Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
CA 02714087 2010-08-04
WO 2009/102537 PCT/US2009/031633
TITLE OF THE INVENTION
INHIBITORS OF CHECKPOINT KINASES
BACKGROUND OF THE INVENTION
Cell cycle checkpoints are regulatory pathways that control the order and
timing
of cell cycle transitions. They ensure that critical events such as DNA
replication and
chromosome segregation are completed in high fidelity. The regulation of these
cell cycle
checkpoints is a critical determinant of the manner in which tumor cells
respond to many
chemotherapies and radiation. Many effective cancer therapies work by causing
DNA damage;
however, resistance to these agents remains a significant limitation in the
treatment of cancer.
Of the several mechanisms of drug resistance, an important one is attributed
to the prevention of
cell cycle progression through the control of critical activation of a
checkpoint pathway. This
arrests the cell cycle to provide time for repair, and induces the
transcription of genes to facilitate
repair, thereby avoiding immediate cell death. By abrogating checkpoint
arrests at, for example,
the G2 checkpoint, it may be possible to synergistically augment tumor cell
death induced by
DNA damage and circumvent resistance.
Human CHK1 plays a role in regulating cell cycle arrest by phosphorylating the
phosphatase cdc25 on Serine 216, which may be involved in preventing
activation of cdc2/cyclin
B and initiating mitosis. Therefore, inhibition of CHK1 should enhance DNA
damaging agents
by initiating mitosis before DNA repair is complete and thereby causing tumor
cell death.
It is an object of the instant invention to provide novel compounds that are
inhibitors of CHK1 (also refered to as Chekl).
It is also an object of the present invention to provide pharmaceutical
compositions that comprise the novel compounds that are inhibitors of CHK1.
It is also an object of the present invention to provide a method for treating
cancer
that comprises administering such inhibitors of CHKI activity.
SUMMARY OF THE INVENTION
The instant invention provides for compounds which comprise substituted
thioquinazolinones that inhibit CHK1 activity. The invention also provides for
compositions
comprising such inhibitory compounds and methods of inhibiting CHKI activity
by
administering the compound to a patient in need of treatment of cancer.
DETAILED DESCRIPTION OF THE INVENTION
The compounds of the instant invention are useful in the inhibition of the
activity
of CHKI. In a first embodiment of this invention, the inhibitors of CHK1
activity are illustrated
by the Formula A:
-1-
CA 02714087 2010-08-04
WO 2009/102537 PCT/US2009/031633
0
N /R2
Z I
3 S N(Ra)n
A N R
O
wherein:
a is0or1;bis0orI;mis0, 1,or2;nis0, 1,2,3,4,5 or 6; and pis 0, 1,2,3,4,
or 6;
5 Ring Z is selected from: aryl, heteroaryl, heterocyclyl, (C4-C8)cycloalkenyl
and
(C4-C8)cycloalkyl;
N
is selected from heteroaryl and heterocyclyl, including spirocyclic
moieties;
R1 is independently selected from: CF3, oxo, (C=O)aObC1-CIO alkyl, (C=O)aOb
aryl, (C=O)aObC2-C 10 alkenyl, (C=O)aObC2-C 10 alkynyl, CO2H, halo, OH, ObC 1-
C6
perfluoroalkyl, (C=O)aNR7R8, CN, (C=O)aObC3-C8 cycloalkyl, S(O)mNR7R8, S(O)m-
(C1-
C l 0)alkyl, and (C=O)aObheterocyclyl, said alkyl, aryl, alkenyl, alkynyl,
cycloalkyl, and
heterocyclyl is optionally substituted with one or more substituents selected
from R6;
R2 is selected from: C1-ClO alkyl, C2-C10 alkenyl, C2-C10 alkynyl, CO2H, C3-
Cg cycloalkyl, aryl and heterocyclyl, said alkyl, alkenyl, alkynyl,
cycloalkyl, aryl and
heterocyclyl is optionally substituted with one or more substituents selected
from R6;
R3 is selected from: H, CF3, oxo, (C=O)aObC 1-C 10 alkyl, (C=O)aOb aryl,
(C=O)aObC2-Cl0 alkenyl, (C=O)aObC2-C10 alkynyl, CO2H, halo, OH, ObCl-C6
perfluoroalkyl, (C=O)aNR7R8, CN, (C=O)aObC3-C8 cycloalkyl, S(O)mNR7R8, S(O)m-
(C1-
C 10)alkyl, and (C=O)aObheterocyclyl, said alkyl, aryl, alkenyl, alkynyl,
cycloalkyl, and
heterocyclyl is optionally substituted with one or more substituents selected
from R6;
R4 is independently selected from: H, CF3, OXO, (C=O)aObC I - C 10 alkyl,
(C=O)aOb aryl, (C=O)aObC2-C10 alkenyl, (C=O)aObC2-C10 alkynyl, CO2H, halo, OH,
ObCI-
C6 perfluoroalkyl, (C=O)aNR7R8, CN, (C=O)aObC3-C8 cycloalkyl, S(O)mNR7R8,
S(O)m-
(C1-CIO)alkyl, and (C=O)aObheterocyclyl, said alkyl, aryl, alkenyl, alkynyl,
cycloalkyl, and
heterocyclyl is optionally substituted with one or more substituents selected
from R6;
R6 is,, CF3, oxo, (C=O)aObC 1-C 10 alkyl, (C=O)aObaryl, C2-C 10 alkenyl, C2-
C 10 alkynyl, (C=O)aOb heterocyclyl, CO2H, halo, CN, OH, ObC 1-C6
perfluoroalkyl,
Oa(C=0)bNR7R8, oxo, CHO, (N=0)R7R8, S(O)mNR7R8, S(O)m-(C 1-C 10)alkyl, SH or
(C=O)aObC3-C8 cycloalkyl, said alkyl, aryl, alkenyl, alkynyl, heterocyclyl,
and cycloalkyl
optionally substituted with one or more substituents selected from R6a;
-2-
CA 02714087 2010-08-04
WO 2009/102537 PCT/US2009/031633
R6a is selected from. CF3, (C=O)aOb(C I -C I O)alkyl, Oa(C I -
C3)perfluoroalkyl,
(CO-C6)alkylene-S(O)rnRa, oxo, OH, halo, CN, (C2-C10)alkenyl, (C2-CIO)alkynyl,
(C3-
C6)cycloalkyl, (CO-C6}alkylene-aryl, (CO-C6)alkylene-heterocyclyl, (C0-
C6)alkylene-N(Rb)2,
C(O)Ra, (C0-C6)alkylene-CO2Ra, C(O)H, and (C0-C6)alkylene-CO2H, said alkyl,
alkenyl,
alkynyl, cycloalkyl, aryl, and heterocyclyl is optionally substituted with up
to three substituents
selected from Rb, OH, (C 1-C6)alkoxy, halogen, CO2H, CN, O(C=O)C 1-C6 alkyl,
oxo, and
N(Rb)2;
R7 and R8 are independently selected from: H, (C=O)ObC 1 -C 10 alkyl,
(C=O)ObC3-C8 cycloalkyl, (C=O)Obaryl, (C=O)Obheterocyclyl, C1-ClO alkyl, aryl,
C2-C10
alkenyl, C2-Cl0 alkynyl, heterocyclyl, C3-C8 cycloalkyl, S(O)mRa, and
(C=O)NRb2, said alkyl,
cycloalkyl, aryl, heterocylyl, alkenyl, and alkynyl is optionally substituted
with one or more
substituents selected from R6a, or R7 and R8 can be taken together with the
nitrogen to which
they are attached to form a monocyclic or bicyclic heterocycle with 3-7
members in each ring
and optionally containing, in addition to the nitrogen, one or two additional
heteroatorns selected
from N, 0 and S, said monocylcic or bicyclic heterocycle optionally
substituted with one or more
substituents selected from R6a;
Ra is H, (C 1 -C6)alkyl, (C3-C6)cycloalkyl, aryl, or heterocyclyl; and
Rb is independently H, (C I -C6)alkyl, aryl, heterocyclyl, (C3-C6)cycloalkyl,
(C=O)OCI-C6 alkyl, (C=O)C1-C6 alkyl or S(O)mRa;
or a pharmaceutically acceptable salt or a stereoisomer thereof
In a second embodiment of this invention, the inhibitors of CHKI activity are
illustrated by the Formula B:
0 (R5)q
~ N
(RI)"
\
CB N 3 S N (84)1,
R
O
wherein:
N
is selected from heteroaryl and heterocyclyl, including spirocyclic
moieties;
gis0,1,2,3,4or5;
R5 is independently selected from: CF3, oxo, (C=O)aObC 1-C l 0 alkyl,
(C=O)aObaryl, C2-CIO alkenyl, C2-C I 0 alkynyl, (C=O)aOb heterocyclyl, CO2H,
halo, CN, OH,
ObC 1-C6 perfluoroalkyl, Oa(C=O)bNR7R8, oxo, CHO, (N=O)R7RS, S(O)mNR7R&, S(O)m-
-3-
CA 02714087 2010-08-04
WO 2009/102537 PCT/US2009/031633
(C1-C10)alkyl, SH and (C=O)aObC3-C8 cycloalkyl, said alkyl, aryl, alkenyl,
alkynyl,
heterocyclyl, and cycloalkyl optionally substituted with one or more
substituents selected from
R6a;
and all substituents and variables are as defined in the first embodiment;
or a pharmaceutically acceptable salt or a stereoisomer thereof
In a third embodiment of this invention, the inhibitors of CHKI activity are
illustrated by the Formula B, wherein:
N
is selected from heteroaryl and heterocyclyl, including spirocyclic
moieties;
aisOorl;bis0orl;misO,l,or2;nisO,1,2,3,4or5; pis0,l,2,3,4,5or
6;andgis0, 1,2,3,4or5;
R1 is independently selected from: CF3, oxo, (C=O)aObCI-CIN alkyl, (C=O)aOb
aryl, (C=O)aObC2-ClO alkenyl, (C=O)aObC2-C1p alkynyl, CO2H, halo, OH, ObCI-C6
perfluoroalkyl, (C=O)aNR7RS, CN, (C=O)aObC3-C8 cycloalkyl, S(O)mNR7R8, S(O)m-
(CI-
C 10)alkyl, and (C=O)aObheterocyclyl, said alkyl, aryl, alkenyl, alkynyl,
cycloalkyl, and
heterocyclyl is optionally substituted with one or more substituents selected
from R6;
R5 is independently selected from. CF3, oxo, (C=O)aObC I -C 10 alkyl,
(C=O)aObaryl, C2-C10 alkenyl, C2-C10 alkynyl, (C=O)aOb heterocyclyl, CO2H,
halo, CN, OH,
ObC1-C6 perfluoroalkyl, Oa(C=O)bNR7R8, oxo, CHO, (N=O)R7R8, S(O)mNR7R8, S(O)m-
(CI-CI0)alkyl, SH and (C=O)aObC3-C8 cycloalkyl, said alkyl, aryl, alkenyl,
alkynyl,
heterocyclyl, and cycloalkyl optionally substituted with one or more
substituents selected from
R6;
R3 is selected from: H, CF3, oxo, (C=O)aObC I -C 10 alkyl, (C=O)aOb aryl,
(C=O)aObC2-Cl0 alkenyl, (C=O)aObC2-C10 alkynyl, CO2H, halo, OH, ObCl-C6
perfluoroalkyl, (C=O)aNR7R8, CN, (C-0)aObC3-C8 cycloalkyl, S(O)mNR7R8, S(O)m-
(C1-
C10)alkyl, and (C=O)aObheterocyclyl, said alkyl, aryl, alkenyl, alkynyl,
cycloalkyl, and
heterocyclyl is optionally substituted with one or more substituents selected
from R6;
R4 is independently selected from: H, CF3, OXO, (C=O)aObC 1 - C 10 alkyl,
(C=O)aOb aryl, (C=O)aObC2-C 10 alkenyl, (C=O)aObC2-C 10 alkynyl, CO2H, halo,
OH, ObC I -
C6 perfluoroalkyl, (C=O)aNR7R8, CN, (C =O)aObC3-C8 cycloalkyl, S(O)1nNR7R8,
S(O)m-
(C1-C10)alkyl, and (C=O)aObheterocyclyl, said alkyl, aryl, alkenyl, alkynyl,
cycloalkyl, and
heterocyclyl is optionally substituted with one or more substituents selected
from R6;
R6 is: CF3, oxo, (C=O)aObCl -C 10 alkyl, (C=O)aObaryl, C2-C10 alkenyl, C2-
C 10 alkynyl, (C=O)aOb heterocyclyl, CO2H, halo, CN, OH, ObC 1-C6
perfluoroalkyl,
Oa(C=O)bNR7R8, oxo, CHO, (N=O)R7RS, S(O)mNR7R8, S(O)m-(CI-C 1Q)alkyl, SH or
(C=O)aObC3-C8 cycloalkyl;
-4-
CA 02714087 2010-08-04
WO 2009/102537 PCT/US2009/031633
R7 and R8 are independently selected from: H, (C=O)ObC 1-C 10 alkyl,
(C=O)ObC3-C8 cycloalkyl, (C=O)Obaryl, (C=O)Obheterocyclyl, C1-C10 alkyl, aryl,
C2-C10
alkenyl, C2-C10 alkynyl, heterocyclyl, C3-C8 cycloalkyl, S(O)mRa, and
(C=O)NRb2, or R7 and
R8 can be taken together with the nitrogen to which they are attached to form
a monocyclic or
bicyclic heterocycle with 3-7 members in each ring and optionally containing,
in addition to the
nitrogen, one or two additional heteroatoms selected from N, 0 and S;
Ra is H or (C 1 -C6)alkyl; and
Rb is independently H or (C 1-C6)alkyl;
or a pharmaceutically acceptable salt or a stereoisomer thereof.
In a fourth embodiment of this invention, the inhibitors of CHK1 activity are
illustrated by the Formula B, wherein:
N
is selected from heteroaryl and heterocyclyl, including spirocyclic
moieties;
aisOorl;bis0orl;mis0,1,or2;nis0,1,2,3,4or5; pisO,1,2,3,4,5or
6; and gis0, 1,2,3,4or5;
R1 is independently selected from: CF3, oxo, (C=O)aObC1-C10 alkyl, (C=O)aOb
aryl, (C=O)aObC2-C 10 alkenyl, (C=O)aObC2-C10 alkynyl, CO2H, halo, OH, ObC 1-
C6
perfluoroalkyl, (C=O)aNR7R8, CN, (C=O)aObC3-Cg cycloalkyl, S(O)mNR7R8, S(O)m-
(C1-
C 10)alkyl, and (C=O)aObheterocyclyl, said alkyl, aryl, alkenyl, alkynyl,
cycloalkyl, and
heterocyclyl is optionally substituted with one or more substituents selected
from R6;
R5 is independently selected from: CF3, OXO, (C=O)aObC 1 -C 10 alkyl,
(C=O)aObaryl, C2-C10 alkenyl, C2-C10 alkynyl, (C=O)aOb heterrocyclyl, CO2H,
halo, CN, OH,
ObC1-C6 perfluoroalkyl, Oa(C=O)bNR7R8, oxo, CHO, (N=O)R7RS, S(O)mNR7R8, S(O)m-
(C1-C1o)alkyl, SH and (C=O)aObC3-C8 cycloalkyl, said alkyl, aryl, alkenyl,
alkynyl,
heterocyclyl, and cycloalkyl optionally substituted with one or more
substituents selected from
R6;
R3 is: C1-C6 alkyl, said alkyl is optionally substituted with one or more
substituents selected from R6;
R4 is independently selected from. H, CF3, oxo, (C=O)aObC 1-C 10 alkyl,
(C=O)aOb aryl, (C=O)aObC2-C10 alkenyl, (C=O)aObC2-C10 alkynyl, CO2H, halo, OH,
ObCI-
C6 perfluoroalkyl, (C=0)aNR7R8, CN, (C=O)aObC3-C8 cycloalkyl, S(O)mNR7R8,
S(O)m-
(C 1-C 10)alkyl, and (C=O)aObheterocyclyl, said alkyl, aryl, alkenyl, alkynyl,
cycloalkyl, and
heterocyclyl is optionally substituted with one or more substituents selected
from R6;
R6 is: CF3, oxo, (C=O)aObC 1-C 10 alkyl, (C=O)aObaryl, C2-C 10 alkenyl, C2-
C10 alkynyl, (C=O)aOb heterocyclyl, CO2H, halo, CN, OH, ObC1-C6
perfluoroalkyl,
-5-
CA 02714087 2010-08-04
WO 2009/102537 PCT/US2009/031633
Oa(C=O)bNR7R8, oxo, CHO, (N=O)R7R8, S(O)mNR7R8, S(O)m-(C 1-C 10)alkyl, SH or
(C=O)aObC3-C8 cycloalkyl;
R7 and R8 are independently selected from: H, (C=O)ObC 1-C 10 alkyl,
(C=O)ObC3-C8 cycloalkyl, (C=O)Obaryl, (C=O)Obheterocyclyl, C1-C10 alkyl, aryl,
C2-C10
alkenyl, C2-Cl0 alkynyl, heterocyclyl, C3-Cg cycloalkyl, S(O)mRa, and
(C=O)NRb2, or R7 and
R8 can be taken together with the nitrogen to which they are attached to form
a monocyclic or
bicyclic heterocycle with 3-7 members in each ring and optionally containing,
in addition to the
nitrogen, one or two additional heteroatoms selected from N, 0 and S;
Ra is H or (C1-C6)alkyl; and
Rb is independently H or (C1-C6)alkyl;
or a pharmaceutically acceptable salt or a stereoisomer thereof.
In a fifth embodiment of this invention, the inhibitors of CHKI activity are
illustrated by the Formula B, wherein:
N
is selected from heteroaryl and heterocyclyl, including spirocyclic
moieties;
ais0orl;bis0orl;mis0, l,or2;nis 1 or2; pis0, 1,2,3,4,5or6;andgis
I or 2;
Rl is independently selected from. H, S(0)2-(CI-C6)alkyl, (C1-C6)alkyl, and
(C=O)O(C1-C6)alkyl;
R5 is independently selected from: H, halo, C3-C8 cycloalkyl, C3-C8
cycloalkenyl, aryl and heterocyclyl, said aryl, cycloalkyl, and heterocyclyl
are optionally
substituted with one or more substituents selected from R6;
R3 is: C 1-C6 alkyl, said alkyl is optionally substituted with one or more
substituents selected from R6;
R4 is independently selected from: H, CF3, oxo, (C=O)aObC I -C 10 alkyl,
(C=O)aOb aryl, (C=O)aObC2-C l 0 alkenyl, (C=O)aObC2-C 10 alkynyl, CO2H, halo,
OH, ObC l -
C6 perfluoroalkyl, (C=0)aNR7R8, CN, (C-O)aObC3-C8 cycloalkyl, S(O)rNR7R8,
S(O)m-
(C 1-C I O)alkyl, and (C=O)aObheterocyclyl, said alkyl, aryl, alkenyl,
alkynyl, cycloalkyl, and
heterocyclyl is optionally substituted with one or more substituents selected
from R6;
R6 is.
CF3, OXO, (C=O)aObC1-C10 alkyl, (C=O)aObaryl, C2-C10 alkenyl, C2-
C 10 alkynyl, (C=O)aOb heterocyclyl, CO2H, halo, CN, OH, ObC 1-C6
perfluoroalkyl,
Oa(C=O)bNR7R8, oxo, CHO, (N=O)R7R8, S(O)mNR7R8, S(O)m-(C1-C1O)alkyl, SH or
(C=O)aObC3-C8 cycloalkyl;
R7 and R8 are independently selected from: H, (C=O)ObC1-C10 alkyl,
(C=O)ObC3-C8 cycloalkyl, (C=0)Obaryl, (C=O)Obheterocyclyl, C1-C10 alkyl, aryl,
C2--C10
alkenyl, C2-CIO alkynyl, heterocyclyl, C3-C8 cycloalkyl, S(O)mRa, and
(C=O)NRb2, or R7 and
-6-
CA 02714087 2010-08-04
WO 2009/102537 PCT/US2009/031633
R8 can be taken together with the nitrogen to which they are attached to form
a monocyclic or
bicyclic heterocycle with 3-7 members in each ring and optionally containing,
in addition to the
nitrogen, one or two additional heteroatoms selected from N, 0 and S;
Ra is H or (C1-C6)alkyl; and
Rb is independently H or (C1-C6)alkyl;
or a pharmaceutically acceptable salt or a stereoisorner thereof
Specific compounds of the instant invention include:
3-(3-chlorophenyl)-2- { [ 1-(2,8-diazaspiro[5.5]under-2-
ylcarbonyl)pentyl]thio} -8-
methylquinazolin-4(3H)-one (1-8);
1-(2- { [3-(3-chlorophenyl)-4-oxo-3,4-dihydroquinazolin-2-yl]methyl
}hexanoyl)piperidine-3-
carboxamide (1-9);
2- {2-[(4-acetylpiperazin-1-y1)carbonyl]hexyl}-3-(3-chlorophenyl)-quinazolin-
4(3H)-one (1-10);
3-(3-chlorophenyl)-2- {2-[(4-phenylpiperazin-1-yl)carbonyl]hexyl}quinazolin-
4(31.2)-one (1-11);
1-(2- {[3 -(3 - eblorophenyl)-4-oxo-3,4-dihydroquinazolin-2-yl]methyl }
hexanoyl)-N,N-
diethylpiperidine-3-carboxamide (1-12);
3-(3-chlorophenyl)-2- {2-[(4-pyridin-2-ylpiperazin-l -yl)carbonyl]hexyl}-
quinazolin-4(3f1)-one
(1-13);
2-(2- { [4-(aminomethyl)-piper idin- l -yl] carbonyl } -hexyl)-3-(3-chloro-
phenyl)quinazolin-4(3H)-
one (1-14);
3-(3-chlorophenyl)-2- {2-[(7-methyl-2,7-diazaspiro[4.4]non-2-
yl)carbonyl]hexyl}quinazolin-
4(3H)-one (1-15);
2-(2- { [4-(1 H-benzimidazol-2-yl)piperidin- l -yl] carbonyl } hexyl)-3-(3 -
chlorophenyl)quinazolin-
4(3H)-one (1-16);
3-(3-chlorophenyl)-2- {2-[(1,1-dioxidothio-ranorpholin-4-yl)-carbonyl]hexyl}
quinazolin-4(3H)-
one (1-17);
3-(3-chlorophenyl)-2-(2- { [4-(methylsulfonyl)-piperazin-l -yl] carbonyl} -
hexyl)quinazolin-4(31-)-
one (1-15);
3-(3-chlorophenyl)-2-(2- { [4-(1 H-imidazol-1-ylrnethyl)piperidin- l -
yl]carbonyl }hexyl)quinazolin-
4(3H)-one (1-19);
3-(3-chlorophenyl)-2-(2- { [4-(methylsulfonyl)-piperi din- l -y1] carbonyl) -
hexyl)quinazolin-4(3H)-
one (1-20);
3-(3-chlorophenyl)-2- [2-(2, 8 -diazaspiro [ 5.5 J-under-2 -ylcarbonyl)-hexyl]
quinazolin-4(3H)-one
(1-21);
3-(3-chlorophenyl)-2-(2- 1[4-(2-fluorophenyl)-piperazin-l-yl] carbonyl } -
hexyl)quinazolin-4(3H)-
one (1-22);
3 -(3 -chlorophenyl)-2--(2- { [4-(4-fluorophenyl)-piperazin-1-y1] carbonyl } -
hexyl)quinazolin-4(3H)-
one (1-23);
-7-
CA 02714087 2010-08-04
WO 2009/102537 PCT/US2009/031633
3-(3-chlorophenyl)-2- {2-[(4,4-diphenylpiperidin-1-yl)carbonyl]hexyl}-
quinazolin-4(3H)-one (1-
24);
3-(3-chlorophenyl)-2- {2 -[(4-pyridin-4-ylpiperidin-l -yl)carbonyl]hexyl}-
quinazolin-4(3H)-one
(1-25);
3-(3-chlorophenyl)-2- {2-[(3,3-diphenylpiperidin-l-yl)carbonyl]hexyl}-
quinazolin-4(3H)-one (1-
26);
3-(3-chlorophenyl)-2-{[ 1-(2,8-diazaspiro[5.5J-undec-2-ylcarbonyl)-
pentyl]thio}-7-(methyl-
sulfonyl)quinazolin-4(3H)-one (1-27);
methyl 3-(3-chloro-phenyl)-2- { [ 1-(2,8-diazaspiro[5.5]undec-2-
ylcarbonyl)pentyl]thio } -4-oxo-
3,4-dihydro-quinazoline-7-carboxylate (1-28);
3 -(3 -chlorophenyl)-2-({ 3-methyl- l -[ (4-pyridin-2-ylpiperazin- l -yl)-
carbonyl]butyl} thio)quinazolin-4(3H)-one (1-29);
3-(3-chlorophenyl)-2-[(1- {[4-(2,3-dihydro-1,4-benzodioxin-5-yl)ppperazin-l -
yl]carbonyl}-3-
methylbutyl)thio]quinazolin-4(3H)-one (1-30);
3-(3-chlorophenyl)-2-[(1-{[4-(4-fluorophenyl)-piperazin-1-yl]carbonyl}-3-
methylbutyl)thio]-
quinazolin-4(3H)-one (1-31);
3-(3 -chlorophenyl)-2-[(1- { [7-(3-chlorophenyl)-2,7-diazaspiro[4.4]non-2-yl]
carbonyl } -3-
methylbutyl)thio]quinazolin-4(3H)-one (1-32);
3-(3-chlorophenyl)-2-[(1- { [7-(2-chlorophenyl)-2,7-diazaspiro[4.4]non-2-
yl]carbonyl}-3-
methylbutyl)thio]quinazolin-4(3H)-one (1-33);
2-({ 1-[(7-benzyl-2,7-diazaspiro[4.4]non-2-yl)carbonyl]-3-methylbutyl }thio)-3-
(3-
chlorophenyl)quinazolin-4(3H)-one (1-34);
3-(3 -chlorophenyl)-2-[(1- { [7-(3-methoxyphenyl)-2,7-di azaspiro[4.4] non-2-
yl ]carbonyl} -3-
methylbutyl)thio]quinazolin-4(3H)-one (1-35);
3-(3-chlorophenyl)-2-[(1-{[7-(4-methoxyphenyl)-2,7-diazaspiro[4.4]non-2-
ylcaarbonyl}-3-
methylbutyl)thio]quinazolin-4(3H)-one (1-36);
1-(2- {[3-(3-chlorophenyl)-4-oxo-3,4-dihydroquinazolin-2-
yl]thio}hexanoyl)piperidine-4-
carboxamide (1-37);
1-[ { [3-(3-chlorophenyl)-4-oxo-3,4-dihydroquinazolin-2-yl]thio}
(phenyl)acetyl]piperidine-4-
carboxamide (1-38);
1-(2- {[3-(3-chlorophenyl)-4-oxo-3,4-dihydroquinazolin-2-yl]thio} -4-
methylpentanoyl)
piperidine-4-carboxamide (1-39);
3-(3-chlorophenyl)-2-[(1- { [7-(2-chlorophenyl)-2,7-diazaspiro [4.4]non-2-
yl]carbonyl }
pentyl)thio]-7-(methylsulfonyl)-quinazolin-4(3H)-one (1-40);
3-(3-chlorophenyl)-2-[(1 _{[7-(4-chlorophenyl)-2,7-diazaspiro[4.4]non-2-
ylccarbonyl}pentyl)
thio]-7-(methylsulfonyl)-quinazolin-4(3H)-one (1-41);
3-(3-chlorophenyl)-2-[(1- { [7-(3-chlorophenyl)-2,7-diazaspiro[4.4]non-2-
yl]carbonyl } pentyl)
thio]-7-(methylsulfonyl)-quinazolin-4(3H)-one (1-42);
-8-
CA 02714087 2010-08-04
