U.S. patent application number 12/874073 was filed with the patent office on 2010-12-23 for enantiomerically pure aminoheteroaryl compounds as protein kinase inhibitors.
This patent application is currently assigned to AGOURON PHARMACEUTICALS, INC.. Invention is credited to Jingrong Jean Cui, Lee Andrew Funk, Lei Jia, Pei-Pei Kung, Jerry Jialun Meng, Mitchell David Nambu, Mason Alan Pairish, Hong Shen, Michelle Tran-Dube.
Application Number | 20100324061 12/874073 |
Document ID | / |
Family ID | 35967909 |
Filed Date | 2010-12-23 |
United States Patent
Application |
20100324061 |
Kind Code |
A1 |
Cui; Jingrong Jean ; et
al. |
December 23, 2010 |
ENANTIOMERICALLY PURE AMINOHETEROARYL COMPOUNDS AS PROTEIN KINASE
INHIBITORS
Abstract
Enantiomerically pure compound of formula 1 ##STR00001## are
provided, as well as methods for their synthesis and use. Preferred
compounds are potent inhibitors of the c-Met protein kinase, and
are useful in the treatment of abnormal cell growth disorders, such
as cancers.
Inventors: |
Cui; Jingrong Jean; (San
Diego, CA) ; Funk; Lee Andrew; (Oceanside, CA)
; Jia; Lei; (San Diego, CA) ; Kung; Pei-Pei;
(San Diego, CA) ; Meng; Jerry Jialun; (San Diego,
CA) ; Nambu; Mitchell David; (San Diego, CA) ;
Pairish; Mason Alan; (San Diego, CA) ; Shen;
Hong; (San Diego, CA) ; Tran-Dube; Michelle;
(La Jolla, CA) |
Correspondence
Address: |
PFIZER INC
10555 SCIENCE CENTER DRIVE
SAN DIEGO
CA
92121
US
|
Assignee: |
AGOURON PHARMACEUTICALS,
INC.
|
Family ID: |
35967909 |
Appl. No.: |
12/874073 |
Filed: |
September 1, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11212331 |
Aug 26, 2005 |
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12874073 |
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60605086 |
Aug 26, 2004 |
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Current U.S.
Class: |
514/252.11 ;
514/253.12; 514/255.05; 514/255.06; 514/318; 514/341; 514/349;
544/357; 544/360; 544/405; 544/408; 546/194; 546/275.4;
546/297 |
Current CPC
Class: |
A61K 31/4418 20130101;
C07D 403/14 20130101; C07D 401/14 20130101; C07D 241/20 20130101;
C07D 403/04 20130101; C07D 213/76 20130101; C07D 401/04 20130101;
A61K 31/4439 20130101; A61K 31/4545 20130101; A61P 35/02 20180101;
C07D 401/12 20130101; A61K 31/497 20130101; A61K 31/496 20130101;
A61K 31/4965 20130101; A61P 35/00 20180101; A61P 43/00 20180101;
C07D 213/73 20130101; A61K 45/06 20130101 |
Class at
Publication: |
514/252.11 ;
544/408; 544/357; 544/360; 546/194; 546/297; 546/275.4; 544/405;
514/255.06; 514/349; 514/253.12; 514/318; 514/341; 514/255.05 |
International
Class: |
A61K 31/497 20060101
A61K031/497; C07D 241/20 20060101 C07D241/20; C07D 403/10 20060101
C07D403/10; C07D 401/10 20060101 C07D401/10; C07D 401/14 20060101
C07D401/14; C07D 213/73 20060101 C07D213/73; C07D 401/04 20060101
C07D401/04; C07D 403/04 20060101 C07D403/04; A61K 31/4965 20060101
A61K031/4965; A61K 31/4412 20060101 A61K031/4412; A61K 31/496
20060101 A61K031/496; A61K 31/4545 20060101 A61K031/4545; A61K
31/4439 20060101 A61K031/4439; A61P 35/00 20060101 A61P035/00 |
Claims
1. A compound of the formula ##STR00127## wherein: Y is CH R.sup.1
is a furan, thiophene, pyrrole, pyrroline, pyrrolidine, dioxolane,
oxazole, thiazole, imidazole, imidazoline, imidazolidine, pyrazole,
