 7.64 (d, J=8.8 Hz, 1H),
7.38-7.25 (m, 3H), 7.17 (s, 1H), 6.92-6.83 (m, 2H), 4.49 (d, J=6.3
Hz, 2H), 3.71 (s, 3H). Anal. Calcd for
C.sub.21H.sub.17Cl.sub.2N.sub.5O: C, 59.17; H, 4.02; N, 16.43%.
Found C, 58.93; H, 4.17; N, 16.12%.
[0271] To a stirred heterogeneous mixture of compound 213 (2.50 g,
5.86 mmol) and DCM (60 ml) was added trifluoroacetic acid (4.49 ml,
58.6 mmol), followed by anisole (1.28 mL, 11.7 mmol), and the
mixture was stirred further at room temperature for 43 h. It was
poured into petroleum ether (600 mL) and stirred at room
temperature for ca. 20 min. Petroleum ether layer was decanted and
discarded. The process was repeated with more petroleum ether (300
mL). To the solid left behind was added 5M NH.sub.3 (80 mL) at
0.degree. C. and stirred at room temperature for 15 min. The solid
was collected and washed successively with water (6.times.10 mL),
petroleum ether-ethyl acetate=3:1 (3.times.20 mL), and dried to
give
N.sup.4-(3,4-dichlorophenyl)pyrido[3,4-d]pyrimidine-4,6-diamine
(222) (1.76 g, 98%), mp 277-280.degree. C.; .sup.1H NMR .delta.
[(CD.sub.3).sub.2SO]9.83 (s, 1H), 8.73 (s, 1H), 8.42 (s, 1H), 8.34
(d, J=2.5 Hz, 1H), 7.92 (dd, J=8.8, 2.5 Hz, 1H), 7.63 (d, J=8.8 Hz,
1H), 7.14 (s, 1H), 6.30 (s, 2H). Anal. Calcd for
C.sub.13H.sub.9Cl.sub.2N.sub.5.0.5H.sub.2O: C, 49.54; H, 3.20; N,
22.22. Found C, 49.70; H, 3.08; N, 22.28.
[0272] To a stirred mixture of CDI (1.98 g, 12.2 mmol) and dry THF
(8 mL), at room temperature and under a nitrogen atmosphere, was
added a solution of 2-(diethoxyphosphoryl)acetic acid (2.18 g, 11.1
mmol) in THF (6 mL). After addition the reaction mixture was
stirred further at 40.degree. C. (bath) for 15 min (whence
evolution of gases ceased). A solution of compound 222 (1.70 g,
5.56 mmol) in a mixed solvents of dry THF (6 mL) and DMA (7 mL) was
added and stirred further at 40.degree. C. The reaction was
monitored by TLC (dichloromethane-MeOH=15:1). After stirred
overnight (17 h), the reaction mixture was poured into water (300
mL) and stirred with petroleum ether (400 mL) at room temperature
for 20 min. Petroleum ether layer was decanted. It was repeated
once more with petroleum ether (400 mL). The solid was collected by
suction filtration; washed with water (5.times.30 mL) and dried to
give diethyl
2-(4-(3,4-dichlorophenylamino)pyrido[3,4-d]pyrimidin-6-ylamino)-2-oxoethy-
lphosphonate (231) (2.56 g, 95%); mp 121-124.degree. C.; .sup.1H
NMR .delta. [(CD.sub.3).sub.2SO]: 10.86 (s, 1H), 10.42 (s, 1H),
9.04 (s, 1H), 8.87 (s, 1H), 8.67 (s, 1H), 8.24 (d, J=2.2 Hz, 1H),
7.89 (dd, J=8.8, 2.2 Hz, 1H), 7.65 (d, J=8.8 Hz, 1H), 4.16-4.04 (m,
4H), 3.32 (d, partially obscured by water peak, 2H), 1.26 (t, J=7.0
Hz, 6H). Anal. Calcd for
C.sub.19H.sub.20Cl.sub.2N.sub.5O.sub.4P.H.sub.2O: C, 45.43; H,
4.42; N, 13.94%. Found C, 45.37; H, 4.20; N, 13.81%.
[0273] To a stirred mixture of 2,2-diethoxy-N,N-dimethylethanamine
(2.08 g, 12.9 mmol) and water (2.2 mL) at room temperature and
under a nitrogen atmosphere was added an aq. 37% HCl (2.16 mL, 25.7
mmol). After addition the mixture was stirred at 40.degree. C.
(bath) for 20 h. It was cooled to 0.degree. C. (bath). This called
solution A. KOH (1.84 g, 32.9 mmol) was dissolved in water (10 mL)
at room temperature under a nitrogen atmosphere. It was cooled to
0.degree. C. (bath). This called solution B. To a stirred
heterogeneous mixture of compound 231 (2.49 g, 5.14 mmol) and THF
(10 mL) at room temperature and under a nitrogen atmosphere was
added minimum amount of DMA (5.5 mL) to give a homogeneous
solution. LiCl (218 mg, 5.14 mmol) was added and stirred at
0.degree. C. (bath) for 15 min. The cold solution B was added and
stirred at 0.degree. C. for 2 min. Then the cold solution A was
added and the final reaction mixture was continued to stir at
0.degree. C. under a nitrogen atmosphere. The reaction was
monitored by TLC (DCM-MeOH=10:1). After 15 min it was found ca. 20%
of compound 231 left. More KOH (0.40 g, 7.14 mmol) was added as a
solid. After stirred further for 15 min. it was poured into water
(300 mL). Petroleum ether (300 mL) was added and stirred at room
temperature for 15 min. Petroleum ether layer was decanted. More
petroleum ether (300 mL) was added and stirred for 15 min. The
solid was collected by suction filtration; washed with water
(4.times.30 mL); dried under reduced pressure over silica gel/KOH
to give
(2E)-N-[4-(3,4-dichloroanilino)pyrido[3,4-d]pyrimidin-6-yl]-4-(dimethylam-
ino)-2-butenamide (4) (2.03 g, 95%) as a beige solid, mp
161-164.degree. C.; HPLC: 98.6% pure; .sup.1H NMR .delta.
[(CD.sub.3).sub.2SO]10.92 (s, 1H), 10.36 (s, 1H), 9.09-8.95 (m,
2H), 8.67 (br s, 1H), 8.27 (br s, 1H), 7.92 (br d, J=7.8 Hz, 1H),
7.65 (d, J=8.8, 1H), 6.88 (dt, J=15.4, 6.0 Hz, 1H), 6.52 (br d,
J=15.4 Hz, 1H), 3.09 (dd, J=6.0, 1.2 Hz, 2H), 2.19 (s, 6H). Anal.
Calcd for C.sub.19H.sub.18Cl.sub.2N.sub.6O.1.2H.sub.2O: C, 51.99;
H, 4.69; N, 19.15. Found C, 51.93; H, 4.59; N, 19.15.
1.1.1.5 Preparation of
(2E)-N-[4-(3-bromo-4-chloroanilino)pyrido[3,4-d]pyrimidin-6-yl]-4-(dimeth-
ylamino)-2-butenamide (5) (Scheme 2)
[0274] A heterogeneous mixture of
6-fluoropyrido[3,4-d]pyrimidin-4(3H)-one (200) (1.50 g, 9.09 mmol),
thionyl chloride (20 mL) and a catalytic amount of DMF (2 drops)
was stirred under reflux for 3 h to give a homogeneous mixture. It
was evaporated under reduced pressure at 40.degree. C. (bath
temperature) to give a light brown solid. To this solid was added a
solution of 3-bromo-4-chloroaniline (2.07 g, 10.0 mmol) in dry DMA
(15 mL). The residue of 3-bromo-4-chloroaniline was washed down
with more DMA (2.times.2 mL). The reaction mixture was stirred at
room temperature for 19 h. It was poured into water (200 mL). The
pH was adjusted to ca. 8 using an aqueous solution of
Na.sub.2CO.sub.3 at room temperature. Petroleum ether (300 mL) was
added and stirred at room temperature for 30 min. The petroleum
ether layer was decanted. It was repeated once more with petroleum
ether. The solid was collected by filtration and washed with water
(4.times.20 mL). It was dried in vacuum over silica gel/KOH
overnight to give
N-(3-bromo-4-chlorophenyl)-6-fluoropyrido[3,4-d]pyrimidin-4-amine
(205) (3.20 g, 99%) as a yellow/orange solid, mp 271-275.degree.
C.; .sup.1H NMR .delta. [(CD.sub.3).sub.2SO]10.15 (s, 1H), 8.99 (s,
1H), 8.79 (s, 1H), 8.45 (d, J=2.5 Hz, 1H), 8.26 (br s, 1H), 7.98
(dd, J=8.8, 2.5 Hz, 1H), 7.69 (d, J=8.8 Hz, 1H). Anal. Calcd for
C.sub.13H.sub.7BrClFN.sub.4: C, 44.16; H, 2.00; N, 15.85. Found C,
44.21; H, 1.87; N, 15.98.
[0275] A mixture of compound 205 (3.09 g, 8.74 mmol) and
4-methoxybenzylamine (11.5 mL, 87.4 mmol) in dry DMSO (20 mL) was
stirred under a nitrogen atmosphere at 58-60.degree. C. (bath
temperature) for a week. The solution was then cooled and petroleum
ether (300 mL) was added. It was stirred at room temperature for 20
min. The layers were allowed to separate and the petroleum ether
layer was decanted. This procedure was repeated once more with
petroleum ether (300 mL). Water (250 mL) was added and the mixture
was stirred at room temperature for 20 h. The yellow/orange solid
was collected by filtration and washed with water (5.times.20 mL),
then with petroleum ether (3.times.30 mL). The sticky yellow/orange
solid was dissolved in acetone (150 mL) at 45.degree. C. (bath
temperature). It was filtered to remove insoluble impurities. To
the filtrate was added water (400 mL) and stirred at room
temperature for 1 h. The solid was collected by suction-filtration,
washed with water (5.times.25 mL), petroleum ether (3.times.30 mL),
and dried in vacuum over silica-gel/KOH to give pure
N.sup.4-(3-bromo-4-chlorophenyl)-N.sup.6-(4-methoxybenzyl)pyrido[3,4-d]py-
rimidine-4,6-diamine (214) (3.01 g, 73%) as a greenish yellow
solid, mp 207-210.degree. C.; .sup.1H NMR .delta.
[(CD.sub.3).sub.2SO.sub.]9.74 (s, 1H), 8.77 (s, 1H), 8.41 (s, 1H),
8.39 (d, J=2.4 Hz, 1H), 7.96 (dd, J=8.8, 2.4 Hz, 1H), 7.64 (d,
J=8.8 Hz, 1H), 7.38-7.25 (m, 3H), 7.17 (s, 1H), 6.92-6.85 (m, 2H),
4.49 (d, J=6.3 Hz, 2H), 3.71 (s, 3H). Anal. Calcd for
C.sub.21H.sub.17BrClN.sub.5O: C, 53.58; H, 3.64; N, 14.88%. Found
C, 53.84; H, 3.55; N, 14.63%.
[0276] To a stirred heterogeneous mixture of compound 214 (2.92 g,
6.20 mmol) and DCM (60 mL) was added trifluoroacetic acid (4.75 mL,
62.0 mmol), followed by anisole (1.36 mL, 12.4 mmol), and the
mixture was stirred further at room temperature for 42 h. It was
poured into petroleum ether (600 mL) and stirred at room
temperature for ca. 20 min. Petroleum ether layer was decanted and
discarded. The process was repeated with more petroleum ether (300
mL). To the solid left behind was added 5M NH.sub.3 (80 mL) at
0.degree. C. and stirred at room temperature for 15 min. The solid
was collected and washed successively with water (6.times.10 mL),
petroleum ether-ethyl acetate=3:1 (3.times.20 mL), and dried to
give
N.sup.4-(3-bromo-4-chlorophenyl)pyrido[3,4-d]pyrimidine-4,6-diamine
(223) (2.17 g, 100%), mp 271-274.degree. C.; .sup.1H NMR .delta.
[(CD.sub.3).sub.2SO]9.82 (s, 1H), 8.72 (s, 1H), 8.44 (d, J=2.3 Hz,
1H), 8.42 (s, 1H), 7.97 (dd, J=8.8, 2.3 Hz, 1H), 7.62 (d, J=8.8 Hz,
1H), 7.14 (s, 1H), 6.30 (s, 2H). Anal. Calcd for
C.sub.13H.sub.9BrClN.sub.5.0.6H.sub.2O: C, 43.20; H, 2.85; N,
19.38. Found C, 43.00; H, 2.95; N, 19.12.
[0277] To a stirred mixture of CDI (1.20 g, 7.42 mmol) and dry THF
(5 mL), at room temperature and under a nitrogen atmosphere, was
added a solution of 2-(diethoxyphosphoryl)acetic acid (1.45 g, 7.42
mmol) in THF (4 mL). After addition the reaction mixture was
stirred further at 40.degree. C. (bath) for 15 min (whence
evolution of gases ceased). A solution of compound 223 (2.00 g,
5.71 mmol) in a mixed solvents of dry THF (6 mL) and DMA (7 mL) was
added and stirred further at 40.degree. C. The reaction was
monitored by TLC (dichloromethane-MeOH=10:1) and found only ca. 60%
reaction occurred after 1 h. Thus, another batch of reagent was
prepared from CDI [0.87 g, 5.37 mmol; dry THF (2.5 mL)] and
2-(diethoxyphosphoryl)acetic acid [0.72 g, 5.37 mmol); THF (3 mL)]
and added to the reaction. It was stirred further for 3 h at
40.degree. C. The reaction mixture was poured into water (300 mL)
and stirred with petroleum ether (400 mL) at room temperature for
20 min. Petroleum ether layer was decanted. It was repeated once
more with petroleum ether (400 mL). The solid was collected by
suction filtration; washed with water (5.times.30 mL) and dried to
give diethyl
2-(4-(3-bromo-4-chlorophenylamino)pyrido[3,4-d]pyrimidin-6-ylamino)-2-oxo-
ethylphosphonate (232) (2.85 g, 94%); mp 113-116.degree. C.;
.sup.1H NMR .delta. [(CD.sub.3).sub.2SO]: 10.86 (s, 1H), 10.40 (s,
1H), 9.04 (s, 1H), 8.87 (s, 1H), 8.67 (s, 1H), 8.34 (d, J=2.3 Hz,
1H), 7.94 (dd, J=8.8, 2.3 Hz, 1H), 7.65 (d, J=8.8 Hz, 1H),
4.16-4.03 (m, 4H), 3.32 (d, partially obscured by water peak, 2H),
1.26 (t, J=7.1 Hz, 6H). Anal. Calcd for
C.sub.19H.sub.20BrClN.sub.5O.sub.4P.H.sub.2O: C, 41.74; H, 4.06; N,
12.81%. Found C, 42.01; H, 3.90; N, 12.78%.
[0278] To a stirred mixture of 2,2-diethoxy-N,N-dimethylethanamine
(2.11 g, 13.1 mmol) and water (2.2 mL) at room temperature and
under a nitrogen atmosphere was added an aq. 37% HCl (2.19 mL, 26.1
mmol). After addition the mixture was stirred at 40.degree. C.
(bath) for 21 h. It was cooled to 0.degree. C. (bath). This called
solution A. KOH (1.87 g, 33.4 mmol) was dissolved in water (10 mL)
at room temperature under a nitrogen atmosphere. It was cooled to
0.degree. C. (bath). This called solution B. To a stirred
heterogeneous mixture of compound 232 (2.76 g, 5.22 mmol) and THF
(10 mL) at room temperature and under a nitrogen atmosphere was
added minimum amount of DMA (5.5 mL) to give a homogeneous
solution. LiCl (221 mg, 5.22 mmol) was added and stirred at
0.degree. C. (bath) for 15 min. The cold solution B was added and
stirred at 0.degree. C. for 2 min. Then the cold solution A was
added and the final reaction mixture was continued to stir at
0.degree. C. under a nitrogen atmosphere. The reaction was
monitored by TLC (DCM-MeOH=10:1). After 35 min. it was poured into
water (300 mL). Petroleum ether (300 mL) was added and stirred at
room temperature for 15 min. Petroleum ether layer was decanted.
More petroleum ether (300 mL) was added and stirred for 15 min. The
solid was collected by suction filtration; washed with water
(4.times.30 mL); dried under reduced pressure over silica gel/KOH
to give
(2E)-N-[4-(3-bromo-4-chloroanilino)pyrido[3,4-d]pyrimidin-6-yl]-4-(dimeth-
ylamino)-2-butenamide (5) (2.37 g, 98%) as a pale yellow solid, mp
168-171; HPLC: 96.8% pure; .sup.1H NMR .delta.
[(CD.sub.3).sub.2SO]10.92 (s, 1H), 10.34 (s, 1H), 9.10-8.95 (m,
2H), 8.67 (br s, 1H), 8.38 (br s, 1H), 7.97 (br d, J=8.3 Hz, 1H),
7.65 (d, J=8.8, 1H), 6.88 (dt, J=15.4, 6.0 Hz, 1H), 6.52 (br d,
J=15.4 Hz, 1H), 3.09 (dd, J=6.0, 1.1 Hz, 2H), 2.19 (s, 6H). Anal.
