U.S. patent application number 15/910708 was filed with the patent office on 2019-02-07 for use of pyrazolopyrimidine derivatives for the treatment of pi3k-delta related disorders.
The applicant listed for this patent is Incyte Corporation, Incyte Holdings Corporation. Invention is credited to Andrew P. Combs, Brent Douty, Joseph Glenn, Chunhong He, Yun-Long Li, Thomas P. Maduskuie, JR., Song Mei, Richard B. Sparks, Wenqing Yao, Eddy W. Yue, Wenyu Zhu.
Application Number | 20190040067 15/910708 |
Document ID | / |
Family ID | 50288319 |
Filed Date | 2019-02-07 |
![](/patent/app/20190040067/US20190040067A1-20190207-C00001.png)
![](/patent/app/20190040067/US20190040067A1-20190207-C00002.png)
![](/patent/app/20190040067/US20190040067A1-20190207-C00003.png)
![](/patent/app/20190040067/US20190040067A1-20190207-C00004.png)
![](/patent/app/20190040067/US20190040067A1-20190207-C00005.png)
![](/patent/app/20190040067/US20190040067A1-20190207-C00006.png)
![](/patent/app/20190040067/US20190040067A1-20190207-C00007.png)
![](/patent/app/20190040067/US20190040067A1-20190207-C00008.png)
![](/patent/app/20190040067/US20190040067A1-20190207-C00009.png)
![](/patent/app/20190040067/US20190040067A1-20190207-C00010.png)
![](/patent/app/20190040067/US20190040067A1-20190207-C00011.png)
View All Diagrams
United States Patent
Application |
20190040067 |
Kind Code |
A1 |
Li; Yun-Long ; et
al. |
February 7, 2019 |
USE OF PYRAZOLOPYRIMIDINE DERIVATIVES FOR THE TREATMENT OF
PI3K-DELTA RELATED DISORDERS
Abstract
The present application provides methods of treating PI3K.delta.
related disorders using compounds of Formula I: ##STR00001## or
pharmaceutically acceptable salts thereof.
Inventors: |
Li; Yun-Long; (Chadds Ford,
PA) ; Yao; Wenqing; (Chadds Ford, PA) ; Combs;
Andrew P.; (Kennett Square, PA) ; Yue; Eddy W.;
(Landenberg, PA) ; Mei; Song; (Wilmington, DE)
; Zhu; Wenyu; (Media, PA) ; Glenn; Joseph;
(Mount Royal, NJ) ; Maduskuie, JR.; Thomas P.;
(Wilmington, DE) ; Sparks; Richard B.;
(Wilmington, DE) ; Douty; Brent; (Fallowfield,
PA) ; He; Chunhong; (Chadds Ford, PA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Incyte Corporation
Incyte Holdings Corporation |
Wilmington
Wilmington |
DE
DE |
US
US |
|
|
Family ID: |
50288319 |
Appl. No.: |
15/910708 |
Filed: |
March 2, 2018 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
14193481 |
Feb 28, 2014 |
9932341 |
|
|
15910708 |
|
|
|
|
61771480 |
Mar 1, 2013 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/519 20130101;
C07D 487/04 20130101; A61P 35/00 20180101; A61K 31/5377
20130101 |
International
Class: |
C07D 487/04 20060101
C07D487/04; A61K 31/5377 20060101 A61K031/5377; A61K 31/519
20060101 A61K031/519 |
Claims
1. A method of treating a disease selected from autoimmune
hemolytic anemia pemphigus, and myelofibrosis in a patient,
comprising administering to said patient a therapeutically
effective amount of a compound of Formula I: ##STR00159## or a
pharmaceutically acceptable salt thereof, wherein: R.sup.2 is
C.sub.1-6 alkyl or C.sub.1-6 haloalkyl; R.sup.4 is halo, OH, CN,
C.sub.1-4 alkyl, C.sub.1-4 haloalkyl, C.sub.1-4 alkoxy, or
C.sub.1-4 haloalkoxy; R.sup.5 is halo, OH, CN, C.sub.1-4 alkyl,
C.sub.1-4 haloalkyl, C.sub.1-4 alkoxy, or C.sub.1-4 haloalkoxy; Cy
is selected from C.sub.3-7 cycloalkyl, 4-7 membered
heterocycloalkyl, phenyl, and 5-6 membered heteroaryl, each of
which is optionally substituted with 1, 2, 3, or 4 independently
selected R.sup.3 groups; each R.sup.3 is independently selected
from Cy.sup.1, --(C.sub.1-3 alkylene)-Cy.sup.1, halo, CN, NO.sub.2,
C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-6
haloalkyl, OR.sup.a1, SR.sup.a1, C(.dbd.O)R.sup.b1,
C(.dbd.O)NR.sup.c1R.sup.d1, C(.dbd.O)OR.sup.a1,
OC(.dbd.O)R.sup.b1OC(.dbd.O)NR.sup.c1R.sup.d1, NR.sup.c1R.sup.d1,
NR.sup.c1C(.dbd.O)R.sup.b1, NR.sup.c1C(.dbd.O)OR.sup.b1,
NR.sup.c1C(.dbd.O)NR.sup.c1R.sup.d1, C(.dbd.NR.sup.e)R.sup.b1,
C(.dbd.NR.sup.e)NR.sup.c1R.sup.d1,
NR.sup.c1C(.dbd.NR.sup.e)NR.sup.c1R.sup.d1,
NR.sup.c1S(.dbd.O)R.sup.b1,
NR.sup.c1S(.dbd.O).sub.2NR.sup.c1R.sup.d1, S(.dbd.O)R.sup.b1,
S(.dbd.O).sub.2R.sup.b1, and S(.dbd.O).sub.2NR.sup.c1R.sup.d1;
wherein said C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl
are each optionally substituted with 1, 2, 3, or 4 independently
selected R.sup.11 groups; each Cy.sup.1 is independently selected
from C.sub.3-7 cycloalkyl, 4-7 membered heterocycloalkyl, phenyl,
and 5-6 membered heteroaryl, each of which is optionally
substituted with 1, 2, 3, or 4 independently selected R.sup.11
groups; each R.sup.a1, R.sup.c1, and R.sup.d1 is independently
selected from H, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.2-6
alkenyl, C.sub.2-6 alkynyl, C.sub.3-7 cycloalkyl, 4-7 membered
heterocycloalkyl, phenyl, and 5-6 membered heteroaryl; wherein said
C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.3-7
cycloalkyl, 4-7 membered heterocycloalkyl, phenyl and 5-6 membered
heteroaryl are each optionally substituted with 1, 2, or 3
independently selected R.sup.11 groups; each R.sup.b1 is
independently selected from C.sub.1-6 alkyl, C.sub.1-6 haloalkyl,
C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.3-7 cycloalkyl, 4-7
membered heterocycloalkyl, phenyl, and 5-6 membered heteroaryl;
wherein said C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl,
C.sub.3-7 cycloalkyl, 4-7 membered heterocycloalkyl, phenyl, and
5-6 membered heteroaryl are each optionally substituted with 1, 2,
or 3 independently selected R.sup.11 groups; or R.sup.c1 and
R.sup.d1 together with the N atom to which they are attached form a
4-, 5-, 6-, or 7 membered heterocycloalkyl group, which is
optionally substituted with --OH or C.sub.1-3 alkyl; each R.sup.e
is independently selected from H, CN, OH, C.sub.1-4 alkyl, and
C.sub.1-4 alkoxy; and each R.sup.11 is independently selected from
OH, NO.sub.2, CN, halo, C.sub.1-3 alkyl, C.sub.2-3 alkenyl,
C.sub.2-3 alkynyl, C.sub.1-3 haloalkyl, cyano-C.sub.1-3 alkyl,
HO--C.sub.1-3 alkyl, C.sub.1-3 alkoxy-C.sub.1-3 alkyl, C.sub.3-7
cycloalkyl, C.sub.1-3 alkoxy, C.sub.1-3 haloalkoxy, amino,
C.sub.1-3 alkylamino, di(C.sub.1-3 alkyl)amino, thio, C.sub.1-3
alkylthio, C.sub.1-3 alkylsulfinyl, C.sub.1-3 alkylsulfonyl,
carbamyl, C.sub.1-3 alkylcarbamyl, di(C.sub.1-3 alkyl)carbamyl,
carboxy, C.sub.1-3 alkylcarbonyl, C.sub.1-4 alkoxycarbonyl,
C.sub.1-3 alkylcarbonylamino, C.sub.1-3 alkylsulfonylamino,
aminosulfonyl, C.sub.1-3 alkylaminosulfonyl, di(C.sub.1-3
alkyl)aminosulfonyl, aminosulfonylamino, C.sub.1-3
alkylaminosulfonylamino, di(C.sub.1-3 alkyl)aminosulfonylamino,
aminocarbonylamino, C.sub.1-3 alkylaminocarbonylamino, and
di(C.sub.1-3 alkyl)aminocarbonylamino.
2. The method of claim 1, wherein R.sup.2 is C.sub.1-3 alkyl or
C.sub.1-3 fluoroalkyl.
3. The method of claim 1, wherein R.sup.2 is methyl, ethyl, or
2,2-difluoromethyl.
4. The method of claim 1, wherein R.sup.2 is methyl.
5. The method of claim 1, wherein R.sup.2 is ethyl.
6. The method of claim 1, wherein R.sup.4 is halo, CN, or C.sub.1-3
alkyl.
7. The method of claim 1, wherein R.sup.4 is F, Cl, CN, or
methyl.
8. The method of claim 1, wherein R.sup.4 is F.
9. The method of claim 1, wherein R.sup.4 is Cl.
10. The method of claim 1, wherein R.sup.4 is CN.
11. The method of claim 1, wherein R.sup.4 is methyl.
12. The method of claim 1, wherein R.sup.5 is halo, CN, or
C.sub.1-3 alkyl.
13. The method of claim 1, wherein R.sup.5 is Cl, CN, or
methyl.
14. The method of claim 1, wherein R.sup.5 is Cl.
15. The method of claim 1, wherein R.sup.5 is CN.
16. The method of claim 1, wherein R.sup.5 is methyl.
17. The method of claim 1, wherein Cy is selected from C.sub.3-6
cycloalkyl, 4-6 membered heterocycloalkyl, phenyl, and 5-6 membered
heteroaryl, each of which is optionally substituted with 1, 2, 3,
or 4 independently selected R.sup.3 groups.
18. The method of claim 1, wherein Cy is 4-6 membered
heterocycloalkyl, each of which is optionally substituted with 1,
2, 3, or 4 independently selected R.sup.3 groups.
19. The method of claim 1, wherein Cy is selected from a
cyclopropyl ring, a phenyl ring, an azetidine ring, a pyrrolidine
ring, a piperidine ring, 3-oxo-morpholin-6-yl,
2-oxo-pyrrolidin-4-yl, 2-oxo-oxazolidin-4-yl,
2-oxo-oxazolidin-5-yl, a pyrazole ring, a pyridine ring, and a
pyrimidine ring, each of which is optionally substituted with 1, 2,
3, or 4 independently selected R.sup.3 groups.
20. The method of claim 1, wherein: each R.sup.3 is independently
selected from Cy.sup.1, --(C.sub.1-3 alkylene)-Cy.sup.1, halo, CN,
C.sub.1-6 alkyl, OR.sup.a1, NR.sup.c1R.sup.d1, C(.dbd.O)R.sup.b1,
C(.dbd.O)OR.sup.a1, C(.dbd.O)NR.sup.c1R.sup.d1, and
S(.dbd.O).sub.2R.sup.b1, wherein said C.sub.1-6 alkyl is optionally
substituted with 1, 2, 3, or 4 independently selected R.sup.11
groups; each Cy.sup.1 is independently C.sub.3-7 cycloalkyl, which
is optionally substituted with 1, 2, 3, or 4 independently selected
R.sup.11 groups; each R.sup.a1, R.sup.c1, and R.sup.d1 is
independently selected from H and C.sub.1-6 alkyl, wherein said
C.sub.1-6 alkyl is optionally substituted with 1, 2, or 3
independently selected R.sup.11 groups; each R.sup.b1 is
independently C.sub.1-6 alkyl, which is optionally substituted with
1, 2, or 3 independently selected R.sup.11 groups; and each
R.sup.11 is independently OH, CN, halo, cyano-C.sub.1-3 alkyl,
C.sub.1-3 haloalkoxy, amino, C.sub.1-3 alkylamino, di(C.sub.1-3
alkyl)amino, C.sub.1-3 alkylcarbonyl, C.sub.1-3 alkoxycarbonyl,
carbamyl, C.sub.1-3 alkylcarbamyl, or di(C.sub.1-3
alkyl)carbamyl.
21. The method of claim 1, wherein: R.sup.2 is C.sub.1-3 alkyl or
C.sub.1-3 fluoroalkyl; R.sup.4 is halo, CN, or C.sub.1-3 alkyl;
R.sup.5 is halo, CN, or C.sub.1-3 alkyl; Cy is selected from
C.sub.3-6 cycloalkyl, 4-6 membered heterocycloalkyl, phenyl, and
5-6 membered heteroaryl, each of which is optionally substituted
with 1, 2, 3, or 4 independently selected R.sup.3 group; each
R.sup.3 is independently selected from Cy.sup.1, --(C.sub.1-3
alkylene)-Cy.sup.1, halo, CN, C.sub.1-6 alkyl, OR.sup.a1,
NR.sup.c1R.sup.d1, C(.dbd.O)R.sup.b1, C(.dbd.O)OR.sup.a1,
C(.dbd.O)NR.sup.c1R.sup.d1, and S(.dbd.O).sub.2R.sup.b1, wherein
said C.sub.1-6 alkyl is optionally substituted with 1, 2, 3, or 4
independently selected R.sup.11 groups; each Cy.sup.1 is
independently C.sub.3-7 cycloalkyl, which is optionally substituted
with 1, 2, 3, or 4 independently selected R.sup.11 groups; each
R.sup.a1, R.sup.c1, and R.sup.d1 is independently selected from H
and C.sub.1-6 alkyl, wherein said C.sub.1-6 alkyl is optionally
substituted with 1, 2, or 3 independently selected R.sup.11 groups;
each R.sup.b1 is independently C.sub.1-6 alkyl, which is optionally
substituted with 1, 2, or 3 independently selected R.sup.11 groups;
and each R.sup.11 is independently OH, CN, halo, cyano-C.sub.1-3
alkyl, C.sub.1-3 haloalkoxy, amino, C.sub.1-3 alkylamino, di(C1-3
alkyl)amino, C.sub.1-3 alkylcarbonyl, C.sub.1-3 alkoxycarbonyl,
carbamyl, C.sub.1-3 alkylcarbamyl, or di(C.sub.1-3
alkyl)carbamyl.
22. The method of claim 1, wherein: R.sup.2 is methyl, ethyl, or
2,2-difluoromethyl; R.sup.4 is F, Cl, CN, or methyl; R.sup.5 is Cl,
CN, or methyl; Cy is selected from C.sub.3-6 cycloalkyl, 4-6
membered heterocycloalkyl, phenyl, and 5-6 membered heteroaryl,
each of which is optionally substituted with 1, 2, 3, or 4
independently selected R.sup.3 group; each R.sup.3 is independently
selected from Cy.sup.1, --(C.sub.1-3 alkylene)-Cy.sup.1, halo, CN,
C.sub.1-6 alkyl, OR.sup.a, NR.sup.c1R.sup.d1, C(.dbd.O)R.sup.b1,
C(.dbd.O)OR.sup.a1, C(.dbd.O)NR.sup.c1R.sup.d1, and
S(.dbd.O).sub.2R.sup.b1, wherein said C.sub.1-6 alkyl is optionally
substituted with 1, 2, 3, or 4 independently selected R.sup.11
groups; each Cy.sup.1 is independently C.sub.3-7 cycloalkyl, which
is optionally substituted with 1, 2, 3, or 4 independently selected
R.sup.11 groups; each R.sup.a1, R.sup.c1, and R.sup.d1 is
independently selected from H and C.sub.1-6 alkyl, wherein said
C.sub.1-6 alkyl is optionally substituted with 1, 2, or 3
independently selected R.sup.11 groups; each R.sup.b1 is
independently C.sub.1-6 alkyl, which is optionally substituted with
1, 2, or 3 independently selected R.sup.11 groups; and each
R.sup.11 is independently OH, CN, halo, cyano-C.sub.1-3 alkyl,
C.sub.1-3 haloalkoxy, amino, C.sub.1-3 alkylamino, di(C.sub.1-3
alkyl)amino, C.sub.1-3 alkylcarbonyl, C.sub.1-3 alkoxycarbonyl,
carbamyl, C.sub.1-3 alkylcarbamyl, or di(C.sub.1-3
alkyl)carbamyl.
23. The method of claim 1, wherein: R.sup.2 is methyl, ethyl, or
2,2-difluoromethyl; R.sup.4 is F, Cl, CN, or methyl; R.sup.5 is Cl,
CN, or methyl; Cy is selected from a cyclopropyl ring, a phenyl
ring, an azetidine ring, a pyrrolidine ring, a piperidine ring,
3-oxo-morpholin-6-yl, 2-oxo-pyrrolidin-4-yl, 2-oxo-oxazolidin-4-yl,
2-oxo-oxazolidin-5-yl, a pyrazole ring, a pyridine ring, and a
pyrimidine ring, each of which is optionally substituted with 1, 2,
3, or 4 independently selected R.sup.3 groups each R.sup.3 is
independently selected from Cy.sup.1, --(C.sub.1-3
alkylene)-Cy.sup.1, halo, C.sub.1-6 alkyl, OR.sup.a1,
NR.sup.c1R.sup.d1, C(.dbd.O)R.sup.b1, C(.dbd.O)OR.sup.a1,
C(.dbd.O)NR.sup.c1R.sup.d1, and S(.dbd.O).sub.2R.sup.b1, wherein
said C.sub.1-6 alkyl is optionally substituted with 1, 2, 3, or 4
independently selected R.sup.11 groups; each Cy.sup.1 is
independently selected from cyclopropyl and cyclobutyl, each of
which is optionally substituted with 1, 2, 3, or 4 independently
selected R.sup.11 groups; each R.sup.a1, R.sup.c1, and R.sup.d1 is
independently selected from H and C.sub.1-4 alkyl; wherein said
C.sub.1-4 alkyl is optionally substituted with 1, 2, or 3
independently selected R.sup.11 groups; each R.sup.b1 is
independently C.sub.1-4 alkyl, which is optionally substituted with
1, 2, or 3 independently selected R.sup.11 groups; each R.sup.11 is
independently OH, CN, halo, cyano-C.sub.1-3 alkyl, C.sub.1-3
alkoxy-C.sub.1-3 alkyl, C.sub.1-3 alkoxy, C.sub.1-3 haloalkoxy,
amino, C.sub.1-3 alkylamino, di(C.sub.1-3 alkyl)amino, C.sub.1-3
alkylcarbonyl, C.sub.1-4 alkoxycarbonyl, carbamyl, C.sub.1-3
alkylcarbamyl, or di(C.sub.1-3 alkyl)carbamyl.
24. The method of claim 1, wherein the compound is a compound of
Formula II: ##STR00160## or a pharmaceutically acceptable salt
thereof.
25. The method of claim 1, wherein the compound is a compound of
Formula III: ##STR00161## or a pharmaceutically acceptable salt
thereof.
26. The method of claim 1, wherein the compound is a compound of
Formula IV: ##STR00162## or a pharmaceutically acceptable salt
thereof, wherein: G is NH, n is 1, and V is O; or G is NH, n is 0,
and V is O or CH.sub.2; or G is O, n is 0 and V is NH.
27. The method of claim 1, wherein the compound is a compound of
Formula IVa: ##STR00163## or a pharmaceutically acceptable salt
thereof.
28. The method of claim 1, wherein the compound is a compound of
Formula IVb: ##STR00164## or a pharmaceutically acceptable salt
thereof.
29. The method of claim 1, wherein the compound is a compound of
Formula IVc: ##STR00165## or a pharmaceutically acceptable salt
thereof.
30. The method of claim 1, wherein the compound is a compound of
Formula IVd: ##STR00166## or a pharmaceutically acceptable salt
thereof.
31. (canceled)
32. The method of claim 1, or a pharmaceutically acceptable salt
thereof, wherein the starred carbon in Formula I: ##STR00167## is a
chiral carbon and said compound or said salt is the
(S)-enantiomer.
33. The method of claim 1, wherein the compound is selected from:
1-{1-[5-Chloro-3-(1-isopropylazetidin-3-yl)-2-methoxy-4-methylphenyl]ethy-
l}-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine;
1-{1-[3-(1-Acetylazetidin-3-yl)-5-chloro-2-methoxy-4-methylphenyl]ethyl}--
3-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine;
1-{1-[5-Chloro-2-methoxy-4-methyl-3-(1-propionylazetidin-3-yl)phenyl]ethy-
l}-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine;
1-(1-{5-Chloro-3-[1-(cyclopropylmethyl)azetidin-3-yl]-2-methoxy-4-methylp-
henyl}ethyl)-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine;
1-{1-[5-chloro-2-methoxy-4-methyl-3-(1-methylazetidin-3-yl)phenyl]ethyl}--
3-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine;
1-{1-[5-Chloro-3-(1-ethylazetidin-3-yl)-2-methoxy-4-methylphenyl]ethyl}-3-
-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine;
1-{1-[5-Chloro-3-(1-isobutylazetidin-3-yl)-2-methoxy-4-methylphenyl]ethyl-
}-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine;
1-{1-[3-(1-sec-butylazetidin-3-yl)-5-chloro-2-methoxy-4-methylphenyl]ethy-
l}-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine;
1-(1-{5-Chloro-2-methoxy-3-[1-(2-methoxyethyl)azetidin-3-yl]-4-methylphen-
yl}ethyl)-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine;
3-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chlor-
o-2-methoxy-6-methylphenyl}-N-methylazetidine-1-carboxamide;
5-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chlor-
o-2-methoxy-6-methylphenyl}-N,N-dimethylpyridine-2-carboxamide;
1-{1-[5-Chloro-4-fluoro-3-(1-isopropylazetidin-3-yl)-2-methoxyphenyl]ethy-
l}-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine;
5-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chlor-
o-2-ethoxy-6-methylphenyl}-N,N-dimethylpyridine-2-carboxamide;
4-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chlor-
o-2-methoxy-6-methylphenyl}-N,N-dimethylpyridine-2-carboxamide;
4-(3-(1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chlor-
o-2-methoxy-6-methylphenyl)-N-methylpicolinamide;
4-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chlor-
o-2-methoxy-6-methylphenyl}-N-(2-hydroxyethyl)pyridine-2-carboxamide;
4-{3-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chlor-
o-2-methoxy-6-methylphenyl}-N-(2-hydroxyethyl)-N-methylpyridine-2-carboxam-
ide;
2-(4-(3-(1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)--
5-chloro-2-methoxy-6-methylphenyl)-1H-pyrazol-1-yl)ethanol;
3'-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5'-chloro-
-3-fluoro-2'-methoxy-N,N,6'-trimethylbiphenyl-4-carboxamide;
3'-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5'-chloro-
-3-fluoro-2'-methoxy-N,6'-dimethylbiphenyl-4-carboxamide;
5-(3-(1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chlor-
o-2-methoxy-6-methylphenyl)-N-(2-hydroxyethyl)picolinamide;
4-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chlor-
o-2-methoxy-6-methylphenyl}-N-(2-hydroxyethyl)-N-methylpyridine-2-carboxam-
ide;
4-{3-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-c-
yano-2-methoxy-6-methylphenyl}-N-(2-hydroxyethyl)-N-methylpyridine-2-carbo-
xamide;
5-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[4,3-c]pyridin-1-yl)ethyl]-5--
chloro-2-methoxy-6-methylphenyl}-N,N-dimethylpyridine-2-carboxamide;
4-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-6-chloro-3-
-ethoxy-2-[5-(methylsulfonyl)pyridin-3-yl]benzonitrile;
5-(3-(1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chlor-
o-6-cyano-2-ethoxyphenyl)-N,N-dimethylpicolinamide
5-{3-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chlor-
o-2-methoxy-6-methylphenyl}-N,N-dimethylnicotinamide
5-{3-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chlor-
o-2-methoxy-6-methylphenyl}-N,N-dimethylpyridine-2-carboxamide;
1-{1-[5-Chloro-4-fluoro-3-(1-isopropylazetidin-3-yl)-2-methoxyphenyl]ethy-
l}-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine;
(2S)-1-(3-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-
-5-chloro-6-fluoro-2-methoxyphenyl}azetidin-1-yl)propan-2-ol;
2-(3-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-ch-
loro-6-fluoro-2-methoxyphenyl}azetidin-1-yl)ethanol;
1-{1-[5-Chloro-4-fluoro-2-methoxy-3-(1-oxetan-3-ylazetidin-3-yl)phenyl]et-
hyl}-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine;
1-{1-[5-Chloro-2-ethoxy-3-(1-isopropylazetidin-3-yl)-4-methylphenyl]ethyl-
}-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine;
2-(3-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-ch-
loro-2-ethoxy-6-methylphenyl}azetidin-1-yl)ethanol;
(2S)-1-(3-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-
-5-chloro-2-ethoxy-6-methylphenyl}azetidin-1-yl)propan-2-ol;
(2S)-1-(3-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-
-5-chloro-2-ethoxy-6-methylphenyl}azetidin-1-yl)-1-oxopropan-2-ol;
(2S)-1-(3-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-
-5-chloro-2-methoxy-6-methylphenyl}azetidin-1-yl)propan-2-ol;
2-(3-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-ch-
loro-2-methoxy-6-methylphenyl}azetidin-1-yl)ethanol;
(3-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chlo-
ro-2-methoxy-6-methylphenyl}azetidin-1-yl)acetonitrile;
1-(1-{5-Chloro-2-methoxy-4-methyl-3-[1-(2,2,2-trifluoroethyl)azetidin-3-y-
l]phenyl}ethyl)-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine;
(2R)-2-(3-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-
-5-chloro-2-methoxy-6-methylphenyl}azetidin-1-yl)-N-methylpropanamide;
2-(3-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-ch-
loro-2-methoxy-6-methylphenyl}azetidin-1-yl)-3,3,3-trifluoropropan-1-ol;
(2R)-3-(3-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-
-5-chloro-2-methoxy-6-methylphenyl}azetidin-1-yl)-1,1,1-trifluoropropan-2--
ol;
(2S)-1-(3-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)eth-
yl]-5-chloro-2-methoxy-6-methylphenyl}azetidin-1-yl)-1-oxopropan-2-ol;
(2R)-1-(3-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-
-5-chloro-2-methoxy-6-methylphenyl}azetidin-1-yl)-1-oxopropan-2-ol;
1-{1-[5-Chloro-2-ethoxy-4-fluoro-3-(1-isopropylazetidin-3-yl)phenyl]ethyl-
}-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine;
1-(3-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-ch-
loro-2-ethoxy-6-fluorophenyl}azetidin-1-yl)-2-methylpropan-2-ol;
1-(1-{5-Chloro-2-ethoxy-4-fluoro-3-[1-(2,2,2-trifluoroethyl)azetidin-3-yl-
]phenyl}ethyl)-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine;
(2S)-1-(3-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-
-5-chloro-2-ethoxy-6-fluorophenyl}azetidin-1-yl)propan-2-ol;
(2R)-2-(3-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-
-5-chloro-2-methoxy-6-methylphenyl}azetidin-1-yl)propan-1-ol;
1-(3-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-ch-
loro-2-methoxy-6-methylphenyl}azetidin-1-yl)-2-methylpropan-2-ol;
(2R)-2-(3-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-
-5-chloro-2-methoxy-6-methylphenyl}azetidin-1-yl)-N,N-dimethylpropanamide;
[1-{3-3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-ch-
loro-2-methoxy-6-methylphenyl}azetidin-1-yl)cyclobutyl]acetonitrile;
1-{1-[5-Chloro-2-methoxy-4-methyl-3-(1-methylpiperidin-4-yl)phenyl]ethyl}-
-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine;
1-(4-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-ch-
loro-2-methoxy-6-methylphenyl}piperidin-1-yl)-2-methylpropan-2-ol;
3-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chlor-
o-2-methoxy-6-methylphenyl}cyclobutanol;
5-(3-(1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chlor-
o-2-methoxy-6-methylphenyl)-N,N-dimethylpicolinamide;
1-[1-(5-Chloro-3-{1-[2-(dimethylamino)ethyl]-1H-pyrazol-4-yl}-2-methoxy-4-
-methylphenyl)ethyl]-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine;
2-[(5-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-c-
hloro-2-methoxy-6-methylphenyl}pyridin-2-yl)amino]ethanol;
2-(5-(3-(1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-ch-
loro-2-methoxy-6-methylphenyl)pyridin-2-yloxy)ethanol;
5-(3-(1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chlor-
o-2-(2,2-difluoroethoxy)-6-methylphenyl)-N,N-dimethylpicolinamide;
5-(3-(1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chlor-
o-2-ethoxy-6-methylphenyl)-N,N-dimethylpicolinamide;
4-(3-(1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chlor-
o-2-methoxy-6-methylphenyl)-N,N-dimethylpicolinamide;
2-(4-(3-(1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-ch-
loro-2-ethoxy-6-methylphenyl)-1H-pyrazol-1-yl)acetamide;
6-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chlor-
o-2-ethoxy-6-methylphenyl}-N,N-dimethylnicotinamide;
5-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-4-methoxy--
2-methyl-3-(1-methyl-1H-pyrazol-4-yl)benzonitrile;
1-(1-(5-chloro-2-methoxy-4-methyl-3-(pyrimidin-5-yl)phenyl)ethyl)-3-methy-
l-1H-pyrazolo[3,4-d]pyrimidin-4-amine;
1-(1-(3-(2-aminopyrimidin-5-yl)-5-chloro-2-methoxy-4-methylphenyl)ethyl)--
3-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine;
4-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-2-azetidin-
-3-yl-6-chloro-3-ethoxybenzonitrile;
4-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-6-chloro-3-
-ethoxy-2-(1-methylazetidin-3-yl)benzonitrile;
4-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-6-chloro-3-
-ethoxy-2-[1-(2-hydroxyethyl)azetidin-3-yl]benzonitrile;
4-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-6-chloro-3-
-ethoxy-2-{1-[(2S)-2-hydroxypropyl]azetidin-3-yl}benzonitrile;
tert-Butyl
2-(3-{3-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-ch-
loro-6-cyano-2-ethoxyphenyl}azetidin-1-yl)-2-methylpropanoate;
4-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-6-chloro-3-
-ethoxy-2-[1-(2-hydroxy-1,1-dimethylethyl)azetidin-3-yl]benzonitrile;
2-(3-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-ch-
loro-6-cyano-2-ethoxyphenyl}azetidin-1-yl)-2-methylpropanamide;
4-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-6-chloro-3-
-ethoxy-2-[1-(2-hydroxy-2-methylpropanoyl)azetidin-3-yl]benzonitrile;
4-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-2-azetidin-
-3-yl-6-chloro-3-methoxybenzonitrile;
4-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-6-chloro-3-
-methoxy-2-(1-methylazetidin-3-yl)benzonitrile;
4-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-6-chloro-2-
-[1-(2-hydroxyethyl)azetidin-3-yl]-3-methoxybenzonitrile;
4-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-6-chloro-2-
-{1-[(2S)-2-hydroxypropyl]azetidin-3-yl}-3-methoxybenzonitrile;
4-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-6-chloro-2-
-[1-(2-hydroxy-1-methylethyl)azetidin-3-yl]-3-methoxybenzonitrile;
2-(1-Acetylazetidin-3-yl)-4-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimi-
din-1-yl)ethyl]-6-chloro-3-methoxybenzonitrile;
4-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-6-chloro-3-
-methoxy-2-[1-(methylsulfonyl)azetidin-3-yl]benzonitrile; Methyl
3-{3-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chlor-
o-6-cyano-2-methoxyphenyl}azetidine-1-carboxylate;
3-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chlor-
o-6-cyano-2-methoxyphenyl}-N-(tert-butyl)azetidine-1-carboxamide;
3-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chlor-
o-6-cyano-2-methoxyphenyl}azetidine-1-carboxamide;
3-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chlor-
o-6-cyano-2-methoxyphenyl}-N,N-dimethylazetidine-1-carboxamide;
1-{1-[4,5-Dichloro-3-(1-ethylazetidin-3-yl)-2-methoxyphenyl]ethyl}-3-meth-
yl-1H-pyrazolo[3,4-d]pyrimidin-4-amine;
4-[1-(4-Amino-5-methyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)ethyl]-6-chloro-3--
ethoxy-2-(1-isopropylazetidin-3-yl)benzonitrile;
4-[-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-2-{1-[(2S-
)-2-hydroxypropyl]azetidin-3-yl}-3-methoxy-6-methylbenzonitrile;
4-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-6-chloro-3-
-ethoxy-2-[6-(1-hydroxy-1-methylethyl)pyridin-3-yl]benzonitrile;
4-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-6-chloro-3-
-ethoxy-2-pyrrolidin-1-ylbenzonitrile;
4-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-6-chloro-3-
-ethoxy-2-(3-methoxyazetidin-1-yl)benzonitrile;
4-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-3-ethoxy-2-
-(1-isopropylazetidin-3-yl)-6-methylbenzonitrile;
4-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-3-ethoxy-2-
-[1-(2-hydroxy-2-methylpropyl)azetidin-3-yl]-6-methylbenzonitrile;
4-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-3-ethoxy-2-
-[1-(2-hydroxy-2-methylpropanoyl)azetidin-3-yl]-6-methylbenzonitrile;
4-{3-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chlor-
o-2-ethoxy-6-methylphenyl}pyrrolidin-2-one;
4-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-6-chloro-3-
-ethoxy-2-(2-oxo-1,3-oxazolidin-5-yl)benzonitrile;
6-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chlor-
o-2-methoxy-6-methylphenyl}morpholin-3-one;
5-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chlor-
o-2-methoxy-6-methylphenyl}-1,3-oxazolidin-2-one;
4-{3-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chlor-
o-2-ethoxy-6-fluorophenyl}pyrrolidin-2-one;
4-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-6-chloro-3-
-ethoxy-2-(5-oxopyrrolidin-3-yl)benzonitrile;
4-{3-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chlor-
o-2-ethoxy-6-fluorophenyl}-1,3-oxazolidin-2-one;
5-{3-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chlor-
o-2-ethoxy-6-fluorophenyl}-1,3-oxazolidin-2-one; and
4-(1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-6-chloro-2-
-(1-(2-hydroxypropyl)azetidin-3-yl)-3-methoxybenzonitrile; or a
pharmaceutically acceptable salt of any of the aforementioned.
34-36. (canceled)
37. The method of claim 1, wherein the compound is
4-{3-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chlor-
o-2-ethoxy-6-fluorophenyl}pyrrolidin-2-one, or a pharmaceutically
acceptable salt thereof.
38. The method of claim 1, wherein the compound is
(S)-4-(3-((S)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-
-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one, or a
pharmaceutically acceptable salt thereof.
39. The method of claim 1, wherein the compound is
(R)-4-(3-((S)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-
-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one, or a
pharmaceutically acceptable salt thereof.
40. The method of claim 1, wherein the compound is
(S)-4-(3-((R)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-
-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one, or a
pharmaceutically acceptable salt thereof.
41. The method of claim 1, wherein the compound is
(R)-4-(3-((R)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-
-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one, or a
pharmaceutically acceptable salt thereof.
42. The method of claim 1, wherein the disease is autoimmune
hemolytic anemia.
43. The method of claim 37, wherein the disease is autoimmune
hemolytic anemia.
44. The method of claim 38, wherein the disease is autoimmune
hemolytic anemia.
45. The method of claim 39, wherein the disease is autoimmune
hemolytic anemia.
46. The method of claim 40, wherein the disease is autoimmune
hemolytic anemia.
47. The method of claim 41, wherein the disease is autoimmune
hemolytic anemia.
48. The method of claim 1, wherein the disease is pemphigus.
49. The method of claim 37, wherein the disease is pemphigus.
50. The method of claim 38, wherein the disease is pemphigus.
51. The method of claim 39, wherein the disease is pemphigus.
52. The method of claim 40, wherein the disease is pemphigus.
53. The method of claim 41, wherein the disease is pemphigus.
54. The method of claim 1, wherein the disease is
myelofibrosis.
55. The method of claim 37, wherein the disease is
myelofibrosis.
56. The method of claim 38, wherein the disease is
myelofibrosis.
57. The method of claim 39, wherein the disease is
myelofibrosis.
58. The method of claim 40, wherein the disease is
myelofibrosis.
59. The method of claim 41, wherein the disease is myelofibrosis.
Description
[0001] This application is a continuation of U.S. Ser. No.
14/193,481, filed Feb. 28, 2014, which claims the benefit of
priority of U.S. Provisional Appl. No. 61/771,480, filed Mar. 1,
2013, which is incorporated herein by reference in its
entirety.
TECHNICAL FIELD
[0002] The present application provides methods of treating
PI3K.delta. related disorders using pyrazolopyrimidine
derivatives.
BACKGROUND
[0003] The phosphoinositide 3-kinases (PI3Ks) belong to a large
family of lipid signaling kinases that phosphorylate
phosphoinositides at the D3 position of the inositol ring (Cantley,
Science, 2002, 296(5573):1655-7). PI3Ks are divided into three
classes (class I, II, and III) according to their structure,
regulation and substrate specificity. Class I PI3Ks, which include
PI3K.alpha., PI3K.beta., PI3K.gamma., and PI3K.delta., are a family
of dual specificity lipid and protein kinases that catalyze the
phosphorylation of phosphatidylinosito-4,5-bisphosphate (PIP.sub.2)
giving rise to phosphatidylinosito-3,4,5-trisphosphate (PIP.sub.3).
PIP.sub.3 functions as a second messenger that controls a number of
cellular processes, including growth, survival, adhesion and
migration. All four class I PI3K isoforms exist as heterodimers
composed of a catalytic subunit (p110) and a tightly associated
regulatory subunit that controls their expression, activation, and
subcellular localization. PI3K.alpha., PI3K.beta., and PI3K.delta.
associate with a regulatory subunit known as p85 and are activated
by growth factors and cytokines through a tyrosine kinase-dependent
mechanism (Jimenez, et al., J Biol Chem., 2002, 277(44):41556-62)
whereas PI3K.gamma. associates with two regulatory subunits (p101
and p84) and its activation is driven by the activation of
G-protein-coupled receptors (Brock, et al., J Cell Biol., 2003,
160(1):89-99). PI3K.alpha. and PI3K.beta. are ubiquitously
expressed. In contrast, PI3K.gamma. and PI3K.delta. are
predominantly expressed in leukocytes (Vanhaesebroeck, et al.,
Trends Biochem Sci., 2005, 30(4):194-204).
[0004] The differential tissue distribution of the PI3K isoforms
factors in their distinct biological functions. Genetic ablation of
either PI3K.alpha. or PI3K.beta. results in embryonic lethality,
indicating that PI3K.alpha. and PI3K.beta. have essential and
non-redundant functions, at least during development
(Vanhaesebroeck, et al., 2005). In contrast, mice which lack
PI3K.gamma. and PI3K.delta. are viable, fertile and have a normal
life span although they show an altered immune system. PI3K.gamma.
deficiency leads to impaired recruitment of macrophages and
neutrophils to sites of inflammation as well as impaired T cell
activation (Sasaki, et al., Science, 2000, 287(5455):1040-6).
PI3K.delta.-mutant mice have specific defects in B cell signaling
that lead to impaired B cell development and reduced antibody
responses after antigen stimulation (Clayton, et al., J Exp Med.
2002, 196(6):753-63; Jou, et al., Mol Cell Biol. 2002,
22(24):8580-91; Okkenhaug, et al., Science, 2002,
297(5583):1031-4).
[0005] The phenotypes of the PI3K.gamma. and PI3K.delta.-mutant
mice suggest that these enzymes may play a role in inflammation and
other immune-based diseases and this is borne out in preclinical
models. PI3K.gamma.-mutant mice are largely protected from disease
in mouse models of rheumatoid arthritis (RA) and asthma (Camps, et
al., Nat Med. 2005, 11(9):936-43; Thomas, et al., Eur J Immunol.
2005, 35(4):1283-91). In addition, treatment of wild-type mice with
a selective inhibitor of PI3K.gamma. was shown to reduce
glomerulonephritis and prolong survival in the MRL-lpr model of
systemic lupus nephritis (SLE) and to suppress joint inflammation
and damage in models of RA (Barber, et al., Nat Med. 2005,
11(9):933-5; Camps, et al., 2005). Similarly, both
PI3K.delta.-mutant mice and wild-type mice treated with a selective
inhibitor of PI3K.delta. have been shown to have attenuated
allergic airway inflammation and hyper-responsiveness in a mouse
model of asthma (Ali, et al., Nature. 2004, 431(7011):1007-11; Lee,
et al., FASEB J. 2006, 20(3):455-65) and to have attenuated disease
in a model of RA (Randis, et al., Eur. J. Immunol., 2008,
38(5):1215-24).
[0006] B cell proliferation has shown to play a major role in the
development of inflammatory autoimmune diseases (Puri, Frontiers in
Immunology (2012), 3(256), 1-16; Walsh, Kidney International (2007)
72, 676-682). For example, B cells support T-cell autoreactivity,
an important component of inflammatory autoimmune dieases. Once
activated and matured, B cells can traffic to sites of inflammation
and recruit inflammatory cells or differentiate to plasmablasts.
Thus, activity of B-cells can be affected by targeting B-cell
stimulatory cytokines, B-cell surface receptors, or via B-cell
depletion. Rituximab--an IgGi .kappa. mouse/human chimeric
monoclonal antibody directed against the B-cell surface receptor
CD20--has been shown to deplete CD20+ B cells. Use of rituximab has
been shown to have efficacy in treating idiopathic thrombocytopenic
purpura, autoimmune hemolytic anemia, or vasculitis. For example,
treatment with rituximab resulted in remission of the disease in
patients suffering from anti-neutrophil cytoplasm antibody
associated (ANCA) systemic vasculitis (AASV) with demonstrated
peripheral B-cell depletion (Walsh, 2007; Lovric, Nephrol Dial
Transplant (2009) 24: 179-185). Similarly, a complete response was
reported in one-third to two-thirds of patients having mixed
cryoglobulinemia vasculitis after treatment with rituximab,
including patients who presented with a severe form of vasculitis
that was resistant or intolerant to other treatments (Cacoub, Ann
Rheum Dis 2008; 67:283-287). Similarly, rituximab has been shown to
have efficacy in treating patients with idiopathic thrombocytopenic
purpura (or immune thrombocytopenic purpura) (Garvey, British
Journal of Haematology, (2008) 141, 149-169; Godeau, Blood (2008),
112(4), 999-1004; Medeo, European Journal of Haematology, (2008)
81, 165-169) and autoimmune hemolytic anemia (Garvey, British
Journal of Haematology, (2008) 141, 149-169).
[0007] PI3K.delta. signaling has been tied to B cell survival,
migration, and activation (Puri, Frontiers in Immunology, 2012,
3(256), 1-16, at pages 1-5; and Clayton, J Exp Med, 2002,
196(6):753-63). For example, PI3K.delta. is required for
antigen-dependent B-cell activation driven by B cell receptor. By
blocking B-cell adhesion, survival, activation, and proliferation,
PI3K.delta. inhibition can impair the ability of B cells to
activate T cells, preventing their activation and reducing
secreation of autoantibodies and pro-inflammatory cytokines. Hence,
by their ability to inhibit B cell activation, PI3K.delta.
inhibitors would be expected to treat B cell mediated diseases that
were treatable by similar methods such as B cell depletion by
rituximab. Indeed, PI3K.delta. inhibitors have been shown to be
useful mouse models of various autoimmune diseases that are also
treatable by rituximab such as arthritis (Puri (2012)). Further,
innate-like B cells, which are linked to autoimmunity are sensitive
to PI3K.delta. activity, as MZ and B-1 cells are nearly absent in
mice lacking the p1106 gene (Puri (2012). PI3K.delta. inhibitors
can reduce trafficking of and activation of MZ and B-1 cells, which
are implicated in autoimmune diseases.
[0008] In addition to their potential role in inflammatory
diseases, all four class I PI3K isoforms may play a role in cancer.
The gene encoding p110.alpha. is mutated frequently in common
cancers, including breast, prostate, colon and endometrial
(Samuels, et al., Science, 2004, 304(5670):554; Samuels, et al.,
Curr Opin Oncol. 2006, 18(1):77-82). Eighty percent of these
mutations are represented by one of three amino acid substitutions
in the helical or kinase domains of the enzyme and lead to a
significant upregulation of kinase activity resulting in oncogenic
transformation in cell culture and in animal models (Kang, et al.,
Proc Natl Acad Sci USA. 2005, 102(3):802-7; Bader, et al., Proc
Natl Acad Sci USA. 2006, 103(5):1475-9). No such mutations have
been identified in the other PI3K isoforms although there is
evidence that they can contribute to the development and
progression of malignancies. Consistent overexpression of
PI3K.delta. is observed in acute myeloblastic leukemia (Sujobert,
et al., Blood, 2005, 106(3):1063-6) and inhibitors of PI3K.delta.
can prevent the growth of leukemic cells (Billottet, et al.,
Oncogene. 2006, 25(50):6648-59). Elevated expression of PI3K.gamma.
is seen in chronic myeloid leukemia (Hickey, et al., J Biol Chem.
2006, 281(5):2441-50). Alterations in expression of PI3K.beta.,
PI3K.gamma. and PI3K.delta. have also been observed in cancers of
the brain, colon and bladder (Benistant, et al., Oncogene, 2000,
19(44):5083-90; Mizoguchi, et al., Brain Pathol. 2004, 14(4):372-7;
Knobbe, et al., Neuropathol Appl Neurobiol. 2005, 31(5):486-90).
Further, these isoforms have all been shown to be oncogenic in cell
culture (Kang, et al., 2006).
[0009] For these reasons, there is a need to develop new PI3K
inhibitors that can be used inflammatory disorders, autoimmune
diseases and cancer. This invention is directed to this need and
others.
SUMMARY
[0010] The present invention provides methods of idiopathic
thrombocytopenic purpura, autoimmune hemolytic anemia, vasculitis,
systemic lupus erythematosus, lupus nephritis, pemphigus,
membranous nephropathy, chronic lymphocytic leukemia (CLL),
Non-Hodgkin lymphoma, hairy cell leukemia, Mantle cell lymphoma,
small lymphocytic lymphoma, follicular lymphoma, lymphoplasmacytic
lymphoma, extranodal marginal zone lymphoma, Hodgkin's lymphoma,
Waldenstrom's macroglobulinemia, prolymphocytic leukemia, acute
lymphoblastic leukemia, myelofibrosis, mucosa-associated lymphatic
tissue (MALT) lymphoma, mediastinal (thymic) large B-cell lymphoma,
lymphomatoid granulomatosis, splenic marginal zone lymphoma,
primary effusion lymphoma, intravascular large B-cell lymphoma,
plasma cell leukemia, extramedullary plasmacytoma, smouldering
myeloma (aka asymptomatic myeloma), monoclonal gammopathy of
undetermined significance (MGUS), activated B-cell like (ABC)
diffuse large B cell lymphoma, or germinal center B cell (GCB)
diffuse large B cell lymphoma in a patient, comprising
administering to said patient a therapeutically effective amount of
a compound of Formula I:
##STR00002##
or a pharmaceutically acceptable salt thereof, wherein R.sup.2,
R.sup.3, R.sup.4, R.sup.5, and Cy are defined infra.
[0011] The present invention also provides a compound described
herein, or a pharmaceutically acceptable salt thereof, for use in
any of the methods described herein.
[0012] The present invention further provides use of a compound
described herein, or a pharmaceutically acceptable salt thereof,
for the manufacture of a medicament for use in any of the methods
described herein.
DESCRIPTION OF DRAWINGS
[0013] FIG. 1 depicts the crystal structure of the compound of
Example 269.
[0014] FIG. 2 depicts the tumor inhibiting effect of twice daily
doses of Example 347 at 0.3, 1, 3, or 10 mg/kg for 14 days in a
Pfeiffer human tumor xenograft model of diffuse large B-cell
lymphoma (y-axis is tumor volume (mm.sup.3.+-.SEM); x-axis is days
post implantation).
DETAILED DESCRIPTION
[0015] The present invention provides a method of treating
idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia,
vasculitis, systemic lupus erythematosus, lupus nephritis,
pemphigus, membranous nephropathy, chronic lymphocytic leukemia
(CLL), Non-Hodgkin lymphoma, hairy cell leukemia, Mantle cell
lymphoma, small lymphocytic lymphoma, follicular lymphoma,
lymphoplasmacytic lymphoma, extranodal marginal zone lymphoma,
Hodgkin's lymphoma, Waldenstrom's macroglobulinemia, prolymphocytic
leukemia, acute lymphoblastic leukemia, myelofibrosis,
mucosa-associated lymphatic tissue (MALT) lymphoma, mediastinal
(thymic) large B-cell lymphoma, lymphomatoid granulomatosis,
splenic marginal zone lymphoma, primary effusion lymphoma,
intravascular large B-cell lymphoma, plasma cell leukemia,
extramedullary plasmacytoma, smouldering myeloma (aka asymptomatic
myeloma), monoclonal gammopathy of undetermined significance
(MGUS), activated B-cell like (ABC) diffuse large B cell lymphoma
(ABC-DLBCL, or germinal center B cell (GCB) diffuse large B cell
lymphoma (GCB-DLBCL) in a patient, comprising administering to said
patient a therapeutically effective amount of a compound of Formula
I:
##STR00003##
or a pharmaceutically acceptable salt thereof, wherein:
[0016] R.sup.2 is C.sub.1-6 alkyl or C.sub.1-6 haloalkyl;
[0017] R.sup.4 is halo, OH, CN, C.sub.1-4 alkyl, C.sub.1-4
haloalkyl, C.sub.1-4 alkoxy, or C.sub.1-4 haloalkoxy;
[0018] R.sup.5 is halo, OH, CN, C.sub.1-4 alkyl, C.sub.1-4
haloalkyl, C.sub.1-4 alkoxy, or C.sub.1-4 haloalkoxy; Cy is
selected from C.sub.3-7 cycloalkyl, 4-7 membered heterocycloalkyl,
phenyl, and 5-6 membered heteroaryl, each of which is optionally
substituted with 1, 2, 3, or 4 independently selected R.sup.3
groups;
[0019] each R.sup.3 is independently selected from Cy.sup.1,
--(C.sub.1-3 alkylene)-Cy.sup.1, halo, CN, NO.sub.2, C.sub.1-6
alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-6 haloalkyl,
OR.sup.a1, SR.sup.a1, C(.dbd.O)R.sup.b1,
C(.dbd.O)NR.sup.c1R.sup.d1, C(.dbd.O)OR.sup.a1, OC(.dbd.O)R.sup.b1,
OC(.dbd.O)NR.sup.c1R.sup.d1, NR.sup.c1R.sup.d1,
NR.sup.c1C(.dbd.O)R.sup.b1, NR.sup.c1C(.dbd.O)OR.sup.b1,
NR.sup.c1C(.dbd.O)NR.sup.c1R.sup.d1, C(.dbd.NR.sup.e)R.sup.b1,
C(.dbd.NR.sup.e)NR.sup.c1R.sup.d1,
NR.sup.c1C(.dbd.NR.sup.e)NR.sup.c1R.sup.d1,
NR.sup.c1S(.dbd.O)R.sup.b1,
NR.sup.c1S(.dbd.O).sub.2NR.sup.c1R.sup.d1, S(.dbd.O)R.sup.b1,
S(.dbd.O).sub.2R.sup.b1, and S(.dbd.O).sub.2NR.sup.c1R.sup.d1;
wherein said C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl
are each optionally substituted with 1, 2, 3, or 4 independently
selected R.sup.11 groups;
[0020] each Cy.sup.1 is independently selected from C.sub.3-7
cycloalkyl, 4-7 membered heterocycloalkyl, phenyl, and 5-6 membered
heteroaryl, each of which is optionally substituted with 1, 2, 3,
or 4 independently selected R.sup.11 groups;
[0021] each R.sup.a1, R.sup.c1, and R.sup.d1 is independently
selected from H, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.2-6
alkenyl, C.sub.2-6 alkynyl, C.sub.3-7 cycloalkyl, 4-7 membered
heterocycloalkyl, phenyl, and 5-6 membered heteroaryl; wherein said
C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.3-7
cycloalkyl, 4-7 membered heterocycloalkyl, phenyl and 5-6 membered
heteroaryl are each optionally substituted with 1, 2, or 3
independently selected R.sup.11 groups;
[0022] each R.sup.b1 is independently selected from C.sub.1-6
alkyl, C.sub.1-6 haloalkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl,
C.sub.3-7 cycloalkyl, 4-7 membered heterocycloalkyl, phenyl, and
5-6 membered heteroaryl; wherein said C.sub.1-6 alkyl, C.sub.2-6
alkenyl, C.sub.2-6 alkynyl, C.sub.3-7 cycloalkyl, 4-7 membered
heterocycloalkyl, phenyl, and 5-6 membered heteroaryl are each
optionally substituted with 1, 2, or 3 independently selected
R.sup.11 groups;
[0023] or R.sup.c1 and R.sup.d1 together with the N atom to which
they are attached form a 4-, 5-, 6-, or 7 membered heterocycloalkyl
group, which is optionally substituted with --OH or C.sub.1-3
alkyl;
[0024] each R.sup.e is independently selected from H, CN, OH,
C.sub.1-4 alkyl, and C.sub.1-4 alkoxy; and
[0025] each R.sup.11 is independently selected from OH, NO.sub.2,
CN, halo, C.sub.1-3 alkyl, C.sub.2-3 alkenyl, C.sub.2-3 alkynyl,
C.sub.1-3 haloalkyl, cyano-C.sub.1-3 alkyl, HO--C.sub.1-3 alkyl,
C.sub.1-3 alkoxy-C.sub.1-3 alkyl, C.sub.3-7 cycloalkyl, C.sub.1-3
alkoxy, C.sub.1-3 haloalkoxy, amino, C.sub.1-3 alkylamino,
di(C.sub.1-3 alkyl)amino, thio, C.sub.1-3 alkylthio, C.sub.1-3
alkylsulfinyl, C.sub.1-3 alkylsulfonyl, carbamyl, C.sub.1-3
alkylcarbamyl, di(C.sub.1-3 alkyl)carbamyl, carboxy, C.sub.1-3
alkylcarbonyl, C.sub.1-4 alkoxycarbonyl, C.sub.1-3
alkylcarbonylamino, C.sub.1-3 alkylsulfonylamino, aminosulfonyl,
C.sub.1-3 alkylaminosulfonyl, di(C.sub.1-3 alkyl)aminosulfonyl,
aminosulfonylamino, C.sub.1-3 alkylaminosulfonylamino, di(C.sub.1-3
alkyl)aminosulfonylamino, aminocarbonylamino, C.sub.1-3
alkylaminocarbonylamino, and di(C.sub.1-3
alkyl)aminocarbonylamino.
[0026] In some embodiments, R.sup.2 is C.sub.1-3 alkyl or C.sub.1-3
fluoroalkyl. In some embodiments, R.sup.2 is methyl, ethyl, or
2,2-difluoromethyl. In some embodiments, R.sup.2 is methyl. In some
embodiments, R.sup.2 is ethyl.
[0027] In some embodiments, R.sup.4 is halo, CN, or C.sub.1-3
alkyl. In some embodiments, R.sup.4 is F, Cl, CN, or methyl. In
some embodiments, R.sup.4 is F. In some embodiments, R.sup.4 is Cl.
In some embodiments, R.sup.4 is CN. In some embodiments, R.sup.4 is
methyl.
[0028] In some embodiments, R.sup.5 is halo, CN, or C.sub.1-3
alkyl. In some embodiments, R.sup.5 is Cl, CN, or methyl. In some
embodiments, R.sup.5 is Cl. In some embodiments, R.sup.5 is CN. In
some embodiments, R.sup.5 is methyl.
[0029] In some embodiments, Cy is selected from C.sub.3-6
cycloalkyl, 4-6 membered heterocycloalkyl, phenyl, and 5-6 membered
heteroaryl, each of which is optionally substituted with 1, 2, 3,
or 4 independently selected R.sup.3 groups. In some embodiments, Cy
is 4-6 membered heterocycloalkyl, each of which is optionally
substituted with 1, 2, 3, or 4 independently selected R.sup.3
groups. In some embodiments, Cy is selected from a cyclopropyl
ring, a phenyl ring, an azetidine ring, a pyrrolidine ring, a
piperidine ring, 3-oxo-morpholin-6-yl, 2-oxo-pyrrolidin-4-yl,
2-oxo-oxazolidin-4-yl, 2-oxo-oxazolidin-5-yl, a pyrazole ring, a
pyridine ring, and a pyrimidine ring, each of which is optionally
substituted with 1, 2, 3, or 4 independently selected R.sup.3
groups.
[0030] In some embodiments:
each R.sup.3 is independently selected from Cy.sup.1, --(C.sub.1-3
alkylene)-Cy.sup.1, halo, CN, C.sub.1-6 alkyl, OR.sup.a1,
NR.sup.c1R.sup.d1, C(.dbd.O)R.sup.b1, C(.dbd.O)OR.sup.a1,
C(.dbd.O)NR.sup.c1R.sup.d1, and S(.dbd.O).sub.2R.sup.b1, wherein
said C.sub.1-6 alkyl is optionally substituted with 1, 2, 3, or 4
independently selected R.sup.11 groups;
[0031] each Cy.sup.1 is independently C.sub.3-7 cycloalkyl, which
is optionally substituted with 1, 2, 3, or 4 independently selected
R.sup.11 groups;
[0032] each R.sup.a1, R.sup.c1, and R.sup.d1 is independently
selected from H and C.sub.1-6 alkyl, wherein said C.sub.1-6 alkyl
is optionally substituted with 1, 2, or 3 independently selected
R.sup.11 groups;
[0033] each R.sup.b1 is independently C.sub.1-6 alkyl, which is
optionally substituted with 1, 2, or 3 independently selected
R.sup.11 groups; and
[0034] each R.sup.11 is independently OH, CN, halo, cyano-C.sub.1-3
alkyl, C.sub.1-3 haloalkoxy, amino, C.sub.1-3 alkylamino,
di(C.sub.1-3 alkyl)amino, C.sub.1-3 alkylcarbonyl, C.sub.1-3
alkoxycarbonyl, carbamyl, C.sub.1-3 alkylcarbamyl, or di(C.sub.1-3
alkyl)carbamyl.
[0035] In some embodiments:
[0036] R.sup.2 is C.sub.1-3 alkyl or C.sub.1-3 fluoroalkyl;
[0037] R.sup.4 is halo, CN, or C.sub.1-3 alkyl;
[0038] R.sup.5 is halo, CN, or C.sub.1-3 alkyl;
[0039] Cy is selected from C.sub.3-6 cycloalkyl, 4-6 membered
heterocycloalkyl, phenyl, and 5-6 membered heteroaryl, each of
which is optionally substituted with 1, 2, 3, or 4 independently
selected R.sup.3 group;
[0040] each R.sup.3 is independently selected from Cy.sup.1,
--(C.sub.1-3 alkylene)-Cy.sup.1, halo, CN, C.sub.1-6 alkyl,
OR.sup.a1, NR.sup.c1R.sup.d1, C(.dbd.O)R.sup.b1,
C(.dbd.O)OR.sup.a1, C(.dbd.O)NR.sup.c1R.sup.d1, and
S(.dbd.O).sub.2R.sup.b1, wherein said C.sub.1-6 alkyl is optionally
substituted with 1, 2, 3, or 4 independently selected R.sup.11
groups;
[0041] each Cy.sup.1 is independently C.sub.3-7 cycloalkyl, which
is optionally substituted with 1, 2, 3, or 4 independently selected
R.sup.11 groups;
[0042] each R.sup.a1, R.sup.c1, and R.sup.d1 is independently
selected from H and C.sub.1-6 alkyl, wherein said C.sub.1-6 alkyl
is optionally substituted with 1, 2, or 3 independently selected
R.sup.11 groups;
[0043] each R.sup.b1 is independently C.sub.1-6 alkyl, which is
optionally substituted with 1, 2, or 3 independently selected
R.sup.11 groups; and
[0044] each R.sup.11 is independently OH, CN, halo, cyano-C.sub.1-3
alkyl, C.sub.1-3 haloalkoxy, amino, C.sub.1-3 alkylamino,
di(C.sub.1-3 alkyl)amino, C.sub.1-3 alkylcarbonyl, C.sub.1-3
alkoxycarbonyl, carbamyl, C.sub.1-3 alkylcarbamyl, or di(C.sub.1-3
alkyl)carbamyl.
[0045] In some embodiments:
[0046] R.sup.2 is methyl, ethyl, or 2,2-difluoromethyl;
[0047] R.sup.4 is F, Cl, CN, or methyl;
[0048] R.sup.5 is Cl, CN, or methyl;
[0049] Cy is selected from C.sub.3-6 cycloalkyl, 4-6 membered
heterocycloalkyl, phenyl, and 5-6 membered heteroaryl, each of
which is optionally substituted with 1, 2, 3, or 4 independently
selected R.sup.3 group;
[0050] each R.sup.3 is independently selected from Cy.sup.1,
--(C.sub.1-3 alkylene)-Cy.sup.1, halo, CN, C.sub.1-6 alkyl,
OR.sup.a1, NR.sup.c1R.sup.d1, C(.dbd.O)R.sup.b1,
C(.dbd.O)OR.sup.a1, C(.dbd.O)NR.sup.c1R.sup.d1, and
S(.dbd.O).sub.2R.sup.b1, wherein said C.sub.1-6 alkyl is optionally
substituted with 1, 2, 3, or 4 independently selected R.sup.11
groups;
[0051] each Cy.sup.1 is independently C.sub.3-7 cycloalkyl, which
is optionally substituted with 1, 2, 3, or 4 independently selected
R.sup.11 groups;
[0052] each R.sup.a1, R.sup.c1, and R.sup.d1 is independently
selected from H and C.sub.1-6 alkyl, wherein said C.sub.1-6 alkyl
is optionally substituted with 1, 2, or 3 independently selected
R.sup.11 groups;
[0053] each R.sup.b1 is independently C.sub.1-6 alkyl, which is
optionally substituted with 1, 2, or 3 independently selected
R.sup.11 groups; and
[0054] each R.sup.11 is independently OH, CN, halo, cyano-C.sub.1-3
alkyl, C.sub.1-3 haloalkoxy, amino, C.sub.1-3 alkylamino,
di(C.sub.1-3 alkyl)amino, C.sub.1-3 alkylcarbonyl, C.sub.1-3
alkoxycarbonyl, carbamyl, C.sub.1-3 alkylcarbamyl, or di(C.sub.1-3
alkyl)carbamyl.
[0055] In some embodiments:
[0056] R.sup.2 is methyl, ethyl, or 2,2-difluoromethyl;
[0057] R.sup.4 is F, Cl, CN, or methyl;
[0058] R.sup.5 is Cl, CN, or methyl;
[0059] Cy is selected from a cyclopropyl ring, a phenyl ring, an
azetidine ring, a pyrrolidine ring, a piperidine ring,
3-oxo-morpholin-6-yl, 2-oxo-pyrrolidin-4-yl, 2-oxo-oxazolidin-4-yl,
2-oxo-oxazolidin-5-yl, a pyrazole ring, a pyridine ring, and a
pyrimidine ring, each of which is optionally substituted with 1, 2,
3, or 4 independently selected R.sup.3 groups.