WO 2009/102537 PCT/US2009/031633
3-(3-chlorophenyl)-7-(methylsulfonyl)-2-({ 1-[(7-phenyl-2,7-diazaspiro[4.4]non-
2-yl)carbonyl]
pentyl} thio)quinazolin-4(3H)-one (1-43);
3-(3-chlorophenyl)-2-[(1- {[7-(3-methoxyphenyl)-2,7-diazaspiro[4.4]non-2-
yl]carbonyl}
pentyl)thio]-7-(methylsulfonyl)-quinazolin-4(3H)-one (1-44);
methyl 3-(3-chlorophenyl)-2-({1-[(3-cyano-3-phenylazetidin-l-
yl)carbonyl]pentyl}-thio)-4-oxo-
3,4-dihydroquinazoline-7-carboxylate (1-45);
methyl 3-(3 -chlorophenyl)-2- { [ 1-(2,6-diazaspiro[3.3]hept-2-
ylcarbonyl)pentyl]thio}-4-oxo-3,4--
dihydro-quinazoline--7-carboxylate (1-46);
3-(3-chlorophenyl)-2-[(1-{[(2R)-2-(trifluoro-methyl) pyrrolidin-1-yl]-
carbonyl} pentyl)thio]-
quinazolin-4(3H)-one (1-47);
3-(3-chlorophenyl)-2-[(1- { [3-(4-fluorobennyl)-2-oxo-l -oxa-8-
azaspiro[4.5]dec-8-yl]carbonyl}
pentyl)thio]quinazolin-4(3H)-one (1-48);
3-(3-chlorophenyl)-2-[(1- { [5-(4-fluorophenyl)-2,5-diazabicyclo[2.2.1 ]hept-2-
yl]carbonyl}
pentyl)-thio]quinazolin-4(3H)-one (1-49);
3-(3-chlorophenyl)-2-[(1- {[5-(3-fluorophenyl)-2,5-diazabicyclo[2.2.1 ]hept-2-
yl]carbonyl}
pentyl)-thio]quinazolin-4(3H)-one (1-50);
3-(3-chlorophenyl)-2-[(1- {[4-(4-methyl-1,2,5-oxadiazol-3-yl)piperazin-1-
yl]carbonyl}pentyl)-
thio]quinazolin-4(3H)-one (1-51);
3-(3-chlorophenyl)-2-[(1- { [3-(4-fluorophenyl)-1-oxa-S-azaspiro[4.5] dec-8-
yl]carbonyl } pentyl)-
thio]quinazolin-4(3H)-one (1-52);
3-(3-chlorophenyl)-2-[(1- { [(3 S,4S)-3,4-difluoropyrrolidin-l -yl] carbonyl }
pentyl)thio]quinazolin-
4(3H)-one (1-53);
3-(3-chlorophenyl)-2- {[2-(2,8-diazaspiro[5.5]undec-2-yl)-1-(1H imidazol-4-
ylrethyl)-2-
oxoethyl]thio}quinazolin-4(3H)-one (1-54);
2-(3-(1 H-imidazol-4-yl)-1-oxo-1-(2,8-diazaspiro[5.5]undecan-2-yl)propan-2-
ylthio)-3-(3-
chlorophenyl)-8-methylquinazolin-4(3H)-one (1-55);
3-(3-chlorophenyl)-8-methyl-2-(1-oxa-1-(4-phenylpiperazin-1-yl)hexan-2-
ylthio)quinazolin-
4(3H)-one (1-56);
3-(3-chlorophenyl)-2-(1-(7-(3-methoxyphenyl)-2,7-diazaspiro[4.4]nonan-2-yl)-1-
oxohexan-2-
ylthio)-8-methylquinazolin-4(3H)-one (1-57);
3-(3-chlorophenyl)-2-(1-(7-(4-chlorophenyl)-2,7-diazaspiro[4.4]nonan-2-yl)-1-
oxohexan-2-
ylthio)-S-methylquinazolin-4(3H)-one (1-58);
3-(3-chlorophenyl)-2-(1-(7-(3-chlorophenyl)-2,7-diazaspiro[4.4]nonan-2-yl)-1-
oxohexan-2-
ylthio)-8-methylquinazolin-4(3H)-one (1-59);
3'-[2-[(1-{[4-(Amino-carbonyl)piperidin-1-yl]carbonyl}pentyl)thio]-4-
oxoquinazolin-3(4H)-
yl]biphenyl-4-carboxylic acid (2-3);
1-(2- {[3-(2'-chlorobiphenyl-3-y1)-4-oxo-3,4-dihydroquinazolin-2-
yl]thio}hexanoyl)piperidine-4-
carboxamide (2-4);
-9-
CA 02714087 2010-08-04
WO 2009/102537 PCT/US2009/031633
1-(2- { [3-(3'-chlorobiphenyl-3-yl)-4-oxo-3,4-dihydroquinazolin-2-
yl]thio)hexanoyl)piperidine-4-
carboxamide (2-5);
1-(2- { [3-(4'-chlorobiphenyl-3-yl)-4-oxo-3,4-dihydroquinazolin-2-yl]thio)
hexanoyl)piperidine-4-
carboxamide (2-6);
1-(2- {[3-(2'-aminobiphenyl-3-yl)-4-oxo-3,4-dihydroquinazolin-2-
yl]thio)hexanoyl)piperidine-4W
carboxamide (2-7);
1-(2- {[3-(3'-aminobiphenyl-3-yl)-4-oxo-3,4-dihydroquinazolin-2-
yl]thio)hexanoyl)piperidine-4-
carboxamide (2-8);
1-(2- { [3-(4'-aminobiphenyl-3-yl)-4-oxo-3,4-dihydroquinazolin-2-
yl]thio}hexanoyl)piperidine-4-
carboxamide (2-9);
1-(2- {[4-oxo-3-(3-pyridin-3-ylphenyl)-3,4-dihydroquinazolin-2-
yl]thio}hexanoyl)piperidine-4-
carboxamide (2-10);
1-(2- { [4-oxo-3-(3-pyridin-4-ylphenyl)-3,4-dihydroquinazolin-2-
yl]thio}hexanoyl)piperidine-4-
carboxamide (2-11);
3'-[2-[(1- {[4-(aminocarbonyl)piperidin-l -yl]carbonyl}pentyl)thio]-4-
oxoquinazolin-3(4H)-
yl]biphenyl-3-carboxylic acid (2-12);
1-[2-({3-[3-(3-fu yl)phenyl]-4-oxo-3,4-dihydroquinazolin-2-
yl}thio)hexanoyl]piperidine-4-
carboxalnide (2-13);
1-[2-({3-[3-(1-methyl-1H-pyrazol-4-yl)phenyl]-4-oxo-3,4-dihydroquinazolin-2-
yl}thio)
hexanoyl]piperidine-4-carboxamide (2-14);
1-(2-- { [3-(3-cyclohex-l -en-l-ylphenyl)-4-oxo-3,4-dihydroquinazolin-2-
y1]thio} hexanoyl)
piperidine-4-carboxamide (2-15);
1-[2-({4-oxo-3 - [3-(2-thienyl)phenyl]-3,4-dihydroquinazolin-2-yl }
thio)hexanoyl]piperidine-4-
carboxamide (2-16);
2-[(1- {[7-(3-chlorophenyl)-2,7-diazaspiro[4.4]non-2-yl]carbonyl}-3-
methylbutyl)thio]-3-[3-(3-
furyl)phenyl]quinazolin-4(3H)-one (2-17);
2-[(1- { [7-(3-chlorophenyl)-2,7-diazaspiro[4.4]non-2-yl]carbonyl } -3-
methylbutyl)thio]-3-(3-
cyclohex-l-en-l-ylphenyl)quinazolin-4(3H)-one (2-18);
1-(2-f [3-(4-fluoro-2-methylbiphenyl-3-yl)-4-oxo-3,4-dihydroquinazolin-2-
yl]thio}hexanoyl)
piperidine-4-carboxamide (2-19);
2-[(1- { [5-(4-fluorophenyl)-2,5-diazabicyclo[2.2.1 ]hept-2-
yl]carbonyl}pentyl)thio]-3-[3-(3-
furyl)phenyl]quinazolin-4(3H)-one (2-20);
2-[(I-{ [3-(4-fluorobenzyl)-2-oxo- l -oxa-8-azaspiro[4.5]dec-8-yl] carbonyl }
pentyl)thio]-3-[3-(3-
furyl)phenyl]quinazolin-4(31-.)-one (2-21);
3-(3-cyclohex- l -en-l-ylphenyl)-2-[(1- { [5-(4-fluorophenyl)-2,5-
diazabicyclo[2.2.1 ]kept-2-
yl]carbonyl}pentyl)thio]quinazolin-4(3H)-one (2-22);
3-(3-cyclohex-l -en-l-ylphenyl)-2-[(1- { [3-(4-fluorobenzyl)-2-oxo-l -oxa-8-
azaspiro[4.5]dec-8-
yl]carbonyl}pentyl)thio]quinazolin-4(3H)-one (2-23);
-10-
CA 02714087 2010-08-04
WO 2009/102537 PCT/US2009/031633
3-(3-cyclohex-l-en- l -ylphenyl)-2-[(1- { [4-(2,4-difluorophenyl)piperazin-l-
yl]carbonyl}pentyl)thio]quinazolin-4(311)-one (2-24);
2-[(1-{[4-(2,4-difluorophenyl)piperazin-l-yl]carbonyl}pentyl)thio]-3-[3-(3-
furyl) phenyl]
quinazolin-4(311)-one (2-25);
3-(3-cyclohex-l-en-l-ylphenyl)-2-{[I -(2,8-diazaspiro[5.5]undec-2-
ylcarbonyl)pentyl]
thio}quinazolin-4(311)-one (2-26);
2- { [3-amino- l -(2,8-diazaspiro[5.5]undec-2-ylcarbonyl)propyl]thio } -3-(3-
chlorophenyl)
quinazolin-4(311)-one (3-7);
2-1[4- amino- I -(2,8-di azaspiro [ 5.5]undec-2-ylcarbonyl)butyl]thio} -3-(3-
chlorophenyl)-8-
methylquinazolin-4(311)-one (3-8);
2- { [5-amino-l-(2,8-diazaspiro[5.5]undec-2-ylcarbonyl)pentyl]tio } -3-(3-
chlorophenyl)
quinazolin-4(311)-one (3-9);
2-[(4-amino-l- {[7-(3-chlorophenyl)-2,7-diazaspiro[4.4]non-2-
yl]carbonyl}butyl)thio]-3-(3-
chlorophenyl)quinazolin-4(311)-one (3-10);
2-(5-amino-l-(7-(3-chlorophenyl)-2,7-diazaspiro[4.4]nonan-2-yl)-1-oxopentan-2-
ylthio)-3-(3-
chlorophenyl)-8-inethylquinazolin-4(3H)-one (3-11);
2-[(5-amino-l - { [7-(3Tchlorophenyl)-2,7-diazaspiro[4.4]non-2-
yl]carbonyl)pentyl)thio]-3-(3-
chlorophenyl)quinazolin-4(311)-one (4-6); and
2- { [ 5-amino- l -(2, 8-diazaspiro [ 5.5 ]undec-2-ylcarbonyl)pentyl] thio } -
3 -(3 -chlorophenyl)-8 -
methylquinazolin-4(311)-one (4-7);
or a pharmaceutically acceptable salt or a stereoisomer thereof
TFA salts of the compounds of the instant invention include:
3-(3-chlorophenyl)-2- { [ 1-(2,8-diazaspiro[5.5]undec-2-
ylcarbonyl)pentyl]thio}-8-
methylquinazolin-4(311)-one (1-8);
1-(2- { [3-(3-chlorophenyl)-4-oxo-3,4-dihydroquinazolin-2-yl]methyl }
hexanoyl)piperidine-3-
carboxamide (1-9);
2- {2-[(4-acetylpiperazin-l -yl)carbonyl]hexyl}-3-(3-chlorophenyl)-quinazolin-
4(311)-one (1-10);
3-(3-chlorophenyl)-2- {2-[(4-phenylpiperazin-1-yl)carbonyl]hexyl} quinazolin-
4(311)-one (1-11);
1-(2-{ [3-(3-chlorophenyl)-4-oxo-3,4-dihydroquinazolin-2-yl]methyl } hexanoyl)-
N,N-
diethylpiperidine-3-carboxarnide (1-12);
3-(3-chlorophenyl)-2- {2-[(4-pyridin-2-ylpiperazin-1-yl)carbonyl]hexyl) -
quinazolin-4(311)-one
(1-13);
2-(2- { [4-(aminom.ethyl)-piperidin-l -yl]carbonyl }-hexyl)-3-(3-chloro-
phenyl)quinazolin-4(3H)-
one (1-14);
3-(3-chlorophenyl)-2-{2-[(7-methyl-2,7-diazaspiro[4.4]non-2-
yl)carbonyl]hexyl}quinazolin-
4(3H)-one (1-15);
2-(2- { [4-(1 H-benzimidazol-2-yl)piperidin-l -yl]carbonyl }hexyl)-3-(3-
chlorophenyl)quinazolin-
4(3H)-one (1-16);
-11-
CA 02714087 2010-08-04
WO 2009/102537 PCT/US2009/031633
3-(3-chlorophenyl)-2- {2-[(1,1-dioxidothio-morpholin-4-yl)-carbonyl]hexyl
}quinazolin-4(3H)-
one (1-17);
3-(3-chlorophenyl)-2-(2- 1[4-(methylsulfonyl)-piperazin-l -yl]carbonyl}-
hexyl)quinazolin-4(3H)-
one (1-18);
3-(3-chlorophenyl)-2-(2- f [4-(1 H-imidazol-1-ylmethyl)piperidin-1-yl]
carbonyl } hexyl)quinazolin-
4(3H)-one (1-19);
3-(3-chlorophenyl)-2-(2- {[4-(i-nethylsulfonyl)-piperidin-1-yl]carbonyl}-
hexyl)quinazolin-4(3H)-
one (1-20);
3-(3-chlorophenyl)-2T [2-(2,8-diazaspiro[5.5]-undec-2-ylcarbonyl)-
hexyl]quinazolin-4(3H)-one
(1-21);
3-(3-chlorophenyl)-2-(2- { [4-(2-fluorophenyl)-piperazin-1-yl]carbonyl } -
hexyl)quinazolin-4(3H)-
one (1-22);
3 -(3 -chlorophenyl)-2-(2- { [4-(4-fluorophenyl)-piperazin-1-yl] carbonyl } -
hexyl)quinazol in-4(3H)-
one (1-23);
3-(3-chlorophenyl)-2-{2-[(4,4-diphenylpiperidin-l-yl)carbonyl]hexyl}-
quinazolin-4(3H)-one (1-
24);
3-(3-chlorophenyl)-2- {2- [(4-pyridin-4-ylpiperidin-1-yl)carbonyl]hexyl } -
quinazolin-4(3H)-one
(1-25);
3-(3-chlorophenyl)-2-{2-[(3,3-diphenylpiperidin-1-yl)carbonyl]hexyl}-
quinazolin-4(311)-one (1-
26);
3-(3-chlorophenyl)-2- { [ 1-(2,8--diazaspiro[5.5]-under-2-ylcarbonyl)-
pentyl]thio} -7-(methyl-
sulfonyl)quinazolin-4(3H)-one (1-27);
methyl 3-(3-chloro-phenyl)-2- { [ 1-(2,8-diazaspiro[5.5]under-2-
ylcarbonyl)pentyl]thio}-4-oxo-
3,4-dihydro-quinazoline-7-carboxylate (1-28);
3-(3-chlorophenyl)-2-({3-m.ethyl-l-[(4-pyridin-2-ylpiperazin-l-yl)-
carbonyl]butyl)thio)quinazolin-4(3H)-one (1-29);
3-(3-chlorophenyl)-2-[(1- { [4-(2,3-dihydro-1,4-benzodioxin-5-yl)piperazin-l -
yl]carbonyl } -3-
methylbutyl)thio]quinazolin-4(3H)-one (1-30);
3-(3-chlorophenyl)-2-[(1- { [4-(4-fluorophenyl)-piperazin-l-yl]carbonyl } -3-
methylbutyl)thio]-
quinazolin-4(311)-one (1-31);
3-(3-chlorophenyl)-2-[(1- f [7-(3-chlorophenyl)-2,7-diazaspiro[4.4]non-2-yl]
carbonyl }-3-
methylbutyl)thio]quinazolin-4(3H)-one (1-32);
3-(3-chlorophenyl)-2-[(1- { [7-(2-chlorophenyl)-2,7-diazaspiro[4.4]non-2-
yl]carbonyl } -3-
methylbutyl)thio]quinazolin-4(3H)-one (1-33);
2-({1-[(7-benzyl-2,7-diazaspiro[4.4]non-2-yl)carbonyl]-3-methylbutyl}thio)-3-
(3-
chlorophenyl)quinazolin-4(3H)-one (1-34);
3-(3-chlorophenyl)-2-[(1- { [7-(3-methoxyphenyl)-2,7-diazaspiro[4.4]non-2-
yl]carbonyl } -3-
methylbutyl)thio]quinazolin-4(3H)-one (1-35);
-12-
CA 02714087 2010-08-04
WO 2009/102537 PCT/US2009/031633
3-(3 -chlorophenyl)-2-[(1- { [7-(4-methoxyphenyl)-2,7-diazaspiro [4.4] non-2-
yl] carbonyl } -3-
methylbutyl)thio]quinazolin-4(3H)-one (1-36);
1-(2- {[3-(3-chlorophenyl)-4-oxo-3,4-dihydroquinazolin-2-
yl]thio}hexanoyl)piperidine-4-
carboxamide (1-37);
1-[ {[3-(3-chlorophenyl)-4-oxo-3,4-dihydroquinazolin-2-yl]thio}
(phenyl)acetyl]piperidine-4-
carboxamide (1-38);
1-(2- { [3-(3-chlorophenyl)-4-oxo-3,4-dihydroquinazolin-2-yl]thio } -4-
methylpentanoyl)
piperidine-4-carboxamide (1-39);
3-(3-chlorophenyl)-2-[(1- { [7-(2-chlorophenyl)-2,7-diazaspiro[4.4]non-2-yl]
carbonyl }
pentyl)thio]-7-(methylsulfonyl)-quiriazolin-4(3H)-one (1-40);
3-(3-chlorophenyl)-2-[(1- { [7-(4-chlorophenyl)-2,7-diazaspiro[4.4]non-2-
yl]carbonyl }pentyl)
thio]-7-(methylsulfonyl)-quinazolin-4(3H)-one (1-41);
3-(3-chlorophenyl)-2-[(1- { [7-(3-chlorophenyl)-2,7-diazaspiro [4.4]non-2-yl]
carbonyl } pentyl)
thio]-7-(methylsulfonyl)-quinazolin-4(3H)-one (1-42);
3--(3-chlorophenyl)-7-(methylsulfonyl)-2-({ 1-[(7-phenyl-2,7-
diazaspiro[4.4]non-2-yl)carbonyl]
pentyl}thio)quinazolin-4(3H)-one (1-43);
3-(3 -chlorophenyl)-2-[(1- { [7-(3-methoxyphenyl)-2,7-diazaspiro [4.4]non-2-
yl] carbonyl )
pentyl)thio]-7-(methylsulfonyl)-quinazolin-4(3H)-one (1-44);
methyl 3-(3-chlorophenyl)-2-({ 1-[(3-cyano-3-phenylazetidin-1-
yl)carbonyl]pentyl } -thio)-4-oxo-
3,4-dihydroquinazoline-7-carboxylate (1-45);
methyl 3-(3-chlorophenyl)-2- {[ 1-(2,6-diazaspiro[3.3]hept-2-
ylcarbonyl)pentyl]thio}-4-oxo-3,4-
dihydro-quinazoline-7-carboxylate (1-46);
3-(3-chlorophenyl)-2-[(1-{[(2R.)-2-(trifluoro-methyl) pyrrolidin-l-yl]-
carbonyl} pentyl)thio]-
quinazolin-4(3H)-one (1-47);
3-(3-chlorophenyl)-2-[(1- {[3-(4-fluoropeenyl)-2-oxo-l -oxa-8-azaspiro[4.5]dec-
8-yl]carbonyl}
pentyl)thio]quinazolin-4(3H)-one (1-48);
3-(3-chlorophenyl)-2-[(1- { [ 5-(4-flnorophenyl)-2,5-diazabicyclo[2.2.1 ]kept-
2-yl]carbonyl }
pentyl)-thio]quinazolin-4(3H)-one (1-49);
3-(3-chlorophenyl)-2-[(1- { [5-(3-flnorophenyl)-2,5-diazabicyclo[2.2.1 ]kept-2-
yl]carbonyl }
pentyl)-thio]quinazolin-4(3H)-one (1-50);
3-(3-chlorophenyl)-2-[(1 - {[4-(4-methyl-1,2,5-oxadiazol-3-yl)piperazin-1-
yl]carbonyl}pentyl)-
thio]quinazolin-4(3H)-one (1-51);
3-(3-chlorophenyl)-2-[(1- {[3-(4-fluorophenyl)-1-oxa-8-azaspiro[4.5]dec-8-
yl]carbonyl}pentyl)-
thio]quinazolin-4(3H)-one (1-52);
3-(3-chlorophenyl)-2-[(I- f [(3S,4S)-3,4-difluoropyrrolidin-1-yl]carbonyl
}pentyl)thio]quinazolin-
4(3H)-one (1-53);
3-(3-chlorophenyl)-2- {[2-(2,8-diazaspiro[5.5]undec-2-yl)-1-(1H-imidazol-4-
ylmethyl)-2-
oxoethyl]thio}quinazolin-4(3H)-one (1-54);
-13-
CA 02714087 2010-08-04
WO 2009/102537 PCT/US2009/031633
2-(3-(1 H-imidazol-4-yl)-1-oxo-1-(2,8-diazaspiro [5.5]undecan-2-yl)propan-2-
ylthio)-3-(3-
chlorophenyl)-8-methylquinazolin-4(3H)-one (1-55);
3-(3-chlorophenyl)-8-methyl-2-(1-oxo-1-(4-phenylpiperazin-l-yl)hexan-2-
ylthio)quinazolin-
4(3H)-one (1-56);
3-(3-chlorophenyl)-2-(1-(7-(3-methoxyphenyl)-2,7-diazaspiro[4.4]nonan-2-yl)-1-
oxohexan-2-
ylthio)-8-methylquinazolin-4(3H)-one (1-57);
3-(3-chlorophenyl)-2-(1-(7-(4-chlorophenyl)-2,7-diazaspiro[4.4]nonan-2-yl)-1-
oxohexan-2-
ylthio)-8-methylquinazolin-4(3H)-one (1-58);
3-(3-chlorophenyl)-2-(1-(7-(3-chlorophenyl)-2,7-diazaspiro[4.4]nonan-2-yl)-1-
oxohexan-2-
ylthio)-8-methylquinazolin-4(3H)-one (1-59);
3 '-[2-[(1- { [4-(Amino-carbonyl)piperidin-l-yl]carbonyl } pentyl)thio]-4-
oxoquinazolin-3(4H)-
yl]biphenyl-4-carboxylic acid (2-3);
1-(2- { [3-(2'-chlorobiphenyl-3-yl)-4-oxo-3,4-dihydroquinazolin-2-
ylthio}hexanoyl)piperidine--4-
carboxamide (2-4);
1-(2- { [3-(3'-chlorobiphenyl-3-yl)-4-oxo-3,4-dihydroquinazolin-2-ylthio }
hexanoyl)piperidine-4-
carboxamide (2-5);
1-(2- { [ 3-(4'-chlorobiphenyl-3-yl)-4-oxo-3,4-dihydroquinazolin-2-ylthio}
hexanoyl)piperidine-4-
carboxamide (2-6);
1-(2- { [3-(2'-aminobiphenyl-3-y1)-4-oxo-3,4-dihydroquinazolin-2-y1]thio }
hexanoyl)piperidine-4-
carboxamide (2-7);
1-(2-f [3-(3'-aminobiphenyl-3-yl)-4-oxo-3,4-dihydroquinazolin-2-
yl]thio}hexanoyl)piperidine-4-
carboxamide (2-8);
1-(2- { [3-(4'-aminobiphenyl-3-yl)-4-oxo-3,4-dihydroquinazolin-2-ylthio }
hexanoyl)piperidine-4-
carboxamide (2-9);
1-(2-{ [4-oxo-3-(3-pyridin-3-ylphenyl)-3,4-dihydroquinazolin-2-y1]thio}
hexanoyl)piperidine-4-
carboxamide (2-10);
1-(2- {[4-oxo-3-(3-pyridin-4-ylphenyl)-3,4-dihydroquinazolin-2-
ylthio}hexanoyl)piperidine-4-
carboxamide (2-11);
3'-[2-[(1- {[4-(aminocarbonyl)piperidin- 1-yl]carbonyl}pentyl)thio]-4-
oxoquinazolin-3(4.I.I)-
yl]biphenyl-3-carboxylic acid (2-12);
1-[2-({3-[3-(3-fiiryl)phenyl]-4-oxo-3,4-dihydroquinazolin-2-yl }
thio)hexanoyl]piperidine-4-
carboxamide (2-13);
1-[2-({3-[3-(1-methyl- lH-pyrazol-4-yl)phenyl]-4-oxo-3,4-dihydroquinazolin-2-
yl) thio)
hexanoyl]piperidine-4-carboxamide (2-14);
1-(2-{ [3-(3-cyclohex-l-en-l-ylphenyl)-4-oxo-3,4-dihydroquinazolin-2-
yl]thio}hexanoyl)
piperidine-4-carboxamide (2-15);
1-[2-({4-oxo-3-[3-(2-thienyl)phenyl]-3,4-dihydroquinazolin-2-yl }
thio)hexanoyl]piperidine-4-
carboxamide (2-16);
-14-
CA 02714087 2010-08-04
WO 2009/102537 PCT/US2009/031633
2-[(1 - { [7-(3-chlorophenyl)-2,7-diazaspiro [4.4]non-2-yl ]carbonyl } -3 -
methylbutyl)thio]-3 -[ 3-(3-
furyl)phenyl]quinazolin-4(3H)-one (2-17);
2-[(1- {[7-(3-chlorophenyl)-2,7-diazaspiro[4.4]non-2-y1]carbonyl }-3-
methylbutyl)thio]-3-(3-
cyclohex- 1-en- l -ylphenyl)quinazolin-4(3H)-one (2-18);
1-(2- { [3-(4'-fluoro-2'-rnethylbiphenyl-3-y1)-4-oxo-3,4-dihydroquinazolin-2-
yl]thio}hexanoyl)
piperidine-4-carboxarnide (2-19);
2-[(1- { [ 5-(4-fluorophenyl)-2, 5-diazabicyclo [2.2.1 ]kept-2-yl] carbonyl }
pentyl)thio] -3-[3 -(3 -
furyl)phenyl]quinazolin-4(3H)-one (2-20);
2-[(1- {[3-(4-fluorobenzyl)-2-oxo-l -oxa-8-azaspiro[4.5]dee-8-
yl]carbonyl}pentyl)thio]-3-[3-(3-
furyl)phenyl]quinazolin-4(3H)-one (2-21);
3-(3-cyclohex.- l -en-l -ylphenyl)-2-[(1- { [5-(4-fluorophenyl)-2,5-
diazabicyclo[2.2.1 ]hept-2-
yl]carbonyl} pentyl)tbio]quinazolin-4(3H)-one (2-22);
3-(3-cyclohex-l -en-l-ylphenyl)-2-[(1- {[3-(4-fluorobenzyl)-2-oxo-l -oxa-8-
azaspiro[4.5] dec-8-
yl]carbonyl} pentyl)thio]quinazolin-4(3H)-one (2-23);
3-(3-cyclohex-1-en-1-ylphenyl)-2-[(1-{[4-(2,4-difluorophenyl)piperazin-l-
yl]carbonyl} pentyl)thio]quinazolin-4(3H)-one (2-24);
2-[(1-{[4-(2,4-difluorophenyl)piperazin-l-yl]carbonyl}pentyl)thio]-3-[3-(3-
furyl) phenyl]
quinazolin-4(3H)-one (2-25);
3-(3-cyclohex-l -en-l -ylphenyl)-2- 1[1-(2,8-diazaspiro[ 5.5]undec-2-
ylcarbonyl)pentyl]
thio}quinazolin-4(3H)-one (2-26);
2- { [3-amino-l -(2,8-diazaspiro[5.5]under-2-ylcarbonyl)propyl]thio}-3-(3-
chlorophenyl)
quinazolin-4(3H)-one (3-7);
2- { [4-amino-l-(2,8-diazaspiro[5.5]under-2-ylcarbonyl)butyl]thio} -3-(3-
chlorophenyl)-8-
methylquinazolin-4(3H)-one (3-8);
2- {[5-amino-l -(2,8-diazaspiro[5.5]under-2-ylcarbonyl)pentyl]thio}-3-(3-
chlorophenyl)
quinazolin-4(3H)-one (3-9);
2-[(4-amino-1- { [7-(3-chlorophenyl)-2,7-diazaspiro[4.4]non-2 -yl] carbonyl
}butyl)thio]-3-(3-
chlorophenyl)quinazolin-4(3H)-one (3-10);
2-(5-amino- 1-(7-(3-chlorophenyl)-2,7-diazaspiro[4.4]nonan-2-yl)-1-oxopentan-2-
ylthio)-3-(3-
chlorophenyl)-8-methylquinazolin-4(3H)-one (3-11);
2-[(5-amino-l- { [7-(3-chlorophenyl)-2,7-diazaspiro[4.4]non-2-
yl]carbonyl}pentyl)thio]-3-(3-
chlorophenyl)quinazolin-4(3H)-one (4-6); and
2- { [5-amino- 1-(2,8-diazaspiro [ 5.5]undee-2-ylcarbonyl)pentyl ]thio } -3-(3
-chlorophenyl)-8-
methylquinazolin-4(3H)-one (4-7);
or a stereoisomer thereof.
The compounds of the present invention may have asymmetric centers, chiral
axes, and chiral planes (as described in: E.L. Eliel and S.H. Wilen,
Stereochemistry of Carbon
Compounds, John Wiley & Sons, New York, 1994, pages 1119-1190), and occur as
racemates,
-15-
CA 02714087 2010-08-04
WO 2009/102537 PCT/US2009/031633
racemic mixtures, and as individual diastereomers, with all possible isomers
and mixtures
thereof, including optical isomers, being included in the present invention.
In addition, the
compounds disclosed herein may exist as tautomers and both tautomeric forms
are intended to be
encompassed by the scope of the invention, even though only one tautomeric
structure is
depicted.
When any variable (e.g. R1, R6, R6a, etc.) occurs more than one time in any
constituent, its definition on each occurrence is independent at every other
occurrence. Also,
combinations of substituents and variables are permissible only if such
combinations result in
stable compounds. Lines drawn into the ring systems from substituents indicate
that the
indicated bond may be attached to any of the substitutable ring atoms. If the
ring system is
bicyclic, it is intended that the bond be attached to any of the suitable
atoms on either ring of the
bicyclic moiety.
It is understood that substituents and substitution patterns on the compounds
of
the instant invention can be selected by one of ordinary skill in the art to
provide compounds that
are chemically stable and that can be readily synthesized by techniques known
in the art, as well
as those methods set forth below, from readily available starting materials.
If a substituent is
itself substituted with more than one group, it is understood that these
multiple groups may be on
the same carbon or on different carbons, so long as a stable structure
results. The phrase
"optionally substituted with one or more substituents" should be taken to be
equivalent to the
phrase "optionally substituted with at least one substituent" and in such
cases the preferred
embodiment will have from zero to three substituents.