Calcd for C.sub.19H.sub.1BBrClN.sub.6O.H.sub.2O: C, 47.57; H, 4.20;
N, 17.52. Found C, 47.80; H, 4.25; N, 17.51.
1.1.1.6 Preparation of
(2E)-N-[4-(4-bromo-3-chloroanilino)pyrido[3,4-d]pyrimidin-6-yl]-4-(dimeth-
ylamino)-2-butenamide (6) (Scheme 2)
[0279] A heterogeneous mixture of
6-fluoropyrido[3,4-d]pyrimidin-4(3H)-one (200) (1.65 g, 10.0 mmol),
thionyl chloride (20 mL) and a catalytic amount of DMF (2 drops)
was stirred under reflux for 3 h to give a homogeneous mixture. It
was evaporated under reduced pressure at 50.degree. C. (bath
temperature) to give a light brown solid. To this solid was added a
solution of 4-bromo-3-chloroaniline (2.27 g, 11.0 mmol) in dry DMA
(15 mL). The residue of 4-bromo-3-chloroaniline was washed down
with more DMA (2.times.2 mL). The reaction mixture was stirred at
room temperature for 18 h. It was poured into water (250 mL). The
pH was adjusted to ca. 8 using an aqueous solution of
Na.sub.2CO.sub.3 at room temperature. Petroleum ether (300 mL) was
added and stirred at room temperature for 1 h. The petroleum ether
layer was decanted. It was repeated once more with petroleum ether.
The solid was collected by filtration and washed with water
(4.times.20 mL). It was dried in vacuum over silica gel/KOH
overnight to give
N-(4-bromo-3-chlorophenyl)-6-fluoropyrido[3,4-d]pyrimidin-4-amine
(206) (3.31 g, 94%) as a beige solid, mp 258-261.degree. C.;
.sup.1H NMR .delta. [(CD.sub.3).sub.2SO]10.17 (s, 1H), 8.99 (s,
1H), 8.79 (s, 1H), 8.35 (br s, 1H), 8.27 (s, 1H), 7.93-7.74 (m,
2H). Anal. Calcd for C.sub.13H.sub.7BrClFN.sub.4: C, 44.16; H,
2.00; N, 15.85. Found C, 43.96; H, 1.96; N, 15.60.
[0280] A mixture of compound 206 (3.22 g, 9.11 mmol) and
4-methoxybenzylamine (11.95 mL, 91.1 mmol) in dry DMSO (24 mL) was
stirred under a nitrogen atmosphere at 70.degree. C. (bath
temperature) for 113 h. The solution was then cooled and stirred
with petroleum ether (300 mL) at room temperature for 20 min. The
layers were allowed to separate and the petroleum ether layer was
decanted. This procedure was repeated once more with petroleum
ether (300 mL). Water (300 mL) was added and the mixture was
stirred at room temperature for 4 h. The solid was collected by
filtration and washed with water (4.times.20 mL). The sticky
yellow/orange solid was dissolved in acetone (80 mL) at 40.degree.
C. (bath temperature). Water (80 mL) was added and stirred at room
temperature for 10 min. The solid was collected by
suction-filtration, washed with acetone/water=1:1 (5.times.20 mL)
and dried in vacuum over silica-gel/KOH to give pure
N.sup.4-(4-bromo-3-chlorophenyl)-N.sup.6-(4-methoxybenzyl)pyrido[3,4-d]py-
rimidine-4,6-diamine (215) (3.63 g, 85%) as a greenish yellow
solid, mp 175-177.degree. C.; .sup.1H NMR .delta.
[(CD.sub.3).sub.2SO]9.77 (s, 1H), 8.77 (s, 1H), 8.42 (s, 1H), 8.30
(br s, 1H), 7.83 (br d, J=8.2, 1H), 7.77 (d, J=8.7 Hz, 1H),
7.43-7.26 (m, 3H), 7.17 (s, 1H), 6.88 (br d, J=8.3 Hz, 2H), 4.49
(d, J=5.6 Hz, 2H), 3.71 (s, 3H). Anal. Calcd for
C.sub.21H.sub.17BrClN.sub.5O.acetone: C, 54.51; H, 4.38; N, 13.24%.
Found C, 54.83; H, 4.31; N, 13.15%.
[0281] To a stirred heterogeneous mixture of compound 215 (3.55 g,
7.56 mmol) and DCM (75 mL) was added trifluoroacetic acid (5.82 mL,
75.6 mmol), followed by anisole (1.66 mL, 15.1 mmol), and the
mixture was stirred further at room temperature for 46 h. It was
poured into petroleum ether (400 mL) and residue washed down with
MeOH. After stirred at room temperature for ca. 20 min. Petroleum
ether layer was decanted and discarded. The process was repeated
with more petroleum ether (400 mL). To the solid left behind was
added 5M NH.sub.3 (100 mL) at 0.degree. C. and stirred at room
temperature for 15 min. The solid was collected and washed with
water (5.times.20 mL) and dried to give
N.sup.4-(4-bromo-3-chlorophenyl)pyrido[3,4-d]pyrimidine-4,6-diamine
(224) (2.41 g, 91%), mp 272-275.degree. C.; .sup.1H NMR .delta.
[(CD.sub.3).sub.2SO]9.83 (s, 1H), 8.73 (s, 1H), 8.42 (s, 1H), 8.34
(d, J=2.0 Hz, 1H), 7.84 (dd, J=8.8, 2.0 Hz, 1H), 7.75 (d, J=8.8 Hz,
1H), 7.14 (s, 1H), 6.31 (s, 2H). Anal. Calcd for
C.sub.13H.sub.9BrClN.sub.5.0.2MeOH: C, 45.15; H, 2.73; N, 19.36.
Found C, 44.84; H, 2.52; N, 19.29.
[0282] To a stirred mixture of CDI (2.97 g, 18.3 mmol) and dry THF
(12 mL), at room temperature and under a nitrogen atmosphere, was
added a solution of 2-(diethoxyphosphoryl)acetic acid (3.2 g, 16.3
mmol) in THF (10 mL). After addition the reaction mixture was
stirred further at 40.degree. C. (bath) for 15 min (whence
evolution of gases ceased). A solution of compound 224 (2.29 g,
6.53 mmol) in a mixed solvents of dry THF (8 mL) and DMA (9 mL) was
added and stirred further at 40.degree. C. The reaction was
monitored by TLC (dichloromethane-MeOH=10:1) and found ca. 80%
reaction occurred after 3 h. Thus, another batch of reagent was
prepared from CDI [0.59 g, 3.66 mmol; dry THF (2.5 mL)] and
2-(diethoxyphosphoryl)acetic acid [0.64 g, 3.26 mmol); THF (2 mL)]
and added to the reaction mixture. It was stirred further for 1 h
at 40.degree. C. The reaction mixture was poured into water (500
mL) and stirred with petroleum ether (500 mL) at room temperature
overnight. Petroleum ether layer was decanted. It was stirred with
more petroleum ether (300 mL) for 10 min. The solid was collected
by suction filtration; washed with water (5.times.30 mL) and dried
to give diethyl
2-(4-(4-bromo-3-chlorophenylamino)pyrido[3,4-d]pyrimidin-6-ylamino)-2-oxo-
ethylphosphonate (233) (3.26 g, 95%); mp 121-124.degree. C.;
.sup.1H NMR .delta. [(CD.sub.3).sub.2SO]: 10.89 (s, 1H), 10.43 (s,
1H), 9.04 (s, 1H), 8.88 (s, 1H), 8.68 (s, 1H), 8.24 (d, J=2.1 Hz,
1H), 7.82 (dd, J=8.8, 2.1 Hz, 1H), 7.78 (d, J=8.8 Hz, 1H),
4.17-4.00 (m, 4H), 3.32 (d, J=21.5, 2H), 1.26 (t, J=7.0 Hz, 6H).
Anal. Calcd for C.sub.19H.sub.20BrClN.sub.5O.sub.4P.0.5H.sub.2O: C,
42.44; H, 3.94; N, 13.02%. Found C, 42.42; H, 3.99; N, 12.98%.
[0283] To a stirred mixture of 2,2-diethoxy-N,N-dimethylethanamine
(2.42 g, 15.0 mmol) and water (2.5 mL) at room temperature and
under a nitrogen atmosphere was added an aq. 37% HCl (2.52 mL, 30.0
mmol). After addition the mixture was stirred at 40.degree. C.
(bath) for 48 h. It was cooled to 0.degree. C. (bath). This called
solution A. KOH (4.30 g, 76.8 mmol) was dissolved in water (13 mL)
at room temperature under a nitrogen atmosphere. It was cooled to
0.degree. C. (bath). This called solution B. To a stirred
heterogeneous mixture of compound 233 (3.17 g, 6.00 mmol) and THF
(13 mL) at room temperature and under a nitrogen atmosphere was
added minimum amount of DMA (6.5 mL) to give a homogeneous
solution. LiCl (254 mg, 6.00 mmol) was added and stirred at
0.degree. C. (bath) for 15 min. The cold solution B was added and
stirred at 0.degree. C. for 2 min. Then the cold solution A was
added and the final reaction mixture was continued to stir at
0.degree. C. under a nitrogen atmosphere. The reaction was
monitored by TLC (DCM-MeOH=20:1). After 40 min. it was poured into
water (300 mL). Petroleum ether (300 mL) was added and stirred at
room temperature for 30 min. Petroleum ether layer was decanted.
More petroleum ether (300 mL) was added and stirred for 15 min. The
solid was collected by suction filtration; washed with water
(5.times.30 mL); dried under reduced pressure over silica gel/KOH
to give
(2E)-N-[4-(4-bromo-3-chloroanilino)pyrido[3,4-d]pyrimidin-6-yl]-4-(dimeth-
ylamino)-2-butenamide (6) (2.71 g, 98%) as beige solid, mp 176-179;
HPLC: 97.8% pure; .sup.1H NMR .delta. [(CD.sub.3).sub.2SO]10.95 (s,
1H), 10.37 (s, 1H), 9.04 (s, 1H), 9.00 (s, 1H), 8.68 (s, 1H), 8.27
(br s, 1H), 7.84 (br d, J=8.3 Hz, 1H), 7.78 (d, J=8.8, 1H), 6.87
(dt, J=15.5, 6.0 Hz, 1H), 6.52 (br d, J=15.5 Hz, 1H), 3.09 (br d,
J=5.4 Hz, 2H), 2.18 (s, 6H). Anal. Calcd for
C.sub.19H.sub.18BrClN.sub.6O.H.sub.2O: C, 47.57; H, 4.20; N, 17.52.
Found C, 47.95; H, 4.16; N, 17.31.
1.1.1.7 Preparation of
(2E)-N-[4-(3,4-dibromoanilino)pyrido[3,4-d]pyrimidin-6-yl]-4-(dimethylami-
no)-2-butenamide (7) (Scheme 2)
[0284] A heterogeneous mixture of
6-fluoropyrido[3,4-d]pyrimidin-4(3H)-one (200) (1.65 g, 10.0 mmol),
thionyl chloride (20 mL) and a catalytic amount of DMF (2 drops)
was stirred under reflux for 5 h to give a homogeneous mixture. It
was evaporated under reduced pressure at 50.degree. C. (bath
temperature) to give a light brown solid. To this solid was added a
solution of 3,4-dibromoaniline (2.76 g, 11.0 mmol) in dry DMA (15
mL). The residue of 3,4-dibromoaniline was washed down with more
DMA (2.times.2 mL). The reaction mixture was stirred at room
temperature for 19 h. It was poured into water (300 mL). The pH was
adjusted to ca. 8 using an aqueous solution of Na.sub.2CO.sub.3 at
room temperature. Petroleum ether (300 mL) was added and stirred at
room temperature for 30 min. The petroleum ether layer was
decanted. It was repeated once more with petroleum ether. The solid
was collected by filtration and washed successively with water
(5.times.25 mL) and petroleum ether/ethyl acetate (10:1)
(4.times.25 mL). It was dried in vacuum over silica gel/KOH
overnight to give
N-(3,4-dibromophenyl)-6-fluoropyrido[3,4-d]pyrimidin-4-amine (207)
(3.50 g, 88%) as a beige solid, mp 262-264.degree. C.; .sup.1H NMR
.delta. [(CD.sub.3).sub.2SO]10.14 (br s, 1H), 8.98 (s, 1H), 8.78
(s, 1H), 8.45 (br s, 1H), 8.26 (s, 1H), 7.91 (br d, J=7.8, 2.5 Hz,
1H), 7.80 (d, J=8.7 Hz, 1H). Anal. Calcd for
C.sub.13H.sub.7Br.sub.2FN.sub.4: C, 39.23; H, 1.77; N, 14.08. Found
C, 39.23; H, 1.79; N, 13.86.
[0285] A mixture of compound 207 (3.40 g, 8.54 mmol) and
4-methoxybenzylamine (11.2 mL, 85.4 mmol) in dry DMSO (22 mL) was
stirred under a nitrogen atmosphere at 70.degree. C. (bath
temperature) for 138 h. The mixture was then cooled and poured into
water (400 mL). Petroleum ether (400 mL) was added and stirred at
room temperature for 30 min. The layers were allowed to separate
and the petroleum ether layer was decanted. This procedure was
repeated with more petroleum ether (400 mL). It was filtered and
washed solid with water (3.times.30 mL). The sticky yellow/orange
solid was stirred with acetone (100 mL) at room temperature for ca.
30 min. to give a fine orange precipitate. Water (200 mL) was added
and stirred further at room temperature for 2 h. The solid was
collected by suction-filtration, washed with water/acetone (2:1)
(5.times.30 mL), and dried in vacuum over silica-gel/KOH to give
pure
N.sup.4-(3,4-dibromophenyl)-N.sup.6-(4-methoxybenzyl)pyrido[3,4-d]pyrimid-
ine-4,6-diamine (216) (4.16 g, 95%) as a greenish yellow solid, mp
166-169.degree. C.; .sup.1H NMR .delta. [(CD.sub.3).sub.2SO]9.72
(s, 1H), 8.77 (s, 1H), 8.42 (s, 1H), 8.40 (br s, 1H), 7.89 (br d,
J=7.7 Hz, 1H), 7.76 (d, J=8.7, Hz, 1H), 7.40-7.25 (m, 3H), 7.17 (s,
1H), 6.88 (br d, J=8.3 Hz, 2H), 4.49 (d, J=5.9 Hz, 2H), 3.71 (s,
3H). Anal. Calcd for C.sub.21H.sub.17Br.sub.2N.sub.5O.0.25hexane:
C, 50.35; H, 3.85; N, 13.05%. Found C, 50.56; H, 3.54; N,
12.87%.
[0286] To a stirred heterogeneous mixture of compound 216 (4.08 g,
7.92 mmol) and DCM (80 mL) was added trifluoroacetic acid (6.07 mL,
79.2 mmol), followed by anisole (1.73 mL, 15.8 mmol), and the
mixture was stirred further at room temperature for 46 h. It was
poured into petroleum ether (500 mL) and stirred at room
temperature for ca. 15 min. Petroleum ether layer was decanted and
discarded. The process was repeated with more petroleum ether (400
mL). The solid left behind was dissolved in minimum MeOH (120 mL)
and 5M NH.sub.3 (150 mL) was added at 0.degree. C. and stirred at
room temperature for 15 min. The solid was collected and washed
successively with water (5.times.20 mL), petroleum ether-ethyl
acetate=5:1 (5.times.20 mL), and dried to give
N.sup.4-(3,4-dibromophenyl)pyrido[3,4-]pyrimidine-4,6-diamine (225)
(2.47 g, 79%), mp 265-268.degree. C.; .sup.1H NMR .delta.
[(CD.sub.3).sub.2SO]9.80 (s, 1H), 8.72 (s, 1H), 8.44 (d, J=2.2 Hz,
1H), 8.42 (s, 1H), 7.90 (dd, J=8.8, 2.2 Hz, 1H), 7.74 (d, J=8.8 Hz,
1H), 7.14 (s, 1H), 6.30 (s, 2H). Anal. Calcd for
C.sub.13H.sub.9Br.sub.2N.sub.5.0.1hexane: C, 40.47; H, 2.60; N,
17.35. Found C, 40.16; H, 2.45; N, 17.20.
[0287] To a stirred mixture of CDI (2.74 g, 16.9 mmol) and dry THF
(13 mL), at room temperature and under a nitrogen atmosphere, was
added a solution of 2-(diethoxyphosphoryl)acetic acid (2.96 g, 15.1
mmol) in THF (10 mL). After addition the reaction mixture was
stirred further at 40.degree. C. (bath) for 15 min (whence
evolution of gases ceased). A solution of compound 225 (2.38 g,
6.02 mmol) in a mixed solvents of dry THF (7 mL) and DMA (8 mL) was
added and stirred further at 40.degree. C. The reaction was
monitored by TLC (dichloromethane-MeOH=15:1). After stirred for 4
h, the reaction mixture was poured into water (250 mL) and stirred
with petroleum ether (350 mL) at room temperature for 20 min.