[0060] each R.sup.3 is independently selected from Cy.sup.1,
--(C.sub.1-3 alkylene)-Cy.sup.1, halo, C.sub.1-6 alkyl, OR.sup.a1,
NR.sup.c1R.sup.d1, C(.dbd.O)R.sup.b1, C(.dbd.O)OR.sup.a1,
C(.dbd.O)NR.sup.c1R.sup.d1, and S(.dbd.O).sub.2R.sup.b1, wherein
said C.sub.1-6 alkyl is optionally substituted with 1, 2, 3, or 4
independently selected R.sup.11 groups;
[0061] each Cy.sup.1 is independently selected from cyclopropyl and
cyclobutyl, each of which is optionally substituted with 1, 2, 3,
or 4 independently selected R.sup.11 groups;
[0062] each R.sup.a1, R.sup.c1, and R.sup.d1 is independently
selected from H and C.sub.1-4 alkyl; wherein said C.sub.1-4 alkyl
is optionally substituted with 1, 2, or 3 independently selected
R.sup.11 groups;
[0063] each R.sup.b1 is independently C.sub.1-4 alkyl, which is
optionally substituted with 1, 2, or 3 independently selected
R.sup.11 groups;
[0064] each R.sup.11 is independently OH, CN, halo, cyano-C.sub.1-3
alkyl, C.sub.1-3 alkoxy-C.sub.1-3 alkyl, C.sub.1-3 alkoxy,
C.sub.1-3 haloalkoxy, amino, C.sub.1-3 alkylamino, di(C.sub.1-3
alkyl)amino, C.sub.1-3 alkylcarbonyl, C.sub.1-4 alkoxycarbonyl,
carbamyl, C.sub.1-3 alkylcarbamyl, or di(C.sub.1-3
alkyl)carbamyl.
[0065] In some embodiments, the compound is a compound of Formula
II:
##STR00004##
or a pharmaceutically acceptable salt thereof.
[0066] In some embodiments, the compound is a compound of Formula
III:
##STR00005##
or a pharmaceutically acceptable salt thereof.
[0067] In some embodiments, the compound is a compound of Formula
IV:
##STR00006##
or a pharmaceutically acceptable salt thereof, wherein:
[0068] G is NH, n is 1, and V is O; or
[0069] G is NH, n is 0, and V is O or CH.sub.2; or
[0070] G is O, n is 0 and V is NH.
[0071] In some embodiments, the compound is a compound of Formula
IVa:
##STR00007##
or a pharmaceutically acceptable salt thereof.
[0072] In some embodiments, the compound is a compound of Formula
IVb:
##STR00008##
or a pharmaceutically acceptable salt thereof.
[0073] In some embodiments, the compound is a compound of Formula
IVc:
##STR00009##
or a pharmaceutically acceptable salt thereof.
[0074] In some embodiments, the compound is a compound of Formula
IVd:
##STR00010##
or a pharmaceutically acceptable salt thereof.
[0075] In some embodiments, the starred carbon in Formula I:
##STR00011##
is a chiral carbon and said compound or said salt is the
(S)-enantiomer.
[0076] In some embodiments, the compounds are those described in
U.S. patent application Ser. No. 13/601,349, filed Aug. 31, 2012
(US Patent Publ. No. 2013/0059835), which is incorporated herein by
reference in its entirety.
[0077] In some embodiments, the method is a method of treating
idiopathic thrombocytopenic purpura (or idiopathic immune
thrombocytopenic purpura) (ITP). In some embodiments, the ITP is
relapsed ITP. In some embodiments, the ITP is refractory ITP.
[0078] In some embodiments, the method is a method of treating
autoimmune hemolytic anemia (AIHA).
[0079] In some embodiments, the method is a method is a method of
treating vasculitis. In some embodiments, the vasculitis is
Behcet's disease, Cogan's syndrome, giant cell arteritis,
polymyalgia rheumatica (PMR), Takayasu's arteritis, Buerger's
disease (thromboangiitis obliterans), central nervous system
vasculitis, Kawasaki disease, polyarteritis nodosa, Churg-Strauss
syndrome, mixed cryoglobulinemia vasculitis (essential or hepatitis
C virus (HCV)-induced), Henoch-Schonlein purpura (HSP),
hypersensitivity vasculitis, microscopic polyangiitis, Wegener's
granulomatosis, or anti-neutrophil cytoplasm antibody associated
(ANCA) systemic vasculitis (AASV). In some embodiments, the method
is a method of treating nephritis.
[0080] In some embodiments, the method of treating non-Hodgkin
lymphoma (NHL) is relapsed or refractory NHL or recucurrent
follicular NHL.
[0081] In som embodiments, the present application provides a
method of treating an aggressive lymphoma (e.g., germinal center B
cell-like (GCB) or activated B cell-like (ABC)) in a patient,
comprising administering a therapeutic amount of any of the
compounds described herein to said patient, or a pharmaceutically
acceptable salt thereof.
[0082] In some embodiments, the present application provides a
method of treating acute myeloid leukemia in a patient, comprising
administering a therapeutic amount of any of the compounds
described herein to said patient, or a pharmaceutically acceptable
salt thereof.
[0083] In some embodiments, the present application provides a
method of treating Burkitt lymphoma in a patient, comprising
administering a therapeutic amount of any of the compounds
described herein to said patient, or a pharmaceutically acceptable
salt thereof.
[0084] It is appreciated that certain features of the invention,
which are, for clarity, described in the context of separate
embodiments, can also be provided in combination in a single
embodiment. Conversely, various features of the invention which
are, for brevity, described in the context of a single embodiment,
can also be provided separately or in any suitable
subcombination.
[0085] At various places in the present specification, divalent
linking substituents are described. It is specifically intended
that each divalent linking substituent include both the forward and
backward forms of the linking substituent. For example,
--NR(CR'R'').sub.n-- includes both --NR(CR'R'').sub.n-- and
--(CR'R'').sub.nNR--. Where the structure clearly requires a
linking group, the Markush variables listed for that group are
understood to be linking groups.
[0086] The term "n-membered" where n is an integer typically
describes the number of ring-forming atoms in a moiety where the
number of ring-forming atoms is n. For example, piperidinyl is an
example of a 6-membered heterocycloalkyl ring, pyrazolyl is an
example of a 5-membered heteroaryl ring, pyridyl is an example of a
6-membered heteroaryl ring, and 1,2,3,4-tetrahydro-naphthalene is
an example of a 10-membered cycloalkyl group.
[0087] As used herein, the phrase "optionally substituted" means
unsubstituted or substituted. As used herein, the term
"substituted" means that a hydrogen atom is removed and replaced by
a substituent. It is to be understood that substitution at a given
atom is limited by valency.
[0088] Throughout the definitions, the term "C.sub.n-m" indicates a
range which includes the endpoints, wherein n and m are integers
and indicate the number of carbons. Examples include C.sub.1-4,
C.sub.1-6, and the like.
[0089] As used herein, the term "C.sub.n-m alkyl", employed alone
or in combination with other terms, refers to a saturated
hydrocarbon group that may be straight-chain or branched, having n
to m carbons. In some embodiments, the alkyl group contains from 1
to 6 carbon atoms, from 1 to 4 carbon atoms, from 1 to 3 carbon
atoms, or 1 to 2 carbon atoms. Examples of alkyl moieties include,
but are not limited to, chemical groups such as methyl, ethyl,
n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl;
higher homologs such as 2-methyl-1-butyl, n-pentyl, 3-pentyl,
n-hexyl, 1,2,2-trimethylpropyl, and the like.
[0090] As used herein, "C.sub.n-m alkenyl" refers to an alkyl group
having one or more double carbon-carbon bonds and having n to m
carbons. In some embodiments, the alkenyl moiety contains 2 to 6, 2
to 4, or 2 to 3 carbon atoms. Example alkenyl groups include, but
are not limited to, ethenyl, n-propenyl, isopropenyl, n-butenyl,
sec-butenyl, and the like.
[0091] As used herein, "C.sub.n-m alkynyl" refers to an alkyl group
having one or more triple carbon-carbon bonds and having n to m
carbons. Example alkynyl groups include, but are not limited to,
ethynyl, propyn-1-yl, propyn-2-yl, and the like. In some
embodiments, the alkynyl moiety contains 2 to 6, 2 to 4, or 2 to 3
carbon atoms.
[0092] As used herein, the term "alkylene", employed alone or in
combination with other terms, refers to a divalent alkyl linking
group. Examples of alkylene groups include, but are not limited to,
ethan-1,2-diyl, propan-1,3-diyl, propan-1,2-diyl, butan-1,4-diyl,
butan-1,3-diyl, butan-1,2-diyl, 2-methyl-propan-1,3-diyl, and the
like.
[0093] As used herein, the term "C.sub.n-m alkoxy", employed alone
or in combination with other terms, refers to a group of formula
--O-alkyl, wherein the alkyl group has n to m carbons. Example
alkoxy groups include methoxy, ethoxy, propoxy (e.g., n-propoxy and
isopropoxy), t-butoxy, and the like. In some embodiments, the alkyl
group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.
[0094] As used herein, the term "C.sub.n-m alkylamino" refers to a
group of formula --NH(alkyl), wherein the alkyl group has n to m
carbon atoms. In some embodiments, the alkyl group has 1 to 6, 1 to
4, or 1 to 3 carbon atoms.
[0095] As used herein, the term "C.sub.n-m alkoxycarbonyl" refers
to a group of formula --C(O)O-- alkyl, wherein the alkyl group has
n to m carbon atoms. In some embodiments, the alkyl group has 1 to
6, 1 to 4, or 1 to 3 carbon atoms.
[0096] As used herein, the term "C.sub.n-m alkylcarbonyl" refers to
a group of formula --C(O)-alkyl, wherein the alkyl group has n to m
carbon atoms. In some embodiments, the alkyl group has 1 to 6, 1 to
4, or 1 to 3 carbon atoms.
[0097] As used herein, the term "C.sub.n-m alkylcarbonylamino"
refers to a group of formula --NHC(O)-alkyl, wherein the alkyl
group has n to m carbon atoms. In some embodiments, the alkyl group
has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.
[0098] As used herein, the term "C.sub.n-m alkylsulfonylamino"
refers to a group of formula --NHS(O).sub.2-alkyl, wherein the
alkyl group has n to m carbon atoms. In some embodiments, the alkyl
group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.
[0099] As used herein, the term "aminosulfonyl" refers to a group
of formula --S(O).sub.2NH.sub.2.
[0100] As used herein, the term "C.sub.n-m alkylaminosulfonyl"
refers to a group of formula --S(O).sub.2NH(alkyl), wherein the
alkyl group has n to m carbon atoms. In some embodiments, the alkyl
group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.
[0101] As used herein, the term "di(C.sub.n-m alkyl)aminosulfonyl"
refers to a group of formula --S(O).sub.2N(alkyl).sub.2, wherein
each alkyl group independently has n to m carbon atoms. In some
embodiments, each alkyl group has, independently, 1 to 6, 1 to 4,
or 1 to 3 carbon atoms.
[0102] As used herein, the term "aminosulfonylamino" refers to a
group of formula --NHS(O).sub.2NH.sub.2.
[0103] As used herein, the term "C.sub.n-m alkylaminosulfonylamino"
refers to a group of formula --NHS(O).sub.2NH(alkyl), wherein the
alkyl group has n to m carbon atoms. In some embodiments, the alkyl
group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.
[0104] As used herein, the term "di(C.sub.n-m
alkyl)aminosulfonylamino" refers to a group of formula
--NHS(O).sub.2N(alkyl).sub.2, wherein each alkyl group
independently has n to m carbon atoms. In some embodiments, each
alkyl group has, independently, 1 to 6, 1 to 4, or 1 to 3 carbon
atoms.
[0105] As used herein, the term "aminocarbonylamino", employed
alone or in combination with other terms, refers to a group of
formula --NHC(O)NH.sub.2.
[0106] As used herein, the term "C.sub.n-m alkylaminocarbonylamino"
refers to a group of formula --NHC(O)NH(alkyl), wherein the alkyl
group has n to m carbon atoms. In some embodiments, the alkyl group
has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.
[0107] As used herein, the term "di(C.sub.n-m
alkyl)aminocarbonylamino" refers to a group of formula
--NHC(O)N(alkyl).sub.2, wherein each alkyl group independently has
n to m carbon atoms. In some embodiments, each alkyl group has,
independently, 1 to 6, 1 to 4, or 1 to 3 carbon atoms.
[0108] As used herein, the term "C.sub.n-m alkylcarbamyl" refers to
a group of formula --C(O)--NH(alkyl), wherein the alkyl group has n
to m carbon atoms. In some embodiments, the alkyl group has 1 to 6,
1 to 4, or 1 to 3 carbon atoms.
[0109] As used herein, the term "thio" refers to a group of formula
--SH.
[0110] As used herein, the term "C.sub.n-m alkylthio" refers to a
group of formula --S-alkyl, wherein the alkyl group has n to m
carbon atoms. In some embodiments, the alkyl group has 1 to 6, 1 to
4, or 1 to 3 carbon atoms.
[0111] As used herein, the term "C.sub.n-m alkylsulfinyl" refers to
a group of formula --S(O)-alkyl, wherein the alkyl group has n to m
carbon atoms. In some embodiments, the alkyl group has 1 to 6, 1 to
4, or 1 to 3 carbon atoms.
[0112] As used herein, the term "C.sub.n-m alkylsulfonyl" refers to
a group of formula --S(O).sub.2-alkyl, wherein the alkyl group has
n to m carbon atoms. In some embodiments, the alkyl group has 1 to
6, 1 to 4, or 1 to 3 carbon atoms.
[0113] As used herein, the term "amino" refers to a group of
formula --NH.sub.2.
[0114] As used herein, the term "carbamyl" to a group of formula
--C(O)NH.sub.2.
[0115] As used herein, the term "carbonyl", employed alone or in
combination with other terms, refers to a --C(O)-- group.
[0116] As used herein, the term "cyano-C.sub.1-3 alkyl" refers to a
group of formula --(C.sub.1-3 alkylene)-CN.
[0117] As used herein, the term "HO--C.sub.1-3 alkyl" refers to a
group of formula --(C.sub.1-3 alkylene)-OH.
[0118] As used herein, the term "C.sub.1-3 alkoxy-C.sub.1-3 alkyl"
refers to a group of formula --(C.sub.1-3 alkylene)-O(C.sub.1-3
alkyl).
[0119] As used herein, the term "carboxy" refers to a group of
formula --C(O)OH.
[0120] As used herein, the term "di(C.sub.n-m-alkyl)amino" refers
to a group of formula --N(alkyl).sub.2, wherein the two alkyl
groups each has, independently, n to m carbon atoms. In some
embodiments, each alkyl group independently has 1 to 6, 1 to 4, or
1 to 3 carbon atoms.
[0121] As used herein, the term "di(C.sub.n-m-alkyl)carbamyl"
refers to a group of formula --C(O)N(alkyl).sub.2, wherein the two
alkyl groups each has, independently, n to m carbon atoms. In some
embodiments, each alkyl group independently has 1 to 6, 1 to 4, or
1 to 3 carbon atoms.
[0122] As used herein, "halo" refers to F, Cl, Br, or I. In some
embodiments, the halo group is F or Cl.
[0123] As used herein, "C.sub.n-m haloalkoxy" refers to a group of
formula --O-haloalkyl having n to m carbon atoms. An example
haloalkoxy group is OCF.sub.3. In some embodiments, the haloalkoxy
group is fluorinated only. In some embodiments, the alkyl group has
1 to 6, 1 to 4, or 1 to 3 carbon atoms.
[0124] As used herein, the term "C.sub.n-m haloalkyl", employed
alone or in combination with other terms, refers to an alkyl group
having from one halogen atom to 2s+1 halogen atoms which may be the
same or different, where "s" is the number of carbon atoms in the
alkyl group, wherein the alkyl group has n to m carbon atoms. In
some embodiments, the haloalkyl group is fluorinated only (e.g., a
"fluoroalkyl" group). In some embodiments, the alkyl group has 1 to
6, 1 to 4, or 1 to 3 carbon atoms.
[0125] As used herein, "cycloalkyl" refers to non-aromatic cyclic
hydrocarbons including cyclized alkyl and/or alkenyl groups.
Cycloalkyl groups can have 3, 4, 5, 6, or 7 ring-forming carbons
(C.sub.3-7). Ring-forming carbon atoms of a cycloalkyl group can be
optionally substituted by oxo or sulfido. Cycloalkyl groups also
include cycloalkylidenes. Example cycloalkyl groups include
cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,
cyclopentenyl, cyclohexenyl, cyclohexadienyl, cycloheptatrienyl,
and the like. In some embodiments, cycloalkyl is cyclopropyl,
cyclobutyl, cyclopentyl, or cyclohexyl. Also included in the
definition of cycloalkyl are moieties that have one or more
aromatic rings fused (i.e., having a bond in common with) to the
cycloalkyl ring, for example, benzo or thienyl derivatives of
cyclopentane, cyclohexane, and the like.
[0126] As used herein, "heteroaryl" refers to a monocyclic aromatic
heterocycle having at least one heteroatom ring member selected
from sulfur, oxygen, and nitrogen. In some embodiments, the
heteroaryl ring has 1, 2, 3, or 4 heteroatom ring members
independently selected from nitrogen, sulfur and oxygen. In some
embodiments, any ring-forming N in a heteroaryl moiety can be an
N-oxide. In some embodiments, the heteroaryl has 5-6 ring atoms and
1 or 2 heteroatom ring members independently selected from
nitrogen, sulfur and oxygen. In some embodiments, the heteroaryl is
a five-membered or six-membered heteroaryl ring.
[0127] A five-membered heteroaryl ring is a heteroaryl with a ring
having five ring atoms wherein one or more (e.g., 1, 2, or 3) ring
atoms are independently selected from N, O, and S. Exemplary
five-membered ring heteroaryls are thienyl, furyl, pyrrolyl,
imidazolyl, thiazolyl, oxazolyl, pyrazolyl, isothiazolyl,
isoxazolyl, 1,2,3-triazolyl, tetrazolyl, 1,2,3-thiadiazolyl,
1,2,3-oxadiazolyl, 1,2,4-triazolyl, 1,2,4-thiadiazolyl,
1,2,4-oxadiazolyl, 1,3,4-triazolyl, 1,3,4-thiadiazolyl, and
1,3,4-oxadiazolyl.
[0128] A six-membered heteroaryl ring is a heteroaryl with a ring
having six ring atoms wherein one or more (e.g., 1, 2, or 3) ring
atoms are independently selected from N, O, and S. Exemplary
six-membered ring heteroaryls are pyridyl, pyrazinyl, pyrimidinyl,
triazinyl and pyridazinyl.
[0129] As used herein, "heterocycloalkyl" refers to non-aromatic
monocyclic heterocycleshaving one or more ring-forming heteroatoms
selected from O, N, or S. Included in heterocycloalkyl are
monocyclic 4-, 5-, 6-, and 7-membered heterocycloalkyl groups.
Example heterocycloalkyl groups include pyrrolidin-2-one,
1,3-isoxazolidin-2-one, pyranyl, tetrahydropuran, oxetanyl,
azetidinyl, morpholino, thiomorpholino, piperazinyl,
tetrahydrofuranyl, tetrahydrothienyl, piperidinyl, pyrrolidinyl,
isoxazolidinyl, isothiazolidinyl, pyrazolidinyl, oxazolidinyl,
thiazolidinyl, imidazolidinyl, azepanyl, 3-oxo-morpholin-6-yl,
2-oxo-pyrrolidin-4-yl, 2-oxo-oxazolidin-4-yl,
2-oxo-oxazolidin-5-yl, and the like. Ring-forming carbon atoms and
heteroatoms of a heterocycloalkyl group can be optionally
substituted by oxo or sulfido (e.g., C(O), S(O), C(S), or
S(O).sub.2, etc.). The heterocycloalkyl group can be attached
through a ring-forming carbon atom or a ring-forming heteroatom. In
some embodiments, the heterocycloalkyl group contains 0 to 3 double
bonds. In some embodiments, the heterocycloalkyl group contains 0
to 2 double bonds. In some embodiments, the heterocycloalkyl has
4-7 or 4-6 ring atoms with 1 or 2 heteroatoms independently
selected from nitrogen, oxygen or sulfur and having one or more
oxidized ring members.
[0130] At certain places, the definitions or embodiments refer to
specific rings (e.g., an azetidine ring, a pyridine ring, etc.).
Unless otherwise indicated, these rings can be attached any ring
member provided that the valency of the atom is not exceeded. For
example, an azetidine ring may be attached at any position of the
ring, whereas an azetidin-3-yl ring is attached at the
3-position.
[0131] The compounds described herein can be asymmetric (e.g.,
having one or more stereocenters). All stereoisomers, such as
enantiomers and diastereomers, are intended unless otherwise
indicated. Compounds of the present invention that contain
asymmetrically substituted carbon atoms can be isolated in
optically active or racemic forms. Methods on how to prepare
optically active forms from optically inactive starting materials
are known in the art, such as by resolution of racemic mixtures or
by stereoselective synthesis. Many geometric isomers of olefins,
C.dbd.N double bonds, and the like can also be present in the
compounds described herein, and all such stable isomers are
contemplated in the present invention. Cis and trans geometric
isomers of the compounds of the present invention are described and
may be isolated as a mixture of isomers or as separated isomeric
forms.
In some embodiments, the compound has the (R)-configuration. In
some embodiments, the compound has the (S)-configuration.
[0132] Resolution of racemic mixtures of compounds can be carried
out by any of numerous methods known in the art. An example method
includes fractional recrystallization using a chiral resolving acid
which is an optically active, salt-forming organic acid. Suitable
resolving agents for fractional recrystallization methods are, for
example, optically active acids, such as the D and L forms of
tartaric acid, diacetyltartaric acid, dibenzoyltartaric acid,
mandelic acid, malic acid, lactic acid or the various optically
active camphorsulfonic acids such as .beta.-camphorsulfonic
acid.
[0133] Other resolving agents suitable for fractional
crystallization methods include stereoisomerically pure forms of
.alpha.-methylbenzylamine (e.g., S and R forms, or
diastereomerically pure forms), 2-phenylglycinol, norephedrine,
ephedrine, N-methylephedrine, cyclohexylethylamine,
1,2-diaminocyclohexane, and the like.
[0134] Resolution of racemic mixtures can also be carried out by
elution on a column packed with an optically active resolving agent
(e.g., dinitrobenzoylphenylglycine). Suitable elution solvent
composition can be determined by one skilled in the art.
[0135] Compounds described herein also include tautomeric forms.
Tautomeric forms result from the swapping of a single bond with an
adjacent double bond together with the concomitant migration of a
proton. Tautomeric forms include prototropic tautomers which are
isomeric protonation states having the same empirical formula and
total charge. Example prototropic tautomers include ketone--enol
pairs, amide--imidic acid pairs, lactam--lactim pairs,
enamine-imine pairs, and annular forms where a proton can occupy
two or more positions of a heterocyclic system, for example, 1H-
and 3H-imidazole, 1H-, 2H- and 4H-1,2,4-triazole, 1H- and
2H-isoindole, and 1H- and 2H-pyrazole. Tautomeric forms can be in
equilibrium or sterically locked into one form by appropriate
substitution.
[0136] Compounds described herein can also include all isotopes of
atoms occurring in the intermediates or final compounds. Isotopes
include those atoms having the same atomic number but different
mass numbers. For example, isotopes of hydrogen include tritium and
deuterium.
[0137] The term, "compound," as used herein is meant to include all
stereoisomers, geometric iosomers, tautomers, and isotopes of the
structures depicted. Compounds herein identified by name or
structure as one particular tautomeric form are intended to include
other tautomeric forms unless otherwise specified.
[0138] All compounds, and pharmaceutically acceptable salts
thereof, can be found together with other substances such as water
and solvents (e.g. hydrates and solvates) or can be isolated.
[0139] In some embodiments, the compounds described herein, or
salts thereof, are substantially isolated. By "substantially
isolated" is meant that the compound is at least partially or
substantially separated from the environment in which it was formed
or detected. Partial separation can include, for example, a
composition enriched in the compounds described herein. Substantial
separation can include compositions containing at least about 50%,
at least about 60%, at least about 70%, at least about 80%, at
least about 90%, at least about 95%, at least about 97%, or at
least about 99% by weight of the compounds described herein, or
salt thereof. Methods for isolating compounds and their salts are
routine in the art.
[0140] The phrase "pharmaceutically acceptable" is employed herein
to refer to those compounds, materials, compositions, and/or dosage
forms which are, within the scope of sound medical judgment,
suitable for use in contact with the tissues of human beings and
animals without excessive toxicity, irritation, allergic response,
or other problem or complication, commensurate with a reasonable
benefit/risk ratio.
[0141] The expressions, "ambient temperature" and "room
temperature" or "rt" as used herein, are understood in the art, and
refer generally to a temperature, e.g. a reaction temperature, that
is about the temperature of the room in which the reaction is
carried out, for example, a temperature from about 20.degree. C. to
about 30.degree. C.
[0142] The present invention also includes pharmaceutically
acceptable salts of the compounds described herein. As used herein,
"pharmaceutically acceptable salts" refers to derivatives of the
disclosed compounds wherein the parent compound is modified by
converting an existing acid or base moiety to its salt form.
Examples of pharmaceutically acceptable salts include, but are not
limited to, mineral or organic acid salts of basic residues such as
amines; alkali or organic salts of acidic residues such as
carboxylic acids; and the like. The pharmaceutically acceptable
salts of the present invention include the conventional non-toxic
salts of the parent compound formed, for example, from non-toxic
inorganic or organic acids. The pharmaceutically acceptable salts
of the present invention can be synthesized from the parent
compound which contains a basic or acidic moiety by conventional
chemical methods. Generally, such salts can be prepared by reacting
the free acid or base forms of these compounds with a
stoichiometric amount of the appropriate base or acid in water or
in an organic solvent, or in a mixture of the two; generally,
non-aqueous media like ether, ethyl acetate, alcohols (e.g.,
methanol, ethanol, iso-propanol, or butanol) or acetonitrile (ACN)
are preferred. Lists of suitable salts are found in Remington's
Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton,
Pa., 1985, p. 1418 and Journal of Pharmaceutical Science, 66, 2
(1977), each of which is incorporated herein by reference in its
entirety.
Methods
[0143] The compounds described herein can modulate activity of one
or more of various kinases including, for example, phosphoinositide
3-kinases (PI3Ks). The term "modulate" is meant to refer to an
ability to increase or decrease the activity of one or more members
of the PI3K family. Accordingly, the compounds described herein can
be used in methods of modulating a PI3K by contacting the PI3K with
any one or more of the compounds or compositions described herein.
In some embodiments, compounds of the present invention can act as
inhibitors of one or more PI3Ks. In further embodiments, the
compounds described herein can be used to modulate activity of a
PI3K in an individual in need of modulation of the receptor by
administering a modulating amount of a compound described herein,
or a pharmaceutically acceptable salt thereof. In some embodiments,
modulating is inhibiting.
[0144] Given that cancer cell growth and survival is impacted by
multiple signaling pathways, the present invention is useful for
treating disease states characterized by drug resistant kinase
mutants. In addition, different kinase inhibitors, exhibiting
different preferences in the kinases which they modulate the
activities of, may be used in combination. This approach could
prove highly efficient in treating disease states by targeting
multiple signaling pathways, reduce the likelihood of
drug-resistance arising in a cell, and reduce the toxicity of
treatments for disease.
[0145] Kinases to which the present compounds bind and/or modulate
(e.g., inhibit) include any member of the PI3K family. In some
embodiments, the PI3K is PI3K.alpha., PI3K.beta., PI3K.gamma., or
PI3K.delta.. In some embodiments, the PI3K is PI3K.gamma. or
PI3K.delta.. In some embodiments, the PI3K is PI3K.gamma.. In some
embodiments, the PI3K is PI3K.delta.. In some embodiments, the PI3K
includes a mutation. A mutation can be a replacement of one amino
acid for another, or a deletion of one or more amino acids. In such
embodiments, the mutation can be present in the kinase domain of
the PI3K.
[0146] In some embodiments, more than one compound described herein
is used to inhibit the activity of one kinase (e.g., PI3K.gamma. or
PI3K.delta.).
[0147] In some embodiments, more than one compound described herein
is used to inhibit more than one kinase, such as at least two
kinases (e.g., PI3K.gamma. and PI3K.delta.).
[0148] In some embodiments, one or more of the compounds is used in
combination with another kinase inhibitor to inhibit the activity
of one kinase (e.g., PI3K.gamma. or PI3K.delta.).
[0149] In some embodiments, one or more of the compounds is used in
combination with another kinase inhibitor to inhibit the activities
of more than one kinase (e.g., PI3K.gamma. or PI3K.delta.), such as
at least two kinases.
[0150] The compounds described herein can be selective. By
"selective" is meant that the compound binds to or inhibits a
kinase with greater affinity or potency, respectively, compared to
at least one other kinase. In some embodiments, the compounds
described herein are selective inhibitors of PI3K.gamma. or
PI3K.delta. over PI3K.alpha. and/or PI3K.beta.. In some
embodiments, the compounds described herein are selective
inhibitors of PI3K.delta. (e.g., over PI3K.alpha., PI3K.beta. and
PI3K.gamma.). In some embodiments, the compounds described herein
are selective inhibitors of PI3K.gamma. (e.g., over PI3K,
PI3K.beta. and PI3K.delta.). In some embodiments, selectivity can
be at least about 2-fold, 5-fold, 10-fold, at least about 20-fold,
at least about 50-fold, at least about 100-fold, at least about
200-fold, at least about 500-fold or at least about 1000-fold.
Selectivity can be measured by methods routine in the art. In some
embodiments, selectivity can be tested at the K.sub.m ATP
concentration of each enzyme. In some embodiments, the selectivity
of compounds described herein can be determined by cellular assays
associated with particular PI3K kinase activity.
[0151] As used herein, the term "contacting" refers to the bringing
together of indicated moieties in an in vitro system or an in vivo
system. For example, "contacting" a PI3K with a compound described
herein includes the administration of a compound of the present
invention to an individual or patient, such as a human, having a
PI3K, as well as, for example, introducing a compound described
herein into a sample containing a cellular or purified preparation
containing the PI3K.
[0152] As used herein, the term "individual" or "patient," used
interchangeably, refers to any animal, including mammals,
preferably mice, rats, other rodents, rabbits, dogs, cats, swine,
cattle, sheep, horses, or primates, and most preferably humans.
[0153] As used herein, the phrase "therapeutically effective
amount" refers to the amount of active compound or pharmaceutical
agent that elicits the biological or medicinal response that is
being sought in a tissue, system, animal, individual or human by a
researcher, veterinarian, medical doctor or other clinician. In
some embodiments, the dosage of the compound, or a pharmaceutically
acceptable salt thereof, administered to a patient or individual is
about 1 mg to about 2 g, about 1 mg to about 1000 mg, about 1 mg to
about 500 mg, about 1 mg to about 100 mg, about 1 mg to 50 mg, or
about 50 mg to about 500 mg.
[0154] As used herein, the term "treating" or "treatment" refers to
one or more of (1) preventing the disease; for example, preventing
a disease, condition or disorder in an individual who may be
predisposed to the disease, condition or disorder but does not yet
experience or display the pathology or symptomatology of the
disease; (2) inhibiting the disease; for example, inhibiting a
disease, condition or disorder in an individual who is experiencing
or displaying the pathology or symptomatology of the disease,
condition or disorder (i.e., arresting further development of the
pathology and/or symptomatology); and (3) ameliorating the disease;
for example, ameliorating a disease, condition or disorder in an
individual who is experiencing or displaying the pathology or
symptomatology of the disease, condition or disorder (i.e.,
reversing the pathology and/or symptomatology) such as decreasing
the severity of disease.
Combination Therapies
[0155] One or more additional pharmaceutical agents such as, for
example, chemotherapeutics, anti-inflammatory agents, steroids,
immunosuppressants, as well as Bcr-Abl, Flt-3, EGFR, HER2, JAK
(e.g., JAK1 or JAK2), c-MET, VEGFR, PDGFR, cKit, IGF-1R, RAF, FAK,
Akt mTOR, PIM, and AKT (e.g., AKT1, AKT2, or AKT3) kinase
inhibitors such as, for example, those described in WO 2006/056399,
or other agents such as, therapeutic antibodies can be used in
combination with the compounds of the present invention for
treatment of PI3K-associated diseases, disorders or conditions. The
one or more additional pharmaceutical agents can be administered to
a patient simultaneously or sequentially.
[0156] Example antibodies for use in combination therapy include
but are not limited to Trastuzumab (e.g. anti-HER2), Ranibizumab
(e.g. anti-VEGF-A), Bevacizumab (trade name Avastin, e.g.
anti-VEGF, Panitumumab (e.g. anti-EGFR), Cetuximab (e.g.
anti-EGFR), Rituxan (anti-CD20) and antibodies directed to
c-MET.
[0157] One or more of the following agents may be used in
combination with the compounds of the present invention and are
presented as a non limiting list: a cytostatic agent, cisplatin,
doxorubicin, taxotere, taxol, etoposide, irinotecan, camptostar,
topotecan, paclitaxel, docetaxel, epothilones, tamoxifen,
5-fluorouracil, methoxtrexate, temozolomide, cyclophosphamide, SCH
66336, R115777, L778,123, BMS 214662, Iressa, Tarceva, antibodies
to EGFR, Gleevec.TM. intron, ara-C, adriamycin, cytoxan,
gemcitabine, Uracil mustard, Chlormethine, Ifosfamide, Melphalan,
Chlorambucil, Pipobroman, Triethylenemelamine,
Triethylenethiophosphoramine, Busulfan, Carmustine, Lomustine,
Streptozocin, Dacarbazine, Floxuridine, Cytarabine,
6-Mercaptopurine, 6-Thioguanine, Fludarabine phosphate,
oxaliplatin, leucovirin, ELOXATIN.TM. Pentostatine, Vinblastine,
Vincristine, Vindesine, Bleomycin, Dactinomycin, Daunorubicin,
Doxorubicin, Epirubicin, Idarubicin, Mithramycin, Deoxycoformycin,
Mitomycin-C, L-Asparaginase, Teniposide 17.alpha.-Ethinylestradiol,
Diethylstilbestrol, Testosterone, Prednisone, Fluoxymesterone,
Dromostanolone propionate, Testolactone, Megestrolacetate,
Methylprednisolone, Methyltestosterone, Prednisolone,
Triamcinolone, Chlorotrianisene, Hydroxyprogesterone,
Aminoglutethimide, Estramustine, Medroxyprogesteroneacetate,
Leuprolide, Flutamide, Toremifene, goserelin, Cisplatin,
Carboplatin, Hydroxyurea, Amsacrine, Procarbazine, Mitotane,
Mitoxantrone, Levamisole, Navelbene, Anastrazole, Letrazole,
Capecitabine, Reloxafine, Droloxafine, Hexamethylmelamine, Avastin,
herceptin, Bexxar, Velcade, Zevalin, Trisenox, Xeloda, Vinorelbine,
Porfimer, Erbitux, Liposomal, Thiotepa, Altretamine, Melphalan,
Trastuzumab, Lerozole, Fulvestrant, Exemestane, Fulvestrant,
Ifosfomide, Rituximab, C225, Campath, Clofarabine, cladribine,
aphidicolon, rituxan, sunitinib, dasatinib, tezacitabine, Smll,
fludarabine, pentostatin, triapine, didox, trimidox, amidox, 3-AP,
MDL-101,731, bendamustine (Treanda), ofatumumab, or GS-1101 (also
known as CAL-101).
[0158] Example chemotherapeutics include proteosome inhibitors
(e.g., bortezomib), thalidomide, revlimid, and DNA-damaging agents
such as melphalan, doxorubicin, cyclophosphamide, vincristine,
etoposide, carmustine, and the like.
[0159] Example steroids include coriticosteroids such as
dexamethasone or prednisone.
[0160] Example Bcr-Abl inhibitors include the compounds, and
pharmaceutically acceptable salts thereof, of the genera and
species disclosed in U.S. Pat. No. 5,521,184, WO 04/005281, and
U.S. Ser. No. 60/578,491.
[0161] Example suitable Flt-3 inhibitors include compounds, and
their pharmaceutically acceptable salts, as disclosed in WO
03/037347, WO 03/099771, and WO 04/046120.
[0162] Example suitable RAF inhibitors include compounds, and their
pharmaceutically acceptable salts, as disclosed in WO 00/09495 and
WO 05/028444.
[0163] Example suitable FAK inhibitors include compounds, and their
pharmaceutically acceptable salts, as disclosed in WO 04/080980, WO
04/056786, WO 03/024967, WO 01/064655, WO 00/053595, and WO
01/014402.
[0164] Example suitable mTOR inhibitors include compounds, and
their pharmaceutically acceptable salts, as disclosed in WO
2011/025889.
[0165] In some embodiments, the compounds of the invention can be
used in combination with one or more other kinase inhibitors
including imatinib, particularly for treating patients resistant to
imatinib or other kinase inhibitors.
[0166] In some embodiments, the compounds of the invention can be
used in combination with a chemotherapeutic in the treatment of
cancer, such as multiple myeloma, and may improve the treatment
response as compared to the response to the chemotherapeutic agent
alone, without exacerbation of its toxic effects. Examples of
additional pharmaceutical agents used in the treatment of multiple
myeloma, for example, can include, without limitation, melphalan,
melphalan plus prednisone [MP], doxorubicin, dexamethasone, and
Velcade (bortezomib). Further additional agents used in the
treatment of multiple myeloma include Bcr-Abl, Flt-3, RAF and FAK
kinase inhibitors. Additive or synergistic effects are desirable
outcomes of combining a PI3K inhibitor of the present invention
with an additional agent. Furthermore, resistance of multiple
myeloma cells to agents such as dexamethasone may be reversible
upon treatment with the PI3K inhibitor of the present invention.
The agents can be combined with the present compound in a single or
continuous dosage form, or the agents can be administered
simultaneously or sequentially as separate dosage forms.
[0167] In some embodiments, a corticosteroid such as dexamethasone
is administered to a patient in combination with the compounds of
the invention where the dexamethasone is administered
intermittently as opposed to continuously.
[0168] In some further embodiments, combinations of the compounds
of the invention with other therapeutic agents can be administered
to a patient prior to, during, and/or after a bone marrow
transplant or stem cell transplant.
Pharmaceutical Formulations and Dosage Forms
[0169] When employed as pharmaceuticals, the compounds described
herein can be administered in the form of pharmaceutical
compositions. These compositions can be prepared in a manner well
known in the pharmaceutical art, and can be administered by a
variety of routes, depending upon whether local or systemic
treatment is desired and upon the area to be treated.
[0170] Administration may be topical (including transdermal,
epidermal, ophthalmic and to mucous membranes including intranasal,
vaginal and rectal delivery), pulmonary (e.g., by inhalation or
insufflation of powders or aerosols, including by nebulizer;
intratracheal or intranasal), oral or parenteral. Parenteral
administration includes intravenous, intraarterial, subcutaneous,
intraperitoneal intramuscular or injection or infusion; or
intracranial, e.g., intrathecal or intraventricular,
administration. Parenteral administration can be in the form of a
single bolus dose, or may be, for example, by a continuous
perfusion pump. Pharmaceutical compositions and formulations for
topical administration may include transdermal patches, ointments,
lotions, creams, gels, drops, suppositories, sprays, liquids and
powders. Conventional pharmaceutical carriers, aqueous, powder or
oily bases, thickeners and the like may be necessary or
desirable.
[0171] This invention also includes pharmaceutical compositions
which contain, as the active ingredient, the compound described
herein or a pharmaceutically acceptable salt thereof, in
combination with one or more pharmaceutically acceptable carriers
(excipients). In some embodiments, the composition is suitable for
topical administration. In making the compositions of the
invention, the active ingredient is typically mixed with an
excipient, diluted by an excipient or enclosed within such a
carrier in the form of, for example, a capsule, sachet, paper, or
other container. When the excipient serves as a diluent, it can be
a solid, semi-solid, or liquid material, which acts as a vehicle,
carrier or medium for the active ingredient. Thus, the compositions
can be in the form of tablets, pills, powders, lozenges, sachets,
cachets, elixirs, suspensions, emulsions, solutions, syrups,
aerosols (as a solid or in a liquid medium), ointments containing,
for example, up to 10% by weight of the active compound, soft and
hard gelatin capsules, suppositories, sterile injectable solutions,
and sterile packaged powders.
[0172] In preparing a formulation, the active compound can be
milled to provide the appropriate particle size prior to combining
with the other ingredients. If the active compound is substantially
insoluble, it can be milled to a particle size of less than 200
mesh. If the active compound is substantially water soluble, the
particle size can be adjusted by milling to provide a substantially
uniform distribution in the formulation, e.g. about 40 mesh.
[0173] The compounds described herein may be milled using known
milling procedures such as wet milling to obtain a particle size
appropriate for tablet formation and for other formulation types.
Finely divided (nanoparticulate) preparations of the compounds
described herein can be prepared by processes known in the art,
e.g., see International App. No. WO 2002/000196.
[0174] Some examples of suitable excipients include lactose,
dextrose, sucrose, sorbitol, mannitol, starches, gum acacia,
calcium phosphate, alginates, tragacanth, gelatin, calcium
silicate, microcrystalline cellulose, polyvinylpyrrolidone,
cellulose, water, syrup, and methyl cellulose. The formulations can
additionally include: lubricating agents such as talc, magnesium
stearate, and mineral oil; wetting agents; emulsifying and
suspending agents; preserving agents such as methyl- and
propylhydroxy-benzoates; sweetening agents; and flavoring agents.
The compositions of the invention can be formulated so as to
provide quick, sustained or delayed release of the active
ingredient after administration to the patient by employing
procedures known in the art.
[0175] The compositions can be formulated in a unit dosage form,
each dosage containing from about 5 to about 1000 mg (1 g), more
usually about 100 to about 500 mg, of the active ingredient. The
term "unit dosage forms" refers to physically discrete units
suitable as unitary dosages for human subjects and other mammals,
each unit containing a predetermined quantity of active material
calculated to produce the desired therapeutic effect, in
association with a suitable pharmaceutical excipient.
[0176] In some embodiments, the compositions of the invention
contain from about 5 to about 50 mg of the active ingredient. One
having ordinary skill in the art will appreciate that this embodies
compositions containing about 5 to about 10, about 10 to about 15,
about 15 to about 20, about 20 to about 25, about 25 to about 30,
about 30 to about 35, about 35 to about 40, about 40 to about 45,
or about 45 to about 50 mg of the active ingredient.
[0177] In some embodiments, the compositions of the invention
contain from about 50 to about 500 mg of the active ingredient. One
having ordinary skill in the art will appreciate that this embodies
compositions containing about 50 to about 100, about 100 to about
150, about 150 to about 200, about 200 to about 250, about 250 to
about 300, about 350 to about 400, or about 450 to about 500 mg of
the active ingredient.
[0178] In some embodiments, the compositions of the invention
contain from about 500 to about 1000 mg of the active ingredient.
One having ordinary skill in the art will appreciate that this
embodies compositions containing about 500 to about 550, about 550
to about 600, about 600 to about 650, about 650 to about 700, about
700 to about 750, about 750 to about 800, about 800 to about 850,
about 850 to about 900, about 900 to about 950, or about 950 to
about 1000 mg of the active ingredient.
[0179] Similar dosages may be used of the compounds described
herein in the methods and uses of the invention.
[0180] The active compound can be effective over a wide dosage
range and is generally administered in a pharmaceutically effective
amount. It will be understood, however, that the amount of the
compound actually administered will usually be determined by a
physician, according to the relevant circumstances, including the
condition to be treated, the chosen route of administration, the
actual compound administered, the age, weight, and response of the
individual patient, the severity of the patient's symptoms, and the
like.
[0181] For preparing solid compositions such as tablets, the
principal active ingredient is mixed with a pharmaceutical
excipient to form a solid preformulation composition containing a
homogeneous mixture of a compound of the present invention. When
referring to these preformulation compositions as homogeneous, the
active ingredient is typically dispersed evenly throughout the
composition so that the composition can be readily subdivided into
equally effective unit dosage forms such as tablets, pills and
capsules. This solid preformulation is then subdivided into unit
dosage forms of the type described above containing from, for
example, about 0.1 to about 1000 mg of the active ingredient of the
present invention.
[0182] The tablets or pills of the present invention can be coated
or otherwise compounded to provide a dosage form affording the
advantage of prolonged action. For example, the tablet or pill can
comprise an inner dosage and an outer dosage component, the latter
being in the form of an envelope over the former. The two
components can be separated by an enteric layer which serves to
resist disintegration in the stomach and permit the inner component
to pass intact into the duodenum or to be delayed in release. A
variety of materials can be used for such enteric layers or
coatings, such materials including a number of polymeric acids and
mixtures of polymeric acids with such materials as shellac, cetyl
alcohol, and cellulose acetate.
[0183] The liquid forms in which the compounds and compositions of
the present invention can be incorporated for administration orally
or by injection include aqueous solutions, suitably flavored
syrups, aqueous or oil suspensions, and flavored emulsions with
edible oils such as cottonseed oil, sesame oil, coconut oil, or
peanut oil, as well as elixirs and similar pharmaceutical
vehicles.
[0184] Compositions for inhalation or insufflation include
solutions and suspensions in pharmaceutically acceptable, aqueous
or organic solvents, or mixtures thereof, and powders. The liquid
or solid compositions may contain suitable pharmaceutically
acceptable excipients as described supra. In some embodiments, the
compositions are administered by the oral or nasal respiratory
route for local or systemic effect. Compositions can be nebulized
by use of inert gases. Nebulized solutions may be breathed directly
from the nebulizing device or the nebulizing device can be attached
to a face mask, tent, or intermittent positive pressure breathing
machine. Solution, suspension, or powder compositions can be
administered orally or nasally from devices which deliver the
formulation in an appropriate manner.
[0185] Topical formulations can contain one or more conventional
carriers. In some embodiments, ointments can contain water and one
or more hydrophobic carriers selected from, for example, liquid
paraffin, polyoxyethylene alkyl ether, propylene glycol, white
Vaseline, and the like. Carrier compositions of creams can be based
on water in combination with glycerol and one or more other
components, e.g. glycerinemonostearate, PEG-glycerinemonostearate
and cetylstearyl alcohol. Gels can be formulated using isopropyl
alcohol and water, suitably in combination with other components
such as, for example, glycerol, hydroxyethyl cellulose, and the
like. In some embodiments, topical formulations contain at least
about 0.1, at least about 0.25, at least about 0.5, at least about
1, at least about 2, or at least about 5 wt % of the compound
described herein. The topical formulations can be suitably packaged
in tubes of, for example, 100 g which are optionally associated
with instructions for the treatment of the select indication, e.g.,
psoriasis or other skin condition.
[0186] The amount of compound or composition administered to a
patient will vary depending upon what is being administered, the
purpose of the administration, such as prophylaxis or therapy, the
state of the patient, the manner of administration, and the like.
In therapeutic applications, compositions can be administered to a
patient already suffering from a disease in an amount sufficient to
cure or at least partially arrest the symptoms of the disease and
its complications. Effective doses will depend on the disease
condition being treated as well as by the judgment of the attending
clinician depending upon factors such as the severity of the
disease, the age, weight and general condition of the patient, and
the like.
[0187] The compositions administered to a patient can be in the
form of pharmaceutical compositions described above. These
compositions can be sterilized by conventional sterilization
techniques, or may be sterile filtered. Aqueous solutions can be
packaged for use as is, or lyophilized, the lyophilized preparation
being combined with a sterile aqueous carrier prior to
administration. The pH of the compound preparations typically will
be between 3 and 11, more preferably from 5 to 9 and most
preferably from 7 to 8. It will be understood that use of certain
of the foregoing excipients, carriers, or stabilizers will result
in the formation of pharmaceutical salts.
[0188] The therapeutic dosage of a compound of the present
invention can vary according to, for example, the particular use
for which the treatment is made, the manner of administration of
the compound, the health and condition of the patient, and the
judgment of the prescribing physician. The proportion or
concentration of a compound described herein in a pharmaceutical
composition can vary depending upon a number of factors including
dosage, chemical characteristics (e.g., hydrophobicity), and the
route of administration. For example, the compounds described
herein can be provided in an aqueous physiological buffer solution
containing about 0.1 to about 10% w/v of the compound for
parenteral administration. Some typical dose ranges are from about
1 rig/kg to about 1 g/kg of body weight per day. In some
embodiments, the dose range is from about 0.01 mg/kg to about 100
mg/kg of body weight per day. The dosage is likely to depend on
such variables as the type and extent of progression of the disease
or disorder, the overall health status of the particular patient,
the relative biological efficacy of the compound selected,
formulation of the excipient, and its route of administration.
Effective doses can be extrapolated from dose-response curves
derived from in vitro or animal model test systems.
[0189] The compositions of the invention can further include one or
more additional pharmaceutical agents such as a chemotherapeutic,
steroid, anti-inflammatory compound, or immunosuppressant, examples
of which are listed herein.
Kits
[0190] The present invention also includes pharmaceutical kits
useful, for example, in the treatment or prevention of
PI3K-associated diseases or disorders, such as cancer, which
include one or more containers containing a pharmaceutical
composition comprising a therapeutically effective amount of a
compound described herein. Such kits can further include, if
desired, one or more of various conventional pharmaceutical kit
components, such as, for example, containers with one or more
pharmaceutically acceptable carriers, additional containers, etc.,
as will be readily apparent to those skilled in the art.
Instructions, either as inserts or as labels, indicating quantities
of the components to be administered, guidelines for
administration, and/or guidelines for mixing the components, can
also be included in the kit.
Synthesis
[0191] Compounds described herein, including salts thereof, can be
prepared using known organic synthesis techniques and can be
synthesized according to any of numerous possible synthetic routes.
In some embodiments, the compounds can be prepared as described in
U.S. patent application Ser. No. 13/601,349, filed Aug. 31, 2012,
which is incorporated herein by reference in its entirety.
[0192] The reactions for preparing compounds described herein can
be carried out in suitable solvents which can be readily selected
by one of skill in the art of organic synthesis. Suitable solvents
can be substantially non-reactive with the starting materials
(reactants), the intermediates, or products at the temperatures at
which the reactions are carried out, e.g., temperatures which can
range from the solvent's freezing temperature to the solvent's
boiling temperature. A given reaction can be carried out in one
solvent or a mixture of more than one solvent. Depending on the
particular reaction step, suitable solvents for a particular
reaction step can be selected by the skilled artisan.
[0193] Preparation of compounds described herein can involve the
protection and deprotection of various chemical groups. The need
for protection and deprotection, and the selection of appropriate
protecting groups, can be readily determined by one skilled in the
art. The chemistry of protecting groups can be found, for example,
in T. W. Greene and P. G. M. Wuts, Protective Groups in Organic
Synthesis, 3.sup.rd Ed., Wiley & Sons, Inc., New York (1999),
which is incorporated herein by reference in its entirety.
[0194] Reactions can be monitored according to any suitable method
known in the art. For example, product formation can be monitored
by spectroscopic means, such as nuclear magnetic resonance
spectroscopy (e.g., .sup.1H or .sup.13C), infrared spectroscopy,
spectrophotometry (e.g., UV-visible), mass spectrometry, or by
chromatographic methods such as high performance liquid
chromatography (HPLC), liquid chromatography-mass spectroscopy
(LCMS), or thin layer chromatography (TLC). Compounds can be
purified by those skilled in the art by a variety of methods,
including high performance liquid chromatography (HPLC)
("Preparative LC-MS Purification: Improved Compound Specific Method
Optimization" Karl F. Blom, Brian Glass, Richard Sparks, Andrew P.
Combs J. Combi. Chem. 2004, 6(6), 874-883, which is incorporated
herein by reference in its entirety) and normal phase silica
chromatography.
[0195] For example, compounds of Formula I can be formed as shown
in Scheme I. The compound (i) can be halogenated with
N-chlorosuccinamide, N-bromosuccinamide or N-iodosuccinamide to
give compound (ii) where X.sup.1.dbd.Cl, Br, or I. The halo group
of (ii) can be coupled to Cy-M, where M is a boronic acid, boronic
ester or an appropriately substituted metal (e.g., Cy-M is
Cy-B(OH).sub.2, Cy-Sn(Bu).sub.4, or Zn-Cy), under standard Suzuki
conditions or standard Stille conditions (e.g., in the presence of
a palladium(0) catalyst, such as
tetrakis(triphenylphosphine)palladium(0) and a base (e.g., a
bicarbonate or carbonate base) or standard Negishi conditions
(e.g., in the presence of a palladium(0) catalyst, such as
tetrakis(triphenylphosphine)palladium(0), to give a derivative of
formula (iii). Alternatively, Cy-M can be a cyclic amine (where M
is H and attached to the amine nitrogen) with coupling to compound
(ii) being performed by heating in base or under Buchwald
conditions (e.g., in the presence of a palladium(0) catalyst, such
as tetrakis(triphenylphosphine)palladium(0) and a base (e.g., an
alkoxide base)) to afford ketone (iii). Reduction of the ketone
(iii) with a suitable reagent, such as sodium tetrahydroborate can
furnish the alcohol (iv) which can be converted to a derivative
bearing a leaving group (v), (e.g., Lg is chloride via reaction
with cyanuric chloride or mesylate via reaction with
methanesulfonic anhydride). Finally, compound (v) can be reacted
with 3-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine ((vi)) under
basic conditions (e.g., NaH or CsCO.sub.3 or K.sub.2CO.sub.3) to
give a compound of Formula I (vii).
##STR00012##
[0196] Alternatively, compounds of Formula I can also be formed as
shown in Scheme II. The ketone compound (i) can be halogenated with
N-chlorosuccinamide, N-bromosuccinamide or N-iodosuccinamide to
give compound (ii) where X.sup.1.dbd.Cl, Br, or I. Ketone (ii) can
be reduced with a suitable reagent, such as sodium
tetrahydroborate, to give an alcohol (iii) which can be converted
to a derivative bearing a leaving group, (e.g., Lg is chloride via
reaction with cyanuric chloride or mesylate via reaction with
methanesulfonic anhydride) and then reacted with a heterocycle to
give a heterocyclic derivative (iv). The enantiomers of compound
(iv) can be separated by chiral chromatography to afford a single
enantiomer of heterocyclic compound (v). Finally, the halo group of
(v) can be coupled to Cy-M, where M is a boronic acid, boronic
ester or an appropriately substituted metal (e.g., Cy-M is
Cy-B(OH)2, Cy-Sn(Bu)4, or Zn-Cy), under standard Suzuki conditions
or standard Stille conditions (e.g., in the presence of a
palladium(0) catalyst, such as
tetrakis(triphenylphosphine)palladium(0) and a base (e.g., a
bicarbonate or carbonate base) or standard Negishi conditions
(e.g., in the presence of a palladium(0) catalyst, such as
tetrakis(triphenylphosphine)palladium(0), to give a derivative of
Formula I (vi).
##STR00013## ##STR00014##
[0197] Compounds of Formula I can also be formed as shown in Scheme
III. The phenol (i) can be alkylated using Mitsunobu conditions
(e.g., R'OH, DEAD, Ph.sub.3P) or standard alkylating conditions
(R'-Lg, Lg=leaving group) to afford ether derivatives (ii),
respectively. The halo group of (ii) can be coupled to Cy-M, where
M is a boronic acid, boronic ester or an appropriately substituted
metal (e.g., Cy-M is Cy-B(OH)2, Cy-Sn(Bu)4, or Zn-Cy), under
standard Suzuki conditions or standard Stille conditions (e.g., in
the presence of a palladium(0) catalyst, such as
tetrakis(triphenylphosphine)palladium(0) and a base (e.g., a
bicarbonate or carbonate base) or standard Negishi conditions
(e.g., in the presence of a palladium(0) catalyst, such as
tetrakis(triphenylphosphine)palladium(0), to give a derivative of
formula (iii). Alternatively, Cy-M can be a cyclic amine (where M
is H and attached to the amine nitrogen) with coupling to compound
(ii) being performed by heating in base or under Buchwald
conditions (e.g., in the presence of a palladium(0) catalyst, such
as tetrakis(triphenylphosphine)-palladium(0) and a base (e.g., an
alkoxide base)) to afford compounds of formula (iii). The ketone
(iii) can be transformed using similar methods as shown in Scheme I
and II to afford compounds of Formula I (iv). Alternatively, the
halo-ketone (ii) can be transformed using similar methods as shown
in Scheme I and II to afford halo intermediate (v). Suzuki, Stille,
Negishi or Buchwald coupling of Cy-M with halo intermediate (v) by
similar methods described in Schemes I and II can also afford
compounds of Formula I (vi).
##STR00015##
[0198] Ketones which can be used in the processes of Scheme I, II
and III, can be formed as shown in Scheme IV below. The carboxylic
acid (i) can be activated with a coupling agent (e.g. HBTU or HATU)
and then reacted with N, O-dimethylhydroxylamine to give a
N-methoxy-N-methylcarboxamide. The phenols can be alkylated using
Mitsunobu conditions (e.g., R.sup.2OH, DEAD, Ph.sub.3P) or standard
alkylating conditions (R.sup.2-Lg, Lg=leaving group) to afford the
ether derivatives (ii), respectively. The halo group of (ii)
(X.sup.1 is halo) can be coupled to Cy-M, where M is a boronic
acid, boronic ester or an appropriately substituted metal (e.g.,
Cy-M is Cy-B(OH)2, Cy-Sn(Bu)4, or Zn-Cy), under standard Suzuki
conditions or standard Stille conditions (e.g., in the presence of
a palladium(0) catalyst, such as
tetrakis(triphenylphosphine)palladium(O) and a base (e.g., a
bicarbonate or carbonate base) or standard Negishi conditions
(e.g., in the presence of a palladium(0) catalyst, such as
tetrakis(triphenylphosphine)palladium(0), to give a derivative of
formula (iii). Alternatively, Cy-M can be a cyclic amine (where M
is H and attached to the amine nitrogen) with coupling to compound
(ii) being performed by heating in base or under Buchwald
conditions (e.g., in the presence of a palladium(0) catalyst, such
as tetrakis(triphenylphosphine)palladium(0) and a base (e.g., an
alkoxide base)) to afford amides (iii). Reaction of compound (iii)
with a Grignard reagent of formula Me-MgX.sup.2 (X.sup.2=halo) can
give ketone (iv). The ketone (iv) can be transformed using similar
methods as shown in Scheme I, II and III to afford compounds of
Formula I.
##STR00016##
[0199] Ketones which can be used in the processes of Scheme I, II
and III, can also be formed as shown in Scheme V below. The halo
group (e.g., X.sup.1.dbd.I) of (i) can be coupled to a zinc reagent
Cy-Zn (e.g., such as tert-butyl 3-iodoazetidine-1-carboxylate with
Zn dust) under standard Knochel/Negishi conditions (e.g., in the
presence of a palladium(0) catalyst, such as tri-(2-furyl)phosphine
and tris(dibenzylideneacetone)dipalladium(0) and 1,2-dibromoethane
and chlorotrimethylsilane) to give a derivative of formula (ii).
The azetidine (ii) can be deprotected (e.g., Pg=Boc, using TFA) and
then reacted under alkylating, acylating or reductive amination
(e.g., R.sup.3X such as R3-Br, R.sup.3COCl, R.sup.3--SO.sub.2Cl,
R.sup.3N.dbd.C.dbd.O or R.sup.3CHO and a reducing agent) conditions
to afford ketone derivatives (iii) which can be converted to
compounds of Formula I (v) by similar methods shown in Schemes I,
II, and III). Alternatively, the ketone (ii) can be reduced with
suitable reagents (NaBH.sub.4 or Corey's chiral CBS catalyst to
give predominantly one isomer of the alcohol), the resulting
alcohol can be converted to a leaving group (e.g., Lg is chloride
via reaction with cyanuric chloride or mesylate via reaction with
methanesulfonic anhydride) and then the chloride or mesylate
reacted with an appropriate heterocycle (e.g., similar to methods
shown in Schemes I, II and III) to afford derivatives of formula
(iv). The protecting group on the amine can be removed under
standard conditions and then reacted under alkylating, acylating or
reductive amination conditions (e.g., R.sup.3X such as R.sup.3--Br,
R.sup.3COCl, R.sup.3--SO.sub.2Cl, R.sup.3N.dbd.C.dbd.O or
R.sup.3CHO and a reducing agent) to give compounds of Formula I
(v).
##STR00017##
[0200] Compound of Formula I can be synthesized from an acid
chloride compound (i) as illustrated in Scheme VI. Condensation of
an acid chloride (i) with malononitrile in the presence of a base,
such as sodium hydride, can give a dicyanoenol intermediate, which
can be O-methylated with an appropriate reagent, such as dimethyl
sulfate in the presence of an appropriate base, such as sodium
bicarbonate, to yield an enol ether (ii). Reaction of enol ether
(ii) with hydrazine dihydrochloride in the presence of a suitable
base, such as triethylamine, can give a pyrazole compound (iii).
Pyrazole compound (iii) can then be reacted with formamide to give
pyrazolopyrimidine (iv). Finally, compound (iv) can be reacted with
appropriate compound bearing a leaving group (v) under basic
conditions to give a compound of Formula I (vi).
##STR00018##
[0201] Compounds of Formula I can also be formed as shown in Scheme
VII. The halo group, X.sup.1, of (i) can be coupled to an alkene
(e.g., acrylate or acrylamide) under standard Heck conditions
(e.g., in the presence of a palladium(II) catalyst, such as
palladium acetate) to give an alkene of formula (ii). Reaction of
alkene (ii) with nitromethane in the presence of DBU can afford the
nitro derivative (iii) which can be reduced under standard
conditions (e.g., NiCl.sub.2/NaBH.sub.4) to give a free amine which
cyclizes to form lactam (iv). The lactam can be alkylated under
standard conditions (e.g., R.sup.3--X.sup.2, where X.sup.2=halo, in
the presence of a base, such as TEA or NaH) to give an
N-alkyl-lactam (v). Compounds of formula (v), and pyrrolidines
derived from the reduction of the lactam (v) with suitable reducing
agents, such as LiAlH.sub.4, can be converted to compounds of
Formula I using conditions described in Schemes I, II and III.
##STR00019## ##STR00020##
[0202] Compounds of Formula I can also be formed as shown in Scheme
VIII. The halo group X.sup.1 of (i) can be coupled to an alkene
boronic acid or ester under standard Suzuki conditions (e.g., in
the presence of a palladium(0) catalyst, such as
tetrakis(triphenylphosphine)palladium(O)) to give an alkene of
formula (ii). Epoxidation of alkene (ii) with mCPBA can afford the
epoxide (iii) which can be reacted with a secondary or primary
amine (amine=NH.sub.2R.sup.3) to give amino compounds of formula
(iv). Secondary or tertiary amine derivatives (iv) can be further
reacted with carbonyldiamidazole or phosgene to form an
oxazolidinone (v) or an acetyl-halide (e.g., chloro-acetylchloride
in the presence of base, such as TEA) to give the N-acyl derivative
which can be converted to the morpholinone derivative (vi) upon
treatment with a base (e.g., NaH). Compounds of formula (iv, v, and
vi) can be deprotected using standard conditions (e.g., compounds
protected with THP groups may be treated with an acid, such as TFA
or HCl) to give compounds of Formula I.