pyrazoline, pyrazolidine, isoxazole, isothiazole, oxadiazole,
triazole, thiadiazole, pyran, pyridine, piperidine, dioxane,
morpholine, dithiane, thiomorpholine, pyridazine, pyrimidine,
pyrazine, piperazine, triazine, trithiane, azitidine or phenyl
group; and each hydrogen in R.sup.1 is optionally substituted by
R.sup.3; each R.sup.3 is independently halogen, C.sub.1-12 alkyl,
C.sub.2-12 alkenyl, C.sub.2-12 alkynyl, C.sub.3-12 cycloalkyl,
C.sub.6-12 aryl, 3-12 membered heteroalicyclic, 5-12 membered
heteroaryl, --S(O).sub.mR.sup.4, --SO.sub.2NR.sup.4R.sup.5,
--S(O).sub.2OR.sup.4, --NO.sub.2, --NR.sup.4R.sup.5, --CN,
--C(O)R.sup.4, --OC(O)R.sup.4, --O(CR.sup.6R.sup.7).sub.nR.sup.4,
--NR.sup.4C(O)R.sup.5, --(CR.sup.6R.sup.7).sub.nC(O)OR.sup.4,
--(CR.sup.6R.sup.7).sub.nOR.sup.4,
--(CR.sup.6R.sup.7).sub.nC(O)NR.sup.4R.sup.5,
--C(.dbd.NR.sup.6)NR.sup.4R.sup.5, --NR.sup.4C(O)NR.sup.5R.sup.6 or
--NR.sup.4S(O).sub.pR.sup.5, each hydrogen in R.sup.3 is optionally
substituted by R.sup.8, and R.sup.3 groups on adjacent atoms may
combine to form a C.sub.6-12 aryl, 5-12 membered heteroaryl,
C.sub.3-12 cycloalkyl or 3-12 membered heteroalicyclic group; each
R.sup.4, R.sup.5, R.sup.6 and R.sup.7 is independently hydrogen,
halogen, C.sub.1-12 alkyl, C.sub.2-12 alkenyl, C.sub.2-12 alkynyl,
C.sub.3-12 cycloalkyl, C.sub.6-12 aryl, 3-12 membered
heteroalicyclic, 5-12 membered heteroaryl; or any two of R.sup.4,
R.sup.5, R.sup.6 and R.sup.7 bound to the same nitrogen atom may,
together with the nitrogen to which they are bound, be combined to
form a 3 to 12 membered heteroalicyclic or 5-12 membered heteroaryl
group optionally containing 1 to 3 additional heteroatoms selected
from N, O, and S; or any two of R.sup.4, R.sup.5, R.sup.6 and
R.sup.7 bound to the same carbon atom may be combined to form a
C.sub.3-12 cycloalkyl, C.sub.6-12 aryl, 3-12 membered
heteroalicyclic or 5-12 membered heteroaryl group; and each
hydrogen in R.sup.4, R.sup.5, R.sup.6 and R.sup.7 is optionally
substituted by R.sup.8; each R.sup.8 is independently halogen,
C.sub.1-12 alkyl. C.sub.2-12 alkenyl, C.sub.2-12 alkynyl,
C.sub.3-12 cycloalkyl, C.sub.6-12 aryl, 3-12 membered
heteroalicyclic, 5-12 membered heteroaryl, --NH.sub.2, --CN, --OH,
--O--C.sub.1-12 alkyl, --O--(CH.sub.2).sub.nC.sub.3-12cycloalkyl,
--O--(CH.sub.2).sub.nC.sub.6-12 aryl, --O--(CH.sub.2).sub.n(3-12
membered heteroalicyclic) or --O--(CH.sub.2).sub.n(5-12 membered
heteroaryl); and each hydrogen in R.sup.8 is optionally substituted
by R.sup.11; each R.sup.11 is independently halogen, C.sub.1-12
alkyl, C.sub.1-12 alkoxy, C.sub.3-12 cycloalkyl, C.sub.6-12 aryl,
3-12 membered heteroalicyclic, 5-12 membered heteroaryl,
--O--C.sub.1-12 alkyl, --O--(CH.sub.2).sub.nC.sub.3-12 cycloalkyl,
--O--(CH.sub.2).sub.nC.sub.6-12 aryl, --O--(CH.sub.2).sub.n(3-12
membered heteroalicyclic), --O--(CH.sub.2).sub.n(5-12 membered
heteroaryl) or --CN, and each hydrogen in R.sup.11 is optionally
substituted by halogen, --OH, --CN, --C.sub.1-12 alkyl which may be
partially or fully halogenated, --O--C.sub.1-12 alkyl which may be
partially or fully halogenated, --CO, --SO or --SO.sub.2; each m is
independently 0, 1 or 2; each n is independently 0, 1, 2, 3 or 4;
each p is independently 1 or 2; or a pharmaceutically acceptable
salt thereof.