Petroleum ether layer was decanted. It was repeated once more with
petroleum ether (200 mL). The solid was collected by suction
filtration; washed with water (5.times.30 mL) and dried to give
diethyl
2-(4-(3,4-dibromophenylamino)pyrido[3,4-d]pyrimidin-6-ylamino)-2-oxoethyl-
phosphonate (234) (3.28 g, 95%); mp 120-124.degree. C.; .sup.1H NMR
.delta. [(CD.sub.3)SO]: 10.89 (s, 1H), 10.41 (s, 1H), 9.04 (s, 1H),
8.88 (s, 1H), 8.67 (s, 1H), 8.34 (d, J=2.5 Hz, 1H), 7.87 (dd,
J=8.8, 2.5 Hz, 1H), 7.77 (d, J=8.8 Hz, 1H), 4.17-4.03 (m, 4H), 3.32
(d, J=21.6 Hz, 2H), 1.26 (t, J=7.0 Hz, 6H). Anal. Calcd for
C.sub.19H.sub.20Br.sub.2N.sub.5O.sub.4, P: C, 39.81; H, 3.52; N,
12.22%. Found C, 39.48; H, 3.78; N, 11.79%.
[0288] To a stirred mixture of 2,2-diethoxy-N,N-dimethylethanamine
(2.25 g, 14.0 mmol) and water (2.4 mL) at room temperature and
under a nitrogen atmosphere was added an aq. 37% HCl (2.35 mL, 28.0
mmol). After addition the mixture was stirred at 45.degree. C.
(bath) for 23 h. It was cooled to 0.degree. C. (bath). This called
solution A. KOH (4.02 g, 71.7 mmol) was dissolved in water (12 mL)
at room temperature under a nitrogen atmosphere. It was cooled to
0.degree. C. (bath). This called solution B. To a stirred
heterogeneous mixture of compound 234 (3.21 g, 5.60 mmol) and THF
(12 mL) at room temperature and under a nitrogen atmosphere was
added minimum amount of DMA (6 mL) to give a homogeneous solution.
LiCl (237 mg, 5.60 mmol) was added and stirred at 0.degree. C.
(bath) for 15 min. The cold solution B was added and stirred at
0.degree. C. for 2 min. Then the cold solution A was added and the
final reaction mixture was continued to stir at 0.degree. C. under
a nitrogen atmosphere. The reaction was monitored by TLC
(DCM-MeOH=25:1). After 1 h it was poured into water (200 mL).
Petroleum ether (200 mL) was added and stirred at room temperature
for 30 min. Petroleum ether layer was decanted. More petroleum
ether (200 mL) was added and stirred for 15 min. The solid was
collected by suction filtration; washed with water (5.times.20 mL);
dried under reduced pressure over silica gel/KOH to give
(2E)-N-[4-(3,4-dibromoanilino)pyrido[3,4-d]pyrimidin-6-yl]-4-(dimethylami-
no)-2-butenamide (7) (2.76 g, 98%) as a beige solid, mp
176-179.degree. C.; HPLC: 97.2% pure; .sup.1H NMR .delta.
[(CD.sub.3).sub.2SO]10.95 (s, 1H), 10.35 (s, 1H), 9.03 (s, 1H),
9.00 (s, 1H), 8.67 (br s, 1H), 8.37 (br s, 1H), 7.89 (br d, J=7.8
Hz, 1H), 7.77 (d, J=8.8, 1H), 6.87 (dt, J=15.4, 6.0 Hz, 1H), 6.52
(br d, J=15.4 Hz, 1H), 3.08 (br d, J=5.3 Hz, 2H), 2.19 (s, 6H).
Anal. Calcd for C.sub.19H.sub.18Br.sub.2N.sub.6O.0.5H.sub.2O: C,
44.30; H, 3.72; N, 16.31. Found C, 44.24; H, 3.81; N, 15.98.
1.1.1.8 Preparation of
(2E)-4-(dimethylamino)-N-[4-(3-ethynyl-4-fluoroanilino)pyrido[3,4-d]pyrim-
idin-6-yl]-2-butenamide (8) (Scheme 2)
[0289] A heterogeneous mixture of
6-fluoropyrido[3,4-d]pyrimidin-4(3H)-one (200) (1.65 g, 10.0 mmol),
thionyl chloride (20 mL) and a catalytic amount of DMF (2 drops)
was stirred under reflux for 1 h to give a homogeneous mixture. It
was evaporated under reduced pressure at 45.degree. C. (bath
temperature) to give a light brown solid. To this solid was added a
solution of 3-ethynyl-4-fluoroaniline (J. Org. Chem., 1981, 46,
2280-2286) (1.49 g, 11.0 mmol) and dry DMA (15 mL). The residue of
3-ethynyl-4-fluoroaniline was washed down with more DMA (2.times.2
mL). The reaction mixture was stirred at room temperature for 45 h.
It was poured into water (300 mL). The pH was adjusted to ca. 9
using an aqueous solution of Na.sub.2CO.sub.3 at room temperature.
Petroleum ether (300 mL) was added and stirred at room temperature
for 30 min. The petroleum ether layer was decanted. It was repeated
once more with petroleum ether (300 mL). The solid was collected by
filtration and washed with water (5.times.50 mL); ethyl
acetate/petroleum ether (1:10) (4.times.50 mL) and dried to give
N-(3-ethynyl-4-fluorophenyl)-6-fluoropyrido[3,4-d]pyrimidin-4-amine
(208) (2.39 g, 85%) as a pale brown solid, mp 223-226.degree. C.;
.sup.1H NMR .delta. [(CD.sub.3).sub.2SO]10.10 (s, 1H), 8.96 (s,
1H), 8.73 (s, 1H), 8.24 (br s, 1H), 8.12 (dd, J=6.4, 2.8 Hz, 1H),
7.96-7.88 (m, 1H), 7.39 (t, J=9.1 Hz, 1H), 4.54 (s, 1H). Anal.
Calcd for C.sub.15H.sub.8F.sub.2N.sub.4.1.8H.sub.2O: C, 57.25; H,
3.72; N, 17.80. Found C, 57.27; H, 3.49; N, 17.90.
[0290] A mixture of compound 208 (2.31 g, 8.19 mmol) and
4-methoxybenzylamine (10.7 mL, 81.9 mmol) in dry DMSO (20 mL) was
stirred under a nitrogen atmosphere at 70.degree. C. (bath
temperature) for 118 h. The mixture was cooled and petroleum ether
(300 mL) was added. It was stirred at room temperature for 15 min.
The layers were allowed to separate and the petroleum ether layer
was decanted. This procedure was repeated with more petroleum ether
(300 mL). Water (300 mL) was added and the mixture was stirred at
room temperature for 20 min. It was filtered and washed sticky
solid with water (5.times.30 mL). The sticky solid was stirred with
warm MeOH (70 mL) for 30 min. After cooled to room temperature, the
solid was collected; washed with cold MeOH (3.times.10 mL) and
dried in vacuum over silica-gel/KOH to give pure
N.sup.4-(3-ethynyl-4-fluorophenyl)-N.sup.6-(4-methoxybenzyl)pyrido[3,4-d]-
pyrimidine-4,6-diamine (217) (0.92 g, 28%) as a yellow/orange
solid, mp 197-199.degree. C. The filtrate was evaporated to dry to
give an orange/amber gum was which applied to a silica column
[ethyl acetate/petroleum ether (2:1)] to afford more compound 217
(0.96 g, 29%). .sup.1H NMR .delta. [(CD.sub.3).sub.2SO]9.66 (s,
1H), 8.74 (s, 1H), 8.36 (s, 1H), 8.08-8.02 (m, 1H), 7.93-7.85 (m,
1H), 7.39-7.13 (m, 5H), 6.88 (br d, J=8.7 Hz, 2H), 4.54-4.44 (m,
3H), 3.71 (s, 3H). Anal. Calcd for
C.sub.23H.sub.18FN.sub.5O.0.15MeOH: C, 68.79; H, 4.64; N, 17.33%.
Found C, 69.09; H, 4.62; N, 16.97%.
[0291] To a stirred heterogeneous mixture of compound 217 (2.12 g,
5.32 mmol) and DCM (55 mL) was added trifluoroacetic acid (3.70 mL,
48.3 mmol), followed by anisole (1.17 mL, 10.6 mmol), and the
mixture was stirred further at room temperature for 78 h. It was
poured into petroleum ether (300 mL) and stirred at room
temperature for ca. 20 min. Petroleum ether layer was decanted and
discarded. The process was repeated with more petroleum ether (300
mL). The solid left behind was dissolved in acetone (25 mL) and 5M
NH.sub.3 (100 mL) added at 0.degree. C. and stirred at room
temperature for 40 min. The solid was collected and washed
successively with acetone/water (1:5) (5.times.20 mL), petroleum
ether/ethyl acetate=3:1 (5.times.20 mL), and dried to give
N.sup.4-(3-ethynyl-4-fluorophenyl)pyrido[3,4-d]pyrimidine-4,6-diamine
(226) (1.31 g, 89%), mp 218-222.degree. C.; .sup.1H NMR .delta.
[(CD.sub.3).sub.2SO]9.72 (s, 1H), 8.70 (s, 1H), 8.36 (s, 1H), 8.10
(dd, J=6.4, 2.7 Hz, 1H), 7.96-7.87 (m, 1H), 7.33 (t, J=9.1 Hz, 1H),
7.13 (s, 1H), 6.26 (s, 2H), 4.50 (s, 1H). Anal. Calcd for
C.sub.15H.sub.10FN.sub.5.0.8H.sub.2O.0.25 ethyl acetate: C, 60.87;
H, 4.34; N, 22.18%. Found C, 60.67; H, 4.15; N, 22.11%.
[0292] To a stirred mixture of CDI (2.01 g, 12.4 mmol) and dry THF
(9 mL), at room temperature and under a nitrogen atmosphere, was
added a solution of 2-(diethoxyphosphoryl)acetic acid (2.18 g, 11.1
mmol) in THF (7 mL). After addition the reaction mixture was
stirred further at 40.degree. C. (bath) for 15 min (whence
evolution of gases ceased). A mixture of compound 226 (1.24 g, 4.44
mmol) in a mixed solvents of dry THF (5 mL) and DMA (6 mL) was
added and stirred further at 40.degree. C. The reaction was
monitored by TLC (dichloromethane-MeOH=30:1). After stirred for 22
h, it was poured into water (200 mL) and the residue washed down
with MeOH. Petroleum ether (300 mL) was added and stirred at room
temperature for 1 h. Petroleum ether layer was decanted. It was
repeated once more with petroleum ether (150 mL). The solid was
collected by suction filtration; washed with water (5.times.20 mL)
and dried to give diethyl
2-(4-(3-ethynyl-4-fluorophenylamino)pyrido[3,4-d]pyrimidin-6-ylam-
ino)-2-oxoethylphosphonate (235) (1.85 g, 91%); mp 103-106.degree.
C.; .sup.1H NMR .delta. [(CD.sub.3).sub.2SO]: 10.84 (s, 1H), 10.33
(s, 1H), 9.01 (s, 1H), 8.86 (s, 1H), 8.61 (s, 1H), 8.01 (dd, J=6.4,
2.7 Hz, 1H), 7.92-7.83 (m, 1H), 7.35 (t, J=9.1 Hz, 1H), 4.51 (s,
1H), 4.17-4.03 (m, 4H), 3.32 (d, partially obscured by water peak,
2H), 1.26 (t, J=7.0 Hz, 6H). Anal. Calcd for
C.sub.21H.sub.21FN.sub.5O.sub.4P.MeOH: C, 53.99; H, 5.15; N,
14.31%. Found C, 54.21; H, 4.94; N, 14.43%.
[0293] To a stirred mixture of 2,2-diethoxy-N,N-dimethylethanamine
(1.56 g, 9.70 mmol) and water (1.6 mL) at room temperature and
under a nitrogen atmosphere was added an aq. 37% HCl (1.62 mL, 19.3
mmol). After addition the mixture was stirred at 45.degree. C.
(bath) for 25 h. It was cooled to 0.degree. C. (bath). This called
solution A. KOH (2.76 g, 49.3 mmol) was dissolved in water (8 mL)
at room temperature under a nitrogen atmosphere. It was cooled to
0.degree. C. (bath). This called solution B. To a stirred
heterogeneous mixture of compound 235 (1.76 g, 3.85 mmol) and THF
(8 mL) at room temperature and under a nitrogen atmosphere was
added minimum amount of DMA (4 mL) to give a homogeneous solution.
LiCl (163 mg, 3.85 mmol) was added and stirred at 0.degree. C.
(bath) for 15 min. The cold solution B was added and stirred at
0.degree. C. for 2 min. Then the cold solution A was added and the
final reaction mixture was continued to stir at 0.degree. C. under
a nitrogen atmosphere. The reaction was monitored by TLC
(DCM-MeOH=10:1). After stirred for 30 min. it was poured into water
(300 mL). Petroleum ether (300 mL) was added and stirred at room
temperature for 10 min. Petroleum ether layer was decanted. More
petroleum ether (300 mL) was added and stirred for 10 min. The
solid was collected by suction filtration; washed with water
(4.times.20 mL); dried under reduced pressure over silica gel/KOH
to give compound 8 (1.38 g, 92%) which was found only 89.7% pure by
HPLC. Thus, the sample was stirred with MeOH (ca. 40 mL) for 30
min. The insoluble material was filtered off and to the filtrates
was added one volume of water to precipitate out the required
product. The solid was collected, washed with water/MeOH=3:1
(3.times.15 mL) and dried to give
(2E)-4-(dimethylamino)-N-[4-(3-ethynyl-4-fluoroanilino)pyrido[3,4-d]pyrim-
idin-6-yl]-2-butenamide (8) (1.28 g, 85%) as a yellow/brown solid,
mp 152-155.degree. C.; .sup.1H NMR .delta.
[(CD.sub.3).sub.2SO]10.93 (s, 1H), 10.29 (s, 1H), 9.02 (s, 1H),
8.99 (s, 1H), 8.61 (s, 1H), 8.04 (dd, J=6.4, 2.7 Hz, 1H), 7.94-7.85
(m, 1H), 7.35 (t, J=9.1 Hz, 1H), 6.87 (dt, J=15.4, 6.0 Hz, 1H),
6.52 (br d, J=15.4 Hz, 1H), 4.53 (s, 1H), 3.09 (br d, J=6.0 Hz,
2H), 2.19 (s, 6H). Anal. Calcd for
C.sub.21H.sub.19FN.sub.6O.0.8H.sub.2O.0.7 MeOH: C, 61.00; H, 5.52;
N, 19.67%. Found C, 61.26; H, 5.25; N, 19.47%.
1.1.1.9 Preparation of
(2E)-N-[4-(4-chloro-3-ethynylanilino)pyrido[3,4-d]pyrimidin-6-yl]-4-(dime-
thylamino)-2-butenamide (9) (Scheme 2)
[0294] A heterogeneous mixture of
6-fluoropyrido[3,4-d]pyrimidin-4(3H)-one (200) (1.65 g, 10.0 mmol),
thionyl chloride (20 mL) and a catalytic amount of DMF (2 drops)
was stirred under reflux for 24 h to give a homogeneous mixture. It
was evaporated under reduced pressure at 45.degree. C. (bath
temperature) to give a light brown solid. To this solid was added a
solution of 3-ethynyl-4-chloroaniline (J. Org. Chem., 1981, 46,
2280-2286) (1.67 g, 11.0 mmol) and dry DMA (15 mL). The residue of
3-ethynyl-4-chloroaniline was washed down with more DMA (2.times.2
mL). The reaction mixture was stirred at room temperature for 24 h.
It was poured into water (300 mL) and the residue washed down with
MeOH. The pH was adjusted to ca. 9 using an aqueous solution of
Na.sub.2CO.sub.3 at room temperature. Petroleum ether (300 mL) was
added and stirred at room temperature for 30 min. The petroleum
ether layer was decanted. It was repeated once more with petroleum
ether (300 mL). The solid was collected by filtration and washed
with water (5.times.25 mL); ethyl acetate/petroleum ether (1:10)
(4.times.25 mL) and dried to give
N-(3-ethynyl-4-chlorophenyl)-6-fluoropyrido[3,4-d]pyrimidin-4-amine
(209) (3.0 g, 100%) as a pale brown solid, mp 218-222.degree. C.;
.sup.1H NMR .delta. [(CD.sub.3).sub.2SO]10.13 (s, 1H), 8.98 (s,
1H), 8.77 (s, 1H), 8.32-8.22 (m, 2H), 7.95 (dd, J=8.8, 2.6 Hz, 1H),
7.61 (d, J=8.8 Hz, 1H), 4.60 (s, 1H). Anal. Calcd for
C.sub.15H.sub.8ClFN.sub.4.0.5H.sub.2O.0.5 MeOH: C, 57.51; H, 3.43;
N, 17.31. Found C, 57.33; H, 3.13; N, 17.10.