##STR00021##
[0203] Compounds of Formula I can also be formed as shown in Scheme
IX. Sharpless amino-hydroxylation of an alkene of formula (i) under
suitable conditions (A or B, as described in JACS, 2001, 123(9),
1862-1871 and J Org. Chem, 2011, 76, 358-372) can give either
amino-hydroxy isomer (ii) or (iii). Compounds (ii) and (iii) can be
reacted with carbonyldiamidazole or phosgene to form an
oxazolidinone (iv), or an acetyl-halide (e.g.,
chloro-acetylchloride in the presence of base, such as TEA) to give
an N-acyl derivative which can be converted to the morpholinone
derivative (v) upon treatment with a base (e.g., NaH). The
alternate amino-hydroxy isomer (iii) can be converted to
oxazolidinone and morpholinone derivatives as shown in Scheme
XV.
##STR00022##
[0204] Compounds of Formula I can be synthesized as shown in Scheme
X. The halo group (e.g., X.sup.1.dbd.Cl, Br, I) of (i) can be
converted to the boronate ester (ii) under standard conditions
(e.g., pinnacle boronate ester in the presence of a palladium(0)
catalyst, such as tetrakis(triphenylphosphine)palladium(0)).
Boronate (ii) can be reacted with an arylhalide or heteroarylhalide
(e.g., R.sup.3--X.sup.2) under Suzuki conditions (e.g., in the
presence of a palladium(0) catalyst, such as
tetrakis(triphenylphosphine)palladium(0) and a base, such as
Na.sub.2CO.sub.3) to give formula (iii). Formula (iii) can be
converted to Formula I using the reaction conditions described in
Schemes I, II or III.
##STR00023##
[0205] Compounds of Formula I, where R.sup.4.dbd.F or CN, can be
formed as shown in Scheme XI. Compound (i) can be acylated with a
suitable acylating reagent (e.g., Me-COCl) to form an ester which
can be rearranged under Lewis acid conditions (e.g., BF.sub.3/HOAc
complex) to afford ketone (ii). Ketone (ii) can be halogenated with
N-chlorosuccinamide, N-bromosuccinamide or N-iodosuccinamide to
give phenol (iii), where X.sup.1.dbd.Cl, Br, or I. Compound (iii)
can be alkylated (e.g. R.sup.2--X and a base, such as NaH or
Na.sub.2CO.sub.3; or under Mitsunobu conditions) to afford the
ether (iv). The fluoro group of (iv) can be displaced (e.g., with
NaCN or KCN) to give cyano derivative (v). The halo group of (v)
can be coupled to Cy-M, where M is a boronic acid, boronic ester or
an appropriately substituted metal (e.g., Cy-M is Cy-B(OH)2,
Cy-Sn(Bu)4, or Zn-Cy), under standard Suzuki conditions or standard
Stille conditions (e.g., in the presence of a palladium(0)
catalyst, such as tetrakis(triphenylphosphine)palladium(0) and a
base (e.g., a bicarbonate or carbonate base) or standard Negishi
conditions (e.g., in the presence of a palladium(0) catalyst, such
as tetrakis(triphenylphosphine)palladium(0)), to give a derivative
of formula (vi). Alternatively, Cy-M can be a cyclic amine (where M
is H and attached to the amine nitrogen) and coupled to compound
(v) by heating in base or under Buchwald conditions (e.g., in the
presence of a palladium(0) catalyst, such as
tetrakis(triphenylphosphine)palladium(0) and a base (e.g., an
alkoxide base)) to afford ketone (vi). Reduction of the ketone (vi)
with a suitable reagent, such as sodium tetrahydroborate or the
Corey CBS reagent can furnish the alcohol which can be converted to
a derivative bearing a leaving group, (e.g., Lg is chloride via
reaction with cyanuric chloride or mesylate via reaction with
methanesulfonic anhydride) and then reacted with
3-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine under basic conditions
(e.g., NaH or CsCO.sub.3 or K.sub.2CO.sub.3) to give a compound of
Formula I (viii). Alternatively, the last two steps can be inverted
so that the ketone (v) can be reduced to give an alcohol which is
converted to a leaving group and displaced with the heterocycle
first and then the Suzuki, Stille, Negishi or Buchwald coupling is
performed to give compounds of Formula I (viii). The fluoro
derivatives (iv) can also be converted to compounds of Formula I by
eliminating the cyanation step in Scheme XI.
##STR00024##
[0206] Compounds of Formula I can also be formed as shown in Scheme
XII. Compound (i) can be acylated with a suitable acylating reagent
(e.g., Me-COCl) to form an ester which can be rearranged under
Lewis acid conditions (e.g., AlCl.sub.3 or BF.sub.3/HOAc complex)
to afford ketone (ii). Halogenation of ketone (ii) using NX.sup.1S
(e.g., NX.sup.1S.dbd.N-chlorosuccinamide, N-bromosuccinamide or
N-iodosuccinamide) can give compound (iii), where X.sup.1.dbd.Cl,
Br, or I. The phenol can be converted to an ether (iv) using
standard conditions (e.g., inorganic base, such as K.sub.2CO.sub.3,
and an alkyl halide, such as Et-I). The halo group of (iv) can be
coupled to R.sup.3-M, where M is a boronic acid, boronic ester or
an appropriately substituted metal (e.g., R.sup.3-M is
R.sup.3--B(OH).sub.2, R.sup.3--Sn(Bu).sub.4, or Zn--R.sup.3 and
R.sup.3 is a substituted or unsubstituted olefin, such as vinyl)
under standard Suzuki conditions or standard Stille conditions
(e.g., in the presence of a palladium(0) catalyst, such as
tetrakis(triphenylphosphine)palladium(0) and a base (e.g., a
bicarbonate or carbonate base) to give a derivative of formula (v).
The alkene can then be dihydroxylated using Sharpless conditions to
afford the diol (vi). Enhancement of one enantiomer of the
secondary alcohol can be achieved using standard Sharpless
asymmetric dihydroxylation methods. The secondary alcohol can be
converted to the N-Boc protected amine via a 6 step process (e.g.
silyl protection (e.g., TBS-Cl and DIEA) of the primary alcohol,
mesylation of the secondary alcohol, displacement of the mesylate
with NaN.sub.3, reduction of the azide with Ph.sub.3P, Boc
protection of the resulting primary amine and then deprotection of
the silyl protecting group on the primary alcohol with TBAF) to
afford amino-alcohol (vii). The amino-alcohol (vii) can be
converted into the oxazolidinone by treatment with phosgene and
subsequent reduction of the ketone with a suitable reagent, such as
sodium tetrahydroborate or sodium borohydride can furnish the
alcohol (viii) which can be converted to a derivative bearing a
leaving group (ix) (e.g., Lg is chloride via reaction with cyanuric
chloride or mesylate via reaction with methanesulfonic anhydride).
Finally, compound (ix) can be reacted with an appropriate
heterocycle (x) (e.g., 3-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine
or 4-aminopyrido[2,3-d]pyrimidin-5(8H)-one) under basic conditions
(e.g., NaH or Cs.sub.2CO.sub.3 or K.sub.2CO.sub.3) to give a
compound of Formula I (xi).
##STR00025##
[0207] The invention will be described in greater detail by way of
specific examples. The following examples are offered for
illustrative purposes, and are not intended to limit the invention
in any manner. Those of skill in the art will readily recognize a
variety of non-critical parameters which can be changed or modified
to yield essentially the same results. The compounds of the
Examples have been found to be PI3K inhibitors according to at
least one assay described herein.
EXAMPLES
[0208] The example compounds below containing one or more chiral
centers were obtained in racemate form or as isomeric mixtures,
unless otherwise specified. Salt stoichiometry which is indicated
any of the products below is meant only to indicate a probable
stoichiometry, and should not be construed to exclude the possible
formation of salts in other stoichiometries. The abbreviations "h"
and "min" refer to hour(s) and minute(s), respectively.
Example 1.
1-{1-[5-Chloro-3-(1-isopropylazetidin-3-yl)-2-methoxy-4-methylp-
henyl]ethyl}-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine
bis(trifluoroacetate)
##STR00026##
[0209] Step 1.
1-(5-Chloro-2-hydroxy-3-iodo-4-methylphenyl)ethanone
[0210] To a stirred solution of
1-(5-chloro-2-hydroxy-4-methylphenyl)ethanone (from Oakwood, 50.0
g, 271 mmol) in acetic acid (300 mL) was added N-iodosuccinimide
(73.1 g, 325 mmol) and the resulting mixture was stirred on a
heating mantle between 60-80.degree. C. over 3.5 hours then cooled
to room temperature and stirred overnight. Water (500 mL) was added
to the mixture in portions, which caused a dark solid to form.
After stirring for 10 minutes, the solids were filtered, washing
with additional water. The light to dark brown solids were dried
under vacuum for 4 hours then air dried over the weekend to give
81.3 g (97%) of the desired product. LCMS calculated for
C.sub.9H.sub.9ClIO.sub.2 (M+H).sup.+: m/z=310.9; Found: 311.0.
.sup.1H NMR (300 MHz, CDCl.sub.3): .delta. 13.21 (s, 1H), 7.71 (s,
1H), 2.65 (s, 3H), 2.63 (s, 3H) ppm.
Step 2. 1-(5-Chloro-3-iodo-2-methoxy-4-methylphenyl)ethanone
[0211] Potassium carbonate (72.4 g, 524 mmol) was added to a
mixture of 1-(5-chloro-2-hydroxy-3-iodo-4-methylphenyl)ethanone
(81.3 g, 262 mmol) and methyl iodide (19.6 mL, 314 mmol) in
N,N-dimethylformamide (250 mL). The mixture was stirred at room
temperature for 4 hours. Water (500 mL) was added and stirred for
15 minutes. The dark solids were filtered and dried in vacuo to
give 42.3 g of the desired product. The filtrate was extracted with
EtOAc (4.times.). The combined filtrates were washed with water
(2.times.) and brine, dried (MgSO.sub.4), filtered and
concentrated. The solids were dried in vacuo to give an additional
37.2 g of the desired product. The product was used without further
purification. LCMS calculated for C.sub.10H.sub.11ClIO.sub.2
(M+H).sup.+: m/z=324.9; Found: 325.0. .sup.1H NMR (300 MHz,
CDCl.sub.3): .delta. 7.62 (s, 1H), 3.78 (s, 3H), 2.65 (s, 3H), 2.62
(s, 3H) ppm.
Step 3. tert-Butyl
3-(3-acetyl-5-chloro-2-methoxy-6-methylphenyl)azetidine-1-carboxylate
[0212] Zinc (1.71 g, 26.2 mmol) was suspended in
N,N-dimethylformamide (45.0 mL) and 1,2-dibromoethane (210 .mu.L,
2.5 mmol) was added. The mixture was heated at 60.degree. C. for 10
minutes and then cooled to room temperature. Chlorotrimethylsilane
(330 .mu.L, 2.6 mmol) was added and stirred at 60.degree. C. for 10
minutes and cooled to room temperature. A solution of tert-butyl
3-iodoazetidine-1-carboxylate (from Oakwood, 6.25 g, 22.1 mmol) in
N,N-dimethylformamide (5.0 mL) was then added and the mixture
stirred at room temperature for 1 hour.
1-(5-chloro-3-iodo-2-methoxy-4-methylphenyl)ethanone (5.00 g, 15.4
mmol), tri-(2-furyl)phosphine (358 mg, 1.54 mmol), and
tris(dibenzylideneacetone)dipalladium(0) (0.70 g, 0.77 mmol) were
added in order and the reaction mixture was warmed to 70.degree. C.
and stirred overnight. The mixture was cooled to room temperature
and partitioned between ethyl acetate (EtOAc) and sat. NH.sub.4Cl
solution. The layers were separated and the aqueous extracted
further with EtOAc (2.times.). The combined organics were washed
with water and brine, dried over MgSO.sub.4, and concentrated. The
residue was purified on silica gel, eluting with 0-30% EtOAc in
hexanes to give 3.0 g (55%) of the desired product as an orange
solid. LCMS calculated for C.sub.18H.sub.24ClNO.sub.4Na
(M+Na).sup.+: m/z=376.1; Found: 376.0. .sup.1H NMR (400 MHz,
CDCl.sub.3): .delta. 7.52 (s, 1H), 4.32, (m, 2H), 4.16 (m, 3H),
3.66 (s, 3H), 2.59 (s, 3H), 2.31 (s, 3H), 1.45 (s, 9H) ppm.
Step 4. tert-Butyl
3-[3-chloro-5-(1-hydroxyethyl)-6-methoxy-2-methylphenyl]azetidine-1-carbo-
xylate
[0213] To a solution of tert-butyl
3-(3-acetyl-5-chloro-2-methoxy-6-methylphenyl)azetidine-1-carboxylate
(1.3 g, 3.7 mmol) in methanol (20 mL) stirring at 0.degree. C. was
added sodium tetrahydroborate (0.167 g, 4.41 mmol). The mixture was
stirred at 0-5.degree. C. for 1 hour. The reaction was quenched
with water and extracted with EtOAc (3.times.). The combined
extracts were dried over MgSO.sub.4, filtered and concentrated to
give 1.3 g (100%) of the desired product. LCMS calculated for
C.sub.18H.sub.26ClNO.sub.4Na (M+Na).sup.+: m/z=378.2; Found: 378.1.
.sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 7.37 (s, 1H), 5.10 (q,
1H), 4.30 (m, 2H), 4.14 (m, 3H), 3.63 (s, 3H), 2.25 (s, 3H), 1.48
(d, 3H), 1.44 (s, 9H) ppm.
Step 5. tert-Butyl
3-[3-chloro-5-(1-chloroethyl)-6-methoxy-2-methylphenyl]azetidine-1-carbox-
ylate
[0214] Cyanuric chloride (from Aldrich, 1.22 g, 6.62 mmol) was
weighed into a flask and N,N-dimethylformamide (0.512 mL, 6.62
mmol) was added. After stirring for a few minutes a solution of
tert-butyl
3-[3-chloro-5-(1-hydroxyethyl)-6-methoxy-2-methylphenyl]azetidine-1-carbo-
xylate (1.5 g, 4.2 mmol) in methylene chloride (30 mL) was added.
The resulting mixture was stirred at room temperature overnight.
Water was added, and then diluted with dichloromethane. The layers
were separated and the organics were washed with sat. NaHCO.sub.3
solution, water, brine, dried over MgSO.sub.4, and concentrated.
The resulting residue was purified on silica gel, eluting with
0-35% EtOAc in hexanes to give the desired product (1.36 g, 86%).
LCMS calculated for C.sub.13H.sub.17ClNO (M-Cl-Boc+H).sup.+:
m/z=238.1; Found: 238.1. .sup.1H NMR (400 MHz, CDCl.sub.3): .delta.
7.46 (s, 1H), 5.44, (q, 1H), 4.32 (m, 2H), 4.18-4.10 (m, 3H), 3.67
(s, 3H), 2.27 (s, 3H), 1.79 (d, 3H), 1.44 (s, 9H) ppm.
Step 6. tert-Butyl
3-{3-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chlor-
o-2-methoxy-6-methylphenyl}azetidine-1-carboxylate
[0215] At room temperature, sodium hydride (0.32 g, 8.0 mmol) was
added to a suspension of
3-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (from ChemBridge, 0.59
g, 4.0 mmol) in N,N-dimethylformamide (20 mL). The resulting
mixture was stirred at room temperature for 25 minutes during which
time the suspension became a nearly clear solution. To the
resultant mixture was added a solution of tert-butyl
3-[3-chloro-5-(1-chloroethyl)-6-methoxy-2-methylphenyl]azetidine-1-carbox-
ylate (1.35 g, 3.61 mmol, from Example 1, step 5) in
N,N-dimethylformamide (10 mL). The mixture was stirred at
50.degree. C. overnight. After cooling, the mixture was diluted
with water and extracted with EtOAc (2.times.). The combined
extracts were washed with water and brine, dried over MgSO.sub.4
and concentrated. The resulting residue was purified on silica gel,
eluted with 0-10% MeOH in dichloromethane to give 1.03 g (59%) of
the desired product as a yellow gum. The racemic products were
applied on a Phenomenex Lux-Cellulose 2 column (21.1.times.250 mm,
5 micron particle size), eluting with 10% ethanol in hexanes at a
flow rate of 18 mL/min, 4 mg/injection, to provide two enantiomers.
The retention time of the first peak was 8.34 min and the retention
time for the second peak was 10.92 min. Peak 1 (463 mg), LCMS
calculated for C.sub.24H.sub.32ClN.sub.6O.sub.3(M+H).sup.+:
m/z=487.2; Found: 487.1. .sup.1H NMR (400 MHz, CDCl.sub.3): .delta.
8.21 (s, 1H), 7.37 (s, 1H), 6.30, (q, 1H), 5.40 (s, 2H), 4.23 (m,
2H), 4.17-4.00 (m, 3H), 3.57 (s, 3H), 2.58 (s, 3H), 2.16 (s, 3H),
1.76 (d, 3H), 1.37 (s, 9H) ppm.
Step 7.
1-[1-(3-Azetidin-3-yl-5-chloro-2-methoxy-4-methylphenyl)ethyl]-3-m-
ethyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine dihydrochloride
[0216] To a solution of tert-butyl
3-{3-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chlor-
o-2-methoxy-6-methylphenyl}azetidine-1-carboxylate (318 mg, 0.653
mmol) (peak 1 from above) in methylene chloride (3.2 mL) was added
4.0 M hydrogen chloride in 1,4-dioxane (1.6 mL, 6.5 mmol). The
resulting mixture was stirred at room temperature for 75 minutes.
The solvents were evaporated and the residue dried in vacuo to give
0.30 g of the desired product as the bis-HCl salt. LCMS calculated
for C.sub.19H.sub.24ClN.sub.6O (M+H).sup.+: m/z=387.2; Found:
387.1.
Step 8.
1-{-[5-Chloro-3-(1-isopropylazetidin-3-yl)-2-methoxy-4-methylpheny-
l]ethyl}-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine
bis(trifluoroacetate)
[0217] To a mixture of
1-[1-(3-azetidin-3-yl-5-chloro-2-methoxy-4-methylphenyl)ethyl]-3-methyl-1-
H-pyrazolo[3,4-d]pyrimidin-4-amine dihydrochloride (58 mg, 0.13
mmol), acetone (18.5 .mu.L, 0.252 mmol) and triethylamine (54.5
.mu.L, 0.391 mmol) in methylene chloride (1.0 mL) was added resin
of sodium triacetoxyborohydride (108 mg, 0.249 mmol). The resulting
mixture was stirred for 3 hours at room temperature. The mixture
was filtered and concentrated. The crude product was purified using
RP-HPLC (XBridge C18 column, eluting with a gradient of
acetonitrile/water containing 0.05% TFA, at flow rate of 30 mL/min)
to give 50 mg (60%) of the desired product as the TFA salt. LCMS
calculated for C.sub.22H.sub.30ClN.sub.6O (M+H).sup.+: m/z=429.2;
Found: 429.1. The product was isolated as a single enantiomer.
.sup.1H NMR (500 MHz, DMSO-d.sub.6): .delta. 8.47 (s, 1H), 7.46 (s,
1H), 6.29 (q, J=6.9 Hz, 1H), 4.52 (m, 2H), 4.21 (m, 1H), 4.15 (t,
J=9.8 Hz, 1H), 4.06 (t, J=9.7 Hz, 1H), 3.53 (s, 3H), 3.39-3.27 (m,
1H), 2.61 (s, 3H), 2.11 (s, 3H), 1.75 (d, J=6.8 Hz, 3H), 1.11 (dd,
J=6.0, 3.8 Hz, 6H) ppm.
Example 2.
1-{1-[3-(1-Acetylazetidin-3-yl)-5-chloro-2-methoxy-4-methylphen-
yl]ethyl}-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine
trifluoroacetate
##STR00027##
[0218] Step 1.
1-[1-(3-azetidin-3-yl-5-chloro-2-methoxy-4-methylphenyl)ethyl]-3-methyl-1-
H-pyrazolo[3,4-d]pyrimidin-4-amine dihydrochloride
[0219] To a solution of the racemic tert-butyl
3-{3-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chlor-
o-2-methoxy-6-methylphenyl}azetidine-1-carboxylate (146 mg, 0.300
mmol) (racemic intermediate from Example 1 Step 6) in methylene
chloride (1.5 mL) was added 4.0 M hydrogen chloride in 1,4-dioxane
(0.75 mL, 3.0 mmol). After stirred at rt for 2 h, the solvents were
evaporated and the resulting residue dried in vacuo to give 138 mg
of the desired product as the HCl salt. LCMS calculated for
C.sub.19H.sub.24ClN.sub.6O (M+H).sup.+: m/z=387.2; Found:
387.1.
Step 2.
1-{1-[3-(1-Acetylazetidin-3-yl)-5-chloro-2-methoxy-4-methylphenyl]-
ethyl}-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine
trifluoroacetate
[0220] To a mixture of
1-[1-(3-azetidin-3-yl-5-chloro-2-methoxy-4-methylphenyl)ethyl]-3-methyl-1-
H-pyrazolo[3,4-d]pyrimidin-4-amine dihydrochloride (20.0 mg, 0.0435
mmol, from Example 2, step 1) and triethylamine (30.3 .mu.L, 0.217
mmol) in methylene chloride (0.20 mL) was added acetyl chloride
(6.18 .mu.L, 0.0870 mmol). The resulting mixture was stirred
overnight at room temperature. The solvents were evaporated and the
crude purified using RP-HPLC (XBridge C18 column, eluting with a
gradient of acetonitrile/water containing 0.05% TFA, at flow rate
of 30 mL/min) to give the desired product as the TFA salt. The
product was isolated as a racemic mixture. LCMS calculated for
C.sub.21H.sub.26ClN.sub.6O.sub.2(M+H).sup.+: m/z=429.2; Found:
429.1. .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta. 8.35 (s, 1H),
7.34 (s, 1H), 6.26 (q, 1H), 4.50 (m, 1H), 4.28-4.20 (m, 2H), 4.01
(m, 1H), 3.88 (m, 1H), 3.52 (s, 3H), 2.58 (s, 3H), 2.18 (s, 3H),
1.75-1.71 (m, 6H) ppm.
Example 3.
1-{1-[5-Chloro-2-methoxy-4-methyl-3-(1-propionylazetidin-3-yl)p-
henyl]ethyl}-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine
trifluoroacetate
##STR00028##
[0222] This compound was prepared using procedures analogous to
those for Example 2, with propanoyl chloride instead of acetyl
chloride. The product was isolated as a racemic mixture. LCMS
calculated for C.sub.22H.sub.28ClN.sub.6O.sub.2(M+H).sup.+:
m/z=443.2; Found: 443.2. .sup.1H NMR (400 MHz, DMSO-d.sub.6)
.delta. 8.30 (s, 1H), 7.33 (s, 1H), 6.25 (q, 1H), 4.49 (m, 1H),
4.27-4.18 (m, 2H), 4.02 (m, 1H), 3.90 (m, 1H), 3.54 (s, 3H), 2.57
(s, 3H), 2.18 (s, 3H), 2.05 (q, 2H), 1.72 (d, 3H), 0.93 (t, 3H)
ppm.
Example 4.
1-(1-{5-Chloro-3-[1-(cyclopropylmethyl)azetidin-3-yl]-2-methoxy-
-4-methylphenyl}ethyl)-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine
bis(trifluoroacetate)
##STR00029##
[0224] This compound was prepared using procedures analogous to
those for Example 1, with racemic
1-[1-(3-azetidin-3-yl-5-chloro-2-methoxy-4-methylphenyl)ethyl]-3-methyl-1-
H-pyrazolo[3,4-d]pyrimidin-4-amine dihydrochloride from Example 2,
Step 1 and cyclopropanecarboxaldehyde (from Aldrich) instead of
acetone. The product was isolated as a racemic mixture. LCMS
calculated for C.sub.23H.sub.30ClN.sub.6O (M+H).sup.+: m/z=441.2;
Found: 441.1. .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta. 8.06 (s,
1H), 7.13 (s, 1H), 5.96 (q, 1H), 4.22 (m, 2H), 4.07 (m, 1H), 3.90
(m, 1H), 3.80 (m, 1H), 3.24 (s, 3H), 2.68 (t, 2H), 2.21 (s, 3H),
1.80 (s, 3H), 1.45 (d, 3H), 0.64 (m, 1H), 0.24 (m, 2H), 0.01 (m,
2H) ppm.
Example 5.
1-{1-[5-chloro-2-methoxy-4-methyl-3-(1-methylazetidin-3-yl)phen-
yl]ethyl}-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine
##STR00030##
[0226] This compound was prepared using procedures analogous to
those for Example 1, with racemic
1-[1-(3-azetidin-3-yl-5-chloro-2-methoxy-4-methylphenyl)ethyl]-3-methyl-1-
H-pyrazolo[3,4-d]pyrimidin-4-amine dihydrochloride from Example 2,
Step 1 and formaldehyde instead of acetone. The crude purified
using RP-HPLC (XBridge C18 column, eluting with a gradient of
acetonitrile/water containing 0.1% ammonium hydroxide, at flow rate
of 30 mL/min) to give the desired product. The product was isolated
as a racemic mixture. LCMS calculated for
C.sub.20H.sub.26ClN.sub.6O (M+H).sup.+: m/z=401.2; Found:
401.2.
Example 6.
1-{1-[5-Chloro-3-(1-ethylazetidin-3-yl)-2-methoxy-4-methylpheny-
l]ethyl}-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine
##STR00031##
[0228] This compound was prepared using procedures analogous to
those for Example 1, with racemic
1-[1-(3-azetidin-3-yl-5-chloro-2-methoxy-4-methylphenyl)ethyl]-3-methyl-1-
H-pyrazolo[3,4-d]pyrimidin-4-amine dihydrochloride from Example 2,
Step 1 and acetaldehyde instead of acetone. The crude purified
using RP-HPLC (XBridge C18 column, eluting with a gradient of
acetonitrile/water containing 0.1% ammonium hydroxide, at flow rate
of 30 mL/min) to give the desired product. The product was isolated
as a racemic mixture. LCMS calculated for
C.sub.21H.sub.28ClN.sub.6O (M+H).sup.+: m/z=415.2; Found:
415.1.
Example 7.
1-{1-[5-Chloro-3-(1-isobutylazetidin-3-yl)-2-methoxy-4-methylph-
enyl]ethyl}-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine
##STR00032##
[0230] This compound was prepared using procedures analogous to
those for Example 1, with racemic
1-[1-(3-azetidin-3-yl-5-chloro-2-methoxy-4-methylphenyl)ethyl]-3-methyl-1-
H-pyrazolo[3,4-d]pyrimidin-4-amine dihydrochloride from Example 2,
Step 1 and isobutyraldehyde instead of acetone. The crude purified
using RP-HPLC (XBridge C18 column, eluting with a gradient of
acetonitrile/water containing 0.1% ammonium hydroxide, at flow rate
of 30 mL/min) to give the desired product. The product was isolated
as a racemic mixture. LCMS calculated for
C.sub.23H.sub.32ClN.sub.6O (M+H).sup.+: m/z=443.2; Found: 443.1.
.sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 8.29 (s, 1H), 7.38 (s,
1H), 6.37 (q, 1H), 5.37 (s, 2H), 4.01 (m, 2H), 3.87 (m, 1H), 3.57
(s, 3H), 3.05 (t, 1H), 2.86 (t, 1H), 2.64 (s, 3H), 2.18 (d, 2H),
2.11 (s, 3H), 1.82 (d, 3H), 1.62 (m, 1H), 0.89 (d, 6H) ppm.
Example 8.
1-{1-[3-(1-sec-butylazetidin-3-yl)-5-chloro-2-methoxy-4-methylp-
henyl]ethyl}-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine
##STR00033##
[0232] This compound was prepared using procedures analogous to
those for Example 1, with racemic
1-[1-(3-azetidin-3-yl-5-chloro-2-methoxy-4-methylphenyl)ethyl]-3-methyl-1-
H-pyrazolo[3,4-d]pyrimidin-4-amine dihydrochloride from Example 2,
Step 1 and 2-butanone instead of acetone. The crude was purified
using RP-HPLC (XBridge C18 column, eluting with a gradient of
acetonitrile/water containing 0.1% ammonium hydroxide, at flow rate
of 30 mL/min) to give the desired product. The product was isolated
as a mixture of diastereomers. LCMS calculated for
C.sub.23H.sub.32ClN.sub.6O (M+H).sup.+: m/z=443.2; Found:
443.1.
Example 9.
1-(1-{5-Chloro-2-methoxy-3-[1-(2-methoxyethyl)azetidin-3-yl]-4--
methylphenyl}ethyl)-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine
##STR00034##
[0234] This compound was prepared using procedures analogous to
those for Example 1, with racemic
1-[1-(3-azetidin-3-yl-5-chloro-2-methoxy-4-methylphenyl)ethyl]-3-methyl-1-
H-pyrazolo[3,4-d]pyrimidin-4-amine dihydrochloride from Example 2,
Step 1 and methoxyacetaldehyde instead of acetone. The crude was
purified using RP-HPLC (XBridge C18 column, eluting with a gradient
of acetonitrile/water containing 0.1% ammonium hydroxide, at flow
rate of 30 mL/min) to give the desired product. The product was
isolated as a racemic mixture. LCMS calculated for
C.sub.22H.sub.30ClN.sub.6O.sub.2(M+H).sup.+: m/z=445.2; Found:
445.2.
Example 10.
3-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chlor-
o-2-methoxy-6-methylphenyl}-N-methylazetidine-1-carboxamide
##STR00035##
[0236] This compound was prepared using procedures analogous to
those for Example 2, with methyl isocyanate instead of acetyl
chloride The crude purified using RP-HPLC (XBridge C18 column,
eluting with a gradient of acetonitrile/water containing 0.1%
ammonium hydroxide, at flow rate of 30 mL/min) to give the desired
product. The product was isolated as a racemic mixture. LCMS
calculated for C.sub.21H.sub.27ClN.sub.7O.sub.2(M+H).sup.+:
m/z=444.2; Found: 444.2.
Example 11.
5-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chlor-
o-2-methoxy-6-methylphenyl}-N,N-dimethylpyridine-2-carboxamide
bis(trifluoroacetate)
##STR00036##
[0237] Step 1.
1-(3-Bromo-5-chloro-2-methoxy-4-methylphenyl)ethanone
[0238] To a stirred solution of
1-(5-chloro-2-methoxy-4-methylphenyl)ethanone (5.00 g, 25.2 mmol,
from Oakwood) in acetic acid (100 mL) was added N-bromosuccinimide
(4.93 g, 27.7 mmol) and the resulting mixture heated at 100.degree.
C. for 18 hours. After cooling to ambient temperature, the reaction
mixture was concentrated in vacuo, then neutralized with sat.
sodium bicarbonate, filtered off insoluble succinimide. The
filtrate was extracted with EtOAc. The combined organic layers were
washed with brine, dried over sodium sulfate, and then concentrated
to dryness under reduced pressure. The residue was purified on
silica gel, eluting with 0 to 50% EtOAc in hexanes, to give the
desired products (2.66 g, 38%). LCMS calculated for
C.sub.10H.sub.11BrClO.sub.2 (M+H).sup.+: m/z=277.0; found: 277.0.
.sup.1H NMR (DMSO-d.sub.6, 300 MHz): .delta. 7.70 (1H, s), 3.77
(3H, s), 2.57 (3H, s), 2.50 (3H, s) ppm.
Step 2.
5-(3-Acetyl-5-chloro-2-methoxy-6-methylphenyl)-N,N-dimethylpyridin-
e-2-carboxamide
[0239] To a mixture of
1-(3-bromo-5-chloro-2-methoxy-4-methylphenyl)ethanone (0.38 g, 1.4
mmol) and
N,N-dimethyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine--
2-carboxamide (from PepTech, 0.46 g, 1.6 mmol) in 1,4-dioxane (6
mL), potassium carbonate (0.38 g, 2.7 mmol) in water (2 mL) was
added. The reaction mixture was bubbled with N.sub.2.
Tetrakis(triphenylphosphine)palladium(0) (0.095 g, 0.082 mmol) was
added and the reaction was stirred overnight at 100.degree. C. The
reaction was diluted with water, extracted with EtOAc. The combined
organic layers were dried over MgSO.sub.4, concentrated and
purified on silica gel (eluting with 0-100% EtOAc in hexanes) to
give the desired product. LCMS calculated for
C.sub.18H.sub.20ClN.sub.2O.sub.3 (M+H).sup.+: m/z=347.1; Found:
347.1.
Step 3.
5-[3-chloro-5-(1-hydroxyethyl)-6-methoxy-2-methylphenyl]-N,N-dimet-
hylpyridine-2-carboxamide
[0240] To a solution of
5-(3-acetyl-5-chloro-2-methoxy-6-methylphenyl)-N,N-dimethylpyridine-2-car-
boxamide (106 mg, 0.306 mmol) in methanol (2 mL) cooled at
0.degree. C. was added sodium tetrahydroborate (14 mg, 0.37 mmol).
The mixture was stirred at room temperature for 1 hour, then
quenched with water, extracted with EtOAc. The organic layers were
dried over MgSO.sub.4 and concentrated to give crude alcohol. LCMS
calculated for C.sub.18H.sub.22ClN.sub.2O.sub.3(M+H).sup.+:
m/z=349.1; Found: 349.1.
Step 4.
5-[3-chloro-5-(1-chloroethyl)-6-methoxy-2-methylphenyl]-N,N-dimeth-
ylpyridine-2-carboxamide
[0241] Cyanuric chloride (85 mg, 0.46 mmol) was added to
N,N-dimethylformamide (0.036 mL, 0.46 mmol) at room temperature.
After the formation of a white solid (10 minutes), methylene
chloride (2 mL) was added, followed by
5-[3-chloro-5-(1-hydroxyethyl)-6-methoxy-2-methylphenyl]-N,N-dimethylpyri-
dine-2-carboxamide (115 mg, 0.330 mmol, from Example 11, step 3).
After the addition, the mixture was stirred at room temperature
overnight. Water was added, and then diluted with dichloromethane.
The organic phase was washed with sat. NaHCO.sub.3 solution, water
and brine, then dried over MgSO.sub.4, concentrated. The residue
was purified on silica gel (eluting with 0 to 80% EtOAc in hexanes)
to give the desired product (76 mg, 63%). LCMS calculated for
C.sub.18H.sub.21Cl.sub.2N.sub.2O.sub.2 (M+H).sup.+: m/z=367.1;
Found: 367.0.
Step 5.
5-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]--
5-chloro-2-methoxy-6-methylphenyl}-N,N-dimethylpyridine-2-carboxamide
bis(trifluoroacetate)
[0242] To a solution of
3-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (6.1 mg, 0.041 mmol)
in N,N-dimethylformamide (0.4 mL) was added sodium hydride (60%,
2.0 mg, 0.082 mmol) at 0.degree. C. and the mixture was stirred at
room temperature for 10 minutes. To the resultant mixture was added
a solution of
5-[3-chloro-5-(1-chloroethyl)-6-methoxy-2-methylphenyl]-N,N-dimethylpyrid-
ine-2-carboxamide (15.0 mg, 0.0408 mmol) in N,N-dimethylformamide
(0.2 mL). The mixture was stirred at room temperature overnight.
The crude mixture was purified on RP-HPLC (XBridge C18 column,
eluting with a gradient of acetonitrile/water containing 0.05% TFA,
at flow rate of 30 mL/min) to give the desired product as bis-TFA
salt. The product was isolated as a racemic mixture. LCMS
calculated for C.sub.24H.sub.27ClN.sub.7O.sub.2(M+H).sup.+:
m/z=480.2; Found: 480.1.
Example 13.
1-{1-[5-Chloro-4-fluoro-3-(1-isopropylazetidin-3-yl)-2-methoxyphenyl]ethy-
l}-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine
bis(trifluoroacetate)
##STR00037##
[0243] Step 1. 1-(5-Chloro-4-fluoro-2-hydroxyphenyl)ethanone
[0244] To 4-chloro-3-fluorophenol (from Aldrich, 20 g, 100 mmol)
was added acetyl chloride (14.1 mL, 199 mmol) under N.sub.2 with
stirring. The resulting mixture turned into a clear solution at
room temperature quickly and it was heated at 60.degree. C. for 2
hours. To the resultant mixture was added aluminum trichloride
(25.0 g, 187 mmol) in portions and the reaction mixture was heated
at 180.degree. C. for 30 minutes. The solids slowly dissolved at
high temperature. The reaction mixture was then cooled to room
temperature while the flask was swirled carefully in order for the
solid to form a thin layer inside the flask and then slowly
quenched with 1.0 N HCl (300 mL) while cooling in an ice-bath and
stirred overnight. The yellow precipitate was washed with water and
dried under vacuum to give the desired product as a yellow solid
(23.8 g), which was directly used in the next step without further
purification.
Step 2. 1-(5-Chloro-4-fluoro-2-hydroxy-3-iodophenyl)ethanone
[0245] A solution of 1-(5-chloro-4-fluoro-2-hydroxyphenyl)ethanone
(23.8 g, 126 mmol) in acetic acid (100 mL) was treated with
N-iodosuccinimide (34.1 g, 151 mmol) and stirred at 70.degree. C.
for 2 hr. The reaction mixture was concentrated, diluted with EtOAc
and quenched with sat. NaHCO.sub.3 solution until the bubbling
stopped. The organic layers were separated, washed with water,
dried over MgSO.sub.4 and stripped to give the desired product
which was used in the next step without further purification.
Step 3. 1-(5-Chloro-4-fluoro-3-iodo-2-methoxyphenyl)ethanone
[0246] 1-(5-Chloro-4-fluoro-2-hydroxy-3-iodophenyl)ethanone (13 g,
41 mmol) was dissolved in N,N-dimethylformamide (41.3 mL). Methyl
iodide (3.9 mL, 62 mmol) was added followed by potassium carbonate
(11 g, 83 mmol). The reaction was heated at 60.degree. C. for 1
hour. The mixture was cooled to room temperature, diluted with
ether. The organic layers were separated and combined, washed with
water, dried over MgSO.sub.4, concentrated and purified on silica
gel (eluting with 0 to 10% EtOAc in hexanes) to give the desired
product (10 g, 70%). LCMS calculated for C.sub.9HsClFIO.sub.2
(M+H).sup.+: m/z=328.9; Found: 328.9.
Step 4. tert-Butyl
3-(3-acetyl-5-chloro-6-fluoro-2-methoxyphenyl)azetidine-1-carboxylate
[0247] Zinc (0.682 g, 10.4 mmol) was suspended with
1,2-dibromoethane (0.0598 mL, 0.694 mmol) in N,N-dimethylformamide
(12 mL). The mixture was heated at 70.degree. C. for 10 minutes and
then cooled to room temperature. Chlorotrimethylsilane (0.088 mL,
0.69 mmol) was added dropwise and stirring was continued for 1
hour. A solution of tert-butyl 3-iodoazetidine-1-carboxylate (2.5
g, 8.7 mmol) in N,N-dimethylformamide (10 mL) was then added and
the mixture was heated at 40.degree. C. for 1 hour before a mixture
of 1-(5-chloro-4-fluoro-3-iodo-2-methoxyphenyl)ethanone (3.0 g, 9.1
mmol), tris(dibenzylideneacetone)dipalladium(0) (0.16 g, 0.17 mmol)
and tri-(2-furyl)phosphine (0.081 g, 0.35 mmol) in
N,N-dimethylformamide (20 mL) was added. The reaction mixture was
warmed to 70.degree. C. and stirred overnight. The mixture was then
cooled to room temperature and partitioned between ether and sat.
NH.sub.4Cl solution. The organic layers were washed with water,
dried over MgSO.sub.4, concentrated and purified on silica gel
(eluting with 0 to 25% EtOAc in hexanes) to give the desired
product (0.8 g). LCMS calculated for C.sub.17H.sub.21ClFNO.sub.4Na
(M+Na).sup.+: m/z=380.1; Found: 380.1.
Step 5. tert-Butyl
3-[3-chloro-2-fluoro-5-(1-hydroxyethyl)-6-methoxyphenyl]azetidine-1-carbo-
xylate
[0248] To a solution of tert-butyl
3-(3-acetyl-5-chloro-6-fluoro-2-methoxyphenyl)azetidine-1-carboxylate
(0.17 g, 0.48 mmol) in methanol (3 mL) cooled at 0.degree. C. was
added sodium tetrahydroborate (0.022 g, 0.57 mmol). The mixture was
stirred at room temperature for 1 hour, then quenched with water,
extracted with EtOAc. The organic layers were combined, dried over
MgSO.sub.4 and concentrated to give the crude alcohol (0.19 g).
LCMS calculated for C.sub.17H.sub.23ClFNO.sub.4Na (M+Na).sup.+:
m/z=382.1; Found: 382.0.
Step 6. tert-Butyl
3-[3-chloro-5-(1-chloroethyl)-2-fluoro-6-methoxyphenyl]azetidine-1-carbox-
ylate
[0249] Cyanuric chloride (140 mg, 0.78 mmol) was added to
N,N-dimethylformamide (0.059 mL, 0.77 mmol) at room temperature.
After the formation of a white solid (ca. 10 minutes), methylene
chloride (4 mL) was added, followed by tert-butyl
3-[3-chloro-2-fluoro-5-(1-hydroxyethyl)-6-methoxyphenyl]azetidine-1-carbo-
xylate (197 mg, 0.547 mmol). After addition, the mixture was
stirred at room temperature overnight. Water was added, and then
diluted with dichloromethane. The organic phases were washed with
sat. NaHCO.sub.3 solution, water and brine, dried over MgSO.sub.4,
and concentrated. The resulting residue was purified on silica gel
(eluting with 0 to 30% EtOAc in hexanes) to give the desired
product (110 mg, 53%).
Step 7. tert-Butyl
3-{3-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chlor-
o-6-fluoro-2-methoxyphenyl}azetidine-1-carboxylate
[0250] To a solution of
3-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (7.9 mg, 0.053 mmol)
in N,N-dimethylformamide (0.6 mL) was added sodium hydride (60%,
2.5 mg, 0.11 mmol) at 0.degree. C. and the mixture was stirred at
room temperature for 10 minutes. To the mixture was added a
solution of tert-butyl
3-[3-chloro-5-(1-chloroethyl)-2-fluoro-6-methoxyphenyl]azetidine-1-carbox-
ylate (20 mg, 0.053 mmol) in N,N-dimethylformamide (0.3 mL). The
reaction mixture was stirred at 35.degree. C. overnight, then
quenched with water, extracted with ether. The combined organic
layers were dried over MgSO.sub.4 and concentrated to afford the
desired product which was used in next step directly. LCMS
calculated for C.sub.23H.sub.29ClFN.sub.6O.sub.3 (M+H).sup.+:
m/z=491.2; Found: 491.1.
Step 8.
1-{1-[5-Chloro-4-fluoro-3-(1-isopropylazetidin-3-yl)-2-methoxyphen-
yl]ethyl}-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine
bis(trifluoroacetate)
[0251] A mixture of tert-butyl
3-{3-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chlor-
o-6-fluoro-2-methoxyphenyl}azetidine-1-carboxylate (14 mg, 0.028
mmol) in methylene chloride (0.2 mL) was treated with 4.0 M
hydrogen chloride in dioxane (0.2 mL, 0.8 mmol) at room temperature
for 1 hour and then the solvent removed to give
1-[1-(3-azetidin-3-yl-5-chloro-4-fluoro-2-methoxyphenyl)ethyl]-3-methyl-1-
H-pyrazolo[3,4-d]pyrimidin-4-amine HCl salt. To a mixture of the
crude HCl salt in acetonitrile (0.1 mL)/methanol (0.1
mL)/tetrahydrofuran (0.1 mL) was added N,N-diisopropylethylamine
(0.1 mL, 0.6 mmol), followed by acetone (0.050 mL, 0.68 mmol). The
mixture was stirred for 30 minutes before the addition of sodium
triacetoxyborohydride (0.030 g, 0.14 mmol). The reaction was
stirred at room temperature overnight, then quenched and purified
on RP-HPLC (XBridge C18 column, eluting with a gradient of
acetonitrile/water containing 0.05% TFA, at flow rate of 30 mL/min)
to give the desired product as TFA salt. The product was isolated
as a racemic mixture. LCMS calculated for
C.sub.21H.sub.27ClFN.sub.6O (M+H).sup.+: m/z=433.2; Found:
433.1.
Example 14.
5-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chlor-
o-2-ethoxy-6-methylphenyl}-N,N-dimethylpyridine-2-carboxamide
bis(trifluoroacetate)
##STR00038##
[0252] Step 1.
1-(5-Chloro-2-ethoxy-3-iodo-4-methylphenyl)ethanone
[0253] 1-(5-Chloro-2-hydroxy-3-iodo-4-methylphenyl)ethanone (18.9
g, 60.9 mmol) (from Example 1, Step 1) was dissolved in
N,N-dimethylformamide (60.8 mL). Iodoethane (7.3 mL, 91 mmol) was
added followed by potassium carbonate (17 g, 120 mmol). The
reaction was heated at 60.degree. C. for 1 hour. The mixture was
cooled to room temperature, diluted with ether. The organic layers
were combined, washed with water, dried over MgSO.sub.4,
concentrated and purified on silica gel (eluting with 0-10% EtOAc
in hexanes) to give the desired product (18.9 g, 91.7%). LCMS
calculated for Cl.sub.1H.sub.13ClIO.sub.2 (M+H).sup.+: m/z=339.0;
Found: 339.0.
Step 2.
5-(3-Acetyl-5-chloro-2-ethoxy-6-methylphenyl)-N,N-dimethylpyridine-
-2-carboxamide
[0254] To a mixture of
1-(5-chloro-2-ethoxy-3-iodo-4-methylphenyl)ethanone (0.69 g, 2.0
mmol) and
N,N-dimethyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine-2-ca-
rboxamide (0.68 g, 2.4 mmol) in 1,4-dioxane (10 mL), potassium
carbonate (0.56 g, 4.1 mmol) in water (3 mL, 200 mmol) was added.
The reaction was bubbled with N.sub.2.
Tetrakis(triphenylphosphine)palladium(0) (0.24 g, 0.20 mmol) was
added and N.sub.2 was bubbled. Reaction was stirred overnight at
95.degree. C. The reaction was diluted with water, extracted with
EtOAc. The combined organic layers were dried over MgSO.sub.4,
concentrated and purified on silica gel (eluting with 0 to 90%
EtOAc in hexanes) to give the desired product (0.6 g, 82%). LCMS
calculated for C.sub.19H.sub.22ClN.sub.2O.sub.3(M+H).sup.+:
m/z=361.1; Found: 361.0.
Step 3.
5-[3-Chloro-6-ethoxy-5-(1-hydroxyethyl)-2-methylphenyl]-N,N-dimeth-
ylpyridine-2-carboxamide
[0255] To a solution of
5-(3-acetyl-5-chloro-2-ethoxy-6-methylphenyl)-N,N-dimethylpyridine-2-carb-
oxamide (0.60 g, 1.7 mmol) in methanol (10 mL) cooled at 0.degree.
C. was added sodium tetrahydroborate (0.075 g, 2.0 mmol). The
mixture was stirred at room temperature for 1 hour, then quenched
with water, extracted with EtOAc. The extracts were dried over
MgSO.sub.4 and concentrated to give crude alcohol (0.6 g). LCMS
calculated for C.sub.19H.sub.24ClN.sub.2O.sub.3(M+H).sup.+:
m/z=363.1; Found: 363.0.
Step 4.
5-[3-Chloro-5-(1-chloroethyl)-6-ethoxy-2-methylphenyl]-N,N-dimethy-
lpyridine-2-carboxamide
[0256] Cyanuric chloride (0.43 g, 2.3 mmol) was added to
N,N-dimethylformamide (0.18 mL, 2.3 mmol) at room temperature.
After the formation of a white solid (10 minutes), methylene
chloride (10 mL) was added, followed by
5-[3-chloro-6-ethoxy-5-(1-hydroxyethyl)-2-methylphenyl]-N,N-dimethylpyrid-
ine-2-carboxamide (0.6 g, 2 mmol). After addition, the mixture was
stirred at room temperature overnight, then diluted with
dichloromethane and washed with sat. NaHCO.sub.3 solution. The
organic layers were dried over MgSO.sub.4, concentrated. The
residue was purified on silica gel (eluting with 0 to 50% EtOAc in
hexanes) to give the desired product (0.58, 90%). LCMS calculated
for C.sub.19H.sub.23Cl.sub.2NO.sub.2 (M+H).sup.+: m/z=381.1; Found:
381.0.
Step 5.
5-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]--
5-chloro-2-ethoxy-6-methylphenyl}-N,N-dimethylpyridine-2-carboxamide
bis(trifluoroacetate)
[0257] To a solution of
3-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (47 mg, 0.31 mmol) in
N,N-dimethylformamide (3 mL) was added sodium hydride (60%, 12.6
mg, 0.524 mmol) at 0.degree. C. and the resultant mixture was
stirred at room temperature for 10 minutes. To the mixture was
added a solution of
5-[3-chloro-5-(1-chloroethyl)-6-ethoxy-2-methylphenyl]-N,N-dimethylpyridi-
ne-2-carboxamide (100 mg, 0.3 mmol, from Example 14, step 4) in
N,N-dimethylformamide (1 mL). The reaction was stirred at
35.degree. C. overnight. The reaction was quenched and applied on
RP-HPLC (XBridge C18 column, eluting with a gradient of
acetonitrile/water containing 0.05% TFA, at flow rate of 30 mL/min)
to give the desired product as bis-TFA salt. The product was
isolated as a racemic mixture. LCMS calculated for
C.sub.25H.sub.29ClN.sub.7O.sub.2(M+H).sup.+: m/z=494.2; Found:
494.1.
Example 16.
4-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chlor-
o-2-methoxy-6-methylphenyl}-N,N-dimethylpyridine-2-carboxamide
##STR00039##
[0258] Step 1. 1-(3-Bromo-5-chloro-2-methoxy-4-methylphenyl)
ethanol
[0259] Sodium tetrahydroborate (0.31 g, 8.1 mmol) was added to a
mixture of 1-(3-bromo-5-chloro-2-methoxy-4-methylphenyl)ethanone
(from Example 11, Step 1) (1.5 g, 5.4 mmol) in methanol (25 mL) at
0.degree. C. and the resultant reaction mixture was stirred at room
temperature for 1 hour. The solvent was removed and the resulting
residue was diluted with ethyl acetate, washed with sat.
NaHCO.sub.3, water, brine, then dried over Na.sub.2SO.sub.4,
filtered and concentrated. The crude product was purified by silica
gel chromatography, eluting with 0 to 40% EtOAc in hexanes (0.30 g,
90%).
Step 2.
4-[3-Chloro-5-(1-hydroxyethyl)-6-methoxy-2-methylphenyl]pyridine-2-
-carbonitrile
[0260] A mixture of
1-(3-bromo-5-chloro-2-methoxy-4-methylphenyl)ethanol (0.30 g, 1.1
mmol),
4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine-2-carbonitrile
(from Combi-Blocks, 0.27 g, 1.2 mmol), sodium carbonate (230 mg,
2.1 mmol) and
[1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II),
complex with dichloromethane (1:1) (100 mg, 0.13 mmol) in
acetonitrile (8 mL)/water (2 mL) was degassed and then refilled
with N.sub.2. The reaction was stirred at 95.degree. C. for 2
hours, then cooled and diluted with ethyl acetate, washed with sat.
NaHCO.sub.3, water, brine, dried over Na.sub.2SO.sub.4, filtered
and concentrated. The crude product was purified by silica gel
chromatography, eluting with 0 to 40% EtOAc in hexanes (0.249 g,
75%). LCMS calculated for
C.sub.16H.sub.16ClN.sub.2O.sub.2(M+H).sup.+: m/z=303.1; Found:
303.0.
Step 3.
4-[3-Chloro-5-(1-chloroethyl)-6-methoxy-2-methylphenyl]pyridine-2--
carbonitrile
[0261] A mixture of cyanuric chloride (170 mg, 0.94 mmol) and
N,N-dimethylformamide (73 .mu.L, 0.94 mmol) was stirred at room
temperature for 10 minutes and then a solution of
4-[3-chloro-5-(1-hydroxyethyl)-6-methoxy-2-methylphenyl]pyridine-2-carbon-
itrile (190 mg, 0.628 mmol) in methylene chloride (4 mL) was added
and the reaction was stirred at room temperature overnight. The
mixture was diluted with methylene chloride, washed with sat.
NaHCO.sub.3, water, brine, dried over Na.sub.2SO.sub.4, filtered
and concentrated. The crude product was used directly in the next
step without purification (121 mg, 60%). LCMS calculated for
C.sub.16H.sub.15Cl.sub.2N.sub.2O (M+H).sup.+: m/z=321.0; Found:
321.0.
Step 4.
4-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]--
5-chloro-2-methoxy-6-methylphenyl}pyridine-2-carbonitrile
[0262] Sodium hydride (20 mg, 0.50 mmol) was added to a mixture of
4-[3-chloro-5-(1-chloroethyl)-6-methoxy-2-methylphenyl]pyridine-2-carboni-
trile (90 mg, 0.28 mmol),
3-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (63 mg, 0.42 mmol) in
N,N-dimethylformamide (4 mL) and the reaction was stirred et
30.degree. C. overnight. The mixture was cooled, treated with water
and then filtered to provide the desired product. LCMS calculated
for C.sub.22H.sub.21ClN.sub.7O (M+H).sup.+: m/z=434.1; Found:
434.2.
Step 5.
4-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]--
5-chloro-2-methoxy-6-methylphenyl}pyridine-2-carboxylic acid
[0263] Sodium hydroxide (1.0 M) in water (0.70 mL, 0.70 mmol) was
added to a mixture of
4-{3-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chlor-
o-2-methoxy-6-methylphenyl}pyridine-2-carbonitrile (0.060 g, 0.14
mmol) in ethanol (1.0 mL) and the resultant mixture was heated at
95.degree. C. for 6 hours. At this time, conc. HCl was added to
adjust pH to .about.3. The solvent was removed and the residue was
used in the next step without further purification. LCMS calculated
for C.sub.22H.sub.22ClN.sub.6O.sub.3(M+H).sup.+: m/z=453.1; Found:
453.2.
Step 6.
4-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]--
5-chloro-2-methoxy-6-methylphenyl}-N,N-dimethylpyridine-2-carboxamide
[0264] 2.0 M Dimethylamine in THF (0.14 mL, 0.28 mmol) was added to
a solution of
4-{3-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chlor-
o-2-methoxy-6-methylphenyl}pyridine-2-carboxylic acid (9.6 mg,
0.021 mmol) and benzotriazol-1-yloxytris(dimethylamino)phosphonium
hexafluorophosphate (10 mg, 0.03 mmol) in N,N-dimethylformamide
(0.7 mL) at room temperature followed by addition of triethylamine
(8.8 .mu.L, 0.064 mmol). The reaction was stirred for 1 hour. The
crude mixture was purified using RP-HPLC (XBridge C18 column,
eluting with a gradient of acetonitrile/water containing 0.1%
ammonium hydroxide, at flow rate of 30 mL/min) to give the desired
product. The product was isolated as a racemic mixture. LCMS
calculated for C.sub.24H.sub.27ClN.sub.7O.sub.2(M+H).sup.+:
m/z=480.2; Found: 480.2.
Example 17.
4-(3-(1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chlor-
o-2-methoxy-6-methylphenyl)-N-methylpicolinamide
##STR00040##
[0266] This compound was prepared using procedures analogous to
those for Example 16, Step 6, with 2.0 M solution of methylamine in
THF replacing 2.0 M dimethylamine in THF. The product was isolated
as a racemic mixture. LCMS calculated for
C.sub.23H.sub.25ClN.sub.7O.sub.2(M+H).sup.+: m/z=466.2; Found:
466.2.
Example 18.
4-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chlor-
o-2-methoxy-6-methylphenyl}-N-(2-hydroxyethyl)pyridine-2-carboxamide
##STR00041##
[0268] This compound was prepared using procedures analogous to
those for Example 16, Step 6, with ethanolamine replacing 2.0 M
dimethylamine in THF. The product was isolated as a racemic
mixture. LCMS calculated for
C.sub.24H.sub.27ClN.sub.7O.sub.3(M+H).sup.+: m/z=496.2; Found:
496.2.
Example 19.
4-{3-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chlor-
o-2-methoxy-6-methylphenyl}-N-(2-hydroxyethyl)-N-methylpyridine-2-carboxam-
ide
##STR00042##
[0270] This compound was prepared using procedures analogous to
those for Example 16, Step 6, with 2-(methylamino)ethanol replacing
2.0 M dimethylamine in THF. The product was isolated as a racemic
mixture. LCMS calculated for
C.sub.25H.sub.29ClN.sub.7O.sub.3(M+H).sup.+: m/z=510.2; Found:
510.2.
Example 20.
2-(4-(3-(1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-ch-
loro-2-methoxy-6-methylphenyl)-1H-pyrazol-1-yl)ethanol
##STR00043##
[0271] Step 1.
3-Bromo-1-chloro-5-(1-chloroethyl)-4-methoxy-2-methylbenzene
[0272] A mixture of cyanuric chloride (1.7 g, 9.2 mmol) and
N,N-dimethylformamide (710 .mu.L, 9.2 mmol) was stirred at room
temperature for 10 minutes and then a solution of
1-(3-bromo-5-chloro-2-methoxy-4-methylphenyl)ethanol (from Example
16, Step 1) (1.72 g, 6.15 mmol) in methylene chloride (34 mL) was
added and the reaction was stirred at room temperature overnight.
The mixture was diluted with methylene chloride, washed with sat.
NaHCO.sub.3, water, brine, dried over Na.sub.2SO.sub.4, filtered
and concentrated. The crude product was purified by silica gel
chromatography, eluting with 0 to 10% EtOAc in hexanes (1.01 g,
60%).
Step 2.
1-[1-(3-Bromo-5-chloro-2-methoxy-4-methylphenyl)ethyl]-3-methyl-1H-
-pyrazolo[3,4-d]pyrimidin-4-amine
[0273] Sodium hydride (36 mg, 0.91 mmol) was added to a mixture of
3-bromo-1-chloro-5-(1-chloroethyl)-4-methoxy-2-methylbenzene (150
mg, 0.503 mmol), 3-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (110
mg, 0.76 mmol) in N,N-dimethylformamide (8 mL) and the reaction was
stirred at 30.degree. C. overnight. The mixture was diluted with
methylene chloride, washed with sat. NaHCO.sub.3, water, brine,
dried over Na.sub.2SO.sub.4, filtered and concentrated. The crude
product was purified by silica gel chromatography, eluting with 0
to 70% EtOAc in CH.sub.2Cl.sub.2 (103 mg, 50%). LCMS calculated for
C.sub.16H.sub.18BrClN.sub.5O (M+H).sup.+: m/z=410.0; Found: 410.
The racemic products were applied on a Phenomenex Lux-Cellulose 1
column (21.1.times.250 mm, 5 micron particle size), eluting with 5%
ethanol in hexanes at a flow rate of 18 mL/min, .about.13
mg/injection, to provide two enantiomers.
Step 3.
1-(2-{[tert-Butyl(dimethyl)silyl]oxy}ethyl)-4-(4,4,5,5-tetramethyl-
-1,3,2-dioxaborolan-2-yl)-1H-pyrazole
[0274] Potassium tert-butoxide (1.0 M) in THF (0.60 mL, 0.60 mmol)
was added to a solution of
4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (0.1 g,
0.5 mmol) in N,N-dimethylformamide (1.5 mL) at 0.degree. C. The
reaction mixture was stirred at room temperature for 5 minutes,
then cooled to 0.degree. C. and treated with
(2-bromoethoxy)(tert-butyl)dimethylsilane (0.2 mL, 0.8 mmol). The
reaction was stirred at room temperature overnight, then diluted
with ethyl acetate, washed with sat. NaHCO.sub.3, water, brine,
dried over Na.sub.2SO.sub.4, filtered and concentrated to provide
the crude product which was purified by silica gel chromatography
eluting with 0 to 30% EtOAc in hexanes. Calculated for
C.sub.17H.sub.34BN.sub.2O.sub.3Si (M+H).sup.+: m/z=353.2; Found:
353.1.
Step 4.
2-(4-(3-(1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethy-
l)-5-chloro-2-methoxy-6-methylphenyl)-1H-pyrazol-1-yl) ethanol
[0275] A mixture of
1-[1-(3-bromo-5-chloro-2-methoxy-4-methylphenyl)ethyl]-3-methyl-1H-pyrazo-
lo[3,4-d]pyrimidin-4-amine (0.026 g, 0.062 mmol) (chiral pure,
first peak from Step 2),
1-(2-{[tert-butyl(dimethyl)silyl]oxy}ethyl)-4-(4,4,5,5-tetramethyl-1,3,2--
dioxaborolan-2-yl)-1H-pyrazole (0.024 g, 0.069 mmol), sodium
carbonate (13 mg, 0.12 mmol) and
[1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II),
complex with dichloromethane (1:1) (6.1 mg, 0.0075 mmol) in
acetonitrile (0.5 mL)/water (0.1 mL) was degassed and then refilled
with N.sub.2. The reaction mixture was stirred at 95.degree. C. for
2 hours, then treated with conc. HCl (0.1 mL) and then stirred at
room temperature for 1 hour. The crude mixture was purified using
RP-HPLC (XBridge C18 column, eluting with a gradient of
acetonitrile/water containing 0.1% ammonium hydroxide, at flow rate
of 30 mL/min) to give the desired product. The product was isolated
as a single enantiomer. LCMS calculated for
C.sub.21H.sub.25ClN.sub.7O.sub.2(M+H).sup.+: m/z=442.2; Found:
442.2.
Example 21.
3'-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5'-chloro-
-3-fluoro-2'-methoxy-N,N,6'-trimethylbiphenyl-4-carboxamide
trifluoroacetate
##STR00044##
[0276] Step 1. Methyl
3'-(1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5'-chloro-
-3-fluoro-2'-methoxy-6'-methylbiphenyl-4-carboxylate
[0277] A mixture of
1-[1-(3-bromo-5-chloro-2-methoxy-4-methylphenyl)ethyl]-3-methyl-1H-pyrazo-
lo[3,4-d]pyrimidin-4-amine (60 mg, 0.15 mmol, chiral pure, first
peak from Example 20, Step 2),
[3-fluoro-4-(methoxycarbonyl)phenyl]boronic acid (from
Combi-Blocks, 0.041 g, 0.20 mmol), sodium carbonate (36 mg, 0.34
mmol) and
[1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II),
complex with dichloromethane (1:1) (6 mg, 0.007 mmol) in
acetonitrile (1.2 mL)/water (0.3 mL) was vacuumed and then refilled
with N.sub.2. The reaction was stirred at 95.degree. C. for 2
hours. Then solvent was removed and the crude mixture was purified
by silica gel chromatography, eluting with 0 to 70% EtOAc in
CH.sub.2Cl.sub.2, to give the desired product (54 mg, 75%). LCMS
calculated for C.sub.24H.sub.24ClFN.sub.5O.sub.3 (M+H).sup.+:
m/z=484.2; Found: 484.1.