It is understood that one or more silicon (Si) atoms can be incorporated into
the
compounds of the instant invention in place of one or more carbon atoms by one
of ordinary skill
in the art to provide compounds that are chemically stable and that can be
readily synthesized by
techniques known in the art from readily available starting materials. Carbon
and silicon differ
in their covalent radius leading to differences in bond distance and the
steric arrangement when
comparing analogous C-element and Si-element bonds. These differences lead to
subtle changes
in the size and shape of silicon-containing compounds when compared to carbon.
One of
ordinary skill in the art would understand that size and shape differences can
lead to subtle or
dramatic changes in potency, solubility, lack of off target activity,
packaging properties, and so
on. (Dials, J. O. et al. Organometallics (2006) 5:1188-1198; Showell, G.A. et
al. Bioorganic &
Medicinal Chemistry Letters (2006) 16:2555-2558).
As used herein, "alkyl" is intended to include both branched and straight-
chain
saturated aliphatic hydrocarbon groups having the specified number of carbon
atoms. For
example, C 1-C 10, as in "C 1-C 10 alkyl" is defined to include groups having
1, 2, 3, d, 5, 6, 7, 8,
9 or 10 carbons in a linear or branched arrangement. For example, "Cl-Cl0
alkyl" specifically
includes methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl, i-butyl, pentyl,
hexyl, heptyl, octyl,
nonyl, decyl, and so on.
-16-
CA 02714087 2010-08-04
WO 2009/102537 PCT/US2009/031633
The term "spirocyclic" means two rings which are conjoined at a single carbon
atom, for example:
XN N
N N N N. z,. N z
N anCFO
N
The term "cycloalkyl" means a monocyclic or bicyclic saturated aliphatic
hydrocarbon group having the specified number of carbon atoms. For example,
"cycloalkyl"
inlcudes cyclopropyl, methyl-cyclopropyl, 2,2-dimethyl-cyclobutyl, 2-ethyl-
cyclopentyl,
cyclohexyl, and so on.
The term "cycloalkenyl" means a monocyclic or bicyclic partially unsaturated
aliphatic hydrocarbon group having the specified number of carbon atoms. For
example,
"cycloalkenyl" inlcudes cyclopropenyl, methyl-cyclopropenyl, 2,2-dimethyl-
cyclobutenyl, 2-
ethyl-cyclopentenyl, cyclohexenyl, and so on.
"Alkoxy" represents either a cyclic or non-cyclic alkyl group of indicated
number
of carbon atoms attached through an oxygen bridge. "Alkoxy" therefore
encompasses the
definitions of alkyl and cycloalkyl above.
If no number of carbon atoms is specified, the term "alkenyl" refers to a non-
aromatic hydrocarbon radical, straight, branched or cyclic, containing from 2
to 10 carbon atoms
and at least one carbon to carbon double bond. Preferably one carbon to carbon
double bond is
present, and up to four non-aromatic carbon-carbon double bonds maybe present.
Thus, "C2-C6
alkenyl" means an alkenyl radical having from 2 to 6 carbon atoms. Alkenyl
groups include
ethenyl, propenyl, butenyl, 2-methylbutenyl and cyclohexenyl. The straight,
branched or cyclic
portion of the alkenyl group may contain double bonds and may be substituted
if a substituted
alkenyl group is indicated.
The term "alkynyl" refers to a hydrocarbon radical straight, branched or
cyclic,
containing from 2 to 10 carbon atoms and at least one carbon to carbon triple
bond. Up to three
carbon-carbon triple bonds may be present. Thus, "C2-C6 alkynyl" means an
alkynyl radical
having from 2 to 6 carbon atoms. Alkynyl groups include ethynyl, propynyl,
butynyl, 3-
methylbutynyl and so on. The straight, branched or cyclic portion of the
alkynyl group may
contain triple bonds and may be substituted if a substituted alkynyl group is
indicated.
In certain instances, substituents may be defined with a range of carbons that
includes zero, such as (CO-C6)alkylene-aryl. If aryl is taken to be phenyl,
this definition would
include phenyl itself as well as -CH2Ph, -CH2CH2Ph, CH(CH3)CH2CH(CH3)Ph, and
so on.
As used herein, "aryl" is intended to mean any stable monocyclic or bicyclic
carbon ring of up to 7 atoms in each ring, wherein at least one ring is
aromatic. Examples of
such aryl elements include phenyl, naphthyl, tetrahydronaphthyl, indanyl,
biphenyl, phenanthryl,
anthryl or acenaphthyl.
-17-
CA 02714087 2010-08-04
WO 2009/102537 PCT/US2009/031633
The term heteroaryl, as used herein, represents a stable monocyclic or
bicyclic
ring of up to 7 atoms in each ring, wherein at least one ring is aromatic and
contains from I to 4
heteroatoms selected from the group consisting of 0, N and S. Heteroaryl
groups within the
scope of this definition include: acridinyl, carbazolyl, cinnolinyl,
quinoxalinyl, pyrrazolyl,
indolyl, benzotriazolyl, furanyl, thienyl, benzothienyl, benzofuranyl,
quinolinyl, isoquinolinyl,
oxazolyl, isoxazolyl, indolyl, pyrazinyl, pyridazinyl, pyridinyl, pyrimidinyl,
pyrrolyl,
tetrahydroquinoline. As with the definition of heterocycle below, "heteroaryl"
is also understood
to include the N-oxide derivative of any nitrogen-containing heteroaryl.
Attachment of a
heteroaryl substituent can occur via a carbon atom or via a heteroatom.
As appreciated by those of skill in the art, "halo" or "halogen" as used
herein is
intended to include chloro (Cl), fluoro (F), bromo (Br) and iodo (I).
The term "heterocycle" or "heterocyclyl" as used herein is intended to mean a
3-
to 10-membered aromatic or nonaromatic heterocycle containing from 1 to 4
heteroatoms
selected from the group consisting of 0, N and S, and includes bicyclic
groups. "Heterocyclyl"
therefore includes the above mentioned heteroaryls, as well as dihydro and
tetrathydro analogs
thereof. Further examples of "heterocyclyl" include: benzoimidazolyl,
benzofuranyl,
benzofurazanyl, benzopyrazolyl, benzotriazolyl, benzothiophenyl, benzoxazolyl,
carbazolyl,
carbolinyl, chromanyl, cinnolinyl, furanyl, imidazolyl, indolinyl, indolyl,
indolazinyl, indazolyl,
isobenzofuranyl, isoindolyl, isoquinolyl, isothiazolyl, isoxazolyl,
naphthpyridinyl, oxadiazolyl,
oxazolyl, oxazoline, isoxazoline, oxetanyl, pyranyl, pyrazinyl, pyrazolyl,
pyridazinyl,
pyridopyridinyl, pyridazinyl, pyridyl, pyrimidyl, pyrrolyl, quinazolinyl,
quinolyl, quinoxalinyl,
tetrahydropyranyl, tetrazolyl, tetrazolopyridyl, thiadiazolyl, thiazolyl,
thienyl, triazolyl,
azetidinyl, 1,4-dioxanyl, hexahydroazepinyl, piperazinyl, piperidinyl,
pyrrolidinyl, morpholinyl,
thiomorpholinyl, dihydrobenzoimidazolyl, dihydrobenzofuranyl,
dihydrobenzothiophenyl,
dihydrobenzoxazolyl, dihydrofuranyl, dihydroimidazolyl, dihydroindolyl,
dihydroisochromenyl,
dihydroisooxazolyl, dihydroisothiazolyl, dihydrooxadiazolyl, dihydrooxazolyl,
dihydropyrazinyl,
dihydropyrazolyl, dihydropyridinyl, dihydropyrimidinyl, dihydropyrrolyl,
dihydroquinolinyl,
dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl, dihydrothienyl,
dihydrotriazolyl,
dihydroazetidinyl, methylenedioxybenzoyl, tetrahydrofuranyl,
tetrahydroisoquinolinyl,
tetrahydroquinolinyl and tetrahydrothienyl, and N-oxides thereof Attachment of
a heterocyclyl
substituent can occur via a carbon atom or via a heteroatom.
The alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclyl
substituents may be unsubstituted or unsubstituted, unless specifically
defined otherwise. For
example, a (C1-C6)alkyl may be substituted with one, two or three substituents
selected from
OH, oxo, halogen, alkoxy, dialkylamino, or heterocyclyl, such as morpholinyl,
piperidinyl, and
so on. In this case, if one substituent is oxo and the other is OH, the
following are included in
the definition: -(C=O)CH2CH(OH)CH3, -(C=O)OH, -CH2(OH)CH2CH(O), and so on.
-18-
CA 02714087 2010-08-04
WO 2009/102537 PCT/US2009/031633
In certain instances, R7 and R8 are defined such that they can be taken
together
with the nitrogen to which they are attached to form a monocyclic or bicyclic
heterocycle with 3-
7 members in each ring and optionally containing, in addition to the nitrogen,
one or two
additional heteroatozns selected from N, 0 and S, said heterocycle optionally
substituted with one
or more substituents selected from R6a. Examples of the heterocycles that can
thus be formed
include the following, keeping in mind that the heterocycle is optionally
substituted with one or
more substituents chosen from R6a:
R6a R6a R6a R6a R6a Rba N R6a Rba
N-' N=~ N=I N\ N,N N\ \ NI \N -N (~
i <N'N <N ~N~ ,N_N ,N~ \-N
R6a R6a R6a R6a
N~~j
N`) -N\ ~ _ N R6a -N~\~ - N
J,
R6a R6a
R6a N
~-N
R6a ~~ R6a R6a R6a R6a
R6a R6a /0
/+ is I /S
N R6a J-N ~S ~-N Sot -Nom) -N~
R6a R6a
R6a N R6a
N~
~ N-N
\ and R6a
R6a R6a
In an embodiment of Formula A, Ring Z is selected from:
a 'cC ,c
O
N
0
and
o N
In an embodiment of Formula A or B, is selected from:
-19-
CA 02714087 2010-08-04
WO 2009/102537 PCT/US2009/031633
D GIN N
N
nN
~N3" N r S ~N~, NJ, N~, Nand
in an embodiment of Formula B:
N
is selected from:
~IN N
.N~-/ ~, N N N N N O "~ n 7 ,~t \ 2
.~N N
N
~ N r--~S
, N
N J J NF Dand N Z.
N N
ais 0or 1;bis 0or l;in is 0,1,or 2;nis 0,1,2,3,4or 5; pis0,1,2,3,4,5or
6; and q is 0, 1, 2,3,4 or 5;
R1 is independently selected from. CF3, oxo, (C=O) aObC 1-C 10 alkyl, (C=O)aOb
aryl, (C=O)aObC2-C10 alkenyl, (C=O)aObC2-C10 alkynyl, CO2H, halo, OH, ObC1-C6
perfluoroalkyl, (C=O)aNR7R8, CN, (C=O)aObC3-C8 cycloalkyl, S(O)mNR7R8, S(O)m-
(Cl-
C i o)alkyl, and (C=O)aObheterocyclyl, said alkyl, aryl, alkenyl, alkynyl,
cycloalkyl, and
heterocyclyl is optionally substituted with one or more substituents selected
from R6;
R5 is independently selected from: CF3, oxo, (C=O)aObC 1 -C 10 alkyl,
(C=O)aObaryl, C2-C10 alkenyl, C2-C10 alkynyl, (C=O)aOb heterocyclyl, CO2H,
halo, CN, OH,
ObCl-C6 perfluoroalkyl, Oa(C=O)bNR7R8, oxo, CHO, (N=O)R7R8, S(O)mNR7R8, S(O)m-
(C1-CI )alkyl, SH and (C=O)aObC3-C8 cycloalkyl, said alkyl, aryl, alkenyl,
alkynyl,
heterocyclyl, and cycloalkyl optionally substituted with one or more
substituents selected from
R6;
R3 is selected from: H, CF3, oxo, (C=O)aObC1-CIO alkyl, (C=O)aOb aryl,
(C=O)aObC2-C 10 alkenyl, (C=O)aObC2-C 10 alkynyl, CO2H, halo, OH, ObC 1-C6
perfluoroalkyl, (C=O)aNR7R8, CN, (C=O)aObC3-C8 cycloalkyl, S(O)mNR7R8, S(O)m-
(Cl-
C i O)alkyl, and (C=O)aObheterocyclyl, said alkyl, aryl, alkenyl, alkynyl,
cycloalkyl, and
heterocyclyl is optionally substituted with one or more substituents selected
from R6;
R4 is independently selected from: H, CF3, oxo, (C=O)aObC I -C 10 alkyl,
(C=O)aOb aryl, (C' O)aObC2-C10 alkenyl, (C=O)aObC2-C10 alkynyl, CO2H, halo,
OH, ObC1-
C6 perfluoroalkyl, (C=O)aNR7R8, CN, (C=O)aObC3-C8 cycloalkyl, S(O)mNR7R8,
S(O)1T1-
-20-
CA 02714087 2010-08-04
WO 2009/102537 PCT/US2009/031633
(C 1-C I O)alkyl, and (C=O)aObheterocyclyl, said alkyl, aryl, alkenyl,
alkynyl, cycloalkyl, and
heterocyclyl is optionally substituted with one or more substituents selected
from R6;
R6 is: CF3, oxo, (C=O)aObC1-CIO alkyl, (C=O)aObaryl, C2-C10 alkenyl, C2-
C10 alkynyl, (C=O)aOb heterocyclyl, CO2H, halo, CN, OH, ObCI-C6
perfluoroalkyl,
Oa(C=O)bNR7R8, oxo, CHO, (N=O)R7R8, S(O)mNR7R$, S(O)m-(C1-CO)alkyl, SH or
(C=O)aObC3-C8 cycloalkyl;
R7 and R8 are independently selected from: H, (C=0)ObC I -C 10 alkyl,
(C=O)ObC3-C8 cycloalkyl, (C=O)Obaryl, (C=O)Obheterocyclyl, C I -C l 0 alkyl,
aryl, C2-CIO
alkenyl, C2-C10 alkynyl, heterocyclyl, C3-C8 cycloalkyl, S(O)mRa, and
(C=O)NRb2, or R7 and
R8 can be taken together with the nitrogen to which they are attached to form
a monocyclic or
bicyclic heterocycle with 3-7 members in each ring and optionally containing,
in addition to the
nitrogen, one or two additional heteroatoms selected from N, 0 and S;
Ra is H or (Cl-C6)alkyl; and
Rb is independently H or (C 1-C6)alkyl;
or a pharmaceutically acceptable salt or a stereoisomer thereof.
In another embodiment of Formula 13-
N
is selected from:
N'
N N N ~. NI N N o
' ?~ =z NN
N
N s
N N~ NJ, NF Nand N
aisOorl;bis0orl;mis0,l,or2;nislor2; pis0,1,2,3,4,5or6;andgis
1 or 2;
RI is independently selected from: H, S(0)2-(Cl-C6)alkyl, (C1-C6)alkyl,
(C=O)O(C 1-C6)alkyl;
R5 is independently selected from: H, halo, C3-C8 cycloalkyl, C3-C8
cycloalkenyl, aryl and heterocyclyl, said aryl, cycloalkyl, and heterocyclyl
are optionally
substituted with one or more substituents selected from R6;
R3 is. CI -C6 alkyl, said alkyl is optionally substituted with one or more
substituents selected from R6;
R4 is independently selected from: H, CF3, oxo, (C=O)aObC 1-C 10 alkyl,
(C=O)aOb aryl, (C=O)aObC2-ClO alkenyl, (C=O)aObC2-ClO alkynyl, C02H, halo, OH,
ObCl-
C6 perfluoroalkyl, (C=O)aNR7R8, CN, (C=O)aObC3-C8 cycloalkyl, S(O)mNR7R8,
S(O)m-
-21-
CA 02714087 2010-08-04
WO 2009/102537 PCT/US2009/031633
(C1-CIO)alkyl, and (C=O)aObheterocyclyl, said alkyl, aryl, alkenyl, alkynyl,
cycloalkyl, and
heterocyclyl is optionally substituted with one or more substituents selected
from R6;
R6 is: CF3, oxo, (C=O)aObC I -C 10 alkyl, (C=O)OObaryl, C2-C10 alkenyl, C2-
C 10 alkynyl, (C=O)aOb heterocyclyl, CO2H, halo, CN, OH, ObC 1-C6
perfluoroalkyl,
Oa(C=O)bNR7R8, oxo, CHO, (N=O)R7R&, S(O)mNR7R8, S(O)m-(C1-C10)alkyl, SH or
(C=O)aObC3-C8 cycloalkyl;
R7 and R8 are independently selected from: H, (C=O)ObC I -C 10 alkyl,
(C=O)ObC3-C8 cycloalkyl, (C=O)Obaryl, (C=O)Obheterocyclyl, C1-C10 alkyl, aryl,
C2-CIO
alkenyl, C2-C10 alkynyl, heterocyclyl, C3-C8 cycloalkyl, S(O)mRa, and
(C=O)NRb2, or R7 and
R8 can be taken together with the nitrogen to which they are attached to form
a monocyclic or
bicyclic heterocycle with 3-7 members in each ring and optionally containing,
in addition to the
nitrogen, one or two additional heteroatoms selected from N, 0 and S;
Ra is H or (C1-C6)alkyl; and
Rb is independently H or (C1-C6)alkyl;
or a pharmaceutically acceptable salt or a stereoisomer thereof.
In another embodiment of Formula B:
N
is selected from:
r N
NI N N f .N N N L)
\ z~ \ . N \N
,
N N
N NF NO N
and
ais0orl;bisOorl;mis0, l,or2;nis 1 or2; pis0, 1,2,3,4,5or6;andgis
1 or 2;
R1 is independently selected from: H, S(0)2-(C1-C6)alkyl, (CI-C6)alkyl,
(C=O)O(C1-C6)alkyl;
R5 is independently selected from. H, halo, C3-C8 cycloalkyl, C3-C8
cycloalkenyl, aryl and heterocyclyl, said aryl, cycloalkyl, and heterocyclyl
are optionally
substituted with one or more substituents selected from R6;
R3 is: C1-C6 alkyl, said alkyl is optionally substituted with NH2;
R4 is independently selected from: H, CF3, oxo, (C=O)aObCl-Cl 0 alkyl,
(C=O)aOb aryl, (C=O)aObC2-C10 alkenyl, (C=O)aObC2-C10 alkynyl, CO2H, halo, OH,
ObCl-
C6 perfluoroalkyl, (C=O)aNR7R8, CN, (C=O)aObC3-C8 cycloalkyl, S(O)mNR7R8,
S(O)m-
(C1-CIO)alkyl, and (C=O)aObheterocyclyl, said alkyl, aryl, alkenyl, alkynyl,
cycloalkyl, and
heterocyclyl is optionally substituted with one or more substituents selected
from R6;
-22-
CA 02714087 2010-08-04
WO 2009/102537 PCT/US2009/031633
R6 is.. CF3, oxo, (C=O)aObC1-C6 alkyl, (C=O)aObaryl, C2-C6 alkenyl, C2-C6
alkynyl, (C=O)aOb heterocyclyl, CO2H, halo, CN, OH, ObCI-C6 perfluoroalkyl,
Oa(C=O)bNR7Rg, oxo, CHO, (N=O)R7R8, S(O)mNR7R8, S(O)m-(C1-C6)alkyl, SH or
(C=O)aObC3-Cg cycloalkyl;
R7 and R8 are independently selected from: H, (C=O)ObC1-C6 alkyl,
(C=O)ObC3-Cg cycloalkyl, (C=O)Obaryl, (C=O)Obheterocyclyl, C1-C6 alkyl, aryl,
C2-C6
alkenyl, C2-C6 alkynyl, heterocyclyl, C3-C8 cycloalkyl, S(O)mRa, and
(C=O)NRb2, or R7 and
R8 can be taken together with the nitrogen to which they are attached to form
a monocyclic or
bicyclic heterocycle with 3-7 members in each ring and optionally containing,
in addition to the
nitrogen, one or two additional heteroatoms selected from N, 0 and S;
Ra is H or (C 1 -C6)alkyl; and
Rb is independently H or (C 1-C6)alkyl;
or a pharmaceutically acceptable salt or a stereoisomer thereof
Included in the instant invention is the free form of compounds of Formula A,
as
well as the pharmaceutically acceptable salts and stereoisomers thereof Some
of the isolated
specific compounds exemplified herein are the protonated salts of amine
compounds. The term
"free form" refers to the amine compounds in non-salt form. The encompassed
pharmaceutically
acceptable salts not only include the isolated salts exemplified for the
specific compounds
described herein, but also all the typical pharmaceutically acceptable salts
of the free form of
compounds of Formula A. The free form of the specific salt compounds described
may be
isolated using techniques known in the art. For example, the free form may be
regenerated by
treating the salt with a suitable dilute aqueous base solution such as dilute
aqueous NaOH,
potassium carbonate, ammonia and sodium bicarbonate. The free forms may differ
from their
respective salt forms somewhat in certain physical properties, such as
solubility in polar solvents,
but the acid and base salts are otherwise pharmaceutically equivalent to their
respective free
forms for purposes of the invention.
The pharmaceutically acceptable salts of the instant compounds can be
synthesized from the compounds of this invention which contain a basic or
acidic moiety by
conventional chemical methods. Generally, the salts of the basic compounds are
prepared either
by ion exchange chromatography or by reacting the free base with
stoichiometric amounts or
with an excess of the desired salt-forming inorganic or organic acid in a
suitable solvent or
various combinations of solvents. Similarly, the salts of the acidic compounds
are formed by
reactions with the appropriate inorganic or organic base.
Thus, pharmaceutically acceptable salts of the compounds of this invention
include the conventional non-toxic salts of the compounds of this invention as
formed by
reacting a basic instant compound with an inorganic or organic acid. For
example, conventional
non-toxic salts include those derived from inorganic acids such as
hydrochloric, hydrobromic,
sulfuric, sulfamic, phosphoric, nitric and the like, as well as salts prepared
from organic acids
-23-
CA 02714087 2010-08-04
WO 2009/102537 PCT/US2009/031633
such as acetic, propionic, succinic, glycolic, stearic, lactic, malic,
tartaric, citric, ascorbic,
pamoic, maleic, hydroxymaleic, phenylacetic, glutarnic, benzoic, salicylic,
sulfanilic, 2-acetoxy-
benzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic,
isethionic,
trifluoroacetic (TFA) and the like.
When the compound of the present invention is acidic, suitable
"pharmaceutically
acceptable salts" refers to salts prepared form pharmaceutically acceptable
non-toxic bases
including inorganic bases and organic bases. Salts derived from inorganic
bases include
aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium,
manganic salts,
manganous, potassium, sodium, zinc and the like. Particularly preferred are
the ammonium,
calcium, magnesium, potassium and sodium salts. Salts derived from
pharmaceutically
acceptable organic non-toxic bases include salts of primary, secondary and
tertiary amines,
substituted amines including naturally occurring substituted amines, cyclic
amines and basic ion
exchange resins, such as arginine, betaine caffeine, choline, N,NI-
dibenzylethylenediamine,
diethylamin, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine,
ethylenediamine,
N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine,
hydrabamine,
isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine,
polyamine resins,
procaine, purines, theobromine, triethylamine, trimethylamine tripropylamine,
tromethamine and
the like.
The preparation of the pharmaceutically acceptable salts described above and
other typical pharmaceutically acceptable salts is more fully described by
Berg et al.,
"Pharmaceutical Salts," J. Pharm. Sci., 1977:66:1-19.
It will also be noted that the compounds of the present invention are
potentially
internal salts or zwitterions, since under physiological conditions a
deprotonated acidic moiety in
the compound, such as a carboxyl group, may be anionic, and this electronic
charge might then
be balanced off internally against the cationic charge of a protonated or
alkylated basic moiety,
such as a quaternary nitrogen atom.
UTILITY
The compounds, compositions and methods provided herein are particularly
deemed useful for the treatment of cancer. Cancers that may be treated by the
compounds,
compositions and methods of the invention include, but are not limited to:
Cardiac: sarcoma
(angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma), myxoma,
rhabdomyoma,
fibroma, lipoma and teratoma; Lung: bronchogenic carcinoma (squamous cell,
undifferentiated
small cell, undifferentiated large cell, adenocarcinoma), alveolar
(bronchiolar) carcinoma,
bronchial adenoma, sarcoma, lymphoma, chondromatous hamartoma, mesothelioma,
non-small
cell; Gastrointestinal: esophagus (squamous cell carcinoma, adenocarcinoma,
Iciomyosarcoma,
lymphoma), stomach (carcinoma, lymphoma, leiomyosarcoma), pancreas (ductal
adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumors,
vipoma), small bowel
(adenocarcinoma, lymphoma, carcinoid tumors, Karposi's sarcoma, leiomyoma,
hemangioma,
-24-
CA 02714087 2010-08-04
WO 2009/102537 PCT/US2009/031633
liporna, neurofibroma, fibroma), large bowel (adenocarcinoma, tubular adenoma,
villous
adenoma, hamartoma, leiomyoma), colon, colorectal, rectal ; Genitourinary
tract: kidney
(adenocarcinoma, Wilm's tumor [nephroblastoma], lymphoma, leukemia), bladder
and urethra
(squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma),
prostate
(adenocarcinoma, sarcoma), testis (seminoma, teratoma, embryonal carcinoma,
teratocarcinoma,
choriocarcinoma, sarcoma, interstitial cell carcinoma, fibroma, fibroadenoma,
adenomatoid
tumors, lipoma); Liver: hepatoma (hepatocellular carcinoma),
cholangiocarcinoma,
hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma; Bone:
osteogenic
sarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma,
chondrosarcoma,
Ewing's sarcoma, malignant lymphoma (reticulum cell sarcoma), multiple
myeloma, malignant
giant cell tumor chordoma, osteochronfroma (osteocartilaginous exostoses),
benign chondroma,
chondroblastoma, chondromyxofibroma, osteoid osteoma and giant cell tumors;
Nervous system:
skull (osteoma, hemangioma, granuloma, xanthoma, osteitis deformans), meninges
(meningioma, meningiosarcoma, gliomatosis), brain (astrocytoma,
medulloblastoma, glioma,
ependymoma, germinoma [pinealoma], glioblastoma multiform, oligodendroglioma,
schwannoma, retinoblastoma, congenital tumors), spinal cord neurofibroma,
meningioma,
glioma, sarcoma); Gynecological. uterus (endometrial carcinoma), cervix
(cervical carcinoma,
pre-tumor cervical dysplasia), ovaries (ovarian carcinoma [serous
cystadenocarcinoma, mutinous
cystadenocarcinoma, unclassified carcinoma], granuoma-thecal cell tumors,
Sertoli-Leydig cell
tumors, dysgerminoma, malignant teratoma), vulva (squamous cell carcinoma,
intraepithelial
carcinoma, adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell
carcinoma, squamous
cell carcinoma, botryoid sarcoma (embryonal rhabdomyosarcoma), fallopian tubes
(carcinoma),
breast; Hematologic: blood (myeloid leukemia [acute and chronic], acute
lymphoblastic
leukemia, chronic lymphocytic leukemia, myeloproliferative diseases, multiple
myeloma,
myelodysplastic syndrome), Hodgkin's disease, non-Hodgkin's lymphoma
[malignant
lymphoma]; Skin: malignant melanoma, basal cell carcinoma, squamous cell
carcinoma,
Karposi's sarcoma, moles dysplastic nevi, lipoma, angioma, dermatofibroma,
keloids, psoriasis;
and Adrenal glands: neuroblastoma. Thus, the term "cancerous cell" as provided
herein, includes
a cell afflicted by any one of the above-identified conditions.
Cancers that may be treated by the compounds, compositions and methods of the
invention include, but are not limited to. breast, prostate, colon,
colorectal, lung, non-small cell
lung, brain, testicular, stomach, ovarian, pancrease, skin, small intestine,
large intestine, throat,
head and neck, oral, bone, liver, bladder, kidney, thyroid and blood.
Cancers that may be treated by the compounds, compositions and methods of the
invention include: breast, prostate, colon, ovarian, colorectal, lung and non-
small cell lung.
Cancers that may be treated by the compounds, compositions and methods of the
invention include: breast, colon, (colorectal) and lung (non-small cell lung).
-25-
CA 02714087 2010-08-04
WO 2009/102537 PCT/US2009/031633
Cancers that may be treated by the compounds, compositions and methods of the
invention include: lymphoma and leukemia.
The compounds of the invention are also useful in preparing a medicament that
is
useful in treating cancer.
The compounds of this invention may be administered to mammals, including
humans, either alone or, in combination with pharmaceutically acceptable
carriers, excipients or
diluents, in a pharmaceutical composition, according to standard
pharmaceutical practice. The
compounds can be administered orally or parenterally, including the
intravenous, intramuscular,
intraperitoneal, subcutaneous, rectal and topical routes of administration.
The pharmaceutical compositions containing the active ingredient may be in a
form suitable for oral use, for example, as tablets, troches, lozenges,
aqueous or oily suspensions,
dispersible powders or granules, emulsions, hard or soft capsules, or syrups
or elixirs.
Compositions intended for oral use may be prepared according to any method
known to the art
for the manufacture of pharmaceutical compositions and such compositions may
contain one or
more agents selected from the group consisting of sweetening agents, flavoring
agents, coloring
agents and preserving agents in order to provide pharmaceutically elegant and
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,
microcrystalline cellulose, sodium crosscarmellose, corn starch, or alginic
acid; binding agents,
for example starch, gelatin, polyvinyl-pyrrolidone or acacia, and lubricating
agents, for example,
magnesium stearate, stearic acid or talc. The tablets may be uncoated or they
may be coated by
known techniques to mask the unpleasant taste of the drug or delay
disintegration and absorption
in the gastrointestinal tract and thereby provide a sustained action over a
longer period. For
example, a water soluble taste masking material such as hydroxypropylmethyl-
cellulose or
hydroxypropylcellulose, or a time delay material such as ethyl cellulose,
cellulose acetate
buryrate may be employed.
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 soluble carrier such as polyethyleneglycol or an oil medium, for
example peanut oil,
liquid paraffin, or olive oil.