[0295] A mixture of compound 209 (3.07 g, 10.3 mmol) and
4-methoxybenzylamine (13.5 mL, 103 mmol) in dry DMSO (27 mL) was
stirred under a nitrogen atmosphere at 70.degree. C. (bath
temperature) for 141 h. The mixture was cooled and petroleum ether
(300 mL) was added. It was stirred at room temperature for 30 min.
The layers were allowed to separate and the petroleum ether layer
was decanted. This procedure was repeated with more petroleum ether
(300 mL). Water (300 mL) was added and the mixture was stirred at
room temperature for 30 min. It was filtered and washed sticky
solid with water (4.times.20 mL). The sticky solid was dissolved in
minimum acetone (50 mL) and water (200 mL) added. The mixture was
stirred at room temperature overnight (22 h). Sticky solid
generated again. Thus, the product was extracted into ethyl
acetate, which after washed with water several times, was dried
(MgSO.sub.4) and evaporated. The crude product was applied to a
silica column (dichloromethane/MeOH=50:1) to give
N.sup.4-(4-chloro-3-ethynylphenyl)-N.sup.6-(4-methoxybenzyl)pyrido[3,4-d]-
pyrimidine-4,6-diamine (218) (2.30 g, 54%) as a greenish yellow
solid, mp 201-203.degree. C.; .sup.1H NMR .delta.
[(CD.sub.3).sub.2SO]9.72 (s, 1H), 8.76 (s, 1H), 8.40 (s, 1H), 8.19
(d, J=2.5 Hz, 1H), 7.96 (dd, J=8.9, 2.5 Hz, 1H), 7.56 (d, J=8.9 Hz,
1H), 7.39-7.28 (m, 3H), 7.17 (s, 1H), 6.93-6.82 (m, 2H), 4.59 (s,
1H), 4.49 (d, J=6.3 Hz, 2H), 3.71 (s, 3H). Anal. Calcd for
C.sub.23H.sub.18ClN.sub.5O.0.18hexane: C, 67.04; H, 4.80; N,
16.23%. Found C, 66.76; H, 4.79; N, 15.96%.
[0296] To a stirred heterogeneous mixture of compound 218 (2.27 g,
5.46 mmol) and DCM (55 mL) was added trifluoroacetic acid (4.2 mL,
54.6 mmol), followed by anisole (1.2 mL, 10.9 mmol), and the
mixture was stirred further at room temperature for 50 h. It was
poured into petroleum ether (400 mL) and stirred at room
temperature for ca. 30 min. Petroleum ether layer was decanted and
discarded. The process was repeated with more petroleum ether (400
mL). The solid left behind was dissolved in acetone (30 mL) and 5M
NH.sub.3 (150 mL) added at 0.degree. C. and stirred at room
temperature for 20 min. The solid was collected and washed
successively with acetone/water (1:5) (5.times.20 mL), petroleum
ether/ethyl acetate=10:1 (5.times.20 mL), and dried to give
N.sup.4-(4-chloro-3-ethynylphenyl)pyrido[3,4-d]pyrimidine-4,6-diamine
(227) (1.54 g, 95%), mp 210-213.degree. C.; .sup.1H NMR .delta.
[(CD.sub.3).sub.2SO]9.77 (s, 1H), 8.71 (s, 1H), 8.40 (s, 1H), 8.24
(d, J=2.6 Hz, 1H), 7.97 (dd, J=8.8, 2.6 Hz, 1H), 7.55 (d, J=8.8 Hz,
1H), 7.14 (s, 1H), 6.29 (s, 2H), 4.56 (s, 1H). Anal. Calcd for
C.sub.15H.sub.10ClN.sub.5.1.3H.sub.2O.0.5 acetone: C, 56.92; H,
4.52; N, 20.11%. Found C, 56.81; H, 4.22; N, 19.82%.
[0297] To a stirred mixture of CDI (2.14 g, 13.2 mmol) and dry THF
(10 mL), at room temperature and under a nitrogen atmosphere, was
added a solution of 2-(diethoxyphosphoryl)acetic acid (2.31 g, 11.8
mmol) in THF (8 mL). After addition the reaction mixture was
stirred further at 40.degree. C. (bath) for 15 min (whence
evolution of gases ceased). A mixture of compound 227 (1.39 g, 4.70
mmol) in a mixed solvents of dry THF (6 mL) and DMA (7 mL) was
added and stirred further at 40.degree. C. The reaction was
monitored by TLC (dichloromethane-MeOH=15:1). After stirred for 3
h, it was poured into water (300 mL) and the residue washed down
with MeOH. Petroleum ether (300 mL) was added and stirred at room
temperature for min. Petroleum ether layer was decanted. It was
repeated once more with petroleum ether (200 mL). The solid was
collected by suction filtration; washed with water (5.times.30 mL)
and dried to give diethyl
2-(4-(4-chloro-3-ethynylphenylamino)pyrido[3,4-d]pyrimidin-6-ylam-
ino)-2-oxoethylphosphonate (236) (2.03 g, 91%); mp 124-127.degree.
C.; .sup.1H NMR .delta. [(CD.sub.3).sub.2SO]: 10.86 (s, 1H), 10.37
(s, 1H), 9.03 (s, 1H), 8.87 (s, 1H), 8.65 (s, 1H), 8.14 (br s, 1H),
7.93 (br d, J=8.8 Hz, 1H), 7.35 (d, J=8.8 Hz, 1H), 4.58 (s, 1H),
4.20-4.00 (m, 4H), 3.30 (d, partially obscured by water peak, 2H),
1.26 (t, J=7.0 Hz, 6H). Anal. Calcd for
C.sub.21H.sub.21ClN.sub.5O.sub.4P.H.sub.2O.0.2 THF: C, 51.72; H,
4.90; N, 13.83%. Found C, 52.06; H, 4.82; N, 13.70%.
[0298] To a stirred mixture of 2,2-diethoxy-N,N-dimethylethanamine
(1.67 g, 10.4 mmol) and water (1.8 mL) at room temperature and
under a nitrogen atmosphere was added an aq. 37% HCl (1.75 mL, 20.8
mmol). After addition the mixture was stirred at 45.degree. C.
(bath) for 27 h. It was cooled to 0.degree. C. (bath). This called
solution A. KOH (2.98 g, 53.2 mmol) was dissolved in water (9 mL)
at room temperature under a nitrogen atmosphere. It was cooled to
0.degree. C. (bath). This called solution B. To a stirred
heterogeneous mixture of compound 236 (1.97 g, 4.16 mmol) and THF
(9 mL) at room temperature and under a nitrogen atmosphere was
added minimum amount of DMA (4.5 mL) to give a homogeneous
solution. LiCl (176 mg, 4.16 mmol) was added and stirred at
0.degree. C. (bath) for 15 min. The cold solution B was added and
stirred at 0.degree. C. for 2 min. Then the cold solution A was
added and the final reaction mixture was continued to stir at
0.degree. C. under a nitrogen atmosphere. The reaction was
monitored by TLC (DCM-MeOH=10:1). After stirred for 35 min. it was
poured into water (300 mL). Petroleum ether (300 mL) was added and
stirred at room temperature for 15 min. Petroleum ether layer was
decanted. More petroleum ether (300 mL) was added and stirred for
10 min. The solid was collected by suction filtration; washed with
water (5.times.20 mL); petroleum ether/ethyl acetate=10:1
(3.times.30 mL); dried under reduced pressure over silica gel/KOH
to give compound 9 (1.61 g, 95%) which was found only 85.2% pure by
HPLC. Thus, the sample was purified by a silica column (ethyl
acetate/MeOH=10:1). The fractions containing the required product
were combined and evaporated to give a yellow/orange solid. The
solid was dissolved in warm ethyl acetate (30 mL) and precipitated
with petroleum ether (90 mL). Solid was collected; washed with
petroleum ether/ethyl acetate=3:1 (3.times.20 mL) and dried to give
(2E)-N-[4-(4-chloro-3-ethynylanilino)pyrido[3,4-d]pyrimidin-6-yl]-
-4-(dimethylamino)-2-butenamide (9) (1.1 g, 65%) as a yellow/orange
solid, mp 173-176.degree. C.; .sup.1H NMR .delta.
[(CD.sub.3).sub.2SO]10.92 (s, 1H), 10.31 (s, 1H), 9.03 (s, 1H),
9.00 (s, 1H), 8.66 (s, 1H), 8.18 (d, J=2.2 Hz, 1H), 7.96 (dd,
J=8.9, 2.2 Hz, 1H), 7.57 (d, J=8.9 Hz, 1H), 6.88 (dt, J=15.4, 6.0
Hz, 1H), 6.52 (br d, J=15.4 Hz, 1H), 4.58 (s, 1H), 3.09 (br d,
J=5.8 Hz, 2H), 2.19 (s, 6H). Anal. Calcd for
C.sub.21H.sub.19ClN.sub.6O.0.8MeOH.0.1ethyl acetate: C, 60.42; H,
5.25; N, 19.04%. Found C, 60.15; H, 4.90; N, 18.76%.
1.1.2 the Synthesis of .alpha.-Methyl Bromide Triggers
1.1.2.1 Preparation of
5-(bromomethyl)-1-methyl-4-nitro-1H-imidazole (239) (Scheme 3)
Method 1
[0299] To a suspension of compound 237 (20.0 g, 157.36 mmol)
(prepared according to the method of Chauviere et al, J. Med. Chem.
2003, 46, 427-440) and K.sub.2CO, (32.62 g, 236.04 mmol) in DMF
(200 mL) at 0.degree. C. was added methyl iodide (14.70 mL, 236.04
mmol) dropwise. The resulting mixture was allowed to warm to room
temperature and then stirred for 2 hours before the excess methyl
iodide was evaporated at room temperature. The precipitate was
removed by filtration and the DMF filtrate was concentrated under
reduced pressure at 45-50.degree. C. The residue obtained was
extracted thoroughly with MeCN/DCM (1:9) and the combined extracts
were filtered through a short column of silica gel. After solvents
were removed the crude was recrystallised from MeCN and toluene to
give compound 238 as an off-white crystalline solid (15.74 g, 71%),
m.p. 161-163.degree. C. .sup.1H NMR (CDCl.sub.3) .delta. 7.33 (s,
1H), 3.65 (s, 3H), 2.63 (s, 3H). Identical to that previously
reported (Hosmane et al, J. Org. Chem., 1985, 50(26), 5892-5).
[0300] A solution compound 238 (4.00 g, 28.34 mmol) and NBS (5.30
g, 29.78 mmol) in MeCN (200 mL) was irradiated at reflux for 2
hours with a 1000 W tungsten halide lamp. Approximately half of the
solvent was removed in vacuo before water (100 mL) was added.
Further concentration under reduced pressure afforded a white
precipitate, which was collected by filtration, washed with water
and dried under vacuum to give
5-(bromomethyl)-1-methyl-4-nitro-1H-imidazole (239) (4.69 g, 75%)
as a white solid, m.p. 130-132.degree. C. .sup.1H NMR (CDCl.sub.3,
400 MHz) .delta. 7.74 (s, 1H), 4.50 (s, 2H), 3.83 (s, 3H).
Identical to that previously reported (Stribbling et al, PCT
International patent publication WO 2008/039087). Analysis found:
C, 27.81; H, 3.27; N, 19.05.
C.sub.5H.sub.6BrN.sub.3O.sub.2.0.04hexane requires: C, 28.16; H,
2.96; N, 18.80. HRMS (FAB+) found: 219.97220, 221.97018 (M+1),
calcd. for C.sub.5H.sub.7.sup.79/81BrN.sub.3O.sub.2: 219.97216,
221.97012.
Method 2
[0301] Compound 237 (120 g, 0.94 mol), K.sub.2CO.sub.3 (259.8 g,
1.88 mol), and acetonitrile (2 L) were charged to a 5-L 3-neck
flask and the mixture was cooled to 0.degree. C. with stirring at
200 rpm. Dimethyl sulphate (DMS) (97.8 mL, 1.03 mol) was slowly
added by syringe pump over 2 hours. After 2 hours, the reaction was
allowed to warm to room temperature and held overnight. The solids
were filtered over celite (120 g) and the 5-L flask was washed with
acetonitrile (200 mL). The solids were washed with acetonitrile
(2.times.500 mL) until all product was removed. The solution was
diluted with 75% brine (1 L) and the acetonitrile was removed by
rotary evaporation. The resulting slurry was extracted with
dichloromethane (4.times.1 L). The combined organic layers were
filtered, before toluene (1 L) was added and the dichloromethane
was removed by rotary evaporation. The resulting slurry was
filtered and the cake washed with toluene (2.times.1 L). The wet
solid was recovered as an off-white solid (121 g). The wet solid
was recrystallized from water (1 L) and the solids washed with
heptane (1 L). The solid was dried in a 40.degree. C. vacuum oven
overnight. Compound 238 was recovered as a white solid (86.6 g,
67%). .sup.1H NMR identical to that described above.
[0302] A 20-L reactor was fitted with N.sub.2, condenser,
temperature probe, and air stirring. The reactor was charged with
compound 238 (100 g, 0.71 mol), 1,3-dibromo-5,5-dimethylhydantoin
(DBDMH) (203.0 g, 0.71 mol), 2,2'-azobisisobutyronitrile (AIBN)
(45.97 g, 0.28 mol), and dichloromethane (6 L). The reaction was
heated at reflux, stirring the homogenous solution at 150 rpm.
After 100 hours, the reaction was held at 20.degree. C. for 36
hours before quenching the reaction with 10% NaHCO.sub.3 (1.5 L)
and 10% Na.sub.2S.sub.2O.sub.5 (1 L) to pH 7 and negative to
KI-starch paper (Note: some gas evolution and a small exotherm
[6.degree. C.] was observed during the addition of
Na.sub.2S.sub.2O.sub.5). The organic layer was separated and
filtered before water (1 L) was added and the dichloromethane was
removed by rotary evaporation. The resulting slurry was filtered to
give a light yellow solid which was re-suspended in toluene (500
mL) at 50.degree. C. for 2.5 hours before cooling to room
temperature. Filtration then gave a solid that was dried at
40.degree. C. in a vacuum oven overnight, before being slurried in
toluene (500 mL) at 100.degree. C. for 1 hour. After cooling to
room temperature the suspension was filtered to give a solid that
was dried in a 40.degree. C. vacuum oven overnight to give
5-(bromomethyl)-1-methyl-4-nitro-1H-imidazole (239) (63.3 g, 38%)
as a light yellow solid. .sup.1H NMR identical to that described
above.
1.1.2.2 Preparation of
5-(bromomethyl)-1,2-dimethyl-4-nitro-1H-imidazole (244) (Scheme
4)
[0303] To a suspension of compound 240 (50.0 g, 393.39 mmol) and
K.sub.2CO.sub.3 (81.55 g, 590.08 mmol) in DMF (300 mL) at 0.degree.
C. was added methyl iodide (36.74 mL, 590.08 mmol) dropwise. The
resulting mixture was allowed to warm to room temperature and then
stirred for 2 hours before the excess methyl iodide was evaporated
at room temperature. The precipitate was removed by filtration and
the DMF filtrate was concentrated under reduced pressure at
45-50.degree. C. The residue obtained was extracted thoroughly with
MeCN/DCM (1:9) and filtered through a short column of silica gel.
After solvents were removed the crude was recrystallised from MeCN
(containing a small amount of MeOH) and toluene to give compound
241 (52.22 g, 94.0%) as a white crystalline solid. .sup.1H NMR
(CDCl.sub.3) .delta. 7.66 (s, 1H), 3.67 (s, 3H), 2.43 (s, 3H).
LR-MS (APCI+ve): m/e 142.5 (M+1). Identical to that previously
reported (Rav-Acha and Cohen, J. Org. Chem. 1981, 46(23),
4717-4720).
[0304] A solution of compound 241 (33.0 g, 233.83 mmol) and t-butyl
dichloroacetate (64.90 g, 350.74 mmol) (prepared from
dichloroacetyl chloride, t-butanol and triethyl amine in DCM) in
DMF (400 mL) was added dropwise to a suspension of potassium
t-butoxide (91.83 g, 818.40 mmol) in DMF (400 mL) at -35 to
-25.degree. C. (dry-ice/MeCN bath). The resulting mixture was
stirred at -25.degree. C. for an additional 20 minutes before being
poured into 0.5N HCl (approximately 1000 mL). A standard ethyl
acetate/aqueous workup and column chromatography on silica gel
eluting with ethyl acetate/hexane (3:2) then gave crude compound
242 as a dark solid (23.83 g, 35%), which was used without further
purification. LR-MS (APCI+ve) m/e 290.5/292.5 (3:1, M+1).
[0305] Compound 242 as prepared above (23.83 g, 82.25 mmol) was
treated with refluxing acetic acid (120 mL) for 45 minutes before
being concentrated to dryness under reduced pressure. A standard
NaHCO.sub.3/DCM workup of the residue followed by column
chromatography on silica gel eluting with ethyl acetate then gave
compound 243 (10.00 g, 64%) as white solid. .sup.1H NMR
(CDCl.sub.3) .delta. 5.03 (s, 2H), 3.67 (s, 3H), 2.46 (s, 3H).