Step 2.
3'-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5'-
-chloro-3-fluoro-2'-methoxy-6'-methylbiphenyl-4-carboxylic acid
[0278] Lithium hydroxide, monohydrate (13 mg, 0.31 mmol) was added
to a solution of methyl
3'-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5'-chloro-
-3-fluoro-2'-methoxy-6'-methylbiphenyl-4-carboxylate made above
(0.030 g, 0.062 mmol) in methanol (0.2 mL)/tetrahydrofuran (0.2
mL)/water (0.09 mL). The reaction was stirred at room temperature
for 1.5 h, then treated with conc. HCl (60 uL) to adjust pH to 2.
The solvent was removed to provide the crude product which was used
in next step without further purification. LCMS calculated for
C.sub.23H.sub.22ClFN.sub.5O.sub.3 (M+H).sup.+: m/z=470.1; Found:
470.2.
Step 3.
3'-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5'-
-chloro-3-fluoro-2'-methoxy-N,N, 6'-trimethylbiphenyl-4-carboxamide
trifluoroacetate
[0279] 2.0 M Dimethylamine in THF (0.1 mL, 0.2 mmol) was added to a
solution of
3'-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5'-chloro-
-3-fluoro-2'-methoxy-6'-methylbiphenyl-4-carboxylic acid (12 mg,
0.026 mmol) made above and
benzotriazol-1-yloxytris(dimethylamino)phosphonium
hexafluorophosphate (20 mg, 0.04 mmol) in N,N-dimethylformamide
(0.7 mL) at room temperature followed by addition of triethylamine
(11 .mu.L, 0.077 mmol). The reaction was stirred for 1 hour,
quenched with water. The crude mixture was applied on RP-HPLC
(XBridge C18 column, eluting with a gradient of acetonitrile/water
containing 0.05% TFA, at flow rate of 30 mL/min) to give the
desired product as TFA salt. The product was isolated as a single
enantiomer. LCMS calculated for
C.sub.25H.sub.27ClFN.sub.6O.sub.2(M+H).sup.+: m/z=497.2; Found:
497.2.
Example 22.
3'-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5'-chloro-
-3-fluoro-2'-methoxy-N,6'-dimethylbiphenyl-4-carboxamide
trifluoroacetate
##STR00045##
[0281] This compound was prepared using procedures analogous to
those for Example 21, Step 3, with 2.0 M methylamine in THF
replacing 2.0 M dimethylamine in THF. The product was isolated as a
single enantiomer. LCMS calculated for
C.sub.24H.sub.25ClFN.sub.6O.sub.2(M+H).sup.+: m/z=483.2; Found:
483.2.
Example 23.
5-(3-(1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chlor-
o-2-methoxy-6-methylphenyl)-N-(2-hydroxyethyl)picolinamide
trifluoroacetate
##STR00046##
[0282] Step 1.
5-[3-Chloro-5-(1-hydroxyethyl)-6-methoxy-2-methylphenyl]pyridine-2-carbon-
itrile
[0283] A mixture of
1-(3-bromo-5-chloro-2-methoxy-4-methylphenyl)ethanol (0.15 g, 0.54
mmol),
5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine-2-carbonitrile
(from Frontier, 0.14 g, 0.59 mmol), sodium carbonate (110 mg, 1.1
mmol) and
[1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II),
complex with dichloromethane (1:1) (52 mg, 0.064 mmol) in
acetonitrile (4 mL)/water (1 mL) was degassed and then refilled
with N.sub.2. The reaction was stirred at 95.degree. C. for 2 h,
cooled, diluted with ethyl acetate, washed with sat. NaHCO.sub.3,
water, brine, and then dried over Na.sub.2SO.sub.4, filtered and
concentrated. The crude product was purified by silica gel
chromatography, eluting with 0 to 40% EtOAc in hexanes, to give the
desired product (114 mg, 70%). LCMS calculated for
C.sub.16H.sub.16ClN.sub.2O.sub.2(M+H).sup.+: m/z=303.1; Found:
303.0.
Step 2.
5-[3-Chloro-5-(1-chloroethyl)-6-methoxy-2-methylphenyl]pyridine-2--
carbonitrile
[0284] A mixture of cyanuric chloride (170 mg, 0.94 mmol) and
N,N-dimethylformamide (73 .mu.L, 0.94 mmol) was stirred at room
temperature for 10 minutes and then a solution of
5-[3-chloro-5-(1-hydroxyethyl)-6-methoxy-2-methylphenyl]pyridine-2-carbon-
itrile (190 mg, 0.628 mmol) in methylene chloride (4 mL) was added
and the reaction was stirred at room temperature overnight. The
mixture was diluted with methylene chloride, washed with sat.
NaHCO.sub.3, water, brine, dried over Na.sub.2SO.sub.4, then
filtered and concentrated. The resultant crude product was used
directly in the next step without further purification (110 mg,
55%). LCMS calculated for C.sub.16H.sub.15Cl.sub.2N.sub.2O
(M+H).sup.+: m/z=321.0; Found: 321.0.
Step 3.
5-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]--
5-chloro-2-methoxy-6-methylphenyl}pyridine-2-carbonitrile
[0285] Sodium hydride (20 mg, 0.50 mmol) was added to a mixture of
5-[3-chloro-5-(1-chloroethyl)-6-methoxy-2-methylphenyl]pyridine-2-carboni-
trile (90 mg, 0.28 mmol),
3-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (63 mg, 0.42 mmol) in
N,N-dimethylformamide (4 mL) and the reaction was stirred at
30.degree. C. overnight. The mixture was treated with water and
then filtered to provide the desired product. LCMS calculated for
C.sub.22H.sub.21ClN.sub.7O (M+H).sup.+: m/z=434.1; Found:
434.2.
Step 4.
5-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]--
5-chloro-2-methoxy-6-methylphenyl}pyridine-2-carboxylic acid
[0286] Sodium hydroxide (1.0 M) in water (0.70 mL, 0.70 mmol) was
added to a mixture of
5-{3-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chlor-
o-2-methoxy-6-methylphenyl}pyridine-2-carbonitrile (0.060 g, 0.14
mmol) in ethanol (1.0 mL). The reaction was heated at 95.degree. C.
for 6 hours, followed by the addition of conc. HCl to adjust pH to
.about.3. The solvent was removed and the resultant residue was
used in the next step without further purification. LCMS calculated
for C.sub.22H.sub.22ClN.sub.6O.sub.3(M+H).sup.+: m/z=453.1; Found:
453.2.
Step 5.
5-(3-(1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)--
5-chloro-2-methoxy-6-methylphenyl)-N-(2-hydroxyethyl)picolinamide
trifluoroacetate
[0287] Ethanolamine (15 .mu.L, 0.25 mmol) was added to a solution
of
5-{3-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chlor-
o-2-methoxy-6-methylphenyl}pyridine-2-carboxylic acid (9.6 mg,
0.021 mmol) and benzotriazol-1-yloxytris(dimethylamino)phosphonium
hexafluorophosphate (10 mg, 0.03 mmol) in N,N-dimethylformamide
(0.7 mL) at room temperature followed by addition of triethylamine
(8.8 .mu.L, 0.064 mmol). The reaction was stirred for 1 hour, and
then quenched with water. The crude mixture was applied on RP-HPLC
(XBridge C18 column, eluting with a gradient of acetonitrile/water
containing 0.05% TFA, at flow rate of 30 mL/min) to give the
desired product as TFA salt. The product was isolated as a racemic
mixture. LCMS calculated for C.sub.24H.sub.27ClN.sub.7O.sub.3
(M+H).sup.+: m/z=496.2; Found: 496.2.
Example 24.
4-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chlor-
o-2-methoxy-6-methylphenyl}-N-(2-hydroxyethyl)-N-methylpyridine-2-carboxam-
ide trifluoroacetate
##STR00047##
[0289] This compound was prepared using procedures analogous to
those for Example 23, with 2-(methylamino)ethanol replacing
ethanolamine. The product was isolated as a racemic mixture. LCMS
calculated for C.sub.25H.sub.29ClN.sub.7O.sub.3 (M+H).sup.+:
m/z=510.2; Found: 510.2.
Example 40.
4-{3-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-cyano-
-2-methoxy-6-methylphenyl}-N-(2-hydroxyethyl)-N-methylpyridine-2-carboxami-
de
##STR00048##
[0291] Catalyst preformation: Anhydrous dimethylacetamide (DMA) was
purged with a gentle stream of N.sub.2 for 30 minutes prior to use.
A 50 mM solution of H.sub.2SO.sub.4 was prepared with 10 mL
dimethylacetamide and 26.8 .mu.L of conc. H.sub.2SO.sub.4 and then
purged with N.sub.2 for 10 minutes. To an 8 mL vial equipped with a
magnetic stir bar and septum cap were added Pd(OAc).sub.2 (22.5 mg,
100 .mu.mol) and
2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl (95.3 mg, 200
.mu.mol). The vial was evacuated and filled with N.sub.2 three
times, purged with a gentle stream of N.sub.2 for 10 minutes.
H.sub.2SO.sub.4 (2.0 mL, 50 mM in DMA) was added, and the catalyst
mixture was stirred in an oil bath at 80.degree. C. for 30 minutes
to give a homogeneous coffee-brown solution.
[0292] The above catalyst (0.05 mL) was added to a mixture of
4-{3-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chlor-
o-2-methoxy-6-methylphenyl}-N-(2-hydroxyethyl)-N-methylpyridine-2-carboxam-
ide (from Example 19) (4.0 mg, 0.0078 mmol), zinc (0.22 mg, 0.0034
mmol) and zinc cyanide (0.92 mg, 0.0078 mmol) in
N,N-dimethylacetamide (0.1 mL). The mixture was degassed and then
the reaction was heated at 120.degree. C. for 1.5 hours. The crude
mixture was applied on RP-HPLC (XBridge C18 column, eluting with a
gradient of acetonitrile/water containing 0.1% ammonium hydroxide,
at flow rate of 30 mL/min) to give the desired product. The product
was isolated as a racemic mixture. LCMS calculated for
C.sub.26H.sub.29N.sub.8O.sub.3 (M+H).sup.+: m/z=501.2; Found:
501.2.
Example 41.
5-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[4,3-c]pyridin-1-yl)ethyl]-5-chloro--
2-methoxy-6-methylphenyl}-N,N-dimethylpyridine-2-carboxamide
bis(trifluoroacetate)
##STR00049##
[0293] Step 1:
N-(2,4-Dimethoxybenzyl)-3-methyl-1H-pyrazolo[4,3-c]pyridin-4-amine
[0294] A solution of 4-chloro-3-methyl-1H-pyrazolo[4,3-c]pyridine
(330 mg, 1.9 mmol) and 1-(2,4-dimethoxyphenyl)methanamine (0.58 mL,
3.9 mmol) in 1-butanol was heated in the microwave at 150.degree.
C. for 40 minutes. Purification via preparative LCMS (XBridge C18
column, eluting with a gradient of acetonitrile/water containing
0.1% ammonium hydroxide, at flow rate of 60 mL/min) gave the
desired product (240 mg, 42%). LCMS for
C.sub.16H.sub.19N.sub.4O.sub.2 (M+H).sup.+: m/z=299.1; Found:
299.2.
Step 2:
5-[3-Chloro-5-(1-{4-[(2,4-dimethoxybenzyl)amino]-3-methyl-1H-pyraz-
olo[4,3-c]pyridin-1-yl}ethyl)-6-methoxy-2-methylphenyl]-N,N-dimethylpyridi-
ne-2-carboxamide
[0295] A solution of
N-(2,4-dimethoxybenzyl)-3-methyl-1H-pyrazolo[4,3-c]pyridin-4-amine
(110 mg, 0.37 mmol) in N,N-dimethylformamide (2 mL) was treated
with sodium hydride (30 mg, 0.75 mmol) and stirred at 20.degree. C.
for 30 minutes. The reaction mixture was treated with a solution of
5-[3-chloro-5-(1-chloroethyl)-6-methoxy-2-methylphenyl]-N,N-dimethylpyrid-
ine-2-carboxamide (130 mg, 0.34 mmol) in N,N-dimethylformamide (1
mL) and heated at 50.degree. C. overnight. The reaction mixture was
diluted with water and extracted with ethyl acetate (2.times.). The
combined organic extracts were washed with water and brine, dried
with magnesium sulfate, filtered, and concentrated to a crude
residue. Purification via preparative LCMS (XBridge C18 column,
eluting with a gradient of acetonitrile/water containing 0.1%
ammonium hydroxide, at flow rate of 60 mL/min) gave the desired
product (110 mg, 49%). LCMS for C.sub.34H.sub.38ClN.sub.6O.sub.4
(M+H).sup.+: m/z=629.3; Found: 629.1.
Step 3:
5-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[4,3-c]pyridin-1-yl)ethyl]-5--
chloro-2-methoxy-6-methylphenyl}-N,N-dimethylpyridine-2-carboxamide
bis(trifluoroacetate)
[0296] A solution of
5-[3-chloro-5-(1-{4-[(2,4-dimethoxybenzyl)amino]-3-methyl-1H-pyrazolo[4,3-
-c]pyridin-1-yl}ethyl)-6-methoxy-2-methylphenyl]-N,N-dimethylpyridine-2-ca-
rboxamide (85 mg, 0.14 mmol) in methylene chloride (2 mL) was
treated with trifluoroacetic acid (2 mL) and stirred at 20.degree.
C. for 3 hours and at 40.degree. C. for 20 minutes. Purification
via preparative LCMS (XBridge C18 column, eluting with a gradient
of acetonitrile/water containing 0.1% trifluoroacetic acid, at flow
rate of 60 mL/min) gave the desired product (44 mg, 46%). The
product was isolated as a racemic mixture. LCMS for
C.sub.25H.sub.28ClN.sub.6O.sub.2(M+H).sup.+: m/z=479.2; Found:
479.0. .sup.1H NMR (300 MHz, DMSO-d.sub.6): .delta. 12.8 (br s,
0.5H), 8.50 (br s, 0.5H), 8.37 (br s, 2H), 7.91-7.86 (m, 0.5H),
7.80-7.75 (m, 0.5H), 7.68-7.58 (m, 3H), 7.17 (d, J=7.3 Hz, 1H),
6.19 (q, J=6.9 Hz, 1H), 3.04 (s, 3H), 3.01 (s, 3H), 2.94 (s, 3H),
2.61 (s, 3H), 2.05 (s, 3H), 1.83 (d, J=6.9 Hz, 3H).
Example 43.
4-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-6-chloro-3-
-ethoxy-2-[5-(methylsulfonyl)pyridin-3-yl]benzonitrile
##STR00050##
[0297] Step 1.
1-(3-bromo-5-chloro-4-fluoro-2-hydroxyphenyl)ethanone
[0298] 1-(5-Chloro-4-fluoro-2-hydroxyphenyl)ethanone (e.g., from
Example 13, step 1) (20.0 g, 101 mmol, 1.00 eq) and a 50% aqueous
sulfuric acid (120 mL) were added to the flask. The resulting
mixture was heated to 60.degree. C. in a water bath with stirring.
N-Bromosuccinimide (21.52 g, 120.9 mmol, 1.20 eq) was added in
three portions [7.0 g+7.0 g+7.52 g] in 8 minute intervals. After
the reaction mixture was heated at 60.degree. C. for 3 hours, the
reaction was complete. The reaction mixture was diluted with water
(160 ml) and dichloromethane (DCM) (300 ml), and the mixture was
stirred for 0.5 hour. The organic layer was separated and the
aqueous layer was extracted with dichloromethane (100 ml). The
combined organic layers were washed with 1 N HCl (100 ml.times.2),
water (100 ml), brine (60 ml), and concentrated under reduced
pressure to afford the crude product (29.1 g) as a yellowish solid.
The crude product was dissolved in HOAc (100 ml) and then diluted
with water (200 ml) under stirring. The resulting mixture was
stirred for 20 min at room temperature and the product was
collected by filtration and dried to give
1-(3-bromo-5-chloro-4-fluoro-2-hydroxyphenyl)ethanone (21.8 g,
80.9%) as a yellowish solid. .sup.1H-NMR (300 MHz, CDCl.sub.3)
.delta. 13.18 (s, 1H, --OH), 7.78 (d, J=7.78 Hz, 1H), 2.63 (s,
3H).
Step 2. 4-Acetyl-2-bromo-6-chloro-3-ethoxybenzonitrile
[0299] 1-(3-Bromo-5-chloro-4-fluoro-2-hydroxyphenyl)ethanone (2.0
g, 7.5 mmol) was combined with potassium cyanide (0.58 g, 9.0 mmol)
in N,N-dimethylformamide (16 mL, 210 mmol) and heated to 85.degree.
C. in an oil bath. After heating for 18 hours, the reaction was
allowed to cool to room temperature and iodoethane (0.90 mL, 11
mmol) and potassium carbonate (2.1 g, 15 mmol) were added. The
reaction was heated to 65.degree. C. and monitored by LC/MS. After
heating for 3 hours the reaction was complete and allowed to cool
to room temperature, then taken up in ethyl acetate and washed with
water, brine, and dried over magnesium sulfate. The resultant
solution was concentrated to give the crude product as a dark oil.
The product was purified by flash column chromatography on silica
gel eluting hexane: ethyl acetate gradient to give
4-acetyl-2-bromo-6-chloro-3-ethoxybenzonitrile (1.15 gm, 50%) as a
solid residue, LCMS calculated for
C.sub.11H.sub.9BrClNO.sub.2(M+H).sup.+: m/z=301.9, 303.9; found:
(no ionization)
Step 3.
2-Bromo-6-chloro-3-ethoxy-4-(1-hydroxyethyl)benzonitrile
[0300] Sodium tetrahydroborate (38 mg, 0.99 mmol) was added to a
mixture of 4-acetyl-2-bromo-6-chloro-3-ethoxybenzonitrile (200 mg,
0.7 mmol) in methanol (5 mL, 100 mmol) at 0.degree. C. The reaction
was stirred at room temperature for 1 hour, concentrated and
partitioned between water and EtOAc. The combined organic layers
were washed with brine, dried over MgSO.sub.4, filtered and
concentrated to give crude
2-bromo-6-chloro-3-ethoxy-4-(1-hydroxyethyl)benzonitrile as a clear
oil (0.15 gm, 100%), LCMS calculated for
CuIHiiBrClNO.sub.2(M+H).sup.+: m/z=303.9, 305.9; found: 304.0,
305.9.
Step 4. 2-Bromo-6-chloro-4-(1-chloroethyl)-3-ethoxybenzonitrile
[0301] Cyanuric chloride (0.11 g, 0.59 mmol) was dissolved in
N,N-dimethylformamide (3 mL, 40 mmol). After stirring for a few
minutes, a solution of
2-bromo-6-chloro-3-ethoxy-4-(1-hydroxyethyl)benzonitrile (150 mg,
0.49 mmol) in methylene chloride (3 mL, 50 mmol) was added. The
resulting mixture was stirred at room temperature overnight. The
reaction was partitioned between water and dichloromethane. The
organic layer was washed with sat. NaHCO.sub.3 solution, water,
brine, dried over MgSO.sub.4, and concentrated. The crude product
was purified by flash column chromatography, eluting a gradient of
0-30% EtOAc/Hexane to give
2-bromo-6-chloro-4-(1-chloroethyl)-3-ethoxybenzonitrile (0.12 gm,
75%) as a semisolid, LCMS calculated for
C.sub.11H.sub.10BrCl.sub.2NO (M+H).sup.+: m/z=323.9, 320.9; found:
(poor ionization).
Step 5.
4-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-2-b-
romo-6-chloro-3-ethoxybenzonitrile
[0302] Sodium hydride (16 mg, 0.41 mmol) was added to a mixture of
3-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (33 mg, 0.22 mmol) in
N,N-dimethylformamide (3 mL, 40 mmol) and was stirred for 10
minutes. 2-bromo-6-chloro-4-(1-chloroethyl)-3-ethoxybenzonitrile
(60 mg, 0.2 mmol) in N,N-dimethylformamide (2 mL) was added and the
reaction was stirred at 50.degree. C. overnight. The mixture was
diluted with methylene chloride, washed with sat'd NaHCO.sub.3,
water, brine, dried over Na.sub.2SO.sub.4, filtered and
concentrated. The product was purified by flash column
chromatography eluting with CH.sub.2Cl.sub.2/MeOH 0-10%, to give
4-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-2-bromo-6--
chloro-3-ethoxybenzonitrile (0.05 gm, 60%) as a solid, LCMS
calculated for C.sub.7H.sub.6BrClN.sub.6O (M+H).sup.+: m/z=437.0,
435.0; found: 436.9, 434.7.
Step 6.
4-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-6-c-
hloro-3-ethoxy-2-[5-(methylsulfonyl)pyridin-3-yl]benzonitrile
[0303] To a mixture of
4-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-2-bromo-6--
chloro-3-ethoxybenzonitrile (20 mg, 0.04 mmol) and
3-(methylsulfonyl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-
e (19 mg, 0.069 mmol) in acetonitrile (2 mL, 40 mmol) was added
sodium carbonate (10 mg, 0.09 mmol) in water (0.5 mL, 30 mmol). The
reaction was degassed with bubbling nitrogen.
[1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) complex
with dichloromethane (1:1) (2 mg, 0.002 mmol) was added and
degassed more with N.sub.2. Reaction was heated at 100.degree. C.
for 2 hours. The crude product was purified on preparative LC-MS
(acetonitrile, water, TFA) to give the desired product (0.004 g,
20%) as white amorphous solid. The product was isolated as a
racemic mixture. LCMS calculated for
C.sub.23H.sub.22ClN.sub.7O.sub.3S (M+H).sup.+: m/z=512.1; found:
512.2. .sup.1H NMR (500 MHz, DMSO) .delta. 9.20 (d, J=2.1 Hz, 1H),
9.12 (d, J=1.9 Hz, 1H), 8.61 (t, J=2.0 Hz, 1H), 8.12 (s, 1H), 7.80
(s, 1H), 6.36 (q, J=7.0 Hz, 1H), 3.54 (dt, J=14.0, 7.0 Hz, 1H),
3.37 (s, 3H), 3.36-3.30 (m, 1H), 2.58 (s, 3H), 1.81 (d, J=7.0 Hz,
3H), 0.92 (t, J=6.9 Hz, 3H).
Example 44.
5-(3-(1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chlor-
o-6-cyano-2-ethoxyphenyl)-N,N-dimethylpicolinamide
##STR00051##
[0305] The title compound was prepared in analogous manor as
Example 43, step 6 but using
N,N-dimethyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)picolinamide
(Peptech, Cat#BE1622) to give the crude product which was purified
on preparative LC-MS (acetonitrile, water, TFA) to give the desired
product (0.005 g, 22%) as white amorphous solid. The product was
isolated as a racemic mixture. LCMS calculated for
C.sub.25H.sub.25ClN.sub.8O.sub.2(M+H).sup.+: m/z=505.1; found:
505.1. .sup.1H NMR (500 MHz, DMSO) .delta. 8.72 (dd, J=2.1, 0.7 Hz,
1H), 8.14-8.12 (m, 1H), 8.11 (s, 1H), 7.75 (s, 1H), 7.71 (dd,
J=8.0, 0.7 Hz, 1H), 6.35 (q, J=7.0 Hz, 1H), 3.61-3.48 (m, 1H),
3.42-3.31 (m, 1H), 3.03 (s, 3H), 2.95 (s, 3H), 2.57 (s, 3H), 1.80
(d, J=7.1 Hz, 3H), 0.92 (t, J=7.0 Hz, 3H).
Example 65.
5-{3-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chlor-
o-2-methoxy-6-methylphenyl}-N,N-dimethylnicotinamide
##STR00052##
[0307] A mixture of
1-[1-(3-bromo-5-chloro-2-methoxy-4-methylphenyl)ethyl]-3-methyl-1H-pyrazo-
lo[3,4-d]pyrimidin-4-amine (25 mg, 0.061 mmol) (chiral pure, first
peak from Example 20, Step 2),
N,N-dimethyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)nicotinamide
(from PepTech) (25 mg, 0.091 mmol), sodium carbonate (13 mg, 0.12
mmol) and [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium
(II), complex with dichloromethane (1:1) (9.9 mg, 0.012 mmol) in
acetonitrile (0.8 mL)/water (0.3 mL) was degassed with N.sub.2 and
then stirred at 95.degree. C. for 2 h. The mixture was filtered and
the filtrate purified by RP-HPLC (XBridge C18 column, eluting with
a gradient of acetonitrile/water containing 0.1% ammonium
hydroxide, at flow rate of 30 mL/min) to give the desired product.
The product was isolated as a single enantiomer. LCMS calculated
for C.sub.24H.sub.27ClN.sub.7O.sub.2(M+H).sup.+: m/z=480.2; Found:
480.2. .sup.1H NMR (500 MHz, DMSO-d.sub.6) .delta. 8.64 (1H, s),
8.54 (1H, br s), 8.13 (1H, s), 7.82 (1H, m), 7.53 (1H, s), 7.42
(2H, br s), 6.28 (1H, q, J=6.5 Hz), 3.22 (3H, s), 2.95 (6H, m),
2.58 (3H, s), 2.04 (3H, s), 1.77 (3H, d, J=6.5 Hz) ppm.
Example 66.
5-{3-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chlor-
o-2-methoxy-6-methylphenyl}-N,N-dimethylpyridine-2-carboxamide
bis(trifluoroacetate)
##STR00053##
[0309] A mixture of
1-[1-(3-bromo-5-chloro-2-methoxy-4-methylphenyl)ethyl]-3-methyl-1H-pyrazo-
lo[3,4-d]pyrimidin-4-amine (25 mg, 0.061 mmol) (chiral pure, first
peak from Example 20, Step 2),
N,N-dimethyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine-2-ca-
rboxamide (25 mg, 0.091 mmol), sodium carbonate (13 mg, 0.12 mmol)
and [1,1'-bis(diphenylphosphino)ferrocene]-dichloropalladium (II),
complex with dichloromethane (1:1) (9.9 mg, 0.012 mmol) in
acetonitrile (0.8 mL)/water (0.3 mL) was degassed with N.sub.2 and
then stirred at 95.degree. C. for 2 hours. After cooling to room
temperature, the mixture was filtered and the filtrate purified on
RP-HPLC (XBridge C18 column, eluting with a gradient of
acetonitrile/water containing 0.05% trifluoroacetic acid, at flow
rate of 30 mL/min) to give the desired product as bis-TFA salt. The
product was isolated as a single enantiomer. LCMS calculated for
C.sub.24H.sub.27ClN.sub.7O.sub.2(M+H).sup.+: m/z=480.2; Found:
480.2. .sup.1H NMR (500 MHz, DMSO-d.sub.6) .delta.: 8.78 (2H, br
s), 8.48 (1H, m), 8.36 (1H, s), 7.86 (1H, br s), 7.65 (1H, br s),
7.58 (1H, s), 6.33 (1H, q, J=7.0 Hz), 3.19 (3H, s), 3.03 (3H, s),
2.97 (3H, s), 2.62 (3H, s), 2.06 (3H, s), 1.81 (3H, d, J=7.0 Hz)
ppm.
Example 67.
1-{1-[5-Chloro-4-fluoro-3-(1-isopropylazetidin-3-yl)-2-methoxyphenyl]ethy-
l}-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine
##STR00054##
[0310] Step 1.
1-[1-(3-Azetidin-3-yl-5-chloro-4-fluoro-2-methoxyphenyl)ethyl]-3-methyl-1-
H-pyrazolo[3,4-d]pyrimidin-4-amine dihydrochloride
[0311] tert-Butyl
3-{3-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chlor-
o-6-fluoro-2-methoxyphenyl}azetidine-1-carboxylate (1.6 g, 3.2
mmol, from Example 13, Step 7) was treated with 4.0 M hydrogen
chloride in dioxane (8.15 mL, 32.6 mmol) in methylene chloride (17
mL) at room temperature for 2 h. The mixture was concentrated to
dryness to give the desired product. LCMS calculated for
C.sub.18H.sub.21ClFN.sub.6O (M+H).sup.+: m/z=391.1; Found:
391.1.
Step 2.
1-{-[5-Chloro-4-fluoro-3-(1-isopropylazetidin-3-yl)-2-methoxypheny-
l]ethyl}-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine
[0312] To a mixture of
1-[1-(3-azetidin-3-yl-5-chloro-4-fluoro-2-methoxyphenyl)ethyl]-3-methyl-1-
H-pyrazolo[3,4-d]pyrimidin-4-amine dihydrochloride (0.90 g, 1.9
mmol, Example 67, step 1), acetone (1.0 mL, 14 mmol) and
triethylamine (2.5 mL, 18 mmol) in methylene chloride (20 mL) was
added sodium triacetoxyborohydride resin (2.5 g, 5.8 mmol). The
mixture was stirred at room temperature for 2 h, then filtered,
washed with water, dried over MgSO.sub.4, filtered and concentrated
to give crude product (870 mg, 100%). LCMS calculated for
C.sub.21H.sub.27ClFN.sub.6O (M+H).sup.+: m/z=433.2; Found:
433.1.
Step 3. Single Enantiomer of
1-{1-[5-chloro-4-fluoro-3-(1-isopropylazetidin-3-yl)-2-methoxyphenyl]ethy-
l}-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine
[0313] Enantiomers of
1-{1-[5-chloro-4-fluoro-3-(1-isopropylazetidin-3-yl)-2-methoxyphenyl]ethy-
l}-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (870 mg, 2.0 mmol)
were separated on a Phenomenex Lux Cellulose-2 column, eluting with
10% ethanol in hexanes, at flow rate of 18 mL/min, and column
loading of .about.8 mg/injection to separate two enantiomers. First
peak retention time 10.9 min; second peak retention time 13.6 min.
The fractions of the 1st peak (110 mg, 13%) were concentrated and
purified using RP-HPLC (XBridge C18 column, eluting with a gradient
of acetonitrile/water containing 0.1% ammonium hydroxide, at flow
rate of 30 mL/min) to give the desired product. The product was
isolated as a single enantiomer. LCMS calculated for
C.sub.21H.sub.27ClFN.sub.6O (M+H).sup.+: m/z=433.2; Found:
433.1.
Example 68.
(2S)-1-(3-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-
-5-chloro-6-fluoro-2-methoxyphenyl}azetidin-1-yl)propan-2-ol
##STR00055##
[0315] To a mixture of
1-[1-(3-azetidin-3-yl-5-chloro-4-fluoro-2-methoxyphenyl)ethyl]-3-methyl-1-
H-pyrazolo[3,4-d]pyrimidin-4-amine dihydrochloride (15 mg, 0.032
mmol, from Example 67, Step 1) and triethylamine (18 .mu.L, 0.13
mmol) in ethanol (0.53 mL) was added (S)-(-)-methyloxirane (6.8
.mu.L, 0.097 mmol). The resulting mixture was heated at 90.degree.
C. for 3 h, then purified on RP-HPLC (XBridge C18 column, eluting
with a gradient of acetonitrile/water containing 0.1% ammonium
hydroxide, at flow rate of 30 mL/min) to give the desired product.
The enantiomers were separated on a Phenomenex Lux Cellulose C-4
column (5 .mu.M, 21.2.times.250 mm), eluting with 20% ethanol in
hexanes, at flow rate of 18 mL/min, to give two enantiomers. First
peak (2.7 mg, 18%) retention time 8.9 min; LCMS calculated for
C.sub.21H.sub.27ClFN.sub.6O.sub.2(M+H).sup.+: m/z=449.2; Found:
449.1. .sup.1H NMR (DMSO-d.sub.6, 500 MHz) .delta. 8.11 (1H, s),
7.42 (1H, d, J=8.5 Hz), 7.25 (2H, br s), 6.21 (1H, q, J=7.5 Hz),
4.28 (1H, d, J=4.0 Hz), 3.82 (3H, m), 3.62 (3H, s), 3.55 (1H, m),
3.05 (1H, m), 2.97 (1H, m), 2.55 (3H, s), 2.28 (2H, m), 1.70 (2H,
d, J=7.5 Hz), 1.00 (3H, d, J=6.0 Hz) ppm. Second peak retention
time 10.0 min.
Example 71.
2-(3-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-ch-
loro-6-fluoro-2-methoxyphenyl}azetidin-1-yl)ethanol
##STR00056##
[0317] To a mixture of
1-[1-(3-azetidin-3-yl-5-chloro-4-fluoro-2-methoxyphenyl)ethyl]-3-methyl-1-
H-pyrazolo[3,4-d]pyrimidin-4-amine dihydrochloride (19 mg, 0.041
mmol, racemic intermediate from Example 67, Step 1) and
triethylamine (28 .mu.L, 0.20 mmol) in methanol (0.1
mL)/acetonitrile (0.1 mL)/tetrahydrofuran (0.1 mL) was added
{[tert-butyl(dimethyl)silyl]oxy}acetaldehyde (39 .mu.L, 0.20 mmol),
followed by sodium triacetoxyborohydride (22 mg, 0.10 mmol). The
resulting mixture was stirred overnight at room temperature. The
mixture was treated with 6.0 M hydrogen chloride in water (0.07 mL,
0.4 mmol) at room temperature for 10 min and then purified on
RP-HPLC (XBridge C18 column, eluting with a gradient of
acetonitrile/water containing 0.1% ammonium hydroxide, at flow rate
of 30 mL/min) to give the desired product (2.5 mg, 13%). The
product was isolated as a racemic mixture. LCMS calculated for
C.sub.20H.sub.25ClFN.sub.6O.sub.2(M+H).sup.+: m/z=435.2; Found:
435.1.
Example 72.
1-{1-[5-Chloro-4-fluoro-2-methoxy-3-(1-oxetan-3-ylazetidin-3-yl)phenyl]et-
hyl}-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine
##STR00057##
[0319] To a mixture of
1-[1-(3-azetidin-3-yl-5-chloro-4-fluoro-2-methoxyphenyl)ethyl]-3-methyl-1-
H-pyrazolo[3,4-d]pyrimidin-4-amine dihydrochloride (19 mg, 0.041
mmol racemic intermediate from Example 67, Step 1) and
triethylamine (28 .mu.L, 0.20 mmol) in methanol (0.1
mL)/acetonitrile (0.1 mL)/tetrahydrofuran (0.1 mL) was added 37%
formaldehyde (15 .mu.L, 0.20 mmol), followed by sodium
triacetoxyborohydride (22 mg, 0.10 mmol). The resulting mixture was
stirred overnight at room temperature. The mixture was purified on
RP-HPLC (XBridge C18 column, eluting with a gradient of
acetonitrile/water containing 0.1% ammonium hydroxide, at flow rate
of 30 mL/min) to give the desired product (1.2 mg, 6.3%). The
product was isolated as a racemic mixture. LCMS calculated for
C.sub.19H.sub.23ClFN.sub.6O (M+H).sup.+: m/z=405.2; Found:
405.1.
Example 94.
1-{1-[5-Chloro-2-ethoxy-3-(1-isopropylazetidin-3-yl)-4-methylphenyl]ethyl-
}-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine
bis(trifluoroacetate)
##STR00058##
[0320] Step 1. Benzyl
3-{3-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chlor-
o-2-ethoxy-6-methylphenyl}azetidine-1-carboxylate
[0321] Cyanuric chloride (200 mg, 1.1 mmol) was added to
N,N-dimethylformamide (0.083 mL, 1.1 mmol) at room temperature.
After the formation of a white solid (ca. 10 minutes), methylene
chloride (5 mL) was added, followed by benzyl
3-[3-chloro-6-ethoxy-5-(1-hydroxyethyl)-2-methylphenyl]azetidine-1-carbox-
ylate (310 mg, 0.77 mmol). After addition, the resultant mixture
was stirred at room temperature overnight. Water was added, and
then diluted with dichloromethane. The organic phases were washed
with sat. NaHCO.sub.3 solution, water and brine, dried over
MgSO.sub.4, concentrated and purified on silica gel (eluting with 0
to 40% EtOAc/hexanes) to give the desired product (140 mg, 43%).
LCMS calculated for C.sub.22H.sub.26Cl.sub.2NO.sub.3 (M+H).sup.+:
m/z=422.1; Found: 422.0.
[0322] A mixture of benzyl
3-[3-chloro-5-(1-chloroethyl)-6-ethoxy-2-methylphenyl]azetidine-1-carboxy-
late (0.375 g, 0.888 mmol),
3-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (0.16 g, 1.1 mmol),
cesium carbonate (0.43 g, 1.3 mmol) and potassium iodide (15 mg,
0.089 mmol) in N,N-dimethylformamide (2.8 mL) was heated at
140.degree. C. for 1 h. The mixture was diluted with ether, and
washed with water. The organic layers were concentrated and
purified on silica gel (eluting with 0 to 100% EtOAc in hexanes) to
give the desired product (0.24 g, 50%). LCMS calculated for
C.sub.28H.sub.32ClN.sub.6O.sub.3(M+H).sup.+: m/z=535.2; Found:
535.0. The enantionmers were separated on a Phenomenex Lux
Cellulose C-2 column (5 .mu.M, 21.2.times.250 mm), eluting with 20%
ethanol in hexanes, at flow rate of 18 mL/min, and column loading
of .about.4.5 mg/injection to separate two enantiomers. First peak
retention time: 21.2 min; second peak retention time: 24.6 min.
Step 2.
1-[1-(3-Azetidin-3-yl-5-chloro-2-ethoxy-4-methylphenyl)ethyl]-3-me-
thyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine
[0323] Benzyl
3-{3-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chlor-
o-2-ethoxy-6-methylphenyl}azetidine-1-carboxylate (170 mg, 0.32
mmol, racemic intermediate) and 5% palladium (80 mg) were combined
in methanol (12 mL), to which was added 0.25 M hydrogen chloride in
water (3.2 mL, 0.79 mmol). The suspension was hydrogenated under
balloon pressure of H.sub.2 at room temperature for 2 h. The
suspension was filtered. The filtrate was neutralized with sat.
NaHCO.sub.3 solution, and extracted with dichloromethane. The
combined organic layers were dried over MgSO.sub.4 and filtered,
concentrated to give the desired product (117 mg, 92%). LCMS
calculated for C.sub.20H.sub.26ClN.sub.6O (M+H).sup.+: m/z=401.2;
Found: 401.1.
Step 3.
1-{1-[5-Chloro-2-ethoxy-3-(1-isopropylazetidin-3-yl)-4-methylpheny-
l]ethyl}-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine
bis(trifluoroacetate)
[0324] Acetone (9.3 .mu.L, 0.13 mmol) was added to
1-[1-(3-azetidin-3-yl-5-chloro-2-ethoxy-4-methylphenyl)ethyl]-3-methyl-1H-
-pyrazolo[3,4-d]pyrimidin-4-amine (10.2 mg, 0.0254 mmol) in
methanol (0.1 mL)/tetrahydrofuran (0.1 mL)/acetonitrile (0.1 mL)
and the mixture was stirred at room temperature for 10 min, before
the addition of sodium triacetoxyborohydride (16 mg, 0.076 mmol).
The reaction mixture was stirred at room temperature for 4 h and
then purified on RP-HPLC (XBridge C18 column, eluting with a
gradient of acetonitrile/water containing 0.05% TFA, at flow rate
of 30 mL/min) to give the desired product as TFA salt (2.3 mg,
22%). The product was isolated as a single enantiomer. LCMS
calculated for C.sub.23H.sub.32ClN.sub.6O (M+H).sup.+: m/z=443.2;
Found: 443.1.
Example 95.
2-(3-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-ch-
loro-2-ethoxy-6-methylphenyl}azetidin-1-yl)ethanol
bis(trifluoroacetate)
##STR00059##
[0326] To a mixture of
1-[1-(3-azetidin-3-yl-5-chloro-2-ethoxy-4-methylphenyl)ethyl]-3-methyl-1H-
-pyrazolo[3,4-d]pyrimidin-4-amine (7.9 mg, 0.020 mmol, racemic
intermediate from Example 94, Step 2) in tetrahydrofuran (0.09
mL)/acetonitrile (0.09 mL)/methanol (0.09 mL) was added
{[tert-butyl(dimethyl)silyl]oxy}acetaldehyde (19 .mu.L, 0.098 mmol)
and the mixture was stirred for 10 min before the addition of
sodium triacetoxyborohydride (12 mg, 0.059 mmol). The resulting
mixture was stirred at room temperature for 4 h, then treated with
6.0 M hydrogen chloride in water (30 .mu.L, 0.2 mmol) for 10 min.
The mixture was purified on RP-HPLC (XBridge C18 column, eluting
with a gradient of acetonitrile/water containing 0.05% TFA, at flow
rate of 30 mL/min) to give the desired product as TFA salt (3.2 mg,
40%). The product was isolated as a racemic mixture. LCMS
calculated for C.sub.22H.sub.30ClN.sub.6O.sub.2(M+H).sup.+:
m/z=445.2; Found: 445.1.
Example 96.
(2S)-1-(3-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-
-5-chloro-2-ethoxy-6-methylphenyl}azetidin-1-yl)propan-2-ol
bis(trifluoroacetate)
##STR00060##
[0327] Step 1. Benzyl
3-{3-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chlor-
o-2-ethoxy-6-methylphenyl}azetidine-1-carboxylate
[0328] The enantionmers from Example 94, Step 1 were separated on a
Phenomenex Lux Cellulose C-2 column (5 .mu.M, 21.2.times.250 mm),
eluting with 20% ethanol in hexanes, at flow rate of 18 mL/min, and
column loading of .about.4.5 mg/injection to separate two
enantiomers. First peak retention time: 21.2 min; second peak
retention time: 24.6 min.
Step 2.
1-[1-(3-Azetidin-3-yl-5-chloro-2-ethoxy-4-methylphenyl)ethyl]-3-me-
thyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine
[0329] Benzyl
3-{3-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chlor-
o-2-ethoxy-6-methylphenyl}azetidine-1-carboxylate (chiral
intermediate from first peak of previous step) was hydrogenated in
the presence of 5% palladium as described in Example 94, Step 2 to
give the desired chiral product. LCMS calculated for
C.sub.20H.sub.26ClN.sub.6O (M+H).sup.+: m/z=401.2; Found:
401.1.
Step 3.
(2S)-1-(3-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl-
)ethyl]-5-chloro-2-ethoxy-6-methylphenyl}azetidin-1-yl)propan-2-ol
bis(trifluoroacetate)
[0330] To a mixture of
1-[1-(3-azetidin-3-yl-5-chloro-2-ethoxy-4-methylphenyl)ethyl]-3-methyl-1H-
-pyrazolo[3,4-d]pyrimidin-4-amine (10 mg, 0.02 mmol, chiral
intermediate from step 2) and triethylamine (9 .mu.L, 0.07 mmol) in
isopropyl alcohol (0.05 mL) was added (S)-(-)-methyloxirane (4.5
.mu.L, 0.064 mmol). The resulting mixture was stirred at 90.degree.
C. overnight, cooled and purified on RP-HPLC (XBridge C18 column,
eluting with a gradient of acetonitrile/water containing 0.05% TFA,
at flow rate of 30 mL/min) to give the desired product as TFA salt
(3.4 mg, 34%). The product was isolated as a single diastereomer.
LCMS calculated for C.sub.23H.sub.32ClN.sub.6O.sub.2(M+H).sup.+:
m/z=459.2; Found: 459.1.
Example 99.
(2S)-1-(3-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-
-5-chloro-2-ethoxy-6-methylphenyl}azetidin-1-yl)-1-oxopropan-2-ol
trifluoroacetate
##STR00061##
[0332] To a mixture of
1-[1-(3-azetidin-3-yl-5-chloro-2-ethoxy-4-methylphenyl)ethyl]-3-methyl-1H-
-pyrazolo[3,4-d]pyrimidin-4-amine (9.8 mg, 0.024 mmol, racemic
intermediate from Example 94, Step 2),
N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium
hexafluorophosphate (14 mg, 0.037 mmol) and triethylamine (10
.mu.L, 0.073 mmol) in N,N-dimethylformamide (0.15 mL) was added 85%
(2S)-2-hydroxypropanoic acid in water (3.2 .mu.L, 0.037 mmol). The
resulting mixture was stirred for 2 h at room temperature. The
mixture was purified on RP-HPLC (XBridge C18 column, eluting with a
gradient of acetonitrile/water containing 0.05% TFA, at flow rate
of 30 mL/min) to give the desired product as trifluoroacetic acid
(TFA) salt (2.9 mg, 29%). The product was isolated as a racemic
mixture. LCMS calculated for
C.sub.23H.sub.30ClN.sub.6O.sub.3(M+H).sup.+: m/z=473.2; Found:
473.1.
Example 102.
(2S)-1-(3-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-
-5-chloro-2-methoxy-6-methylphenyl}azetidin-1-yl)propan-2-ol
##STR00062##
[0334] To a mixture of
1-[1-(3-azetidin-3-yl-5-chloro-2-methoxy-4-methylphenyl)ethyl]-3-methyl-1-
H-pyrazolo[3,4-d]pyrimidin-4-amine dihydrochloride (21 mg, 0.046
mmol) (Example 1, step 7, chiral intermediate from peak 1) and
triethylamine (20 .mu.L, 0.1 mmol) in isopropyl alcohol (0.10 mL)
was added (S)-(-)-methyloxirane (3.2 .mu.L, 0.046 mmol). The
resulting mixture was stirred at 90.degree. C. After 90 min,
additional (S)-(-)-methyloxirane (6.4 uL) was added and stirred at
90.degree. C. overnight. After cooling, the mixture was diluted
with methanol and purified using RP-HPLC (XBridge C18 column,
eluting with a gradient of acetonitrile/water containing 0.1%
ammonium hydroxide, at flow rate of 30 mL/min) to give 6 mg (30%)
of the product. The product was isolated as a single diastereomer.
LCMS calculated for C.sub.22H.sub.30ClN.sub.6O.sub.2(M+H).sup.+:
m/z=445.2; Found: 445.2.
Example 104.
2-(3-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-ch-
loro-2-methoxy-6-methylphenyl}azetidin-1-yl)ethanol
##STR00063##
[0336] To a mixture of
1-[1-(3-azetidin-3-yl-5-chloro-2-methoxy-4-methylphenyl)ethyl]-3-methyl-1-
H-pyrazolo[3,4-d]pyrimidin-4-amine dihydrochloride (20 mg, 0.04
mmol) (Example 1, step 7, chiral intermediate from peak 1),
{[tert-butyl(dimethyl)silyl]oxy}acetaldehyde (8.3 mg, 0.048 mmol),
and triethylamine (19 .mu.L, 0.14 mmol) in methylene chloride (0.3
mL) was added sodium triacetoxyborohydride resin (38 mg, 0.087
mmol). The resulting mixture was stirred overnight at room
temperature. The mixture was filtered and concentrated. The crude
product was dissolved in tetrahydrofuran (1 mL) and cooled to
0.degree. C. 1.0 M Tetra-n-butylammonium fluoride in THF (0.44 mL,
0.44 mmol) was added and warmed to room temperature. After 3 h, the
solvents were evaporated. The crude was purified using RP-HPLC
(XBridge C18 column, eluting with a gradient of acetonitrile/water
containing 0.1% ammonium hydroxide, at flow rate of 30 mL/min) to
give 8.1 mg (40%) of the desired product. The product was isolated
as a single enantiomer. LCMS calculated for
C.sub.21H.sub.28ClN.sub.6O.sub.2(M+H).sup.+: m/z=431.2; Found:
431.3.
Example 105.
(3-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chlo-
ro-2-methoxy-6-methylphenyl}azetidin-1-yl)acetonitrile
##STR00064##
[0338] To a mixture of
1-[1-(3-azetidin-3-yl-5-chloro-2-methoxy-4-methylphenyl)ethyl]-3-methyl-1-
H-pyrazolo[3,4-d]pyrimidin-4-amine dihydrochloride (16 mg, 0.035
mmol, chiral intermediate from peak 1 of Example 1, Step 7) and
triethylamine (14 .mu.L, 0.10 mmol) in acetonitrile (0.7 mL) was
added bromoacetonitrile (2.7 .mu.L, 0.038 mmol). The resulting
mixture was stirred at room temperature for 2.5 h. The mixture was
diluted with acetonitrile and purified by using RP-HPLC (XBridge
C18 column, eluting with a gradient of acetonitrile/water
containing 0.05% TFA, at flow rate of 30 mL/min) to give the
desired product as the TFA salt. The pure fractions were partially
evaporated and then made basic by the addition of 1 N NaOH. The
aqueous mixture was extracted with dichloromethane (2.times.). The
extracts were dried (MgSO4), filtered, and concentrated. The solid
was dried in vacuo to give 6.9 mg (46%) of the desired product. The
product was isolated as a single enantiomer. LCMS calculated for
C.sub.21H.sub.25ClN.sub.7O (M+H).sup.+: m/z=426.2; Found:
426.0.
Example 108.
1-(1-{5-Chloro-2-methoxy-4-methyl-3-[1-(2,2,2-trifluoroethyl)azetidin-3-y-
l]phenyl}ethyl)-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine
##STR00065##
[0340] A mixture of
1-[1-(3-azetidin-3-yl-5-chloro-2-methoxy-4-methylphenyl)ethyl]-3-methyl-1-
H-pyrazolo[3,4-d]pyrimidin-4-amine dihydrochloride (15 mg, 0.024
mmol, chrial intermediate from first peak of Example 1, step 7),
2,2,2-trifluoroethyl trifluoromethanesulfonate (6.8 mg, 0.029 mmol)
and triethylamine (12 .mu.L, 0.085 mmol) in methylene chloride (0.3
mL) was stirred over a weekend at room temperature. The solvents
were evaporated and the crude purified using RP-HPLC (XBridge C18
column, eluting with a gradient of acetonitrile/water containing
0.1% ammonium hydroxide, at flow rate of 30 mL/min) to give 4.5 mg
(39%) of the desired product. The product was isolated as a single
enantiomer. LCMS calculated for C.sub.21H.sub.25ClF.sub.3N.sub.6
(M+H).sup.+: m/z=469.2; Found: 469.1.
Example 110.
(2R)-2-(3-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-
-5-chloro-2-methoxy-6-methylphenyl}azetidin-1-yl)-N-methylpropanamide
trifluoro acetate
##STR00066##
[0342] A mixture of
1-[1-(3-azetidin-3-yl-5-chloro-2-methoxy-4-methylphenyl)ethyl]-3-methyl-1-
H-pyrazolo[3,4-d]pyrimidin-4-amine dihydrochloride (26 mg, 0.067
mmol, chrial intermediate from peak 1 of Example 1, Step 7),
(2R)-2-bromopropanoic acid (7.3 .mu.L, 0.081 mmol) and
triethylamine (19 .mu.L, 0.13 mmol) in acetonitrile (0.8 mL) was
stirred overnight at room temperature. The reaction was not
complete so it was heated to 50.degree. C. After 4 h, the solvents
were evaporated. To the crude residue was added methylammonium
chloride (4.5 mg, 0.067 mmol), N,N-dimethylformamide (0.2 mL),
triethylamine (19 .mu.L, 0.13 mmol), and
benzotriazol-1-yloxytris(dimethylamino)phosphonium
hexafluorophosphate (45 mg, 0.10 mmol). The resulting mixture was
stirred overnight at room temperature. The reaction mixture was
added to a vial containing sat. NaHCO.sub.3 and extracted with
EtOAc (2.times.). The organics were dried (MgSO.sub.4), filtered,
and concentrated. The crude was purified using RP-HPLC (XBridge C18
column, eluting with a gradient of acetonitrile/water containing
0.05% TFA, at flow rate of 30 mL/min) to give 1.4 mg (3.6%) of the
desired product as the TFA salt. The product was isolated as a
single diastereomer. LCMS calculated for
C.sub.23H.sub.31ClN.sub.7O.sub.2(M+H).sup.+: m/z=472.2; Found:
472.2.
Example 113.
2-(3-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-ch-
loro-2-methoxy-6-methylphenyl}azetidin-1-yl)-3,3,3-trifluoropropan-1-ol
##STR00067##
[0344] To a mixture of
1-[1-(3-azetidin-3-yl-5-chloro-2-methoxy-4-methylphenyl)ethyl]-3-methyl-1-
H-pyrazolo[3,4-d]pyrimidin-4-amine dihydrochloride (20 mg, 0.04
mmol, chrial intermediate from peak 1 of Example 1, step 7) and
triethylamine (19 .mu.L, 0.13 mmol) in acetonitrile (0.6 mL) was
added 2-bromo-3,3,3-trifluoropropan-1-ol (from Synquest Labs, 9.2
mg, 0.048 mmol). N,N-dimethylformamide (0.3 mL) was added, which
created a clear solution that was stirred at 70.degree. C.
overnight. The mixture was diluted water and purified using RP-HPLC
(XBridge C18 column, eluting with a gradient of acetonitrile/water
containing 0.1% ammonium hydroxide, at flow rate of 30 mL/min) to
give 6.6 mg (30%) of the desired product. The product was isolated
as a mixture of diastereomers. LCMS calculated for
C.sub.22H.sub.27ClF.sub.3N.sub.6O.sub.2 (M+H).sup.+: m/z=499.2;
Found: 499.1.
Example 115.
(2R)-3-(3-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-
-5-chloro-2-methoxy-6-methylphenyl}azetidin-1-yl)-1,1,1-trifluoropropan-2--
ol
##STR00068##
[0346] A mixture of
1-[1-(3-azetidin-3-yl-5-chloro-2-methoxy-4-methylphenyl)ethyl]-3-methyl-1-
H-pyrazolo[3,4-d]pyrimidin-4-amine dihydrochloride (20 mg, 0.044
mmol, chrial intermediate from peak 1 of Example 1, Step 7),
(2R)-2-(trifluoromethyl)oxirane (9.4 .mu.L, 0.11 mmol), and
triethylamine (18 .mu.L, 0.13 mmol) in ethanol (0.3 mL) was heated
in a microwave at 120.degree. C. for 25 min. The mixture was
diluted with MeOH and purified by RP-HPLC (XBridge C18 column,
eluting with a gradient of acetonitrile/water containing 0.1%
ammonium hydroxide, at flow rate of 30 mL/min) to give 6.2 mg (28%)
of the desired product. The product was isolated as a single
enantiomer. LCMS calculated for
C.sub.22H.sub.27ClF.sub.3N.sub.6O.sub.2 (M+H).sup.+: m/z=499.2;
Found: 499.1.
Example 118.
(2S)-1-(3-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-
-5-chloro-2-methoxy-6-methylphenyl}azetidin-1-yl)-1-oxopropan-2-ol
##STR00069##
[0348] To a mixture of
1-[1-(3-azetidin-3-yl-5-chloro-2-methoxy-4-methylphenyl)ethyl]-3-methyl-1-
H-pyrazolo[3,4-d]pyrimidin-4-amine dihydrochloride (15 mg, 0.033
mmol, chrial intermediate from Example 1, Step 7, peak 1), mixture
of (2S)-2-hydroxypropanoic acid (4.3 .mu.L, 0.049 mmol) (L-lactic
acid, 85% aq.) and triethylamine (14 .mu.L, 0.098 mmol) in
N,N-dimethylformamide (0.2 mL) was added
N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium
hexafluorophosphate (19 mg, 0.049 mmol). The resulting mixture was
stirred overnight at room temperature. The mixture was diluted with
MeOH and purified using RP-HPLC (XBridge C18 column, eluting with a
gradient of acetonitrile/water containing 0.1% ammonium hydroxide,
at flow rate of 30 mL/min) to give 3.0 mg (20%) of the desired
product. The product was isolated as a single enantiomer. LCMS
calculated for C.sub.22H.sub.28ClN.sub.6O.sub.3(M+H).sup.+:
m/z=459.2; Found: 459.2.
Example 121.
(2R)-1-(3-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-
-5-chloro-2-methoxy-6-methylphenyl}azetidin-1-yl)-1-oxopropan-2-ol
trifluoroacetate
##STR00070##
[0350] This compound was prepared using procedures analogous to
those for Example 118 (starting from chiral material from Example
1, Step 7, peak 1), with (R)-2-hydroxypropanoic acid instead of
(2S)-2-hydroxypropanoic acid (4.3 .mu.L, 0.049 mmol) and
benzotriazol-1-yloxytris(dimethylamino)phosphonium
hexafluorophosphate instead of
N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium
hexafluorophosphate. The crude was purified using RP-HPLC (XBridge
C18 column, eluting with a gradient of acetonitrile/water
containing 0.05% TFA, at flow rate of 30 mL/min) to give the
desired product as the TFA salt. The product was isolated as a
single enantiomer. LCMS calculated for
C.sub.22H.sub.28ClN.sub.6O.sub.3(M+H).sup.+: m/z=459.2; Found:
459.2.
Example 139. Enantiomers of
1-{1-[5-Chloro-2-ethoxy-4-fluoro-3-(1-isopropylazetidin-3-yl)phenyl]ethyl-
}-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine
##STR00071##
[0351] Step 1.
1-(5-Chloro-2-ethoxy-4-fluoro-3-iodophenyl)ethanone
[0352] This compound was prepared according to the procedure of
Example 13 Step 3, using
1-(5-chloro-4-fluoro-2-hydroxy-3-iodophenyl)ethanone and iodoethane
as the starting materials. LCMS calculated for
C.sub.10H.sub.10ClFI.sub.2 (M+H).sup.+: m/z=342.9; Found:
342.9.
Step 2. tert-Butyl
3-(3-acetyl-5-chloro-2-ethoxy-6-fluorophenyl)azetidine-1-carboxylate
[0353] A round-bottom flask equipped with a magnetic stir bar and a
rubber septum was charged with lithium chloride (3.9 g, 91 mmol).
The flask was heated at 140.degree. C. for 10 min under high vacuum
and backfilled with nitrogen after cooling to room temperature.
Zinc (6.0 g, 91 mmol) was added and the flask was heated at
140.degree. C. for 10 min under high vacuum and backfilled with
nitrogen after cooling to room temperature. Tetrahydrofuran (THF)
(38 mL) and 1,2-dibromoethane (233 .mu.L, 2.70 mmol) were added via
syringe. The mixture was heated at 60.degree. C. for 10 min and
then cooled to room temperature. Chlorotrimethylsilane (68 .mu.L,
0.54 mmol) and iodine (69 mg, 0.27 mmol) in THF (1 mL) were added
and the resulting mixture was stirred at 60.degree. C. for 10 min
then cooled to room temperature. A solution of tert-butyl
3-iodoazetidine-1-carboxylate (12.17 g, 42.99 mmol) in THF (10 mL)
was then added and the mixture stirred at 40.degree. C. for 1 h and
at room temperature for 1 h. Another flask charged with
1-(5-chloro-2-ethoxy-4-fluoro-3-iodophenyl)ethanone (13.0 g, 38.0
mmol), palladium acetate (170 mg, 0.76 mmol),
2'-(dicyclohexylphosphino)-N,N,N',N'-tetramethylbiphenyl-2,6-diamine
(660 mg, 1.5 mmol), and toluene (35 mL) was evacuated under high
vacuum and backfilled with nitrogen. The mixture was cooled to
0.degree. C. and the zinc reagent made above was added slowly via
syringe. After addition, the reaction was heated to 50.degree. C.
overnight. The reaction solution was partitioned between EtOAc and
sat. NH.sub.4Cl solution. The layers were separated and the aqueous
extracted further with EtOAc (2.times.). The combined organics were
washed with water, brine, then dried over MgSO4, and concentrated.
The crude mixture was purified on silica gel column to give the
desired product as an orange oil (6.3 g, 45%). LCMS calculated for
C.sub.18H.sub.23ClFNO.sub.4Na (M+Na).sup.+: m/z=394.1; Found:
394.1.
Step 3. tert-Butyl
3-[3-chloro-6-ethoxy-2-fluoro-5-(1-hydroxyethyl)phenyl]azetidine-1-carbox-
ylate
[0354] This compound was prepared according to the procedure of
Example 13 Step 5, using tert-butyl
3-(3-acetyl-5-chloro-2-ethoxy-6-fluorophenyl)azetidine-1-carboxylate
and sodium tetrahydroborate as the starting materials. LCMS
calculated for C.sub.18H.sub.25ClFNO.sub.4Na (M+Na).sup.+:
m/z=396.1; Found: 396.1.
Step 4. tert-Butyl
3-[3-chloro-5-(1-chloroethyl)-6-ethoxy-2-fluorophenyl]azetidine-1-carboxy-
late
[0355] This compound was prepared according to the procedure of
Example 13 step 6, using tert-butyl
3-[3-chloro-6-ethoxy-2-fluoro-5-(1-hydroxyethyl)phenyl]azetidine-1-carbox-
ylate (racemic) and cyanuric chloride as the starting
materials.
Step 5. tert-Butyl
3-{3-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chlor-
o-2-ethoxy-6-fluorophenyl}azetidine-1-carboxylate
[0356] To a mixture of 3-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine
(1.10 g, 7.37 mmol), cesium carbonate (3.2 g, 10 mmol) and
potassium iodide (111 mg, 0.670 mmol) in DMF (20 mL) was added
tert-butyl
3-[3-chloro-5-(1-chloroethyl)-6-ethoxy-2-fluorophenyl]azetidine-1-carboxy-
late (2.63 g, 6.70 mmol) and the mixture was stirred at 90.degree.
C. for 3 h. The solvent was removed in vacuo. The residue was
diluted with ethyl acetate and water. Aqueous layer was extracted
with ethyl acetate twice. The combined organic layers were washed
with water, brine, dried over Na.sub.2SO.sub.4, filtered and
concentrated. The residue was purified on silica gel column
(eluting with 100% ethyl acetate) to give the desired product as a
foam (2.15 g, 63%). LCMS calculated for
C.sub.24H.sub.31ClFN.sub.6O.sub.3(M+H).sup.+: m/z=505.2; Found:
505.2.
Step 6.
1-[1-(3-Azetidin-3-yl-5-chloro-2-ethoxy-4-fluorophenyl)ethyl]-3-me-
thyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine dihydrochloride
[0357] To a solution of tert-butyl
3-{3-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chlor-
o-2-ethoxy-6-fluorophenyl}azetidine-1-carboxylate (275 mg, 0.544
mmol) in dichloromethane (2.4 mL) was added 4.0 M hydrogen chloride
in dioxane (1.1 mL, 4.4 mmol). The reaction solution was stirred at
room temperature for 6 h. The solvent was removed under reduced
pressure to give the desired product as a white solid (250 mg,
96%). LCMS calculated for C.sub.19H.sub.23ClFN.sub.6O (M+H).sup.+:
m/z=405.2; Found: 405.1.
Step 7.