2. The compound of claim 1, wherein R.sup.1 is pyrazole, or a
pharmaceutically acceptable salt thereof.
3. The compound of claim 2, wherein R.sup.3 is 3-12 membered
heteroalicyclic, or a pharmaceutically acceptable salt thereof.
4. An enantiomerically pure compound selected from the group
consisting of
5-bromo-3-[(R)-1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-pyrazin-2-ylam-
ine;
5-iodo-3-[(R)1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-pyridin-2-ylami-
ne;
5-bromo-3-[1(R)-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-pyridin-2-ylami-
ne;
4-{5-amino-6-[(R)-1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-pyrazin-2-y-
l}-benzoic acid;
(4-{5-amino-6-[(R)-1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-pyrazin-2-yl}-
-phenyl)-piperazin-1-yl-methanone;
4-(4-{5-amino-6-[(R)-1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-pyrazin-2-y-
l}-benzoyl)-piperazine-1-carboxylic acid tert-butyl ester;
3-[(1R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]-5-[4-(piperazin-1-ylcarbon-
yl)phenyl]pyridin-2-amine;
4-{6-amino-5-[(1R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]pyridin-3-yl}-N--
[2-(dimethylamino)ethyl]-N-methylbenzamide;
(4-{6-amino-5-[(1R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]pyridin-3-yl}ph-
enyl)methanol;
4-{6-amino-5-[(1R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]pyridin-3-yl}-N--
[3-(dimethylamino)propyl]-N-methylbenzamide; tert-butyl
4-(4-{6-amino-5-[(1R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]pyridin-3-yl}-
benzoyl)piperazine-1-carboxylate;
3-[(R)-1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-5-[1-(1-methyl-piperidin--
4-yl)-1H-pyrazol-4-yl]-pyridin-2-ylamine;
1-[4-(4-{6-amino-5-[(R)-1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-pyridin--
3-yl}-pyrazol-1-yl)-piperidin-1-yl]-2-hydroxy-ethanone;
3-[(R)-1-(2,6-dichloro-3-fluoro-phenyl)ethoxy]-5-(1-piperidin-4-yl-1H-pyr-
azol-4-yl)-pyridin-2-ylamine;
3-[(R)-1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-5-(1-piperidin-4-yl-1H-py-
razol-4-yl)-pyrazin-2-ylamine;
3-[(R)-1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-5-(1H-pyrazol-4-yl)-pyraz-
in-2-ylamine;
1-[4-(4-{5-amino-6-[(R)-1-(2,6-dichloro-3-fluorophenyl)-ethoxy]-pyrazin-2-
-yl}-pyrazol-1-yl)-piperidin-1-yl]-2-hydroxy-ethanone;
3-[(R)-1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-5-[1-(1-methyl-piperidin--
4-yl)-1H-pyrazol-4-yl]-pyrazin-2-ylamine;
1-[4-(4-{5-amino-6-[(R)-1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-pyrazin--
2-yl}-pyrazol-1-yl)-piperidin-1-yl]-2-dimethylamino-ethanone;
3-[(R)-1-(2-chloro-3,6-difluoro-phenyl)-ethoxy]-5-(1-piperidin-4-yl-1H-py-
razol-4-yl)-pyridin-2-ylamine; or a pharmaceutically acceptable
salt thereof.
5. A pharmaceutical composition comprising a compound of claim 1,
or a pharmaceutically acceptable salt thereof, and a
pharmaceutically acceptable carrier.
Description
[0001] This is a Continuation of U.S. application Ser. No.
11/212,331, filed Aug. 26, 2005, which claims the benefit of U.S.
Provisional Application No. 60/605,086 filed on Aug. 26, 2004, the
contents of which is hereby incorporated by reference in its
entirety.
FIELD OF THE INVENTION
[0002] The invention relates generally to novel chemical compounds
and methods. More particularly, the invention provides
enantiomerically pure aminoheteroaryl compounds, particularly
aminopyridines and aminopyrazines, having protein tyrosine kinase
activity, and methods of synthesizing and using such compounds.
Preferred compounds are c-Met inhibitors useful for the treatment
of abnormal cell growth, such as cancers.