LR-MS (APCI+ve): m/e 190.4/192.4 (3:1, M+1).
[0306] A suspension of compound 243 (10.00 g, 52.74 mmol) and LiBr
(4.80 g, 55.20 mmol) in ethyl acetate (500 mL) was heated at reflux
for 4 hours before being subjected to a standard ethyl
acetate/aqueous workup. The solid thus obtained was treated once
again with LiBr/ethyl acetate as above. The crude product was then
precipitated from DCM/i-Pr.sub.2O by the addition of hexane, to
give 5-(bromomethyl)-1,2-dimethyl-4-nitro-1H-imidazole (244) (11.46
g, 93%) as an off-white solid. .sup.1H NMR (CDCl.sub.3) 4.88 (s,
2H), 3.64 (s, 3H), 2.46 (s, 3H). Anal. Calcd for
C.sub.5H.sub.6BrN.sub.3O.sub.2.0.04hexane: C, 28.16; H, 2.96; N,
18.80%. Found: C, 27.81; H, 3.27; N, 19.05%. LR-MS (APCI+ve): m/e
234.4/236.4 (1:1, M+1).
1.1.2.3 Preparation of
5-(bromomethyl)-2-ethyl-1-methyl-4-nitro-1H-imidazole (250) (Scheme
5)
[0307] To a suspension of compound 238 (12.65 g, 90.00 mmol) in
chloroform (100 mL), was added bromine (5.53 mL, 108.00 mmol),
slowly. The resulting mixture was then stirred for 2 hours before
water (130 mL) was added. The chloroform was then removed by
distillation and the resulting precipitate was collected by
filtration, washed with water and dried under vacuum to give
compound 245 (15.50 g, 79%) as a white solid, m.p. 180-181.degree.
C., identical to the reported value (Pyman and Timmis, J. Chem.
Soc., Trans., 1923, 123, 494-503). .sup.1H NMR (CDCl.sub.3) 3.63
(s, 3H), 2.69 (s, 3H). Anal. Calcd for
C.sub.5H.sub.6BrN.sub.3O.sub.2: C, 27.29; H, 2.75; N, 19.10%.
Found: C, 27.56; H, 2.83; N, 19.10%. LR-MS (APCI+ve): m/e
220.3/222.3 (1:1, M+1).
[0308] A solution of compound 245 (2.20 g, 10.0 mmol) and
N-bromosuccinimide (NBS) (1.96 g, 11.0 mmol) in acetonitrile (100
mL) was radiated at reflux for 2 hours by a 1000 W tungsten halide
lamp. Approximately half of the solvent was then removed by rotary
evaporator before the same volume of water was added. Further
evaporation afforded a white precipitate, which was collected by
filtration, washed with water and dried under vacuum to give
compound 246 (2.84 g, 95%) as white solid. .sup.1H NMR (CDCl.sub.3,
400 MHz) d 4.88 (s, 2H), 3.74 (s, 3H). Analysis found: C, 20.36; H,
1.74; N, 13.98. C.sub.5H.sub.5Br.sub.2N.sub.3O.sub.2 requires: C,
20.09; H, 1.69; N, 14.06. LR-MS (+): m/e 234.4/236.4 (1:1, M+1).
298.3/300.3/302.3 (1:2:1, M+1).
[0309] To a solution of compound 246 (2.80 g, 9.36 mmol) in DMF (30
mL) was added anhydrous sodium acetate (1.92 g, 23.4 mmol). The
mixture was stirred for 2 hours at room temperature then given a
standard aqueous ethyl acetate workup, to give compound 247 (2.54
g, 98%) as white solid, m. p. 110-112.degree. C. .sup.1H NMR
(CDCl.sub.3, 400 MHz) d 5.50 (s, 2H), 3.74 (s, 3H), 2.11 (s, 3H).
Analysis found: C, 30.48; H, 2.82; N, 15.13.
C.sub.7H.sub.8BrN.sub.3O.sub.4 requires: C, 30.24; H, 2.90; N,
15.11. LR-MS (+): m/e 278.4/280.4 (1:1, M+1).
[0310] A mixture of compound 247 (1.90 g, 6.83 mmol), tetraethyltin
(5.42 mL, 27.34 mmol) and tetrakis(triphenylphosphine)palladium
(790 mg, 0.68 mmol) in NMP (20 mL) was heated at 110-120.degree. C.
for 5 hours before undergoing a standard aqueous ethyl acetate
workup. The crude product obtained was purified by flash column
chromatography eluting with MeCN/DCM (1:5) before being
precipitated from DCM by the addition of hexane, to give compound
248 (1.04 g, 67%) as a white solid, m.p. 71-73.degree. C. .sup.1H
NMR (CDCl.sub.3, 400 MHz) .delta. 5.48 (s, 2H), 3.64 (s, 3H), 2.76
(q, J=7.43 Hz, 2H), 2.10 (s, 3H), 1.37 (t, J=7.43 Hz, 3H). Analysis
found: C, 48.11; H, 5.90; N, 18.23%.
C.sub.9H.sub.13N.sub.3O.sub.4.0.04hexane requires: C, 48.11; H,
5.92; N, 18.22%. LR-MS (+): m/e 228.5 (M+1).
[0311] To the solution of compound 248 (1.25 g, 5.50 mmol) in MeOH
(10 mL) was added dry K.sub.2CO.sub.3 (1.52 g, 11.0 mmol). After
stirring for 20 minutes the solvent was removed at reduced pressure
and the residue was dissolved in DCM, filtered through a layer of
silica gel and washed with ethyl acetate. The filtrate was
concentrated to give white crystals, which were collected by
filtration and washed with a mixture of ethyl acetate/hexane (1:1)
to give compound 249 (949 mg, 93%) as white crystalline solid, m.p.
153-155.degree. C. .sup.1H NMR (CDCl.sub.3, 400 MHz) .delta. 4.96
(d, J=6.80 Hz, 2H), 3.67 (s, 3H), 2.79 (t, J=6.80 Hz, 1H), 2.74 (q,
J=7.50 Hz, 2H), 1.36 (t, J=7.50 Hz, 3H). Analysis found: C, 45.71;
H, 6.07; N, 22.87%. C.sub.7H.sub.11N.sub.3O.sub.3 requires: C,
45.40; H, 5.99; N, 22.68%. LR-MS (+): m/e 186.5 (M+1).
[0312] To the solution of compound 249 (685 mg, 3.70 mmol) in DCM
(30 mL) at 0.degree. C. was added triethylamine (0.773 mL, 5.55
mmol), followed by MsCl (0.344 mL, 4.44 mmol) dropwise. After
stirring for 45 minutes, the mixture was washed twice with
saturated aqueous ammonium chloride and once with brine before
being dried over anhydrous sodium sulphate and filtered through
celite. Concentration of the filtrate in vacuo gave a white solid
(971 mg) which was found by .sup.1H NMR to be a mixture of mesylate
and .alpha.-methyl chloride (3:1) and used directly in the next
step. A solution of this solid (968 mg) in THF (50 mL) was treated
LiBr (6.39 g, 86.85 mmol) at reflux for 0.5 hour. The solvent was
then removed under reduced pressure and the resulting residue was
distributed between water and ethyl acetate. The organic phase was
washed with water twice and brine once before being dried over
anhydrous sodium sulphate and filtered through celite. The solvent
was removed in vacuo to give
5-(bromomethyl)-2-ethyl-1-methyl-4-nitro-1H-imidazole (250) (851
mg, 93%) as white solid, m.p. 91-93.degree. C. .sup.1H NMR
(CDCl.sub.3, 400 MHz) .delta. 4.88 (s, 2H), 3.65 (s, 3H), 2.76 (q,
J=7.60 Hz, 2H), 1.37 (t, J=7.60 Hz, 3H). Analysis found: C, 34.41;
H, 4.07; N, 16.96%. C.sub.7H.sub.10BrN.sub.3O.sub.2.0.04EtOAc
requires: C, 34.18; H, 4.13; N, 16.70%. LR-MS (+): m/e 248.4/250.4
(1:1, M+1).
1.1.2.4 Preparation of
5-(bromomethyl)-1-methyl-4-nitro-1H-imidazole-2-carbonitrile (264)
(Scheme 7)
Method 1
[0313] To a solution of compound 246 (1.40 g, 4.68 mmol) in DMA (14
mL) containing several drops of water, was added K.sub.2CO.sub.3
(647 mg, 4.68 mmol). The resulting solution was stirred over night
before a standard ethyl acetate workup, followed by silica gel
column chromatography eluting with MeCN/DCM (5:95-15:85), gave
compound 262 (330 mg, 30%) as an off-white solid. .sup.1H NMR
(.sup.6d-DMSO, 400 MHz) .delta. 5.56 (t, J=5.8 Hz, 1H), 4.86 (d,
J=5.8 Hz, 2H), 3.70 (s, 3H). LR-MS (+): m/e 236.5/238.5 (1:1,
M+1).
[0314] A mixture of compound 262 (300 mg, 1.27 mmol), Zn(CN).sub.2
(90 mg, 0.76 mmol), Zinc powder (10 mg, 0.15 mmol),
Pd.sub.2(dba).sub.3 (23 mg, 0.025 mmol) and dppf (28 mg, 0.051
mmol) in DMA (3 mL) was stirred under nitrogen at 120.degree. C.
for 3.5 hours. A standard aqueous NH.sub.4Cl/ethyl acetate workup
followed by silica gel column chromatography eluting with ethyl
acetate/hexanes (1:1 to 2:1) then gave compound 263 (180 mg) as an
off-white solid, which was found by .sup.1H NMR to contain a small
amount of unreacted starting material 262 and was used directly in
the next step. .sup.1H NMR (CDCl.sub.3, 400 MHz) .delta. 5.09 (d,
J=6.7 Hz, 2H), 4.00 (s, 3H), 2.49 (t, J=6.7 Hz, 1H).
[0315] To the solution of compound 263 (173 mg, ca. 0.93 mmol) in
THF (10 mL) at 0.degree. C. was added MsCl (0.088 mL, 1.14 mmol),
followed by DIPEA (0.182 mL, 1.04 mmol) dropwise. After stirring
for 1 hour, the reaction mixture was subjected to a standard
aqueous NH.sub.4Cl/ethyl acetate workup to give a yellow oil (237
mg; mixture of mesylate and .alpha.-methyl chloride by .sup.1H NMR)
that was used directly. To a solution of this oil (235 mg, ca. 0.90
mmol) in THF (10 mL) was added LiBr (1.57 g, 18.06 mmol). After 0.5
hr heating at reflux the solvent was removed in vacuo and the
residue was subjected to a standard aqueous NH.sub.4Cl/ethyl
acetate workup. The crude product was further purified by silica
gel column chromatography eluting with ethyl acetate/hexanes (1:4
to 1:2) to give
5-(bromomethyl)-1-methyl-4-nitro-1H-imidazole-2-carbonitrile (264)
(65 mg, 21% over three steps) as a pink oil. .sup.1H NMR
(CDCl.sub.3, 400 MHz) .delta. 4.86 (s, 2H), 3.95 (s, 3H). LR-MS
(+): m/e 277.6/279.6 (1:1, M+1+MeOH).
Method 2
[0316] A mixture of compound 245 (1.10 g, 5.00 mmol), Zn(CN).sub.2
(352 mg, 3.00 mmol), zinc powder (39 mg, 0.60 mmol),
Pd.sub.2(dba).sub.3 (92 mg, 0.10 mmol) and dppf (111 mg, 0.20 mmol)
in DMA (10 mL) was stirred under nitrogen at 120.degree. C. for 3
hours. The reaction was then diluted with water and given a
standard ethyl acetate workup, followed by silica gel column
chromatography eluting with ethyl acetate/hexanes (3:4 then 1:1) to
give compound 265 (657 mg, 79%) as an off-white solid, m.p.
99-101.degree. C. .sup.1H NMR (CDCl.sub.3) .delta. 3.84 (s, 3H),
2.72 (s, 3H). Analysis found: C, 43.64; H, 3.58; N, 33.86.
C.sub.6H.sub.6N.sub.4O.sub.2 requires: C, 43.38; H, 3.64; N,
33.72.
[0317] A mixture of compound 265 (166 mg, 1.00 mmol),
1,3-dibromo-5,5-dimethylhydantoin (DBDMH) (286 mg, 1.00 mmol),
2,2'-azobisisobutyronitrile (AIBN) (66 mg, 0.40 mmol) in
dichloromethane (10 mL) was heated at reflux for 5 days before
being concentrated at reduced pressure. The resulting residue was
purified by silica gel column chromatography eluting with ethyl
acetate/hexanes (1:2 then 1:1) to give
5-(bromomethyl)-1-methyl-4-nitro-1H-imidazole-2-carbonitrile (264)
(137 mg, 56%) as colourless oil. .sup.1H NMR (CDCl.sub.3, 400 MHz)
.delta. 4.86 (s, 2H), 3.95 (s, 3H). Analysis found: C, 30.17; H,
1.99; N, 22.25. C.sub.6H.sub.5BrN.sub.4O.sub.2.0.07EtOAc requires:
C, 30.03; H, 2.23; N, 22.30. HRMS (ESI+, .sup.79/81Br) found: m/
266.9490/268.9475 (M+Na), calcd. for
C.sub.6H.sub.5.sup.79/81BrN.sub.4NaO.sub.2.sup.+:
266.9488/268.9468.
1.1.2.5 Preparation of
5-(bromomethyl)-1-methyl-4-nitro-1H-imidazole-2-carboxamide (266)
(Scheme 7)
[0318] A solution of
5-(bromomethyl)-1-methyl-4-nitro-1H-imidazole-2-carbonitrile (264)
(70 mg, 0.29 mmol) in 90% H.sub.2SO (1 mL) was heated at
65-70.degree. C. for 1 hour, before being diluted with water and
given a standard ethyl acetate workup to give
5-(bromomethyl)-1-methyl-4-nitro-1H-imidazole-2-carboxamide (266)
(67 mg, 84%), as white solid, m.p. 220-222.degree. C. .sup.1H NMR
[(CD.sub.3).sub.2SO].delta. 8.18 (s, 1H), 7.87 (s, 1H), 5.05 (s,
2H), 4.02 (s, 3H). HR-MS (APCI+, .sup.79/81Br) found: m/
262.9770/264.9752 (M+1), calcd. for
C.sub.6H.sub.8.sup.79/81BrN.sub.4O.sub.3.sup.+:
262.9774/264.9754.
1.1.2.6 Preparation of
5-(bromomethyl)-1-methyl-4-nitro-2-(1-propynyl)-1H-imidazole (270)
(Scheme 8)
[0319] A mixture of compound 247 (500 mg, 1.80 mmol),
tributyl(1-propynyl)tin (1.64 mL, 5.39 mmol) and
tetrakis(triphenylphosphine)palladium (416 mg, 0.36 mmol) in NMP
(15 mL) was heated at 80.degree. C. overnight (14 hours) before
undergoing a standard aqueous-ethyl acetate workup. The crude
product obtained was further purified by flash column
chromatography eluting with MeCN/DCM (gradient from 1:20 to 1:5) to
give compound 267 (147 mg, 34%) as white solid, .sup.1H NMR
(CDCl.sub.3, 400 MHz) .delta. 5.47 (s, 2H), 3.77 (s, 3H), 2.14 (s,
3H), 2.10 (s, 3H). LR-MS (+): m/le 238.5 (M+1); followed by
compound 268 (105 mg, 30%) also as white solid, .sup.1H NMR
(CDCl.sub.3, 400 MHz) .delta. 4.96 (d, J=7.0 Hz, 2H), 3.79 (s, 3H),
2.60 (t, J=7.0 Hz, 1H), 2.14 (s, 3H). LR-MS (+): m/e 196.5 (M+1).
Compound 268 was obtained quantitatively by treating compound 267
with K.sub.2CO.sub.3 in MeOH.
[0320] To a solution of compound 268 (110 mg, 0.56 mmol) in DCM (10
mL) at 0.degree. C. was added triethylamine (0.118 mL, 0.84 mmol)
followed by MsCl (0.052 mL, 0.68 mmol) dropwise. After 30 minutes
at 0.degree. C. and 30 minutes at room temperature, the mixture was
washed twice with saturated aqueous ammonium chloride and brine,
before being dried over anhydrous sodium sulphate and filtered
through celite. Concentration under reduced pressure gave
compound(s) 269 (145 mg, .about.94%) as an off-white solid, which
was found by .sup.1H NMR to be a mixture of mesylate and chloride
(3.6:1) and was used without further purification. .sup.1H NMR
(CDCl.sub.3, 400 MHz) for the mesylate: .delta. 5.62 (s, 2H), 3.81
(s, 3H), 3.13 (s, 3H), 2.15 (s, 3H); for the chloride: .delta. 5.02
(s, 2H), 3.79 (s, 3H), 2.14 (s, 3H). LR-MS (+): 274.5 (M+1 of the
mesylate); 214.4/216.4 (3:1, M+1 of the chloride).