1-{-[5-Chloro-2-ethoxy-4-fluoro-3-(1-isopropylazetidin-3-yl)phenyl-
]ethyl}-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine
[0358] To a mixture of
1-[1-(3-azetidin-3-yl-5-chloro-2-ethoxy-4-fluorophenyl)ethyl]-3-methyl-1H-
-pyrazolo[3,4-d]pyrimidin-4-amine dihydrochloride (49 mg, 0.10
mmol), acetone (8.28 .mu.L, 0.113 mmol), and triethylamine (44.3
.mu.L, 0.318 mmol) in dichloromethane (0.67 mL) was added sodium
triacetoxyborohydride resin (89 mg, 0.20 mmol). The resulting
mixture was stirred overnight at room temperature. The mixture was
filtered and concentrated and then purified by preparative LCMS
(XBridge C18 column, eluting with a gradient of acetonitrile/water
containing 0.05% TFA, at flow rate of 60 mL/min) to give the
racemic product. LCMS: found m/z=447.2 (M+H).sup.+. The racemic
mixture was separated by chiral HPLC (column IA, eluting with 5%
ethanol/95% hexanes, at flow rate 18 mL/min) to give two peaks
(isomer 1: 9.5 mg, 21%; isomer 2: 9.2 mg, 20%).
[0359] Isomer 1 (first to elute, retention time: 4.4 min): 1H NMR
(400 MHz, DMSO-d.sub.6): .delta..quadrature. 8.10 (s, 1H), 7.45 (d,
1H), 6.21 (m, 1H), 3.70 (m, 5H), 2.91 (m, 2H), 2.53 (s, 3H), 2.17
(m, 1H), 1.66 (d, 3H), 1.31 (t, 3H), 0.81 (m, 6H) ppm; LCMS
calculated for C.sub.22H.sub.29ClFN.sub.6O (M+H).sup.+: m/z=447.2;
Found: 447.2.
[0360] Isomer 2 (second to elute, retention time: 19.5 min): LCMS
calculated for C.sub.22H.sub.29ClFN.sub.6O (M+H).sup.+: m/z=447.2;
Found: 447.2.
Example 140.
1-(3-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-ch-
loro-2-ethoxy-6-fluorophenyl}azetidin-1-yl)-2-methylpropan-2-ol
##STR00072##
[0362] To a mixture of
1-[1-(3-azetidin-3-yl-5-chloro-2-ethoxy-4-fluorophenyl)ethyl]-3-methyl-1H-
-pyrazolo[3,4-d]pyrimidin-4-amine dihydrochloride (20 mg, 0.042
mmol, racemic intermediate from Example 139, Step 6) and
triethylamine (18 .mu.L, 0.12 mmol) in ethanol (1 mL) was added
oxirane, 2,2-dimethyl-(6.98 .mu.L, 0.0837 mmol). The resulting
mixture was heated at 120.degree. C. in microwave reactor for 45
min. The reaction was diluted with methanol and purified on RP-HPLC
(XBridge C18 column, eluting with a gradient of acetonitrile/water
containing 0.1% ammonium hydroxide, at flow rate of 30 mL/min) to
give the desired product as white a solid (3.4 mg, 17%). The
product was isolated as a racemic mixture. LCMS calculated for
C.sub.23H.sub.31ClFN.sub.6O.sub.2 (M+H).sup.+: m/z=477.2; Found:
477.3.
Example 141.
1-(1-{5-Chloro-2-ethoxy-4-fluoro-3-[1-(2,2,2-trifluoroethyl)azetidin-3-yl-
]phenyl}ethyl)-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine
##STR00073##
[0364] To a mixture of
1-[1-(3-azetidin-3-yl-5-chloro-2-ethoxy-4-fluorophenyl)ethyl]-3-methyl-1H-
-pyrazolo[3,4-d]pyrimidin-4-amine dihydrochloride (19 mg, 0.040
mmol, racemic intermediate from Example 139, Step 6) and
triethylamine (20 .mu.L, 0.14 mmol) in dichloromethane (0.5 mL) was
added 2,2,2-trifluoroethyl trifluoromethanesulfonate (11 mg, 0.048
mmol). The resulting mixture was stirred overnight at room
temperature. The solvents were evaporated under reduced pressure
and the crude mixture purified on RP-HPLC (XBridge C18 column,
eluting with a gradient of acetonitrile/water containing 0.1%
ammonium hydroxide, at flow rate of 30 mL/min) to give the desired
product (3.8 mg, 19%). The product was isolated as a racemic
mixture. LCMS calculated for C.sub.21H.sub.24ClF.sub.4N.sub.6O
(M+H).sup.+: m/z=487.2; Found: 487.1.
Example 149.
(2S)-1-(3-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-
-5-chloro-2-ethoxy-6-fluorophenyl}azetidin-1-yl)propan-2-ol
##STR00074##
[0365] Step 1. Enantiomers of tert-Butyl
3-{3-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chlor-
o-2-ethoxy-6-fluorophenyl}azetidine-1-carboxylate
[0366] The racemic mixture was separated by chiral HPLC (column IA,
eluting with 5% ethanol/95% hexanes, flow rate 18 mL/min) to give
two peaks; Isomer 1 (first to elute): Retention time: 16.8 min;
LCMS calculated for C.sub.24H.sub.31ClFN.sub.6O.sub.3(M+H).sup.+:
m/z=505.2; Found: 505.2; Isomer 2 (second to elute): Retention
time: 19.5 min; LCMS calculated for
C.sub.24H.sub.31ClFN.sub.6O.sub.3 (M+H).sup.+: m/z=505.2; Found:
505.2.
Step 2
1-[1-(3-Azetidin-3-yl-5-chloro-2-ethoxy-4-fluorophenyl)ethyl]-3-met-
hyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine dihydrochloride
[0367] This compound was prepared using procedures analogous to
those for Example 139 step 6 with tert-butyl
3-{3-[(1S)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5--
chloro-2-ethoxy-6-fluorophenyl}azetidine-1-carboxylate (first peak
from chiral separation) as starting material. LCMS calculated for
C.sub.19H.sub.23ClFN.sub.6O (M+H).sup.+: m/z=405.2; Found:
405.1.
Step 3.
(2S)-1-(3-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl-
)ethyl]-5-chloro-2-ethoxy-6-fluorophenyl}azetidin-1-yl)propan-2-ol
[0368] To a mixture of
1-[1-(3-azetidin-3-yl-5-chloro-2-ethoxy-4-fluorophenyl)ethyl]-3-methyl-1H-
-pyrazolo[3,4-d]pyrimidin-4-amine dihydrochloride (46 mg, 0.11
mmol) (from isomer 1) and triethylamine (50 .mu.L, 0.4 mmol) in
isopropyl alcohol (0.3 mL) was added (S)-(-)-methyloxirane (16
.mu.L, 0.23 mmol). The resulting mixture was stirred at 90.degree.
C. for 3 h. After cooling, the mixture was diluted with
acetonitrile and purified by RP-HPLC (XBridge C18 column, eluting
with a gradient of acetonitrile/water containing 0.1% ammonium
hydroxide, at flow rate of 30 mL/min) to give the desired product
(12 mg, 23%). The product was isolated as a single diastereomer.
.sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta. 8.05 (s, 1H), 7.38 (d,
1H), 6.15 (m, 1H), 4.26 (d, 1H), 3.76-3.60 (m, 6H), 2.99 (m, 2H),
2.48 (s, 3H), 2.22 (m, 2H), 1.62 (d, 3H), 1.25 (t, 3H), 0.93 (d,
3H) ppm; LCMS calculated for
C.sub.22H.sub.29ClFN.sub.6O.sub.2(M+H).sup.+: m/z=463.2; Found:
463.2.
Example 156.
(2R)-2-(3-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-
-5-chloro-2-methoxy-6-methylphenyl}azetidin-1-yl)propan-1-ol
##STR00075##
[0369] Step 1. Methyl (2S)-2-bromopropanoate
[0370] DMF (28 .mu.L, 0.36 mmol) was added to a mixture of
(2S)-2-bromopropanoic acid (0.552 g, 3.61 mmol) and oxalyl chloride
(0.61 mL, 7.2 mmol) in dichloromethane (4.6 mL) at 0.degree. C. The
reaction mixture was stirred at room temperature overnight. The
solvent was removed in vacuo. The residue was dissolved in
dichloromethane and treated with methanol (1.5 mL, 36 mmol) and
pyridine (0.44 mL, 5.4 mmol). The reaction solution was stirred at
room temperature for 2 h. The reaction solution was quenched with
saturated sodium bicarbonate solution and washed with brine, dried
over Na.sub.2SO.sub.4, filtered and concentrated to give the
desired product (0.51 g, 85%).
Step 2. Methyl
(2R)-2-(3-{3-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-
-5-chloro-2-methoxy-6-methylphenyl}azetidin-1-yl)propanoate
[0371] To a solution of
1-[1-(3-azetidin-3-yl-5-chloro-2-methoxy-4-methylphenyl)ethyl]-3-methyl-1-
H-pyrazolo[3,4-d]pyrimidin-4-amine hydrochloride (20.1 mg, 0.0475
mmol, chiral intermediate from Example 1, Step 7) in acetonitrile
(1 mL) was added triethylamine (23 .mu.L, 0.17 mmol) and methyl
(2S)-2-bromopropanoate (9.5 mg, 0.057 mmol). The reaction solution
was stirred at room temperature for 4 h. The solvent was removed to
give the desired product (6.2 mg, 28%). LCMS calculated for
C.sub.23H.sub.30ClN.sub.6O.sub.3(M+H).sup.+: m/z=473.2; Found:
473.3.
Step 3.
(2R)-2-(3-{3-[(1S)-1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-
-1-yl)ethyl]-5-chloro-2-methoxy-6-methylphenyl}azetidin-1-yl)propan-1-ol
[0372] A solution of methyl
(2R)-2-(3-{3-[(1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl-
]-5-chloro-2-methoxy-6-methylphenyl}azetidin-1-yl)propanoate (6.2
mg, 0.013 mmol) in dichloromethane (0.5 mL) was treated with 1.0 M
diisobutylaluminum hydride in toluene (0.1 mL, 0.1 mmol) at
0.degree. C. for 3 h. The reaction was quenched with methanol and
purified with preparative RP-HPLC (XBridge C18 column, eluting with
a gradient of acetonitrile/water containing 0.1% ammonium
hydroxide, at flow rate of 30 mL/min) to give the desired product
(0.8 mg, 14%). The product was isolated as a single diastereomer.
LCMS calculated for C.sub.22H.sub.30ClN.sub.6O.sub.2(M+H).sup.+:
m/z=445.2; Found: 445.1.
Example 158.
1-(3-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-ch-
loro-2-methoxy-6-methylphenyl}azetidin-1-yl)-2-methylpropan-2-ol
##STR00076##
[0374] This compound was prepared using procedures analogous to t
Example 140 with
1-[1-(3-azetidin-3-yl-5-chloro-2-methoxy-4-methylphenyl)ethyl]-3-
-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine hydrochloride (chiral
intermediate from Example 1, Step 7) and oxirane, 2,2-dimethyl--as
starting materials. The product was isolated as single enatiomer.
LCMS calculated for C.sub.23H.sub.32ClN.sub.6O.sub.2(M+H).sup.+:
m/z=459.2; Found: 459.1 .sup.1H NMR (300 MHz, DMSO-d.sub.6):
.delta. 8.04 (s, 1H), 7.23 (bs, 2H), 7.16 (s, 1H), 6.14 (m, 1H),
3.96 (s, 1H), 3.85 (m, 3H), 3.45 (s, 3H), 2.94 (m, 1H), 2.80 (m,
1H), 2.49 (s, 3H), 2.14 (s, 2H), 2.00 (s, 3H), 1.63 (d, 3H), 0.98
(s, 6H) ppm.
Example 159.
(2R)-2-(3-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-
-5-chloro-2-methoxy-6-methylphenyl}azetidin-1-yl)-N,N-dimethylpropanamide
##STR00077##
[0375] Step 1.
(2R)-2-(3-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-
-5-chloro-2-methoxy-6-methylphenyl}azetidin-1-yl)propanoic acid
[0376] To a solution of methyl
(2R)-2-(3-{3-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-
-5-chloro-2-methoxy-6-methylphenyl}azetidin-1-yl)propanoate (chiral
intermediate from example 156 step 2) (13 mg, 0.027 mmol) in
acetonitrile (0.6 mL) and water (0.2 mL) was added lithium
hydroxide (2.4 mg, 0.10 mmol). The reaction mixture was stirred at
room temperature overnight. The reaction solution was diluted with
ethyl acetate and 1 M HCl solution. The organic layer was separated
and dried over Na.sub.2SO.sub.4, filtered and concentrated to give
the desired product (10.2 mg, 83%). LCMS calculated for
C.sub.22H.sub.28ClN.sub.6O.sub.3(M+H).sup.+: m/z=459.2; Found:
459.1.
Step 2.
(2R)-2-(3-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl-
)ethyl]-5-chloro-2-methoxy-6-methylphenyl}azetidin-1-yl)-N,N-dimethylpropa-
namide
[0377] To a solution of
(2R)-2-(3-{3-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-
-5-chloro-2-methoxy-6-methylphenyl}azetidin-1-yl)propanoic acid (4
mg, 0.009 mmol) and
benzotriazol-1-yloxytris(dimethylamino)phosphonium
hexafluorophosphate (4 mg, 0.009 mmol) in DMF (0.3 mL) at room
temperature was added triethylamine (4 .mu.L, 0.03 mmol) and
dimethylamine hydrochloride (0.9 mg, 0.01 mmol). The reaction
mixture was stirred for 1 h, then diluted with methanol and
purified by preparative RP-HPLC (XBridge C18 column, eluting with a
gradient of acetonitrile/water containing 0.1% ammonium hydroxide,
at flow rate of 30 mL/min) to give the desired product (2.7 mg,
63%). The product was isolated as a single diastereomer. LCMS
calculated for C.sub.24H.sub.33ClN.sub.7O.sub.2(M+H).sup.+:
m/z=486.2; Found: 486.1. .sup.1H NMR (300 MHz, DMSO-d.sub.6):
.delta..quadrature. 8.09 (s, 1H), 7.23 (s, 1H), 6.18 (m, 1H), 3.78
(m, 3H), 3.50 (s, 3H), 3.01 (s, 3H), 3.0-2.9 (m, 3H), 2.77 (s, 3H),
2.54 (s, 3H), 2.06 (s, 3H), 1.67 (d, 3H), 0.98 (d, 3H) ppm.
Example 161.
[1-(3-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-c-
hloro-2-methoxy-6-methylphenyl}azetidin-1-yl)cyclobutyl]acetonitrile
##STR00078##
[0379] To a solution of
1-[(1-(3-azetidin-3-yl-5-chloro-2-methoxy-4-methylphenyl)ethyl]-3-methyl--
1H-pyrazolo[3,4-d]pyrimidin-4-amine dihydrochloride (10 mg, 0.022
mmol, chiral intermediate from Example 1, Step 7) in acetonitrile
(0.1 mL) was added cyclobutylideneacetonitrile (4.1 mg, 0.044
mmol), followed by 1,8-diazabicyclo[5.4.0]undec-7-ene (13 .mu.L,
0.087 mmol). The resulting mixture was stirred at room temperature
overnight. The reaction mixture was diluted with acetonitrile and
purified by preparative RP-HPLC (XBridge C18 column, eluting with a
gradient of acetonitrile/water containing 0.1% ammonium hydroxide,
at flow rate of 30 mL/min) to give the desired product (4.3 mg,
41%). The product was isolated as a single enantiomer. LCMS
calculated for C.sub.25H.sub.31ClN.sub.7O (M+H).sup.+: m/z=480.2;
Found: 480.0.
Example 163.
1-{1-[5-Chloro-2-methoxy-4-methyl-3-(1-methylpiperidin-4-yl)phenyl]ethyl}-
-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine
##STR00079##
[0380] Step 1. tert-Butyl
4-(3-acetyl-5-chloro-2-methoxy-6-methylphenyl)piperidine-1-carboxylate
[0381] This compound was prepared using procedures analogous to
those for Example 139 step 2 with
1-(5-chloro-3-iodo-2-methoxy-4-methylphenyl)ethanone and tert-butyl
4-iodopiperidine-1-carboxylate as starting materials. LCMS
calculated for C.sub.20H.sub.28ClNO.sub.4Na (M+Na).sup.+:
m/z=404.1; Found: 404.1.
Step 2. tert-Butyl
4-[3-chloro-5-(1-hydroxyethyl)-6-methoxy-2-methylphenyl]piperidine-1-carb-
oxylate
[0382] This compound was prepared according to the procedure of
Example 13 step 5, using of tert-butyl
4-(3-acetyl-5-chloro-2-methoxy-6-methylphenyl)piperidine-1-carboxylate
and sodium tetrahydroborate as the starting materials. LCMS
calculated for C.sub.20H.sub.30ClNO.sub.4Na (M+Na).sup.+:
m/z=406.1; Found: 406.1.
Step 3. tert-Butyl
4-[3-chloro-5-(1-chloroethyl)-6-methoxy-2-methylphenyl]piperidine-1-carbo-
xylate
[0383] This compound was prepared according to the procedure of
Example 13 step 6, using tert-butyl
4-[3-chloro-5-(1-hydroxyethyl)-6-methoxy-2-methylphenyl]piperidine-1-carb-
oxylate (racemic) and cyanuric chloride as the starting materials.
.sup.1H NMR (400 MHz, CDCl.sub.3): .delta. 7.44 (s, 1H), 5.46 (m,
1H), 4.23 (bs, 2H), 3.73 (s, 3H), 3.29 (bs, 1H), 2.78 (bs, 2H),
2.40 (s, 3H), 2.27-2.09 (m, 2H), 1.78 (d, 3H), 1.63 (m, 2H), 1.43
(s, 9H) ppm.
Step 4. tert-Butyl
4-{3-[1-(4-amino-3-methyl-H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chloro-
-2-methoxy-6-methylphenyl}piperidine-1-carboxylate
[0384] This compound was prepared according to the procedure of
Example 139 step 5, using of tert-butyl
4-[3-chloro-5-(1-chloroethyl)-6-methoxy-2-methylphenyl]piperidine-1-carbo-
xylate and 3-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine as the
starting materials. LCMS calculated for
C.sub.26H.sub.36ClN.sub.6O.sub.3(M+H).sup.+: m/z=515.3; Found:
515.2.
Step 5.
1-[1-(5-Chloro-2-methoxy-4-methyl-3-piperidin-4-ylphenyl)ethyl]-3--
methyl-H-pyrazolo[3,4-d]pyrimidin-4-amine dihydrochloride
[0385] This compound was prepared according to the procedure of
Example 139 step 6, using of tert-butyl
4-{3-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chlor-
o-2-methoxy-6-methylphenyl}piperidine-1-carboxylate as the starting
material. LCMS calculated for C.sub.21H.sub.28ClN.sub.6O
(M+H).sup.+: m/z=415.2; Found: 415.2.
Step 6.
1-{-[5-Chloro-2-methoxy-4-methyl-3-(1-methylpiperidin-4-yl)phenyl]-
ethyl}-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine
[0386] This compound was prepared according to the procedure of
Example 139 step 7, using of
1-[1-(5-chloro-2-methoxy-4-methyl-3-piperidin-4-ylphenyl)ethyl]-3-methyl--
1H-pyrazolo[3,4-d]pyrimidin-4-amine dihydrochloride and
formaldehyde as the starting materials. The product was isolated as
a racemic mixture. LCMS calculated for C.sub.22H.sub.30ClN.sub.6O
(M+H).sup.+: m/z=429.2; Found: 429.1.
Example 164.
1-(4-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-ch-
loro-2-methoxy-6-methylphenyl}piperidin-1-yl)-2-methylpropan-2-ol
##STR00080##
[0388] This compound was prepared using procedures analogous to
those for Example 140 with
1-[1-(5-chloro-2-methoxy-4-methyl-3-piperidin-4-ylphenyl)ethyl]-3-methyl--
1H-pyrazolo[3,4-d]pyrimidin-4-amine dihydrochloride (racemic
intermediate from Example 163, Step 5) and oxirane, 2,2-dimethyl-
as starting materials. The product was isolated as a racemic
mixture. LCMS calculated for
C.sub.25H.sub.36ClN.sub.6O.sub.2(M+H).sup.+: m/z=487.3; Found:
487.3. .sup.1H NMR (300 MHz, DMSO-d.sub.6): .delta. 8.05 (s, 1H),
7.24 (bs, 2H), 7.22 (s, 1H), 6.16 (m, 1H), 4.01 (bs, 1H), 3.67 (s,
3H), 2.97 (m, 3H), 2.49 (s, 3H), 2.32 (s, 3H), 2.15-2.04 (m, 6H),
1.63 (d, 3H), 1.40 (m, 2H), 1.03 (s, 6H) ppm.
Example 166.
3-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chlor-
o-2-methoxy-6-methylphenyl}cyclobutanol trifluoroacetate
##STR00081##
[0389] Step 1.
1-(5-Chloro-2-methoxy-4-methyl-3-vinylphenyl)ethanone
[0390] A mixture of
1-(5-chloro-3-iodo-2-methoxy-4-methylphenyl)ethanone (1.0 g, 3.2
mmol, from Example 1, Step 2),
4,4,5,5-tetramethyl-2-vinyl-1,3,2-dioxaborolane (0.66 mL, 3.9
mmol), [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II),
complex with dichloromethane (1:1) (0.26 g, 0.32 mmol) and
potassium carbonate (1.3 g, 9.4 mmol) in 1,4-dioxane (10 mL) and
water (5 mL) was degassed with N.sub.2 and heated at 80.degree. C.
overnight. After cooled to room temperature, the reaction mixture
was diluted with water and ethyl acetate. The organic layer was
washed with brine, dried over MgSO.sub.4, concentrated and purified
on a silica gel column (eluting with 0 to 10% EtOAc in hexanes) to
give the desired product (0.60 g, 82%). LCMS calculated for
C.sub.12H.sub.14ClO.sub.2 (M+H).sup.+: m/z=225.1; Found: 225.1
Step 2.
3-(3-Acetyl-5-chloro-2-methoxy-6-methylphenyl)cyclobutanone
[0391] To a solution of
1-(5-chloro-2-methoxy-4-methyl-3-vinylphenyl)ethanone (530 mg, 2.4
mmol) in ether (10 mL) was added zinc-copper couple (1.8 g, 14
mmol). The reaction mixture was heated at 40.degree. C. and a
solution of trichloroacetyl chloride (1.4 mL, 13 mmol) and
phosphoryl chloride (1.2 mL, 13 mmol) in 1,2-dimethoxyethane (3 mL)
was added slowly over 2 h. After addition, the reaction mixture was
stirred under reflux overnight. The reaction was quenched with
saturated NaHCO.sub.3 solution and diluted with ether. The organic
layer was washed with brine, dried over Na.sub.2SO.sub.4, filtered
and concentrated. The residue and zinc (0.31 g, 4.7 mmol) in acetic
acid (10 mL) was stirred at room temperature for 2 h and then
reflux overnight. Another portion of zinc was added and reflux for
another 4 h. The mixture was diluted with water and extracted with
ether. The organic phase was washed successively with a saturated
NaHCO.sub.3 solution, water and brine, then dried over MgSO.sub.4
and concentrated. The crude material was purified with flash
chromatography (eluting with 0 to 30% ethyl acetate in hexanes) to
give the desired product (0.17 g, 27%). LCMS calculated for
C.sub.14H.sub.16ClO.sub.3 (M+H).sup.+: m/z=267.1; Found: 267.0.
Step 3.
3-[3-Chloro-5-(1-hydroxyethyl)-6-methoxy-2-methylphenyl]cyclobutan-
ol
[0392] This compound was prepared according to the procedure of
Example 13 step 5, using of
3-(3-acetyl-5-chloro-2-methoxy-6-methylphenyl)cyclobutanone and
sodium tetrahydroborate as the starting materials. LCMS calculated
for C.sub.14H.sub.19ClO.sub.3Na (M+Na).sup.+: m/z=293.1; Found:
293.1.
Step 4.
3-[3-Chloro-5-(1-chloroethyl)-6-methoxy-2-methylphenyl]cyclobutano-
l
[0393] To a solution of
3-[3-chloro-5-(1-hydroxyethyl)-6-methoxy-2-methylphenyl]cyclobutanol
(170 mg, 0.628 mmol) in dimethyl sulfoxide (1 mL) was added
cyanuric chloride (64 mg, 0.34 mmol). After stirred overnight, the
reaction mixture was diluted with ether and water. The aqueous
layer was extracted with ethyl acetate once. The combined organic
extracts were washed with brine, dried over Na.sub.2SO.sub.4,
filtered and concentrated. The crude was purified with silica gel
column to give the desired product (39.6 mg, 22%). LCMS calculated
for C.sub.14H.sub.18ClO.sub.2 (M-Cl).sup.+: m/z=253.1; Found:
253.2.
Step 5.
3-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]--
5-chloro-2-methoxy-6-methylphenyl}cyclobutanol trifluoroacetate
[0394] This compound was prepared according to the procedure of
Example 139 step 5, using of
3-[3-chloro-5-(1-chloroethyl)-6-methoxy-2-methylphenyl]cyclobutanol
and 3-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine as the starting
materials. The product was isolated as a racemic mixture. LCMS
calculated for C.sub.20H.sub.25ClN.sub.5O.sub.2(M+H).sup.+:
m/z=402.2; Found: 402.2.
Example 167.
5-(3-(1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chlor-
o-2-methoxy-6-methylphenyl)-N,N-dimethylpicolinamide
bis(2,2,2-trifluoroacetate)
##STR00082##
[0395] Step 1.
1-(3-Bromo-5-chloro-2-methoxy-4-methylphenyl)ethanone
[0396] To a stirred solution of
1-(5-chloro-2-methoxy-4-methylphenyl)ethanone (5.00 g, 25.2 mmol,
from Oakwood) in acetic acid (100 mL) was added N-bromosuccinimide
(4.93 g, 27.7 mmol) and the resulting mixture heated at 100.degree.
C. for 18 hours. After cooling to ambient temperature, the reaction
mixture was concentrated in vacuo, then neutralized with sat.
sodium bicarbonate, filtered off insoluble succinimide. The
filtrate was extracted with EtOAc. The combined organic layers were
washed with brine, dried over sodium sulfate, and then concentrated
to dryness under reduced pressure. The residue was purified on
silica gel, eluting with 0 to 50% EtOAc in hexanes, to give the
desired products (2.66 g, 38%). LCMS calculated for
C.sub.10H.sub.11BrClO.sub.2 (M+H).sup.+: m/z=277.0; found: 277.0.
.sup.1H NMR (DMSO-d.sub.6, 300 MHz): .delta. 7.70 (1H, s), 3.77
(3H, s), 2.57 (3H, s), 2.50 (3H, s) ppm.
Step 2. 1-(3-Bromo-5-chloro-2-methoxy-4-methylphenyl)ethanol
[0397] Sodium tetrahydroborate (0.31 g, 8.1 mmol) was added to a
mixture of 1-(3-bromo-5-chloro-2-methoxy-4-methylphenyl)ethanone
(1.5 g, 5.4 mmol) in methanol (25 mL) at 0.degree. C. and the
resultant reaction mixture was stirred at room temperature for 1
hour. The solvent was removed and the resulting residue was diluted
with ethyl acetate, washed with sat. NaHCO.sub.3, water, brine,
then dried over Na.sub.2SO.sub.4, filtered and concentrated. The
crude product was purified by silica gel chromatography, eluting
with 0 to 40% EtOAc in hexanes, to give the desired product (0.30
g, 90%).
Step 3.
3-Bromo-1-chloro-5-(1-chloroethyl)-4-methoxy-2-methylbenzene
[0398] A mixture of cyanuric chloride (1.7 g, 9.2 mmol) and
N,N-dimethylformamide (710 .mu.L, 9.2 mmol) was stirred at room
temperature for 10 minutes and then a solution of
1-(3-bromo-5-chloro-2-methoxy-4-methylphenyl)ethanol (from Example
16, Step 1) (1.72 g, 6.15 mmol) in methylene chloride (34 mL) was
added and the reaction was stirred at room temperature overnight.
The mixture was diluted with methylene chloride, washed with sat.
NaHCO.sub.3, water, brine, dried over Na.sub.2SO.sub.4, filtered
and concentrated. The crude product was purified by silica gel
chromatography, eluting with 0 to 10% EtOAc in hexanes, to give the
desired product (1.01 g, 60%).
Step 4.
1-[1-(3-Bromo-5-chloro-2-methoxy-4-methylphenyl)ethyl]-3-methyl-1H-
-pyrazolo[3,4-d]pyrimidin-4-amine
[0399] A mixture of
3-bromo-1-chloro-5-(1-chloroethyl)-4-methoxy-2-methylbenzene (150
mg, 0.503 mmol), 3-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (110
mg, 0.76 mmol, ACES Phanna Product List, item #47024), potassium
iodide (9.0 mg, 0.05 mmol) and cesium carbonate (330 mg, 1.0 mmol)
in N,N-dimethylformamide (4 mL) and was stirred at 140.degree. C.
for 1 h. The mixture was diluted with methylene chloride, washed
with sat. NaHCO.sub.3, water, brine, dried over Na.sub.2SO.sub.4,
filtered and concentrated. The crude product was purified by silica
gel chromatography, eluting with 0 to 70% EtOAc in
CH.sub.2Cl.sub.2, to give the desired product (103 mg, 50%). LCMS
calculated for C.sub.16H.sub.18BrClN.sub.5O (M+H).sup.+: m/z=410.0;
Found: 410.2. The racemic products were applied on a Phenomenex
Lux-Cellulose 1 column (21.1.times.250 mm, 5 micron particle size),
eluting with 5% ethanol in hexanes at a flow rate of 18 mL/min,
.about.13 mg/injection, to provide two enantiomers. Peak 1,
retention time: 12.35 min; Peak 2, retention time: 14.98 min.
Step 5.
5-(3-(1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)--
5-chloro-2-methoxy-6-methylphenyl)-N,N-dimethylpicolinamide
bis(2,2,2-trifluoroacetate)
[0400] A mixture of
1-[1-(3-bromo-5-chloro-2-methoxy-4-methylphenyl)ethyl]-3-methyl-1H-pyrazo-
lo[3,4-d]pyrimidin-4-amine (25 mg, 0.061 mmol) (first peak from
previous step chiral separation),
N,N-dimethyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine-2-ca-
rboxamide (25 mg, 0.091 mmol, from PepTech Corp. Encyclopedia of
Amino Acid Analogs and Boronic Acids, item #BE1622-1), sodium
carbonate (13 mg, 0.12 mmol) and
[1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium (II),
complex with dichloromethane (1:1) (9.9 mg, 0.012 mmol) in
acetonitrile (0.8 mL)/water (0.3 mL) was degassed with N.sub.2 and
then stirred at 95.degree. C. for 2 h. After cooling to room
temperature, the mixture was filtered and the filtrate purified on
RP-HPLC (XBridge C18 column, eluting with a gradient of
acetonitrile/water containing 0.05% trifluoroacetic acid, at flow
rate of 30 mL/min) to give the desired product as bis-TFA salt (2.9
mg, 6.7%). The product was isolated as a single enantiomer. LCMS
calculated for C.sub.24H.sub.27ClN.sub.7O.sub.2(M+H).sup.+:
m/z=480.2; Found: 480.2. .sup.1H NMR (500 MHz, DMSO-d.sub.6)
.delta.: 8.78 (2H, br s), 8.48 (1H, m), 8.36 (1H, s), 7.86 (1H, br
s), 7.65 (1H, br s), 7.58 (1H, s), 6.33 (1H, q, J=7.0 Hz), 3.19
(3H, s), 3.03 (3H, s), 2.97 (3H, s), 2.62 (3H, s), 2.06 (3H, s),
1.81 (3H, d, J=7.0 Hz) ppm.
Example 183.
1-[1-(5-Chloro-3-{1-[2-(dimethylamino)ethyl]-1H-pyrazol-4-yl}-2-methoxy-4-
-methylphenyl)ethyl]-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine
##STR00083##
[0401] Step 1.
1-(2-Chloroethyl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyra-
zole
[0402] A mixture of
4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (0.39
g, 2.0 mmol), 1-bromo-2-chloroethane (0.3 mL, 3 mmol) and cesium
carbonate (1.3 g, 4.0 mmol) in acetonitrile (6 mL) was stirred at
75.degree. C. for 5 h. The mixture was diluted with ethyl acetate,
washed with sat. NaHCO.sub.3, water, brine, dried over
Na.sub.2SO.sub.4, filtered and concentrated and the product (0.45
g, 88%) was purified by chromatography eluting with hexanes/EtOAc
(max. EtOAc 30%). LCMS calculated for
C.sub.11H.sub.19BClN.sub.2O.sub.2(M+H).sup.+: m/z=257.1; Found:
257.0
Step 2.
N,N-Dimethyl-2-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-
-pyrazol-1-yl]ethanamine
[0403] A mixture of
1-(2-chloroethyl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyra-
zole (0.10 g, 0.39 mmol), sodium iodide (58 mg, 0.39 mmol) and 2.0
M dimethylamine in THF (1.0 mL, 2.0 mmol) in N,N-dimethylformamide
(0.5 mL) was stirred at 80.degree. C. overnight. The solvent was
removed to provide the desired product which was used in the next
step. LCMS calculated for
Cl.sub.3H.sub.25BN.sub.3O.sub.2(M+H).sup.+: m/z=266.2; Found:
266.3.
Step 3.
1-[1-(5-chloro-3-{1-[2-(dimethylamino)ethyl]-H-pyrazol-4-yl}-2-met-
hoxy-4-methylphenyl)ethyl]-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine
[0404] A mixture of
1-[1-(3-bromo-5-chloro-2-methoxy-4-methylphenyl)ethyl]-3-methyl-1H-pyrazo-
lo[3,4-d]pyrimidin-4-amine (Peak 1 from Example 167, step 4, 10 mg,
0.024 mmol),
N,N-dimethyl-2-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-
-pyrazol-1-yl]-ethanamine (8.6 mg, 0.036 mmol), sodium carbonate
(5.2 mg, 0.049 mmol) and
[1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II),
complex with dichloromethane (1:1) (4.0 mg, 0.0049 mmol) in
acetonitrile (0.5 mL)/water (0.1 mL) was vacuumed and the refilled
with N.sub.2 and the stirred at 95.degree. C. for 2 h. The crude
was purified using RP-HPLC (XBridge C18 column, eluting with a
gradient of acetonitrile/water containing 0.1% ammonium hydroxide,
at flow rate of 30 mL/min) to give the desired product (3.1 mg,
28%). The product was isolated as a single enantiomer. LCMS
calculated for C.sub.23H.sub.30ClN.sub.8O (M+H).sup.+: m/z=469.2;
Found: 469.2.
Example 184.
2-[(5-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-c-
hloro-2-methoxy-6-methylphenyl}pyridin-2-yl)amino]ethanol
##STR00084##
[0405] Step 1.
1-{1-[5-Chloro-3-(6-fluoropyridin-3-yl)-2-methoxy-4-methylphenyl]ethyl}-3-
-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine
[0406] A mixture of
1-[1-(3-bromo-5-chloro-2-methoxy-4-methylphenyl)ethyl]-3-methyl-1H-pyrazo-
lo[3,4-d]pyrimidin-4-amine (Peak 1 from Example 167, step 4, 25.0
mg, 0.06 mmol),
2-fluoro-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine
(20. mg, 0.088 mmol), sodium carbonate (12 mg, 0.12 mmol) and
[1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II),
complex with dichloromethane (1:1) (9.5 mg, 0.012 mmol) in
acetonitrile (1 mL)/water (0.3 mL) was degassed with N.sub.2 and
the stirred at 95.degree. C. for 2 h. The mixture was diluted with
methylene chloride, washed with sat. NaHCO.sub.3, water, brine,
dried over Na.sub.2SO.sub.4, filtered and concentrated. The product
was purified by chromatography eluting with CH.sub.2Cl.sub.2/MeOH
(max. MeOH 5%). LCMS calculated for C.sub.21H.sub.21ClFN.sub.6O
(M+H).sup.+: m/z=427; Found: 427.2.
Step 2.
2-[(5-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)eth-
yl]-5-chloro-2-methoxy-6-methylphenyl}pyridin-2-yl)amino]ethanol
[0407] A mixture of
1-{1-[5-chloro-3-(6-fluoropyridin-3-yl)-2-methoxy-4-methylphenyl]ethyl}-3-
-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (10 mg, 0.023 mmol) and
ethanolamine (0.10 mL) in 1-butanol (1 mL) was stirred at
130.degree. C. for 5 h. The crude was purified using RP-HPLC
(XBridge C18 column, eluting with a gradient of acetonitrile/water
containing 0.1% ammonium hydroxide, at flow rate of 30 mL/min) to
give the desired product (1.6 mg, 15%). The product was isolated as
a single enantiomer. LCMS calculated for
C.sub.23H.sub.27ClN.sub.7O.sub.2 (M+H).sup.+: m/z=468.2; Found:
468.2.
Example 188.
2-(5-(3-(1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-ch-
loro-2-methoxy-6-methylphenyl)pyridin-2-yloxy)ethanol
##STR00085##
[0409] Sodium hydride (20 mg, 0.5 mmol) was added to 1,2-ethanediol
(0.5 mL, 9 mmol) and the mixture was stirred at room temperature
for 10 min. At this time
1-{1-[5-chloro-3-(6-fluoropyridin-3-yl)-2-methoxy-4-methylphenyl]ethyl}-3-
-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (10 mg, 0.023 mmol) was
added and then the reaction was stirred at 110.degree. C.
overnight. The crude was purified using RP-HPLC (XBridge C18
column, eluting with a gradient of acetonitrile/water containing
0.1% ammonium hydroxide, at flow rate of 30 mL/min) to give the
desired product (1.8 mg, 17%). The product was isolated as a single
enantiomer. LCMS calculated for
C.sub.23H.sub.26ClN.sub.6O.sub.3(M+H).sup.+: m/z=469.2; Found:
469.1.
Example 189.
5-(3-(1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chlor-
o-2-(2,2-difluoroethoxy)-6-methylphenyl)-N,N-dimethylpicolinamide
bis(2,2,2-trifluoroacetate)
##STR00086##
[0410] Step 1.
5-(3-(1-(4-Amino-3-methyl-H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-
-2-hydroxy-6-methylphenyl)-N,N-dimethylpicolinamide
[0411] 1.0 M Boron tribromide in CH.sub.2Cl.sub.2 (250 .mu.L, 0.25
mmol) was added to a mixture of
5-{3-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chlor-
o-2-methoxy-6-methylphenyl}-N,N-dimethylpyridine-2-carboxamide
(Example 167, step 5, (first peak) 60 mg, 0.13 mmol) in methylene
chloride (1.2 mL) at -78.degree. C. and then the reaction was
warmed to room temperature. At this time conc. HCl (0.1 mL) was
added and the mixture was stirred for 4 h. The reaction was
quenched by the addition of sat. NaHCO.sub.3. The mixture was then
extracted with methylene chloride. The combined extracts were
washed with brine, dried and concentrated to give the desired crude
product (40 mg, 68%) which was used in the next step without
further purification. LCMS calculated for
C.sub.23H.sub.25ClN.sub.7O.sub.2(M+H).sup.+: m/z=466.2; Found:
466.2.
Step 2.
5-[3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]--
5-chloro-2-(2,2-difluoroethoxy)-6-methylphenyl]-N
N-dimethylpyridine-2-carboxamide
[0412] Diisopropyl azodicarboxylate (13 .mu.L, 0.064 mmol) was
added to a mixture of
5-{3-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chlor-
o-2-hydroxy-6-methylphenyl}-N,N-dimethylpyridine-2-carboxamide
(15.0 mg, 0.0322 mmol), 2,2-difluoroethanol (7.9 mg, 0.096 mmol,
from Alfa Aesar, item # B22201) and triphenylphosphine (17 mg,
0.064 mmol) in tetrahydrofuran (0.5 mL) at 0.degree. C. and then
the reaction was stirred at room temperature for 24 h. The crude
was purified on RP-HPLC (XBridge C18 column, eluting with a
gradient of acetonitrile/water containing 0.05% trifluoroacetic
acid, at flow rate of 30 mL/min) to give the desired product as
bis-TFA salt (1.6 mg, 6.6%). The product was isolated as a single
enantiomer. LCMS calculated for
C.sub.25H.sub.27ClF.sub.2N.sub.7O.sub.2 (M+H).sup.+: m/z=530.2;
Found: 530.2.
Example 192.
1-[1-(5-Chloro-3-cyclopropyl-2-methoxy-4-methylphenyl)ethyl]-3-methyl-1H--
pyrazolo[3,4-d]pyrimidin-4-amine
##STR00087##
[0414] To a microwave vial was added
1-[1-(3-bromo-5-chloro-2-methoxy-4-methylphenyl)ethyl]-3-methyl-1H-pyrazo-
lo[3,4-d]pyrimidin-4-amine (15 mg, 0.037 mmol, from peak 1 from
Example 167, step 4), potassium cyclopropyltrifluoroborate (8 mg,
0.06 mmol, from Frontier Scientific, item # C10298), potassium
phosphate (23 mg, 0.11 mmol), and
tetrakis(triphenylphosphine)palladium (4.2 mg, 0.0036 mmol) and
then toluene (0.3 mL)/water (0.1 mL). The vial was sealed and
degassed with N.sub.2 three times. The reaction was heat at
110.degree. C. for 20 h. The crude was purified using RP-HPLC
(XBridge C18 column, eluting with a gradient of acetonitrile/water
containing 0.1% ammonium hydroxide, at flow rate of 30 mL/min) to
give the desired product (1.1 mg, 8%). The product was isolated as
a single enantiomer. LCMS calculated for C.sub.19H.sub.23ClN.sub.5O
(M+H).sup.+: m/z=372.2; Found: 372.2.
Example 195.
5-(3-(1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chlor-
o-2-ethoxy-6-methylphenyl)-N,N-dimethylpicolinamide
bis(2,2,2-trifluoroacetate)
##STR00088##
[0415] Step 1.
1-(3-Bromo-5-chloro-2-ethoxy-4-methylphenyl)ethanone
[0416] Into a round bottom flask was placed
1-(3-bromo-5-chloro-2-hydroxy-4-methylphenyl)ethanone (6.0 g, 23
mmol) in anhydrous DMF (22.8 mL). Potassium carbonate (6.3 g, 46
mmol) was then added followed by iodoethane (2.73 mL, 34.2 mmol).
The resulting suspension was stirred at 60.degree. C. for 2 h. The
mixture was poured into 100 mL water and extracted with 200 mL of
ethyl ether. The organic layers were separated, combined and washed
with water and saturated NaCl solution, dried over anhydrous sodium
sulfate, filtered, and concentrated to 6.0 g of tan oil. LCMS
calculated for C.sub.11H.sub.13BrClO.sub.2 (M+H).sup.+: m/z=293.0;
Found: 293.0.
Step 2. 1-(3-Bromo-5-chloro-2-ethoxy-4-methylphenyl)ethanol
[0417] Sodium tetrahydroborate (0.31 g, 8.1 mmol) was added to a
mixture of 1-(3-bromo-5-chloro-2-ethoxy-4-methylphenyl)ethanone
(1.5 g, 5.4 mmol) in methanol (25 mL) at 0.degree. C. and the
resultant reaction mixture was stirred at room temperature for 1
hour. The solvent was removed and the resulting residue was diluted
with ethyl acetate, washed with sat. NaHCO.sub.3, water, brine,
then dried over Na.sub.2SO.sub.4, filtered and concentrated. The
crude product was purified by silica gel chromatography, eluting
with 0 to 30% EtOAc in hexanes (0.30 g, 90%).
Step 3.
3-Bromo-1-chloro-5-(1-chloroethyl)-4-ethoxy-2-methylbenzene
[0418] A mixture of cyanuric chloride (1.7 g, 9.2 mmol) and
N,N-dimethylformamide (710 .mu.L, 9.2 mmol) was stirred at room
temperature for 10 minutes and then a solution of
1-(3-bromo-5-chloro-2-ethoxy-4-methylphenyl)ethanol (1.72 g, 6.15
mmol) in methylene chloride (34 mL) was added and the reaction was
stirred at room temperature overnight. The mixture was diluted with
methylene chloride, washed with sat. NaHCO.sub.3, water, brine,
dried over Na.sub.2SO.sub.4, filtered and concentrated. The crude
product was purified by silica gel chromatography, eluting with 0
to 10% EtOAc in hexanes (1.01 g, 60%).
Step 4.
1-(1-(3-Bromo-5-chloro-2-ethoxy-4-methylphenyl)ethyl)-3-methyl-1H--
pyrazolo[3,4-d]pyrimidin-4-amine
[0419] A mixture of
3-bromo-1-chloro-5-(1-chloroethyl)-4-ethoxy-2-methylbenzene (150
mg, 0.50 mmol), 3-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (110
mg, 0.76 mmol), potassium iodide (9 mg, 0.05 mmol) and cesium
carbonate (330 mg, 1.0 mmol) in N,N-dimethylformamide (4 mL) was
stirred at 140.degree. C. for 1 h. The mixture was diluted with
methylene chloride, washed with sat. NaHCO.sub.3, water, brine,
dried over Na.sub.2SO.sub.4, filtered and concentrated. The crude
product was purified by silica gel chromatography, eluting with 0
to 70% EtOAc in CH.sub.2Cl.sub.2 (103 mg, 50%). LCMS calculated for
C.sub.17H.sub.20BrClN.sub.5O (M+H).sup.+: m/z=423.1; Found: 423.0.
The racemic products were applied on a Phenomenex Lux-Cellulose 1
column (21.1.times.250 mm, 5 micron particle size), eluting with 4%
ethanol in hexanes at a flow rate of 18 mL/min, .about.13
mg/injection, to provide two enantiomers. Peak 1, retention time:
8.64 min; Peak 2, retention time: 10.64 min.
Step 5.
5-(3-(1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)--
5-chloro-2-ethoxy-6-methylphenyl)-N,N-dimethylpicolinamide
bis(2,2,2-trifluoroacetate)
[0420] A mixture of
1-[1-(3-bromo-5-chloro-2-ethoxy-4-methylphenyl)ethyl]-3-methyl-1H-pyrazol-
o[3,4-d]pyrimidin-4-amine (25 mg, 0.061 mmol) (first peak from
previous step chiral separation),
N,N-dimethyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine-2-ca-
rboxamide (25 mg, 0.09 mmol), sodium carbonate (13 mg, 0.12 mmol)
and [1,1'-bis(diphenylphosphino)-ferrocene]dichloropalladium (II),
complex with dichloromethane (1:1) (9.9 mg, 0.012 mmol) in
acetonitrile (0.8 mL)/water (0.3 mL) was degassed with N.sub.2 and
then stirred at 95.degree. C. for 2 hours. After cooling to room
temperature, the mixture was filtered and the filtrate purified on
RP-HPLC (XBridge C18 column, eluting with a gradient of
acetonitrile/water containing 0.05% trifluoroacetic acid, at flow
rate of 30 mL/min) to give the desired product as bis-TFA salt (2.3
mg, 5%). The product was isolated as a single enantiomer. LCMS
calculated for C.sub.25H.sub.29ClN.sub.7O.sub.2(M+H).sup.+:
m/z=494.2; Found: 494.2.
Example 200.
4-(3-(1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chlor-
o-2-methoxy-6-methylphenyl)-N,N-dimethylpicolinamide
bis(2,2,2-trifluoro acetate)
##STR00089##
[0421] Step 1.
4-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chlor-
o-2-methoxy-6-methylphenyl}pyridine-2-carbonitrile
[0422] A mixture of
1-[1-(3-bromo-5-chloro-2-methoxy-4-methylphenyl)ethyl]-3-methyl-1H-pyrazo-
lo[3,4-d]pyrimidin-4-amine (peak 1 from Example 167, step 4, 322
mg, 0.76 mmol),
4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine-2-carbonit-
rile (210 mg, 0.91 mmol, from Combi-Blocks Catalog, item #
PN-0143), sodium carbonate (130 mg, 1.2 mmol) and
[1,1'-bis(diphenylphosphino)ferrocene]-dichloropalladium(II),
complex with dichloromethane (1:1) (99 mg, 0.12 mmol) in
acetonitrile (5 mL)/water (2 mL) was degassed with N.sub.2 and the
reaction was stirred at 95.degree. C. for 2 h. The mixture was
diluted with methylene chloride, washed with sat. NaHCO.sub.3,
water, brine, dried over Na.sub.2SO.sub.4, filtered and
concentrated. The product (0.28 g, 85%) was purified by
chromatography eluting with CH.sub.2Cl.sub.2/MeOH (max. MeOH 6%).
LCMS calculated for C.sub.22H.sub.21ClN.sub.7O (M+H).sup.+:
m/z=434.1; Found: 434.1.
Step 2.
4-(3-(1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)--
5-chloro-2-methoxy-6-methylphenyl)picolinic acid
dihydrochloride
[0423] 1.0 M Sodium hydroxide (2.9 mL, 2.9 mmol) was added to a
mixture of
4-{3-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chlor-
o-2-methoxy-6-methylphenyl}pyridine-2-carbonitrile (0.250 g, 0.576
mmol) in ethanol (4.0 mL) and the resulting mixture was heated at
95.degree. C. for 6 h. At this time, conc. HCl was added to adjust
the pH to .about.3. The solvent was removed and the residue was
used in the next step without further purification. LCMS calculated
for C.sub.22H.sub.22ClN.sub.6O.sub.3(M+H).sup.+: m/z=453.1; Found:
453.2.
Step 3.
4-(3-(1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)--
5-chloro-2-methoxy-6-methylphenyl)-N,N-dimethylpicolinamide
bis(2,2,2-trifluoroacetate)
[0424] 2.0 M Dimethylamine in THF (2.0 mL, 4.0 mmol) was added to a
solution of
4-{3-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chlor-
o-2-methoxy-6-methylphenyl}pyridine-2-carboxylic acid (250 mg,
0.552 mmol) and benzotriazol-1-yloxytris(dimethylamino)phosphonium
hexafluorophosphate (370 mg, 0.83 mmol) in N,N-dimethylformamide (4
mL) at 0.degree. C. followed by adding triethylamine (0.23 mL, 1.6
mmol). The reaction was stirred for 1 h. The crude mixture was
purified on RP-HPLC (XBridge C18 column, eluting with a gradient of
acetonitrile/water containing 0.05% trifluoroacetic acid, at flow
rate of 30 mL/min) to give the desired product as bis-TFA salt. The
product was isolated as a single enantiomer. LCMS calculated for
C.sub.24H.sub.27ClN.sub.7O.sub.2(M+H).sup.+: m/z=480.2; Found:
480.2. .sup.1H NMR (DMSO-d.sub.6, 500 MHz) .delta. 8.67 (br s, 1H),
8.36 (s, 1H), 7.58 (s, 1H), 7.41 (m, 2H), 6.32 (q, 2H), 3.20 (s,
3H), 3.00 (s, 3H), 2.94 (s, 3H), 2.62 (s, 3H), 2.03 (s, 3H), 1.80
(d, 3H) ppm.
Example 203.
2-(4-(3-(1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-ch-
loro-2-ethoxy-6-methylphenyl)-1H-pyrazol-1-yl)acetamide
##STR00090##
[0425] Step 1. tert-Butyl
[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazol-1-yl]acetate
[0426] 1.0 M Potassium tert-butoxide in THF (2.4 mL, 2.4 mmol) was
added to a solution of
4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (0.39
g, 2.0 mmol) in N,N-dimethylformamide (6.0 mL) at 0.degree. C. The
reaction mixture was stirred at room temperature for 5 min. After
cooled to 0.degree. C., to the mixture was added t-butyl
bromoacetate (0.5 mL, 3 mmol). The reaction was stirred at room
temperature for 2 h, then diluted with ethyl acetate, washed with
sat. NaHCO.sub.3, water, brine, dried over Na.sub.2SO.sub.4,
filtered and concentrated. The product (0.5 g, 81%) was purified by
chromatography eluting with hexanes/EtOAc (max. EtOAc 30%). LCMS
calculated for C.sub.15H.sub.26BN.sub.2O.sub.4(M+H).sup.+:
m/z=309.2; Found: 309.1.
Step 2. tert-Butyl
(4-{3-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chlo-
ro-2-ethoxy-6-methylphenyl}-1H-pyrazol-1-yl)acetate
[0427] A mixture of
1-[1-(3-bromo-5-chloro-2-ethoxy-4-methylphenyl)ethyl]-3-methyl-1H-pyrazol-
o[3,4-d]pyrimidin-4-amine (70 mg, 0.16 mmol) (first peak from
Example 195, step 4), tert-butyl
[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazol-1-yl]acetate
(65 mg, 0.21 mmol), sodium carbonate (30. mg, 0.28 mmol) and
[1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II),
complex with dichloromethane (1:1) (23 mg, 0.028 mmol) in
acetonitrile (3 mL)/water (0.7 mL) was degassed with N.sub.2 and
then stirred at 95.degree. C. for 2 h. The mixture was diluted with
methylene chloride, washed with sat. NaHCO.sub.3, water, brine,
dried over Na.sub.2SO.sub.4, filtered and concentrated. The product
(65 mg, 78%) was purified by chromatography eluting with
CH.sub.2Cl.sub.2/MeOH (max. MeOH 5%). LCMS calculated for
C.sub.26H.sub.33ClN.sub.7O.sub.3 (M+H).sup.+: m/z=526.2; Found:
526.3.
Step 3.
(4-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-
-5-chloro-2-ethoxy-6-methylphenyl}-1H-pyrazol-1-yl)acetic acid bis
trifluoroacetate
[0428] Trifluoroacetic acid (0.5 mL) was added to a solution of
tert-butyl
(4-{3-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chlo-
ro-2-ethoxy-6-methylphenyl}-1H-pyrazol-1-yl)acetate (0.065 g, 0.12
mmol) in methylene chloride (0.5 mL). The reaction was stirred at
room temperature for 4 h. The solvent was removed to provide the
crude product which was used in the next step. LCMS calculated for
C.sub.22H.sub.25ClN.sub.7O.sub.3(M+H).sup.+: m/z=470.2; Found:
470.1.
Step 4.
2-(4-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethy-
l]-5-chloro-2-ethoxy-6-methylphenyl}-1H-pyrazol-1-yl)acetamide
[0429] Ammonium carbonate (20 mg, 0.21 mmol) was added to a
solution of
(4-{3-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chlo-
ro-2-ethoxy-6-methylphenyl}-1H-pyrazol-1-yl)acetic acid bis
trifluoroacetate (10 mg, 0.021 mmol) and
benzotriazol-1-yloxytris(dimethylamino)phosphonium
hexafluorophosphate (10 mg, 0.03 mmol) in N,N-dimethylformamide
(0.7 mL) at room temperature followed by triethylamine (8.8 .mu.L,
0.064 mmol). The reaction was stirred for 1 h. The crude was
purified using RP-HPLC (XBridge C18 column, eluting with a gradient
of acetonitrile/water containing 0.1% ammonium hydroxide, at flow
rate of 30 mL/min) to give the desired product (2.5 mg, 25%). The
product was isolated as a single enantiomer. LCMS calculated for
C.sub.22H.sub.26ClN.sub.8O.sub.2(M+H).sup.+: m/z=469.2; Found:
469.2.
Example 208.
6-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chlor-
o-2-ethoxy-6-methylphenyl}-N,N-dimethylnicotinamide
bis(trifluoroacetate)
##STR00091##
[0430] Step 1.
1-{1-[5-Chloro-2-ethoxy-4-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborola-
n-2-yl)phenyl]ethyl}-3-methyl-H-pyrazolo[3,4-d]pyrimidin-4-amine
[0431]
1-[1-(3-Bromo-5-chloro-2-ethoxy-4-methylphenyl)ethyl]-3-methyl-1H-p-
yrazolo[3,4-d]pyrimidin-4-amine (0.050 g, 0.12 mmol, Peak 1 from
Example 195, step 4) was combined in a microwave vial with
potassium acetate (0.035 g, 0.35 mmol) and
4,4,5,5,4',4',5',5'-octamethyl-[2,2']bi[[1,3,2]dioxaborolanyl]
(0.060 g, 0.24 mmol) in dimethyl sulfoxide (0.44 mL) at room
temperature. This was degassed with nitrogen and then
[1,1'-bis(diphenylphosphino)ferrocene]-dichloropalladium(II),
complex with dichloromethane (1:1) (0.01 g, 0.01 mmol) was added.
The reaction was heated in an oil bath to 105.degree. C. overnight.
This was allowed to cool, then taken up in ethyl acetate and washed
with water, brine, dried over magnesium sulfate and concentrated.
The product (15 mg, 20%) was purified by chromatography eluting
with CH.sub.2Cl.sub.2/MeOH (max. MeOH 10%). LCMS calculated for
C.sub.23H.sub.32BClN.sub.5O.sub.3(M+H).sup.+: m/z=472.2; Found:
472.3.
Step 2.
6-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]--
5-chloro-2-ethoxy-6-methylphenyl}-N,N-dimethylnicotinamide
bis(trifluoroacetate)
[0432] A mixture of
1-{1-[5-chloro-2-ethoxy-4-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborola-
n-2-yl)phenyl]ethyl}-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine
(15 mg, 0.032 mmol), 6-chloro-N,N-dimethylnicotinamide (12 mg,
0.064 mmol), sodium carbonate (9.0 mg, 0.085 mmol) and
[1,1'-bis(diphenylphosphino)ferrocene]-dichloropalladium(II),
complex with dichloromethane (1:1) (6.9 mg, 0.0085 mmol) in
acetonitrile (0.9 mL)/water (0.2 mL) was degassed with N.sub.2 and
then stirred at 95.degree. C. overnight. The crude was purified
using RP-HPLC (XBridge C18 column, eluting with a gradient of
acetonitrile/water containing 0.05% trifluoroacetic acid, at flow
rate of 30 mL/min) to give the desired product as TFA salt (2 mg,
9%). The product was isolated as a single enantiomer. LCMS
calculated for C.sub.25H.sub.29ClN.sub.7O.sub.2 (M+H).sup.+:
m/z=494.2; Found: 494.2.
Example 209.
5-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-4-methoxy--
2-methyl-3-(1-methyl-1H-pyrazol-4-yl)benzonitrile
##STR00092##
[0434] Pre-formed catalyst (0.05 mL, from Example 40) was added to
a mixture
1-{1-[5-chloro-2-methoxy-4-methyl-3-(1-methyl-1H-pyrazol-4-yl)phe-
nyl]ethyl}-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (7.7 mg,
0.019 mmol), zinc (0.54 mg, 0.0082 mmol) and zinc cyanide (2.2 mg,
0.019 mmol) in N,N-dimethylacetamide (0.3 mL). The mixture was
degassed with nitrogen 3 times. The reaction was heated at
120.degree. C. for 1.5 h. The crude was purified using RP-HPLC
(XBridge C18 column, eluting with a gradient of acetonitrile/water
containing 0.1% ammonium hydroxide, at flow rate of 30 mL/min) to
give the desired product (2.1 mg, 27%). The product was isolated as
a single enantiomer. LCMS calculated for C.sub.21H.sub.23N.sub.8O
(M+H).sup.+: m/z=403.2; Found: 403.2.
[0435] Experimental procedures and LCMS mass spectral data (MS) for
the compounds below are summarized in Table 1.
TABLE-US-00001 TABLE 1 ##STR00093## Ex. MS No. Name R.sup.2 R.sup.4
R.sup.5 R.sup.3 Salt Proc..sup.1 [M + H].sup.+ 168 1-(1-(5-
chloro-2- methoxy-4- methyl-3- (pyrimidin-5- yl)phenyl)eth-
yl)-3-methyl- 1H- pyrazolo[3,4- Me Me Cl ##STR00094## 2TFA 167
425.1 d]pyrimidin- 4-amine.sup.2 169 1-(1-(3-(2- amino-
pyrimidin-5- yl)-5-chloro- 2-methoxy-4- methylphen- yl)ethyl)-3-
methyl-1H- pyrazolo[3,4- d]pyrimidin- 4-amine.sup.2 Me Me Cl
##STR00095## 167 425.1 .sup.1Synthesized according to the
experimental procedure of compound listed; .sup.2Compound isolated
as a single enantiomer.
Example 212.
4-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-2-azetidin-
-3-yl-6-chloro-3-ethoxybenzonitrile
##STR00096##
[0436] Step 1.
1-(5-Chloro-2-ethoxy-4-fluoro-3-iodophenyl)ethanone
[0437] The desired compound was prepared according to the procedure
of Example 13, step 3 to form a racemic intermediate, using
iodoethane instead of iodomethane as the starting material in 90%
yield. .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.68 (d, J=8.3 Hz,
1H), 3.94 (q, J=7.0 Hz, 2H), 2.61 (s, 3H), 1.48 (t, J=7.0 Hz, 3H).
LCMS for C.sub.10H.sub.10ClFIO.sub.2 (M+H).sup.+: m/z=342.9, 344.9;
Found: 342.9, 344.8.
Step 2. 4-Acetyl-6-chloro-3-ethoxy-2-iodobenzonitrile
[0438] A solution of
1-(5-chloro-2-ethoxy-4-fluoro-3-iodophenyl)ethanone (7.3 g, 21
mmol) in N,N-dimethylformamide (80 mL) was treated with potassium
cyanide (2.1 g, 32 mmol) and stirred at 40.degree. C. for 5 h. The
reaction mixture was diluted with ethyl acetate and poured into
saturated sodium bicarbonate solution/water (1:1). The organic
layer was separated, washed with saturated sodium bicarbonate
solution, dried with magnesium sulfate, filtered, and concentrated
to give a crude brown oil. The crude material was purified by flash
column chromatography using ethyl acetate in hexanes (0%-30%) to
give the desired product (6.1 g, 81%) as a yellow solid. .sup.1H
NMR (400 MHz, CDCl.sub.3) .delta. 7.57 (s, 1H), 3.93 (q, J=7.0 Hz,
2H), 2.61 (s, 3H), 1.47 (t, J=7.0 Hz, 3H). LCMS for
Cl.sub.1H.sub.10ClINO.sub.2 (M+H).sup.+: m/z=349.9; Found:
349.9.
Step 3. tert-Butyl
3-(3-acetyl-5-chloro-6-cyano-2-ethoxyphenyl)azetidine-1-carboxylate
[0439] Zinc (4.60 g, 70.3 mmol) and oven dried Celite (870 mg) was
added to a flask and the flask was heated with a heat gun while
under high-vac for 5 min and then back-filled with nitrogen.
N,N-Dimethylacetamide (57 mL) was added, followed by
1,2-dibromoethane (430 .mu.L, 5.0 mmol) and the mixture was heated
at 70.degree. C. for 10 min and then cooled to room temperature.
The reaction mixture was treated with chlorotrimethylsilane (630
.mu.L, 5.0 mmol) dropwise and stirred at room temperature for 1 h.
The reaction mixture was treated with a solution of tert-butyl
3-iodoazetidine-1-carboxylate (18 g, 62 mmol) in
N,N-dimethylacetamide (28 mL) dropwise (internal temperature was
kept below 40.degree. C. with a water bath) and heated at
40.degree. C. for 2 h. The zinc-iodo reagent (transferred via
canula) was filtered through a plastic filter (that was
appropriately sealed to avoid atmospheric exposure) directly into a
clean, dry flask that was flushed with nitrogen. The reaction
mixture was treated with tris(dibenzylideneacetone)dipalladium(0)
(720 mg, 0.79 mmol) and tri-(2-furyl)phosphine (370 mg, 1.6 mmol)
and degassed with nitrogen for a few minutes. The reaction mixture
was treated with a solution of
4-acetyl-6-chloro-3-ethoxy-2-iodobenzonitrile (14 g, 41 mmol) in
N,N-dimethylacetamide (130 mL) (degassed with nitrogen) quickly and
heated at 70.degree. C. for 2 h. The reaction mixture was poured
into saturated ammonium chloride solution and extracted with ethyl
acetate (3.times.300 mL). The combined organic extracts were washed
with water (4.times.500 mL) and brine (1.times.500 mL), dried with
magnesium sulfate, filtered, and concentrated to a crude dark oil.