BACKGROUND
[0003] The hepatocyte growth factor (HGF) receptor (c-MET or HGFR)
receptor tyrosine kinase (RTK) has been shown in many human cancers
to be involved in oncogenesis, tumor progression with enhanced cell
motility and invasion, as well as metastasis (see, e.g., Ma, P. C.,
Maulik, G., Christensen, J. & Salgia, R. (2003b). Cancer
Metastasis Rev, 22, 309-25; Maulik, G., Shrikhande, A., Kijima, T.,
Ma, P. C., Morrison, P. T. & Salgia, R. (2002b). Cytokine
Growth Factor Rev, 13, 41-59). c-MET (HGFR) can be activated
through overexpression or mutations in various human cancers
including small cell lung cancer (SCLC) (Ma, P. C., Kijima, T.,
Maulik, G., Fox, E. A., Sattler, M., Griffin, J. D., Johnson, B. E.
& Salgia, R. (2003a). Cancer Res, 63, 6272-6281).
[0004] c-MET is a receptor tyrosine kinase that is encoded by the
Met proto-oncogene and transduces the biological effects of
hepatocyte growth factor (HGF), which is also referred to as
scatter factor (SF). Jiang et al., Crit. Rev. Oncol. Hematol. 29:
209-248 (1999). c-MET and HGF are expressed in numerous tissues,
although their expression is normally confined predominantly to
cells of epithelial and mesenchymal origin, respectively. c-MET and
HGF are required for normal mammalian development and have been
shown to be important in cell migration, cell proliferation and
survival, morphogenic differentiation, and organization of
3-dimensional tubular structures (e.g., renal tubular cells, gland
formation, etc.). In addition to its effects on epithelial cells,
HGF/SF has been reported to be an angiogenic factor, and c-MET
signaling in endothelial cells can induce many of the cellular
responses necessary for angiogenesis (proliferation, motility,
invasion).
[0005] The c-MET receptor has been shown to be expressed in a
number of human cancers. c-Met and its ligand, HGF, have also been
shown to be co-expressed at elevated levels in a variety of human
cancers (particularly sarcomas). However, because the receptor and
ligand are usually expressed by different cell types, c-MET
signaling is most commonly regulated by tumor-stroma (tumor-host)
interactions. Furthermore, c-MET gene amplification, mutation, and
rearrangement have been observed in a subset of human cancers.
Families with germline mutations that activate c-MET kinase are
prone to multiple kidney tumors as well as tumors in other tissues.
Numerous studies have correlated the expression of c-MET and/or
HGF/SF with the state of disease progression of different types of
cancer (including lung, colon, breast, prostate, liver, pancreas,
brain, kidney, ovaries, stomach, skin, and bone cancers).
Furthermore, the overexpression of c-MET or HGF have been shown to
correlate with poor prognosis and disease outcome in a number of
major human cancers including lung, liver, gastric, and breast.
c-MET has also been directly implicated in cancers without a
successful treatment regimen such as pancreatic cancer, glioma, and
hepatocellular carcinoma.
[0006] Examples of c-MET (HGFR) inhibitors, their synthesis and
use, can be found in U.S. patent application Ser. No. 10/786,610,
entitled "Aminoheteroaryl Compounds as Protein Kinase Inhibitors",
filed Feb. 26, 2004, and corresponding international application
PCT/US2004/005495 of the same title, filed Feb. 26, 2004, the
disclosures of which are incorporated herein by reference in their
entireties.
[0007] It would be desirable to have novel c-MET (HGFR) inhibitors
and methods of using such inhibitors for the treatment of abnormal
cell growth, such as cancer.