[0321] Mixture 269 (145 mg, .about.0.53 mmol) was treated with LiBr
(922 mg, 10.61 mmol) in refluxing THF (10 mL) for 30 minutes. The
THF was then removed in vacuo and the resulting residue was
distributed between water and ethyl acetate. The organic phase was
washed with water and brine, dried over anhydrous sodium sulphate
and filtered through celite, before being concentrated in vacuo.
The crude product thus obtained was purified by flash column
chromatography eluting with ethyl acetate/hexane (1:1) to give
5-(bromomethyl)-1-methyl-4-nitro-2-(1-propynyl)-1H-imidazole (270)
(95 mg, 69%) as white solid. .sup.1H NMR (CDCl.sub.3, 400 MHz)
.delta. 4.86 (s, 2H), 3.76 (s, 3H), 2.14 (s, 3H). LR-MS (+): m/e
258.5/260.5 (1:1, M+1).
1.1.3 the Synthesis of Quaternary Ammonium Salt Prodrugs
[0322] Method A: Preparation of Quaternary Ammonium Salt Prodrugs
in N-metyl-2-pyrolidinone (NMP) Followed by Acetonitrile
Precipitation.
[0323] To a solution of the dimethylamine-bearing kinase inhibitor
of Formula I in NMP at room temperature was added the
.alpha.-methyl bromide trigger (1.0-1.2 eq.). The resulting mixture
was stirred overnight (.about.15 hours). Acetonitrile was then
added to the reaction mixture dropwise with continued stirring
until a precipitate started to form. The resulting mixture was then
stirred for a further 2 hours before the precipitate was collected
by filtration or by centrifugation, washed with acetonitrile, ethyl
acetate and hexane. Drying under vacuum then gave the quaternary
ammonium salt prodrug as a white or off-white solid. If necessary,
the product was further purified by recrystallisation from NMP and
MeCN.
1.1.3.1 Preparation of
(2E)-4-[(4-{3-chloro-4-[(3-chlorobenzyl)oxy]anilino}pyrido[3,4-d]pyrimidi-
n-6-yl)amino]-N,N-dimethyl-N-[(1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-4-
-oxo-2-buten-1-ammonium bromide (12) (Scheme 10)
[0324] Reaction of compound 1 (500 mg, 0.96 mmol) in NMP (1.2 mL)
with .alpha.-methyl bromide 239 (231 mg, 1.05 mmol) according to
Method A gave
(2E)-4-[(4-{3-chloro-4-[(3-chlorobenzyl)oxy]anilino}pyrido[3,4-d]pyrimidi-
n-6-yl)amino]-N,N-dimethyl-N-[(1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-4-
-oxo-2-buten-1-ammonium bromide (12) (517 mg, 73%), m.p.
182-186.degree. C. (dec). .sup.1H NMR [(CD.sub.3)SO].delta. 11.28
(s, 1H), 10.25 (s, 1H), 9.04 (s, 1H), 8.98 (s, 1H), 8.61 (s, 1H),
8.14 (s, 1H), 7.98 (d, J=2.5 Hz, 1H), 7.74-7.72 (dd, J=8.9, 2.5 Hz,
1H), 7.56 (s, 1H), 7.47-7.40 (m, 3H), 7.28 (d, J=9.1 Hz, 1H),
7.06-6.98 (m, 1H), 6.80 (d, J=15.2 Hz, 1H), 5.26 (s, 2H), 5.05 (br,
2H), 4.44 (d, J=7.1 Hz, 2H), 3.88 (s, 3H), 3.13 (s, 6H). Analysis
found: C, 48.93; H, 3.92; N, 16.27.
C.sub.31H.sub.30BrCl.sub.2N.sub.9O.sub.4.H.sub.2O.0.1EtOAc
requires: C, 48.96; H, 4.29; N, 16.37.
1.1.3.2 Preparation of
(2E)-4-[(4-{[1-(3-fluorobenzyl)-1H-indazol-5-yl]amino}pyrido[3,4-d]pyrimi-
din-6-yl)amino]-N,N-dimethyl-N-[(1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-
-4-oxo-2-buten-1-ammonium bromide (13) (Scheme 10)
[0325] Reaction of compound 2 (500 mg, 1.01 mmol) in NMP (1.8 mL)
with .alpha.-methyl bromide 239 (244 mg, 1.11 mmol) according to
Method A gave
(2E)-4-[(4-{[1-(3-fluorobenzyl)-H-indazol-5-yl]amino}pyrido[3,4-d]pyrimid-
in-6-yl)amino]-N,N-dimethyl-N-[(1-methyl-4-nitro-1H-imidazol-5-yl)methyl]--
4-oxo-2-buten-1-ammonium bromide (13) (538 mg, 74%), m.p.
183-187.degree. C. (dec). .sup.1H NMR [(CD.sub.3).sub.2SO].delta.
11.27 (s, 1H), 10.35 (s, 1H), 9.02 (s, 2H), 8.56 (s, 1H), 8.17-8.14
(m, 3H), 7.75-7.69 (m, 2H), 7.40-7.35 (m, 1H), 7.12-6.99 (m, 4H),
6.80 (d, J=15.2 Hz, 1H), 5.71 (s, 2H), 5.05 (br, 2H), 4.44 (d,
J=7.0 Hz, 2H), 3.87 (s, 3H), 3.13 (s, 6H). Analysis found: C,
51.71; H, 4.53; N, 20.33.
C.sub.32H.sub.31BrFN.sub.11O.sub.3.1.5H.sub.2O.0.1 EtOAc requires:
C, 51.72; H, 4.66; N, 20.48.
1.1.3.3 Preparation of
(2E)-N-[(1,2-dimethyl-4-nitro-1H-imidazol-5-yl)methyl]-4-[(4-{[1-(3-fluor-
obenzyl)-1H-indazol-5-yl]amino}pyrido[3,4-d]pyrimidin-6-yl)amino]-N,N-dime-
thyl-4-oxo-2-buten-1-ammonium bromide (14) (Scheme 11)
[0326] Reaction of compound 2 (100 mg, 0.20 mmol) in NMP (0.5 mL)
with .alpha.-methyl bromide 244 (52 mg, 0.22 mmol) according to
Method A gave
(2E)-N-[(1,2-dimethyl-4-nitro-1H-imidazol-5-yl)methyl]-4-[(4-{[1-(3-fluor-
obenzyl)-1H-indazol-5-yl]amino}pyrido[3,4-d]pyrimidin-6-yl)amino]-N,N-dime-
thyl-4-oxo-2-buten-1-ammonium bromide (14) (93 mg, 63%), m.p.
188-192.degree. C. (dec). .sup.1H NMR [(CD.sub.3).sub.2SO].delta.
11.27 (s, 1H), 10.38 (s, 1H), 9.03 (s, 2H), 8.56 (s, 1H), 8.17 (s,
2H), 7.76-7.68 (m, 2H), 7.40-7.35 (m, 1H), 7.12-6.99 (m, 4H), 6.80
(d, J=15.2 Hz, 1H), 5.71 (s, 2H), 5.05 (br, 2H), 4.43 (d, J=6.8 Hz,
2H), 3.75 (s, 3H), 3.11 (s, 6H), 2.44 (s, 3H). Analysis found: C,
50.81; H, 4.76; N, 19.42.
C.sub.33H.sub.33BrFN.sub.11O.sub.3.2.9H.sub.2O requires: C, 50.63;
H, 5.00; N, 19.64.
1.1.3.4 Preparation of
(2E)-4-({4-[3-chloro-4-(2-pyridinylmethoxy)anilino]pyrido[3,4-d]pyrimidin-
-6-yl}amino)-N,N-dimethyl-N-[(1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-4--
oxo-2-buten-1-ammonium bromide (15) (Scheme 10)
[0327] Reaction of compound 3 (500 mg, 1.02 mmol) in NMP (1 mL)
with .alpha.-methyl bromide 239 (225 mg, 1.02 mmol) according to
Method A gave
(2E)-4-({4-[3-chloro-4-(2-pyridinylmethoxy)anilino]pyrido[3,4-d]pyrimidin-
-6-yl}amino)-N,N-dimethyl-N-[(1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-4--
oxo-2-buten-1-ammonium bromide (15) (610 mg, 84%), m.p.
189-191.degree. C. (dec). .sup.1H NMR [(CD.sub.3).sub.2SO].delta.
11.28 (s, 1H), 10.25 (s, 1H), 9.04 (s, 2H), 8.98 (s, 1H), 8.61-8.60
(m, 2H), 8.14 (s, 1H), 7.99 (d, J=2.5 Hz, 1H), 7.91-7.87 (dt,
J=1.6, 7.7 Hz, 1H), 7.74-7.71 (dd, J=2.5, 8.94 Hz, 1H), 7.59 (d,
J=7.8 Hz, 1H), 7.39-7.36 (dd, J=5.3, 7.0 Hz, 1H), 7.29 (d, J=9.0
Hz, 1H), 7.06-6.98 (m, 1H), 6.80 (d, J=15.2 Hz, 1H), 5.31 (s, 2H),
5.05 (br, 2H), 4.44 (d, J=7.1 Hz, 2H), 3.88 (s, 3H), 3.11 (s, 6H).
Analysis found: C, 48.65; H, 4.34; N, 18.58.
C.sub.30H.sub.30BrClN.sub.10O.sub.4.1.8H.sub.2O requires: C, 48.54;
H, 4.56, N, 18.87.
1.1.3.5 Preparation of
(2E)-4-{[4-(3,4-dichloroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N,N-di-
methyl-N-[(1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-4-oxo-2-buten-1-ammon-
ium bromide (16) (Scheme 10)
[0328] Reaction of compound 4 (500 mg, 1.20 mmol) in NMP (1.2 mL)
with .alpha.-methyl bromide 239 (290 mg, 1.32 mmol) according to
Method A gave
(2E)-4-{[4-(3,4-dichloroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N,N-di-
methyl-N-[(1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-4-oxo-2-buten-1-ammon-
ium bromide (16) (648 mg, 85%), m.p. 191-195.degree. C. (dec).
.sup.1H NMR [(CD.sub.3).sub.2SO].delta. 11.32 (s, 1H), 10.41 (s,
1H), 9.09 (s, 1H), 9.02 (s, 1H), 8.71 (s, 1H), 8.27 (d, J=2.4 Hz,
1H), 8.14 (s, 1H), 7.93-7.90 (dd, J=8.8, 2.4 Hz, 1H), 7.67 (d,
J=8.8 Hz, 1H), 7.07-6.99 (m, 1H), 6.80 (d, J=15.2 Hz, 1H), 5.06
(br, 2H), 4.45 (d, J=7.2 Hz, 2H), 3.88 (s, 3H), 3.13 (s, 6H).
Analysis found: C, 43.86; H, 3.82; N, 18.79.
C.sub.24H.sub.24BrCl.sub.2N.sub.9O.sub.3.H.sub.2O.0.1EtOAc
requires: C, 44.13; H, 4.07; N, 18.98.
1.1.3.6 Preparation of
(2E)-4-{[4-(3-bromo-4-chloroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N,-
N-dimethyl-N-[(1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-4-oxo-2-buten-1-a-
mmonium bromide (17) (Scheme 10)
[0329] Reaction of compound 5 (500 mg, 1.08 mmol) in NMP (1.2 mL)
with .alpha.-methyl bromide 239 (262 mg, 1.19 mmol) according to
Method A gave
(2E)-4-{[4-(3-bromo-4-chloroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N,-
N-dimethyl-N-[(1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-4-oxo-2-buten-1-a-
mmonium bromide (17) (650 mg, 88%), m.p. 200-204.degree. C. (dec).
.sup.1H NMR [(CD.sub.3).sub.2SO].delta. 11.32 (s, 1H), 10.40 (s,
1H), 9.08 (s, 1H), 9.02 (s, 1H), 8.70 (s, 1H), 8.37 (d, J=2.5 Hz,
1H), 8.14 (s, 1H), 7.98-7.95 (dd, J=8.8, 2.5 Hz, 1H), 7.66 (d,
J=8.8 Hz, 1H), 7.06-6.99 (m, 1H), 6.80 (d, J=15.3 Hz, 1H), 5.05
(br, 2H), 4.44 (d, J=7.2 Hz, 2H), 3.88 (s, 3H), 3.13 (s, 6H).
Analysis found: C, 41.16; H, 3.67; N, 17.55.
C.sub.24H.sub.24Br.sub.2ClN.sub.9O.sub.3.H.sub.2O.0.1 EtOAc
requires: C, 41.36; H, 3.81; N, 17.79.
1.1.3.7 Preparation of
(2E)-4-{[4-(4-bromo-3-chloroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N,-
N-dimethyl-N-[(1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-4-oxo-2-buten-1-a-
mmonium bromide (18) (Scheme 10)
[0330] Reaction of compound 6 (700 mg, 1.52 mmol) in NMP (1.5 mL)
with .alpha.-methyl bromide 239 (367 mg, 1.67 mmol) according to
Method A gave
(2E)-4-{[4-(4-bromo-3-chloroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N,-
N-dimethyl-N-[(1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-4-oxo-2-buten-1-a-
mmonium bromide (18) (875 mg, 85%), m.p. 209-213.degree. C. (dec).
.sup.1H NMR [(CD.sub.3).sub.2SO].delta. 11.35 (s, 1H), 10.43 (s,
1H), 9.09 (s, 1H), 9.03 (s, 1H), 8.71 (s, 1H), 8.27 (d, J=2.1 Hz,
1H), 8.15 (s, 1H), 7.86-7.78 (m, 2H), 7.07-6.99 (m, 1H), 6.80 (d,
J=15.3 Hz, 1H), 5.05 (br, 2H), 4.44 (d, J=6.8 Hz, 2H), 3.87 (s,
3H), 3.12 (s, 6H). Analysis found: C, 40.97; H, 3.68; N, 18.03.
C.sub.24H.sub.24Br.sub.2ClN.sub.9O.sub.3.1.2H.sub.2O requires: C,
40.98; H, 3.78; N, 17.92.
1.1.3.8 Preparation of
(2E)-4-{[4-(3,4-dibromoanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N,N-dim-
ethyl-N-[(1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-4-oxo-2-buten-1-ammoni-
um bromide (19) (Scheme 10)
[0331] Reaction of compound 7 (700 mg, 1.38 mmol) in NMP (1.5 mL)
with .alpha.-methyl bromide 239 (335 mg, 1.52 mmol) according to
Method A gave
(2E)-4-{[4-(3,4-dibromoanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}-N,N-dim-
ethyl-N-[(1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-4-oxo-2-buten-1-ammoni-
um bromide (19) (840 mg, 84%), m.p. 215-219.degree. C. (dec).
.sup.1H NMR [(CD.sub.3).sub.2SO].delta. 11.35 (s, 1H), 10.41 (s,
1H), 9.09 (s, 1H), 9.02 (s, 1H), 8.71 (s, 1H), 8.37 (d, J=2.1 Hz,
1H), 8.14 (s, 1H), 7.91-7.88 (dd, J=8.8, 2.2 Hz, 1H), 7.79 (d,
J=8.8 Hz, 1H), 7.07-7.01 (m, 1H), 6.80 (d, J=15.2 Hz, 1H), 5.04
(br, 2H), 4.44 (d, J=7.1 Hz, 2H), 3.87 (s, 3H), 3.12 (s, 6H).
Analysis found: C, 38.79; H, 3.34; N, 16.70.
C.sub.24H.sub.24Br.sub.3N.sub.9O.sub.3.H.sub.2O requires: C, 38.73;
H, 3.52; N, 16.94.
1.1.3.9 Preparation of
(2E)-4-{[4-(3-ethynyl-4-fluoroanilino)pyrido[3,4-]pyrimidin-6-yl]amino}-N-
,N-dimethyl-N-[(1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-4-oxo-2-buten-1--
ammonium bromide (20) (Scheme 10)
[0332] Reaction of compound 8 (586 mg, 1.50 mmol) in NMP (1 mL)
with .alpha.-methyl bromide 239 (363 mg, 1.65 mmol) according to
Method A gave
(2E)-4-{[4-(3-ethynyl-4-fluoroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}--
N,N-dimethyl-N-[(1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-4-oxo-2-buten-1-
-ammonium bromide (20) (645 mg, 70%/0), m.p. 198.degree. C. (dec).
.sup.1H NMR [(CD.sub.3).sub.2SO].delta. 11.31 (s, 1H), 10.36 (s,
1H), 9.06 (s, 1H), 9.00 (s, 1H), 8.65 (s, 1H), 8.14 (s, 1H), 8.03
(br, 1H), 7.88 (br, 1H), 7.36 (t, J=9.1 Hz, 1H), 7.06-6.99 (m, 1H),
6.80 (d, J=15.2 Hz, 1H), 5.06 (br, 2H), 4.53 (s, 1H), 4.45 (d,
J=7.0 Hz, 2H), 3.88 (s, 3H), 3.13 (s, 6H). Analysis found: C,
48.61; H, 4.37; N, 18.78.