Aqueous suspensions contain the active material in admixture with excipients
suitable for the manufacture of aqueous suspensions. Such excipients are
suspending agents, for
example sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethyl-
cellulose,
sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia;
dispersing or wetting
agents may be a naturally-occurring phosphatide, for example lecithin, or
condensation products
-26-
CA 02714087 2010-08-04
WO 2009/102537 PCT/US2009/031633
of 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 a 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, saccharin or
aspartame.
Oily suspensions may be formulated by suspending the active ingredient in a
vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil,
or in 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 a palatable oral preparation. These
compositions may be
preserved by the addition of an anti-oxidant such as butylated hydroxyanisol
or alpha-tocopherol.
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. These
compositions may be preserved by the addition of an anti-oxidant such as
ascorbic acid.
The pharmaceutical compositions of the invention may also be in the form of an
oil-in-water emulsion. 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 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, flavouring
agents, preservatives and antioxidants.
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, flavoring and coloring agents and antioxidant.
The pharmaceutical compositions may be in the fonn of sterile injectable
aqueous
solutions. Among the acceptable vehicles and solvents that may be employed are
water, Ringer's
solution and isotonic sodium chloride solution.
The sterile injectable preparation may also be a sterile injectable oil-in-
water
microemulsion where the active ingredient is dissolved in the oily phase. For
example, the
-27-
CA 02714087 2010-08-04
WO 2009/102537 PCT/US2009/031633
active ingredient may be first dissolved in a mixture of soybean oil and
lecithin. The oil solution
then introduced into a water and glycerol mixture and processed to form a
microemulsion.
The injectable solutions or microemulsions may be introduced into a patient's
blood-stream by local bolus injection. Alternatively, it may be advantageous
to administer the
solution or microemulsion in such a way as to maintain a constant circulating
concentration of
the instant compound. In order to maintain such a constant concentration, a
continuous
intravenous delivery device may be utilized. An example of such a device is
the Deltec CADD-
PLUSTM model 5400 intravenous pump.
The pharmaceutical compositions maybe in the form of a sterile injectable
aqueous or oleagenous suspension for intramuscular and subcutaneous
administration. This
suspension maybe formulated according to the known art using those suitable
dispersing or
wetting agents and suspending agents which have been mentioned above. The
sterile injectable
preparation may also be a sterile injectable solution or suspension in a non-
toxic parenterally-
acceptable diluent or solvent, for example as a solution in 1,3-butane diol.
In addition, sterile,
fixed oils are conventionally employed as a solvent or suspending medium. For
this purpose any
bland fixed oil may be employed including synthetic mono- or diglycerides. In
addition, fatty
acids such as oleic acid find use in the preparation of injectables.
Compounds of the instant invention 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, glycerinated gelatin, hydrogenated
vegetable oils, mixtures
of polyethylene glycols of various molecular weights and fatty acid esters of
polyethylene glycol.
For topical use, creams, ointments, jellies, solutions or suspensions, etc.,
containing the compound of the instant invention are employed. (For purposes
of this
application, topical application shall include mouth washes and gargles.)
The compounds for the present invention can be administered in intranasal form
via topical use of suitable intranasal vehicles and delivery devices, or via
transdermal routes,
using those forms of transdermal skin patches well known to those of ordinary
skill in the art.
To be administered in the form of a transdermal delivery system, the dosage
administration will,
of course, be continuous rather than intermittent throughout the dosage
regimen. Compounds of
the present invention may also be delivered as a suppository employing bases
such as cocoa
butter, glycerinated gelatin, hydrogenated vegetable oils, mixtures of
polyethylene glycols of
various molecular weights and fatty acid esters of polyethylene glycol.
When a composition according to this invention is administered into a human
subject, the daily dosage will normally be determined by the prescribing
physician with the
dosage generally varying according to the age, weight, and response of the
individual patient, as
well as the severity of the patient's symptoms.
-28-
CA 02714087 2010-08-04
WO 2009/102537 PCT/US2009/031633
The dosage regimen utilizing the compounds of the instant invention can be
selected in accordance with a variety of factors including type, species, age,
weight, sex and the
type of cancer being treated; the severity (i.e., stage) of the cancer to be
treated; the route of
administration; the renal and hepatic function of the patient; and the
particular compound or salt
thereof employed. An ordinarily skilled physician or veterinarian can readily
determine and
prescribe the effective amount of the drug required to treat, for example, to
prevent, inhibit (fully
or partially) or arrest the progress of the disease. For example, compounds of
the instant
invention can be administered in a total daily dose of up to 1000 mg.
Compounds of the instant
invention can be administered once daily (QD), or divided into multiple daily
doses such as twice
daily (BID), and three times daily (TID). Compounds of the instant invention
can be
administered at a total daily dosage of up to 1000 mg, e.g., 200 mg, 300 mg,
400 mg, 600 mg,
800 mg or 1000 mg, which can be administered in one daily dose or can be
divided into multiple
daily doses as described above.
In addition, the administration can be continuous, i.e., every day, or
intermittently.
The terms "intermittent" or "intermittently" as used herein means stopping and
starting at either
regular or irregular intervals. For example, intermittent administration of a
compound of the
instant invention may be administration one to six days per week or it may
mean administration
in cycles (e,g. daily administration for two to eight consecutive weeks, then
a rest period with no
administration for up to one week) or it may mean administration on alternate
days.
In addition, the compounds of the instant invention may be administered
according to any of the schedules described above, consecutively for a few
weeks, followed by a
rest period. For example, the compounds of the instant invention may be
administered according
to any one of the schedules described above from two to eight weeks, followed
by a rest period
of one week, or twice daily at a dose of 100 - 500 mg for three to five days a
week. In another
particular embodiment, the compounds of the instant invention may be
administered three times
daily for two consecutive weeks, followed by one week of rest.
Any one or more of the specific dosages and dosage schedules of the compounds
of the instant invention, may also be applicable to any one or more of the
therapeutic agents to be
used in the combination treatment (hereinafter refered to as the "second
therapeutic agent").
Moreover, the specific dosage and dosage schedule of this second therapeutic
agent can further vary, and the optimal dose, dosing schedule and route of
administration will be
determined based upon the specific second therapeutic agent that is being
used.
Of course, the route of administration of the compounds of the instant
invention is
independent of the route of administration of the second therapeutic agent. In
an embodiment,
the administration for a compound of the instant invention is oral
administration. In another
embodiment, the administration for a compound of the instant invention is
intravenous
administration. Thus, in accordance with these embodiments, a compound of the
instant
invention is administered orally or intravenously, and the second therapeutic
agent can be
-29-
CA 02714087 2010-08-04
WO 2009/102537 PCT/US2009/031633
administered orally, parenterally, intraperitoneally, intravenously,
intraarterially, transdermally,
sublingually, intramuscularly, rectally, transbuccally, intranasally,
liposomally, via inhalation,
vaginally, intraoccularly, via local delivery by catheter or stent,
subcutaneously, intraadiposally,
intraarticularly, intrathecally, or in a slow release dosage form.
In addition, a compound of the instant invention and second therapeutic agent
may
be administered by the same mode of administration, i.e. both agents
administered e.g. orally, by
IV. However, it is also within the scope of the present invention to
administer a compound of
the instant invention by one mode of administration, e.g. oral, and to
administer the second
therapeutic agent by another mode of administration, e.g. IV or any other ones
of the
administration modes described hereinabove.
The first treatment procedure, administration of a compound of the instant
invention, can take place prior to the second treatment procedure, i.e., the
second therapeutic
agent, after the treatment with the second therapeutic agent, at the same time
as the treatment
with the second therapeutic agent, or a combination thereof. For example, a
total treatment
period can be decided for a compound of the instant invention. The second
therapeutic agent can
be administered prior to onset of treatment with a compound of the instant
invention or following
treatment with a compound of the instant invention. In addition, anti-cancer
treatment can be
administered during the period of administration of a compound of the instant
invention but does
not need to occur over the entire treatment period of a compound of the
instant invention.
The instant compounds are also useful in combination with therapeutic,
chemotherapeutic and anti-cancer agents. Combinations of the presently
disclosed compounds
with therapeutic, chemotherapeutic and anti-cancer agents are within the scope
of the invention.
Examples of such agents can be found in Cancer Principles and Practice of
Oncology by V.T.
Devita and S. Hellman (editors), 6th edition (February 15, 2001), Lippincott
Williams & Wilkins
Publishers. A person of ordinary skill in the art would be able to discern
which combinations of
agents would be useful based on the particular characteristics of the drugs
and the cancer
involved. Such agents include the following: estrogen receptor modulators,
androgen receptor
modulators, retinoid receptor modulators, cytotoxic/cytostatic agents,
antiproliferative agents,
prenyl-protein transferase inhibitors, HMG-CoA reductase inhibitors and other
angiogenesis
inhibitors, HIV protease inhibitors, reverse transcriptase inhibitors,
inhibitors of cell proliferation
and survival signaling, bisphosphonates, aromatase inhibitors, siRNA
therapeutics, y-secretase
inhibitors, agents that interfere with receptor tyrosine kinases (RTKs) and
agents that interfere
with cell cycle checkpoints. The instant compounds are particularly useful
when co-administered
with radiation therapy.
"Estrogen receptor modulators" refers to compounds that interfere with or
inhibit
the binding of estrogen to the receptor, regardless of mechanism. Examples of
estrogen receptor
modulators include, but are not limited to, tamoxifen, raloxifene, idoxifene,
LY353381,
LY117081, toremifene, fulvestrant, 4-[7-(2,2-dimethyl-l-oxopropoxy-4-methyl-2-
[4-[2-(1-
-30-
CA 02714087 2010-08-04
WO 2009/102537 PCT/US2009/031633
piperidinyl)ethoxy]phenyl]-2H-1-benzopyran-3-yl]-phenyl-2,2-
dimethylpropanoate, 4,4'-
dihydroxybenzophenone-2,4-dinitrophenyl-hydrazone, and SH646.
"Androgen receptor modulators" refers to compounds which interfere or inhibit
the binding of androgens to the receptor, regardless of mechanism. Examples of
androgen
receptor modulators include finasteride and other 5a-reductase inhibitors,
nilutamide, flutamide,
bicalutamide, liarozole, and abiraterone acetate.
"Retinoid receptor modulators" refers to compounds which interfere or inhibit
the
binding of retinoids to the receptor, regardless of mechanism. Examples of
such retinoid
receptor modulators include bexarotene, tretinoin, 13-cis-retinoic acid, 9-cis-
retinoic acid, a-
difluoromethylornithine,1LX23-7553, trans-N-(4'-hydroxyphenyl) retinamide, and
N-4-
carboxyphenyl retinamide.
"Cytotoxic/cytostatic agents" refer to compounds which cause cell death or
inhibit
cell proliferation primarily by interfering directly with the cell's
functioning or inhibit or interfere
with cell myosis, including alkylating agents, tumor necrosis factors,
intercalators, hypoxia
activatable compounds, microtubule inhibitors/microtubule-stabilizing agents,
inhibitors of
mitotic kinesins, histone deacetylase inhibitors, inhibitors of kinases
involved in mitotic
progression, inhibitors of kinases involved in growth factor and cytokine
signal transduction
pathways, antimetabolites, biological response modifiers, hormonal/anti-
hormonal therapeutic
agents, haematopoietic growth factors, monoclonal antibody targeted
therapeutic agents,
topoisomerase inhibitors, proteosome inhibitors, ubiquitin ligase inhibitors,
and aurora kinase
inhibitors.
Examples of cytotoxic/cytostatic agents include, but are not limited to,
sertenef,
cachectin, ifosfamide, tasonermin, lonidamine, carboplatin, altretamine,
prednimustine,
dibromodulcitol, ranimustine, fotemustine, nedaplatin, oxaliplatin,
temozolomide, heptaplatin,
estramustine, improsulfan tosilate, trofosfamide, nimustine, dibrospidium
chloride, pumitepa,
lobaplatin, satraplatin, profiromycin, cisplatin, irofulven, dexifosfamide,
cis-aminedichloro(2-
methyl-pyridine)platinum, benzylguanine, glufosfamide, GPXI 00, (trans, trans,
trans)-bis-mu-
(hexane-1,6-diamine)-mu-[diamine-platinum(II)]bis[diamine(chloro)platinum
(11)]tetrachloride,
diarizidinylspermine, arsenic trioxide, 1-(11-dodecylamino-l0-hydroxyundecyl)-
3,7-
dimethylxanthine, zorubicin, idarubicin, daunorubicin, bisantrene,
mitoxantrone, pirarubicin,
pinafide, valrubicin, amrubicin, antineoplaston, 3'-deamino-3'-morpholino-13-
deoxo-10-
hydroxycarminomycin, annamycin, galarubicin, elinafide, MEN10755, 4-demethoxy-
3-deamino-
3-aziridinyl-4-methylsulphonyl-daunorubicin (see WO 00/50032), Raf kinase
inhibitors (such as
Bay43-9006) and mTOR inhibitors (such as Wyeth's CC1-779).
An example of a hypoxia activatable compound is tirapazamine.
Examples of proteosome inhibitors include but are not limited to lactacystin
and
MLN-341 (Velcade).
-31-
CA 02714087 2010-08-04
WO 2009/102537 PCT/US2009/031633
Examples of microtubule inhibitorslmicrotubule-stabilising agents include
paclitaxel, vindesine sulfate, 3',4'-didehydro-4'-deoxy-8'-
norvincaleukoblastine, docetaxol,
rhizoxin, dolastatin, mivobulin isethionate, auristatin, cemadotin, RPRI
09881, BMS 184476,
vinflunine, cryptophycin, 2,3,4,5,6-pentafluoro-N-(3-fluoro-4-methoxyphenyl)
benzene
sulfonamide, anhydrovinblastine, N,N-dimethyl-L-valyl-L-valyl-N-methyl-L-valyl-
L-prolyl-L-
proline-t-butylainide, TDX258, the epothilones (see for example U.S. Pat. Nos.
6,284,781 and
6,288,237) and BMS188797. In an embodiment the epothilones are not included in
the
microtubule inhibitorslmicrotubule-stabilising agents.
Some examples of topoisomerase inhibitors are topotecan, hycaptamine,
irinotecan, rubitecan, 6-ethoxypropionyl-3',4'-O-exo-benzylidene-chartreusin,
9-methoxy-N,N-
dimethyl-5-nitropyrazolo[3,4,5-kl]acridine-2-(6H) propanamine, 1-amino-9-ethyl-
5-fluoro-2,3-
dihydro-9-hydroxy-4-methyl-1 H,12H-benzo[de]pyrano[3',4' :b,7]-indolizino[
1,2b]quinoline-
10,13(9H,15H)dione, lurtotecan, 7-[2-(N-isopropylamino)ethyl]-
(20S)camptothecin, BNP1350,
BNPI1100, BN80915, BN80942, etoposide phosphate, teniposide, sobuzoxane, 2-
dimethylamino-2'-deoxy-etoposide, GL331, N- [2-(dimethylamino)ethyl] -9-
hydroxy-5,6-
dimethyl-6H-pyrido[4,3-b]carbazole-1-carboxamide, asulacrine, (5a, 5aB,
8aa,9b)-9-[2-[N-[2-
(dimethylamino)ethyl]-N-methylamino] ethyl] -5-[4-hydrooxy-3,5-
dimethoxyphenyl]-
5,5a,6,8,8a,9-hexohydrofuro(3',4':6,7)naphtho(2,3-d)-1,3-dioxol-6-one, 2,3-
(methylenedioxy)-5-
methyl-7-hydroxy-8-methoxybenzo[c]-phenanthridinium, 6,9-bis[(2-
aminoethyl)amino]benzo[g]isoguinoline-5,10-dione, 5-(3-aminopropylamino)-7,10-
dihydroxy-2-
(2-hydroxyethylaminomethyl)-6H-pyrazolo[4,5,1-de]acridin-6-one, N-[1-
[2(diethylamino)ethylamino]-7-mnethoxy-9-oxo-9H-thioxanthen-4-
ylmethyl]formamide, N-(2-
(dimethylam.ino)ethyl)acridine-4-carboxamide, 6-[[2-
(dimethylamino)ethyl]amino] -3-hydroxy-
7H-indeno[2,1-c] quinolin-7-one, and dimesna.
Examples of inhibitors of mitotic kinesins, and in particular the human
mitotic
kinesin KSP, are described in Publications W003/039460, W003/050064,
W003/050122,
W003/049527, W003/049679, W003/049678, W004/039774, W003/079973, W003/09921 1,
W003/105855, W003/106417, W004/037171, W004/058148, W004/058700, W004/126699,
W005/018638, W005/019206, W005/019205, W005/018547, W005/017190,
US2005/0176776. In an embodiment inhibitors of mitotic kinesins include, but
are not limited to
inhibitors of KSP, inhibitors of MKLP I, inhibitors of CENP-E, inhibitors of
MCAK and
inhibitors of Rab6-KIFL.
Examples of "histone deacetylase inhibitors" include, but are not limited to,
SAHA, TSA, oxamflatin, PXD101, MG98 and scriptaid. Further reference to other
histone
deacetylase inhibitors may be found in the following manuscript; Miller, T.A.
et al. J. Med.
Chem. 46(24):5097-5116 (2003).
"Inhibitors of kinases involved in mitotic progression" include, but are not
limited
to, inhibitors of aurora kinase, inhibitors of Polo-like kinases (PLK; in
particular inhibitors of
-32-
CA 02714087 2010-08-04
WO 2009/102537 PCT/US2009/031633
PLK-1), inhibitors of bub-1 and inhibitors of bub-Rl. An example of an "aurora
kinase
inhibitor" is VX-680.
"Antiproliferative agents" includes antisense RNA and DNA oligonucleotides
such as G3139, ODN698, RVASKRAS, GEM231, and INX3001, and antimetabolites such
as
enocitabine, carmofur, tegafur, pentostatin, doxifluridine, trimetrexate,
fludarabine, capecitabine,
galocitabine, cytarabine ocfosfate, fosteabine sodium hydrate, raltitrexed,
paltitrexid, emitefur,
tiazofurin, decitabine, nolatrexed, pemetrexed, nelzarabine, 2'-deoxy-2'-
methylidenecytidine, 2'-
fluoromethylene-2'-deoxycytidine, N-[5-(2,3-dihydro-benzofuryl)sulfonyl]-N'-
(3,4-
dichlorophenyl)urea, N6--[4-deoxy-4-[N2-[2(E),4(E)-
tetradecadienoyl]glycylamino]-L-glycero-B-
L-manno-heptopyranosyl] adenine, aplidine, ecteinascidin, troxacitabine, 4-[2-
amino-4-oxo-
4,6,7,8-tetrahydro-3H-pyrimidino[5,4-b] [ 1,4]thiazin-6-yl-(S)-ethyl]-2,5-
thienoyl-L-glutamic
acid, aminopterin, 5-flurouracil, alanosine, 11-acetyl-8-(carbamoyloxymethyl)-
4-formyl-6-
methoxy-14-oxa-1,11 -diazatetracyclo(7.4.1Ø0)-tetradeca-2,4,6-trien-9-yl
acetic acid ester,
swainsonine, lometrexol, dexrazoxane, methioninase, 2'-cyano-2'-deoxy-N4-
palmitoyl-l-B-D-
arabino furanosyl cytosine, 3-aminopyridine-2-carboxaldehyde thiosemicarbazone
and
trastuzumab.
Examples of monoclonal antibody targeted therapeutic agents include those
therapeutic agents which have cytotoxic agents or radioisotopes attached to a
cancer cell specific
or target cell specific monoclonal antibody. Examples include Bexxar.
"HMG-CoA reductase inhibitors" refers to inhibitors of 3-hydroxy-3-
methylglutaryl-CoA reductase. Examples of HMG-CoA reductase inhibitors that
may be used
include but are not limited to lovastatin (MEVACOR ; see U.S. Patent Nos.
4,231,938,
4,294,926 and 4,319,039), simvastatin (ZOCOR ; see U.S. Patent Nos. 4,444,784,
4,820,850
and 4,916,239), pravastatin (PRAVACHOL ; see U.S. Patent Nos. 4,346,227,
4,537,859,
4,410,629, 5,030,447 and 5,180,589), fluvastatin (LESCOL ; see U.S. Patent
Nos. 5,354,772,
4,911,165, 4,929,437, 5,189,164, 5,118,853, 5,290,946 and 5,356,896),
atorvastatin (LIPITOR ;
see U.S. Patent Nos. 5,273,995, 4,681,893, 5,489,691 and 5,342,952) and
cerivastatin (also
known as rivastatin and BAYCHOL ; see US Patent No. 5,177,080). The structural
formulas of
these and additional HMG-CoA reductase inhibitors that may be used in the
instant methods are
described at page 87 of M. Yalpani, "Cholesterol Lowering Drugs", Chemistry &
Industry, pp.
85-89 (5 February 1996) and US Patent Nos. 4,782,084 and 4,885,314. The term
HMG-CoA
reductase inhibitor as used herein includes all pharmaceutically acceptable
lactone and open-acid
forms (i.e., where the lactone ring is opened to form the free acid) as well
as salt and ester forms
of compounds which have HMG-CoA reductase inhibitory activity, and therefor
the use of such
salts, esters, open-acid and lactone forms is included within the scope of
this invention.
"Prenyl-protein transferase inhibitor" refers to a compound which inhibits any
one
or any combination of the prenyl-protein transferase enzymes, including
farnesyl-protein
-33-
CA 02714087 2010-08-04
WO 2009/102537 PCT/US2009/031633
transferase (FPTase), geranylgeranyl-protein transferase type I (GGPTase-1),
and geranylgeranyl-
protein transferase type-IT (GGPTase-II, also called Rab GGPTase).
Examples of prenyl-protein transferase inhibitors can be found in the
following
publications and patents: WO 96/30343, WO 97/18813, WO 97/21701, WO 97/23478,
WO
97/38665, WO 98/28980, WO 98/29119, WO 95/32987, U.S. Patent No. 5,420,245,
U.S. Patent
No. 5,523,430, U.S. Patent No. 5,532,359, U.S. Patent No. 5,510,510, U.S.
Patent No. 5,589,485,
U.S. Patent No. 5,602,098, European Patent Publ. 0 618 221, European Patent
Pub!. 0 675 112,
European Patent Publ. 0 604 181, European Patent Publ. 0 696 593, WO 94/19357,
WO
95/08542, WO 95/11917, WO 95/12612, WO 95/12572, WO 95/10514, U.S. Patent No.
5,661,152, WO 95/10515, WO 95/10516, WO 95/24612, WO 95/34535, WO 95/25086, WO
96/05529, WO 96/06138, WO 96/06193, WO 96/16443, WO 96/21701, WO 96/21456, WO
96/22278, WO 96/24611, WO 96/24612, WO 96/05168, WO 96/05169, WO 96/00736,
U.S.
Patent No. 5,571,792, WO 96/17861, WO 96/33159, WO 96/34850, WO 96/34851, WO
96/30017, WO 96/30018, WO 96/30362, WO 96/30363, WO 96/31111, WO 96/31477,
WO 96/31478, WO 96/31501, WO 97/00252, WO 97/03047, WO 97/03050, WO 97/04785,
WO
97/02920, WO 97/17070, WO 97/23478, WO 97/26246, WO 97/30053, WO 97/44350, WO
98/02436, and U.S. Patent No. 5,532,359. For an example of the role of a
prenyl-protein
transferase inhibitor on angiogenesis see European J. of Cancer, Vol. 35, No.
9, pp.1394-1401
(1999).
"Angiogenesis inhibitors" refers to compounds that inhibit the formation of
new
blood vessels, regardless of mechanism. Examples of angiogenesis inhibitors
include, but are
not limited to, tyrosine kinase inhibitors, such as inhibitors of the tyrosine
kinase receptors Fit-1
(VEGFRI) and Flk-l/KDR (VEGFR2), inhibitors of epidermal-derived, fibroblast-
derived, or
platelet derived growth factors, MMP (matrix metalloprotease) inhibitors,
integrin blockers,
interferon-a, interleukin- 12, pentosan polysulfate, cyclooxygenase
inhibitors, including
nonsteroidal anti-inflammatories (NSAtDs) like aspirin and ibuprofen as well
as selective
cyclooxy-genase-2 inhibitors like celecoxib and rofecoxib (PNAS, Vol. 89, p.
7384 (1992); JNCI,
Vol. 69, p. 475 (1982); Arch. Opthalmol., Vol. 108, p.573 (1990); Anat. Rec.,
Vol. 238, p. 68
(1994); FEBS Letters, Vol. 372, p. 83 (1995); Clin, Orthop. Vol. 313, p. 76
(1995); J. Mol.
Endocrinol., Vol. 16, p.107 (1996); Jpn. J. Pharmacol., Vol. 75, p. 105
(1997); Cancer Res.,
Vol. 57, p. 1625 (1997); Cell, Vol. 93, p. 705 (1998); Intl. J. Mol. Med.,
Vol. 2, p. 715 (1998); J.
Biol. Chem., Vol. 274, p. 9116 (1999)), steroidal anti-inflammatories (such as
corticosteroids,
mineralocorticoids, dexamethasone, prednisone, prednisolone, methylpred,
betamethasone),
carboxyamidotriazole, combretastatin A-4, squalamine, 6-O-chloroacetyl-
carbonyl)-fumagillol,
thalidomide, angiostatin, troponin-1, angiotensin II antagonists (see
Fernandez et al., J. Lab.
Clin. Med. 105:141-145 (1985)), and antibodies to VEGF (see, Nature
Biotechnology, Vol. 17,
pp.963-968 (October 1999); Kim et al., Nature, 362, 841-844 (1993); WO
00/44777; and WO
00/61186).
-34-
CA 02714087 2010-08-04
WO 2009/102537 PCT/US2009/031633
Other therapeutic agents that modulate or inhibit angiogenesis and may also be
used in combination with the compounds of the instant invention include agents
that modulate or
inhibit the coagulation and fibrinolysis systems (see review in Clin. Chem.
La. Med. 38:679-692
(2000)). Examples of such agents that modulate or inhibit the coagulation and
fibrinolysis
pathways include, but are not limited to, heparin (see Thromb. Haemost. 80:10-
23 (1998)), low
molecular weight heparins and carboxypeptidase U inhibitors (also known as
inhibitors of active
thrombin activatable fibrinolysis inhibitor [TAFIa)) (see Thrombosis Res.
101:329-354 (2001)).
TAFIa inhibitors have been described in U.S. Ser. Nos. 60/310,927 (filed
August 8, 2001) and
60/349,925 (filed January 18, 2002).
"Agents that interfere with cell cycle checkpoints" refer to compounds that
inhibit
protein kinases that transduce cell cycle checkpoint signals, thereby
sensitizing the cancer cell to
DNA damaging agents. Such agents include inhibitors of ATR, ATM, the CHK1 and
CHK2
kinases and cdk and cdc kinase inhibitors and are specifically exemplified by
7-
hydroxystaurosporin, flavopiridol, CYC202 (Cyclacel) and BMS-387032.
"Agents that interfere with receptor tyrosine kinases (RTKs)" refer to
compounds
that inhibit RTKs and therefore mechanisms involved in oncogenesis and tumor
progression.
Such agents include inhibitors of c-Kit, Eph, PDGF, F1t3 and c-Met. Further
agents include
inhibitors of RTKs as described by Bume-Jensen and Hunter, Nature, 411:355-
365, 2001.
"Inhibitors of cell proliferation and survival signalling pathway" refer to
compounds that inhibit signal transduction cascades downstream of cell surface
receptors. Such
agents include inhibitors of serine/threonine kinases (including but not
limited to inhibitors of
Akt such as described in WO 02/083064, WO 02/083139, WO 02/083140, US 2004-
0116432,
WO 02/083138, US 2004-0102360, WO 03/086404, WO 03/086279, WO 03/086394, WO
03/084473, WO 03/086403, WO 2004/041162, WO 2004/096131, WO 2004/096129, WO
2004/096135, WO 2004/096130, WO 2005/100356, WO 2005/100344, US 2005/029941,
US
2005/44294, US 2005/43361, 60/734188, 60/652737, 60/670469), inhibitors of Raf
kinase (for
example BAY-43-9006 ), inhibitors of MEK (for example CI-1040 and PD-098059),
inhibitors
of mTOR (for example Wyeth CCI-779), and inhibitors of P13K (for example
LY294002).
As described above, the combinations with NSAID's are directed to the use of
NSAID's which are potent COX-2 inhibiting agents. For purposes of this
specification an
NSAID is potent if it possesses an IC50 for the inhibition of COX-2 of 1 M or
less as measured
by cell or microsomal assays.
The invention also encompasses combinations with NSAID's which are selective
COX-2 inhibitors. For purposes of this specification NSAID's which are
selective inhibitors of
COX-2 are defined as those which possess a specificity for inhibiting COX-2
over COX-1 of at
least 100 fold as measured by the ratio of IC50 for COX-2 over IC50 for COX-1
evaluated by
cell or microsomal assays. Such compounds include, but are not limited to
those disclosed in
U.S. Patent 5,474,995, U.S. Patent 5,861,419, U.S. Patent 6,001,843, U.S.
Patent 6,020,343, U.S.
-35-
CA 02714087 2010-08-04
WO 2009/102537 PCT/US2009/031633
Patent 5,409,944, U.S. Patent 5,436,265, U.S. Patent 5,536,752, U.S. Patent
5,550,142, U.S.
Patent 5,604,260, U.S. 5,698,584, U.S. Patent 5,710,140, WO 94/15932, U.S.
Patent 5,344,991,
U.S. Patent 5,134,142, U.S. Patent 5,380,738, U.S. Patent 5,393,790, U.S.
Patent 5,466,823, U.S.
Patent 5,633,272 and U.S. Patent 5,932,598, all of which are hereby
incorporated by reference.