The crude material was purified by flash column chromatography
using ethyl acetate in hexanes (5%-45%) to give the desired product
(14 g, 88%). .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.46 (s,
1H), 4.42-4.20 (m, 5H), 3.80 (q, J=7.0 Hz, 2H), 2.59 (s, 3H), 1.44
(s, 9H), 1.37 (t, J=7.0 Hz, 3H). LCMS for
C.sub.15H.sub.16ClN.sub.2O.sub.4 ([M-(t-Bu)+H]+H).sup.+: m/z=323.1;
Found: 323.0.
Step 4. tert-Butyl
3-[3-chloro-2-cyano-6-ethoxy-5-(1-hydroxyethyl)phenyl]azetidine-1-carboxy-
late
[0440] A solution of
(3aS)-1-methyl-3,3-diphenyltetrahydro-3H-pyrrolo[1,2-c][1,3,2]oxazaborole
(9.7 g, 35 mmol) in tetrahydrofuran (100 mL) was treated with 1.0 M
borane-THF complex in tetrahyrofuran (42 mL, 42 mmol) and stirred
at 20.degree. C. for 15 min. The reaction mixture was cooled to
-30.degree. C. and treated with a solution of tert-butyl
3-(3-acetyl-5-chloro-6-cyano-2-ethoxyphenyl)azetidine-1-carboxylate
(13 g, 35 mmol) in tetrahydrofuran (110 mL) slowly. The flask
containing the starting material ketone was rinsed with additional
tetrahydrofuran (20 mL) and added to the reaction mixture. The
reaction mixture was warmed to 0.degree. C. over a period of 30 min
and stirred at 0.degree. C. for 15 min. The reaction mixture was
quenched with water at 0.degree. C., poured into saturated sodium
bicarbonate solution, and extracted with ethyl acetate. The aqueous
layer was separated and extracted with ethyl acetate. The combined
organic layers were washed with water and brine, dried with
magnesium sulfate, filtered, and concentrated to a crude dark oil.
The crude material was purified by flash column chromatography
using ethyl acetate in hexanes (0%-70%) to give the desired product
(10.4 g, 78%) as a yellow foam as a 98:2 mixture of enantiomers
(Retention times=7.73 min and 9.41 min; ChiralPak AD-H column,
4.6.times.150 mm, 5 micron particle size, eluting with 5% ethanol
in hexanes at 1 ml/min). .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.
7.56 (s, 1H), 5.15-5.07 (m, 1H), 4.41-4.17 (m, 5H), 3.74 (q, J=7.0
Hz, 2H), 2.12 (d, J=3.7 Hz, 1H), 1.49-1.37 (m, 15H). LCMS for
C.sub.15H.sub.18ClN.sub.2O.sub.4 ([M-(t-Bu)+H]+H).sup.+: m/z=325.1;
Found: 325.1.
Step 5. tert-Butyl
3-{3-[1-(4-amino-3-methyl-H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chloro-
-6-cyano-2-ethoxyphenyl}azetidine-1-carboxylate
[0441] A solution of tert-butyl
3-[3-chloro-2-cyano-6-ethoxy-5-(1-hydroxyethyl)phenyl]azetidine-1-carboxy-
late (98:2 mixture of enantiomers from step 4) (10 g, 27 mmol) in
methylene chloride (260 mL) at 0.degree. C. was treated with
triethylamine (11 mL, 82 mmol) followed by methanesulphonic
anhydride (7.1 g, 41 mmol) and stirred at 0.degree. C. for 15 min.
The reaction mixture was diluted with dichloromethane and washed
with water and brine, dried with magnesium sulfate, filtered, and
concentrated to give the crude mesylate that was used without
further purification. A solution of the crude mesylate intermediate
in N,N-dimethylformamide (140 mL) was treated with cesium carbonate
(13 g, 41 mmol) and 3-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine
(4.7 g, 31 mmol) and heated at 60.degree. C. for 1 h. The reaction
mixture was diluted with water and extracted with ethyl acetate
(3.times.250 mL). The combined organic layers were washed with
water and brine, dried with magnesium sulfate, filtered, and
concentrated to a crude oil. The crude material was purified by
flash column chromatography (100% dichloromethane to 70%
acetonitrile containing 3% methanol/30% dichloromethane) to give
the desired product (8.7 g, 62% for 2 steps) as a yellow foam as a
95:5 mixture of enantiomers (RT=4.29 min and 6.00 min; Phenomenex
Lux Cellulose C-1 column, 4.6.times.150 mm, 5 micron particle size,
eluting with 15% ethanol in hexanes at 1 ml/min). This material was
separated by chiral HPLC (Phenomenex Lux Cellulose C-1 column,
21.2.times.250 mm, 5 micron particle size, eluting with 15% ethanol
in hexanes at 10 ml/min) to give 7.0 g of the desired peak 1
material (retention time of 8.20 min). .sup.1H NMR (300 MHz,
CDCl.sub.3) .delta. 8.24 (s, 1H), 7.51 (s, 1H), 6.32 (q, J=7.1 Hz,
1H), 5.48 (br s, 2H), 4.40-4.18 (m, 5H), 4.05-3.93 (m, 1H),
3.81-3.65 (m, 1H), 2.64 (s, 3H), 1.81 (d, J=7.1 Hz, 3H), 1.48 (t,
J=7.0 Hz, 3H), 1.43 (s, 9H). LCMS for
C.sub.25H.sub.31ClN.sub.7O.sub.3(M+H).sup.+: m/z=512.2; Found:
512.3.
Step 6.
4-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-2-a-
zetidin-3-yl-6-chloro-3-ethoxybenzonitrile
[0442] A solution of tert-butyl
3-{3-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chlor-
o-6-cyano-2-ethoxyphenyl}azetidine-1-carboxylate (peak 1 enantiomer
from step 5) (2.2 g, 4.2 mmol) in methylene chloride (11 mL) was
treated with trifluoroacetic acid (11 mL) dropwise and stirred at
room temperature for 30 min. The reaction mixture was concentrated
to an oil that was reconcentrated from ethanol (2.times.) to give a
residue. This material was dissolved in a minimum amount of
methanol, added dropwise to ice cooled saturated sodium bicarbonate
solution (100 ml), and extracted several times with 2:1
dichloromethane/isopropanol to give the desired product (1.8 g,
quantitative) that was used without further purification. A small
amount of the desired product was purified by preparative LCMS
(XBridge C18 column, eluting with a gradient of acetonitrile/water
containing 0.1% ammonium hydroxide, at flow rate of 60 mL/min) to
give the desired product. The product was isolated as a single
enantiomer. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 8.11 (s,
1H), 7.47 (s, 1H), 6.23 (q, J=7.0 Hz, 1H), 4.37-4.26 (m, 1H),
3.91-3.61 (m, 6H), 2.54 (s, 3H), 1.71 (d, J=7.1 Hz, 3H), 1.32 (t,
J=7.0 Hz, 3H). LCMS for C.sub.20H.sub.23ClN.sub.7O (M+H).sup.+:
m/z=412.2; Found: 412.1.
Example 213.
4-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-6-chloro-3-
-ethoxy-2-(1-methylazetidin-3-yl)benzonitrile
##STR00097##
[0444] A solution of
4-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-2-azetidin-
-3-yl-6-chloro-3-ethoxybenzonitrile (chiral intermediate in Example
212, Step 6) (0.30 g, 0.73 mmol) in methanol (7.3 mL) was treated
with formaldehyde (37% in water) (0.54 mL, 7.3 mmol) and this was
stirred at room temperature for 5 min. The reaction mixture was
treated with sodium cyanoborohydride (0.092 g, 1.5 mmol) and
stirred at room temperature for 2 h. The reaction mixture was
diluted with methanol and purified by preparative LCMS (XBridge C18
column, eluting with a gradient of acetonitrile/water containing
0.1% ammonium hydroxide, at flow rate of 60 mL/min) to give the
desired product (0.16 g, 50%). The product was isolated as a single
enantiomer. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 8.11 (s,
1H), 7.48 (s, 1H), 6.27-6.18 (m, 1H), 4.10-3.98 (m, 1H), 3.96-3.86
(m, 2H), 3.83-3.74 (m, 1H), 3.72-3.64 (m, 1H), 3.10-2.98 (m, 2H),
2.54 (s, 3H), 2.20 (s, 3H), 1.71 (d, J=6.9 Hz, 3H), 1.32 (t, J=6.7
Hz, 3H). LCMS for C.sub.21H.sub.25ClN.sub.7O (M+H).sup.+:
m/z=426.2; Found: 426.2.
Example 219.
4-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-6-chloro-3-
-ethoxy-2-[1-(2-hydroxyethyl)azetidin-3-yl]benzonitrile
##STR00098##
[0446] A solution of
4-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-2-azetidin-
-3-yl-6-chloro-3-ethoxybenzonitrile (300 mg, 0.74 mmol, chiral
intermediate from Example 212) in tetrahydrofuran (14 mL) was
treated with triethylamine (260 .mu.L, 1.8 mmol) followed by
2-bromoethanol (63 .mu.L, 0.89 mmol) dropwise and stirred at
60.degree. C. for 6 h. The reaction mixture was treated with
additional 2-bromoethanol (26 .mu.L, 0.37 mmol) and stirred at
60.degree. C. for another 6 h. The reaction mixture was poured into
saturated sodium bicarbonate solution and extracted with ethyl
acetate. The organic layer was concentrated and purified by
preparative LCMS (XBridge C18 column, eluting with a gradient of
acetonitrile/water containing 0.1% ammonium hydroxide, at flow rate
of 60 mL/min) to give the desired product (0.15 g, 44%). The
product was isolated as a single enantiomer. .sup.1H NMR (400 MHz,
DMSO-d.sub.6) .delta. 8.19 (s, 1H), 7.56 (s, 1H), 6.36-6.25 (m,
1H), 4.48 (br s, 1H), 4.19-4.07 (m, 1H), 4.04-3.94 (m, 2H),
3.91-3.82 (m, 1H), 3.81-3.72 (m, 1H), 3.20-3.08 (m, 2H), 2.62 (s,
2H), 2.57 (s, 3H), 1.79 (d, J=6.8 Hz, 3H), 1.40 (t, J=6.6 Hz, 3H).
LCMS for C.sub.22H.sub.27ClN.sub.7O.sub.2(M+H).sup.+: m/z=456.2;
Found: 456.1.
Example 220.
4-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-6-chloro-3-
-ethoxy-2-{1-[(2S)-2-hydroxypropyl]azetidin-3-yl}benzonitrile
##STR00099##
[0448] A solution of
4-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-2-azetidin-
-3-yl-6-chloro-3-ethoxybenzonitrile (50 mg, 0.12 mmol, chiral
intermediate from example 212) in ethanol (1.7 mL) was treated with
(S)-(-)-methyloxirane (21 .mu.L, 0.30 mmol) and heated in the
microwave at 125.degree. C. for 15 min. The reaction mixture was
diluted with methanol and purified by preparative LCMS (XBridge C18
column, eluting with a gradient of acetonitrile/water containing
0.1% ammonium hydroxide, at flow rate of 60 mL/min) to give the
desired product (27 mg, 47%). The product was isolated as a single
diastereomer. .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 8.11 (s,
1H), 7.48 (s, 1H), 6.23 (q, J=6.9 Hz, 1H), 4.35 (d, J=4.5 Hz, 1H),
4.13-3.99 (m, 1H), 3.97-3.88 (m, 2H), 3.85-3.63 (m, 2H), 3.61-3.51
(m, 1H), 3.15-2.99 (m, 2H), 2.55 (s, 3H), 2.28 (d, J=5.9 Hz, 2H),
1.71 (d, J=7.0 Hz, 3H), 1.32 (t, J=6.9 Hz, 3H), 1.00 (d, J=6.2 Hz,
3H). LCMS for C.sub.23H.sub.29ClN.sub.7O.sub.2(M+H).sup.+:
m/z=470.2; Found: 470.2.
Example 236. tert-Butyl
2-(3-{3-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-ch-
loro-6-cyano-2-ethoxyphenyl}azetidin-1-yl)-2-methylpropanoate
##STR00100##
[0450] A solution of
4-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-2-azetidin-
-3-yl-6-chloro-3-ethoxybenzonitrile (0.38 g, 0.92 mmol, chiral
intermediate from Example 212) in N,N-dimethylformamide (4. 6 mL)
was treated with potassium carbonate (0.51 g, 3.7 mmol) followed by
tert-butyl 2-bromo-2-methylpropanoate (0.86 mL, 4.6 mmol) and
heated at 60.degree. C. for 3 h. The reaction mixture was poured
into water and extracted with ethyl acetate. The organic layer was
separated, dried with magnesium sulfate, filtered, and concentrated
to a crude oil. The crude material was purified by flash column
chromatography using methanol in dichloromethane (0%-10%) to give
the desired product (0.43 g, 83%). The product was isolated as a
single enantiomer. .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 8.10
(s, 1H), 7.44 (s, 1H), 6.22 (q, J=6.8 Hz, 1H), 4.12-3.97 (m, 1H),
3.88-3.70 (m, 4H), 3.62-3.48 (m, 2H), 2.54 (s, 3H), 1.70 (d, J=7.0
Hz, 3H), 1.33 (t, J=6.9 Hz, 3H), 1.17 (s, 9H), 1.05 (s, 6H). LCMS
for C.sub.28H.sub.37ClN.sub.7O.sub.3(M+H).sup.+: m/z=554.3; Found:
554.3.
Example 237.
4-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-6-chloro-3-
-ethoxy-2-[1-(2-hydroxy-1,1-dimethylethyl)azetidin-3-yl]benzonitrile
##STR00101##
[0451] Step 1.
2-(3-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-ch-
loro-6-cyano-2-ethoxyphenyl}azetidin-1-yl)-2-methylpropanoic acid
bis(trifluoroacetate)
[0452] tert-Butyl
2-(3-{3-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-ch-
loro-6-cyano-2-ethoxyphenyl}azetidin-1-yl)-2-methylpropanoate (0.36
g, 0.65 mmol, chiral intermediate from Example 236) was dissolved
in a premixed solution of trifluoroacetic acid (3.2 mL)/water
(0.065 mL) and stirred at room temperature for 3 h and at
50.degree. C. for 30 min. The reaction mixture was concentrated and
reconcentrated from acetonitrile (2.times.) to give the desired
product as a gum. This gum was treated with a small amount of
methyl-tert-butylether that was swirled until a solid formed. The
methyl-tert-butylether was decanted and the residue was
concentrated to give the desired product (0.51 g, 109%) that was
used without further purification. LCMS for
C.sub.24H.sub.29ClN.sub.7O.sub.3(M+H).sup.+: m/z=498.2; Found:
498.3.
Step 2.
4-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-6-c-
hloro-3-ethoxy-2-[1-(2-hydroxy-1,1-dimethylethyl)azetidin-3-yl]benzonitril-
e
[0453] A solution of
2-(3-{3-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-ch-
loro-6-cyano-2-ethoxyphenyl}azetidin-1-yl)-2-methylpropanoic acid
bis(trifluoroacetate) (0.10 g, 0.16 mmol) in tetrahydrofuran (0.9
mL) was cooled to -25.degree. C., treated with 4-methylmorpholine
(0.072 mL, 0.65 mmol) and isobutyl chloroformate (0.085 mL, 0.65
mmol), and stirred at -15.degree. C. for 15 min. The reaction
mixture was filtered though a disposable filter cartridge into a
separate round bottom flask. This solution was then cooled to
-20.degree. C. and a solution of sodium tetrahydroborate (0.031 g,
0.82 mmol) in a minimum amount of water was added dropwise. The
reaction mixture was stirred at -15.degree. C. for 30 min, poured
into water, and extracted with ethyl acetate. The organic layer was
separated, concentrated, diluted with methanol, and purified by
preparative LCMS (XBridge C18 column, eluting with a gradient of
acetonitrile/water containing 0.1% ammonium hydroxide, at flow rate
of 30 mL/min) to give the desired product (3.5 mg, 4%). The product
was isolated as a single enantiomer. .sup.1H NMR (300 MHz,
DMSO-d.sub.6) .delta. 8.11 (s, 1H), 7.50 (s, 1H), 7.35 (br s, 2H),
6.23 (q, J=6.7 Hz, 1H), 4.44-4.35 (m, 1H), 4.04-3.88 (m, 1H),
3.86-3.73 (m, 1H), 3.72-3.57 (m, 3H), 3.12 (d, J=4.7 Hz, 2H), 2.54
(s, 3H), 1.71 (d, J=6.9 Hz, 3H), 1.31 (t, J=6.9 Hz, 3H), 0.80 (s,
6H). LCMS for C.sub.24H.sub.31ClN.sub.7O.sub.2(M+H).sup.+:
m/z=484.2; Found: 484.2.
Example 239.
2-(3-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-ch-
loro-6-cyano-2-ethoxyphenyl}azetidin-1-yl)-2-methylpropanamide
##STR00102##
[0455] A solution of
2-(3-{3-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-ch-
loro-6-cyano-2-ethoxyphenyl}azetidin-1-yl)-2-methylpropanoic acid
bis(trifluoroacetate) (0.05 g, 0.069 mmol, chiral intermediate from
Example 237, Step 1) and 2.0 M ammonia in ethanol (0.17 mL, 0.34
mmol) in N,N-dimethylformamide (1 mL) was treated with
triethylamine (0.048 mL, 0.35 mmol) and
benzotriazol-1-yloxytris(dimethylamino)phosphonium
hexafluorophosphate (0.046 g, 0.10 mmol) and stirred at room
temperature for 1 h. The reaction mixture was quenched with a few
drops of water, diluted with methanol, and purified by preparative
LCMS (XBridge C18 column, eluting with a gradient of
acetonitrile/water containing 0.1% ammonium hydroxide, at flow rate
of 60 mL/min) to give the desired product (25 mg, 73%). The product
was isolated as a single enantiomer. .sup.1H NMR (400 MHz,
DMSO-d.sub.6) .delta. 8.11 (s, 1H), 7.51 (s, 1H), 7.23 (s, 1H),
6.98 (s, 1H), 6.23 (q, J=7.0 Hz, 1H), 4.09-3.96 (m, 1H), 3.84-3.61
(m, 4H), 3.39-3.34 (m, 1H), 3.32-3.28 (m, 1H), 2.54 (s, 3H), 1.71
(d, J=7.0 Hz, 3H), 1.31 (t, J=6.9 Hz, 3H), 1.02 (s, 6H). LCMS for
C.sub.24H.sub.30ClN.sub.8O.sub.2(M+H).sup.+: m/z=497.2; Found:
497.3.
Example 247.
4-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-6-chloro-3-
-ethoxy-2-[1-(2-hydroxy-2-methylpropanoyl)azetidin-3-yl]benzonitrile
##STR00103##
[0457] A solution of
4-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-2-azetidin-
-3-yl-6-chloro-3-ethoxybenzonitrile (0.04 g, 0.097 mmol, chiral
intermediate from Example 212) and propanoic acid,
2-hydroxy-2-methyl- (0.012 g, 0.12 mmol) in N,N-dimethylformamide
(0.54 mL) was treated with triethylamine (0.034 mL, 0.24 mmol)
followed by O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate (0.048 g, 0.13 mmol) and stirred at room
temperature for 30 min. The reaction mixture was diluted with
methanol and acetonitrile and purified by preparative LCMS (XBridge
C18 column, eluting with a gradient of methanol/water containing
0.1% ammonium hydroxide, at flow rate of 60 mL/min) to give the
desired product (7 mg, 14%). The product was isolated as a single
enantiomer. .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 8.11 (s,
1H), 7.54 (d, J=4.5 Hz, 1H), 6.25 (q, J=7.2 Hz, 1H), 5.08 (s, 1H),
4.88-4.77 (m, 1H), 4.73-4.60 (m, 1H), 4.50-4.35 (m, 1H), 4.29-4.09
(m, 2H), 3.85-3.73 (m, 2H), 2.55 (s, 3H), 1.73 (d, J=7.0 Hz, 3H),
1.37 (t, J=6.3 Hz, 3H), 1.26 (s, 3H), 1.22 (s, 3H). LCMS for
C.sub.24H.sub.29ClN.sub.7O.sub.3(M+H).sup.+: m/z=498.2; Found:
498.2.
Example 261.
4-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-2-azetidin-
-3-yl-6-chloro-3-methoxybenzonitrile
##STR00104##
[0458] Step 1. 4-Acetyl-6-chloro-2-iodo-3-methoxybenzonitrile
[0459] A solution of
1-(5-chloro-4-fluoro-3-iodo-2-methoxyphenyl)ethanone (intermediate
from Example 13, Step 3) (18 g, 54 mmol) in N,N-dimethylformamide
(200 mL) was treated with potassium cyanide (5.2 g, 81 mmol) and
stirred at 40.degree. C. for 6 h. The reaction mixture was diluted
with ethyl acetate and poured into saturated sodium bicarbonate
solution/water (1:1). The organic layer was separated, washed with
saturated sodium bicarbonate solution, dried with magnesium
sulfate, filtered, and concentrated to give a crude brown oil. The
crude material was purified by flash column chromatography using
ethyl acetate in hexanes (0%-30%) to give the desired product (11
g, 61%) as a yellow solid. .sup.1H NMR (300 MHz, CDCl.sub.3)
.delta. 7.60 (s, 1H), 3.81 (s, 3H), 2.62 (s, 3H). LCMS for
C.sub.10H.sub.8ClINO.sub.2 (M+H).sup.+: m/z=335.9; Found:
335.9.
Step 2. tert-Butyl
3-(3-acetyl-5-chloro-6-cyano-2-methoxyphenyl)azetidine-1-carboxylate
[0460] Zinc (5.0 g, 77 mmol) and oven dried Celite (520 mg) was
added to a flask and the flask was heated with a heat gun while
under high-vac for 5 min and then back-filled with nitrogen. N,
N-dimethylacetamide (53 mL) was added, followed by
1,2-dibromoethane (400 .mu.L, 4.6 mmol) and the mixture was heated
at 70.degree. C. for 15 min and then cooled to room temperature.
The reaction mixture was treated with chlorotrimethylsilane (580
.mu.L, 4.6 mmol) dropwise and stirred at room temperature for 1 h.
The reaction mixture was treated with a solution of tert-butyl
3-iodoazetidine-1-carboxylate (16 g, 58 mmol) in
N,N-dimethylacetamide (26 mL) dropwise (internal temperature was
kept below 40.degree. C. with a water bath) and heated at
40.degree. C. for 2 h. The zinc-iodo reagent (transferred via
canula) was filtered through a plastic filter (that was
appropriately sealed to avoid atmospheric exposure) directly into a
clean, dry flask that was flushed with nitrogen. The reaction
mixture was treated with tris(dibenzylideneacetone)dipalladium(0)
(670 mg, 0.73 mmol) and tri-(2-furyl)phosphine (340 mg, 1.5 mmol)
and degassed with nitrogen for a few minutes. The reaction mixture
was treated with a solution of
4-acetyl-6-chloro-2-iodo-3-methoxybenzonitrile (13 g, 39 mmol) in
N,N-dimethylacetamide (120 mL) (degassed with nitrogen) quickly and
heated at 70.degree. C. for 2 h. The reaction mixture was poured
into saturated ammonium chloride solution and extracted with ethyl
acetate (3.times.300 mL). The combined organic extracts were washed
with water (4.times.500 mL) and brine (1.times.500 mL), dried with
magnesium sulfate, filtered, and concentrated to a crude dark oil.
The crude material was purified by flash column chromatography
using ethyl acetate in hexanes (5%-40%) to give the desired product
(12 g, 85%). .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 7.79 (s,
1H), 4.39-4.29 (m, 1H), 4.28-4.11 (m, 4H), 3.68 (s, 3H), 2.58 (s,
3H), 1.38 (s, 9H).
Step 3. tert-Butyl
3-[3-chloro-2-cyano-5-(1-hydroxyethyl)-6-methoxyphenyl]azetidine-1-carbox-
ylate
[0461] A solution of
(3aS)-1-methyl-3,3-diphenyltetrahydro-3H-pyrrolo[1,2-c][1,3,2]oxazaborole
(4.3 g, 16 mmol) in tetrahydrofuran (46 mL) was treated with 1.0 M
borane-THF complex in tetrahyrofuran (19 mL, 19 mmol) and stirred
at 20.degree. C. for 15 min. The reaction mixture was cooled to
-30.degree. C. and treated with a solution of tert-butyl
3-(3-acetyl-5-chloro-6-cyano-2-methoxyphenyl)azetidine-1-carboxylate
(5.7 g, 16 mmol) in tetrahydrofuran (49 mL) slowly. The flask
containing the starting material ketone was rinsed with additional
tetrahydrofuran (9 mL) and added to the reaction mixture. The
temperature of the reaction was -20.degree. C. after the addition
was complete. The reaction mixture was warmed to -5.degree. C. over
a period of 30 min. The reaction mixture was quenched with water at
0.degree. C., poured into saturated sodium bicarbonate solution,
and extracted with ethyl acetate. The aqueous layer was separated
and extracted with ethyl acetate. The combined organic layers were
washed with water and brine, dried with magnesium sulfate,
filtered, and concentrated to a crude dark oil. The crude material
was purified by flash column chromatography using ethyl acetate in
hexanes (0%-100%) to give the desired product (5.5 g, 97%) as a
beige foam as a 97:3 mixture of enantiomers (Retention times=12.19
min and 13.18 min; Phenomenex Lux Cellulose C-2 column,
4.6.times.150 mm, 5 micron particle size, eluting with 8% ethanol
in hexanes at 1 ml/min). .sup.1H NMR (400 MHz, DMSO-d.sub.6)
.delta. 7.62 (s, 1H), 5.48 (d, J=4.6 Hz, 1H), 5.00-4.90 (m, 1H),
4.43-4.31 (m, 1H), 4.30-4.10 (m, 4H), 3.66 (s, 3H), 1.38 (s, 9H),
1.29 (d, J=6.4 Hz, 3H). LCMS for C.sub.14H.sub.16ClN.sub.2O.sub.4
([M-(t-Bu)+H]+H).sup.+: m/z=311.1; Found: 311.1.
Step 4. tert-Butyl
3-{3-[1-(4-amino-3-methyl-H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chloro-
-6-cyano-2-methoxyphenyl}azetidine-1-carboxylate
[0462] A solution of tert-butyl
3-[3-chloro-2-cyano-5-(1-hydroxyethyl)-6-methoxyphenyl]azetidine-1-carbox-
ylate (8.6 g, 23 mmol) (97:3 mixture of enantiomers from step 3) in
methylene chloride (220 mL) at 0.degree. C. was treated with
triethylamine (8.2 mL, 59 mmol) followed by methanesulphonic
anhydride (6.1 g, 35 mmol) and stirred at 0.degree. C. for 15 min.
The reaction mixture was diluted with dichloromethane and washed
with water and brine, dried with magnesium sulfate, filtered, and
concentrated to give the crude mesylate that was used without
further purification. A solution of the crude mesylate intermediate
in N,N-dimethylformamide (82 mL) was cooled to 0.degree. C.,
treated with sodium hydride (1.2 g, 30 mmol) (60% in mineral oil),
and stirred at 0.degree. C. for 30 min. The reaction mixture was
treated with a solution of tert-butyl
3-(3-chloro-2-cyano-6-methoxy-5-{1-[(methylsulfonyl)oxy]ethyl}phenyl)azet-
idine-1-carboxylate (11 g, 24 mmol) in N,N-dimethylformamide (170
mL) dropwise over a period of 10 min and stirred at 0.degree. C.
for 30 min and heated at 50.degree. C. for 1 h. The reaction
mixture was diluted with water and saturated sodium bicarbonate
solution and extracted with ethyl acetate (3.times.200 mL). The
combined organic extracts were washed with water (4.times.150 mL)
and brine, dried with magnesium sulfate, filtered, and concentrated
to a crude oil. The crude material was purified by flash column
chromatography (2% methanol/98% dichloromethane to 7% methanol/93%
dichloromethane [the dichloromethane contained 0.5% triethylamine])
to give the desired product (9.1 g, 77% for 2 steps) as a 9:1
mixture of enantiomers. This material was separated by chiral HPLC
(retention times=5.81 min and 8.94 min; Chiracel AD-H column,
20.times.250 mm, 5 micron particle size, eluting with 10% ethanol
in hexanes at 18 ml/min, 10 mg/inj) to give 6.9 g of the desired
peak 1 material. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 8.11
(s, 1H), 7.52 (s, 1H), 6.25 (q, J=7.0 Hz, 1H), 4.45-4.33 (m, 1H),
4.27-4.13 (m, 4H), 3.70 (s, 3H), 2.55 (s, 3H), 1.73 (d, J=7.1 Hz,
3H), 1.37 (s, 9H). LCMS for C.sub.20H.sub.21ClN.sub.7O.sub.3
([M-(t-Bu)+H]+H).sup.+: m/z=442.1; Found: 442.1.
Step 5.
4-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-2-a-
zetidin-3-yl-6-chloro-3-methoxybenzonitrile
[0463] A solution of tert-butyl
3-{3-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chlor-
o-6-cyano-2-methoxyphenyl}azetidine-1-carboxylate (1.7 g, 3.3 mmol)
in methylene chloride (30 mL) was treated with trifluoroacetic acid
(20 mL) and stirred at room temperature for 20 min. The reaction
mixture was concentrated to give a residue that was diluted with
methanol (50 mL) and saturated sodium bicarbonate solution (50 mL).
This aqueous solution was diluted with brine (50 mL) and extracted
with a 5:1 mixture of dichloromethane/isopropanol (5.times.100 mL).
The combined organic extracts were dried over sodium sulfate and
concentrated to give the desired product (1.4 g, 97%). The product
was isolated as a single enantiomer. .sup.1H NMR (300 MHz,
DMSO-d.sub.6) .delta. 8.11 (s, 1H), 7.46 (s, 1H), 7.34 (br s, 2H),
6.24 (q, J=6.9 Hz, 1H), 4.40-4.26 (m, 1H), 3.90-3.68 (m, 4H), 3.63
(s, 3H), 2.55 (s, 3H), 1.72 (d, J=7.1 Hz, 3H). LCMS for
C.sub.19H.sub.21ClN.sub.7O (M+H).sup.+: m/z=398.1; Found:
398.1.
Example 262.
4-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-6-chloro-3-
-methoxy-2-(1-methylazetidin-3-yl)benzonitrile
##STR00105##
[0465] A solution of
4-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-2-azetidin-
-3-yl-6-chloro-3-methoxybenzonitrile (chiral intermediate from
Example 261) (50 mg, 0.13 mmol) in methanol (3 mL) was treated with
sodium cyanoborohydride (20 mg, 0.31 mmol) followed by formaldehyde
(37% in water) (37 .mu.L, 0.50 mmol) and stirred at room
temperature for 20 min. The reaction mixture was quenched with
acetic acid (170 .mu.L, 2.9 mmol), diluted with methanol, and
purified by preparative LCMS (XBridge C18 column, eluting with a
gradient of acetonitrile/water containing 0.1% ammonium hydroxide,
at flow rate of 60 mL/min) to give the desired product (30 mg,
58%). The product was isolated as a single enantiomer. .sup.1H NMR
(300 MHz, DMSO-d.sub.6) .delta. 8.11 (s, 1H), 7.46 (s, 1H), 7.37
(br s, 2H), 6.23 (q, J=7.0 Hz, 1H), 4.10-3.96 (m, 1H), 3.95-3.85
(m, 2H), 3.63 (s, 3H), 3.05-2.94 (m, 2H), 2.55 (s, 3H), 2.18 (s,
3H), 1.72 (d, J=7.1 Hz, 3H). LCMS for C.sub.20H.sub.23ClN.sub.7O
(M+H).sup.+: m/z=412.2; Found: 412.1.
Example 268.
4-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-6-chloro-2-
-[1-(2-hydroxyethyl)azetidin-3-yl]-3-methoxybenzonitrile
##STR00106##
[0467] A solution of
4-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-2-azetidin-
-3-yl-6-chloro-3-methoxybenzonitrile (chiral intermediate from
Example 261) (400 mg, 1.0 mmol) in tetrahydrofuran (14 mL) was
treated with triethylamine (350 .mu.L, 2.5 mmol) and 2-bromoethanol
(85 .mu.L, 1.2 mmol) and stirred at 60.degree. C. overnight. The
reaction mixture was concentrated, diluted with methanol, and
purified by preparative LCMS (XBridge C18 column, eluting with a
gradient of acetonitrile/water containing 0.1% ammonium hydroxide,
at flow rate of 60 mL/min) to give the desired product (0.14 g,
31%). The product was isolated as a single enantiomer. .sup.1H NMR
(400 MHz, DMSO-d.sub.6) .delta. 8.11 (s, 1H), 7.46 (s, 1H), 6.24
(q, J=6.9 Hz, 1H), 4.41 (t, J=5.4 Hz, 1H), 4.12-4.03 (m, 1H),
3.97-3.88 (m, 2H), 3.64 (s, 3H), 3.38-3.34 (m, 2H), 3.09-3.01 (m,
2H), 2.55 (s, 3H), 2.41 (t, J=5.9 Hz, 2H), 1.72 (d, J=7.0 Hz, 3H).
LCMS for C.sub.21H.sub.25ClN.sub.7O.sub.2(M+H).sup.+: m/z=442.2;
Found: 442.2.
[0468] The compounds of Example 268 and 269 were synthesized from
the same chiral intermediate in Example 261. According to the
crystal structure determination in Example 269, the stereochemistry
at the carbon at the 1-position of the ethan-1,1-diyl group is S.
Because the compound of Example 268 was synthesized from the same
chiral intermediate as Example 269, one of ordinary skill in the
art would expect that the carbon at the 1-position of the
ethan-1,1-diyl group of Example 268 is also in the S-configuration.
Accordingly, it is believed that the compound of Example 268 is
(S)-4-(1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-
-6-chloro-2-(1-(2-hydroxyethyl)azetidin-3-yl)-3-methoxybenzonitrile.
Example 269.
4-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-6-chloro-2-
-{1-[(2S)-2-hydroxypropyl]azetidin-3-yl}-3-methoxybenzonitrile
##STR00107##
[0470] A solution of
4-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-2-azetidin-
-3-yl-6-chloro-3-methoxybenzonitrile (chiral intermediate from
Example 261) (2.5 g, 6.3 mmol) in ethanol (130 mL) was treated with
(S)-(-)-methyloxirane (1.1 mL, 16 mmol) and heated in the microwave
at 120.degree. C. for 25 min. The reaction mixture was concentrated
to give a residue that was purified by flash column chromatography
using methanol in dichloromethane (0%-10%; methanol contained 0.5%
triethylamine) and by preparative LCMS (XBridge C18 column, eluting
with a gradient of acetonitrile/water containing 0.1% ammonium
hydroxide, at flow rate of 60 mL/min) to give the desired product
(0.76 g, 26%). The product was isolated as a single diastereomer.
.sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 8.11 (s, 1H), 7.46 (s,
1H), 7.34 (br s, 2H), 6.23 (q, J=7.0 Hz, 1H), 4.35 (br s, 1H),
4.14-3.99 (m, 1H), 3.98-3.87 (m, 2H), 3.64 (s, 3H), 3.60-3.52 (m,
1H), 3.13-2.99 (m, 2H), 2.55 (s, 3H), 2.28 (d, J=5.9 Hz, 2H),
1.75-1.69 (m, 3H), 1.00 (d, J=6.2 Hz, 3H). LCMS for
C.sub.22H.sub.27ClN.sub.7O.sub.2(M+H).sup.+: m/z=456.2; Found:
456.2.
Crystal Structure Determination for the Compound of Example 269
C22,H26,N7,O2,CL1+H.sub.2O
[0471] CRYSTAL DATA: C22 H28 Cl F0 N7 O3, from ACN/water,
colorless, needle, .about.0.500.times.0.070.times.0.050 mm,
monoclinic, C2, a=25.941(7) .ANG., b=4.9767(13) .ANG., c=17.787(5)
.ANG., beta=101.967(4).degree., Vol=2246.3(10) .ANG..sup.3, Z=4,
T=-100.degree. C., Formula weight=473.96, Density=1.401 g/cm.sup.3,
(Mo)=0.21 mm.sup.-1 DATA COLLECTION: Bruker SMART APEX-II CCD
system, MoKalpha radiation, standard focus tube, anode power=50
kV.times.42 mA, crystal to plate distance=5.0 cm, 512.times.512
pixels/frame, beam center=(256.13, 253.14), total frames=704,
oscillation/frame=0.50.degree., exposure/frame=120.1 sec/frame,
SAINT integration, hkl min/max=(-27, 34, -6, 6, -23, 11), data
input to shelx=7578, unique data=5186, two-theta range=3.20 to
56.740, completeness to two-theta 56.74=99.70%, R(int-xl)=0.0331,
SADABS correction applied. SOLUTION AND REFINEMENT: Structure
solved using XS(Shelxtl), refined using shelxtl software package,
refinement by full-matrix least squares on F.sup.2, scattering
factors from Int. Tab. Vol C Tables 4.2.6.8 and 6.1.1.4, number of
data=5186, number of restraints=2, number of parameters=313,
data/parameter ratio=16.57, goodness-of-fit on F.sup.2=1.02, R
indices[I>4sigma(I)] R1=0.0524, wR2=0.1033, R indices(all data)
R1=0.0826, wR2=0.1162, max difference peak and hole=0.294 and
-0.221 e/.ANG..sup.3, refined flack parameter=0.05(8), All of the
hydrogen atoms except the NH2 and water hydrogens have been
idealized using a riding model. RESULTS: The asymmetric unit
contains one molecule and one water molecule as shown in FIG. 1
with thermal ellipsoids drawn to the 50% probability level. The
predicted structure is confirmed. The absolute configuration is
determined based upon the known S configuration at C21. The
configuration at C7 is determined to be S. The flack parameter also
confirms the correct configuration. Based on the crystal structure,
the compound of Example 269 is believed to be
4-((S)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-6-chlo-
ro-2-(1-((S)-2-hydroxypropyl)azetidin-3-yl)-3-methoxybenzonitrile.
The crystal structure is shown in FIG. 1.
Examples 272 and 273. Diastereoisomers of
4-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-6-chloro-2-
-[1-(2-hydroxy-1-methylethyl)azetidin-3-yl]-3-methoxybenzonitrile
##STR00108##
[0473] A solution of
4-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-2-azetidin-
-3-yl-6-chloro-3-methoxybenzonitrile (40 mg, 0.10 mmol) in methanol
(2 mL) was treated with sodium cyanoborohydride (16 mg, 0.25 mmol)
followed by acetol (28 .mu.L, 0.40 mmol) and stirred at room
temperature for 1 h. The reaction mixture was quenched with acetic
acid (100 .mu.L, 1.8 mmol), diluted with methanol, and purified by
preparative LCMS (XBridge C18 column, eluting with a gradient of
acetonitrile/water containing 0.1% ammonium hydroxide, at flow rate
of 60 mL/min) to give the desired products as a mixture of
diastereoisomers. This mixture of diastereoisomers was separated by
chiral HPLC (RT=3.70 min and 6.58 min; Phenomenex Lux Cellulose C-4
column, 21.2.times.250 mm, 5 micron particle size, eluting with 20%
ethanol in hexanes at 18 ml/min, 5 mg/inj) to give the desired peak
1 isomer (compound 272) (19 mg, 41%) and peak 2 isomer (compound
273) (23 mg, 50%) Peak 1: .sup.1H NMR (300 MHz, DMSO-d.sub.6)
.delta. 8.11 (s, 1H), 7.47 (s, 1H), 7.34 (br s, 2H), 6.24 (q, J=6.9
Hz, 1H), 4.43 (t, J=5.2 Hz, 1H), 4.07-3.82 (m, 3H), 3.64 (s, 3H),
3.31-3.24 (m, 1H), 3.17-3.06 (m, 2H), 3.06-2.97 (m, 1H), 2.55 (s,
3H), 2.21-2.11 (m, 1H), 1.72 (d, J=7.1 Hz, 3H), 0.81 (d, J=6.3 Hz,
3H). LCMS for C.sub.22H.sub.27ClN.sub.7O.sub.2(M+H).sup.+:
m/z=456.2; Found: 456.2. Peak 2: .sup.1H NMR (300 MHz,
DMSO-d.sub.6) .delta. 8.11 (s, 1H), 7.47 (s, 1H), 7.35 (br s, 2H),
6.24 (q, J=7.0 Hz, 1H), 4.43 (t, J=5.5 Hz, 1H), 4.06-3.91 (m, 2H),
3.89-3.79 (m, 1H), 3.64 (s, 3H), 3.30-3.24 (m, 1H), 3.15-3.00 (m,
3H), 2.55 (s, 3H), 2.21-2.10 (m, 1H), 1.72 (d, J=7.1 Hz, 3H), 0.82
(d, J=6.2 Hz, 3H). LCMS for
C.sub.22H.sub.27ClN.sub.7O.sub.2(M+H).sup.+: m/z=456.2; Found:
456.2.
Example 281.
2-(1-Acetylazetidin-3-yl)-4-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimi-
din-1-yl)ethyl]-6-chloro-3-methoxybenzonitrile
##STR00109##
[0475] A solution of
4-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-2-azetidin-
-3-yl-6-chloro-3-methoxybenzonitrile (chiral intermediate from
Example 261) (60 mg, 0.15 mmol) in tetrahydrofuran (2 mL) at
0.degree. C. was treated with triethylamine (53 .mu.L, 0.38 mmol)
followed by acetyl chloride (13 .mu.L, 0.18 mmol) and stirred at
20.degree. C. overnight. The reaction mixture was diluted with
methanol and purified by preparative LCMS (XBridge C18 column,
eluting with a gradient of acetonitrile/water containing 0.1%
ammonium hydroxide, at flow rate of 60 mL/min) to give the desired
product (39 mg, 59%). The product was isolated as a single
enantiomer. .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 8.11 (s,
1H), 7.52 (d, J=2.5 Hz, 1H), 7.36 (br s, 2H), 6.26 (q, J=7.0 Hz,
1H), 4.57-4.36 (m, 3H), 4.30-4.21 (m, 1H), 4.18-4.08 (m, 1H), 3.71
(d, J=3.1 Hz, 3H), 2.55 (s, 3H), 1.78-1.71 (m, 6H). LCMS for
C.sub.21H.sub.23ClN.sub.7O.sub.2(M+H).sup.+: m/z=440.2; Found:
440.1.
Example 285.
4-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-6-chloro-3-
-methoxy-2-[1-(methylsulfonyl)azetidin-3-yl]benzonitrile
##STR00110##
[0477] A solution of
4-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-2-azetidin-
-3-yl-6-chloro-3-methoxybenzonitrile (chiral intermediate from
Example 261) (40 mg, 0.10 mmol) in dichloromethane (1 mL) was
treated with triethylamine (35 .mu.L, 0.25 mmol), cooled to
0.degree. C., treated with methanesulfonyl chloride (9.3 .mu.L,
0.12 mmol) and stirred at 0.degree. C. for 1 h. The reaction
mixture was diluted with methanol and purified by preparative LCMS
(XBridge C18 column, eluting with a gradient of acetonitrile/water
containing 0.1% ammonium hydroxide, at flow rate of 60 mL/min) to
give the desired product (20 mg, 42%). The product was isolated as
a single enantiomer. .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta.
8.12 (s, 1H), 7.55 (s, 1H), 7.35 (br s, 2H), 6.25 (q, J=7.0 Hz,
1H), 4.54-4.40 (m, 1H), 4.27-4.12 (m, 4H), 3.68 (s, 3H), 3.01 (s,
3H), 2.55 (s, 3H), 1.74 (d, J=7.1 Hz, 3H). LCMS for
C.sub.20H.sub.23ClN.sub.7O.sub.3S (M+H).sup.+: m/z=476.1; Found:
476.1.
Example 289. Methyl
3-{3-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chlor-
o-6-cyano-2-methoxyphenyl}azetidine-1-carboxylate
##STR00111##
[0479] A solution of
4-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-2-azetidin-
-3-yl-6-chloro-3-methoxybenzonitrile (chiral intermediate from
Example 261) (20 mg, 0.05 mmol) in dichloromethane (1 mL) was
treated with triethylamine (20 .mu.L, 0.14 mmol) followed by methyl
chloroformate (4.7 .mu.L, 0.06 mmol) and stirred at room
temperature for 1 h. The reaction mixture was diluted with methanol
and purified by preparative LCMS (XBridge C18 column, eluting with
a gradient of acetonitrile/water containing 0.1% ammonium
hydroxide, at flow rate of 60 mL/min) to give the desired product
(12 mg, 52%). The product was isolated as a single enantiomer.
.sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 8.11 (s, 1H), 7.51 (s,
1H), 7.34 (br s, 2H), 6.25 (q, J=7.0 Hz, 1H), 4.53-4.38 (m, 1H),
4.36-4.17 (m, 4H), 3.71 (s, 3H), 3.55 (s, 3H), 2.55 (s, 3H), 1.73
(d, J=7.1 Hz, 3H). LCMS for
C.sub.21H.sub.23ClN.sub.7O.sub.3(M+H).sup.+: m/z=456.2; Found:
456.1.
Example 292.
3-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chlor-
o-6-cyano-2-methoxyphenyl}-N-(tert-butyl)azetidine-1-carboxamide
##STR00112##
[0481] A solution of
4-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-2-azetidin-
-3-yl-6-chloro-3-methoxybenzonitrile (chiral intermediate from
Example 261) (20 mg, 0.05 mmol) in N,N-dimethylformamide (1 mL) was
treated with triethylamine (20 .mu.L, 0.14 mmol) followed by
2-isocyanato-2-methyl-propane (7.2 .mu.L, 0.063 mmol) and stirred
at room temperature overnight. The reaction mixture was diluted
with methanol and purified by preparative LCMS (XBridge C18 column,
eluting with a gradient of acetonitrile/water containing 0.1%
ammonium hydroxide, at flow rate of 60 mL/min) to give the desired
product (16 mg, 64%). The product was isolated as a single
enantiomer. LCMS for C.sub.24H.sub.30ClN.sub.8O.sub.2(M+H).sup.+:
m/z=497.2; Found: 497.2.
Example 293.
3-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chlor-
o-6-cyano-2-methoxyphenyl}azetidine-1-carboxamide
##STR00113##
[0483] A solution of
3-{3-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chlor-
o-6-cyano-2-methoxyphenyl}-N-(tert-butyl)azetidine-1-carboxamide
(chiral intermediate from Example 292) (16 mg, 0.032 mmol) in
trifluoroacetic acid (2 mL) was heated in the microwave at
120.degree. C. for 10 min. The reaction mixture was diluted with
methanol and purified by preparative LCMS (XBridge C18 column,
eluting with a gradient of acetonitrile/water containing 0.1%
ammonium hydroxide, at flow rate of 60 mL/min) to give the desired
product (7 mg, 50%). The product was isolated as a single
enantiomer. .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 8.12 (s,
1H), 7.62 (s, 1H), 7.35 (br s, 2H), 6.28 (q, J=6.9 Hz, 1H), 5.70
(br s, 1H), 4.62-4.49 (m, 1H), 4.34-4.20 (m, 1H), 3.83 (s, 3H),
3.78-3.49 (m, 2H), 2.55 (s, 3H), 1.73 (d, J=7.0 Hz, 3H). LCMS for
C.sub.20H.sub.22ClN.sub.8O.sub.2(M+H).sup.+: m/z=441.2; Found:
441.1.
Example 296.
3-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chlor-
o-6-cyano-2-methoxyphenyl}-N,N-dimethylazetidine-1-carboxamide
##STR00114##
[0485] A solution of
4-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-2-azetidin-
-3-yl-6-chloro-3-methoxybenzonitrile (chiral intermediate from
Example 261) (40 mg, 0.10 mmol) in N,N-dimethylformamide (2 mL) was
treated with triethylamine (40 .mu.L, 0.29 mmol) followed by
p-nitrophenyl chloroformate (23 .mu.L, 0.13 mmol) and stirred at
room temperature for 1 h. The reaction mixture was diluted with
methanol and purified by preparative LCMS (XBridge C18 column,
eluting with a gradient of acetonitrile/water containing 0.1%
ammonium hydroxide, at flow rate of 60 mL/min) to give the desired
product that was used immediately. A solution of the p-nitrophenyl
carbamate intermediate in tetrahydrofuran (1 mL) was treated with
triethylamine (15 .mu.L, 0.11 mmol) followed by a solution of 1.0 M
dimethylamine in tetrahydrofuran (150 .mu.L, 0.15 mmol) and heated
in a sealed tube at 60.degree. C. for 2 h. The reaction mixture was
concentrated, diluted with methanol and purified by preparative
LCMS (XBridge C18 column, eluting with a gradient of
acetonitrile/water containing 0.1% ammonium hydroxide, at flow rate
of 60 mL/min) to give the desired product (13 mg, 28%). The product
was isolated as a single enantiomer. .sup.1H NMR (300 MHz,
DMSO-d.sub.6) .delta. 8.11 (s, 1H), 7.49 (s, 1H), 7.36 (br s, 2H),
6.25 (q, J=7.0 Hz, 1H), 4.44-4.23 (m, 3H), 4.22-4.10 (m, 2H), 3.69
(s, 3H), 2.76 (s, 6H), 2.55 (s, 3H), 1.73 (d, J=7.1 Hz, 3H). LCMS
for C.sub.22H.sub.26ClN.sub.8O.sub.2(M+H).sup.+: m/z=469.2; Found:
469.1.
Example 298.
1-{1-[4,5-Dichloro-3-(1-ethylazetidin-3-yl)-2-methoxyphenyl]ethyl}-3-meth-
yl-1H-pyrazolo[3,4-d]pyrimidin-4-amine
##STR00115##
[0486] Step 1. 1-(4,5-Dichloro-2-hydroxyphenyl)ethanone
[0487] A solution of 3,4-dichlorophenol [AK Scientific] (30 g, 18
mmol) in acetyl chloride (19 mL, 270 mmol) was stirred at
60.degree. C. for 2 h. The reaction mixture was cooled to
20.degree. C., treated with aluminum trichloride (37 g, 280 mmol)
portionwise, and heated at 180.degree. C. for 30 min. The reaction
mixture was cooled to 20.degree. C. and the solution hardened into
a solid block that was not easy to break apart. This material was
cooled to 0.degree. C. and quenched slowly with 1 M HCl in
portions. The solid block of material slowly broke apart with
enough HCl and this heterogenous mixture was stirred at 20.degree.
C. overnight to ensure uniformity. The solid was filtered, washed
with copious amounts of water, and dried under vacuum to give the
desired product (38 g, quantitative) as a tan solid.
Step 2. 1-(4,5-Dichloro-2-hydroxy-3-iodophenyl)ethanone
[0488] A solution of 1-(4,5-dichloro-2-hydroxyphenyl)ethanone (12
g, 59 mmol) in acetic acid (70 mL) was treated with
N-iodosuccinimide (16 g, 71 mmol) and stirred at 90.degree. C. for
18 h. The reaction mixture was treated with additional
N-iodosuccinimide (8 g, 36 mmol) and stirred at 90.degree. C. for 4
h. The reaction mixture was concentrated, diluted with ethyl
acetate, and quenched with saturated sodium bicarbonate until the
bubbling stopped. The organic layer was separated and the aqueous
was re-extracted with ethyl acetate. The combined organic layers
were dried and concentrated to give a brown solid. This material
was recrystallized from methanol to give desired product (9.0 g,
46%) as a tan solid. .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.
13.36 (s, 1H), 7.85 (s, 1H), 2.65 (s, 3H). LCMS for
C.sub.8H.sub.6Cl.sub.2IO.sub.2 (M+H).sup.+: m/z=330.9, 332.9;
Found: 330.8, 332.9.
Step 3. 1-(4,5-Dichloro-3-iodo-2-methoxyphenyl)ethanone
[0489] A solution of
1-(4,5-dichloro-2-hydroxy-3-iodophenyl)ethanone (16 g, 47 mmol) and
potassium carbonate (17 g, 120 mmol) in N,N-dimethylformamide (40
mL) was treated with methyl iodide (6.4 mL, 100 mmol) and stirred
at 60.degree. C. for 1 h. The reaction mixture was diluted with
water and extracted with ethyl acetate (2.times.). The combined
organic layers were dried with magnesium sulfate, filtered, and
concentrated to give a crude solid. The crude material was purified
by flash column chromatography using ethyl acetate in hexanes
(5%-30%) to give the desired product (14 g, 84%) as an orange
solid. .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.69 (s, 1H), 3.79
(s, 3H), 2.60 (s, 3H). LCMS for C.sub.9H.sub.8Cl.sub.2IO.sub.2
(M+H).sup.+: m/z=344.9, 346.9; Found: 344.8, 346.9.
Step 4. tert-Butyl
3-(3-acetyl-5,6-dichloro-2-methoxyphenyl)azetidine-1-carboxylate
[0490] Zinc (4.5 g, 69 mmol) was suspended with 1,2-dibromoethane
(420 .mu.L, 4.9 mmol) in N, N-dimethylformamide (54 mL). The
mixture was heated at 70.degree. C. for 10 min and then cooled to
room temperature. Chlorotrimethylsilane (620 .mu.L, 4.9 mmol) was
added dropwise and stirring was continued for 1 h. A solution of
tert-butyl 3-iodoazetidine-1-carboxylate (17 g, 61 mmol) in
N,N-dimethylformamide (30 mL) was then added and the mixture was
heated at 40.degree. C. for 1 h before a mixture of
1-(4,5-dichloro-3-iodo-2-methoxyphenyl)ethanone (14 g, 41 mmol),
tris(dibenzylideneacetone)dipalladium(0) (710 mg, 0.77 mmol) and
tri-(2-furyl)phosphine (360 mg, 1.6 mmol) in N,N-dimethylformamide
(120 mL) was added quickly. The reaction mixture was stirred
overnight at room temperature. The reaction mixture was then
partitioned between ethyl acetate and saturated ammonium chloride
solution. The organic layer was washed with water, dried with
magnesium sulfate, filtered, and concentrated to a crude residue
that was purified by flash column chromatography using ethyl
acetate in hexanes (0%-25%) to give the desired product (12 g,
77%). LCMS for C.sub.17H.sub.21Cl.sub.2NO.sub.4Na (M+Na).sup.+:
m/z=396.1; Found: 396.0.
Step 5. tert-Butyl
3-[2,3-dichloro-5-(1-hydroxyethyl)-6-methoxyphenyl]azetidine-1-carboxylat-
e
[0491] A solution of tert-butyl
3-(3-acetyl-5,6-dichloro-2-methoxyphenyl)azetidine-1-carboxylate
(9.6 g, 26 mmol) in methanol (240 mL) at 0.degree. C. was treated
with sodium tetrahydroborate (1.9 g, 51 mmol) portionwise over 5
min and stirred at 0.degree. C. for 30 min. The reaction mixture
was quenched with acetic acid (7.3 mL, 130 mmol) at 0.degree. C.
and treated with saturated sodium bicarbonate solution (.about.50
mL). The reaction mixture was concentrated to remove most of the
methanol (to .about.60 mL), poured into saturated sodium
bicarbonate solution (150 ml), and extracted with ethyl acetate
(2.times.200 mL). The combined organic extracts were washed with
water and brine, dried over sodium sulfate, filtered, and
concentrated to give the desired product (9.6 g, quantitative) that
was used without further purification. LCMS for
C.sub.13H.sub.16Cl.sub.2NO.sub.4 ([M-(t-Bu)+H]+H).sup.+: m/z=320.0;
Found: 320.0.
Step 6. tert-Butyl
3-[2,3-dichloro-5-(1-chloroethyl)-6-methoxyphenyl]azetidine-1-carboxylate
[0492] N,N-Dimethylformamide (0.92 mL, 12 mmol) was added to solid
cyanuric chloride (2.2 g, 12 mmol) at room temperature (DMF is
absorbed by the solid). The mixture was allowed to stand for 10
min, treated with methylene chloride (60 mL), and stirred for a few
minutes to break up the solid. The reaction mixture was treated
with a solution of tert-butyl
3-[2,3-dichloro-5-(1-hydroxyethyl)-6-methoxyphenyl]azetidine-1-carboxylat-
e (3.0 g, 8.0 mmol) in methylene chloride (30 mL) and stirred at
35-40.degree. C. for 2 h. The reaction mixture was treated with
additional N,N-dimethylformamide (1 mL) and stirred at
35-40.degree. C. for 4 h. The reaction required another treatment
of N,N-dimethylformamide (1 mL) with stirring at 35-40.degree. C.
overnight to proceed to completion. The reaction mixture was
diluted with water and dichloromethane. The organic phase was
separated and washed with saturated sodium bicarbonate solution,
water and brine, dried over magnesium sulfate, filtered, and
concentrated to a crude residue. The crude material was purified by
flash column chromatography using ethyl acetate in hexanes (5%-40%)
to give the desired product (2.8 g, 90%). LCMS for
C.sub.13H.sub.15Cl.sub.3NO.sub.3 ([M-(t-Bu)+H]+H).sup.+: m/z=338.0,
340.0; Found: 337.9, 339.9.
Step 7. tert-Butyl
3-{3-[1-(4-amino-3-methyl-1H-pyrazolo[4,3-c]pyridin-1-yl)ethyl]-5,6-dichl-
oro-2-methoxyphenyl}azetidine-1-carboxylate
[0493] A solution of tert-butyl
3-[2,3-dichloro-5-(1-chloroethyl)-6-methoxyphenyl]azetidine-1-carboxylate
(1.0 g, 2.5 mmol) and 3-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine
(0.43 g, 2.9 mmol) in N,N-dimethylformamide (23 mL) was treated
with cesium carbonate (1.2 g, 3.8 mmol) and potassium iodide (42
mg, 0.25 mmol) and heated at 100.degree. C. for 10 h. The reaction
mixture was diluted with ethyl acetate (75 mL) and water (75 mL).
The aqueous layer was separated and reextracted with ethyl acetate
(2.times.50 mL). The combined organic layers were washed with
water, saturated sodium bicarbonate solution, and brine, dried over
magnesium sulfate, filtered, and concentrated to a crude residue.
The crude material was purified by flash column chromatography
using methanol in dichloromethane (0%-10%) to give the desired
product (0.97 g, 75%). LCMS for
C.sub.23H.sub.29Cl.sub.2N.sub.6O.sub.3 (M+H).sup.+: m/z=507.2,
509.2; Found: 507.0, 509.0.
Step 8.
1-[1-(3-Azetidin-3-yl-4,5-dichloro-2-methoxyphenyl)ethyl]-3-methyl-
-1H-pyrazolo[3,4-d]pyrimidin-4-amine
[0494] A solution of tert-butyl
3-{3-[1-(4-amino-3-methyl-1H-pyrazolo[4,3-c]pyridin-1-yl)ethyl]-5,6-dichl-
oro-2-methoxyphenyl}azetidine-1-carboxylate (0.97 g, 1.9 mmol) in
methylene chloride (20 mL) was treated with trifluoroacetic acid
(10 mL) and stirred at 20.degree. C. for 30 min. The reaction
mixture was concentrated and the residue was diluted with methanol
(.about.20 mL) and treated with saturated sodium bicarbonate
solution (to pH.about.8). The reaction mixture was concentrated to
remove the methanol. The oil that was suspended in the aqueous
layer was extracted into a 5:1 mixture of
dichloromethane/isopropanol, dried over magnesium sulfate,
filtered, and concentrated to give the desired product (0.77 g,
99%) that was used in the next step without further purification.
LCMS for C.sub.18H.sub.21Cl.sub.2N.sub.6O (M+H).sup.+: m/z=407.1,
409.1; Found: 407.0, 409.0.
Step 9.
1-{1-[4,5-Dichloro-3-(1-ethylazetidin-3-yl)-2-methoxyphenyl]ethyl}-
-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine
[0495] A solution of
1-[1-(3-azetidin-3-yl-4,5-dichloro-2-methoxyphenyl)ethyl]-3-methyl-1H-pyr-
azolo[3,4-d]pyrimidin-4-amine (40 mg, 0.098 mmol) in methanol (2.6
mL) was treated with sodium cyanoborohydride (15 mg, 0.25 mmol)
followed by acetaldehyde (22 .mu.L, 0.39 mmol) and stirred at
20.degree. C. for 20 min. The reaction mixture was quenched with
acetic acid (130 .mu.L, 2.3 mmol), diluted with methanol, and
purified by preparative LCMS (XBridge C18 column, eluting with a
gradient of acetonitrile/water containing 0.1% ammonium hydroxide,
at flow rate of 60 mL/min) to give the desired product as a mixture
of enantiomers. This racemic mixture was separated by chiral HPLC
(RT=18.6 min and 22.0 min; Phenomenex Lux Cellulose C-4 column,
21.2.times.250 mm, 5 micron particle size, eluting with 5% ethanol
in hexanes at 18 ml/min, 2.5 mg/inj) to give the desired peak 1
isomer (11 mg, 26%). .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta.
8.11 (s, 1H), 7.45 (s, 1H), 7.33 (br s, 2H), 6.21 (q, J=6.9 Hz,
1H), 3.98-3.77 (m, 3H), 3.57 (s, 3H), 2.92-2.83 (m, 1H), 2.79-2.72
(m, 1H), 2.55 (s, 3H), 2.35-2.22 (m, 2H), 1.70 (d, J=7.1 Hz, 3H),
0.86 (t, J=7.1 Hz, 3H). LCMS for C.sub.20H.sub.25Cl.sub.2N.sub.6O
(M+H).sup.+: m/z=435.1; Found: 435.0.
Example 307.
4-[1-(4-Amino-5-methyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)ethyl]-6-chloro-3--
ethoxy-2-(1-isopropylazetidin-3-yl)benzonitrile
##STR00116##
[0496] Step 1. tert-Butyl
3-{3-[1-(4-amino-5-methyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)ethyl]-5-chloro-
-6-cyano-2-ethoxyphenyl}azetidine-1-carboxylate
[0497] The desired compound was prepared according to the procedure
of Example 212, step 5 (chiral intermediate), using
5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-amine [ACES Pharma] instead
of 3-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine as the starting
material in 18% yield. .sup.1H NMR (500 MHz, CDCl.sub.3) .delta.
8.13 (s, 1H), 6.93 (br s, 1H), 6.79 (s, 1H), 6.17 (q, J=7.1 Hz,
1H), 5.24 (s, 2H), 4.40-4.27 (m, 4H), 4.27-4.18 (m, 1H), 4.03-3.92
(m, 1H), 3.80-3.70 (m, 1H), 2.43 (s, 3H), 1.74 (d, J=7.1 Hz, 3H),
1.43 (s, 9H), 1.40 (t, J=7.0 Hz, 3H). LCMS for
C.sub.26H.sub.32ClN.sub.6O.sub.3(M+H).sup.+: m/z=511.2; Found:
511.2.
Step 2.