C.sub.26H.sub.25BrFN.sub.9O.sub.3.2H.sub.2O.0.2EtOAc requires: C,
48.47; H, 4.64; N, 18.98.
1.1.3.10 Preparation of
(2E)-4-{[4-(4-chloro-3-ethynylanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}--
N,N-dimethyl-N-[(1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-4-oxo-2-buten-1-
-ammonium bromide (21) (Scheme 10)
[0333] Reaction of compound 9 (500 mg, 1.23 mmol) in NMP (1.2 mL)
with .alpha.-methyl bromide 239 (297 mg, 1.35 mmol) according to
Method A gave
(2E)-4-{[4-(4-chloro-3-ethynylanilino)pyrido[3,4-d]pyrimidin-6-yl]amino}--
N,N-dimethyl-N-[(1-methyl-4-nitro-1H-imidazol-5-yl)methyl]-4-oxo-2-buten-1-
-ammonium bromide (21) (630 mg, 82%), m.p. 199-202.degree. C.
(dec). .sup.1H NMR [(CD.sub.3).sub.2SO].delta. 11.34 (s, 1H), 10.39
(s, 1H), 9.08 (s, 1H), 9.02 (s, 1H), 8.69 (s, 1H), 8.17 (d, J=2.5
Hz, 1H), 8.14 (s, 1H), 7.96-7.93 (dd, J=8.9, 2.5 Hz, 1H), 7.59 (d,
J=8.9 Hz, 1H), 7.06-6.99 (m, 1H), 6.80 (d, J=15.2 Hz, 1H), 5.05
(br, 2H), 4.60 (s, 1H), 4.44 (d, J=7.0 Hz, 2H), 3.87 (s, 3H), 3.12
(s, 6H). Analysis found: C, 48.16; H, 4.22; N, 19.19.
C.sub.26H.sub.25BrClN.sub.9O.sub.3.1.2H.sub.2O.0.1EtOAc requires:
C, 48.24; H, 4.32; N, 19.18.
2. Efficacy of the Compounds
2.1 Cell Growth Inhibitory Activity
2.1.1. Pan-erbB Kinase Inhibitors
[0334] Kinase inhibitors of Formula I (compounds 1-9) were tested
for their ability to inhibit the proliferation of three human
carcinoma cell lines, selected to provide a comparison with
literature precedent: A431 (epidermoid), which overexpresses erbB1
(EGFR); H1975 (non-small-cell lung), which overexpresses erbB1
L858R/T790M a double mutant form of erbB1 that is known to confer
resistance to the approved reversible erbB1 inhibitor erlotinib and
SKOV3 (ovarian), which over express erbB2 (HER2). The cells were
exposed to test compounds for either 24 hours under oxic conditions
or for 4 hours under anoxia followed by 20 hours under oxic
conditions. They were then washed free of drug and incubated for a
further 4 days, before being stained for cellular growth with
sulforhodamine B. The concentration of compound required to inhibit
cellular growth by 50% relative to untreated control wells, termed
the IC50 value, was calculated.
Results are summarized in Table 1.
TABLE-US-00001 TABLE 1 Inhibition of cellular proliferation in
A431, H1975 and SKOV3 cells Cellular Growth Inhibition IC.sub.50
(.mu.M).sup.a Compound A431 H1975 SKOV3 Number Oxic.sup.b
Anoxic.sup.c HCR.sup.d Oxic.sup.b Anoxic.sup.c HCR.sup.d Oxic.sup.b
Anoxic.sup.c HCR.sup.d 1 0.28 0.221 1.2 1.2 0.88 2.5 1.8 0.69 1.8 2
0.13 0.09 1.0 0.9 1.0 1.4 0.78 0.9 3.3 3 0.12 0.14 1.4 0.51 0.73
0.8 0.71 1.13 1.7 4 0.009 0.015 0.9 0.42 0.56 1.1 0.36 0.55 0.9 5
0.009 0.010 1.0 0.29 0.50 0.9 0.30 0.45 0.9 6 0.005 0.005 2.0 0.15
0.22 0.7 0.20 0.55 0.7 7 0.005 0.008 0.8 0.18 0.27 1.0 0.37 0.60
1.2 8 0.07 0.12 1.8 1.3 1.4 1.4 1.8 1.8 1.7 9 0.02 0.016 2.2 0.40
0.56 0.5 0.50 0.96 1.7 Footnotes for Table 1 .sup.acompound
dose-response curves were determined at 10 concentrations. Cells
received a 24 hour exposure to test compounds before being washed
(x3) with drug-free media. The IC50 (umol/L) values are the
concentrations required to inhibit cell growth by 50% relative to
untreated controls. Values are the average of 2-8 independent
determinations (% CV <20 in all cases). .sup.bExperiment
performed entirely under oxic conditions. .sup.cThe initial 4 hours
of the 24 hour drug exposure was performed under anoxic conditions.
.sup.dHypoxic Cytotoxicity Ratio = fold change in
intra-experimental IC50 for cells receiving 4 hours of anoxia
relative to cells that received only oxic conditions.
[0335] Irreversible erbB1, 2, 4 inhibitors 1, 2, 3, 4, 5, 6, 7, 8
and 9 more potently inhibited proliferation of aerobic A431 cells
(IC50s=0.28 to 0.005 umol/L) than H1975 (IC50s=1.3 to 0.15 umol/L)
and SKOV3 (IC50s=1.8 to 0.2 umol/L) cells and did not show any
significant change in potency when the cells received 4 hours of
anoxia with intraexperimental HCR ranging from 0.5 to 3.3 for all
compounds across the three cell lines.
2.1.2 Prodrugs of Pan-erbB Inhibitors
[0336] Selected prodrug compounds of Formula III (compounds 12-21)
were tested for their ability to inhibit the proliferation of three
human carcinoma cell lines, selected to provide a comparison with
literature precedent: A431 (epidermoid), which overexpresses erbB1
(EGFR); H1975 (non-small-cell lung), which overexpresses erbB1
L858R/T790M a double mutant form of erbB1 that is known to confer
resistance to the approved reversible erbB1 inhibitor erlotinib and
SKOV3 (ovarian), which over express erbB2 (HER2). The cells were
exposed to test compounds for either 24 hours under oxic conditions
or for 4 hours under anoxia followed by 20 hours under oxic
conditions. They were then washed free of drug and incubated for a
further 4 days, before being stained for cellular growth with
sulforhodamine B. The concentration of compound required to inhibit
cellular growth by 50% relative to untreated control wells, termed
the IC50 value, was calculated.
Results are summarized in Table 2.
TABLE-US-00002 TABLE 2 Inhibition of cellular proliferation in
A431, H1975 and SKOV3 cells Cellular Growth Inhibition IC.sub.50
(.mu.M).sup.a Compound A431 H1975 SKOV3 Number Oxic.sup.b
Anoxic.sup.c HCR.sup.d Oxic.sup.b Anoxic.sup.c HCR.sup.d Oxic.sup.b
Anoxic.sup.c HCR.sup.d 12 2.9 0.37 34 36 2.4 14 47 8.1 26 13 6.2
0.24 52 92 3.3 42 315 1.6 528 14 5.7 0.063 96 20 1.2 26 32 1.10 42
15 10 0.30 47 26 0.43 69 159 0.60 295 16 0.33 0.015 24 32 0.40 89
40 0.53 170 17 0.15 0.019 8 11 0.48 22 19 1.1 38 18 0.50 0.012 50
13.3 0.20 66 13 0.60 24 19 0.42 0.025 23 15 0.23 75 29 0.42 91 20
3.9 0.13 56 118 3.3 41 194 4.4 15 21 0.85 <0.01 >85 18 0.60
18 47 1.13 73 Footnotes for Table 2 .sup.acompound dose-response
curves were determined at 10 concentrations. Cells received a 24
hour exposure to test compounds before being washed (x3) with
drug-free media. The IC50 (umol/L) values are the concentrations
required to inhibit cell growth by 50% relative to untreated
controls. Values are the average of 2-8 independent determinations
(% CV <20 in all cases). .sup.bExperiment performed entirely
under oxic conditions. .sup.cThe initial 4 hours of the 24 hour
drug exposure was performed under anoxic conditions. .sup.dHypoxic
Cytotoxicity Ratio = fold change in intra-experimental IC50 for
cells receiving 4 hours of anoxia relative to cells that received
only oxic conditions.
[0337] All of the prodrugs (12, 13, 14, 15, 16, 17, 18, 19, 20 and
21) of Table 2 were significantly more potent at inhibiting the
growth of all three cell lines after the cells received 4 hours of
anoxia. The hypoxic cytotoxicity ratios (HCR) ranged from 8 to 96
in A431 cells, 14 to 89 in H1975 cells and 15 to 528 in SKOV3
cells, consistent with hypoxia-selective reduction of the
4-nitroimidazole reductive trigger, followed by trigger
fragmentation to release an irreversible erbB1, 2, 4 inhibitor.
2.2. Cellular Enzyme Inhibitory Activities
[0338] The compounds 3, 5 and 6 were tested for their ability to
inhibit the autophosphorylation of erbB1 (EGFR), and p44/42 MAPK
(Erk1/2) in EGF-stimulated A431 cells by Western immunoblotting
measurement of phospho-erbB1 and phospho-Erk1/2 status.
[0339] A431 cells were seeded into 6 well plates (with
.alpha.MEM+5% FCS). The following day, plates were washed once with
serum-free medium and grown for 18 hours in serum-free medium
(.alpha.MEM) before being exposed to a range of inhibitor or
prodrug concentrations for 1 hour and then stimulated for 15
minutes with 100 ng/ml epidermal growth factor (EGF receptor
ligand). Next cells were washed with ice cold PBS and lysed in
modified RIPA buffer on ice (30 min). Lysates were vortexed and
clarify by spinning before protein concentrations of samples was
determined by BCA assay. For western blot analysis 5 .mu.g of total
protein/well was loaded on a NuPAGE 4-12% gel and run at 150V (1 h)
before transfer to 0.45 .mu.m nitrocellulose membrane followed by
blocking for 1 hr with 2% BSA in TBS-Tween 0.1%. All antibodies
were diluted in 2% BSA TBS-Tween 0.1%. To detect phospho-EGFR (Tyr
1068) the monoclonal antibody (1:500; Cell Signalling #2234) was
incubated overnight at 4.degree. C. and binding was detected with
goat-anti-Rabbit-IgG-HRP conjugated secondary antibody (1:5000;
Santa Cruz #SC2054; 3 h RT). To detect downstream phospho-p44/42
MAPK (Erk 1/2) (Thr202/Tyr 204) the monoclonal antibody (Cell
Signalling #4370) was used at a 1:500 dilution. The blot was then
treated with Goat anti-rabbit IgG-HRP conjugated secondary antibody
(Santa Cruz Biotechnology, Inc. sc-2054) for three hours at a ratio
of 1:5000. To normalise for any inaccuracy in gel loading, each
blot was probed with anti-.beta.-actin antibody (Abacus ALS
#MAB-1501R, 1:2000 dilution). The following day, the blot was
treated with Goat anti-mouse IgG-HRP conjugated secondary antibody
(Santa Cruz Biotechnology, Inc. sc-2055) for one hour at a 1:5000
ratio. Protein bands were visualized using Amersham ECL Plus
Western Blotting Detection Reagent (GE Healthcare RPN2132).
Densitometry was determined using Image J software. Values
normalized to .beta.-actin were plotted on SigmaPlot 11.0.
[0340] Compounds 3, 5 and 6 were shown to be potent inhibitors of
cellular erbB1 (FIGS. 12A, 13A and 14A, respectively) with IC50s of
0.0483, 0.0395 and 0.0250 .mu.M, respectively. In addition,
suppression of EGFR phosphorylation in A431 cells was accompanied
by coordinate loss of downstream p44/42 MAPK (Erk 1/2)
phosphorylation (Thr202/Tyr 204) indicating modulation of the
signalling network governed by EGFR activity (FIGS. 12B, 13B and
14B, respectively). In contrast the quaternary ammonium salt
derivative prodrug 15 was 110-fold less effective (IC50 of 5.29
.mu.M; FIG. 15A and FIG. 16) at inhibiting erbB1
autophosphorylation in intact A431 cells than its respective
inhibitor, compound 3 (FIG. 12A). A similar loss of potency was
observed for p44/42 MAPK (Erk 1/2) phosphorylation (Thr202/Tyr 204)
(IC50 of 0.179 .mu.M and 8.9 .mu.M for compound 3 and prodrug 15,
respectively (compare FIG. 12B and FIG. 15B) indicating less
effective modulation of the downstream signalling network governed
by EGFR activity. When the respective band densitometry values were
plotted for the dose-dependent inhibition of phospho-EGFR
(corrected for .beta.-actin), it is evident that prodrug 15 is
deactivated 110-fold relative to compound 3. This loss of cellular
erbB1 inhibitory potency for the prodrugs is attributed primarily
to cellular exclusion of the prodrugs due to the presence of a
positively charged quaternary ammonium salt.
2.3 Radiolytic Reduction
[0341] Electron-affinic prodrugs can be selectively reduced by
1-electron processes in the hypoxic regions of solid tumours, in
contrast to under normoxic conditions in normal tissues, to form or
release a cytotoxic effector (Brown and Wilson, Nature Rev. Cancer,
2004, 4, 437-447). The prodrug should contain a trigger moiety
possessing a 1-electron reduction potential, E(1), of between -0.6V
to -0.2 V and preferably between -0.5 V to -0.3V vs. NHE. The E(1)
values of many compounds can be obtained from the literature, (for
example, Wardman, P. J. Phys. Chem. Ref. Data, 1989, 18, 1637-1755)
or determined by a number of methods. The pulse radiolysis method,
for example, measures the equilibrium constant between the radical
anions of the prodrugs, formed upon their 1-electron reduction, and
reference standards such as viologen and quinone compounds, from
which data the E(1) values of the compounds can be calculated.
(Meisel and Czapski. J. Phys. Chem., 1975, 79, 1503-1509.) The E(1)
values of prodrugs 13, 15-19 were measured by the pulse radiolysis
method and determined to range between -0.428V and -0.417V (Table
3). All are considered to possess appropriate E(1) values to enable
enzymatic formation of their radical anions in a biological
context.
[0342] Prodrugs possessing appropriate E(1) values can be tested
for their ability to release effector moieties by a number of
methods, following the radiolysis of the prodrugs in solution. For
example, mass spectrometry (MS) and/or high performance liquid
chromatography (HPLC) before and after radiolysis identifies the
starting compound and the products formed as a result of the
radiolysis. Several 1-electron reductants can be produced upon the
radiolysis of solutions containing different solutes. For example
the CO.sub.2..sup.- radical, formed in .gamma.-irradiated solutions
containing formate ions, possesses a low E(1) of -1.90 V (Schwarz
et al, Inorg. Chem., 1985, 24, 433-439) and undergoes facile
electron transfer to compounds of higher E(1). Under the radiation
conditions employed, a concentration of 0.66 .mu.M in 1-electron
reducing equivalents (the CO.sub.2..sup.- radical) are produced per
Gy (J kg.sup.-1) of absorbed radiation dose. (Mulazzani et al, J.
Phys. Chem., 1980, 90, 5347-5352.) By comparing the loss in prodrug
concentration with the concentration of reducing equivalents
produced upon the radiolysis of the solution, it is possible to
determine whether one or multi-electron reduction is required for
complete loss of each prodrug. Typically, evidence for 1-electron
removal of a prodrug is sought after 0.95 reducing equivalents are
transferred to the prodrug, to minimise multi-electron reduction of
the same prodrug molecule. In the case of 1-electron removing a
prodrug, this often indicates fragmentation of its radical anion.
This conclusion is further supported by combined HPLC-MS
identification of the released cytotoxic effector and the products
arising from the transient benzyl-type radical (e.g. the methyl
nitroaromatic compound (MNA) formed by H-atom abstraction). This
has been shown to occur in the case of certain related arylmethyl
quaternary nitrogen mustards. (Anderson et al. J. Phys. Chem.,
1997, 101, 9704-9709; Wilson et al. Radiat. Res., 1998, 149,
237-245.) The data obtained for prodrug 17 is consistent with its
consumption at the 1-electron reduction level (>50% loss of
prodrug at the 0.95 reducing equivalents level) with the released
effector (compound 5) detected by HPLC-MS, Table 3.
[0343] It is desirable that the reductive prodrugs are selected to
have controlled fragmentation rate constants upon 1-electron
reduction of the trigger moiety. Whilst fast fragmentation to
release high concentrations of the cytotoxic effectors in the
hypoxic regions of tumour cells is desirable, this not so for
normal tissue cells under normoxia. The rate constant of the back
oxidation of the 1-electron reduced nitroarene-based prodrugs by
oxygen, O.sub.2, which effectively inhibits the release of the
effector, is given by the expression:--(Wardman et al, Biochem.
Soc. Symp., 1995, 61, 171-194; Anderson et al, Org. Biomol. Chem.