Inhibitors of COX-2 that are particularly useful in the instant method of
treatment
are: 3-phenyl-4-(4-(methylsulfonyl)phenyl)-2-(5H)-furanone; and
5-chloro-3-(4-methylsulfonyl)phenyl-2-(2-methyl-5-pyridinyl)pyridine; or a
pharmaceutically
acceptable salt thereof.
Compounds that have been described as specific inhibitors of COX-2 and are
therefore useful in the present invention include, but are not limited to, the
following: parecoxib,
BEXTRA and CELEBREX or a pharmaceutically acceptable salt thereof
Other examples of angiogenesis inhibitors include, but are not limited to,
endostatin, ukrain, ranpirnase,1M862, 5-methoxy-4-[2-methyl-3-(3-methyl-2-
butenyl)oxiranyl]-
1-oxaspiro[2,5]oct-6-yl(chloroacetyl)carbamate, acetyldinanaline, 5-amino- l-
[[3,5-dichloro-4-(4-
chlorobenzoyl)phenyl]methyl]-1H-1,2,3-triazole-4-carboxamide,CM101,
squalamine,
combretastatin, RP14610, NX31838, sulfated mannopentaose phosphate, 7,7-
(carbonyl-
bis[imino-N-methyl-4,2-pyrrolocarbonylimino [N-methyl-4,2-pyrrol e]-
carbonylimino] -bis-(1,3 -
naphthalene disulfonate), and 3-[(2,4-dimethylpyrrol-5-yl)methylene]-2-
indolinone (SU5416).
As used above, "integrin blockers" refers to compounds which selectively
antagonize, inhibit or counteract binding of a physiological ligand to the
avJ33 integrin, to
compounds which selectively antagonize, inhibit or counteract binding of a
physiological ligand
to the av(35 integrin, to compounds which antagonize, inhibit or counteract
binding of a
physiological ligand to both the av[33 integrin and the avj35 integrin, and to
compounds which
antagonize, inhibit or counteract the activity of the particular integrin(s)
expressed on capillary
endothelial cells. The term also refers to antagonists of the avj36, av(38, al
(31, a2(31, a5p1,
96!31 and a6j34 integrins. The term also refers to antagonists of any
combination of av33,
av(35, av(36, av138, al J31, a2J31, a0l, a601 and U44 integrins.
Some specific examples of tyrosine kinase inhibitors include N-
(trifluoromethylphenyl)-5-methylisoxazol-4-carboxamide, 3-[(2,4-dimethylpyrrol-
5-
yl)methylidenyl)indolin-2-one, 17-(allylamino)-17-demethoxygeldanamycin, 4-(3-
chloro-4-
fluorophenylamino)-7-methoxy-6-[3-(4-morpholinyl)propoxyl]quinazoline, N-(3-
ethynylphenyl)-
6,7-bis(2-methoxyethoxy)-4-quinazolinamine, BTBX1382, 2,3,9,10,11,12-hexahydro-
10-
(hydroxymethyl)-10-hydroxy-9-methyl-9,12-epoxy-1 H-diindolo[ 1,2,3-fg:3',2',
I'-kl]pyrrolo[3,4-
i][1,6]benzodiazocin-l-one, SH268, genistein, ST1571, CEP2563, 4-(3-
chlorophenylamino)-5,6-
dimethyl-7H-pyrrolo[2,3-d]pyrimidinemethane sulfonate, 4-(3-bromo-4-
hydroxyphenyl)amino-
6,7-dimethoxyquinazoline, 4-(4'-hydroxyphenyl)amino-6,7-dimethoxyquinazoline,
SU6668,
ST157 I A, N-4-chlorophenyl-4-(4-pyridylmethyl)-1-phthalazinamine, and EMD
121974.
-36-
CA 02714087 2010-08-04
WO 2009/102537 PCT/US2009/031633
Combinations with compounds other than anti-cancer compounds are also
encompassed in the instant methods. For example, combinations of the instantly
claimed
compounds with PPAR-y (i.e., PPAR-gamma) agonists and PPAR-3 (i.e., PPAR-
delta) agonists
are useful in the treatment of certain malingnancies. PPAR-y and PPAR- are the
nuclear
peroxisorne proliferator-activated receptors y and S. The expression of PPAR-y
on endothelial
cells and its involvement in angiogenesis has been reported in the literature
(see J. Cardiovase.
Pharmacol. 1998; 31:909-913; J. Biol. Chem. 1999;274:9116-9121; Invest.
Ophthalmol Vis. Sci.
2000; 41:2309-2317). More recently, PPAR-y agonists have been shown to inhibit
the
angiogenic response to VEGF in vitro; both troglitazone and rosiglitazone
maleate inhibit the
development of retinal neovascularization in mice. (Arch. Ophthamol. 2001;
119:709-717).
Examples of PPAR-y agonists and PPAR- yla agonists include, but are not
limited to,
thiazolidinediones (such as DRF2725, CS-011, troglitazone, rosiglitazone, and
pioglitazone),
fenofibrate, gemfibrozil, clofibrate, GW2570, SB219994, AR-H039242, JTT-501,
MCC-555,
GW2331, GW409544, NN2344, KRP297, NP0110, DRF4158, NN622, G1262570, PNU182716,
DRF552926, 2-[(5,7-dipropyl-3-trifluoromethyl-1,2-benzisoxazol-6-yl)oxy]-2-
methylpropionic
acid (disclosed in USSN 09/782,856), and 2(R)-7-(3-(2-chloro-4-(4-
fluorophenoxy)
phenoxy)propoxy)-2-ethylchromane-2-carboxylic acid (disclosed in USSN
60/235,708 and
60/244,697).
Another embodiment of the instant invention is the use of the presently
disclosed
compounds in combination with gene therapy for the treatment of cancer. For an
overview of
genetic strategies to treating cancer see Hall et al (Am. J. Hum. Genet.
61:785-789, 1997) and
Kufe et al (Cancer Medicine, 5th Ed, pp 876-889, BC Decker, Hamilton 2000).
Gene therapy
can be used to deliver any tumor suppressing gene. Examples of such genes
include, but are not
limited to, p53, which can be delivered via recombinant virus-mediated gene
transfer (see U.S.
Patent No. 6,069,134, for example), a uPA/uPAR antagonist ("Adenovirus-
Mediated Delivery of
a uPA/uPAR Antagonist Suppresses Angiogenesis-Dependent Tumor Growth and
Dissemination
in Mice," Gene Therapy, August 1998;5(8):1105-13), and interferon gamma (J.
Immunol.
2000;164:217-222).
The compounds of the instant invention may also be administered in combination
with an inhibitor of inherent multidrug resistance (MDR), in particular MDR
associated with
high levels of expression of transporter proteins. Such MDR inhibitors include
inhibitors of p-
glycoprotein (P-gp), such as LY335979, XR9576, OC144-093, R101922, VX853 and
PSC833
(valspodar).
A compound of the present invention may be employed in conjunction with anti-
emetic agents to treat nausea or emesis, including acute, delayed, late-phase,
and anticipatory
emesis, which may result from the use of a compound of the present invention,
alone or with
radiation therapy. For the prevention or treatment of emesis, a compound of
the present
invention may be used in conjunction with other anti-emetic agents, especially
neurokinin- 1
-37-
CA 02714087 2010-08-04
WO 2009/102537 PCT/US2009/031633
receptor antagonists, 5HT3 receptor antagonists, such as ondansetron,
granisetron, tropisetron,
and zatisetron, GABAB receptor agonists, such as baclofen, a corticosteroid
such as Decadron
(dexamethasone), Kenalog, Aristocort, Nasalide, Preferid, Benecorten or others
such as disclosed
in U.S.Patent Nos. 2,789,118, 2,990,401, 3,048,581, 3,126,375, 3,929,768,
3,996,359, 3,928,326
and 3,749,712, an antidopaminergic, such as the phenothiazines (for example
prochlorperazine,
fluphenazine, thioridazine and mesoridazine), metoclopramide or dronabinol. In
another
embodiment, conjunctive therapy with an anti-emesis agent selected from a
neurokinin-1
receptor antagonist, a 5HT3 receptor antagonist and a corticosteroid is
disclosed for the treatment
or prevention of emesis that may result upon administration of the instant
compounds.
Neurokinin-1 receptor antagonists of use in conjunction with the compounds of
the present invention are fully described, for example, in U.S. Patent Nos.
5,162,339, 5,232,929,
5,242,930, 5,373,003, 5,387,595, 5,459,270, 5,494,926, 5,496,833, 5,637,699,
5,719,147;
European Patent Publication Nos. EP 0 360 390, 0 394 989, 0 428 434, 0 429
366, 0 430 771, 0
436 334, 0 443 132, 0 482 539, 0 498 069, 0 499 313, 0 512 901, 0 512 902, 0
514 273, 0 514
274, 0 514 275, 0 514 276, 0 515 681, 0 517 589, 0 520 555, 0 522 808, 0 528
495, 0 532 456, 0
533 280, 0 536 817, 0 545 478, 0 558 156, 0 577 394, 0 585 913,0 590 152, 0
599 538, 0 610
793, 0 634 402, 0 686 629, 0 693 489, 0 694 535, 0 699 655, 0 699 674, 0 707
006, 0 708 101, 0
709 375, 0 709 376, 0 714 891, 0 723 959, 0 733 632 and 0 776 893; PCT
International Patent
Publication Nos. WO 90/05525, 90/05729, 91/09844, 91/18899, 92/01688,
92/06079, 92/12151,
92/15585, 92/17449, 92/20661, 92/20676, 92/21677, 92/22569, 93/00330,
93/00331, 93/01159,
93/01165, 93/01169, 93/01170, 93/06099, 93/09116, 93/10073, 93/14084,
93/14113, 93/18023,
93/19064, 93/21155, 93/21181, 93/23380, 93/24465, 94/00440, 94/01402,
94/02461, 94/02595,
94/03429, 94/03445, 94/04494, 94/04496, 94/05625, 94/07843, 94/08997,
94/10165, 94/10167,
94/10168, 94/10170, 94/11368, 94/13639, 94/13663, 94/14767, 94/15903,
94/19320, 94/19323,
94/20500, 94/26735, 94/26740, 94/29309, 95/02595, 95/04040, 95/04042,
95/06645, 95/07886,
95/07908, 95/08549, 95/11880, 95/14017, 95/15311, 95/16679, 95/17382,
95/18124, 95118129,
95/19344, 95/20575, 95/21819, 95/22525, 95/23798, 95/26338, 95/28418,
95/30674, 95/30687,
95/33744, 96/05181, 96/05193, 96/05203, 96/06094, 96/07649, 96/10562,
96/16939, 96/18643,
96/20197, 96/21661, 96/29304, 96/29317, 96/29326, 96/29328, 96/31214,
96/32385, 96/37489,
97/01553, 97/01554, 97/03066, 97/08144, 97/14671, 97/17362, 97/18206,
97/19084, 97/19942
and 97/21702; and in British Patent Publication Nos. 2 266 529, 2 268 931, 2
269 170, 2 269
590, 2 271 774, 2 292 144, 2 293 168, 2 293 169, and 2 302 689. The
preparation of such
compounds is fully described in the aforementioned patents and publications,
which are
incorporated herein by reference.
In an embodiment, the neurokinin-1 receptor antagonist for use in conjunction
with the compounds of the present invention is selected from: 2-(R)-(1-(R)-
(3,5-
bis(trifluoromethyl)phenyl)ethoxy)-3-(S)-(4-fluorophenyl)-4-(3-(5-oxo-l H,4H-
1,2,4-
-38-
CA 02714087 2010-08-04
WO 2009/102537 PCT/US2009/031633
triazolo)methyl)morpholine, or a pharmaceutically acceptable salt thereof,
which is described in
U.S. Patent No. 5,719,147.
A compound of the instant invention may also be administered with an agent
useful in the treatment of anemia. Such an anemia treatment agent is, for
example, a continuous
erythropoiesis receptor activator (such as epoetin alfa).
A compound of the instant invention may also be administered with an agent
useful in the treatment of neutropenia. Such a neutropenia treatment agent is,
for example, a
hematopoietic growth factor which regulates the production and function of
neutrophils such as a
human granulocyte colony stimulating factor, (G-CSF). Examples of a G-CSF
include
filgrastim.
A compound of the instant invention may also be administered with an
immunologic-enhancing drug, such as levamisole, isoprinosine and Zadaxin.
A compound of the instant invention may also be useful for treating or
preventing
cancer in combination with P450 inhibitors including: xenobiotics, quinidine,
tyramine,
ketoconazole, testosterone, quinine, methyrapone, caffeine, phenelzine,
doxorubicin,
troleandomycin, cyclobenzaprine, erythromycin, cocaine, furafyline,
cimetidine,
dextromethorphan, ritonavir, indinavir, amprenavir, diltiazem, terfenadine,
verapamil, cortisol,
itraconazole, mibefradil, nefazodone and nelfinavir.
A compound of the instant invention may also be useful for treating or
preventing
cancer in combination with Pgp and/or BCRP inhibitors including: cyclosporin
A, PSC833,
GF120918, cremophorEL, fumitremorgin C, Ko132, Ko134, Iressa, Imatnib
mesylate, EKI-785,
Cl1033, novobiocin, diethylstilbestrol, tamoxifen, resperpine, VX-710,
tryprostatin A,
flavonoids, ritonavir, saquinavir, nelfinavir, omeprazole, quinidine,
verapamil, terfenadine,
ketoconazole, nifidepine, FK506, amiodarone, X.129576, indinavir, amprenavir,
cortisol,
testosterone, LY335979, OC144-093, erythromycin, vincristine, digoxin and
talinolol.
A compound of the instant invention may also be useful for treating or
preventing
cancer, including bone cancer, in combination with bisphosphonates (understood
to include
bisphosphonates, diphosphonates, bisphosphonic acids and diphosphonic acids).
Examples of
bisphosphonates include but are not limited to: etidronate (Didronel),
pamidronate (Aredia),
alendronate (Fosamax), risedronate (Actonel), zoledronate (Zometa),
ibandronate (Boniva),
incadronate or cimadronate, clodronate, EB-1053, minodronate, neridronate,
piridronate and
tiludronate including any and all pharmaceutically acceptable salts,
derivatives, hydrates and
mixtures thereof.
A compound of the instant invention may also be useful for treating or
preventing
breast cancer in combination with aromatase inhibitors. Examples of aromatase
inhibitors
include but are not limited to: anastrozole, letrozole and exemestane.
A compound of the instant invention may also be useful for treating or
preventing
cancer in combination with siRNA therapeutics.
-39-
CA 02714087 2010-08-04
WO 2009/102537 PCT/US2009/031633
The compounds of the instant invention may also be administered in combination
with y-secretase inhibitors and/or inhibitors of NOTCH signaling. Such
inhibitors include
compounds described in WO 01/90084, WO 02/30912, WO 01/70677, WO 03/013506, WO
02/36555, WO 03/093252, WO 03/093264, WO 03/093251, WO 03/093253, WO
2004/039800,
WO 2004/039370, WO 2005/030731, WO 2005/014553, USSN 10/957,251, WO
2004/089911,
WO 02/081435, WO 02/081433, WO 03/018543, WO 2004/03 1 1 37, WO 2004/03 1 1
39, WO
2004/031138, WO 2004/101538, WO 2004/101539 and WO 02/47671 (including LY-
450139).
A compound of the instant invention may also be useful for treating or
preventing
cancer in combination with PARP inhibitors.
A compound of the instant invention may also be useful for treating cancer in
combination with the following therapeutic agents: abarelix (Plenaxis depot );
aldesleukin
(Prokine ); Aldesleukin (Proleukin ); Alemtuzumabb (Campath ); alitretinoin
(Panretin );
allopurinol (Zyloprim(D); altretamine (Hexalen ); amifostine (Ethyol );
anastrozole
(Arimidex ); arsenic trioxide (Trisenox ); asparaginase (Elspar(M);
azacitidine (Vidaza );
bevacuzimab (Avastin ); bexarotene capsules (Targretin ); bexarotene gel
(Targretin );
bleomycin (Blenoxane ); bortezomib (Velcade ); busulfan intravenous (Busulfex
); busulfan
oral (Myleran ); calusterone (Methosarb ); capecitabine (Xeloda ); carboplatin
(Paraplatin );
carmustine (BCNU , BiCNU ); carmustine (Gliadel ); carmustine with
Polifeprosan 20
Implant (Gliadel Wafer ); celecoxib (Celebrex ); cetuximab (Erbitux );
chlorambucil
(Leukeran ); cisplatin (Platinol ); cladribine (Leustatin , 2-CdA );
clofarabine (Clolar );
cyclophosphamide (Cytoxan , Neosar ); cyclophosphamide (Cytoxan Injection );
cyclophosphamide (Cytoxan Tablet ); cytarabine (Cytosar-U ); cytarabine
liposomal
(DepoCyt ); dacarbazine (DTIC-Dome ); dactinomycin, actinomycin D (Cosmegen );
Darbepoetin alfa (Aranesp ); daunorubicin liposomal (DanuoXome );
daunorubicin,
daunomycin (Daunorubicin ); daunorubicin, daunomycin (Cerubidine ); Denileukin
diftitox
(Ontak ); dexrazoxane (Zinecard ); docetaxel (Taxotere ); doxorubicin
(Adriamycin PFS );
doxorubicin (Adriamycin , Rubex ); doxorubicin (Adriamycin PFS Injection );
doxorubicin
liposomal (Doxil ); DROMOSTANOLONE PROPIONATE (DROMOSTANOLONE );
DROMOSTANOLONE PROPIONATE (MASTERONE INJECTION ); Elliott's B Solution
(Elliott's B Solution ); epirubicin (Ellence ); Epoetin alfa (epogen );
erlotinib (Tarceva );
estramustine (Emcyt ); etoposide phosphate (Etopophos ); etoposide, VP- 16
(Vepesid );
exemestane (Aromasin ); Filgrastim (Neupogen ); floxuridine (intraarterial)
(FUDR );
fludarabine (Fludara ); fluorouracil, 5-FU (Adrucil ); fulvestrant (Faslodex
); gefitinib
(Iressa ); gemcitabine (Gemzar ); gemtuzumab ozogamicin (Mylotarg ); goserelin
acetate
(Zoladex Implant ); goserelin acetate (Zoladex ); histrelin acetate (Histrelin
implant );
hydroxyurea (Hydrea ); Ibritumomab Tiuxetan (Zevalin ); idarubicin (Idamycin
); ifosfamide
(IFEX ); imatinib mesylate (Gleevec ); interferon alfa 2a (Roferon A );
Interferon alfa-2b
(Intron A ); irinotecan (Camptosar ); lenalidomide (Revlimid(D); letrozole
(Femara );
-40-
CA 02714087 2010-08-04
WO 2009/102537 PCT/US2009/031633
leucovorin (Wellcovorin , Leucovorin ); Leuprolide Acetate (Eligard );
levamisole
(Ergamisol ); lomustine, CCNU (CeeBU ); meclorethamine, nitrogen mustard
(Mustargen );
megestrol acetate (Megace ); melphalan, L-PAM (Alkeran ); mercaptopurine, 6-MP
(Purinethol ); mesna (Mesnex ); mesna (Mesnex tabs ); methotrexate
(Methotrexate );
methoxsalen (Uvadex(&); mitomycin C (Mutamycin ); mitotane (Lysodren );
mitoxantrone
(Novantrone ); nandrolone phenpropionate (Durabolin-50 ); nelarabine (Arranon
);
Nofetumomab (Verluma ); Oprelvekin (Neumega ); oxaliplatin (Eloxatin );
paclitaxel
(Paxene ); paclitaxel (Taxol ); paclitaxel protein-bound particles (Abraxane
); palifermin
(Kepivance ); pamidronate (Aredia(@); pegademase (Adagen (Pegademase Bovine)
);
pegaspargase (Oncaspar ); Pegfilgrastim (Neulasta ); pemetrexed disodium
(Alimta );
pentostatin (Nipent ); pipobroman (Vercyte ); plicamycin, mithramycin
(Mithracin );
porfimer sodium (Photofrin ); procarbazine (Matulane ); quinacrine (Atabrine
); Rasburicase
(Elitek ); Rituximab (Rituxan ); sargramostim (Leukine ); Sargramostim
(Prokine );
sorafenib (Nexavar ); streptozocin (Zanosar(&); sunitinib maleate (Sutent );
talc (Sclerosol );
tamoxifen (Nolvadex ); temozolomide (Temodar ); teniposide, VM-26 (Vurn.on );
testolactone (Teslac ); thioguanine, 6-TG (Thioguanine ); thiotepa (Thioplex
); topotecan
(Hycamtin ); toremifene (Fareston ); Tositumomab (Bexxar ); Tositumomab/I-131
tositumomab (Bexxar ); Trastuzumab (Herceptin ); tretinoin, ATRA (Vesanoid );
Uracil
Mustard (Uracil Mustard Capsules ); valrubicin (Valstar ); vinblastine (Velban
); vincristine
(Oncovin ); vinorelbine (Navelbine ); vorinostat (Zolinza ) and zoledronate
(Zometa ).
Thus, the scope of the instant invention encompasses the use of the instantly
claimed compounds in combination with a second compound selected from: an
estrogen receptor
modulator, an androgen receptor modulator, a retinoid receptor modulator, a
cytotoxic/cytostatic
agent, an antiproliferative agent, a prenyl-protein transferase inhibitor, an
HMG-CoA reductase
inhibitor, an HIV protease inhibitor, a reverse transcriptase inhibitor, an
angiogenesis inhibitor,
PPAR-y agonists, PPAR-6 agonists, an inhibitor of inherent multidrug
resistance, an anti-emetic
agent, an agent useful in the treatment of anemia, an agent useful in the
treatment of neutropenia,
an immunologic-enhancing drug, an inhibitor of cell proliferation and survival
signaling, a
bisphosphonate, an aromatase inhibitor, an siRNA therapeutic, y-secretase
and/or NOTCH
inhibitors, agents that interfere with receptor tyrosine kinases (RTKs), an
agent that interferes
with a cell cycle checkpoint, and any of the therapeutic agents listed above.
The terra "administration" and variants thereof (e.g., "administering" a
compound)
in reference to a compound of the invention means introducing the compound or
a prodrug of the
compound into the system of the animal in need of treatment. When a compound
of the
invention or prodrug thereof is provided in combination with one or more other
active agents
(e.g., a cytotoxic agent, etc.), "administration" and its variants are each
understood to include
concurrent and sequential introduction of the compound or prodrug thereof and
other agents.
-41-
CA 02714087 2010-08-04
WO 2009/102537 PCT/US2009/031633
As used herein, the term "composition" is intended to encompass a product
comprising the specified ingredients in the specified amounts, as well as any
product which
results, directly or indirectly, from combination of the specified ingredients
in the specified
amounts.
The term "therapeutically effective amount" as used herein means that amount
of
active compound or pharmaceutical agent that elicits the biological or
medicinal response in a
tissue, system, animal or human that is being sought by a researcher,
veterinarian, medical doctor
or other clinician.
The term "treating cancer" or "treatment of cancer" refers to administration
to a
mammal afflicted with a cancerous condition and refers to an effect that
alleviates the cancerous
condition by killing the cancerous cells, but also to an effect that results
in the inhibition of
growth and/or metastasis of the cancer.
In an embodiment, the angiogenesis inhibitor to be used as the second compound
is selected from a tyrosine kinase inhibitor, an inhibitor of epidermal-
derived growth factor, an
inhibitor of fibroblast-derived growth factor, an inhibitor of platelet
derived growth factor, an
MMP (matrix metalloprotease) inhibitor, an integrin blocker, interferon-a,
interleukin-12,
pentosan polysulfate, a cyclooxygenase inhibitor, carboxyamidotriazole,
combretastatin A-4,
squalamine, 6-O-chloroacetyl-carbonyl)-fumagillol, thalidomide, angiostatin,
troponin-l, or an
antibody to VEGF. In an embodiment, the estrogen receptor modulator is
tamoxifen or
raloxifene.
Also included in the scope of the claims is a method of treating cancer that
comprises administering a therapeutically effective amount of a compound of
the instant
invention in combination with radiation therapy and/or in combination with a
second compound
selected from: an estrogen receptor modulator, an androgen receptor modulator,
a retinoid
receptor modulator, a cytotoxiccytostatic agent, an antiproliferative agent, a
prenyl-protein
transferase inhibitor, an HMG-CoA reductase inhibitor, an HIV protease
inhibitor, a reverse
transcriptase inhibitor, an angiogenesis inhibitor, PPAR-y agonists, PPAR-6
agonists, an
inhibitor of inherent multidrug resistance, an anti-emetic agent, an agent
useful in the treatment
of anemia, an agent useful in the treatment of neutropenia, an immunologic-
enhancing drug, an
inhibitor of cell proliferation and survival signaling, a bisphosphonate, an
aromatase inhibitor, an
siRNA therapeutic, y-secretase and/or NOTCH inhibitors, agents that interfere
with receptor
tyrosine kinases (RTKs), an agent that interferes with a cell cycle
checkpoint, and any of the
therapeutic agents listed above.
And yet another embodiment of the invention is a method of treating cancer
that
comprises administering a therapeutically effective amount of a compound of
the instant
invention in combination with paclitaxel or trastuzumab.
-42-
CA 02714087 2010-08-04
WO 2009/102537 PCT/US2009/031633
The invention further encompasses a method of treating or preventing cancer
that
comprises administering a therapeutically effective amount of a compound of
the instant
invention in combination with a COX-2 inhibitor.
The instant invention also includes a phannaceutical composition useful for
treating or preventing cancer that comprises a therapeutically effective
amount of a compound of
the instant invention and a second compound selected from: an estrogen
receptor modulator, an
androgen receptor modulator, a retinoid receptor modulator, a
cytotoxic/cytostatic agent, an
antiproliferative agent, a prenyl-protein transferase inhibitor, an HMG-CoA
reductase inhibitor,
an HIV protease inhibitor, a reverse transcriptase inhibitor, an angiogenesis
inhibitor, a PPAR-y
agonist, a PPAR-b agonist, an inhibitor of cell proliferation and survival
signaling, a
bisphosphonate, an aromatase inhibitor, an siRNA therapeutic, y-secretase
and/or
NOTCHinhibitors, agents that interfere with receptor tyrosine kinases (RTKs),
an agent that
interferes with a cell cycle checkpoint, and any of the therapeutic agents
listed above.
All patents, publications and pending patent applications identified are
hereby
incorporated by reference.
Abbreviations that may be used in the description of the chemistry and in the
Schemes that follow are: Ac20 (acetic anhydride); AcOH (acetic acid); AEBSF (p-
aminoethylbenzenesulfonyl fluoride); BSA (bovine serum albumin); BuLi (n-Butyl
lithium);
CDC13 (chloroform-d); Cul (copper iodide); CuSO4 (copper sulfate); DBU (1,8-
diazabicyclo[5.4.0]undec-7-ene); DCE (dichloroethane); DCM (dichloromethane);
DEAD
(diethyl azodicarboxylate); DIPEA (diisopropylethylamine); DMF (N,N-
dimethylformamide);
DMP (Dess-Martin periodinane); DMSO (dimethyl sulfoxide); DPPA
(diphenylphosphoryl
azide); DTT (dithiothreitol); EDTA (ethylene-diamine-tetra-acetic acid); EGTA
(ethylene-
glycol-tetra-acetic acid); Et20 (diethylether); EtOAc (ethyl acetate); EtOH
(ethanol); HOAc
(acetic acid); HPLC (high-performance liquid chromatography); FIRMS (high
resolution mass
spectrum); LAH (lithium aluminum hydride); LCMS (liquid chromatograph-mass
spectrometer);
LHMDS (lithium bis(trimethylsilyl)amide); LRMS (low resolution mass spectrum);
mCPBA (3-
chloroperoxybenzoic acid); MeOH (methanol); MP-B(CN)H3 (Macroporous
eyanoborohydride);
NaHCO3 (sodium bicarbonate); Na2SO4 (sodium sulfate); Na(OAc)3BH (sodium
triacetoxyborohydride); NH4OAc (ammonium acetate); NBS (N-bromosuccinamide);
NMP (1-
methyl-2-pyrrolidinone); NMR (nuclear magnetic resonance); PBS (phosphate
buffered saline);
PCR (polymerase chain reaction); Pd(dppf) ([1,1'-
bis(diphenylphosphino)ferrocene] palladium);
Pd(Ph3)4 (palladium(0) tetrakis-triphenylphosphine); POC13 (phosphorous
oxychloride); PS-
DIEA (polystyrene diisopropylethylamine); PS-PPh3 (polystyrene-triphenyl
phosphine); PTSA
(para-toluene sulfonic acid); ); PyBOP (benzotriazol-l-yl-oxy-
tris(pyrrolidino)phosphonium
hexafluorophosphate); Pyr (pyridine); Selectfluor (1-chloromethyl-4-fluoro-1,4-
diazoniabicyclo2.2.2]octane bis(tetrafluoroborate); RP HPLC (reverse phase
high-performance
liquid chromatography); TBAF (tetrabutylammonium fluoride); t-BuOH (tert-
butanol); THE
-43-
CA 02714087 2010-08-04
WO 2009/102537 PCT/US2009/031633
(tetrahydrofuran); Tf (trifluoromethanesulfonyl); TFA (trifluoroacteic acid);
and TMSCH2N2
(trimethylsilyldiazomethane).
The compounds of this invention may be prepared by employing reactions as
shown in the following Reaction Schemes, in addition to other standard
manipulations that are
known in the literature or exemplified in the experimental procedures. The
illustrative Reaction
Schemes below, therefore, are not limited by the compounds listed or by any
particular
substituents employed for illustrative purposes. Substituent numbering as
shown in the Reaction
Schemes do not necessarily correlate to that used in the claims and often, for
clarity, a single
substituent is shown attached to the compound where multiple substituents are
optionally
allowed under the definitions of Formula A hereinabove.
Reactions used to generate the compounds of this invention are prepared by
employing reactions as shown in Reaction Schemes A-C.