4-[1-(4-Amino-5-methyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)ethyl]-2-az-
etidin-3-yl-6-chloro-3-ethoxybenzonitrile
[0498] The desired compound was prepared according to the procedure
of Example 212, step 6, using tert-butyl
3-{3-[1-(4-amino-5-methyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)ethyl]-5-chloro-
-6-cyano-2-ethoxyphenyl}azetidine-1-carboxylate instead of
tert-butyl
3-{3-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chlor-
o-6-cyano-2-ethoxyphenyl}azetidine-1-carboxylate as the starting
material in 99% yield. LCMS for C.sub.21H.sub.24ClN.sub.6O
(M+H).sup.+: m/z=411.2; Found: 411.1.
Step 3.
4-[1-(4-Amino-5-methyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)ethyl]-6-ch-
loro-3-ethoxy-2-(1-isopropylazetidin-3-yl)benzonitrile
[0499] The desired compound was prepared according to the procedure
of Example 213 using
4-[1-(4-amino-5-methyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)ethyl]-2-azetidin--
3-yl-6-chloro-3-ethoxybenzonitrile instead of
4-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-2-azetidin-
-3-yl-6-chloro-3-ethoxybenzonitrile and acetone instead of
formaldehyde as the starting materials in 65% yield. The product
was isolated as a single enantiomer. .sup.1H NMR (300 MHz, dmso)
.delta. 7.95 (s, 1H), 7.19 (s, 1H), 7.16-7.13 (m, 1H), 6.58 (s,
2H), 6.11 (q, J=7.1 Hz, 1H), 4.04-3.67 (m, 5H), 3.04-2.92 (m, 2H),
2.36 (s, 3H), 2.27-2.12 (m, 1H), 1.69 (d, J=7.1 Hz, 3H), 1.30 (t,
J=6.9 Hz, 3H), 0.85 (dd, J=6.1, 1.8 Hz, 6H). LCMS for
C.sub.24H.sub.30ClN.sub.6O (M+H).sup.+: m/z=453.2; Found:
453.3.
Example 315.
4-[-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-2-{1-[(2S-
)-2-hydroxypropyl]azetidin-3-yl}-3-methoxy-6-methylbenzonitrile
##STR00117##
[0500] Step 1: 4-Acetyl-5-hydroxy-2-methylbenzonitrile
[0501] The 1-(4-bromo-2-hydroxy-5-methylphenyl)ethanone (8.5 g, 37
mmol, Alfa Aesar catalog# H29125) was combined with zinc cyanide
(8.7 g, 74 mmol) in N,N-dimethylformamide (75 mL) degassed with
nitrogen and the tris(dibenzylideneacetone)dipalladium(0) (1.0 g,
1.1 mmol) and
(9,9-dimethyl-9H-xanthene-4,5-diyl)bis(diphenylphosphine) (1.5 g,
2.6 mmol) were added. The reaction was degassed again with nitrogen
and heated to 120.degree. C. and monitored by LC/MS. After heating
for 18 h, the reaction was complete, the reaction was allowed to
cool to room temperature, taken up in ethyl acetate and washed with
water (2.times.), brine, dried over magnesium sulfate and
concentrated to give the crude product as a dark amber oil. The
product was purified by FCC on silica gel eluting hexane: ethyl
acetate gradient to give 4-acetyl-5-hydroxy-2-methylbenzonitrile as
a solid (6.3 g, 98%). LCMS calculated for C.sub.10H.sub.10NO.sub.2
(M+H).sup.+: m/z=176.1; found: 176.2.
Step 2: 4-Acetyl-3-hydroxy-2-iodo-6-methylbenzonitrile
[0502] The 4-acetyl-5-hydroxy-2-methylbenzonitrile (6.7 g, 38 mmol)
was dissolved in acetic acid (80 mL) and the N-Iodosuccinimide (10.
g, 46 mmol) was added. The reaction was heated to 80.degree. C. in
an oil bath and monitored by LC/MS. After heating for 4 hrs the
reaction was complete. This was allowed to cool and was
concentrated in vacuo to give a dark oil. The oil was taken up in
ethyl acetate and washed with water, sodium bicarbonate (3.times.,
until remained slightly basic), brine, dried over magnesium sulfate
and concentrated to give the crude product as a dark oil. The
product was purified by FCC on silica gel eluting hexane: ethyl
acetate gradient to give
4-acetyl-3-hydroxy-2-iodo-6-methylbenzonitrile as pale yellow solid
(7.2 g, 62%). LCMS calculated for C.sub.10H.sub.9INO.sub.2
(M+H).sup.+: m/z=301.9; found: 301.9.
Step 3: 4-Acetyl-2-iodo-3-methoxy-6-methylbenzonitrile
[0503] The 4-acetyl-3-hydroxy-2-iodo-6-methylbenzonitrile (5.0 g,
17 mmol) was dissolved in N,N-dimethylformamide (50 mL) and the
potassium carbonate (4.6 g, 33 mmol) and methyl iodide (2.1 mL, 33
mmol) were added. The reaction was heated to 60.degree. C. and
monitored by LC/MS. After heating for 2 hrs the reaction was
complete. This was allowed to cool, diluted with ethyl acetate (300
mL) and filtered to remove the remaining solids. The organic layer
was washed with water (3.times.), brine, dried over magnesium
sulfate and concentrated to give the crude product as a dark solid.
The product was purified by FCC on silica gel eluting hexane: ethyl
acetate gradient to give
4-acetyl-3-methoxy-2-iodo-6-methylbenzonitrile as a pale yellow
crystalline solid (5.0 g, 96%). LCMS calculated for
C.sub.11H.sub.11INO.sub.2 (M+H).sup.+: m/z=315.9; found: 316.0.
Step 4: tert-butyl
3-(3-acetyl-6-cyano-2-methoxy-5-methylphenyl)azetidine-1-carboxylate
[0504] Zinc (1.70 g, 26.0 mmol) and celite (oven dried, 500 mg)
were ground together in a flask until the solids appeared
homogenous, the flask was heated with a heat gun while under
high-vac for 5 minutes and then back-filled with nitrogen. The
solids were suspended in N,N-dimethylacetamide (4.2 mL) and
1,2-dibromoethane (0.13 mL, 1.5 mmol) was added. The reaction
mixture was heated at 70.degree. C. for 30 min and then cooled to
room temperature. Chlorotrimethylsilane (0.16 mL, 1.3 mmol) was
added dropwise and stirring was continued for 2 hrs at room
temperature. A solution of tert-butyl 3-iodoazetidine-1-carboxylate
(2.70 g, 9.52 mmol) in N,N-dimethylacetamide (4.35 mL) was then
added slowly and the resulting mixture was heated at 50.degree. C.
for 2 hrs. The zinc-iodo reagent was allowed to cool to room
temperature and was taken up in a syringe and filtered through a
PTFE filter (adapted with a needle) directly into a suspension of
tris(dibenzylideneacetone)dipalladium(0) (0.111 g, 0.121 mmol) and
tri-(2-furyl)phosphine (0.056 g, 0.24 mmol) and
4-acetyl-2-iodo-3-methoxy-6-methylbenzonitrile (2.0 g, 6.3 mmol) in
N,N-dimethylacetamide (19.6 mL) pre-degassed by bubbling N.sub.2.
The reaction mixture was degassed with nitrogen again and heated to
70.degree. C. After heating for 30 minutes the reaction was
complete by LC/MS. This was allowed to cool, taken up in ethyl
acetate and washed with water, brine, dried over magnesium sulfate
and concentrated to give the crude product as an oil. The product
was purified by FCC on silica gel eluting hexane; ethyl acetate
gradient to give tert-butyl
3-(3-acetyl-6-cyano-2-methoxy-5-methylphenyl)azetidine-1-carboxylate
as a clear oil. (1.8 g, 82%). LCMS calculated for
C.sub.15H.sub.17N.sub.2O.sub.4 (M+H).sup.+: m/z=289.1; found:
289.1.
Step 5: tert-butyl
3-[2-cyano-5-(1-hydroxyethyl)-6-methoxy-3-methylphenyl]azetidine-1-carbox-
ylate
[0505] The tert-butyl
3-(3-acetyl-6-cyano-2-methoxy-5-methylphenyl)azetidine-1-carboxylate
(2.2 g, 6.4 mmol) was dissolved in methanol (20 mL) and cooled in
ice bath. The sodium tetrahydroborate (0.26 g, 7.0 mmol) was added
portionwise and the reaction was monitored by LC/MS. After stirring
for 1 h the reaction was complete. This was diluted with ethyl
acetate and water. The combined organic layer was washed with
water, saturated sodium bicarbonate, brine, dried over magnesium
sulfate and concentrated to give crude tert-butyl
3-[2-cyano-5-(1-hydroxyethyl)-6-methoxy-3-methylphenyl]azetidine-1-carbox-
ylate as a yellow foam (2.1 g, 99%). LCMS calculated for
C.sub.15H.sub.19N.sub.2O.sub.4 (M+H).sup.+: m/z=291.1; found:
291.1.
Step 6: tert-butyl
3-[3-(1-chloroethyl)-6-cyano-2-methoxy-5-methylphenyl]azetidine-1-carboxy-
late
[0506] The tert-butyl
3-[2-cyano-5-(1-hydroxyethyl)-6-methoxy-3-methylphenyl]azetidine-1-carbox-
ylate (2.1 g, 6.4 mmol) was taken up in methylene chloride (50.0
mL) and N,N-dimethylformamide (0.59 mL), cooled in an ice bath and
the thionyl chloride (0.56 mL, 7.7 mmol) was added slowly. After
stirring for 2 hrs the reaction was complete by LC/MS and was
partitioned between ethyl acetate and water. The combined organic
layer was washed with water saturated sodium bicarbonate, brine,
dried over magnesium sulfate and concentrated to give crude
tert-butyl
3-[3-(1-chloroethyl)-6-cyano-2-methoxy-5-methylphenyl]azetidine-1-carboxy-
late as an oil (2.2 g, 100%). LCMS calculated for
C.sub.15H.sub.18ClN.sub.2O.sub.3(M+H).sup.+: m/z=309.1; found:
309.1.
Step 7: tert-butyl
3-{3-[-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-6-cyan-
o-2-methoxy-5-methylphenyl}azetidine-1-carboxylate
[0507] The tert-butyl
3-[3-(1-chloroethyl)-6-cyano-2-methoxy-5-methylphenyl]azetidine-1-carboxy-
late (2.3 g, 6.3 mmol) was dissolved in N,N-dimethylformamide (68
mL) with cesium carbonate (4.1 g, 13 mmol) and
3-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (1.4 g, 9.4 mmol) and
was heated in an oil bath to 80.degree. C. The reaction was stirred
for 18 hrs and allowed to cool to room temperature. The reaction
mixture was taken up in ethyl acetate, filtered, washed with water,
brine, dried over magnesium sulfate and concentrated to give the
crude product. The product was purified by FCC on silica gel
eluting a (hexane: 10% ethanol ethyl acetate) gradient to give
tert-butyl
3-{3-[-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-6-cyan-
o-2-methoxy-5-methylphenyl}azetidine-1-carboxylate as a semisolid
(1.5 g, 50%). LCMS calculated for C.sub.25H.sub.32N.sub.7O.sub.3
(M+H).sup.+: m/z=478.2; found: 478.2. The enantiomers were
separated by Chiral column HPLC using: Phenomenex LUX Cellulose
Column, 21.1.times.250 mm, 5 micron, 15% ethanol in hexane, 18
mL/min.about.5 mg/injection to give: First peak retention time: 2.1
minutes, tert-butyl
3-{3-[-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-6-cyan-
o-2-methoxy-5-methylphenyl}azetidine-1-carboxylate; Second peak
retention time: 3.9 minutes, tert-butyl
3-{3-[-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-6-cyan-
o-2-methoxy-5-methylphenyl}azetidine-1-carboxylate.
Step 8:
4-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-2-a-
zetidin-3-yl-3-methoxy-6-methylbenzonitrile
bis(trifluoroacetate)
[0508] The tert-butyl
3-{3-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-6-cyano-
-2-methoxy-5-methylphenyl}azetidine-1-carboxylate (0.35 g, 0.73
mmol) (Step 7, peak 1) was dissolved in methylene chloride (3.0 mL)
and trifluoroacetic acid (1.0 mL) at room temperature. After
stirring for 1 h the reaction was complete by LC/MS. The reaction
was concentrated in vacuo to give
4-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-2-azetidin-
-3-yl-3-methoxy-6-methylbenzonitrile (bis(trifluoroacetate) as a
viscous amber oil (0.50 g, 100%). LCMS calculated for
C.sub.20H.sub.24N.sub.7O (M+H).sup.+: m/z=378.2; found: 378.2.
Step 9:
4-[-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-2--
{1-[(2S)-2-hydroxypropyl]azetidin-3-yl}-3-methoxy-6-methylbenzonitrile
[0509] The
4-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]--
2-azetidin-3-yl-3-methoxy-6-methylbenzonitrile
bis(trifluoroacetate) (0.074 g, 0.10 mmol) was dissolved in ethanol
(3.0 mL) and DIPEA (0.071 mL, 0.41 mmol) and the
(S)-(-)-methyloxirane (0.0071 g, 0.12 mmol) was added. The reaction
was heated in a sealed tube to 90.degree. C. and monitored by
LC/MS. After heating for 6 hrs the reaction was purified without
workup by prep HPLC on a C-18 column eluting water: acetonitrile
gradient buffered pH 10 to give the title compound as a white
amorphous solid (0.018 g, 40%). The product was isolated as a
single enantiomer. LCMS calculated for
C.sub.23H.sub.30N.sub.7O.sub.2 (M+H).sup.+: m/z=436.2; found:
436.3. .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 8.09 (s, 1H),
7.21 (s, 1H), 6.22 (q, J=7.1 Hz, 1H), 4.34 (d, J=4.5 Hz, 1H),
4.09-3.83 (m, 3H), 3.60 (s, 3H), 3.58-3.51 (m, 1H), 3.12-2.95 (m,
2H), 2.55 (s, 3H), 2.33 (s, 3H), 2.27 (d, J=5.9 Hz, 2H), 1.71 (d,
J=7.1 Hz, 3H), 1.00 (d, J=6.2 Hz, 3H).
Example 316.
4-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-6-chloro-3-
-ethoxy-2-[6-(1-hydroxy-1-methylethyl)pyridin-3-yl]benzonitrile
##STR00118##
[0510] Step 1. 5-bromo-N-methoxy-N-methylpyridine-2-carboxamide
[0511] N, O-dimethylhydroxylamine hydrochloride (500 mg, 5 mmol)
was added to a mixture of
N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium
hexafluorophosphate (1400 mg, 3.7 mmol), N,N-diisopropylethylamine
(1000 .mu.L, 7 mmol) and 5-bromopyridine-2-carboxylic acid (500 mg,
2 mmol, Frontier Scientific catalog# B1704) in
N,N-dimethylformamide (10 mL). The reaction mixture was stirred
overnight at room temperature and was complete by LC/MS. The
reaction was partitioned between water and EtOAc. The combined
organic layer was washed with brine, dried over MgSO.sub.4,
filtered and concentrated to give the crude product. The product
was purified on by FCC on silica gel eluting a hexane: EtOAc
(0-30%) gradient to give
5-bromo-N-methoxy-N-methylpyridine-2-carboxamide clear oil (0.50 g,
60%). LCMS calculated for C.sub.8H.sub.10BrN.sub.2O.sub.2
(M+H).sup.+: m/z=244.9, 246.9; found: 244.9, 246.9.
Step 2. 1-(5-bromopyridin-2-yl)ethanone
[0512] Methylmagnesium chloride 3.0 M in THF (0.5 mL) was added
dropwise to a mixture of
5-bromo-N-methoxy-N-methylpyridine-2-carboxamide (200 mg, 0.8 mmol)
in tetrahydrofuran (10 mL) at 0.degree. C. After stirring for 1 hr
at room temperature, the reaction was quenched with 1 N NH.sub.4Cl
and was extracted with EtOAc. The combined organic layer was washed
with brine and dried over MgSO.sub.4, concentrated to give the
crude product 1-(5-bromopyridin-2-yl)ethanone (0.15 g, 90%). LCMS
calculated for C.sub.7H.sub.7BrNO (M+H).sup.+: m/z=199.9, 201.9;
found: 199.9, 201.9.
Step 3. 2-(5-bromopyridin-2-yl)propan-2-ol
[0513] Methylmagnesium chloride 3.0 M in THF (0.3 mL) was added
dropwise to a mixture of 1-(5-bromopyridin-2-yl)ethanone (100 mg,
0.5 mmol) in tetrahydrofuran (10 mL) at 0.degree. C. After stirring
for 1 h at room temperature, the reaction was quenched with 1 N
NH.sub.4Cl and was extracted with EtOAc. The combined organic layer
was washed with brine and dried over MgSO.sub.4, concentrated to
give crude 2-(5-bromopyridin-2-yl)propan-2-ol (0.1 g, 100%). LCMS
calculated for C.sub.8H.sub.11BrNO (M+H).sup.+: m/z=215.9, 217.9;
found: 215.8, 217.8.
Step 4. [6-(1-hydroxy-1-methylethyl)pyridin-3-yl]boronic acid
[0514] A mixture of 2-(5-bromopyridin-2-yl)propan-2-ol (70 mg, 0.3
mmol),
4,4,5,5,4',4',5',5'-octamethyl-[2,2']bi[[1,3,2]dioxaborolanyl] (90.
mg, 0.36 mmol),
[1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II),
complex with dichloromethane (1:1) (10 mg, 0.01 mmol), and
potassium acetate (100 mg, 1 mmol) in 1,4-dioxane (5 mL) was heated
at 120.degree. C. overnight. The reaction was complete by LC/MS,
was concentrated in vacuo to give crude
[6-(1-hydroxy-1-methylethyl)pyridin-3-yl]boronic acid. LCMS
calculated for CsH.sub.13BNO.sub.3 (M+H).sup.+: m/z=182.1; found:
182.1.
Step 5.
4-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-6-c-
hloro-3-ethoxy-2-[6-(1-hydroxy-1-methylethyl)pyridin-3-yl]benzonitrile
bis(2,2,2-trifluoroacetate)
[0515] Sodium carbonate (10 mg, 0.09 mmol) in water (0.5 mL) was
added to a mixture of
4-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-2-bromo-6--
chloro-3-ethoxybenzonitrile (20 mg, 0.04 mmol, racemic intermediate
from Example 43, Step 5) and
[6-(1-hydroxy-1-methylethyl)pyridin-3-yl]boronic acid (12 mg, 0.069
mmol, Example 306, Step 4) in acetonitrile (1 mL). The reaction
mixture was degassed with N.sub.2 and
[1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II),
complex with dichloromethane (1:1) (2 mg, 0.002 mmol) was added.
The reaction was degassed with N.sub.2 again and heated to
100.degree. C. for 1 h. The reaction was allowed to cool to room
temperature and was purified without workup by prep HPLC on a C-18
column eluting a water; acetonitrile gradient buffered with TFA to
give the title compound as white amorphous solid. The product was
isolated as a racemic mixture. LCMS calculated for
C.sub.25H.sub.27ClN.sub.7O.sub.2(M+H).sup.+: m/z=492.1; found:
492.1. .sup.1H NMR (500 MHz, DMSO-d.sub.6) .delta. 8.60 (d, J=2.0
Hz, 1H), 8.22 (s, 1H), 7.96 (dd, J=8.2, 2.3 Hz, 1H), 7.80 (d, J=8.3
Hz, 1H), 7.73 (s, 1H), 6.36 (q, J=7.0 Hz, 1H), 3.52-3.40 (m, 1H),
3.40-3.30 (m, 1H), 2.59 (s, 3H), 1.80 (d, J=7.0 Hz, 3H), 1.48 (d,
J=2.3 Hz, 6H), 0.88 (t, J=7.0 Hz, 3H).
Example 318.
4-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-6-chloro-3-
-ethoxy-2-pyrrolidin-1-ylbenzonitrile
##STR00119##
[0516] Step 1. 4-acetyl-6-chloro-3-ethoxy-2-iodobenzonitrile
[0517] The 4-acetyl-6-chloro-3-ethoxy-2-iodobenzonitrile was
prepared by analogous methods described in Example 43, Step 1 and
Step 2, but using N-iodosuccinimide. LCMS calculated for
C.sub.11H.sub.10ClINO.sub.2 (M+H).sup.+: m/z=349.9; found:
350.0
Step 2.
4-acetyl-6-chloro-3-ethoxy-2-pyrrolidin-1-ylbenzonitrile
[0518] The 4-acetyl-6-chloro-3-ethoxy-2-iodobenzonitrile (0.20 g,
0.57 mmol) was combined with pyrrolidine (0.052 mL, 0.63 mmol) in
N,N-dimethylformamide (2.0 mL) with cesium carbonate (0.19 g, 0.57
mmol) and heated to 120.degree. C. in a sealed tube. After heating
for 18 hrs the reaction was allowed to cool, taken up in ethyl
acetate, washed with water, brine, dried over magnesium sulfate and
concentrated to give the crude product as a dark oil. The product
was purified by FCC on silica gel eluting with hexane: ethyl
acetate gradient to give
4-acetyl-6-chloro-3-ethoxy-2-pyrrolidin-1-ylbenzonitrile as an oil
(0.045 g, 27%). LCMS calculated for
C.sub.15H.sub.18ClN.sub.2O.sub.2(M+H).sup.+: m/z=293.1; found
293.1.
Step 3.
6-chloro-3-ethoxy-4-(1-hydroxyethyl)-2-pyrrolidin-1-ylbenzonitrile
[0519] The 4-acetyl-6-chloro-3-ethoxy-2-pyrrolidin-1-ylbenzonitrile
(0.045 g, 0.15 mmol) was dissolved in methanol (3 mL) and cooled in
an ice bath. The sodium tetrahydroborate (0.0058 g, 0.15 mmol) was
added and the reaction was monitored by LC/MS. After stirring for 1
h, the reaction was taken up in ethyl acetate and washed with
water, sodium bicarbonate, brine and dried over magnesium sulfate
to give crude
6-chloro-3-ethoxy-4-(1-hydroxyethyl)-2-pyrrolidin-1-ylbenzonitrile
as a clear oil (0.045 g, 100%). LCMS calculated for
C.sub.15H.sub.20ClN.sub.2O.sub.2(M+H).sup.+: m/z=295.1; found
295.1.
Step 4.
6-chloro-4-(1-chloroethyl)-3-ethoxy-2-pyrrolidin-1-ylbenzonitrile
[0520] The
6-chloro-3-ethoxy-4-(1-hydroxyethyl)-2-pyrrolidin-1-ylbenzonitr-
ile (0.045 g, 0.15 mmol) was taken up in methylene chloride (3.0
mL) and N,N-dimethylformamide (0.002 mL, 0.03 mmol) and cooled in
an ice bath. The thionyl chloride (0.017 mL, 0.23 mmol) was added
and the reaction was monitored by LC/MS. After stirring for 2 hrs
the reaction was complete. The reaction was then taken up in ethyl
acetate, washed with sodium bicarbonate, brine, dried over
magnesium sulfate and concentrated to give crude
6-chloro-4-(1-chloroethyl)-3-ethoxy-2-pyrrolidin-1-ylbenzonitrile
as a yellow oil (0.048 g, 100%). LCMS calculated for
C.sub.15H.sub.19Cl.sub.2N.sub.2O (M+H).sup.+: m/z=313.1; found
313.1.
Step 5.
4-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-6-c-
hloro-3-ethoxy-2-pyrrolidin-1-ylbenzonitrile
[0521] The
6-chloro-4-(1-chloroethyl)-3-ethoxy-2-pyrrolidin-1-ylbenzonitri- le
(0.048 g, 0.15 mmol, racemic mixture) was combined with
3-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (0.034 g, 0.23 mmol)
and cesium carbonate (0.10 g, 0.31 mmol) in N,N-dimethylformamide
(3.0 mL) and heated in an oil bath to 85.degree. C. After heating
for 18 hrs the reaction was complete. The crude reaction was
purified with out work up by prep HPLC on a C-18 column eluting
water: acetonitrile gradient buffered pH 10 to give the title
compound as a white amorphous solid (0.012 g, 18%). The product was
isolated as a racemic mixture. LCMS calculated for
C.sub.21H.sub.25ClN.sub.7O (M+H).sup.+: m/z=426.1; found 426.1.
.sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 8.11 (s, 1H), 6.91 (s,
1H), 6.25 (q, J=7.1 Hz, 1H), 3.71 (dp, J=15.7, 8.1, 7.2 Hz, 4H),
3.49-3.35 (m, 2H), 2.55 (s, 3H), 2.00-1.76 (m, 4H), 1.70 (d, J=7.1
Hz, 3H), 1.34 (t, J=7.0 Hz, 3H).
Example 319.
4-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-6-chloro-3-
-ethoxy-2-(3-methoxyazetidin-1-yl)benzonitrile
##STR00120##
[0522] Step 1.
4-acetyl-6-chloro-3-ethoxy-2-(3-methoxyazetidin-1-yl)benzonitrile
[0523] To a mixture of
4-acetyl-6-chloro-3-ethoxy-2-iodobenzonitrile (50 mg, 0.1 mmol,
Example 318, Step 1), 3-methoxyazetidine hydrochloride (21 mg, 0.17
mmol Chem-Impex catalog#20140) and cesium carbonate (70. mg, 0.21
mmol) in 1,4-dioxane (4 mL) was added
(9,9-dimethyl-9H-xanthene-4,5-diyl)bis(diphenylphosphine) (40 mg,
0.07 mmol) and tris(dibenzylideneacetone)dipalladium (0) (60 mg,
0.07 mmol). The reaction mixture was degassed with N.sub.2. The
reaction was heated at 80.degree. C. for 2 hrs and was monitored by
LC/MS. The reaction was allowed to cool to room temperature, was
diluted with water and extracted with EtOAc. The combined organic
layers were washed with brine, dried over MgSO4, filtered and
concentrated to give the crude product. The product was purified by
FCC on silica gel eluting (hexanes: EtOAc 0-70%) gradient to give
to
4-acetyl-6-chloro-3-ethoxy-2-(3-methoxyazetidin-1-yl)benzonitrile
as clear oil (0.030 g, 70%). LCMS calculated for
C.sub.15H.sub.18ClN.sub.2O.sub.3(M+H).sup.+: m/z=309.1; found:
309.1.
Step 2.
6-chloro-3-ethoxy-4-(1-hydroxyethyl)-2-(3-methoxyazetidin-1-yl)ben-
zonitrile
[0524]
4-Acetyl-6-chloro-3-ethoxy-2-(3-methoxyazetidin-1-yl)benzonitrile
(30 mg, 0.1 mmol was dissolved in methanol (5 mL) cooled to
0.degree. C. and sodium tetrahydroborate (5.5 mg, 0.14 mmol) was
added. Reaction was stirred for 1 h at 0.degree. C. The reaction
was partitioned between EtOAc and water. The combined organic layer
was washed with water and saturated NaHCO.sub.3, brine, dried over
Na.sub.2SO.sub.4, filtered and concentrated to give crude
6-chloro-3-ethoxy-4-(1-hydroxyethyl)-2-(3-methoxyazetidin-1-yl)benzonitri-
le (0.030 g, 100%). LCMS calculated for
C.sub.15H.sub.20ClN.sub.2O.sub.3 (M+H).sup.+: m/z=311.1; found:
311.1.
Step 3.
6-chloro-4-(1-chloroethyl)-3-ethoxy-2-(3-methoxyazetidin-1-yl)benz-
onitrile
[0525]
6-chloro-3-ethoxy-4-(1-hydroxyethyl)-2-(3-methoxyazetidin-1-yl)benz-
onitrile (30 mg, 0.1 mmol) (racemic mixture) was dissolved in
methylene chloride (5 mL) and N,N-dimethylformamide (100 .mu.L, 1
mmol). Thionyl chloride (18 .mu.L, 0.24 mmol) was added dropwise at
room temperature and the reaction was stirred for 2 hrs. The
reaction was diluted with EtOAc, washed with water and saturated
NaHCO.sub.3, brine, dried over Na.sub.2SO.sub.4, filtered and
concentrated to give the crude
6-chloro-4-(1-chloroethyl)-3-ethoxy-2-(3-methoxyazetidin-1-yl)benzonitril-
e (0.030 g, 100%). LCMS calculated for
C.sub.15H.sub.19Cl.sub.2N.sub.2O.sub.3 (M+H).sup.+: m/z=329.1;
found: 329.1.
Step 4.
4-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-6-c-
hloro-3-ethoxy-2-(3-methoxyazetidin-1-yl)benzonitrile
[0526] Cesium carbonate (50 mg, 0.2 mmol) was added to a mixture of
3-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (16 mg, 0.10 mmol) and
6-chloro-4-(1-chloroethyl)-3-ethoxy-2-(3-methoxyazetidin-1-yl)benzonitril-
e (30 mg, 0.09 mmol) in N,N-dimethylformamide (3 mL, 40 mmol) and
the reaction was stirred at 80.degree. C. overnight. The mixture
was diluted with EtOAc, washed with water, brine, dried over
Na.sub.2SO.sub.4, filtered and concentrated the crude product. The
product was purified was purified by prep HPLC on a C-18 column
eluting water: acetonitrile gradient buffered pH 10 to give the
title compound as a white amorphous solid (0.007 g, 20%). The
product was isolated as a racemic mixture. LCMS calculated for
C.sub.21H.sub.25ClN.sub.7O.sub.2(M+H).sup.+: m/z=442.1; found:
442.1. H NMR (400 MHz, DMSO-d.sub.6) .delta. 8.11 (s, 1H), 6.80 (s,
1H), 6.18 (d, J=7.1 Hz, 1H), 4.58-4.44 (m, 2H), 4.18 (m, 1H),
4.13-4.01 (m, 2H), 3.81-3.62 (m, 2H), 3.23 (s, 3H), 2.55 (s, 3H),
1.69 (d, J=7.1 Hz, 3H), 1.35 (t, J=7.0 Hz, 3H).
Example 320.
4-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-3-ethoxy-2-
-(1-isopropylazetidin-3-yl)-6-methylbenzonitrile
##STR00121##
[0527] Step 1:
4-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-2-azetidin-
-3-yl-3-ethoxy-6-methylbenzonitrile bis(trifluoroacetate)
[0528] Using methods described in Example 315 but using ethyl
iodide in Step 3, instead of methyl iodide, the intermediate
4-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-2-azetidin-
-3-yl-3-ethoxy-6-methylbenzonitrile bis(trifluoroacetate) was
prepared. LCMS calculated for C.sub.21H.sub.26N.sub.7O (M+H).sup.+:
m/z=392.2; found: 392.2.
Step 2.
4-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-3-e-
thoxy-2-(1-isopropylazetidin-3-yl)-6-methylbenzonitrile
[0529] To a mixture of
4-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-2-azetidin-
-3-yl-3-ethoxy-6-methylbenzonitrile (70 mg, 0.2 mmol) in methanol
(50 mL) was added acetone (0.1 mL, 2 mmol) and sodium
cyanoborohydride (17 mg, 0.27 mmol). The reaction was stirred at
room temperature for 1 h, and was complete by LC/MS. The reaction
was quenched with water and was extracted with EtOAc. The combined
organic layer was washed with brine, dried over MgSO.sub.4,
filtered and concentrated to give the crude product. The product
was purified by prep HPLC on a C-18 column eluting water:
acetonitrile gradient buffered pH 10 to give the title compound as
a white amorphous solid (0.030 g, 40%). The product was isolated as
a racemic mixture. LCMS calculated for C.sub.24H.sub.32N.sub.7O
(M+H).sup.+: m/z=434.2; found: 434.3. .sup.1H NMR (300 MHz,
CD.sub.3OD) .delta. 8.17 (s, 1H), 7.35 (s, 1H), 6.37 (q, J=7.1 Hz,
1H), 4.17-3.98 (m, 4H), 3.90-3.71 (m, 3H), 2.65 (s, 3H), 2.46 (s,
4H), 1.84 (d, J=7.1 Hz, 3H), 1.42 (t, J=7.0 Hz, 3H), 1.03 (dd,
J=6.2, 1.4 Hz, 6H).
Example 321.
4-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-3-ethoxy-2-
-[1-(2-hydroxy-2-methylpropyl)azetidin-3-yl]-6-methylbenzonitrile
##STR00122##
[0531] The
4-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]--
2-azetidin-3-yl-3-ethoxy-6-methylbenzonitrile (0.055 g, 0.14 mmol,
chiral intermediate from Example 320, Step 1) was combined with
tetrahydrofuran (22 mL), DIPEA (0.049 mL, 0.28 mmol) and oxirane,
2,2-dimethyl- (0.018 mL, 0.21 mmol) at room temperature. The
reaction was heated to 95.degree. C. and allowed to stir overnight.
The reaction was allowed to cool to room temperature and was
purified without workup by prep HPLC on a C-18 column eluting
water: acetonitrile gradient buffered pH 10 to give the title
compound as a white amorphous solid (0.035 g, 50%). The product was
isolated as a single enantiomer. LCMS calculated for
C.sub.25H.sub.34N.sub.7O.sub.2 (M+H).sup.+: m/z=464.3; found:
464.3. .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 8.09 (s, 1H),
7.23 (s, 1H), 6.21 (q, J=6.8 Hz, 1H), 4.00 (m, 4H), 3.81-3.54 (m,
2H), 3.15 (m, 2H), 2.53 (s, 3H), 2.33 (s, 3H), 2.27 (bs, 2H), 1.70
(d, J=7.1 Hz, 3H), 1.30 (t, J=6.9 Hz, 3H), 1.04 (s, 6H).
Example 322.
4-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-3-ethoxy-2-
-[1-(2-hydroxy-2-methylpropanoyl)azetidin-3-yl]-6-methylbenzonitrile
##STR00123##
[0533] The
4-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]--
2-azetidin-3-yl-3-ethoxy-6-methylbenzonitrile (0.075 g, 0.10 mmol,
chiral intermediate from Example 320, Step 1) was dissolved in
N,N-dimethylformamide (3.0 mL) and DIPEA (0.089 mL, 0.51 mmol) and
the propanoic acid, 2-hydroxy-2-methyl- (0.013 g, 0.12 mmol) and
N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium
hexafluorophosphate (0.058 g, 0.15 mmol) were added. The reaction
was stirred at room temperature for 18 hrs and was complete by
LC/MS. The product was purified without workup by prep HPLC on a
C-18 column eluting water: acetonitrile gradient buffered to pH 10
to give the title compound as a white amorphous solid (0.025 g,
51%). The product was isolated as a single enantiomer. LCMS
calculated for C.sub.25H.sub.32N.sub.7O.sub.3 (M+H).sup.+:
m/z=478.2; found: 478.2. .sup.1H NMR (300 MHz, DMSO-d.sub.6)
.delta. 8.10 (s, 1H), 7.29 (s, 1H), 6.24 (q, J=6.8 Hz, 1H), 5.07
(s, 1H), 4.90-4.75 (m, 1H), 4.73-4.58 (m, 1H), 4.39 (p, J=8.5 Hz,
1H), 4.30-4.05 (m, 2H), 3.75 (d, J=7.1 Hz, 2H), 2.54 (s, 3H), 2.38
(s, 3H), 1.72 (d, J=6.9 Hz, 3H), 1.35 (t, J=6.1 Hz, 3H), 1.26 (s,
3H), 1.23 (s, 3H).
Examples 310 and 311. Diastereoisomers of
4-{3-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chlor-
o-2-ethoxy-6-methylphenyl}pyrrolidin-2-one
##STR00124##
[0534] Step 1.
1-(5-Chloro-2-ethoxy-3-iodo-4-methylphenyl)ethanol
[0535] The desired compound was prepared according to the procedure
of Example 212, step 4 (racemic mixture), using
1-(5-chloro-2-ethoxy-3-iodo-4-methylphenyl)ethanone instead of
tert-butyl
3-(3-acetyl-5-chloro-6-cyano-2-ethoxyphenyl)azetidine-1-carboxylate
as the starting material in 94% yield as a 96:4 mixture of
enantiomers (RT=3.56 min and 4.28 min; Chiral Technologies
ChiralPak AD-H column, 20.times.250 mm, 5 micron particle size,
eluting with 5% ethanol in hexanes at 1 ml/min). LCMS for
C.sub.11H.sub.13ClIO (M-(OH)).sup.+: m/z=323.0; Found: 322.9.
Step 2.
1-[1-(5-Chloro-2-ethoxy-3-iodo-4-methylphenyl)ethyl]-3-methyl-1H-p-
yrazolo[3,4-d]pyrimidin-4-amine
[0536] The desired compound was prepared according to the procedure
of Example 212, step 5, using
1-(5-Chloro-2-ethoxy-3-iodo-4-methylphenyl)ethanol (96:4 mixture
from step 1) instead of tert-butyl
3-[3-chloro-2-cyano-6-ethoxy-5-(1-hydroxyethyl)phenyl]azetidine-1-carboxy-
late as the starting material in 32% yield as a single enantiomer
(peak 1 desired, retention time=3.39 min; ChiralPak IA column,
20.times.250 mm, 5 micron particle size, eluting with 3% ethanol in
hexanes at 18 ml/min). LCMS for C.sub.17H.sub.20ClIN.sub.5O
(M+H).sup.+: m/z=472.0; Found: 472.0.
Step 3. Methyl
(2E)-3-{3-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5--
chloro-2-ethoxy-6-methylphenyl}acrylate
[0537] A suspension of
1-[1-(5-chloro-2-ethoxy-3-iodo-4-methylphenyl)ethyl]-3-methyl-1H-pyrazolo-
[3,4-d]pyrimidin-4-amine (peak 1 single isomer from step 2) (0.61
g, 1.3 mmol) in acetonitrile (7.4 mL) in a sealed tube was degassed
with nitrogen and treated with triphenylphosphine (0.048 g, 0.18
mmol), methyl acrylate (0.41 mL, 4.5 mmol), and palladium acetate
(0.029 g, 0.13 mmol) followed by triethylamine (0.54 mL, 3.9 mmol)
and heated at 100.degree. C. for 16 h. The reaction mixture was
cooled to room temperature, filtered, and the solids washed with
acetonitrile. The filtrate was concentrated to a residue. The crude
material was purified by flash column chromatography using ethyl
acetate (containing 3% methanol) in hexanes (0%-100%) to give the
desired product (0.40 g, 72%). LCMS for
C.sub.21H.sub.25ClN.sub.5O.sub.3 (M+H).sup.+: m/z=430.2; Found:
430.2.
Step 4. Diastereoisomers of methyl
3-{3-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chlor-
o-2-ethoxy-6-methylphenyl}-4-nitrobutanoate
[0538] A solution of methyl
(2E)-3-{3-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5--
chloro-2-ethoxy-6-methylphenyl}acrylate (0.40 g, 0.93 mmol) in
nitromethane (6.3 mL) was treated with
1,8-diazabicyclo[5.4.0]undec-7-ene (0.14 mL, 0.93 mmol) and stirred
at 90.degree. C. for 22 h. The reaction mixture was concentrated,
diluted with methanol, and purified by preparative LCMS (XBridge
C18 Column, eluting with a gradient of acetonitrile in water with
0.1% trifluoroacetic acid, at flow rate of 60 mL/min). The LCMS
fractions were concentrated to remove acetonitrile, treated with
solid sodium bicarbonate, and extracted into ethyl acetate. The
ethyl acetate was concentrated to give the desired product (0.22 g,
48%) as a mixture of diastereoisomers. LCMS for
C.sub.22H.sub.28ClN.sub.6O.sub.5(M+H).sup.+: m/z=491.2; Found:
491.2.
Step 5. Diastereoisomers of
4-{3-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chlor-
o-2-ethoxy-6-methylphenyl}pyrrolidin-2-on
[0539] A solution of methyl
3-{3-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chlor-
o-2-ethoxy-6-methylphenyl}-4-nitrobutanoate (0.089 g, 0.18 mmol) in
methanol (1.3 mL) was treated with nickel chloride hexahydrate
(0.087 g, 0.36 mmol) was and stirred for 5 min. The reaction
mixture was cooled to 0.degree. C., treated with sodium
tetrahydroborate (0.073 g, 1.9 mmol) in four portions, and stirred
at room temperature for 30 min. The reaction mixture was heated at
60.degree. C. for 1.5 h, cooled to room temperature, diluted with
saturated sodium bicarbonate solution (10 mL) and dichloromethane
(25 mL), and filtered through Celite. The Celite was washed with
dichloromethane and the filtrate was transferred to a separatory
funnel. The organic layer was separated, washed with brine, dried
over sodium sulfate, filtered, and concentrated to residue. The
crude residue was diluted with methanol and purified by preparative
LCMS (XBridge C18 column, eluting with a gradient of
acetonitrile/water containing 0.1% ammonium hydroxide, at flow rate
of 60 mL/min) to give the desired peak 1 diastereoisomer (16 mg,
21%) and peak 2 diastereoisomer (19 mg, 24%). Peak 1 (compound
310): .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 8.10 (s, 1H),
7.89 (s, 1H), 7.34 (s, 1H), 6.21 (q, J=7.1 Hz, 1H), 4.38-4.22 (m,
1H), 3.93-3.80 (m, 1H), 3.79-3.67 (m, 1H), 3.65-3.55 (m, 1H),
3.28-3.20 (m, 1H), 2.54 (s, 3H), 2.29 (dd, J=17.5, 8.3 Hz, 1H),
2.21 (s, 3H), 1.70 (d, J=7.0 Hz, 3H), 1.40 (t, J=6.9 Hz, 3H). LCMS
for C.sub.21H.sub.26ClN.sub.6O.sub.2(M+H).sup.+: m/z=429.2; Found:
429.2. Peak 2 (compound 311): H NMR (300 MHz, DMSO-d.sub.6) .delta.
8.11 (s, 1H), 7.89 (s, 1H), 7.33 (s, 1H), 6.20 (q, J=7.1 Hz, 1H),
4.38-4.22 (m, 1H), 3.90-3.68 (m, 2H), 3.65-3.56 (m, 1H), 3.28-3.17
(m, 1H), 2.54 (s, 3H), 2.32 (dd, J=17.3, 8.5 Hz, 1H), 2.21 (s, 3H),
1.69 (d, J=7.0 Hz, 3H), 1.39 (t, J=6.9 Hz, 3H). LCMS for
C.sub.21H.sub.26ClN.sub.6O.sub.2(M+H).sup.+: m/z=429.2; Found:
429.2.
Example 323.
4-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-6-chloro-3-
-ethoxy-2-(2-oxo-1,3-oxazolidin-5-yl)benzonitrile
##STR00125##
[0540] Step 1. 4-Acetyl-6-chloro-3-ethoxy-2-vinylbenzonitrile
[0541] A mixture of 4-acetyl-6-chloro-3-ethoxy-2-iodobenzonitrile
(1.3 g, 3.6 mmol), 4,4,5,5-tetramethyl-2-vinyl-1,3,2-dioxaborolane
(740 .mu.L, 4.3 mmol),
[1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II),
complex with dichloromethane (1:1) (100 mg, 0.20 mmol) and
potassium carbonate (1.5 g, 11 mmol) in 1,4-dioxane (20 mL) and
water (10 mL) was heated at 80.degree. C. overnight. The mixture
was cooled to room temperature and extracted with ethyl acetate.
The extracts were washed with brine, dried over sodium sulfate,
filtered and concentrated. Purification on silica gel using ethyl
acetate in hexanes (0-20%) gave the desired compound, 780 mg, 87%.
LCMS calculated for Cl.sub.3H.sub.13ClNO.sub.2 (M+H).sup.+:
m/z=250.1; found: 250.1. .sup.1H NMR (400 MHz, DMSO-d.sub.6):
.delta. 7.78 (s, 1H), 6.83 (m, 1H), 6.10 (m, 1H), 5.83 (m, 1H),
3.84 (m, 2H), 2.58 (s, 3H), 1.22 (m, 3H).
Step 2. tert-Butyl
[2-(3-acetyl-5-chloro-6-cyano-2-ethoxyphenyl)-2-hydroxyethyl]carbamate
[0542] 0.2 M Osmium tetraoxide in water (0.5 mL) was added to a
solution of tert-butyl [(4-chlorobenzoyl)oxy]carbamate (Ref.
Lawrence Harris, J. Org. Chem, 2011, 76, 358-372). (0.91 g, 3.3
mmol) in acetonitrile (10 mL) and stirred for 10 minutes.
4-Acetyl-6-chloro-3-ethoxy-2-vinylbenzonitrile (0.56 g, 2.2 mmol)
as a solution in acetonitrile (10 mL) was added to the carbamate
solution followed by the addition of water (2 mL) and the reaction
was stirred for 3 hours at room temperature. The reaction was
quenched with saturated 10 M dipotassium disulfite in water (12 mL)
and stirred for 5 minutes. Water was added and the reaction mixture
was extracted with ethyl acetate. The extracts were washed with
saturated sodium bicarbonate solution, brine and dried over sodium
sulfate, filtered and evaporated. Purification on silica gel using
ethyl acetate in hexane (0-100%) gave the desired compound as a
racemic mixture, 610 mg, 72%. LCMS calculated for
C.sub.18H.sub.24ClN.sub.2O.sub.5(M+H).sup.+: m/z=383.1; found:
383.1. .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta. 7.62 (s, 1H),
7.03 (br s, 1H), 5.68 (br s, 1H), 3.96 (m, 1H), 3.69 (m, 1H), 3.31
(m, 1H), 3.19 (m, 1H), 2.60 (s, 3H), 1.30 (m, 12H).
Step 3.
4-Acetyl-6-chloro-3-ethoxy-2-(2-oxo-1,3-oxazolidin-5-yl)benzonitri-
le
[0543] tert-Butyl
[2-(3-acetyl-5-chloro-6-cyano-2-ethoxyphenyl)-2-hydroxyethyl]carbamate
(290 mg, 0.76 mmol) (racemic mixture from step 2) was treated with
4.0 M hydrogen chloride in 1,4-dioxane (6.1 mL) for 15 minutes and
the mixture was evaporated. The residue was dissolved in
tetrahydrofuran (2.3 mL) and N,N-diisopropylethylamine (0.66 mL,
3.8 mmol). N,N-carbonyldiimidazole (250 mg, 1.5 mmol) was added and
the reaction mixture was refluxed at 70.degree. C. overnight. The
reaction mixture was evaporated. Purification on silica gel using
ethyl acetate in hexane (0-100%) gave the desired compound as a
racemic mixture, 110 mg, 47%. LCMS calculated for
C.sub.14H.sub.14ClN.sub.2O.sub.4(M+H).sup.+: m/z=309.1; found:
309.1. .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta. 8.00 (br s,
1H), 7.93 (s, 1H), 5.99 (m, 1H), 3.89 (m, 1H), 3.81 (m, 2H), 3.52
(m, 1H), 2.58 (s, 3H), 1.23 (m, 3H).
Step 4.
6-Chloro-3-ethoxy-4-(1-hydroxyethyl)-2-(2-oxo-1,3-oxazolidin-5-yl)-
benzonitrile
[0544] Sodium tetrahydroborate (19 mg, 0.50 mmol) was added to a
mixture of
4-acetyl-6-chloro-3-ethoxy-2-(2-oxo-1,3-oxazolidin-5-yl)benzonitrile
(100 mg, 0.34 mmol) (racemic mixture from step 3) in methanol (1.6
mL, 38 mmol) at 0.degree. C. and the reaction mixture was stirred
at room temperature for 10 minutes and evaporated. The residue was
diluted with ethyl acetate, washed with 1 N HCl, brine, dried over
sodium sulfate, filtered and concentrated to give the desired
compound as a mixture of four diastereomers, 58 mg, 55%. LCMS
calculated for C.sub.14H.sub.16ClN.sub.2O.sub.4(M+H).sup.+:
m/z=311.1; found: 311.1.
Step 5.
6-Chloro-4-(1-chloroethyl)-3-ethoxy-2-(2-oxo-1,3-oxazolidin-5-yl)b-
enzonitrile
[0545] To a mixture of
6-chloro-3-ethoxy-4-(1-hydroxyethyl)-2-(2-oxo-1,3-oxazolidin-5-yl)benzoni-
trile (58 mg, 0.19 mmol) (mixture of four diastereomers from step
4), N,N-dimethylformamide (36 .mu.L) in methylene chloride (1 mL),
thionyl chloride (40 .mu.L, 0.56 mmol) was added and the mixture
was stirred at room temperature for 20 minutes The mixture was
diluted with methylene chloride, washed with saturated sodium
bicarbonate, water, brine, dried over sodium sulfate, filtered and
concentrated to give the desired compound as a mixture of four
diastereomers, 55 mg, 91%. LCMS calculated for
C.sub.14H.sub.15Cl.sub.2N.sub.2O.sub.3 (M+H).sup.+: m/z=329.0;
found: 329.1.
Step 6.
4-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-6-c-
hloro-3-ethoxy-2-(2-oxo-1,3-oxazolidin-5-yl)benzonitrile
[0546] Cesium Carbonate (0.11 g, 0.34 mmol) was added to a mixture
of 3-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (30 mg, 0.20 mmol)
(mixture of four diastereomers from step 5) in
N,N-dimethylformamide (0.91 mL) and stirred for 10 minutes. To the
mixture was added
6-chloro-4-(1-chloroethyl)-3-ethoxy-2-(2-oxo-1,3-oxazolidin-5-yl)benzonit-
rile (56 mg, 0.17 mmol) in N,N-dimethylformamide (1.0 mL) and the
reaction was stirred at 90.degree. C. for 1 hour. Purification by
preparative LCMS (pH 10) using RP-HPLC (XBridge C18 column, eluting
with a gradient of acetonitrile/water containing 0.1% ammonium
hydroxide, at flow rate of 30 mL/min) gave the desired compounds as
Peak 1 (racemic mixture of two diastereomers) LCMS calculated for
C.sub.20H.sub.21ClN.sub.7O.sub.3(M+H).sup.+: m/z=442.1; found:
442.1. .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta. 8.17 (s, 1H),
8.00 (br s, 1H), 7.79 (s, 1H), 6.25 (m, 1H), 5.92 (m, 1H), 3.90 (m,
3H), 3.57 (m, 1H), 2.58 (s, 3H), 1.75 (m, 3H), 1.40 (m, 3H); Peak 2
(racemic mixture of 2 diastereomers): LCMS calculated for
C.sub.20H.sub.21ClN.sub.7O.sub.3(M+H).sup.+: m/z=442.1; found:
442.1. .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta. 8.12 (s, 1H),
8.00 (br s, 1H), 7.71 (s, 1H), 6.23 (m, 1H), 5.96 (m, 1H), 3.85 (m,
3H), 3.58 (m, 1H), 2.58 (s, 3H), 1.75 (m, 3H), 1.40 (m, 3H).
[0547] Chiral purification of Peak 2 (racemic mixture of two
diastereomers) on Phenomenex Lux Cellulose-1, 21.2.times.250 mm, 5
micron particle size at 18 mL/min using 20% ethanol in hexanes gave
Peak 3 and Peak 4. Peak 3, retention time=12.22 minutes (single
enantiomer): LCMS calculated for
C.sub.20H.sub.21ClN.sub.7O.sub.3(M+H).sup.+: m/z=442.1; found:
442.1. .sup.1H NMR (400 MHz, DMSO-d.sub.6): .delta. 8.12 (s, 1H),
7.98 (br s, 1H), 7.71 (s, 1H), 6.23 (m, 1H), 5.96 (m, 1H), 3.85 (m,
3H), 3.58 (m, 1H), 2.58 (s, 3H), 1.75 (m, 3H), 1.40 (m, 3H). Peak
4, retention time=16.25 minutes (single enantiomer). LCMS
calculated for C.sub.20H.sub.21ClN.sub.7O.sub.3(M+H).sup.+:
m/z=442.1; found: 442.1. .sup.1H NMR (400 MHz, DMSO-d.sub.6):
.delta. 8.12 (s, 1H), 7.98 (br s, 1H), 7.71 (s, 1H), 6.23 (m, 1H),
5.96 (m, 1H), 3.85 (m, 3H), 3.58 (m, 1H), 2.58 (s, 3H), 1.75 (m,
3H), 1.40 (m, 3H).
Example 324.
6-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chlor-
o-2-methoxy-6-methylphenyl}morpholin-3-one
##STR00126##
[0548] Step 1.
1-(5-Chloro-2-methoxy-4-methyl-3-vinylphenyl)ethanone
[0549] A mixture of
1-(3-bromo-5-chloro-2-methoxy-4-methylphenyl)ethanone (2.6 g, 9.5
mmol), 4,4,5,5-tetramethyl-2-vinyl-1,3,2-dioxaborolane (1.9 mL, 11
mmol), [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II),
complex with dichloromethane (1:1) (400 mg, 0.5 mmol) and potassium
carbonate (4.0 g, 29 mmol) in 1,4-dioxane (60 mL), and water (30
mL). The resulting mixture was heated at 80.degree. C. for 3 hours.
The mixture was cooled to room temperature and extracted with ethyl
acetate. Purification on a silica gel using ethyl acetate in
hexanes (0-20%) gave the desired compound, 2.0 g, 94%. LCMS
calculated for C.sub.12H.sub.14ClO.sub.2 (M+H).sup.+: m/z=225.1;
found: 225.1.
Step 2. tert-Butyl
[2-(3-acetyl-5-chloro-2-methoxy-6-methylphenyl)-2-hydroxyethyl]carbamate
[0550] 0.2 M Osmium tetraoxide in water (1 mL) was added to a
solution of tert-butyl [(4-chlorobenzoyl)oxy]carbamate (2.0 g, 7.2
mmol) (Ref. Lawrence Harris, J. Org. Chem, 2011, 76, 358-372) in
acetonitrile (22 mL) and stirred for 10 minutes.
1-(5-Chloro-2-methoxy-4-methyl-3-vinylphenyl)ethanone (1.1 g, 4.8
mmol) as a solution in acetonitrile (22 mL) was added to the
carbamate solution followed by the addition of water (5 mL). The
reaction was stirred for 3 hours at room temperature. The reaction
was quenched with saturated 10 M dipotassium disulfite in water (25
mL) and stirred for 5 minutes. Water was added to the reaction and
the mixture was extracted with ethyl acetate. The organic extracts
were washed with saturated sodium bicarbonate solution, brine,
dried over sodium sulfate and evaporated under reduced pressure.
Purification on silica gel using ethyl acetate in hexane (0-100%)
gave the desired compound as a racemic mixture, 1.2 g, 69%. LCMS
calculated for C.sub.17H.sub.24ClNO.sub.5Na (M+Na).sup.+:
m/z=380.1; found: 380.1. .sup.1H NMR (500 MHz, DMSO-d.sub.6):
.delta. 7.48 (s, 1H), 6.80 (m, 1H), 5.50 (br s, 1H), 5.20 (br s,
1H), 3.83 (s, 3H), 3.32 (m, 1H), 3.22 (m, 1H), 2.59 (s, 3H), 2.55
(s, 3H), 1.32 (s, 9H).
[0551] Chiral purification on ChiralPak AD-H, 20.times.250 mm
(Chiral Technologies), 5 micron particle size, at flow rate of 18
mL/min using 8% ethanol in hexanes gave the Peak 1 (single
enantiomer) (retention time=9.86 minutes) and Peak 2 (single
enantiomer) (retention time=11.47 minutes).
Step 3.
N-[2-(3-Acetyl-5-chloro-2-methoxy-6-methylphenyl)-2-hydroxyethyl]--
2-chloroacetamide
[0552] tert-Butyl
[2-(3-acetyl-5-chloro-2-methoxy-6-methylphenyl)-2-hydroxyethyl]carbamate
(170 mg, 0.47 mmol) (Peak 1 from step 2) was treated with 4.0 M
hydrogen chloride in 1,4-dioxane (12 mL) for 15 minutes. The
solvents were evaporated, methylene chloride (6 mL) and
triethylamine (200 .mu.L, 1.4 mmol) were added and the mixture
cooled to 0.degree. C. Chloroacetyl chloride (45 .mu.L, 0.56 mmol)
was added slowly and was stirred for 10 minutes at 0.degree. C. The
solvents were evaporated to dryness. Water was added and the
mixture was extracted with ethyl acetate. The combined extracts
were washed with brine, dried over sodium sulfate, and concentrated
to give the crude residue as a single enantiomer. LCMS calculated
for Cl.sub.4H.sub.17Cl.sub.2NO.sub.4Na (M+Na).sup.+: m/z=356.1;
found: 356.1.
Step 4. 6-(3-Acetyl-5-chloro-2-methoxy-6-methylphenyl)
morpholin-3-one
[0553] To a solution of
N-[2-(3-acetyl-5-chloro-2-methoxy-6-methylphenyl)-2-hydroxyethyl]-2-chlor-
oacetamide (170 mg, 0.50 mmol) (single enantiomer from step 3) in
tetrahydrofuran (4 mL) cooled at 0.degree. C., a mixture of sodium
hydride (60% dispersion in mineral oil; 39 mg, 1.0 mmol) was added
and stirred for 1 hour. The reaction was quenched with water and
extracted with ethyl acetate. The combined extracts were washed
with brine, dried over sodium sulfate, and concentrated to give the
crude residue as a single enantiomer, 61 mg, 41%. LCMS calculated
for C.sub.14H.sub.17ClNO.sub.4 (M+H).sup.+: m/z=298.1; found:
298.1.
Step 5.
6-[3-Chloro-5-(1-hydroxyethyl)-6-methoxy-2-methylphenyl]morpholin--
3-one
[0554] To a solution of
6-(3-acetyl-5-chloro-2-methoxy-6-methylphenyl)morpholin-3-one (27
mg, 0.090 mmol) (single enantiomer from step 4) in methanol (2 mL)
was added sodium tetrahydroborate (6.8 mg, 0.18 mmol) at 0.degree.
C. and stirred for 1 hour. Purification by preparative LCMS (pH 10)
gave the desired compound as a racemic mixture of two
diastereomers, 20 mg, 76%. LCMS calculated for
C.sub.14H.sub.17ClNO.sub.3 (M-OH).sup.+: m/z=282.1; found:
282.1.
Step 6.
6-[3-Chloro-5-(1-chloroethyl)-6-methoxy-2-methylphenyl]morpholin-3-
-one
[0555] A mixture of thionyl chloride (15 .mu.L, 0.21 mmol) and
N,N-dimethylformamide (10.0 .mu.L) was stirred at room temperature
for 10 minutes. A solution of
6-[3-chloro-5-(1-hydroxyethyl)-6-methoxy-2-methylphenyl]morpholin-3-one
(19.0 mg, 0.0634 mmol) (racemic mixture of two diastereomers from
step 5) in methylene chloride (1.0 mL) was added and the mixture
was stirred at room temperature overnight. The mixture was diluted
with methylene chloride, washed with saturated sodium bicarbonate,
water, brine, dried over sodium sulfate, filtered and concentrated
to give the desired compound as a racemic mixture of two
diastereomers, 19 mg, 94%. LCMS calculated for
C.sub.14H.sub.17ClNO.sub.3 (M-C1).sup.+: m/z=282.1; found:
282.1.
Step 7.
6-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]--
5-chloro-2-methoxy-6-methylphenyl}morpholin-3-one
[0556] A mix of
6-[3-chloro-5-(1-chloroethyl)-6-methoxy-2-methylphenyl]morpholin-3-one
(19.0 mg, 0.0597 mmol) (racemic mixture of two diastereomers from
step 6) 3-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (11 mg, 0.072
mmol), cesium carbonate (29 mg, 0.090 mmol) and potassium iodide
(0.99 mg, 0.006 mmol) in N,N-dimethylformamide (0.19 mL) was heated
at 140.degree. C. for 1 hour. The mixture was diluted with ether,
washed with water, concentrated and purified by preparative LCMS
(pH 10) using RP-HPLC (XBridge C18 column, eluting with a gradient
of acetonitrile/water containing 0.1% ammonium hydroxide, at flow
rate of 30 mL/min) to give 2.5 mg, 10% of Peak 1 (single
enantiomer, retention time 10.15 min): LCMS calculated for
C.sub.20H.sub.24ClN.sub.6O.sub.3(M+H).sup.+: m/z=431.2; found:
431.1, and 2.7 mg, 10% of Peak 2 (single enantiomer, retention time
10.76 min): LCMS calculated for
C.sub.20H.sub.24ClN.sub.6O.sub.3(M+H).sup.+: m/z=431.2; found:
431.1.
Example 325.
5-{3-[1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chlor-
o-2-methoxy-6-methylphenyl}-1,3-oxazolidin-2-one
##STR00127##
[0557] Step 1.
5-(3-Acetyl-5-chloro-2-methoxy-6-methylphenyl)-1,3-oxazolidin-2-one
[0558] To a solution of tert-butyl
[2-(3-acetyl-5-chloro-2-methoxy-6-methylphenyl)-2-hydroxyethyl]carbamate
(140 mg, 0.40 mmol) (Peak 1, single enantiomer from step 2, Example
324) in tetrahydrofuran (2.5 mL), N,N-diisopropylethylamine (0.35
mL, 2.0 mmol) and N,N-carbonyldiimidazole (130 mg, 0.80 mmol). The
reaction was refluxed at 70.degree. C. for 10 minutes. The reaction
was evaporated to dryness. Purification on silica gel using (0-50%)
ethyl acetate in hexane gave the desired compound as a single
enantiomer, 78 mg, 69%. LCMS calculated for
C.sub.13H.sub.15ClNO.sub.4 (M+H).sup.+: m/z=284.1; found:
284.1.
Step 2.
5-[3-Chloro-5-(1-hydroxyethyl)-6-methoxy-2-methylphenyl]-1,3-oxazo-
lidin-2-one
[0559] To a solution of
5-(3-acetyl-5-chloro-2-methoxy-6-methylphenyl)-1,3-oxazolidin-2-one
(21 mg, 0.072 mmol) (single enantiomer from step 1) in methanol (1
mL) was added sodium tetrahydroborate (5.5 mg, 0.14 mmol) at
0.degree. C. The mixture was stirred at 0.degree. C. for 1 hour. It
was diluted with methanol and purified on preparative LCMS using pH
10 buffer to give the desired compound as a racemic mixture of two
diastereomers, 17 mg, 83%. LCMS calculated for
C.sub.13H.sub.15ClNO.sub.3 (M-OH).sup.+: m/z=268.1; found:
268.1.
Step 3.
5-[3-Chloro-5-(1-chloroethyl)-6-methoxy-2-methylphenyl]-1,3-oxazol-
idin-2-one
[0560] A mixture of cyanuric chloride (16 mg, 0.084 mmol) and
N,N-dimethylformamide (15 .mu.L) was stirred at room temperature
for 10 minutes. A solution of
5-[3-chloro-5-(1-hydroxyethyl)-6-methoxy-2-methylphenyl]-1,3-oxazolidin-2-
-one (16 mg, 0.056 mmol) (racemic mixture of two diastereomers from
step 2) in methylene chloride (0.3 mL) was added and the reaction
was stirred at room temperature overnight. Thionyl chloride (12
.mu.L, 0.17 mmol) was added and stirred for 10 min. The mixture was
diluted with methylene chloride, washed with saturated sodium
bicarbonate, water, brine, dried over sodium sulfate, filtered and
concentrated to give the desired compound as a racemic mixture of
two diastereomers, 17 mg, 100%. LCMS calculated for
C.sub.13H.sub.16Cl.sub.2NO.sub.3 (M+H).sup.+: m/z=304.0; found:
304.1.