2005, 3, 2167-2174)
log O.sub.2/M.sup.-1
s.sup.-1=(4.6.+-.0.1)-(5.0.+-.0.2).times.E(1)C/C..sup.-
where E(1)C/C..sup.- is the 1-electron reduction potential of the
prodrug. This means, for over the preferred range in E(1), -0.5 V
to -0.3 V, the pseudo 1.sup.st-order rate constants for this back
oxidation in normal cells (which can be, under physiological
conditions, as low as 10 .mu.M in oxygen concentration) decrease
from 130 s.sup.-1 to 13 s.sup.-1. Hence, over the preferred range
in E(1) of the prodrug, the ranges in fragmentation rate to allow
for sufficient back-oxidation in normoxic tissue and therefore to
impart hypoxia-selective fragmentation to the prodrug (e.g. 5:1),
is most preferably in the region of ca. 2-30 s.sup.-1 for prodrugs
in the upper range of desirable E(1) and ca. 20-300 s.sup.-1 for
prodrugs in the lower range of desirable E(1). The rate constants
for fragmentation, frag, of the 1-electron reduced prodrugs can be
measured using pulse radiolysis to observe the time-resolved
formation of the absorption spectrum of the benzyl-type radical
produced upon fragmentation of the radical anion. (Anderson et al,
J. Phys. Chem. A, 1997, 101, 9704-9709.) The frag values of
prodrugs 13, 15-17 were measured by pulse radiolysis and are
presented in Table 3. All of the prodrugs possess fragmentation
rates upon 1-electron reduction under hypoxia in the desirable
range, consistent with them showing acceptable hypoxic cytotoxicity
ratios (HCRs) in vitro in A431, H1975 and SKOV3 cell-based
anti-proliferative assays (Table 3).
TABLE-US-00003 TABLE 3 Radiolytic reduction of selected prodrugs by
the CO.sub.2.sup..- radical. % Loss of Detection Prodrug
E(1)/V.sup.a kfrag..sup.b/s.sup.-1 prodrug.sup.c of MNA.sup.d 13
-0.417 60 .+-. 10 15 -0.423 60 .+-. 10 16 -0.418 60 .+-. 10 17
-0.425 70 .+-. 10 70 Yes 18 -0.428 19 -0.425 Footnotes for Table 3
.sup.aDetermined against methylviologen, E(1)MV.sup.2+/MV.sup.+. =
-447 .+-. 7 mV. .sup.bPulse radiolysis data for the formation of
the benzyl-type radicals absorbing in the 360-390 nm region.
.sup.cMeasurements made by HPLC-MS at 0.95 reducing equivalents;
>50% indicates fragmentation upon 1-electron reduction.
.sup.dDetection of methyl nitroaromatic (MNA) by HPLC-MS.
2.3.1 Radiolytic Reduction Experimental
[0344] The relative activities of example prodrugs in solution to
release effectors, upon the introduction of reducing equivalents,
were determined by the use of a .sup.60Co .gamma.-ray irradiator.
Prodrugs were dissolved in Millipore water (containing added 50 mM
sodium formate buffered at pH 7 by 5 mM sodium phosphate) at a
concentration of 50 .mu.M or below. Solutions, contained in
air-tight glassware continuously saturated with N.sub.2O gas for 30
mins prior to radiolysis at a dose rate of 7.5 Gy min.sup.-1,
previously determined using Fricke dosimetry (Fricke and Hart,
"Chemical Dosimetry" in Radiation Dosimery Vol. 2, Attrix, F. H.;
Roesch, W. C.; Tochilin, E. (Eds.), Academic Press, New York, 1966,
pp 167-239.) Under the radiation conditions employed above, a
concentration of 0.66 .mu.M in 1-electron reducing equivalents (the
CO.sub.2..sup.- radical) are produced per Gy (Mulazzani et al, J.
Phys. Chem., 90, 5347-5352, 1980) and the prodrugs, (P), are
reduced by electron transfer.
.gamma.-radiation+H.sub.2O.fwdarw.e.sub.aq.sup.-+H.+.OH+H.sub.3O.sup.+
e.sub.aq.sup.-+N.sub.2O.fwdarw..OH+OH.+N.sub.2
.OH/H.+HCOO--.fwdarw.H.sub.2O/H.sub.2+CO.sub.2..sup.-
CO.sub.2..sup.-+P.fwdarw.P..sup.-+CO.sub.2
[0345] The loss of prodrug 17 and formation of its effector 5 was
monitored by HPLC-mass spectrophotometry (MS) in duplicate
irradiated samples. The percentage loss in the concentration of the
prodrugs and formation of the effectors at the 0.95 reducing
equivalents level was determined. In addition, the detection of the
methyl-nitroaromatic from prodrug 17 was recorded. Prodrugs
exhibiting >50% loss in concentration at the 0.95 reducing
equivalents level, indicate 1-electron stoichiometry.
[0346] Pulse radiolysis was used to monitor the 1-electron
reduction and stability of the compounds in real time. A linear
accelerator delivering short pulses of high energy electrons (2-3
Gy in 200 ns of 4 MeV) equipped with a fast spectophotometric
detection system was used. (Anderson et al, J. Phys. Chem. A, 101,
9704-9709, 1997). Prodrugs were dissolved in N.sub.2O-saturated
solutions containing formate ions, as above, which, following pulse
radiolysis, resulted in the rapid formation of the radical anions
of the compounds within a few microseconds. The rate of
fragmentation was determined by analysing kinetic transients at
wavelengths corresponding to the formation of the benzyl-type
radical of the trigger moiety. (Bays et al, J. Am. Chem. Soc., 105,
320-324, 1983; Anderson et al, J. Phys. Chem. A, 101, 9704-9709,
1997).
2.4 In Vivo Efficacy of Compounds of the Invention
Methods
[0347] Specific pathogen-free female NIH-III nude mice, derived
from breeding mice supplied by Charles River Laboratories
(Wilmington, Mass.), were housed in groups of 4-6 in a
temperature-controlled room (22.+-.2.degree. C.) with a 12-hour
light/dark cycle and were fed ad libitum water and a standard
rodent diet (Harlan Teklad diet 2018i). All animals were uniquely
identifiable by ear tag number.
[0348] Freshly harvested cell suspensions were subcutaneously
inoculated (100 .mu.L) on the right flank with 5.times.10.sup.6
H1975 or A431 cells in PBS. Mean tumour diameter was averaged from
the longest diameter (length) multiplied by the perpendicular
measurement (width). Tumour volume (mm) was calculated using the
formula (L.times.w.sup.2).times..pi./6 (where; L=length and w=width
in mm of the carcinoma).
Growth Delay Experimental Procedure
[0349] Treatment was initiated when the tumours reached a volume of
approximately 250 mm.sup.3, as determined by calliper measurement.
All drugs were given by intraperitoneal injection at dosing volumes
of 10-20 ml/kg. Mice were dosed at the MTD over a q3dx4, q5dx4 or
q7dx4 schedule with tumour growth measured by callipers every 3-5
days over the 30-day duration of the study. Mice were culled if
they developed signs of toxicity or if bodyweight loss exceeded 20%
of starting weight. All animal experiments followed protocols
approved by the Animal Ethics Committee of The University of
Auckland.
[0350] Tumour bearing mice were assigned randomly to treatment
groups when tumour diameter reached treatment size. Animals were
rejected if xenografts show evidence of: (i) attachment to
underlying muscle (due to risk of local invasion), (ii) signs of
ulceration, or (iii) indolent tumour growth. Drug administration
begins on the day of assignment.
[0351] During and after treatment, tumour size and body weights
were measured regularly. Animals were culled if (i) the average
diameter of the tumour exceeds 15 mm (survival endpoint), (ii) body
weight loss exceeds 20% of pre-treatment value, (iii) there is
evidence of prolonged or excessive morbidity, or (iv) tumour
ulceration occurred. The experiment was terminated at day 21 (A431
tumours) or day 30 (H1975 tumours) after treatment initiation.
Efficacy Analysis
[0352] The time for individual tumours to increase in volume by 4
fold relative to treatment day-1 (RTV.sup.4) was recorded. The
median RTV.sup.4 is calculated for each group and the difference in
RTV.sup.4 between control and treatment groups is described as the
Tumour Growth Delay (TGD) in days. RTV.sup.4 values normalise for
any bias in tumour treatment volume on day 0. Kaplan-Meier plots
were constructed and median survival was calculated (TTE.sub.50).
The statistical significance of any differences in overall survival
time taken to reach RTV.sup.4 between treatment groups and control
was analysed by Log Rank P statistical test.
Toxicity
[0353] Weight loss nadirs (time independent maxima) were recorded
for each treatment group. Any signs of treatment related morbidity
were documented. Acceptable toxicity was defined as no mean group
weight loss of over 15% during the test, no individual weight loss
over 20% and no individual weight loss over 10% in any 24 h period.
All unscheduled deaths were recorded.
Results
[0354] Median tumour growth curves following treatment are
presented in FIGS. 1-11. Summary tables of the effect of treatment
on toxicity and efficacy are presented in Tables 4-6 below.
[0355] Overall, nine kinase inhibitors and nine prodrugs were
administered to mice with human H1975 or A431 tumour xenografts.
The mean (.+-.SD) tumour volume at treatment initiation was 245
mm.sup.3.+-.60 mm.sup.3 for H1975 tumours and 268 mm.sup.3.+-.124
mm.sup.3 for A431 tumours.
[0356] At tolerated dose levels, all kinase inhibitors delayed
tumour growth. This tumour growth delay was particularly
significant for compounds 2-9 when administered at q3dx4
(P<0.05, log-rank test) in both H1975 (FIGS. 1-3) and A431 (FIG.
4) tumours. There was only minor bodyweight loss across all groups,
although there was 1 death following treatment with compounds 5 and
7 in H1975 xenograft mice (Table 4).
TABLE-US-00004 TABLE 4 Summary of treatment toxicity and efficacy
parameters for kinase inhibitors Drug Dose.sup.a related Weight
Loss Log Compound (.mu.mol/kg) Schedule Cell line N deaths.sup.b
Nadir (%).sup.c TGD.sup.d Rank.sup.e 1 23.7 q3dx4 H1975 3 0 -3.6
.+-. 3.0 12.5 ns 2 56.2 q3dx4 H1975 3 0 -7.9 .+-. 0.6 100 <0.05
3 100 q3dx4 H1975 3 0 -4.7 .+-. 0.4 150 <0.05 4 56.2 q3dx4 H1975
3 0 -9.7 .+-. 4.0 188 <0.05 5 75 q3dx4 H1975 3 1 -8.5 .+-. 0.6
175 <0.05 5 75 q3dx4 A431 3 0 -6.3 .+-. 4.5 >250 <0.05 6
42.2 q3dx4 A431 3 0 -1.5 .+-. 0.8 >250 <0.05 7 56.2 q3dx4
H1975 3 1 -4.9 .+-. 2.4 156 <0.05 8 100 q3dx4 H1975 3 0 -3.3
.+-. 3.0 87.5 <0.05 9 75 q3dx4 A431 3 0 -1.7 .+-. 1.7 >250
<0.05 Footnotes for Table 4 .sup.aAdministered in lactate buffer
(pH 4) by intraperitoneal injection (<0.02 ml/g); .sup.ball
animal deaths considered likely to be drug related; .sup.cMean
weight loss nadir (time independent maxima) relative to day 0
weight (%) for each individual; .sup.dTumour Growth Delay
calculated as % increase in time required to reach 4 times initial
treatment volume (RTV.sup.4; relative to day 0 volume) relative to
control growth; .sup.eKaplan-Meier Log Rank survival analysis of
compound treated relative to buffer treated control assuming a
survival endpoint of RTV.sup.4.
[0357] Similarly, the prodrugs (compounds 12, 13, 15-21) all
significantly delayed tumour growth in H1975 (FIG. 5-7) or A431
(FIG. 8) xenograft models after treatment at a q3dx4 schedule
(P<0.05, log-rank test). Again, there was only minor bodyweight
loss in the mice in all treatment groups, although there was 1
death following treatment with compound 19 (Table 5).
TABLE-US-00005 TABLE 5 Summary of treatment toxicity and efficacy
parameters for prodrugs Drug Dose.sup.a related Weight Loss Log
Compound (.mu.mol/kg) Schedule Cell line N deaths.sup.b Nadir
(%).sup.c TGD.sup.d Rank.sup.e 12 133 q3dx4 H1975 3 0 -7.8 .+-. 0.6
87.5 <0.05 13 75 q3dx4 H1975 3 0 -4.1 .+-. 2.5 138 <0.05 15
133 q3dx4 H1975 3 0 -9.4 .+-. 3.3 >250 <0.05 16 100 q3dx4
H1975 3 0 -10.1 .+-. 0.7 188 <0.05 17 178 q3dx4 H1975 3 0 -12.8
.+-. 2.3 >275 <0.05 17 178 q3dx4 A431 3 0 -3.6 .+-. 1.8
>250 <0.05 18 178 q3dx4 A431 3 0 -4.1 .+-. 0.4 >250
<0.05 19 75 q3dx4 H1975 4 1 -2.7 .+-. 0.6 138 <0.05 20 178
q3dx4 H1975 3 0 -4.4 .+-. 2.3 150 <0.05 21 178 q3dx4 A431 3 0
-4.3 .+-. 2.3 >250 <0.05 Footnotes for Table 5
.sup.aAdministered in lactate buffer (pH 4) by intraperitoneal
injection (<0.02 ml/g); .sup.ball animal deaths considered
likely to be drug related; .sup.cMean weight loss nadir (time
independent maxima) relative to day 0 weight (%) for each
individual; .sup.dTumour Growth Delay calculated as % increase in
time required to reach 4 times initial treatment volume (RTV.sup.4;
relative to day 0 volume) relative to control growth;
.sup.eKaplan-Meier Log Rank survival analysis of compound treated
relative to buffer treated control assuming a survival endpoint of
RTV.sup.4.
[0358] Administration of prodrugs 15 and 17, alongside their
cognate kinase inhibitors, 3 and 5 respectively, revealed an
extended period of growth delay in H1975 tumours for the prodrugs
compared to the kinase inhibitors (FIGS. 9-10).
[0359] Prodrug 17 was tested at multiple dosing schedules: q3dx4,
q5dx4 and q7dx4 at its q3dx4 MTD. At all 3 dosing schedules,
compound 17 significantly delayed tumour growth in H1975 tumours
compared to controls (FIG. 11). There were no statistically
significant differences in tumour growth between the 3 dosing
schedules. Bodyweight loss was greatest after q3dx4 dosing and
minimal after q7dx4 dosing (Table 6). However, 1 death was observed
in the q7dx4 treatment group. It is not clear if this death was
drug-related or not. Compound 17 was also administered at q3dx4 and
q5dx4 at its q3dx4 MTD to A431 xenograft mice, with both dosing
schedules causing similar delays in A431 tumour growth and similar
losses in animal bodyweight (Table 6).
TABLE-US-00006 TABLE 6 Summary of treatment toxicity and efficacy
parameters for 17 at q3dx4, q5dx4 and q7dx4 schedules Drug
Dose.sup.a related Weight Loss Log Compound (.mu.mol/kg) Schedule
Cell line N deaths.sup.b Nadir (%).sup.c TGD.sup.d Rank.sup.e 17
178 Q3dx4 H1975 3 0 -12.8 .+-. 2.3 >275 <0.05 17 178 Q5dx4
H1975 3 0 -7.6 .+-. 0.8 225 <0.05 17 178 Q7dx4 H1975 3 1 -1.9
.+-. 0.3 194 <0.05 17 178 Q3dx4 A431 3 0 -3.6 .+-. 1.8 >250
<0.05 17 178 Q5dx4 A431 3 0 -4.6 .+-. 0.8 >250 <0.05
Footnotes for Table 6 .sup.aAdministered in lactate buffer (pH 4)
by intraperitoneal injection (<0.02 ml/g); .sup.ball animal
deaths considered likely to be drug related; .sup.cMean weight loss
nadir (time independent maxima) relative to day 0 weight (%) for
each individual; .sup.dTumour Growth Delay calculated as % increase
in time required to reach 4 times initial treatment volume
(RTV.sup.4; relative to day 0 volume) relative to control growth;
.sup.eKaplan-Meier Log Rank survival analysis of compound treated
relative to buffer treated control assuming a survival endpoint of
RTV.sup.4.
[0360] Overall, the in vitro and in vivo activity data illustrate
the effectiveness of the compounds of the invention as kinase
inhibitors. The compounds are therefore suitable for use in
kinase-inhibitory therapy. This particularly the case with the
reductive prodrugs and cancer therapy as tumours commonly have
hypoxic regions. The prodrugs are reduced under hypoxia to release
the parent kinase inhibitor and produce a tumour-targeted
effect.
Prodrugs 15, 17 and 18 are considered particularly promising
therapeutic candidates.
[0361] While the present invention is broadly as described above,
those persons skilled in the art will appreciate that the specific
description is illustrative only and that variations may be made
without departing from the invention as defined in the following
claims.
[0362] All publications referenced above are incorporated herein in
their entirety.
* * * * *