SYNOPSIS OF REACTION SCHEMES
As shown in Reaction Scheme I, the substituted antrhranilic acid (A-1) can
undergo a condensation with substituted isothiocyanates to provide
mercaptopyrimidinone (A-2).
Compounds A-2 can be further derivatized by coupling to cc-halo carboxylic
acids to furnish
compounds denoted by A-3.
Reaction Scheme A
0
R2
O OH RN S O N R2 XOH is
R(R,)s NH2 SH A-1 A-2 A-3 R,3OH
R4 O
Reaction Scheme II illustrates the preparation of thioquinazolinones (B-1) by
amide coupling of substituted mercaptopyrimidinone (A-3) with various amines
(B-2).
Reaction Scheme B
0 0
R2 R5 R2
N~ HN, N,
(RI)5 fA R6 (R,),5 A I
N S B-2 N S R5
A-3 R3 - /OH - B-1 R3 R
4 O 4 O
As shown in Reaction Scheme III, Suzuki Coupling of iodide C-1 with boronic
ester (C-2) can afford compounds denoted by C-3.
-44-
CA 02714087 2010-08-04
WO 2009/102537 PCT/US2009/031633
Reaction Scheme C
O
0
Suzuki Coupling
N I R7
Pd(Q) N
(R1)s A (Ri)5 A J
N S R5 R70 O N' S R5
C-1 R3 N`R6 BO C-3 R3(N,R6
R4, R4
C-2
EXAMPLES
Examples provided are intended to assist in a further understanding of the
invention. Particular materials employed, species and conditions are intended
to be further
illustrative of the invention and not limitative of the reasonable scope
thereof. The reagents
utilized in synthesizing the compounds depicted in the following Tables are
either commercially
available or are readily prepared by one of ordinary skill in the art.
SCHEME I
r
O -5 0 \ O \
I
DH EtOH N CI 1.4 O l I N CI
\ NH 75C NSH Et3N, DMF
75C
1-1 1-2 CI 1-3 1-5 OH
O
NBOC O p
1-6 ON CI N \ CI
NH S TFA N
PyBOP, EtA 1-7 N DCM 1_g
N r h Y
DMF, rt N O
BOC H
3- 3-chloro hen 1-2-merca to-8-meth 1 uinazolin-4 3 -one (1-31
2-m-nino-3-methylbenzoic acid (1-1) (3 g, 21.88 mmol, 1.0 equiv) was dissolved
in EtOH (44 mL), and heated to 75 T. 3-Chlorophenyl isothiocyanate (1-2) was
added in 0.2 eq.
portions over the course of 78 hours until disappearance of benzoic acid. The
mixture was
cooled, concentrated, and the slurry was filtered and washed with cold ethanol
to afford pure 3-
(3-chlorophenyl)-2-mercapto-8-methylquinazolin-4(3H)-one (1-3). LRMS m/z (M+H)
303.0
found, 303.1 required.
2- { (3-(3-chlorophenyl)-8-methyl-4-oxo-3,4-dihydroquinazolin-2-yl]thio }
hexanoic acid (1-5)
3-(3-Chlorophenyl)-2,3-dihydro-2-thioxo-4(IH)-8-methylquinazolinone (1-3,
145 mg, 0.50 mmol, 1.0 equiv), 2-bromohexanoic acid (1-4, 93 j.tL, 0.65 mmol,
1.3 equiv.), and
triethylamine (420 4L, 3.0 mmol, 6 equiv.) were dissolved in anhydrous DMF (1
mL) and
stirred at 75 C. When LCMS indicated the reaction was complete, the crude
reaction mixture
was partitioned between ethyl acetate and 0.1 M HCI, the aqueous phase drained
and the organic
-45-
CA 02714087 2010-08-04
WO 2009/102537 PCT/US2009/031633
layer washed twice more with 0.1 M HCl followed by brine to yield the product
(1-5). LRMS
m/z (M+H) 417.0 found, 417.1 required.
tent-Butyl 8-(2- { [3-(3-chlorophenyl)-8-methyl-4-oxo-3,4-dihydroquinazolin-2-
1 thin hexano 1 -2 8-diazas iro 5.5 undecane-2-carbox late 1-7
2-{[3-(3-Chlorophenyl)-- 8_methyl-4-oxo-3,4-dihydroquinazolin-2-
yl]thio}hexanoic acid (1-5, 174 mg, 0.43 mmol, 1.0 equiv.), tent-butyl 2,8-
diazaspiro[5.5]undecane-2-carboxylate (1-6, 220 mg, 0.864 mmol, 2 equiv.),
PyBOP (337 mg,
0.648 mmol, 1.5 equiv.) and triethylamine (0.6 mL, 4.32 mmol, 10 equiv.) were
dissolved in
DMF (1 mL) and stirred at rt. Once LCMS showed reaction was complete, the
reaction mixture
was filtered and purified with reverse phase HPLC (H20/ CH3CN gradient w/ 0.1
% TFA present)
to yield the product (1-7). LRMS m/z (M+H) 653.1 found, 653.3 required
3-(3-chlorophenyl)-2- { [ 1-(2,8-diazaspiro[5.5]undec-2-
ylcarbonyl)pentyl]thio} -8-
meth 1 uinazolin-4 3 -one (1-8)
tent-Butyl 8-(2- { [3-(3-chlorophenyl)-8-methyl-4-oxo-3,4-dihydroquinazolin-2-
yl]thio}hexanoyl)-2,8-diazaspiro[5.5]undecane-2-carboxylate (1-7, 200 mg, 0.31
mmol,
1.0 equiv.) was dissolved in DCM (2 mL). Trifluoroacetic acid (1 mL) was added
and the
mixture stirred at room temperature. Once LCMS showed reaction was complete,
toluene was
added, the solvent and TFA were removed at reduced pressure and the resulting
crude was
filtered and purified with reverse phase HPLC (H20/ CH3CN gradient w/ 0.1 %
TFA present) to
yield the product. 1H NMR (500 MHz, CDC13) S 8.09 (d, 111, J= 8.0 Hz), 7.61
(d, 1H, J= 7.0
Hz), 7.46 (m, 2H), 7.32 (m, 2H), 7.21 (m, 1H), 3.73 (m, 2H), 3.42 (br in, 1H),
3.23 (m, 1H), 2.92
(m, 111), 2.77 (m, 114), 2.73 (d, I H, J= 13 Hz), 2.67 (s, 2H), 2.60 (in, 3H),
2.50 (br dd, 1H, J=
4.5, 12Hz), 2.08 (m, 1H), 1.85 (m, lH), 1.59 (br m, 4H), 1.46 (m, 2H), 1.36
(br t, 4H, J= 4.0
Hz), 1.25 (s, 2H), 0.88 (t, 3H, J= 7.0 Hz). LRMS m/z (M+H) 553.1 found, 553.3
required.
The compounds shown in Table 1 were synthesized as TFA salts according to
Scheme 1.
Table 1
Cmp Structure Name LRMS m/z (M+H)
1-9 1-(2-{[3-(3- LRMS m/z (M+H)
0 chlorophenyl)-4-oxo-3,4- 513.0 found, 513.1
N Ci dihydroquinazolin-2-yl] required.
NS methyl}hexanoyl)piperidi
N NH2 ne-3-carboxamide
r~~o 0
-46-
CA 02714087 2010-08-04
WO 2009/102537 PCT/US2009/031633
1-10 2- {2-[(4-acetylpiperazin- LRMS m/z (M+H)
0 I 1-yl)carbonyl]hexyl}-3- 513.0 found, 513.1
cl (3-chlorophenyl)- required.
NHS rN quinazolin-4(3H)-one
NJ
1_11 C 3-(3-chlorophenyl)-2-{2- LRMS m/z (M+H)
N C! [(4-phenylpiperazin-1- 547.0 found, 547.1
N s iN yl)carbonyl]hexyl}quinaz required.
N olin-4(3H)-one
0
1-12 O ~ 1-(2-{[3-(3- LRMS m/z (M+H)
cl N Cl chlorophenyl)-4-oxo-3,4- 569.1 found, 569.2
N~S ( dihydroquinazolin-2- required.
N N yl]methyl}hexanoyl)-
0 NN diethylpiperidine-3-
carboxamide
1-13 3-(3-chlorophenyl)-2-{2- LRMS m/z (M+H)
0 I [(4-pyridin-2-ylpiperazin- 548.0 found, 548..1
N c~ N 1-yl)carbonyl]hexyl}- required.
Ns ' rN quinazolin-4(3H)-one
N,_,)
O
1-14 2-(2- {[4-(aminomethyl)- LRMS m/z (M+H)
0 piperidin-l-yl]carbonyl) - 499.1 found, 499.1
i I Cl
N NH2 hexyl)-3-(3-chloro- required.
N s phenyl)quinazolin-4(3H)-
N one
O
1-15 3-(3-chlorophenyl)-2-{2- LRMS m/z (M+H)
0 [(7-methyl-2,7-diazaspiro 525.0 found, 525.1
i I N a CI [4.4]non-2-yl)carbonyl] required.
N )~'5 hexyl) quinazolin-4(3H)-
Nr \ one
O
-47-
CA 02714087 2010-08-04
WO 2009/102537 PCT/US2009/031633
1-16 2-(2-{[4-(1H- LRMS m/z (M+H)
0 / benzimidazol-2- 586.0 found, 586.2
N L Cl N yl)piperidin-l- required.
N~S N yl]carbonyl}hexyl)-3-(3-
N H chlorophenyl)quinazolin-
O 4(311)-one
1-17 3-(3-chlorophenyl)-2-{2-- LRMS m/z (M+H)
0 [(1,1-dioxidothio- 520.0 found, 520.1
N CI 0 morpholin 4 yl)- required.
N~S carbonyl ]hexyl}quinazoli
N n-4(311)-one
J
O
1-18 3-(3-chlorophenyl)-2-(2- LRMS m/z (M+H)
0 {[4-(methylsulfonyl)- 549.0 found, 549.1
/ N CI 0 piperazin 1 yl]carbonyl}- required.
NS N' 0 hexyl)quinazolin-4(3H)-
NJ one
O
1-19 3-(3-chlorophenyl)-2-(2- LRMS m/z (M+H)
0 N {[4-(lH iinidazol-l- 549.0 found, 549.1
N Cl N ylmethyl)piperidm- l - required.
LN~S yl]carbonyl}hexyl)quinaz
N olin-4(3R)-one
O
1-20 3-(3-chlorophenyl)-2-(2- LRMS m/z (M+H)
O {[4-(methylsulfonyl)- 548.0 found, 548.1
N Cl0piperidin 1 yl]carbonyl}- required.
hexyl)quinazolin-4(3H)-
N S Na one
O
- 48-
CA 02714087 2010-08-04
WO 2009/102537 PCT/US2009/031633
1-21 3-(3-chlorophenyl)-2-[2- LRMS m/z (M+H)
O (2,8-diazaspiro[5.5] = 539.0 found, 539.1
N CI under-2 ylcarbonyl) required.
hexyl]quinazolin-4(3H)-
r"- ~~
N S
N one
H
rf~'Yo N
1-22 3-(3-chlorophenyl)-2-(2- LRMS m/z (M+H)
O a~'-IICI {[4-(2-fluorophenyl)- 565.0 found, 565.1
N / piperazin 1 yl]carbonyl } required.
N~S N 1 hexyl)quinazolin-4(3H)-
N J F one
O
1-23 3-(3-chlorophenyl)-2-(2- LRMS m/z (M+H)
O / {[4-(4-fluorophenyl)- 565.0 found, 565.1
1
/ N CI F piperazin-1-yl]earbonyl}- required.
hex l uinazolin-4 3 -
N S rN y )q
N J one
O
1-24 3-(3-chlorophenyl)-2-{2- LRMS m/z (M+H)
O / [(4,4-diphenylpiperidin- 622.1 found, 622.2
N CI \ 1-yl)carbonyl]hexyl}- required.
NS N quinazolin-4(3R)-one
/
rl' 0
1-25 3-(3-chlorophenyl)-2-{2- LRMS m/z (M+H)
O / I [(4-pyridin-4-ylpiperidin- 547.0 found, 547.1
N Cl 1-yl)carbonyl]hexyl}- required.
N'J'S quinazolin-4(3H)-one
-49-
CA 02714087 2010-08-04
WO 2009/102537 PCT/US2009/031633
1-26 3-(3-chlorophenyl)-2-(2- LRMS m/z (M+H)
0 [(3,3-diphenylpiperidin- 622.1 found, 622.2
ON) / C! 1-yl)carbonyl]hexyl} required.
NS \ quinazolin-4(3H)-one
N
O
rf, 1-27 3-(3-chlorophenyl)-2- LRMS m/z (M+H)
0 / I {[1-(2,8-diazaspiro[5.5]- 617.1 found, 617.2
e"' CI undec-2-ylcarbonyl)- required.
O.~ S pentyl]thio}-7-(methyl-
0 sulfonyl)quinazolin-
O N 4(3H)-one
rf, H
1-28 methyl 3-(3-chloro- LRMS m/z (M+H)
O phenyl)-2-{[1-(2,8- - - 597.2 found, 597.2
J/ ' CI diazaspiro[5.5]undec 2 required.
O 1 N S ylcarbonyl)pentyl]thio}-
O N 4-oxo-3,4-dihydro-
N quinazoline-7-
H carboxylate
1-29 3-(3-chlorophenyl)-2- LRMS m/z (M+H)
O ({3-methyl-1 [(4-pyridin- 548.1 found, 548.2
N CI N 2-ylpiperazin- 1 yl} required.
NS N carbonyl]butyl}thio)quin
NJ azolin-4(3H)-one
O
1-30 3-(3-chlorophenyl)-2-[(1- LRMS m/z (M+H)
O {[4-(2,3-dihydro-1,4- 548.1 found, 548.2
N CI benzodioxin 5 required.
NLS N O yl)piperazin-1-
N J OJ yl]carbonyl}-3-
p methylbutyl)thio]quinazo
lin-4 3 -one
-50-
CA 02714087 2010-08-04
WO 2009/102537 PCT/US2009/031633
1-31 0 3-(3-chlorophenyl)-2-[(1- LRMS m/z (M+H)
N Cl / {[4-(4-fluorophenyl)- 565.2 found, 565.2
i erazin-l- 1 carbon 1 - required.
N S ~N pp y] y} N 3-methylbutyl)thio]-
O quinazolin-4(3H)-one
1-32 O / 3-(3-chlorophenyl)-2-[(1- LRMS m/z (M+H)
N CI {[7-(3-chlorophenyl)-2,7- 621.0 found, 621.2
NS diazaspiro[4.4]non-2- required.
NOON yl]carbonyl}-3-
O methylbutyl)thio]quinazo
lin-4(3H)-one
k
1-33 0 3-(3-chlorophenyl)-2-[(1- LRMS m/z (M+H)
Cl { [7-(2-chlorophenyl)-2,7- 621.0 found, 621.2
N" S diazaspiro[4.4]non-2- required.
r:)C N Cl yl]carbonyl}-3-
0 methylbutyl)thio]quinazo
lin-4 3 -one
1-34 0 2-({l -[(7-benzyl-2,7- LRMS m/z (M+H)
N CI diazaspiro[4.4]non-2- 601.2 found, 601.2
N~S yl)carbonyl]-3- required.
N methylbutyl}thin)-3-(3-
chlorophenyl)quinazolin-
1 4(3R)-one
1-35 0 / 3-(3-chlorophenyl)-2-[(1- LRMS m/z (M+H)
N {[7 (3-methoxyphenyl)- 617.3 found, 617.2
N 2,7-diazaspiro[4.4]non-2- required.
N - f ON yllcarbonyl}_3-
0 { \ methylbutyl)thio]quinazo
9 lin-4(3H)-one
1-36 0 / 3-(3-chlorophenyl)-2-[(1- LRMS m/z (M+H)
/ I N Cl {[7-(4-methoxyphenyl)- 617.2 found, 617.2
N~k S 2,7-diazaspiro[4.4]non-2- required.
N yl]carbonyl}-3-
O methylbutyl)thio]quinazo
lin-4(3H)-one
O-
-51 -
CA 02714087 2010-08-04
WO 2009/102537 PCT/US2009/031633
1-37 O 1-(2-{[3-(3- LRMS m/z (M+H)
Cl N NH2 chlorophenyl)-4--oxo-3,4- 513.0 found, 513.2
Lk N' S O dihydroquinazolin-2- required.
N yl]thio}hexanoyl)piperidi
O ne-4-carboxamide
rf, 1-38 O 1-[{3-(3-chlorophenyl)- LRMS m/z (M+H)
Cl NH2 4-oxo-3,4- 513.0 found, 513.2
N S p dihydroquinazolin-2- required.
N yl]thio} (phenyl)acetyl]pi
0--lyo peridine-4-carboxamide
1-39 O 1-(2-{[3-(3- LRMS m/z (M+H)
N aCl NH2 chlorophenyl)-4-oxo-3,4- 513.0 found, 513.2
NxS O dihydroquinazolin-2- required.
yl]thio}-4-
O methylpentanoyl)piperidi
ne-4-carboxamide
1-40 O 3-(3-chlorophenyl)-2-[(1- LRMS m/z (M+H)
CI {[7-(2-chlorophenyl)-2,7- 699.0 found, 699.1
O, N S diazaspiro[4.4]non-2- required.
O No CI yl]carbonyl}pentyl)thio]-
O 7-(methylsulfonyl)-
uinazolin-4(3R)-one
1-41 0 3-(3-chlorophenyl)-2-[(1- LRMS m/z (M+H)
\ CI {[7-(4-chlorophenyl)-2,7- 699.0 found, 699.1
o N s diazaspiro[4.4]non-2- required.
0 N`' ONN yl]carbonyl}pentyl)thio]-
0 7-(methylsulfonyl)-
CI quinazolin-4(3H)-one
1-42 0 3-(3-chlorophenyl)-2-[(1- LRMS m/z (M+H)
N CI {[7-(3-chlorophenyl)-2,7- 699.0 found, 699.1
N"~'s diazaspiro[4.4]non-2- required.
o o NOON CI yl]carbonyl}pentyl)thio]-
7-(m.ethylsulfonyl)-
uinazolin-4(3H)-one
-52-
CA 02714087 2010-08-04
WO 2009/102537 PCT/US2009/031633
1-43 0 3-(3-chlorophenyl)-7- LRMS m/z (M+H)
CI (methylsulfonyl)-2-({1- 665.0 found, 665.2
0 N" 5 [(7-phenyl-2,7- required.
O N diazaspiro[4.4]non-2-
o yl)carbonyl]pentyl}thio)q
uinazolin-4 3 -one
1-44 0 1 3-(3-chlorophenyl)-2-[(1- LRMS m/z (M+H)
N \ ci {[7-(3-methoxyphenyl)- 695.1 found, 695.2
N~S S 2,7-diazaspiro[4.4]non-2- required.
o N / yl]carbonyl}pentyl)thio]-
1 o
0
7-(methylsulfonyl)-
quinazoli n-4(31])-one
1-45 0 methyl 3-(3- LRMS m/z (M+H)
N Cl chlorophenyl)-2-((l-[(3- 601.1 found, 601.1
%I 5 CN cyan-3-phenylazetidin- required.
N
N \ / 1-yl)carbonyl]pentyl}-
0 thio)-4-oxo-3,4-
dihydroquinazol ine-7-
carboxlate
rfl 1-46 0 1 methyl 3-(3- LRMS m/z (M+H)
N CI chlorophenyl)-2- { [ 1-(2,6- 541.0 found, 541.1
o N';~ S /JNH diazaspiro[3.3]hept-2- required.
NCI YlcarbonY1)pentY1]thia}-
0 4-oxo-3,4-dihydro-
quinazoline-7-
carboxylate
1-47 0 3-(3-chlorophenyl)-2-[(1- LRMS m/z (M+H)
N CI {[(2R)-2-(trifluoro- 524.0 found, 523.1
N methyl) pyrrolidin-1-yl]- required.
N[D carbonyl} pentyl)thio]-
0 FTF quinazolin-4(3H)-one
F
-53-
CA 02714087 2010-08-04
WO 2009/102537 PCT/US2009/031633
1-48 0 0 3-(3-chlorophenyl)-2-[(1- LRMS m/z (M+H)
N c[ 0 {[3-(4-fluorobennyl)-2- 648.1 found, 648.1
NS oxo-l-oxa-8- required.
N / \ azaspiro[4.5]dec-8-
0 - yl]carbonyl}pentyl)thio]q
uinazolin-4(3H)-one
F 1-49 0 / 3-(3-chlorophenyl)-2-[(1- LRMS m/z (M+H)
/ I N CI {[5-(4-fluorophenyl)-2,5- 577.1 found, 576.2
N~S diazabicyclo[2.2.1 ]hept- required.
N,--A- 2-y1]carbonyl}pentyl)-
N F thio]quinazolin-4(3H)-
0 one
1.50 0 3-(3-chlorophenyl)-2-[(1- LRMS m/z (M+H)
N CI {[5-(3-fluorophenyl)-2,5- 577.1 found, 576.2
N S F diazabicyclo[2.2.1 ]hept- required.
N - 2-yl]carbonyl}pentyl)-
0 N \ / thio]quinazolin-4(3H)-
one
1-51 0 3-(3-chlorophenyl)-2-[(1- LRMS m/z (M+H)
\ N Cl N-/o,N {[4-(4-methyl-1,2,5- 553.0 found, 552.2
NS ~N" ~( oxadiazol-3-yl)piperazin- required.
NJ ` 1-yl]carbonyl) pentyl)-
0 thio]quinazolin-4(3H)-
one
1-52 F 3-(3-chlorophenyl)-2-[(1- LRMS m/z (M+H)
0 az~-Icl {[3-(4-fluorophenyl)-1- 620.1 found, 619.2
N oxa-8-azaspiro[4.5]dec- required.
N~S 8-yl]carbonyl}pentyl)-
N 0 thio]quinazolin-4(3H)-
one
0
-54-
CA 02714087 2010-08-04
WO 2009/102537 PCT/US2009/031633
1-53 0 3-(3-chlorophenyl)-2-[(1- LRMS m/z (M+H)
NN CI {[(3S,4S)-3,4- 492.0 found, 492.1
N S difluoropyrrolidin-1- required.
YNO-F yl]carbonyl}pentyl)thio]q
0 uinazolin-4(3H)-one
1-54 CI 3-(3-chlorophenyl)-2- LRMS m/z (M+H)
0 / {[2-(2,8- 563.0 found, 563.2
eNS N diazaspiro[5,5]undec-2- required.
:Nr yl)-1-(1H-imidazol-4-
NH ylmethyl)-2-
oxoethyl]thio} quinazolin
N
-4(3H)-one
N
H
1-55 CI 2-(3-(1H-imidazol-4-yl)- LRMS m/z (M+H)
0 / 1-oxo-1-(2,8- 577.0 found, 577.2
JN > diazaspiro[5.5]undecan- required.
N N~ 2-yl)propan-2-ylthio)-3-
0 - NH (3-chlorophenyl)-8-
methylquinazolin-4(3H)-
N
one
N
1-56 0 3-(3-chlorophenyl)-8- LRMS m/z (M+H)
N CI methyl-2-(1-oxo-1-(4- 561.1 found, 561.2
N phenylpiperazin-l- required.
1N
N yl)hexan-2-
0 ylthio)quinazolin-4(3H)-
one
1-57 0 3-(3-chlorophenyl)-2-(1- LRMS m/z (M+H)
NI C9 (7-(3-methoxyphenyl)- 631.1 found, 631.2
N 2,7-diazaspiro[4.4] required.
N40 nonan-2-yl)-1-oxohexan-
0 2-ylthio)-8-methyl
quinazolin-4(3H)-one
-0
-55-
CA 02714087 2010-08-04
WO 2009/102537 PCT/US2009/031633
1-58 0 3-(3-chlorophenyl)-2-(1- LRMS m/z (M+H)
NO- CI (7-(4-chlorophenyl)-2,7- 635.0 found, 635.2
N diazaspiro[4.4]nonan-2- required.
NN yl)-1-oxohexan-2-ylthio)-
00 8-methylquinazolin-
Ci 4(3H)-one
1-59 0 3-(3-chlorophenyl)-2-(1- LRMS mlz (M+H)
CI (7-(3-chlorophenyl)-2,7- 635.0 found, 635.2
N diazaspiro[4.4]nonan-2- required.
N r/ ON yl)-1-oxohexan-2-ylthio)-
0 ] _\ C 8-methyl quinazolin-
4(3H)-one
SCHEME 2
CO2H
H02C O
N S NH2 PdCJ2(dppf), CsCO3 N S NH2
N THF, MW, 120 C, 1 h N
O 2-1 rf"O 2-3
3'-[2-[(1- { [4-(Amino-carbonyl)piperidin- l -yl] carbonyl }pentyl)thio]..4 .
oxo uinazolin-3 4 - 1 bi hen l-4-carbox lic acid (2-3)
1-(2- { [3 -(3-iodophenyl)-4-oxo-3,4-dihydroquinazolin-2-
yl]thio}hexanoyl)piperidine-4-carboxamide (2-1, 30 mg, 0.05 mmol, 1.0 equiv.),
4-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)- benzoic acid (2-2, 24 mg, 0.1 mmol, 2
equiv.), PdCI2(dppf)
(4 mg, 0.005 mmol, 0.1 equiv.) and 2M aqueous Cs2CO3 (0.15 mL, 0.3 mmol, 6
equiv.) were
suspended in THE (0.4 mL) and the reaction mixture was irradiated in microwave
at 120 C for
60 minutes. The crude mixture was treated with Quadrapure overnight, filtered,
concentrated
and purified with reverse phase HPLC (H20/ CH3CN gradient w/ 0.1 % TFA
present) to yield 3'-
[2-[(1- { [4-(amino-carbonyl)piperidin-1-yl]carbonyl } pentyl)thio]-4-
oxoquinazolin-3 (4H)-
yl]biphenyl-4-carboxylic acid (2-3). LRMS m/z (M+H) 599.1 found, 599.2
required.
-56-
CA 02714087 2010-08-04
WO 2009/102537 PCT/US2009/031633
The compounds shown in Table 2 were synthesized as TA salts according to
Scheme 2.
Table 2
Crisp Structure Name LRMS m/z (M+H)
2-4 1-(2-{[3-(2'- LRMS m/z (M+H)
CI chlorobiphenyl-3-yl)-4- 589.1 found, 589.2
O oxo-3,4- required.
dihydroquinazolin-2-
N NH2
yl]thio}hexanoyl)piperidi
N'S O ne-4-carboxamide
N
O
2-5 CI 1-(2-{[3-(3'- LRMS rn/z (M+H)
chlorobiphenyl-3-yl)-4- 589.1 found, 589.2
oxo-3,4- required.
0
dihydroquinazolin-2-
'N' NH2 yl]thio}hexanoyl)piperidi
NhS O ne-4-carboxamide
O
2-6 C) 1-(2-{[3-(4'- LRMS rn/z (M+H)
chlorobiphenyl-3-yl)-4- 589.1 found, 589.2
oxo-3,4- required.
O dihydroquinazolin-2-
yl]thio}hexanoyl)piperidi
NH2
ne-4-carboxamide
N S O
N
J---I O
2-7 1-(2-{[3-(2'- LRMS m/z (M+H)
NH2 aminobiphenyl-3-yl)-4- 570.2 found, 570.2
0 oxo-3,4- required.
dihydroquinazolin-2-
\ N NH2 yl]thio)hexanoyl)piperidi
NS O ne-4-carboxamide
N
O
-57-
CA 02714087 2010-08-04
WO 2009/102537 PCT/US2009/031633
2-8 NH2 LRMS m/z (M+H)
1 aminobiphenyl-3-yl)-4- 570.2 found, 570.2
oxo-3,4- required.
O /
dihydroquinazolin-2-
N NH2 yl]thio}hexanoyl)piperidi
NS 0 ne-4-carboxamide
J--l 0
2-9 NH2 LRMS m/z (M+H)
aminobiphenyl-3-yl)-4- 570.2 found, 570.2
oxo-3,4- required.
O dihydroquinazolin-2-
yl]thio}hexanoyl)piperidi
N NH2 ne-4-carboxamide
N S 0
J--lYO
------------ -
2-10 N 1-(2-{[4-oxo-3-(3- LRMS m/z (M+H) 11 pyridin-3-ylphenyl)-3,4- 556.2
found, 556.3
O dihydroquinazolin-2- required.
yl]thio}hexanoyl)piperidi
N NH2
ne-4-carboxamide
N S O
J-11YO
2-11 N 1-(2-{[4-oxo-3-(3- LRMS m/z (M+H)
1 pyridin-4-ylphenyl)-3,4- 556.2 found, 556.3
dihydroquinazolin-2- required.
0 /
yl]thio}hexanoyl)piperidi
N NH2 ne-4-earboxamide
N';~ S 0
N
-58-
CA 02714087 2010-08-04
WO 2009/102537 PCT/US2009/031633
2-12 0 3'-[2-[(1-{[4- LRMS m/z (M+H)
OH (arinocarbonyl)piperidin 599.2 found, 599.2
-1- required.
O yl]carbonyl}pentyl)thio]-
4-oxoquinazolin-3(4H)-
NH2
yl]biphenyl-3-carboxylic
N S O acid
J--Il
2-13 0 1-[2-({3-[3-(3- LRMS m/z (M+H)
furyl)phenyl]-4-oxo-3,4- 545.2 found, 545.2
0 dihydroquinazolin-2- required.
N NH yl}thio)hexanoyl]piperidi
2
ne-4-carboxamide
N"S 0
J--,YO
2-14 N_N/ 1-[2-({3-[3-(l-methyl- LRMS m/z (M+H)
1H pyrazol-4-yl)phenyl]- 559.2 found, 559.3
4-oxo-3,4- required.