Step 4.
5-{3-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]--
5-chloro-2-methoxy-6-methylphenyl}-1,3-oxazolidin-2-one
[0561] A mixture of
5-[3-chloro-5-(1-chloroethyl)-6-methoxy-2-methylphenyl]-1,3-oxazolidin-2--
one (17 mg, 0.056 mmol) (racemic mixture of two diastereomers from
step 3) 3-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (10 mg, 0.067
mmol), cesium carbonate (27 mg, 0.084 mmol) and potassium iodide
(0.93 mg, 0.0056 mmol) in N,N-dimethylformamide (0.18 mL) was
heated at 140.degree. C. for 1 hour. The mixture was diluted with
ether, washed with water, concentrated and purified by preparative
LCMS (pH 10) to give the desired compound as a racemic mixture of
two diastereomers, 2.2 mg, 9%; LCMS calculated for
C.sub.19H.sub.22ClN.sub.6O.sub.3(M+H).sup.+: m/z=417.1; found:
417.1.
Examples 345-348. Diastereoisomers of
4-{3-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chlor-
o-2-ethoxy-6-fluorophenyl}pyrrolidin-2-one
##STR00128##
[0562] Step 1.
1-(5-Chloro-2-ethoxy-3-iodo-4-methylphenyl)ethanol
##STR00129##
[0564] A solution of
1-(5-chloro-2-ethoxy-4-fluoro-3-iodophenyl)ethanone (20.0 g, 58.4
mmol; Example 212, step 1) and 1,2-ethanediol (6.5 mL, 120 mmol) in
toluene (190 mL) was treated with p-toluenesulfonic acid
monohydrate (1.1 g, 5.8 mmol). The flask was fitted with a
Dean-Stark trap that was filled with sieves, and refluxed for 3 h.
The reaction mixture was cooled and added to ice cooled saturated
sodium bicarbonate solution (250 mL) and extracted with ethyl
acetate. The organic layer was washed with brine, dried over sodium
sulfate, filtered, and concentrated to a crude orange oil. The
crude material was purified by flash column chromatography using
ethyl acetate in hexanes (0%-20%) to give the desired product (22
g, 99%). LCMS for C.sub.12H.sub.14ClFIO.sub.3 (M+H).sup.+:
m/z=387.0; Found: 386.9.
Step 2. Ethyl
(2E)-3-[3-chloro-6-ethoxy-2-fluoro-5-(2-methyl-1,3-dioxolan-2-yl)phenyl]a-
crylate
##STR00130##
[0566] A mixture of
2-(5-chloro-2-ethoxy-4-fluoro-3-iodophenyl)-2-methyl-1,3-dioxolane
(22 g, 58 mmol) (from Step 1), ethyl
(2E)-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)acrylate (16
mL, 70 mmol), and potassium carbonate (24 g, 170 mmol) in
1,4-dioxane (230 mL) and water (110 mL) was degassed with nitrogen
for 10 min. The reaction mixture was treated with [1,1'-bis
(diphenylphosphino)ferrocene]dichloropalladium(II), complex with
dichloromethane (1:1) (2.4 g, 2.9 mmol), degassed with nitrogen for
another 10 min, and heated at 80.degree. C. for 2 h. The reaction
mixture was filtered through Celite and washed with ethyl acetate
(300 mL). The filtrate was poured into water (400 mL). The aqueous
layer was separated and extracted with additional ethyl acetate
(300 mL). The combined organic extracts were washed with brine,
dried over sodium sulfate, filtered, and concentrated to a crude
brown solid. The crude material was purified by flash column
chromatography using ethyl acetate in hexanes (0%-30%) to give the
desired product (20 g, 96%). .sup.1H NMR (400 MHz, CDCl.sub.3)
.delta. 7.74 (d, J=16.5 Hz, 1H), 7.56 (d, J=8.6 Hz, 1H), 6.70 (dd,
J=16.5, 0.9 Hz, 1H), 4.26 (q, J=7.1 Hz, 2H), 4.10-3.99 (m, 2H),
3.91 (q, J=7.0 Hz, 2H), 3.87-3.76 (m, 2H), 1.73 (s, 3H), 1.44 (t,
J=7.0 Hz, 3H), 1.33 (t, J=7.1 Hz, 3H). LCMS for
C.sub.17H.sub.21ClFO.sub.5 (M+H).sup.+: m/z=359.1; Found:
359.1.
Step 3. Ethyl
3-[3-chloro-6-ethoxy-2-fluoro-5-(2-methyl-1,3-dioxolan-2-yl)phenyl]-4-nit-
robutanoate
##STR00131##
[0568] A solution ethyl
(2E)-3-[3-chloro-6-ethoxy-2-fluoro-5-(2-methyl-1,3-dioxolan-2-yl)phenyl]a-
crylate (10 g, 28 mmol) (from Step 2) in nitromethane (100 mL) was
treated with 1,8-diazabicyclo[5.4.0]undec-7-ene (4.6 mL, 31 mmol)
and stirred at 60.degree. C. for 15 h. The reaction mixture was
poured into water (400 mL) and extracted with ethyl acetate
(2.times.300 mL). The combined organic extracts were washed with
brine, dried over sodium sulfate, filtered, and concentrated to a
crude orange oil. The crude material was purified by flash column
chromatography using ethyl acetate in hexanes (0%-30%) to give the
desired product as a mixture of enantiomers (10.4 g, 89%). .sup.1H
NMR (400 MHz, CDCl.sub.3) .delta. 7.52 (d, J=9.1 Hz, 1H), 4.82
(ddd, J=12.5, 7.6, 1.4 Hz, 1H), 4.68 (dd, J=12.5, 7.2 Hz, 1H),
4.54-4.40 (m, 1H), 4.15-3.90 (m, 6H), 3.89-3.75 (m, 2H), 2.85 (ddd,
J=16.0, 8.6, 1.4 Hz, 1H), 2.73 (dd, J=16.1, 6.2 Hz, 1H), 1.70 (s,
3H), 1.47 (t, J=7.0 Hz, 3H), 1.21 (t, J=7.1 Hz, 3H). LCMS for
C.sub.18H.sub.24ClFNO.sub.7 (M+H).sup.+: m/z=420.1; Found:
420.1.
Step 4. Enantiomers
4-[3-chloro-6-ethoxy-2-fluoro-5-(2-methyl-1,3-dioxolan-2-yl)phenyl]pyrrol-
idin-2-one
##STR00132##
[0570] A suspension of ethyl
3-[3-chloro-6-ethoxy-2-fluoro-5-(2-methyl-1,3-dioxolan-2-yl)phenyl]-4-nit-
robutanoate (1.0 g, 2.4 mmol) (from Step 3) in ethanol (16 mL) was
warmed to dissolve the solid. The solution was cooled back to
ambient temperature, degassed with nitrogen, and treated with a
slurry of 2800 Raney Nickel in water (1.5 mL). The reaction mixture
was degassed again with nitrogen and hydrogenated with a balloon of
hydrogen for 3 h. The reaction mixture was filtered through Celite
and concentrated to give the intermediate amino ester (0.93 g,
100%). The intermediate amino ester was dissolved in toluene (12
mL) and heated at 110.degree. C. for 12 h. The reaction mixture was
cooled to ambient temperature, at which point a solid precipitated
from solution. This mixture was cooled to 0.degree. C., stirred for
30 min, filtered, washed with cold toluene, and dried to give the
desired product as a mixture of enantiomers (0.61 g, 75%). LCMS for
C.sub.16H.sub.20ClFNO.sub.4 (M+H).sup.+: m/z=344.1; Found: 344.1.
The mixture of enantiomers was separated by chiral HPLC to give the
individual enantiomers as peak 1 and peak 2 (RT=5.39 min and 7.01
min, respectively; Phenomenex Lux Cellulose C-1, 21.2.times.250 mm,
5 micron particle size, eluting with 20% ethanol in hexanes at 18
mL/min).
Step 5. Enantiomers of
4-(3-acetyl-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one
##STR00133##
[0572] The separated enantiomers from step 4 were each processed
individually to the final compounds. A solution of
4-[3-chloro-6-ethoxy-2-fluoro-5-(2-methyl-1,3-dioxolan-2-yl)phenyl]pyrrol-
idin-2-one (1.7 g, 5.0 mmol) (from Step 4) in methanol (17 mL) was
treated with 6.0 M hydrogen chloride in water (11 mL, 69 mmol)
dropwise and stirred 20.degree. C. for 30 min. The reaction mixture
was added dropwise to ice cooled saturated sodium bicarbonate
solution (75 ml) and extracted with ethyl acetate (2.times.100 ml).
The combined organic extracts were washed with brine, dried over
sodium sulfate, filtered, and concentrated to give the desired
products [from peak 1 (1.5 g, 99%); from peak 2 (1.5 g, 99%)] that
were used without further purification. From peak 1: .sup.1H NMR
(400 MHz, DMSO-d.sub.6) .delta. 7.84 (s, 1H), 7.70 (d, J=8.6 Hz,
1H), 4.16-3.99 (m, 1H), 3.83 (q, J=7.0 Hz, 2H), 3.65-3.54 (m, 1H),
3.30-3.23 (m, 1H), 2.55 (s, 3H), 2.33 (dd, J=16.8, 8.4 Hz, 1H),
1.30 (t, J=7.0 Hz, 3H). LCMS for C.sub.14H.sub.16ClFNO.sub.3
(M+H).sup.+: m/z=300.1; Found: 300.0. From peak 2: .sup.1H NMR (400
MHz, DMSO-d.sub.6) .delta. 7.84 (s, 1H), 7.70 (d, J=8.6 Hz, 1H),
4.13-4.00 (m, 1H), 3.87-3.77 (m, 2H), 3.65-3.55 (m, 1H), 3.31-3.23
(m, 1H), 2.55 (s, 3H), 2.32 (ddd, J=16.9, 8.4, 1.6 Hz, 1H), 1.30
(t, J=7.0 Hz, 3H). LCMS for C.sub.14H.sub.16ClFNO.sub.3
(M+H).sup.+: m/z=300.1; Found: 300.1.
Step 6. Diastereoisomers of
4-[3-chloro-6-ethoxy-2-fluoro-5-(1-hydroxyethyl)phenyl]pyrrolidin-2-one
##STR00134##
[0574] The enantiomers from step 5 were each processed individually
to the final products. A solution of
4-(3-acetyl-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one
(0.402 g, 1.34 mmol) (from Step 5) in anhydrous methanol (6.7 mL)
under an atmosphere of nitrogen at 0.degree. C. was treated with
sodium tetrahydroborate (0.10 g, 2.7 mmol) and stirred at 0.degree.
C. for 30 min. The reaction mixture was quenched with water at
0.degree. C. and poured into water (50 mL)/ethyl acetate (100 mL)
while stirring. The mixture was warmed to ambient temperature and
the aqueous layer was separated and extracted with additional ethyl
acetate (50 mL). The combined organic extracts were washed with
brine, dried over sodium sulfate, filtered, and concentrated to
give white foams. The crude material were purified by flash column
chromatography using acetonitrile (containing 7% methanol) in
dichloromethane (0%-100%) to give the desired products as mixtures
of diastereoisomers [from peak 1 (0.40 g, 99%); from peak 2 (0.40
g, 99%)]. From peak 1: LCMS for C.sub.14H.sub.18ClFNO.sub.3
(M+H).sup.+: m/z=302.1; Found: 302.0. From peak 2: LCMS for
C.sub.14H.sub.18ClFNO.sub.3 (M+H).sup.+: m/z=302.1; Found:
302.1.
Step 7. Diastereoisomers of
4-[3-chloro-5-(1-chloroethyl)-6-ethoxy-2-fluorophenyl]pyrrolidin-2-one
##STR00135##
[0576] The mixture of diastereoisomers from step 6 were each
processed individually to the final products. A solution of
4-[3-chloro-6-ethoxy-2-fluoro-5-(1-hydroxyethyl)phenyl]pyrrolidin-2-one
(0.41 g, 1.4 mmol) (from Step 6) in methylene chloride (12 mL) was
treated with N,N-dimethylformamide (0.011 mL, 0.14 mmol) followed
by thionyl chloride (0.21 mL, 2.9 mmol) dropwise and stirred at
20.degree. C. for 30 min. The reaction mixture was added dropwise
to ice cooled saturated sodium bicarbonate solution and extracted
with dichloromethane. The organic layer was separated and washed
with brine, dried over sodium sulfate, filtered, and concentrated
to give the desired products [from peak 1 (0.38 g, 87%); from peak
2 (0.39 g, 89%)] along with 17-18% of the styrene that formed from
chloride elimination. These mixtures were used without further
purification. From peak 1: LCMS for
C.sub.14H.sub.17Cl.sub.2FNO.sub.2 (M+H).sup.+: m/z=320.1; Found:
320.0. From peak 2: LCMS for C.sub.14H.sub.17Cl.sub.2FNO.sub.2
(M+H).sup.+: m/z=320.1; Found: 320.0.
Step 8. Diastereoisomers of
4-{3-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chlor-
o-2-ethoxy-6-fluorophenyl}pyrrolidin-2-one
##STR00136##
[0578] The mixture of diastereoisomers from step 7 were each
processed individually to the final products. A mixture of
4-[3-chloro-5-(1-chloroethyl)-6-ethoxy-2-fluorophenyl]pyrrolidin-2-one
(0.36 g, 1.1 mmol) (from Step 7),
3-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (0.19 g, 1.3 mmol),
cesium carbonate (0.54 g, 1.7 mmol) and potassium iodide (18 mg,
0.11 mmol) in N,N-dimethylformamide (7.4 mL) was heated at
100.degree. C. for 4.5 h. The reaction mixture was poured into
water (30 ml) and extracted with ethyl acetate (3.times.50 mL) to
give a mixture of diastereoisomer
((S)-4-(3-((S)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl-
)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one;
(R)-4-(3-((S)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-
-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one;
(S)-4-(3-((R)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-
-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one; and
(R)-4-(3-((R)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-
-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one). The mixture of
diastereoisomers were purified by preparative LCMS (XBridge C18
column, eluting with a gradient of acetonitrile/water containing
0.1% ammonium hydroxide, at flow rate of 60 mL/min) to give the
desired products [from peak 1 were isolated peak A (compound 345)
(0.13 g, 54%) and peak B (compound 346) (0.11 g, 46%); from peak 2
were isolated peak A (compound 347) (0.15 g, 63%) and peak B
(compound 348) (0.14 g, 55%)]. Compound 346: .sup.1H NMR (300 MHz,
DMSO-d.sub.6) .delta. 8.12 (s, 1H), 7.82 (s, 1H), 7.52 (d, J=8.5
Hz, 1H), 7.30 (br s, 1H), 6.23 (q, J=7.0 Hz, 1H), 4.05-3.90 (m,
1H), 3.88-3.78 (m, 2H), 3.63-3.53 (m, 1H), 3.29-3.20 (m, 1H), 2.54
(s, 3H), 2.38-2.21 (m, 1H), 1.70 (d, J=7.1 Hz, 3H), 1.39 (t,
J.sup.=6.9 Hz, 3H). LCMS for
C.sub.20H.sub.23ClFN.sub.6O.sub.2(M+H).sup.+: m/z=433.2; Found:
433.1. Compound 347: .sup.1H NMR (500 MHz, DMSO-d.sub.6) .delta.
8.12 (s, 1H), 7.77 (s, 1H), 7.53 (d, J=8.5 Hz, 1H), 7.26 (br s,
2H), 6.24 (q, J=7.0 Hz, 1H), 4.04-3.94 (m, 1H), 3.93-3.85 (m, 1H),
3.84-3.77 (m, 1H), 3.61-3.53 (m, 1H), 3.27-3.22 (m, 1H), 2.54 (s,
3H), 2.30 (dd, J=18.1, 8.6 Hz, 1H), 1.71 (d, J=7.1 Hz, 3H), 1.40
(t, J=6.9 Hz, 3H). LCMS for
C.sub.20H.sub.23ClFN.sub.6O.sub.2(M+H).sup.+: m/z=433.2; Found:
433.1.
Examples 349-352. Diastereoisomers of
4-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-6-chloro-3-
-ethoxy-2-(5-oxopyrrolidin-3-yl)benzonitrile
##STR00137##
[0579] Step 1. Enantiomers of
4-acetyl-6-chloro-3-ethoxy-2-(5-oxopyrrolidin-3-yl)benzonitrile
##STR00138##
[0581] A racemic mixture of
4-(3-acetyl-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one (0.20
g, 0.67 mmol) (from Example 345, Step 5) and sodium cyanide (0.057
g, 1.2 mmol) in dimethyl sulfoxide (1.5 mL) was stirred at
80.degree. C. for 3 h. The reaction mixture was poured into water
(35 mL) and extracted with ethyl acetate (2.times.50 mL). The
combined organic extracts were washed with brine, dried over sodium
sulfate, filtered, and concentrated to give a crude residue. The
crude material was purified by flash column chromatography using
ether (containing 10% methanol) in hexanes (0%-100%) to give the
desired product (0.15 g, 71%) as a mixture of enantiomers. LCMS for
C.sub.15H.sub.16ClN.sub.2O.sub.3(M+H).sup.+: m/z=307.1; Found:
307.0. The mixture of enantiomers was separated by chiral HPLC to
give the individual enantiomers as peak 1 and peak 2 (RT=5.00 min
and 10.4 min; Phenomenex Lux Cellulose C-2, 21.2.times.250 mm, 5
micron particle size, eluting with 60% ethanol in hexanes at 18
mL/min).
Step 2. Diastereoisomers of
6-chloro-3-ethoxy-4-(1-hydroxyethyl)-2-(5-oxopyrrolidin-3-yl)benzonitrile
##STR00139##
[0583] The enantiomers from step 1 were each processed individually
to the final products. A solution of
4-acetyl-6-chloro-3-ethoxy-2-(5-oxopyrrolidin-3-yl)benzonitrile
(from peak 1: 0.83 g, 2.7 mmol; from peak 2: 0.86 g, 2.8 mmol) in
anhydrous methanol (14 mL) under an atmosphere of nitrogen at
0.degree. C. was treated with sodium tetrahydroborate (0.20 g, 5.4
mmol) and stirred at 0.degree. C. for 30 min. The reaction mixture
was quenched with water at 0.degree. C. and poured into water (50
mL)/ethyl acetate (100 mL) while stirring. The mixture was warmed
to ambient temperature and the aqueous layer was separated and
extracted with additional ethyl acetate (50 mL). The combined
organic extracts were washed with brine, dried over sodium sulfate,
filtered, and concentrated to give the desired products as mixtures
of diastereoisomers [from peak 1 (0.83 g, 99%); from peak 2 (0.87
g, 99%)]. From peak 1: LCMS for
C.sub.15H.sub.18ClN.sub.2O.sub.3(M+H).sup.+: m/z=309.1; Found:
309.1. From peak 2: LCMS for
C.sub.15H.sub.18ClN.sub.2O.sub.3(M+H).sup.+: m/z=309.1; Found:
309.1.
Step 3. Diastereoisomers of
6-chloro-4-(1-chloroethyl)-3-ethoxy-2-(5-oxopyrrolidin-3-yl)benzonitrile
##STR00140##
[0585] The mixture of diastereoisomers from step 2 were each
processed individually to the final products. A solution of
6-chloro-3-ethoxy-4-(1-hydroxyethyl)-2-(5-oxopyrrolidin-3-yl)benzonitrile
(from peak 1: 0.83 g, 2.7 mmol; from peak 2: 0.87 g, 2.8 mmol) in
methylene chloride (23 mL) was treated with N,N-dimethylformamide
(0.021 mL, 0.27 mmol) followed by thionyl chloride (0.490 mL, 6.72
mmol) dropwise and stirred at 20.degree. C. for 2 h. The reaction
mixture was added dropwise to ice cooled saturated sodium
bicarbonate solution and extracted with dichloromethane. The
organic layer was separated and washed with brine, dried over
sodium sulfate, filtered, and concentrated to give the desired
products as mixtures of diastereoisomers [from peak 1 (0.85 g,
97%); from peak 2 (0.90 g, 98%)]. These mixtures were used without
further purification. From peak 1: LCMS for
C.sub.15H.sub.17Cl.sub.2N.sub.2O.sub.2 (M+H).sup.+: m/z=327.1;
Found: 327.1. From peak 2: LCMS for
C.sub.15H.sub.17Cl.sub.2N.sub.2O.sub.2 (M+H).sup.+: m/z=327.1;
Found: 327.1.
Step 4. Diastereoisomers of
4-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-6-chloro-3-
-ethoxy-2-(5-oxopyrrolidin-3-yl)benzonitrile
##STR00141##
[0587] The mixture of diastereoisomers from step 3 were each
processed individually. A mixture of
6-chloro-4-(1-chloroethyl)-3-ethoxy-2-(5-oxopyrrolidin-3-yl)benzonitrile
(from peak 1: 0.85 g, 2.6 mmol; from peak 2: 0.89 g, 2.7 mmol),
3-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (0.46 g, 3.1 mmol),
cesium carbonate (1.3 g, 3.9 mmol) and potassium iodide (43 mg,
0.26 mmol) in N,N-dimethylformamide (17 mL, 220 mmol) was heated at
90.degree. C. for 3 h. The reaction mixture was poured into water
(100 mL)/ethyl acetate (100 mL) and filtered through Celite to
remove black solids. The aqueous layer was separated and extracted
with ethyl acetate (2.times.100 mL). The combined organic extracts
were washed with brine, dried over sodium sulfate, filtered, and
concentrated to give white foams. The crude material were purified
by flash column chromatography using methanol in dichloromethane
(0%-20%) to give the desired products as mixtures of
diastereoisomers [from peak 1 (0.49 g, 43%); from peak 2 (0.53 g,
44%)]. Analytical chiral HPLC analysis of the diastereoisomers from
peak 1 revealed a mixture of four peaks instead of the desired two
due to epimerization. Analysis of the diastereoisomers from peak 2
also revealed four peaks. Both sets of mixtures were combined and
purified via chiral HPLC to give four individual peaks (RT=6.41
min, 8.13 min, 9.93 min, 14.4 min; Phenomenex Lux Cellulose C-2,
21.2.times.250 mm, 5 micron particle size, eluting with 60% ethanol
in hexanes at 18 mL/min). The compounds of peak 1 (compound 351),
peak 2 (compound 349), peak 3 (compound 352), and peak 4 (compound
350) were then tested in the assays of Example A3 and B2. Compound
349: .sup.1H NMR (500 MHz, DMSO-d.sub.6) .delta. 8.12 (s, 1H), 7.88
(s, 1H), 7.58 (s, 1H), 7.30 (br s, 2H), 6.26 (q, J=7.0 Hz, 1H),
4.32-4.20 (m, 1H), 4.00-3.91 (m, 1H), 3.90-3.81 (m, 1H), 3.65-3.59
(m, 1H), 3.49-3.42 (m, 1H), 2.55 (s, 3H), 1.74 (d, J=7.0 Hz, 3H),
1.43 (t, J=6.9 Hz, 3H). LCMS for
C.sub.21H.sub.23ClN.sub.7O.sub.2(M+H).sup.+: m/z=440.2; Found:
440.2. Compound 352: .sup.1H NMR (500 MHz, DMSO-d.sub.6) .delta.
8.12 (s, 1H), 7.88 (s, 1H), 7.56 (s, 1H), 7.30 (br s, 2H), 6.26 (q,
J=7.0 Hz, 1H), 4.32-4.19 (m, 1H), 3.97-3.82 (m, 2H), 3.67-3.59 (m,
1H), 3.49-3.40 (m, 1H), 2.59-2.52 (m, 3H), 1.73 (d, J=7.0 Hz, 3H),
1.42 (t, J=6.9 Hz, 3H). LCMS for
C.sub.21H.sub.23ClN.sub.7O.sub.2(M+H).sup.+: m/z=440.2; Found:
440.2.
Examples 353 and 354. Diastereomers of
4-{3-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chlor-
o-2-ethoxy-6-fluorophenyl}-1,3-oxazolidin-2-one
##STR00142##
[0588] Step 1:
1-(5-Chloro-2-ethoxy-4-fluoro-3-vinylphenyl)ethanone
##STR00143##
[0590] A mixture of
1-(5-chloro-2-ethoxy-4-fluoro-3-iodophenyl)ethanone (13.3 g, 38.8
mmol) (from Example 139, Step 1),
4,4,5,5-tetramethyl-2-vinyl-1,3,2-dioxaborolane (7.9 mL, 46 mmol),
[1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II),
complex with dichloromethane (1:1) (1.0 g, 1.0 mmol) and potassium
carbonate (16 g, 120 mmol) in 1,4-dioxane (200 mL) and water (100
mL) was heated at 80.degree. C. for 2 hours. The mixture was cooled
to rt and extracted with ethyl acetate. The extracts were washed
with brine, dried over sodium sulfate, filtered and concentrated.
Purification on silica gel using ethyl acetate in hexanes (0-30%)
gave the desired compound, 7.0 g, 74%. LCMS calculated for
C.sub.12H.sub.13ClFO.sub.2 (M+H).sup.+: m/z=243.0; found:
243.1.
Step 2:
1-[5-Chloro-3-(1,2-dihydroxyethyl)-2-ethoxy-4-fluorophenyl]ethanon-
e
##STR00144##
[0592] AD-mix-alpha (5.8 g, 7.3 mmol) (Aldrich #392758) was stirred
in tert-butyl alcohol (21 mL) with water (21 mL) for 15 minutes.
1-(5-chloro-2-ethoxy-4-fluoro-3-vinylphenyl)ethanone (1.0 g, 4.1
mmol) (from Step 1) was added and the suspension was stirred for 16
hours. Sodium sulfite (6.2 g, 49 mmol) was added and the suspension
was stirred for 15 minutes. The reaction mixture was extracted with
ethyl acetate. The extracts were washed with brine and dried over
sodium sulfate, filtered and evaporated. Purification on silica gel
using ethyl acetate in hexanes (0-80%) gave the desired compound as
a racemic mixture, 900 mg, 80%. Chiral purification on Phenomenex
Lux Cellulose C-2, 21.2.times.250 mm (Chiral Technologies), 5
micron particle size, at flow rate of 18 mL/min using 20% ethanol
in hexanes gave peak 1 (single enantiomer) (retention time=7.88
minutes) and peak 2 (single enantiomer) (retention time=11
minutes); the desired enantiomer was peak 2. LCMS calculated for
C.sub.12H.sub.13ClFO.sub.3 (M-OH).sup.+: m/z=259.1; found:
259.1.
Step 3:
1-[3-(2-{[tert-Butyl(dimethyl)silyl]oxy}-1-hydroxyethyl)-5-chloro--
2-ethoxy-4-fluorophenyl]ethanone
##STR00145##
[0594]
1-[5-Chloro-3-(1,2-dihydroxyethyl)-2-ethoxy-4-fluorophenyl]ethanone
(700 mg, 2 mmol) (from Step 2, peak 2) was stirred in
1,2-dichloroethane (6 mL) with N,N-diisopropylethylamine (4.0 mL,
23 mmol) and a 1.0 M solution of tert-butyldimethylsilyl chloride
in 1,2-dichloroethane (7.6 mL) was added. The mixture was heated to
80.degree. C. for 3 hours and cooled to rt. Evaporation and
purification on silica gel using ethyl acetate in hexanes (0-50%)
gave the desired compound 800 mg, 80%. LCMS calculated for
C.sub.18H.sub.28ClFO.sub.4SiNa (M+Na).sup.+: m/z=413.1; found:
413.1.
Step 4:
1-(3-Acetyl-5-chloro-2-ethoxy-6-fluorophenyl)-2-{[tert-butyl(dimet-
hyl)silyl]oxy}ethyl methanesulfonate
##STR00146##
[0596]
1-[3-(2-{[tert-Butyl(dimethyl)silyl]oxy}-1-hydroxyethyl)-5-chloro-2-
-ethoxy-4-fluorophenyl]ethanone (700 mg, 2.0 mmol) (from Step 3)
was stirred in 1,2-dichloroethane (15 mL) with triethylamine (2.0
mL, 14 mmol) and methanesulfonic anhydride (670 mg, 3.8 mmol) at rt
for 1.5 hours. The mixture was poured into brine and extracted with
dichloromethane. The extracts were dried over sodium sulfate,
filtered and evaporated to give the desired compound 830 mg, 100%.
LCMS calculated for C.sub.18H.sub.27ClFO.sub.3Si (M-OMs).sup.+:
m/z=373.1; found: 373.1.
Step 5:
1-[3-(1-Azido-2-{[tert-butyl(dimethyl)silyl]oxy}ethyl)-5-chloro-2--
ethoxy-4-fluorophenyl]ethanone
##STR00147##
[0598]
1-(3-Acetyl-5-chloro-2-ethoxy-6-fluorophenyl)-2-{[tert-butyl(dimeth-
yl)silyl]oxy}ethyl methanesulfonate (0.83 g, 1.77 mmol) (from Step
4) was stirred in dimethyl sulfoxide (10 mL) and sodium azide (0.12
g, 1.8 mmol) was added. The mixture was heated to 50.degree. C. for
1 hour and cooled to rt. The mixture was poured into brine and
extracted with ethyl acetate. The extracts were dried over sodium
sulfate, filtered and evaporated to give the desired compound 736
mg, 100%. LCMS calculated for C.sub.18H.sub.27ClFN.sub.3O.sub.3SiNa
(M+Na).sup.+: m/z=438.1; found: 438.1.
Step 6:
1-[3-(1-Amino-2-{[tert-butyl(dimethyl)silyl]oxy}ethyl)-5-chloro-2--
ethoxy-4-fluorophenyl]ethanone
##STR00148##
[0600]
1-[3-(1-Azido-2-{[tert-butyl(dimethyl)silyl]oxy}ethyl)-5-chloro-2-e-
thoxy-4-fluorophenyl]ethanone (750 mg, 1.8 mmol) (from Step 5) was
stirred in tetrahydrofuran (10 mL) with water (0.33 mL) and
triphenylphosphine was added. The mixture was heated to 60.degree.
C. for 2 hours and cooled to rt. Brine was added and the mixture
was extracted with ethyl acetate. The extracts were dried over
sodium sulfate, filtered and evaporated to give the desired
compound 700 mg, 100%. LCMS calculated for
C.sub.18H.sub.30ClFNO.sub.3Si (M+H).sup.+: m/z=390.2; found:
390.2.
Step 7: tert-Butyl
(1-(3-acetyl-5-chloro-2-ethoxy-6-fluorophenyl)-2-{[tert-butyl(dimethyl)si-
lyl]oxy}ethyl)carbamate
##STR00149##
[0602]
1-[3-(1-Amino-2-{[tert-butyl(dimethyl)silyl]oxy}ethyl)-5-chloro-2-e-
thoxy-4-fluorophenyl]ethanone (700 mg, 2.0 mmol) (from Step 6) was
stirred in tetrahydrofuran (30 mL) with di-tert-butyldicarbonate
(780 mg, 3.6 mmol) and N,N-diisopropylethylamine (0.94 mL, 5.4
mmol) was added. The mixture was stirred at rt for 30 minutes.
Brine was added and the mixture was extracted with ethyl acetate.
The extracts were dried over sodium sulfate, filtered and
evaporated. Purification on silica gel using ethyl acetate in
hexanes (0-30%) gave the desired compound 550 mg, 60%. LCMS
calculated for C.sub.23H.sub.37ClFNO.sub.5SiNa (M+Na).sup.+:
m/z=512.2; found: 512.2.
Step 8: tert-Butyl
[1-(3-acetyl-5-chloro-2-ethoxy-6-fluorophenyl)-2-hydroxyethyl]carbamate
##STR00150##
[0604] Tert-Butyl
(1-(3-acetyl-5-chloro-2-ethoxy-6-fluorophenyl)-2-{[tert-butyl(dimethyl)si-
lyl]oxy}ethyl)carbamate (500 mg, 1.0 mmol) (from Step 7) was
stirred in tetrahydrofuran (10 mL) and a 1.0 M solution of
tetra-n-butylammonium fluoride in tetrahydrofuran (1.5 mL) was
added. The mixture was stirred at rt for 30 minutes and evaporated.
Purification on silica gel using ethyl acetate in hexanes (0-50%)
gave the desired compound 238 mg, 60%. LCMS calculated for
C.sub.17H.sub.23ClFNO.sub.5Na (M+Na).sup.+: m/z=398.1; found:
398.1.
Step 9:
4-(3-Acetyl-5-chloro-2-ethoxy-6-fluorophenyl)-1,3-oxazolidin-2-one
##STR00151##
[0606] tert-Butyl
[1-(3-acetyl-5-chloro-2-ethoxy-6-fluorophenyl)-2-hydroxyethyl]carbamate
(234 mg, 0.62 mmol) (from Step 8) was dissolved in
1,2-dichloroethane (12 mL) and a solution of 2.0 M phosgene in
toluene (0.93 mL) was added. The mixture was heated to 80.degree.
C. for 1.5 hours. Evaporation and purification on silica gel using
ethyl acetate in hexanes (0-85%) gave the desired compound, 175 mg,
93%. LCMS calculated for C.sub.13H.sub.14ClFNO.sub.4 (M+H).sup.+:
m/z=302.1; found: 302.1.
Step 10:
4-[3-chloro-6-ethoxy-2-fluoro-5-(1-hydroxyethyl)phenyl]-1,3-oxazo-
lidin-2-one
##STR00152##
[0608]
4-(3-Acetyl-5-chloro-2-ethoxy-6-fluorophenyl)-1,3-oxazolidin-2-one
(175 mg, 0.58 mmol) was stirred in methanol (10 mL) at 0.degree. C.
and sodium tetrahydroborate (33 mg, 0.87 mmol) was added. The
mixture was stirred at rt for 1 hour and evaporated. Water was
added and the mixture was extracted with ethyl acetate. The
extracts were washed with brine, dried over sodium sulfate,
filtered and evaporated to give an approximate 1:1 mixture of
diastereomers, 175 mg, 99%. LCMS calculated for
C.sub.13H.sub.15ClFNO.sub.4Na (M+Na).sup.+: m/z=326.1; found:
326.1.
Step 11:
4-[3-chloro-5-(chloroethyl)-6-ethoxy-2-fluorophenyl]-1,3-oxazolid-
in-2-one
##STR00153##
[0610]
4-[3-chloro-6-ethoxy-2-fluoro-5-(1-hydroxyethyl)phenyl]-1,3-oxazoli-
din-2-one (150 mg, 0.49 mmol) (from Step 10) was stirred in
dichloromethane (4 mL) with N,N-dimethylformamide (96 .mu.L) and
thionyl chloride (110 .mu.L, 1.5 mmol) was added. The mixture was
evaporated. Water was added and the mixture was extracted with
ethyl acetate. The extracts were washed with brine, dried over
sodium sulfate, filtered and evaporated to give the desired
compound, 159 mg, 100%.
Step 12:
4-{3-[1-(4-amino-3-methyl-H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]--
5-chloro-2-ethoxy-6-fluorophenyl}-1,3-oxazolidin-2-one
##STR00154##
[0612]
4-[3-chloro-5-(chloroethyl)-6-ethoxy-2-fluorophenyl]-1,3-oxazolidin-
-2-one (160 mg, 0.50 mmol) (from Step 11) was stirred in
N,N-dimethylformamide (21 mL) with cesium carbonate (324 mg, 0.99
mmol) and 3-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (89 mg, 0.60
mmol) was added. The mixture was heated to 80.degree. C. for 1.5
hours and cooled to rt. The mixture was diluted with water and
extracted with ethyl acetate. The extracts were washed with brine,
dried over sodium sulfate, filtered and evaporated. Purification by
preparative LCMS (pH 10) using RP-HPLC (XBridge C18 column, eluting
with a gradient of acetonitrile/water containing 0.1% ammonium
hydroxide, at flow rate of 30 mL/min) separated the two
diastereomers (peak 1 [compound 353] Rt=4.9 min. and peak 2
[compound 354] Rt=5.6 min.); providing compound 354 as the desired
single enantiomer, 28 mg, 13%. peak 2: LCMS calculated for
C.sub.19H.sub.21ClFN.sub.6O.sub.3 (M+H).sup.+: m/z=435.1; found:
435.1. .sup.1H NMR (300 MHz, CD.sub.3OD): .delta. 8.15 (s, 1H),
7.62 (m, 1H), 6.31 (m, 1H), 5.39 (m, 1H), 4.79 (m, 1H), 4.40 (m,
1H), 3.95 (m, 1H), 3.80 (m, 1H), 2.60 (s, 3H), 1.80 (m, 3H), 1.40
(m, 3H).
Examples 355-358. Diastereomers of
5-{3-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]-5-chlor-
o-2-ethoxy-6-fluorophenyl}-1,3-oxazolidin-2-one
##STR00155##
[0613] Step 1: tert-Butyl
[2-(3-acetyl-5-chloro-2-ethoxy-6-fluorophenyl)-2-hydroxyethyl]carbamate
##STR00156##
[0615] 0.2 M Osmium tetraoxide in water (10 mL) was added to a
solution of tert-butyl [(4-chlorobenzoyl)oxy]carbamate (Lawrence
Harris, J Org. Chem, 2011, 76, 358-372). (19 g, 70 mmol) in
acetonitrile (210 mL) and stirred for 10 minutes.
1-(5-chloro-2-ethoxy-4-fluoro-3-vinylphenyl)ethanone (11.2 g, 46
mmol) (from Example 353, Step 1) as a solution in acetonitrile (210
mL) was added to the carbamate solution followed by the addition of
water (50 mL) and the reaction was stirred for 3 hours at room
temperature. The reaction was quenched with saturated 10 M
dipotassium disulfite in water (240 mL) and stirred for 5 minutes.
Water was added and the reaction mixture was extracted with ethyl
acetate. The extracts were washed with saturated sodium bicarbonate
solution, brine and dried over sodium sulfate, filtered and
evaporated. Purification on silica gel using ethyl acetate in
hexanes (0-100%) gave the desired compound as a racemic mixture,
16.6 g, 95%. LCMS calculated for C.sub.17H.sub.23ClFNO.sub.5Na
(M+Na).sup.+: m/z=398.1; found: 398.0.
Step 2:
5-{3-[1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl]--
5-chloro-2-ethoxy-6-fluorophenyl}-1,3-oxazolidin-2-one
##STR00157##
[0617] The desired single enantiomer (peak 3) was prepared using
the same procedure as Example 353 (steps 8-12), except that the
intermediate from step 1 in this example was racemic and thus the
final separation of the four diastereomers occurred in step 12.
Chiral purification on Phenomenex Lux Cellulose C-4, 21.times.250
mm (Chiral Technologies), 5 micron particle size, at flow rate of
18 mL/min using 30% ethanol in hexanes gave the peak 1: compound
355 (single enantiomer) (retention time=12.7 minutes), peak 2:
compound 356 (single enantiomer) (retention time=14.2 minutes),
peak 3: compound 357 (single enantiomer) (retention time=20.3
minutes), and peak 4: compound 358 (single enantiomer) (retention
time=28.9 minutes); the most active enantiomer was peak 3. LCMS
calculated for C.sub.19H.sub.21ClFN.sub.6O.sub.3(M+H).sup.+:
m/z=435.1; found: 435.1. .sup.1H NMR (500 MHz, DMSO-d.sub.6):
.delta. 8.15 (s, 1H), 7.81 (s, 1H), 7.71 (d, 1H), 7.26 (bs, 1H),
6.23 (m, 1H), 5.84 (t, 1H), 3.92 (m, 1H), 3.83 (m, 1H), 2.52 (s,
3H), 1.75 (d, 3H), 1.40 (m, 3H).
Examples 361-363. Diastereomers of
4-(1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-6-chloro-2-
-(1-(2-hydroxypropyl)azetidin-3-yl)-3-methoxybenzonitrile
[0618] Based on the stereochemistry of Example 269, the
stereochemistry of each diastereomer is believed to be
4-((R)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-6-chlo-
ro-2-(1-((S)-2-hydroxypropyl)azetidin-3-yl)-3-methoxybenzonitrile
(Example 361),
4-((S)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)--
6-chloro-2-(1-((R)-2-hydroxypropyl)azetidin-3-yl)-3-methoxybenzonitrile
(Example 362), and
4-((R)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-6-chlo-
ro-2-(1-((R)-2-hydroxypropyl)azetidin-3-yl)-3-methoxybenzonitrile
(Example 363) (structures shown below)
##STR00158##
Synthesis of Example 361
[0619] To
(R)-4-(1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethy-
l)-2-(azetidin-3-yl)-6-chloro-3-methoxybenzonitrile (6.00 g, 14.3
mmol) was added methanol (72 mL). To the resulting suspension was
added (S)-(-)-methyloxirane (2.01 mL, 28.6 mmol) at room
temperature and the mixture was stirred at room temperature for 19
h. Additional (S)-(-)-methyloxirane (0.50 mL, 7.2 mmol) was added
and the stirring was continued for an additional hour. To the
reaction mixture was added water (280 mL) and the cloudy solution
was stirred. The mixture was extracted with methylene chloride (300
mL.times.4). The organic layer was combined and washed with brine
(50 mL) and concentrated. The crude product was purified by silica
column chromatography eluted with MeOH (contained about 0.5%
ammonium hydroxide) in methylene chloride. The fractions contained
product were collected and evaporated to dryness. This residue was
further purified by preparative HPLC to give the title compound. A
sample of the title compound was analyzed by NMR spectroscopy and
mass spectrometry and gave the following data. .sup.1H NMR (500
MHz, DMSO) .delta. 8.11 (s, 1H), 7.47 (s, 1H), 7.30 (br s, 2H),
6.24 (q, J=7.0 Hz, 1H), 4.32 (br s, 1H), 4.07 (m, 1H), 3.94 (m,
2H), 3.65 (s, 3H), 3.59 (m, 1H), 3.08 (m, 2H), 2.56 (s, 3H),
2.38-2.19 (m, 2H), 1.73 (d, J=7.1 Hz, 3H), 1.00 (d, J=6.2 Hz, 3H)
ppm. LCMS for C.sub.22H.sub.27ClN.sub.7O.sub.2(M+H).sup.+:
m/z=456.2; found: 456.2.
Synthesis of Example 362
[0620] To
(S)-4-(1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethy-
l)-2-(azetidin-3-yl)-6-chloro-3-methoxybenzonitrile (293.0 mg, 0.73
mmol) was added methanol (3.7 mL). To the resulting suspension was
added (R)-(+)-methyloxirane 103 .mu.L, 1.46 mmol) at room
temperature and the mixture was stirred at room temperature for 19
h. Additional (R)-(+)-methyloxirane (51.3 .mu.L, 0.73 mmol) was
added and the stirring was continued for additional 2.5 hours. To
the reaction mixture was added water (14 mL) and the cloudy
solution was stirred. The mixture was extracted with methylene
chloride (4.times.16 mL). The organic layer was combined and washed
with brine (50 mL) and concentrated. The crude product was purified
by silica column chromatography, eluted with MeOH (contained about
0.5% ammonium hydroxide) in methylene chloride. The fractions
contained product were collected and evaporated to dryness. This
residue was further purified by preparative HPLC to give the title
compound. A sample of the title compound was analyzed by NMR
spectroscopy and mass spectrometry and gave the following data.
.sup.1H NMR (500 MHz, DMSO) .delta. 8.11 (s, 1H), 7.47 (s, 1H),
7.30 (br s, 2H), 6.24 (q, J=7.0 Hz, 1H), 4.37 (br s, 1H), 4.09 (m,
2H), 3.93 (m, 2H), 3.65 (s, 3H), 3.59 (m, 1H), 3.12 (m, 2H), 2.56
(s, 3H), 2.39-2.26 (m, 2H), 1.73 (d, J=7.1 Hz, 3H), 1.00 (d, J=6.2
Hz, 3H) ppm. LCMS for C.sub.22H.sub.27ClN.sub.7O.sub.2(M+H).sup.+:
m/z=456.2; found: 456.2.
Synthesis of Example 363
[0621] To
(R)-4-(1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethy-
l)-2-(azetidin-3-yl)-6-chloro-3-methoxybenzonitrile (6.0 g, 14.3
mmol) was added methanol (72 mL). To the resulting suspension was
added (R)-(+)-methyloxirane (2.01 mL, 28.6 mmol) at room
temperature and the mixture was stirred at room temperature for 18
h. To the reaction mixture was added water (280 mL) and the cloudy
solution was stirred. The mixture was extracted with methylene
chloride (300 mL.times.4). The organic layer was combined and
washed with brine (50 mL) and concentrated. The crude product was
purified by silica column chromatography, eluted with MeOH
(contained about 0.5% ammonium hydroxide) in methylene chloride.
The fractions contained product were collected and evaporated to
dryness. This residue was further purified by preparative HPLC to
give the title compound. A sample of the title compound was
analyzed by NMR spectroscopy and mass spectrometry and gave the
following data. .sup.1H NMR (500 MHz, DMSO) .delta. 8.11 (s, 1H),
7.46 (s, 1H), 7.29 (br s, 2H), 6.24 (q, J=7.0 Hz, 1H), 4.31 (d,
J=4.2 Hz, 1H), 4.11-4.00 (m, 1H), 3.98-3.90 (m, 1H), 3.65 (s, 3H),
3.61-3.53 (m, 2H), 3.07 (m, 2H), 2.56 (s, 3H), 2.28 (d, J=5.9 Hz,
2H), 1.73 (d, J=7.1 Hz, 3H), 1.00 (d, J=6.2 Hz, 3H) ppm.
[0622] Three HPLC methods were developed to separate the
stereoisomers from the compound of Example 269. Method A was
developed to separate the diastereomer Example 361 from Example
269. The retention times of Example 361 from Example 269 are 15.7
min and 11.5 min respectively. Chromatographic conditions are
described in Table B1.
TABLE-US-00002 TABLE B1 Column Phenomenex Cellulose 3 (250 mm, 4.6
mm, 5 micron) Mobile Phase 89.9% hexane/10% ethanol/0.1%
diethylamine (pre-mixed) Flow Rate 1 mL/min Run Time 30 min
Detection Wavelength 247 nm Quantitation Peak area ratio
[0623] Method B was developed to separate the diastereomer Example
362 from Example 269. The retention times of Example 362 from
Example 269 are 26.4 min and 21.7 min respectively. Chromatographic
conditions are described in Table B2.
TABLE-US-00003 TABLE B2 Column Phenomenex Cellulose 4 (250 mm, 4.6
mm, 5 micron) Mobile Phase 84.9% hexane/15% ethanol/0.1%
diethylamine (pre-mixed) Flow Rate 1 mL/min Run Time 40 min
Detection Wavelength 247 nm Quantitation Peak area ratio
[0624] Method C was developed to separate the three stereoisomers
Example 361, Example 362 and Example 363 from Example 269. The
stereoisomers Example 361, Example 362 and Example 363 elute at
retention time 12.9 min as a broad band while Example 269 elutes at
retention time 14.3 min. An estimation of the level of the
enantiomer, Example 363 can be made by a combination of data from
Methods A, B, and C. Chromatographic conditions are described in
Table B3.
TABLE-US-00004 TABLE B3 Column Phenomenex Cellulose 1 (250 mm, 4.6
mm, 5 micron) Mobile Phase 88% hexanes, 12% ethanol (conatins 0.1%
diethylamine) Flow Rate 1 mL/min Run Time 25 min Detection
Wavelength 247 nm Quantitation Peak area ratio
Example A1: PI3K Enzyme Assay
[0625] PI3-Kinase luminescent assay kit including lipid kinase
substrate, D-myo-phosphatidylinositol 4,5-bisphosphate
(PtdIns(4,5)P2)D (+)-sn-1,2-di-O-octanoylglyceryl, 3-O-phospho
linked (PIP2), biotinylated I(1,3,4,5)P4, PI(3,4,5)P3 Detector
Protein is purchased from Echelon Biosciences (Salt Lake City,
Utah). AlphaScreen.TM. GST Detection Kit including donor and
acceptor beads was purchased from PerkinElmer Life Sciences
(Waltham, Mass.). PI3K.delta. (p110.delta./p85.alpha.) is purchased
from Millipore (Bedford, Mass.). ATP, MgCl.sub.2, DTT, EDTA, HEPES
and CHAPS are purchased from Sigma-Aldrich (St. Louis, Mo.).
AlphaScreen.TM. Assay for PI3K.delta.
[0626] The kinase reaction are conducted in 384-well REMP plate
from Thermo Fisher Scientific in a final volume of 40 .mu.L.
Inhibitors are first diluted serially in DMSO and added to the
plate wells before the addition of other reaction components. The
final concentration of DMSO in the assay is 2%. The PI3K assays are
carried out at room temperature in 50 mM HEPES, pH 7.4, 5 mM
MgCl.sub.2, 50 mM NaCl, 5 mM DTT and CHAPS 0.04%. Reactions are
initiated by the addition of ATP, the final reaction mixture
consisted of 20 .mu.M PIP2, 20 .mu.M ATP, 1.2 nM PI3K.delta. are
incubated for 20 minutes. 10 .mu.L of reaction mixture are then
transferred to 5 .mu.L 50 nM biotinylated I(1,3,4,5)P4 in quench
buffer: 50 mM HEPES pH 7.4, 150 mM NaCl, 10 mM EDTA, 5 mM DTT, 0.1%
Tween-20, followed with the addition of 10 .mu.L AlphaScreen.TM.
donor and acceptor beads suspended in quench buffer containing 25
nM PI(3,4,5)P3 detector protein. The final concentration of both
donor and acceptor beads is 20 mg/ml. After plate sealing, the
plate are incubated in a dark location at room temperature for 2
hours. The activity of the product is determined on Fusion-alpha
microplate reader (Perkin-Elmer). IC.sub.50 determination is
performed by fitting the curve of percent control activity versus
the log of the inhibitor concentration using the GraphPad Prism 3.0
software.
Example A2: PI3K Enzyme Assay
[0627] Materials:
[0628] Lipid kinase substrate, phosphoinositol-4,5-bisphosphate
(PIP2), are purchased from Echelon Biosciences (Salt Lake City,
Utah). PI3K isoforms .alpha., .beta., .delta. and .gamma. are
purchased from Millipore (Bedford, Mass.). ATP, MgCl.sub.2, DTT,
EDTA, MOPS and CHAPS are purchased from Sigma-Aldrich (St. Louis,
Mo.).
[0629] The kinase reaction are conducted in clear-bottom 96-well
plate from Thermo Fisher Scientific in a final volume of 24 .mu.L.
Inhibitors are first diluted serially in DMSO and added to the
plate wells before the addition of other reaction components. The
final concentration of DMSO in the assay is 0.5%. The PI3K assays
are carried out at room temperature in 20 mM MOPS, pH 6.7, 10 mM
MgCl.sub.2, 5 mM DTT and CHAPS 0.03%. The reaction mixture is
prepared containing 50 .mu.M PIP2, kinase and varying concentration
of inhibitors. Reactions are initiated by the addition of ATP
containing 2.2 .mu.Ci [.gamma.-.sup.33P]ATP to a final
concentration of 1000 .mu.M. The final concentration of PI3K
isoforms .alpha., .beta., .delta. and .gamma. in the assay were
1.3, 9.4, 2.9 and 10.8 nM, respectively. Reactions are incubated
for 180 minutes and terminated by the addition of 100 .mu.L of 1 M
potassium phosphate pH 8.0, 30 mM EDTA quench buffer. A 100 .mu.L
aliquot of the reaction solution are then transferred to 96-well
Millipore MultiScreen IP 0.45 gm PVDF filter plate (The filter
plate is prewetted with 200 .mu.L 100% ethanol, distilled water,
and 1 M potassium phosphate pH 8.0, respectively). The filter plate
is aspirated on a Millipore Manifold under vacuum and washed with
18.times.200 .mu.L wash buffer containing 1 M potassium phosphate
pH 8.0 and 1 mM ATP. After drying by aspiration and blotting, the
plate is air dried in an incubator at 37.degree. C. overnight.
Packard TopCount adapter (Millipore) is then attached to the plate
followed with addition of 120 .mu.L Microscint 20 scintillation
cocktail (Perkin Elmer) in each well. After the plate sealing, the
radioactivity of the product is determined by scintillation
counting on Topcount (Perkin-Elmer). IC.sub.50 determination is
performed by fitting the curve of percent control activity versus
the log of the inhibitor concentration using the GraphPad Prism 3.0
software.
Example A3: PI3K.delta. Scintillation Proximity Assay
Materials
[0630] [.gamma.-.sup.33P]ATP (10 mCi/mL) was purchased from
Perkin-Elmer (Waltham, Mass.). Lipid kinase substrate,
D-myo-Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2)D
(+)-sn-1,2-di-O-octanoylglyceryl, 3-O-phospho linked (PIP2), CAS
204858-53-7, was purchased from Echelon Biosciences (Salt Lake
City, Utah). PI3K.delta. (p110.delta./p85.alpha.) was purchased
from Millipore (Bedford, Mass.). ATP, MgCl.sub.2, DTT, EDTA, MOPS
and CHAPS were purchased from Sigma-Aldrich (St. Louis, Mo.). Wheat
Germ Agglutinin (WGA) YSi SPA Scintillation Beads was purchased
from GE healthcare life sciences (Piscataway, N.J.).
[0631] The kinase reaction was conducted in polystyrene 384-well
matrix white plate from Thermo Fisher Scientific in a final volume
of 25 .mu.L. Inhibitors were first diluted serially in DMSO and
added to the plate wells before the addition of other reaction
components. The final concentration of DMSO in the assay was 0.5%.
The PI3K assays were carried out at room temperature in 20 mM MOPS,
pH 6.7, 10 mM MgCl.sub.2, 5 mM DTT and CHAPS 0.03%. Reactions were
initiated by the addition of ATP, the final reaction mixture
consisted of 20 .mu.M PIP2, 20 .mu.M ATP, 0.2 .mu.Ci
[.gamma.-.sup.33P] ATP, 4 nM PI3K.delta.. Reactions were incubated
for 210 min and terminated by the addition of 40 .mu.L SPA beads
suspended in quench buffer: 150 mM potassium phosphate pH 8.0, 20%
glycerol. 25 mM EDTA, 400 .mu.M ATP. The final concentration of SPA
beads was 1.0 mg/mL. After the plate sealing, plates were shaken
overnight at room temperature and centrifuged at 1800 rpm for 10
minutes, the radioactivity of the product was determined by
scintillation counting on Topcount (Perkin-Elmer). IC.sub.50
determination was performed by fitting the curve of percent control
activity versus the log of the inhibitor concentration using the
GraphPad Prism 3.0 software. IC.sub.50 data for the Examples is
presented in Table 2 as determined by Assay A3. IC.sub.50 data for
Examples 361 and 363 is shown in Table 3 as determined by Assay
A2.
TABLE-US-00005 TABLE 2 Example # PI3K.delta. SPA IC.sub.50 (nM)* 1
+ 2 + 3 + 4 + 5 + 6 + 7 + 8 + 9 + 10 + 11 + 13 + 14 + 16 + 17 + 18
+ 19 + 20 (1.sup.st peak) + 20 (2.sup.nd peak) +++ 21 + 22 + 23 +
24 + 40 ++ 41 +++ 43 + 44 + 65 + 66 + 67 (1.sup.st peak) + 68
(1.sup.st peak) + 71 + 72 + 94 + 95 + 96 + 99 + 102 + 104 + 105 +
108 + 110 + 113 + 115 + 118 + 121 + 139 (1.sup.st peak) + 140 + 141
+ 149 + 156 + 158 + 159 + 161 + 163 + 164 + 166 + 167 + 168 + 169 +
183 + 184 + 188 + 189 + 192 ++ 195 + 200 + 203 + 208 + 209 ++ 212 +
213 + 219 + 220 + 236 + 237 + 239 + 247 + 261 + 262 + 268 + 269 +
272 + 273 + 281 + 285 + 289 + 292 + 293 + 296 + 298 (1.sup.st peak)
+ 307 + 315 + 316 + 318 + 319 + 320 + 321 (1st peak) + 322
(1.sup.st peak) + 310 + 311 + 323 (1.sup.st peak) + 323 (2.sup.nd
peak) + 323 (3.sup.rd peak) +++ 323 (4.sup.th peak) + 324 (1.sup.st
peak) +++ 324 (2.sup.nd peak) + 325 + 345 +++ 346 + 347 + 348 +++
349 + 350 +++++ 351 +++ 352 + 353 +++++ 354 + 355 +++ 356 +++ 357 +
358 +++++ 362 +
TABLE-US-00006 TABLE 3 Example # PI3K.delta. IC.sub.50 (nM)* 361
+++++ 363 +++
* column symbols (for Tables 2 and 3): + refers to <10 nM ++
refers to >10 nM to 50 nM +++ refers to >50 nM to 200 nM ++++
refers to >200 nM to 500 nM +++++ refers to >500 nM
Example B1: B Cell Proliferation Assay
[0632] To acquire B cells, human PBMC are isolated from the
peripheral blood of normal, drug free donors by standard density
gradient centrifugation on Ficoll-Hypague (GE Healthcare,
Piscataway, N.J.) and incubated with anti-CD19 microbeads (Miltenyi
Biotech, Auburn, Calif.). The B cells are then purified by positive
immunosorting using an autoMacs (Miltenyi Biotech) according to the
manufacture's instruction.
[0633] The purified B cells (2.times.10.sup.5/well/200 .mu.L) are
cultured in 96-well ultra-low binding plates (Corning, Corning,
N.Y.) in RPMI1640, 10% FBS and goat F(ab')2 anti-human IgM (10
.mu.g/ml) (Invitrogen, Carlsbad, Calif.) in the presence of
different amount of test compounds for three days.
[.sup.3H]-thymidine (1 .mu.Ci/well) (PerkinElmer, Boston, Mass.) in
PBS is then added to the B cell cultures for an additional 12 hours
before the incorporated radioactivity is separated by filtration
with water through GF/B filters (Packard Bioscience, Meriden,
Conn.) and measured by liquid scintillation counting with a
TopCount (Packard Bioscience).
Example B2: Pfeiffer Cell Proliferation Assay
[0634] Pfeiffer cell line (diffuse large B cell lymphoma) are
purchased from ATCC (Manassas, Va.) and maintained in the culture
medium recommended (RPMI and 10% FBS). To measure the
anti-proliferation activity of the compounds, the Pfeiffer cells
are plated with the culture medium (2.times.10.sup.3 cells/well/per
200 .mu.l) into 96-well ultra-low binding plates (Corning, Corning,
N.Y.), in the presence or absence of a concentration range of test
compounds. After 3-4 days, [.sup.3H]-thymidine (1 .mu.Ci/well)
(PerkinElmer, Boston, Mass.) in PBS is then added to the cell
culture for an additional 12 hours before the incorporated
radioactivity is separated by filtration with water through GF/B
filters (Packard Bioscience, Meridenj, Conn.) and measured by
liquid scintillation counting with a TopCount (Packard Bioscience).
IC.sub.50 data for select compounds is presented in Table 4.
TABLE-US-00007 TABLE 4 Example # Pfeiffer IC.sub.50 (nM)* 67
(1.sup.st peak) + 68 (1.sup.st peak) + 96 + 102 + 104 ++ 121 ++ 139
(1.sup.st peak) + 140 + 149 + 163 ++ 167 + 195 + 200 + 213 + 219 +
220 + 262 + 268 + 269 + 315 + 354 + 357 + 346 + 347 + 349 + *column
symbols: + refers to .ltoreq.10 nM ++ refers to >10 nM to 50
nM
Example C: Akt Phosphorylation Assay
[0635] Ramos cells (B lymphocyte from Burkitts lymphoma) are
obtained from ATCC (Manassas, Va.) and maintained in RPMI1640 and
10% FBS. The cells (3.times.10.sup.7 cells/tube/3 mL in RPMI) are
incubated with different amounts of test compounds for 2 hrs at
37.degree. C. and then stimulated with goat F(ab')2 anti-human IgM
(5 .mu.g/mL) (Invitrogen) for 17 minutes in a 37.degree. C. water
bath. The stimulated cells are spun down at 4.degree. C. with
centrifugation and whole cell extracts are prepared using 300 .mu.L
lysis buffer (Cell Signaling Technology, Danvers, Mass.). The
resulting lysates are sonicated and supernatants are collected. The
phosphorylation level of Akt in the supernatants are analyzed by
using PathScan phospho-Akt1 (Ser473) sandwich ELISA kits (Cell
Signaling Technology) according to the manufacturer's
instruction.
Example D: Pfeiffer Model of Lymphoma
[0636] Methods:
[0637] Female SCID mice, (5 to 8 weeks of age, Charles River
Laboratories, Wilmington, Mass.) were inoculated with 1.times.107
tumor cells (Pfeiffer, ATCC #CRL-2632, Manassas, Va.) and matrigel
(BD Biosciences #354234) in 0.2 mL sterile saline. The inoculation
was performed subcutaneously on the flank. Tumor tissue fragments
(approximately 3 mm.times.3 mm) were collected 3 to 6 weeks after
the inoculation of cultured cells and implanted subcutaneously in
lieu of cellular inoculation. Tissue fragments were implanted as
solid pieces using blunt-tip forceps. The treatment of tumor
bearing mice was started 15 to 25 days after tumor inoculation,
depending upon the tumor size. Animals were sorted to obtain
roughly equivalent mean tumor volumes in each group. Minimum mean
tumor volume in all groups was 150 mm3 on the first day of
treatment and groups consisted of 7 animals. Experimental
therapeutic agent, Example 347, was administered to mice orally
(PO). Treatment frequency was 2 times daily for a minimum of 14
days for efficacy. The size of subcutaneous tumors was measured 2
to 3 times weekly using a digital caliper. The tumor volume was
calculated by measuring the tumor in 2 dimensions and utilizing the
equation: Volume=[Length.times.(Width2)]/2; where the larger number
was length, and the smaller number width. If multiple tumors were
formed, the final volume was the sum of the individual tumors
subject to the same equation: eg, 2 tumors;
Volume={[L1.times.(W1)2]/2}+{[L2.times.(W2)2]/2}. Effects on tumor
growth were reported as percent tumor growth inhibition (% TGI).
Percent TGI was calculated with the equation: (1-(Tx vol./control
vol.))*100, where control volume was the vehicle or untreated tumor
volume on a given day, and Tx volume was any treatment group tumor
volume on that same day. Statistical differences between treatment
and vehicle controls were assessed using ANOVA: Single Factor
test.
[0638] Results:
[0639] Example 347 was evaluated as a single agent in the Pfeiffer
human tumor xenograft model of diffuse large B-cell lymphoma, a
subtype of NHL. Pfeiffer cancer cells were shown to be sensitive to
the anti-proliferative effects of Example 347 in vitro. Therefore,
a tumor model was established based on subcutaneous inoculation of
tumor cells into immune compromised SCID mice and tumor-bearing
mice received twice daily oral doses of vehicle or Example 347 at
0.3, 1, 3, or 10 mg/kg for 14 days. Example 347 treatment inhibited
tumor growth by 22%, 24%, 36%, and 58% (percent tumor growth
inhibition) with increasing dose (FIG. 2).
[0640] Various modifications of the invention, in addition to those
described herein, will be apparent to those skilled in the art from
the foregoing description. Such modifications are also intended to
fall within the scope of the appended claims. Each reference,
including all patent, patent applications, and publications, cited
in the present application is incorporated herein by reference in
its entirety.
* * * * *