O
dihydroquinazolin-2-
N NH2 yl}thio)hexanoyl]piperidi
N_;~ S O ne-4-carboxamide
N
O
2-15 1-(2-{[3-(3-cyclohex-l- LRMS m/z (M+H)
en-l -ylphenyl)-4-oxo- 559.2 found, 559.3
O 3,4-dihydroquinazolin-2- required.
yl]thio}hexanoyl)piperidi
N NH2
ne-4-carboxamide
~ N~S O
N
3
O
59-
CA 02714087 2010-08-04
WO 2009/102537 PCT/US2009/031633
2-16 S LRMS na/z (M+H)
1-(2-({4-oxo-3-[3-(2- 561.1 found, 561.2
0 / thienyl)phenyl]W3,4- required.
N NH2 dihydroquinazolin-2-
N" ';~ S 0 yl)thio)hexanoyl]piperidi
N ne-4-carboxamide
O
J--l
2-17 0 2-[(1-{[7-(3- LRMS rn/z (M+H)
chlorophenyl)-2,7- 653.3 found, 653.3
0 diazaspiro[4.4]non-2- required.
N yl] carbonyl } -3-
/
N~S methylbutyl)thio]-3-[3-
N4 (3-
N furyl)phenyl]quinazolin-
O
4(3H)-one
CI
2-18 2-[(1-{[7-(3- LRMS rn/z (M+H)
/ chlorophenyl)-2,7- 667.3 found, 667.3
0 / diazaspiro[4.4]non-2- required.
yl]carbonyl}-3-
methylbutyl)thio]-3-(3-
N S cyclohex-l -en-1-
N
ylphenyl)quinazol in-
O 4(3H)-one
CI
2-19 F 1-(2- { [3-(4'-fluoro-2'- LRMS m/z (M+H)
methylbiphenyl-3-yl)-4- 587.3 found, 587.3
oxo-3,4- required.
0 dihydroquinazolin-2-
yl]thio } hexanoyl)piperidi
/ I \ NH2
ne-4-carboxamide
N S O
N
J--lYO
-60-
CA 02714087 2010-08-04
WO 2009/102537 PCT/US2009/031633
2-20 0 / 2-[(1-{[5-(4- LRMS m/z (M+x)
N fluorophenyl)-2,5- 609.3 found, 609.3
N~S -- O diazabicyclo[2.2.1]hept- required.
2-
J yl]carbonyl}pentyl)thio]-
o 3[3(3
-
furyl)phenyl ] quinazolin-
4 3 -one
2-21 0 2-[(1-([3-(4- LRMS m/z (M+H)
fluorobenzyl)-2-oxo-1- 680.3 found, 680.3
0 oxa-8-azaspiro[4.5]dec- required.
N 0 8-
NS O yl]carbonyl}pentyl)thio]-
N 3-[3-(3-
furyl)phenyl]quinazolin-
F 4(3.H)-one
2-22 0 / 3-(3-cyclohex-l-en-1- LRMS m1z (M+H)
N ylphenyl)-2-[(1-{[5-(4- 623.3 found, 623.3
NS fluorophenyl)-2,5- required.
N--\ diazabicyelo[2.2.1]hept-
N 2-
o
yl] carbonyl } pentyl)thio] q
uinazolin-4 3 -one
2-23 3-(3-cyclohex-l-en-1- LRMS m/z (M+H)
ylphenyl)-2-[(1-{[3-(4- 694.3 found, 694.3
0 fluorobenzyl)-2-oxo-1- required.
N O oxa-8-azaspiro[4.5]dec-
O 8-
N" yl] carbonyl } pentyl)thio]q
N uinazolin-4(3H)-one
O
F
61-
CA 02714087 2010-08-04
WO 2009/102537 PCT/US2009/031633
2-24 3-(3-cyclohex-l-en-1- LRMS m/z (M+H)
/ ylphenyl)-2-[(1-{[4-(2,4- 629.3 found, 629.3
O difluorophenyl)piperazin- required.
~
\ \ yl]carbonyl}pentyl)thio]q
N S N uinazolin-4(3H)-one
O
2-25 / O 2-[(1-{[4-(2,4- LRMS m/z (M+H)
difluorophenyl)piperazin- 615.2 found, 614.3
O 1- required.
F yl]carbonyl}pentyl)thio]-
N
NS N 3-[3-(3-
F furyl)phenyl]quinazolin-
4(3H)-one
O
2-26 3-(3-cyclohex-l-en-1- LRMS m/z (M+H)
ylphenyl)-2-{[1-(2,8- 585.4 found, 585.3
0 diazaspiro[5.5]undec-2- required.
ylcarbonyl)pentyl]thio}q
\ N uinazolin-4(3R)-one
N-5~ S
O nN
H
-62-
CA 02714087 2010-08-04
WO 2009/102537 PCT/US2009/031633
SCHEME 3
Q - o
O \ Toluene PC13, Br2
HZNOH I / O reflex, 16h OH
3-1 3-2 Q O 3-3
O I
~ / O O O \ I Et375GMP I N \ Cf
N C[ N Q
N~~ \ I r
O Br OH NSH N
3 4 1-3 3- HO
O a~--Cl O
N CI
QQ16Q6 N Q \ I
HN NBOC 1. N2H4, EtOH I
N N S
PyBOP, Et3N, O 2. Tf=A, DGM N f-~f DM F, rt N O BOC
I 3-HZN O H
3-7
4- (1,3 -dioxo- 1 3-dih dro-2H-isoindol-2- 1 butanoic acid (3-3)
4-Aminobutanoic acid (3-1, 2 g, 19 mmol, 1.0 equiv.) and phthalie anhydride (3-
2, 2.87 g, 19 mmol, 1.0 eq.) were placed in a round bottomed flask equipped
with a condenser.
Toluene was added (39 mL) and the reaction mixture was heated to reflex until
disappearance of
the starting material. The solvent was removed under reduced pressure to yield
4-(1,3-dioxo-1,3-
dihydro-2.--I isoindol-2-yl)butanoic acid (3-3).
2-bromo-4- (1,3-dioxo-1 3-dihdro-2H-isoindol-2- 1 butanoic acid 3-4
4-(1,3-dioxo-1,3-dihydro-2H isoindol-2-yl)butanoic acid (3-3, 4g, 17 mmol),
bromine (3g, 18.9 mmol, 1.1 eq) and PC13 (236 mg, 1.7 mmol, 0.1 eq.) were
placed in a round
bottomed flask equipped with a reflux condenser, and the mixture was heated at
70 C until
complete disappearance of starting material. The volatiles were evaporated and
the product was
carried forward without purification. LRMS m/z (M+H) 313.9 found, 313.0
required.
2- {3-(3-chlorophenyl)-8-methyl-4-oxo-3,4-dihydroquinazolin-2-ylthio}- 4-( 1,3-
dioxo-1 3-dih dro-2- H-isoindol-2- 1 butanoic acid (3-5)
3-(3-chlorophenyl)-2-mercapto-8-methylquinazolin-4(3H)-one (1-3) was prepared
as described before. 3-(3-chlorophenyl)-2-mercapto-8-methylquinazolin-4(3H)-
one (1-3,
211 mg, 0.70 mmol, 1.0 equiv), 2-bromo-4-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-
yl)butanoic
acid (3-4, 284 mg, 0.91 mmol, 1.3 equiv.), and triethylamine (588 L, 4.2
mmol, 6 equiv.) were
dissolved in anhydrous DMF (1 mL) and stirred at 75 C. When LCMS indicated
the reaction
was complete, the crude reaction mixture was partitioned between ethyl acetate
and 0.1 M HCI,
-63-
CA 02714087 2010-08-04
WO 2009/102537 PCT/US2009/031633
the aqueous phase drained and the organic layer washed twice more with 0.1 M
HCl followed by
brine to yield the product (3-5). LRMS m/z (M+H) 533.8 found, 533.1 required.
tent-butyl 8-[2- {[3-(3-chlorophenyl)-8-methyl-4-oxo-3,4-dihydroquinazolin-2-
yl]thio}-4-(l,3-dioxo-1,3-dihydro--2H isoindol-2-yl)butanoyl]-2,8-
diazas iro 5.5 undecane-2-carbox late 3-6
2-{[3-(3-chlorophenyl)-8-methyl-4-oxo-3,4-dihydroquinazolin-2-yl]thio}- 4-(1,3-
dioxo-1,3-dihydro-2- H-isoindol-2-yl)butanoic acid (3-5, 331 mg, 0.43 minol,
1.0 equiv.), tert-
butyl 2,8-diazaspiro[5.5]undecane-2-carboxylate (1-6, 220 mg, 0.864 mmol, 2
equiv.), PyBOP
(337 mg, 0.648 mmol, 1.5 equiv.) and triethylamine (0.6 mL, 4.32 mmol, 10
equiv.) were
dissolved in DMF (1 mL) and stirred at it Once LCMS showed reaction was
complete, the
reaction mixture was filtered and purified with reverse phase HPLC (H201 CH3CN
gradient w/
0A% TFA present) to yield the product. LRMS m/z (M+H) 770.0 found, 770.3
required.
tert-Butyl 8-(4-amino-2- { [3-(3-chlorophenyl)-8-methyl-4-oxo-3,4-
dihydroquinazolin-2-yl]thio}butanoyl)-2,8-diazaspiro[ 5.5]undecane-2-
carbox late
tert-Butyl 8-[2- {[3-(3-chlorophenyl)-8-methyl-4-oxo-3,4-dihydroquinazolin-2-
yl]thio}-4-(1,3-dioxo-1,3-dihydro-21"J"isoindol-2-yl)butanoyl]-2,8-
diazaspiro[5.5]undecane-2-
carboxylate (3-6, 90 mg, 0.117 rmnol, 1.0 equiv.) was dissolved in ethanol
(0.585 mL). Excess
IM hydrazine in THE (20 eq) was added and the mixture was stirred at rt. Once
LCMS showed
reaction was complete, the reaction mixture was concentrated, dissolved in
DMSO and purified
with reverse phase HPLC (H20/ CH3CN gradient w/ 0.1 % TFA present) to yield
the product.
LRMS m/z (M+H) 640.0 found, 640.2 required
2-1[3 -amino- 1-(2,8-diazaspiro[5.5 ]undec-2-ylcarbonyl)propyl]thio} -3-(3-
chlorohen l uinazolin-4 3I -one (3-7)
tert-Butyl 8-(4-amino-2-{[3-(3-chlorophenyl)-8-methyl-4-oxo-3,4-
dihydroquinazolin-2-yl]thio}butanoyl)-2,8-diazaspiro[5.5]undecane-2-
carboxylate (128 mg, 0.2
mmol, 1.0 equiv.) was dissolved in DCM (lmL). Trifluoroacetic acid (0.5 mL)
was added and
the mixture stirred at room temperature . Once LCMS showed reaction was
complete, toluene
was added, the solvent and TFA were removed at reduced pressure and the
resulting crude was
filtered and purified with reverse phase HPLC (H20/ CH3CN gradient w/ 0.1 %
TFA present) to
yield the product (3-7). 'H NMR (500 MHz, CDC13) b 8.13 (br s, 2H), 8.09 (d,
1H, J= 8.0 Hz),
7.63 (t, 1H, J= 7.0 Hz), 7.48 (m, 2H), 7.32 (m, 3H), 3.85 (m, 1H), 3.27 (m,
2H), 3.07 (m, 4H),
2.88 (br in, 6H), 2.55 (s, 3H), 2.36 (m, 2H), 1.88 (m, 1H), 1.64 (m, 5H), 1.37
(d, 1H, J= 10.5
Hz). LRMS nz/z (M+H) 539.9 found, 540.2 required.
The compounds shown in Table 3 were synthesized as TFA salts according to
Scheme 3.
-64-
CA 02714087 2010-08-04
WO 2009/102537 PCT/US2009/031633
Table 3
3-8 0 2-{[4-amino-1-(2,8- LRMS m/z (M+H)
N Cl diazaspiro[5.5]undec-2- 554.1 found, 554.2
N S ylcarbonyl)butyl]thio}-3- required.
N NH (3-chlorophenyl)-8-
methylquinazolin-4(3H)-
N H2 one
3.9 0 2-{[5-amino-l-(2,8- LRMS m/z (M+H)
01( AN CI diazaspiro[5.5]undec-2- 554.1 found, 554.3
N S ylcarbonyl)pentyl]thio) - required.
N NH 3-(3-
0 chlorophenyl)quinazolin-
4(3H)-one
HN
3-10 Cf 2-[(4-amino-1-{[7-(3- LRMS m/z (M+H)
0 / chlorophenyl)-2,7- 622.0 found, 622.2
I diazaspiro[4.4]non-2- required.
N
yl]carbonyl}butyl)thio]-
N N 3_(3--9 H2N p chlorophenyl)quinazolin-
CI 4(3H)-one
3-11 CI 2-(5-amino-l-(7-(3- LRMS rnlz (M+H)
0 / I chlorophenyl)-2,7- 636.0 found, 636.2
N diazaspiro[4.4]nonan-2- required.
N~ S yl)-1-ox.opentan-2-
NrN ylthio)-3-(3-
H2N o chlorophenyl)-8-
Cl nnethylquinazolin-4(3H)-
one
-65-
CA 02714087 2010-08-04
WO 2009/102537 PCT/US2009/031633
SCHEME 4
O I /
N Cf 0
C~A (NOSH 4-2 N CI HN N CI
DMF, 60C 4 4
CT)OH / N S N3
4-1 Br 2. NaN3, DMF, 60C 4-3 HO PySOP, Et3N,
DMF, rt
0
o o ~
~ CI PPh3, Tol, rt N~ ct
N" S N S
N CIN
N3 N NHa N
0
45 0 4-6
6-azido-2- {[3-(3-chlorophenyl)-4-oxo-3,4-dihydroquinazolin-2-yl]thio}hexanoic
acid (4-3)
2,6-Dibromoxanoic acid (4-1, 300 mg, 1.0 mmol, 1.0 equiv.) and 2-(3-
chlorophenyl)-2,7-diazaspiro[4.4]nonane (4-4, mg, 0.9 mmal, 0.9 eq.) were
placed in a. round
bottomed flask. DMF was added (20 mL) and the reaction mixture was heated to
40 C until
disappearance of the starting material, at which point excess of NaN3 was
added. Stirring was
continued until completion of the reaction as judged by LC-MS. The reaction
mixture was
carefully partitioned between Et20 and a 0.1N aqueous solution ofHC1, the
organic solvent
separated, dried and removed under reduced pressure. The crude was purified
with reverse phase
HPLC (H20/ CH3CN gradient w/ 0.1% TFA present) to yield the product. LRMS rn/z
(M+H)
770.0 found, 770.3 required. to yield 6-azido-2-{[3-(3-chlorophenyl)-4-oxo-3,4-
dihydroquinazolin-2-yljthio}hexanoic acid (4-3).
2-[(5-azido-l - { [7-(3-chlorophenyl)-2,7-diazaspiro [4.4]non-2-
1 carbon 1 ent 1 thin -3- 3-chloro hen 1 uinazolin-4 3 -one (4-5)
6-Azido-2- {[3-(3-chlorophenyl)-4-oxo-3,4-dihydroquinazolin-2-yl]thio}hexanoic
acid (4-3, 15 mg, 0.034 mmol, 1.0 equiv.), tent-butyl 2,8-
diazaspiro[5.5]undecane-2-carboxylate
(4-4, 9 mg, 0.864 nimol, 1.2 equiv.), PyBOP (21 mg, 0.041 mmol, 1.2 equiv.)
and triethylamine
(0.05 mL, 0.34 mmol, 10 equiv.) were dissolved in DMF (0.2 mL) and stirred at
rt. Once LCMS
showed reaction was complete, the reaction mixture was filtered and purified
with reverse phase
HPLC (H20/ CH3CN gradient w/ 0.1 % TFA present) to yield the product. LRMS m/z
(M+H)
662.1 found, 662.2 required.
2-[(5-amino-l- { [7-(3-chlorophenyl)-2,7-diazaspiro[4.4]non-2-
lcarbon 1 ent 1 thio -3- 3-chloro hen 1 uinazolin-4 3 -one 4-6
To 2-[(5-azido-l-{[7-(3-chlorophenyl)-2,7-diazaspiro[4.4]non-2-
yl]carbonyl }pentyl)thio]-3-(3-chlorophenyl)quinazolin-4(3FI)-one-(10 mg,
0.016 mmol,
1.0 equiv.) was added a 1M solution of PMe3 in toluene and the mixture stirred
at room
-66-
CA 02714087 2010-08-04
WO 2009/102537 PCT/US2009/031633
temperature. Once LCMS showed reaction was complete, the volatiles were
removed at reduced
pressure and the resulting crude was purified with reverse phase HPLC (H20/
CH3CN gradient
w/ 0.1% TFA present) to yield the product. LRMS m/z (M+H) 636.0 found, 636.2
required.
The compound shown in Table 4 was synthesized as a TFA salt according to
Scheme 4.
Table 4
4-7 0 2-{[5-amino-l-(2,8- LRMS m/z (M+H)
N CI diazaspiro[5.5]undec-2- 568.1 found, 568.2
N,
S ylcarbonyl)pentyl]thio}- required.
N NH 3-(3-chlorophenyl)-8-
0 methylquinazolin-4(3H)-
one
HN
EXAMPLES 1-8
Examples are provided below to further illustrate different features and
advantages of the present invention. The examples also illustrate useful
methodology for
practicing the invention. These examples do not limit the claimed invention.
EXAMPLE 1: Identification of CHKlsvl Using Real-time PCR
To facilitate the determination of compound inhibitory properties, it is
desirable to
identify variants of the "normal" splicing of exon regions encoding CHKI. In
particular,
naturally occurring splicing variations resulting in the loss of the C-
terminal regulatory domain
of CHKI were sought. Deletion of the C-terminus confers greater kinase
activity to CHKI
(Chen et al., 2000, Cell 100:681-692; Katsuragi and Sagata, 2004, Mol. Biol.
Cell. 15:1680-
1689). Exons 2-8 encode the catalytic kinase domain and exon 9 encodes the
linker region. The
SQ and C-terminal regulatory domains lie within exons 10-13 (Sanchez et al.,
1997, 277:1497-
1501; Katsuragi and Sagata, 2004, Mol. Biol. Cell. 15:1680-1689). Real-time
PCR experiments
and RT-PCR have been used to identify and confirm the presence of novel splice
variants of
human CHKI mRNA. A naturally occurring splice variant which encodes a C-
terminal
truncation of the CHKI inhibitory domain was identified, cloned, expressed and
purified for use
in a CHKI kinase assay of utility for the determination of compound inhibitory
properties.
RT-PCR
The structure of CHKI mRNA in the region corresponding to exons 8 to I I was
determined for RNA extracted from human testis using an RT-PCR based assay.
Total RNA
isolated from human testis was obtained from BD Biosciences Clontech (Palo
Alto, CA). RT-
PCR primers were selected that were complementary to sequences in exon 8 and
exon I I of the
reference exon coding sequences in CHKI (NM_001274). Based upon the nucleotide
sequence
of CHKI mRNA, the CHKI exon 8 and exon I I primer set (hereafter CHKI&_~1
primer set) was
-67-
CA 02714087 2010-08-04
WO 2009/102537 PCT/US2009/031633
expected to amplify a 478 base pair amplicon representing the "reference" CHKI
mRNA region.
The CHK1$_11 primer set was expected to amplify a 300 base pair amplicon in a
transcript that
possessed alternative splicing of exon 9 to exon 11. The CHK1 exon 8 forward
primer has the
sequence: 5' ATCAGCAAGAATTACCATTCCAGACATC 3' (SEQ ID NO 1); and the CHKI
exon 1 I reverse primer has the sequence: 5' CATACAACI T'"tTCTTCCATTGATAGCCC
3'
(SEQ ID NO 2).
Total RNA from human testis was subjected to a one-step reverse transcription-
PCR amplification protocol using the Qiagen, Inc. (Valencia, CA), One-Step RT-
PCR kit, using
the following cycling conditions:
1) 50 C for 30 minutes;
2) 95 C for 15 minutes;
3) 35 cycles of
94 C for 30 seconds;
63.5 C for 40 seconds;
72 C for 50 seconds; then
72 C for 10 minutes.
RT-PCR amplification products (amplicons) were size fractionated on a 2%
agarose gel. Selected fragments representing 250 to 350 base pair amplicons
were manually
extracted from the gel and purified with a Qiagen Gel Extraction Kit. The
purified amplicon
fragments were reamplified with the CHKI8.11 primer set, and these amplicons
were size
fractionated on an agarose gel. Fragments representing 250 to 350 base pair
amplicons were
manually extracted from the gel and purified with a Qiagen Gel Extraction Kit.
The purified
amplicon fragments were reamplified with the CHK18_11 primer set once more.
Following size
fractionation on an agarose gel and manual extraction of the 250 to 350 base
pair amplicons, the
purified amplicon fragments (Qiagen Gel Extraction Kit) were cloned into an
Invitrogen pCR2.1
vector using the reagents and instructions provided with the TOPO TA cloning
kit (Invitrogen,
Carlsbad, CA). Clones were then plated in pools of 440 colonies per plate,
onto 15 plates, for a
total of 6600 clones. DNA was extracted from the pooled 440 colonies from each
plate and used
as template for real-time PCR.
Real-time PCR/TAQman
To determine the presence of an alternatively spliced isoform to the CHKI
reference protein (NP_001265), a real-time PCR assay was used.
TAQman primers and probes used to detect the CHKIsv1 isoform were designed
and synthesized as pre-set mixtures (Applied Biosystems, Foster City, CA). The
sequences of
the TAQman primers and probes used to detect the CHKI reference form (SEQ ID
NOs 3, 4, and
5) and CHK1sv1 isoform (SEQ ID NOs 6, 7, and 8) are shown in Table 1. Splice
junction
specific probes were labeled with the 6-FAM fluorophore at the 5' end (FAM)
and a non-
fluorescent quencher at the 3' end (NFQ). Real-time PCR was performed on human
testis eDNA
-68-
CA 02714087 2010-08-04
WO 2009/102537 PCT/US2009/031633
using the TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City,
CA). The
TAQman reaction contained:
96-well format 384-well format
12.5 l 5 p.l TAQman Universal MasterMix
1.25 p.l 0.5 p.l Primer-probe mix
6.25 p.l 2.5 .d H2O
5 p l 2111 DNA
Table 1. Primers and probes used to detect CHKI isoforms.
Name SEQ ID Sequence Specificity
NO
CHKI reference SEQ ID NO GTTACTTGGCACCCCAGGA CHKI
forward primer 3 reference
CHKI reference SEQ ID NO CHKI
reverse primer 4 CATCCAATTTGGTAAAGAATCGTGTCA reference
CHKI reference probe SEQ ID NO FAM-TCCTCACAGAACCCC-NFQ CHK1
5 reference
CHK1svl forward SEQ ID NO GCACATTCAATCCAATTTGGACTTCT CHKI svl
primer 6
CHK1svI reverse SEQ ID NO CATCCAATTTGGTAAAGAATCGTGTCAT CHKlsvl
primer 7
CHKlsv1 probe SEQ IDNO FAM-CAGTGCTTCTAGAACCC-NFQ CHKIsv1
8
The TAQman reactions were performed on an ABI Prism 7900HT Sequence
Detection System (Applied Biosystems, Foster City, CA). The thermocycling
conditions were
50 C for 2 minutes, 95 C for 10 minutes, and 40 cycles of 95 C for 15 seconds
and 60 C for
I minute. Data analysis of the fluorescence emission was performed by the
Sequence Detector
Software (SDS) (Applied Biosystems, Foster City, CA).
Results of the TAQman assay indicated that pooled DNA from 13 out of 15 plates
appeared to possess clones that represented an alternative exon 9 to exon 1 I
splice junction.
DNA from one of these positive pools, representing 440 colonies, was used to
transform bacterial
host cells. Clones were plated in pools of 55 colonies per plate onto 12
plates total. The
colonies on each of the 12 plates were again pooled and used for a TAQman
assay. Pooled DNA
from I out of 12 plates appeared to possess a clone that represented an
alternative exon 9 to
exon 1 I splice junction. The 55 colonies on this positive plate were
individually screened using
a TAQman assay, and one clone was identified as possessing an alternative exon
9 to exon 1 I
splice junction. This positive clone was then sequenced from each end using
the CHKI exon 8
forward primer (SEQ ID NO 1) and a different exon 1 I reverse primer with the
sequence
5' TGCATCCAATTTGGTAAAGAATCG 3' (SEQ ID NO 9).
-69-
CA 02714087 2010-08-04
WO 2009/102537 PCT/US2009/031633
Sequence analysis of the clone revealed that it matched the expected sequence
for
alternative splicing of exon 9 of the CHK1 heteronuclear RNA to exon 11; that
is the coding
sequence of exon 10 is completely absent.
EXAMPLE 2: Cloning of CHKIsvl
Real-time PCR, RT-PCR, and sequencing data indicate that in addition to the
normal CHKI reference mRNA sequence, NM_001274, encoding CHKI protein,
NP_001265, a
novel splice variant form of CHKI mRNA also exist in testis tissue and MOLT-4,
and Daudi cell
lines.
Clones having a nucleotide sequence comprising the CHKI svl splice variant
identified in Example 1 were isolated using recombination-mediated plasmid
construction in
yeast. A set of two primer pairs was used to amplify and clone the entire mRNA
coding
sequences of CHK]svl. In the case of CHKI svl , real-time quantitative PCR
analysis indicated
that transcripts of this splice variant form were present at very low levels.
In order to clone
CHKIsv1, clones containing coding sequences of the reference CHKI (NM__001274)
were
altered by an additional recombination step in yeast with 80 base pair linkers
that were designed
to create the desired exon 9 to exon i 1 splice junction.
A 5' "forward" primer and a 3' "reverse" primer were designed for isolation of
full length clones corresponding to CHKIsvI. The 5' "forward" CHKIsv1 primer
was designed
to have the nucleotide sequence of 5' TTACTGGCTTATCGAAATTAATACGACTCACTATAG
GGAGGAGTCATGGCAGTGCCCTTTGT 3' (SEQ ID NO 10) and to have sequences
complementary to exon 2 of the CHKI mRNA (NM_001274). The 3' "reverse" CHK1svl
primer was designed to have the nucleotide sequence of 5' TA GAA GGCA CA GTCGA
GGCTGA
TCAGCGGGTTTAAACTCATGCATCCAATTTGGTAAAGAATCG 3' (SEQ ID NO 11) and to
have sequences complementary to exon 11 of the CHKI mRNA (NM_001274). The
40 nucleotides at the 5' ends of the primer sequences indicated in italics are
"tails" that were
incorporated into the PCR amplicons and facilitated subsequent plasmid
recombination events in
yeast. These CHKIsvl "forward" and "reverse" primers were expected to amplify
coding
sequences of the reference CHKI mRNA (NM_001274), which was then used in a
subsequent
recombinational cloning step to create CIKIsvl-specific sequence.
RT-PCR
The CI-IK1svI cDNA sequence was cloned using a combination of reverse
transcription (RT) and polymerase chain reaction (PCR). More specifically,
about 25 ng of
MOLT-4 cell line mRNA (BD Biosciences Clontech, Palo Alto, CA) was reverse
transcribed
using Superscript 11(Gibco/Invitrogen, Carlsbad, CA) and oligo d(T) primer
(RESGEN/hnvitrogen, Huntsville, AL) according to the Superscript Il
manufacturer's
instructions. For PCR, I l of the completed RT reaction was added to 40 it of
water, 5 l of
10X buffer, 1 j l of dNTPs and 1 l of enzyme from a Clontech (Palo Alto, CA)
Advantage 2
PCR kit. PCR was done in a Gene Amp PCR System 9700 (Applied Biosystems,
Foster City,
-70-
CA 02714087 2010-08-04
WO 2009/102537 PCT/US2009/031633
CA) using the CHKIsvI "forward" and "reverse" primers for CHKIsvI (SEQ ID NOs
10,11).
After an initial 94 C denaturation of 1 minute, 35 cycles of amplification
were performed using a
30 second denaturation at 94 C followed by a 40 second annealing at 63.5 C and
a 50 second
synthesis at 72 C. The 35 cycles of PCR were followed by a 10 minute extension
at 72 C. The
50 l reaction was then chilled to 4 C. 10 t1 of the resulting reaction
product was run on a
1% agarose (Invitrogen, Ultra pure)'gel stained with 0.3 q,g/ml ethidium
bromide (Fisher
Biotech, Fair Lawn, NJ). Nucleic acid bands in the gel were visualized and
photographed on a
UV light box to determine if the PCR had yielded products of the expected
size, in the case of
the CHKI mRNA, a product of about 1243 base pairs. The remainder of the 50 gl
PCR reactions
from MOLT-4 cells was purified using the QlAquik Gel extraction Kit (Qiagen,
Valencia, CA)
following the QlAquik PCR Purification Protocol provided with the kit. About
50 111 of product
obtained from the purification protocol was concentrated to about 6 PI by
drying in a Speed Vac
Plus (SC1 IOA, from Savant, Holbrook, NY) attached to a Universal Vacuum
System 400 (also
from Savant) for about 30 minutes on medium heat.
Cloning and assembly of CHKI svI full-length clones and yeast transformation
Assembly of the full length CI-IKIsvI clone by homologous recombination
cloning in yeast was performed using a cycloheximide-based counterselection
scheme similar to
that described previously by Raymond et al. (2002, Genome Res. 12:190-197).
Assembly of the full-length CHKI svl full length clone by homologous
recombination between the 1243 base pair CHKI amplicon, produced using the
CHKI svl
forward and reverse "tailed" primers described earlier, and the expression
vector was performed
by simultaneous transformation of these pieces into yeast cells. A subsequent
recombination step
with 80 base pair oligonucleotide linkers created the CHKI sv1 exon 9 to exon
I 1 splice junction.
All yeast transformation steps described in subsequent paragraphs were
performed by
electroporation (Raymond et al., 2002 Genome Res. 12:190-197).
1 g of the 1243 base pair CHKI purified amplicon was cloned directly into
100 ng of Srf1-digested pCMR1I by cotransformation of 100 p.1 of yeast strain
CMY1-5 (Mata,
URA3A, CYH2R). Ura , cycloheximide resistant colonies were selected on Ura-
deficient media
plates containing 1 g/ml cycloheximide (Sigma, St. Louis, MO). Standard yeast
media were
used (Sherman, 1991, Methods Enzymol. 194:3-21). Total DNA from yeast cell
culture
containing the CHKI clone was used to transform E. coli to chloramphenicol
(Sigma, St. Louis,
MO) resistance to prepare a large quantity of the recombinant plasmid as
described in Hoffman
and Winston (1987 Gene 57:267-72). The colonies were picked from the plates
into 2 ml of 2X
LB media. These liquid cultures were incubated overnight at 37 C. Plasmid DNA
was extracted
from these cultures using the Qiagen (Valencia, CA) Qiaquik Spin Miniprep kit.
-71-
CA 02714087 2010-08-04
WO 2009/102537 PCT/US2009/031633
Table 2. Composition pCMRII_plasmid
Nucleotide Functional description of sequence
coordinates
1 - 6013 Copy-controlTM E. coli origin of replication from pCCIFOS
(Epicentre Technologies, Madison, WI).
6014 - 7884 Yeast URA3 gene, ARS4 autonomously replicating sequence and
CEN6 centromere from pRS316 (Sikorski and Hieter, 1989).
7885 - 8825 Mammalian CMV promoter from InVitrogen (Carlsbad, CA) vector
pcDNA3.1lmyc-HIS A.
8826 - 10,774 Yeast CYH2 gene amplified from strain BY4709 (Brachmann et al.
1998)
10,775 - 10,782 Engineered Srfl restriction site.
10,783 - 13,556 Mammalian poly-adenylation sites, selectable markers, SV40
origin,
etc. from pcDNA3.1/myc-HIS A.
13,557 - 13,596 DNA sequence from InVitrogen vector pENTR11.
13,597 - 14,561 pCMRI I - specific; chloramphenicol resistance gene from
pCCIFOS.
To construct the CHKlsvl clone, 1 g of 80 base pair linkers shown in Table 3
(SEQ ID NOs 12, 13) that spans the region of the alternative splicing of exon
9 to exon 11, and
100 ng of BamHI-digested CHKIIpCMR11 clone were used to cotransform. 100 l of
a
cycloheximide sensitive yeast strain. The overlapping DNA between the linkers
and
CHKI/pCMR1I clone dictates that most yeast transformants will possess the
correctly assembled
construct. Ura , cycloheximide resistant colonies were selected for subsequent
preparation and
transformation of E. coli. Plasmid DNA extracted from E. coli was analyzed by
restriction
digest to confirm the presence of the alternative splicing of exon 9 to exon
11 in the CHKIsvJ
clone. Eight CHKIsvI clones were sequenced to confirm identity, and the clones
possessing the
appropriate sequences are used for protein expression in multiple systems.
Table 3. Linkers used to create exon 9 to exon 11 splice junction for CHKIsvI
clone
SEQ ID NO Linker Sequence_
SEQ ID NO 12 AATCCAATTTGGACTTCTCTCCAGTAAACAGTGCTTCTAGAACC
CCTGGCAGCGGTTGGTCAAAAGAATGACACGATTCT
SEQ ID NO 13 AGAATCGTGTCATTCT ITGACCAACCGCTGCCAGGGGTTCTAG
AAGCACTGTTTACTGGAGAGAAGTCCAAATTGGATT
-72-
CA 02714087 2010-08-04
WO 2009/102537 PCT/US2009/031633
Summa of CHK1svl of ucleotide
The polynucleotide coding sequence of CHK1 sv1 mRNA (Seq ID NO 14)
contains an open reading frame that encodes a CHK1 svl protein (SEQ ID NO 15)
similar to the
reference CHK1 protein (NP_001265), but lacking amino acids encoded by a 178
base pair
region corresponding to exons 10 of the full length coding sequence of
reference CHKI mRNA
(NM_001274). The deletion of the 178 base pair region results in a shift of
the protein
translation reading frame in comparison to the reference CHKI protein reading
frame, creating a
carboxy terminal peptide region that is unique to CHKI svl (italicized in Seq
ID NO 15). The
frameshift also creates a premature termination codon 29 nucleotides
downstream of the
exon 9/exon 11 splice junction. Therefore, the CHKlsvl protein is missing an
internal 59 amino
acid region corresponding to the amino acid region encoded by exon 10 and is
also lacking the
amino acids encoded by the nucleotides downstream of the premature stop codon
as compared to
the reference CHK1 (NP 001265). Exon 10 encodes the SQ/TQ domains of CHK1, and
exons 11-13 encode the autoinhibitory region (Sanchez et al., 1997, Science
277:1497-1501;
Katsuragi and Sagata, 2004, Mol. Biol. Cell. 15:1680-1689). While deletion of
the autoinhibitory
region confers constitutive activity to the CHK1 kinase domain, when the SQ/TQ
domains are
also removed, CHKI enzymatic activity decreases (Ng et al., 2004, J. Biol.
Chem. 279:8808-
8819).
Table 4. Nucleotide coding sequence and coded of e tide for CHKIsvI
Seq ID ATGGCAGTGCCCTTTGTGGAAGACTGGGACTTGGTGCAAACCCTGGG
NO 14 AGAAGGTGCCTATGGAGAAGTTCAACTTGCTGTGAATAGAGTAACTG
AAGAAGCAGTCGCAGTGAAGATTGTAGATATGAAGCGTGCCGTAGAC
TGTCCAGAAAATATTAAGAAAGAGATCTGTATCAATAAAATGCTAAA
TCATGAAAATGTAGTAAAATTCTATGGTCACAGGAGAGAAGGCAATA
TCCAATATTTATTTCTGGAGTACTGTAGTGGAGGAGAGCTTTTTGACA
GAATAGAGCCAGACATAGGCATGCCTGAACCAGATGCTCAGAGATTC
TTCCATCAACTCATGGCAGGGGTGGTTTATCTGCATGGTATTGGAATA
ACTCACAGGGATATTAAACCAGAAAATCTTCTGTTGGATGAAAGGGA
TAACCTCAAA.ATCTCAGACTTTGGCTTGGCAACAGTATTTCGGTATAA
TAATCGTGAGCGTTTGTTGAACAAGATGTGTGGTACTTTACCATATGT
TGCTCCAGAACTTCTGAAGAGAAGAGAATTTCATGCAGAACCAGTTG
ATGTTTGGTCCTGTGGAATAGTACTTACTGCAATGCTCGCTGGAGAAT
TGCCATGGGACCAACCCAGTGACAGCTGTCAGGAGTATTCTGACTGG
AAAGA CATACCTCAACCCTTGGAAAAAAATCGATTCTGC
TCCTCTAGCTCTGCTGCATAAAATCTTAGTTGAGAATCCATCAGCAAG
AATTACCATTCCAGACATCAAAAAAGATAGATGGTACAACAAACCCC
TCAAGAAAGGGGCAA.AAAGGCCCCGAGTCACTTCAGGTGGTGTGTCA
GAGTCTCCCAGTGGATTT T'CTAAGCACATTCAATCCAATTTGGACTTCT
-73-
CA 02714087 2010-08-04
WO 2009/102537 PCT/US2009/031633
CTCCAGTAAACAGTGCTTCTAGAACCCCTGGCAGCGGTTGGTCAAAAG
AATGA
Seq ID MAVPFVEDWDLVQTLGEGAYGEVQLAVNRVTEEAVAVKIVDMK-RA.VD
NO15 CPENIKKEICINKMLNHENVVKFYGHRREGNIQYLFLEYCSGGELFDRIEP
DIGMPEPDAQRFFHQLMAGV VYLHGIGITHRDIKKPENLLLDERDNLKISDF
GLATVFRYNNRERLLNKMCGTLPYVAPELLKRREFHAEPVDVWSCGIVL
TAMLAGELP W DQP SD S CQEYSD WKEKKTYLNP WKKIDSAPLALLHKILV
ENP SARIT1PDIKKDRWYNKPLKKGAK.RPRVTS GGV SESP S GFSKHIQSNL
DFSPVNSASR TPGSGWSKE
EXAMPLE 3: Expression of CHK1svI Protein
The baculovirus gene expression vector system permits protein expression
insect
cells, which are inexpensive and easy to maintain. The proteins produced are
of similar quality
to that in mammalian cells (Miller, 1988, Biotechnology 10:457-465; Miller,
1989, Bioassays
11:91-95). Methods of protein expression using the baculovirus expression
vectors in insect
cells are known in the art and techniques are discussed in O'Reilly et al.,
Baculovirus Expression
Vectors - A Laboratory Manual, W. H. Freeman and Co., New York, 1992 and
Baculovirus
Expression Vector System Instruction Manual, 6th edition, Pharmingen, San
Diego, 1999.
Cloning CHK1sv1 for Insect Cell Expression
To create a CHKlsvl/baculovirus transfer vector construct, the
CHK1svllpCMR11 clone (see Example 2) was used as template for PCR to amplify
the coding
sequence of CHKIsv1 (SEQ ID NO 14) using the primers listed in Table 5 (SEQ ID
NOs 16,
17). The primer represented by SEQ ID NO 16 contains an optimal translation
initiation
sequence immediately upstream of the ATG start colon and an upstream EcoRl
restriction site
that become incorporated into the amplicon. The primer represented by SEQ ID
NO 17 contains
sequence encoding six histidine residues C-terminal to the CHK1 svl coding
sequence as well as
an Eagl restriction site that become incorporated into the CHKlsvlamplicon.
The CHK1sv1
amplicon was run on a I% agarose gel. A selected amplicon fragment of the
expected size, in
the case of CHKlsvl, a product of about 994 base pairs, was manually extracted
from the gel and
purified with a Qiagen Gel Extraction Kit. The purified amplicon fragment was
digested with
EcoRl and EagI. The EcoRl/Eagl-digested amplicon was ligated into the
baculovirus transfer
vector pVLl 393 (Pharmingen, San Diego, CA) which had been digested with EcoRI
and Eagl
and dephosphorylated with alkaline phosphatase. The CHKIsvllpVLI393 construct
was then
transformed into E. coli strain DH5a. Plasmid DNA extracted from selected from
ampicillin
resistant colonies was sequenced to confirm identity, and the clones
possessing the appropriate
sequences were used for protein expression in insect cells.
-74-
CA 02714087 2010-08-04
WO 2009/102537 PCT/US2009/031633
Table 5. Primers used to clone CHKIsvl into baculovirus transfer vector VLI393
SEQ ID NO Primer Sequence
SEQ ID NO 16 CCCGGAATTCACCATGGCAGTGCCCTTTGTGGAAGACTGG
SEQ ID NO 17 TGTGTCCGGCCGTCAGTGATGGTGATGGTGATGTTCTTTTGACC
AACCGCTGCC
Insect Cell Expression of CHKIsvl
The CHK]svl/pVLI393 construct was co-transfected with linearized AcNPV
BaculoGold DNA (Pharmingen, San Diego, CA) into SF9 insect cells (Invitrogen,
Carlsbad,
CA). Individual recombinant viruses were selected by end point dilution. Virus
clones were
amplified to obtain high titer stocks. These virus stocks were used for
protein expression tests in
small scale SF9 cultures to verify production of the CHKI svl recombinant
protein. Transfected
SF9 cell lysates were analyzed by polyacrylamide gel electrophoresis for
CHKIsvl protein
expression. The CHKI svl protein was visualized by Cominassie staining or by
Western blotting
using an anti-CHKI antibody (G4 antibody; Santa Cruz Biotechnology, Inc).
Based on
expression, an individual virus was selected for larger scale CHK1svl
expression. For
recombinant protein expression on the liter scale, SF9 suspension cultures
were grown at 27 C in
Ex-cell 401 serum-free media (JRH Scientific, Lenexa, KS) and were infected
with a
recombinant virus stock using a multiplicity of infection of 0.3 virus per
cell. The infected SF9
culture was harvested 72 hour following virus transfection, and pelleted by
centrifugation.
Pellets were stored at --70 C.
Purification of CHKI svl Recombinant Protein
Insect cell pellets were lysed with B-PER protein extraction reagent (Pierce,
Rockford, IL) containing I M microcystin (Sigma, St. Louis, MO), 10 p.M
cypermethrin (EMD
Biosciences, San Diego, CA), and EDTA-free Protease Inhibitor Cocktail (Roche
Diagnostics,
Mannheim, Germany) (I tablet/50 ml lysis buffer). All manipulations during
protein purification
were performed at 4 C. Cells were resuspended in the lysis buffer were stirred
for 45 minutes.
DNAseI (Roche) was then added to a final concentration of 200 U/ml and the
cell suspension
was stirred for an additional 30 minutes. The lysed cell suspension was
centrifuged for
30 minutes at 30,000 g. The lysis supernatant was decanted and centrifuged for
30 minutes at
30,000 g. For each 10 ml of cleared supernatant, 1 ml bed volume of Talon
metal affinity resin
(Clontech, Palo Alto, CA) was added, and the suspension was stirred for 45
minutes. The
affinity resin/lysate suspension was centrifuged at 5000 g for 3 minutes and
then the supernatant
was discarded. The affinity resin was washed 4X with Buffer A (50 AM Tris, pH
8.0; 250 mM
NaCl) using 5X volumes of the resin. The washed resin was resuspended as a 2X
slurry in
Buffer A and packed into a chromatography column. The resin-packed column was
washed with
6X bed volumes of Buffer A. CHK1sv1-His-tagged protein is eluted from the
column using a
step-wise gradient of imidazole in Buffer A. Imidazole concentrations in the
2X bed volumen
fractions were 5, 10, 20, 30, 40, 50, and 60 mM. Elution fractions were
concentrated using the
-75-
CA 02714087 2010-08-04
WO 2009/102537 PCT/US2009/031633
Amicon Ultra 15 Centrifugal Filter Device, 30,000 Nominal Molecular Weight
Limit (Millipore,
Billerica, MA). The concentrated enzyme fractions were diluted 50% in glycerol
and stored at -
20 C. Fractions were analyzed for the presence of CHK1svl-His-tagged protein
using
polyacrylamide gel electrophoresis followed by Coommassie staining and Western
blotting using
an anti-CHKI antibody (G4 antibody; Santa Cruz Biotechnology, hic). The CHKI
svl kinase
activity of the column fractions was determined using the kinase assay
described in the following
section.
EXAMPLE 4: CHKlsvl Kinase Assa
CHKlsvl activity was assayed in vitro using a synthetic peptide substrate. The
phosphopeptide product was quantitated using a Homogenous Time-Resolved
Fluorescence
(HTRF) assay system (Park et al., 1999, Anal. Biochem. 269:94-104). The
reaction mixture
contained 40 mM HEPES, pH 7.3; 100 mM NaCl; 10 mM MgCl2; 2 mM dithiothreitol;
1%
BSA; 0.1 mM ATP; 0.5 E.iM peptide substrate; and 0.1 nM CHKI sv1 enzyme in a
final volume
of 40 lil. The peptide substrate has the amino acid sequence amino
terminus-GGRARTSSFAEPG-carboxy terminus (SynPep, Dublin CA) (SEQ ID NO 18) and
is
biotinylated at the N-terminus. The kinase reaction was incubated for 30
minutes at 22 C, and
then terminated with 60 l Stop/Detection Buffer (40 mM HEPES, pH 7.3; 10 mM
EDTA;
0.125% Triton X-100; 1.25% BSA; 250 nM PhycoLink Streptavidin-Allophycocyanin
(APC)
Conjugate (Prozyme, San Leandro, CA); and 0.75 nM GSK3a anti-phosphoserine
antibody (Cell
Signaling Technologies, Beverly, MA; Cat# 9338) labeled with europium-chelate
(Perkin Elmer,
Boston, MA). The reaction was allowed to equilibrate for 2 hours at 22 C, and
relative
fluorescent units were read on a Discovery plate reader (Packard Biosciences).
Inhibitor
compounds are assayed in the reaction described above, to determine compound
IC50s. I pL of
compound dissolved in DMSO was added to each 40 L reaction in a half-log
dilution series
covering a range of 1 nM to 100 ?.M. Relative phospho substrate formation,
read as HTRF
fluorescence units, is measured over the range of compound concentrations and
a titration curve
generated using a four parameter sigmoidal fit.
Specific compounds of the instant invention were tested in the assay described
above and were found to have IC50 of < 50 M against substrate.
EXAMPLE 5: Inhibition of CHKI Auto has ho lation in Cells
Inhibitor compounds are assayed for their ability to inhibit CHKI in cells by
monitoring CHKI autophosphorylation in response to DNA damage. H1299 cells
(ATCC,
Manassas, VA) are grown in culture medium: RPMI 1640 supplemented with 10%
fetal bovine
serum; 10 mM HEPES; 2 mM L-glutamine; Ix non-essential amino acids; and
penicillin-
streptomycin. Cells from T-75 flasks are pooled, counted, seeded into 6 well
dishes at 200,000
cells per well in 2 ml media, and incubated. Serial dilution series of
compounds in DMSO or
DMSO control are added to each well from a 1000x working stock in DMSO and
incubated for 2
hr at 37 C. Following the 2-hr incubation period, IOOnM camptothecin (EMD
Biosciences, San
-76-
CA 02714087 2010-08-04
WO 2009/102537 PCT/US2009/031633
Diego, CA) is added from a 200x working stock in PBS to all drug-treated cells
(except one of
the high dose wells) and one DMSO control well. After a 4 hour incubation with
camptothecin,
each well is washed once with ice-cold PBS and 300 L of lysis buffer (50 mM
Tris (pH 8.0),
150 mM NaCl, 50 mM NaF, I % NP-40, 0.5% Deoxycholic acid, 0.1 % SDS, 0.5 p.M
Na3VO4
and 1X Protease Inhibitor Cocktail Complete - without EDTA (Roche Diagnostics,
Mannheim,
Germany)) is added to each well. Plates are shaken at 4 C for 10-15 min and
lysates are then
transferred to 1.5 ml microcentrifuge tubes and frozen at -80 C. Lysates are
thawed on ice and
cleared by centrifugation at 15,000 x g for 20 min and the supernatants are
transferred to clean
tubes.
Samples (20p.L) are prepared for gel electrophoresis by addition of 5 tL of 5x
sample loading buffer and heat-denaturation for 5 min at 100 C. Samples are
electorphoresed in
Tris/Glycine SDS-polyacrylamide gels (10%) and proteins are transferred onto
PVDF. Blots are
then blocked for 1 hr in 3% BSA in TBS and probed using an antibody against
phospho-Ser-296
CHK1 (Cell Signaling Technologies - Cat #2346). Bound antibody is visualized
using a
horseradish peroxidase conjugated secondary antibody (goat anti-rabbit Jackson
Labs - Cat# 111-
035-046) and enhanced chemiluminescence (ECL-plus, Amersham, Piscataway, NJ).
After
stripping of the first antibody set by incubation in 62.5 mM Tris HC1 pH 6.7,
2% SDS and 2-
mercaptoethanol to 100 gM for 30 min at 55 C, blots are re-probed for total
CHK1, using a
CHK1 monoclonal antibody (Santa Cruz Biotechnology Inc., Cat# SC-8408). The
CHK1
monoclonal is detected using a a sheep anti-mouse IgG coupled to horseradish
peroxidase
(Amersham Biosciences, Piscataway, NJ, Cat##NA931) and enhanced
chemiluminescence (ECL-
plus, Amersham). ECL exposed films are scanned and the intensity of specific
bands is
quantitated with ImageQuant software. Titrations are evaluated for level of
phospho-CHK1
(Ser296) signal normalized to total CHKI and IC50 values are calculated,
EXAMPLE 6: Functional Activity of Inhibitors in Checkpoint Escape Assay
DNA damage arrest
To measure functional activity of CHKI inhibitors in cells, compounds are
assayed for their ability to abrogate DNA damage induced cell cycle arrest.
The assay determines
cell phospho-nucleolin levels as a measure of the quantity of cells entering M-
phase after cell
cycle arrest brought on by the DNA damaging agent camptothecin.
HI299 cells (ATCC, Manassas VA) are seeded at a density of 5000 cells/well in
RPMI640 media supplemented with 10% fetal bovine serum. After incubation for
24 hours at
37 C at 5% C02, camptothecin is added to a final concentration of 200 nM and
incubated for 16
hours. An equal volume of a test compound serial dilution series in growth
media plus 200nM
camptothecin and 332nM nocodozole (final concentration: 50ng/ml) is added and
incubation at
37 C is continued for 8 hours. Media is removed from the wells and 50 PL lysis
buffer (20 mM
HEPES, pH7.5, 150 mM NaCl, 50 mM NaF, I% Triton X-100, 1'0% Glycerol, I x
Proteinase
Inhibitor Cocktail (Roche Diagnostics, Mannheim Germany), I jd/rnl DNase I
(Roche
-77-
CA 02714087 2010-08-04
WO 2009/102537 PCT/US2009/031633
Diagnostics), 300 M Sodium Orthovanadate, 1 pM Microcystin (Sigma, St. Louis,
MO) added.
The plate with lysis buffer is shaken for 30 min at 4 C and frozen (-70 C) for
20 min. Levels of
phosphonucleolin in the cell lysates is measured using the IGEN Origen
technology (BioVeris
Corp., Gaithersburg, MD).
Detection of phosphonucleolin in cell l sates
4E2 anti-nucleolin antibody (Research Diagnostics Inc., Flanders, NJ) was
biotinylated using Origen Biotin-LC-NHS-Ester (BioVeris Corp.) using the
protocol described by
the manufacturer. Goat anti-mouse antibody (Jackson Immuno Research, West
Grove, PA) was
ruthenylated employing a ruthenylation kit (BioVeris Corp.; cat# 110034)
according to the
protocol described by the manufacturer. To each well of a 96-well plate is
added 25 L of
antibody buffer (phospho buffered saline pH7.2, I% bovine serum albumin, 0.5%
Tween-20)
containing 2 g/ml biotynylated 4E2 anti-nucleolin antibody and 0.4mg/ml
streptavidin coated
paramagnetic Dynabeads (BioVeris Corp.) along with 25 L of cell lysate
(above). The antibodies
and lysate are incubated with shaking for 1 hr at room temperature. Next, 50
ng of anti-
phosphonucleolin TG3 antibody (Applied NeuroSolutions Inc., Vernon Hills, IL)
in a volume of
50 L of antibody buffer (above) are added to each well of the lysate mix and
incubation is
continued for 30 min at room temperature. Lastly, 25 L of a 240ng/ml solution
of the
ruthenylated goat anti-mouse antibody in antibody buffer is added to each well
and incubation
continued for 3 hours at room temperature. The lysate antibody mixtures are
read in a BioVeris
M-series M8 analyser and EC50s for compound dependent increases in phosphor-
nucleolin are
determined.
EXAMPLE 7: Other Biological Assays.
CHKI Expression and Purification: Recombinant human CHKI can be expressed
as a fusion protein with glutathione S-transferase at the amino-terminus (GST-
CHKI) using
standard baculovirus vectors and a (Bac-to-Bac ) insect cell expression system
purchased from
GIBCOTM Invitrogen. Recombinant protein expressed in insect cells can be
purified using
glutathione sepharose (Amersham Biotech) using standard procedures described
by the
manufacturer.
CHKI Fluorescense Polarization Assays: CHKI kinase inhibitors can be
identified using fluorescence polarization to monitor kinase activity. This
assay utilizes 10 nM
GST-CHKI and contains 5 mM 2-(N-Morpholino)ethanesulfonic acid (MES, pH 6.5),
5 mM
magnesium chloride (MgCl2), 0.05% Tween -20, I M adenosine 5' triphosphate
(ATP), 2 mM
1,4-Dithio-DL-threitol (DTT), I M peptide substrate (Biotin-ILSRRPSYRKII ND-
free acid)
(SEQ ID NO: 19), 10 nM peptide substrate tracer (Fluoreseine-GSRRP-pS-YRKI-
free acid) (pS
= phosphorylated-Serine) (SEQ ID NO. 20), 60 ng anti-phospho-CREB(S 133) mouse
monoclonal IgG purified on Protein G sepharose from crude mouse ascites
purchased from Cell
Signalling Technologies (Beverly, MA), 4% dimethyl sulfoxide (DMSO) and 30 M
inhibitor
compound. Reactions are incubated at room temperature for 140 minutes and
terminated by
-78-
CA 02714087 2010-08-04
WO 2009/102537 PCT/US2009/031633
addition of 25 mM EDTA (pH 8.0). Stopped reactions are incubated for 120
minutes at room
temperature and fluorescence polarization values determined using a Molecular
Devices/LJL
Biosystems AnalystTM AD (Sunnyvale, CA) with standard fluorescine settings.
CHK1 SPA Filtration Assay: Assays (25 l) contain 10 nM GST-CHKI, 10 mM
MES, 2 mM DTT, 10 mM MgC12, 0.025% Tween -20, 1 uM peptide substrate (Biotin-
ILSRRPSYRKILND-free acid) (SEQ ID NO: 19), 1 p.M ATP, 0.1 p.Ci 33P-'y-ATP (New
England Nuclear, NEN) and are reacted for 90 minutes at room temperature.
Reactions are
terminated by adding 55 pl of phosphate buffered saline containing 50 mM EDTA,
6.9 mM
ATP, 0.5 mg Scintilation proximity assay (SPA) beads (Amersham Biosciences).
Peptide
substrate is allowed to bind beads for 10 minutes at room temperature followed
by filtration on a
Packard GF/B Unifilter plate and washed with phosphate buffered saline. Dried
plates may are
sealed with TopsealTM (NEN) and 33p incorporated to peptide substrate using a
Packard
Topcount scintillation counter with standard settings for 33p.
CHK1 FlashPlate Kinase Assay: Assays (25 pd) contain 8.7 GST-CHK1, 10
mM MES, 0.1 mM ethylene glycol-bis( 3-aminoethylether)-N,N,N',N'-tetracetic
acid (EGTA, pH
8.0), 2 mM DTT, 0.05% Tween 20, 3 p.M peptide substrate (Biotin-
1LSRRPSYRK.B.,ND-free
acid) (SEQ ID NO: 19), 1 pM ATP, 0.4 [Xi 33P-y-ATP (NEN) and 4% DMSO.
Reactions are
incubated for 30 minutes at room temperature, terminated with 50 l of 50 mM
EDTA. 90 p,l of
reaction is transferred to streptavidin-coated FlashPlates (NEN) and
incubated for I hour at
room temperature. Plates are washed with phosphate buffered saline containing
0.01% Tween-
20 and 10 mM sodium pyrophosphate. Plates are dried, sealed with TopsealTM
(NEN) and an
amount of 33p incorporated into the peptide substrate measured using a Packard
Topcount
NXTTM scintillation counter with standard settings.
CHKI DELFIA Kinase Assay: Assays (25 pd) utilize 6.4 mM GST-CHK1
containing 25 mM Tris, pH 8.5, 20% glycerol, 50 mM sodium chloride (NaCI), 0.1
Surfact-
Amps 20, 1 pM peptide substrate (Biotin-GLYRSPSMPEN-amide) (SEQ ID NO: 21), 2
mM
DTT, 4% DMSO, 12.5 jiM ATP, 5 mM MgCl and are reacted for 30 minutes at room
temperature. Reactions are terminated with 100 pd Stop buffer containing I%
BSA, 10 mM Tris,
pH 8.0, 150 mM NaCl and 100 inM EDTA. Stopped reactions (100 pl) are
transferred to 96 well
neutravidin plates (Pierce) to capture the biotin-peptide substrate during a
30 minute room
temperature incubation. Wells are washed and reacted with 100 p1 PerkinElmer
Wallac Assay
Buffer containing 21.5 ng/ml anti-phospho-Ser2l6-Cdc25c rabbit polyclonal
antibody from Cell
Signalling Technology (Beverly, MA) and 292 ng/ml europium labeled anti-rabbit-
IgG for 1 hour
at room temperature. Wells are washed and europium released from the bound
antibody by
addition of Enhancement Solution (100 l) (PerkinElmer Wallac) and detected
using a Wallac
Victor2TM using standard manufacturer settings.
Compounds of the present invention may be tested in the CHKI FlashPlate
Kinase Assay described above.
-79-
CA 02714087 2010-08-04
WO 2009/102537 PCT/US2009/031633
WST Assay: HT29, HCT116 (5000 cells/well) or other cells are seeded (75 gl) to
96 well clear bottom plates at densities which provide linear growth curves
for 72 hours. Cells
are cultured under sterile conditions in appropriate media and for HT29 and
HCT116 this media
is McCoy's 5A containing 10% Fetal Bovine Serum (FBS). Following the initial
seeding of
cells, cells are incubated at 37 C, 5% C02 from 17 to 24 hours at which time
the appropriate
DNA damaging agents (camptothicins, 5-fluorouracil and etoposide) are added at
increasing
concentrations to a point which is capable of causing at least 80% cell
killing within 48 hours.
Final volume of all DNA damaging agent and compound additions are 25 1.
Assays contain
<1% DMSO final. At the same time as DNA damaging agent addition, CHK1
inhibitor
compound is added at fixed concentrations to each DNA damaging agent titration
to observe
enhancement of cell killing. Cell viability/cell killing under the conditions
described above are
determined by addition of WST reagent (Roche) according to the manufacturer at
47 hours
following DNA damage and CHK1 inhibitor compound addition and following a 3.5
hour or 2.5
hour incubation at 37 C, 5% C02 wherein OD450 is measured.
Compounds of the present invention may be tested in the assays described
above.
EXAMPLE 8: Other Biological Assqys
Other assays that may be utilized to determine biological activity of the
instant
compounds include assays found in the following publications: WO 04/080973, WO
02/070494,
and WO 03/101444.
-80-