SUMMARY
[0008] In one embodiment, the invention provides an
enantiomerically pure compound of formula 1
##STR00002##
wherein:
[0009] Y is N or CR.sup.12;
[0010] R.sup.1 is selected from hydrogen, halogen, C.sub.6-12 aryl,
5-12 membered heteroaryl, C.sub.3-12 cycloalkyl, 3-12 membered
heteroalicyclic, --O(CR.sup.6R.sup.7).sub.nR.sup.4, --C(O)R.sup.4,
--C(O)OR.sup.4, --CN, --NO.sub.2, --S(O).sub.mR.sup.4,
--SO.sub.2NR.sup.4R.sup.5, --C(O)NR.sup.4R.sup.5,
--NR.sup.4C(O)R.sup.5, --C(.dbd.NR.sup.6)NR.sup.4R.sup.5, C.sub.1-8
alkyl, C.sub.2-8 alkenyl, and C.sub.2-8 alkynyl; and each hydrogen
in R.sup.1 is optionally substituted by one or more R.sup.3
groups;
[0011] R.sup.2 is hydrogen, halogen, C.sub.1-12 alkyl, C.sub.2-12
alkenyl, C.sub.2-12 alkynyl, C.sub.3-12 cycloalkyl, C.sub.6-12
aryl, 3-12 membered heteroalicyclic, 5-12 membered heteroaryl,
--S(O).sub.mR.sup.4, --SO.sub.2NR.sup.4R.sup.5,
--S(O).sub.2OR.sup.4, --NO.sub.2, --NR.sup.4R.sup.5,
--(CR.sup.6R.sup.7).sub.nOR.sup.4, --CN, --C(O)R.sup.4,
--OC(O)R.sup.4, --O(CR.sup.6R.sup.7).sub.nR.sup.4,
--NR.sup.4C(O)R.sup.5, --(CR.sup.6R.sup.7).sub.nC(O)OR.sup.4,
--(CR.sup.6R.sup.7).sub.nNCR.sup.4R.sup.5,
--C(.dbd.NR.sup.6)NR.sup.4R.sup.5, --NR.sup.4C(O)NR.sup.5R.sup.6,
--NR.sup.4S(O).sub.pR.sup.5 or --C(O)NR.sup.4R.sup.5, and each
hydrogen in R.sup.2 is optionally substituted by R.sup.8;
[0012] each R.sup.3 is independently halogen, C.sub.1-12 alkyl,
C.sub.2-12 alkenyl, C.sub.2-12 alkynyl, C.sub.3-12 cycloalkyl,
C.sub.6-12 aryl, 3-12 membered heteroalicyclic, 5-12 membered
heteroaryl, --S(O).sub.mR.sup.4, --SO.sub.2NR.sup.4R.sup.5,
--S(O).sub.2OR.sup.4, --NO.sub.2, --NR.sup.4R.sup.5,
--(CR.sup.6R.sup.7).sub.nOR.sup.4, --CN, --C(O)R.sup.4,
--OC(O)R.sup.4, --O(CR.sup.6R.sup.7).sub.nR.sup.4,
--NR.sup.4C(O)R.sup.5, --(CR.sup.6R.sup.7).sub.nC(O)OR.sup.4,
--(CR.sup.6R.sup.7).sub.nOR.sup.4,
--(CR.sup.6R.sup.7).sub.nC(O)NR.sup.4R.sup.5,
--(CR.sup.6R.sup.7).sub.nNCR.sup.4R.sup.5,
--C(.dbd.NR.sup.6)NR.sup.4R.sup.5, --NR.sup.4C(O)NR.sup.5R.sup.6,
--NR.sup.4S(O).sub.pR.sup.5 or --C(O)NR.sup.4R.sup.5, each hydrogen
in R.sup.3 is optionally substituted by R.sup.8, and R.sup.3 groups
on adjacent atoms may combine to form a C.sub.6-12 aryl, 5-12
membered heteroaryl, C.sub.3-12 cycloalkyl or 3-12 membered
heteroalicyclic group;
[0013] each R.sup.4, R.sup.5, R.sup.6 and R.sup.7 is independently
hydrogen, halogen, C.sub.1-12 alkyl, C.sub.2-12 alkenyl, C.sub.2-12
alkynyl, C.sub.3-12 cycloalkyl, C.sub.6-12 aryl, 3-12 membered
heteroalicyclic, 5-12 membered heteroaryl; or any two of R.sup.4,
R.sup.5, R.sup.6 and R.sup.7 bound to the same nitrogen atom may,
together with the nitrogen to which they are bound, be combined to
form a 3 to 12 membered heteroalicyclic or 5-12 membered heteroaryl
group optionally containing 1 to 3 additional heteroatoms selected
from N, O, and S; or any two of R.sup.4, R.sup.5, R.sup.6 and
R.sup.7 bound to the same carbon atom may be combined to form a
C.sub.3-12 cycloalkyl, C.sub.6-12 aryl, 3-12 membered
heteroalicyclic or 5-12 membered heteroaryl group; and each
hydrogen in R.sup.4, R.sup.5, R.sup.6 and R.sup.7 is optionally
substituted by R.sup.8;
[0014] each R.sup.8 is independently halogen, C.sub.1-12 alkyl,
C.sub.2-12 alkenyl, C.sub.2-12 alkynyl, C.sub.3-12 cycloalkyl,
C.sub.6-12 aryl, 3-12 membered heteroalicyclic, 5-12 membered
heteroaryl, --NH.sub.2, --CN, --OH, --O--C.sub.1-12 alkyl,
--O--(CH.sub.2).sub.nC.sub.3-12 cycloalkyl,
--O--(CH.sub.2).sub.nC.sub.6-12 aryl, --O--(CH.sub.2).sub.n(3-12
membered heteroalicyclic) or --O--(CH.sub.2).sub.n(5-12 membered
heteroaryl); and each hydrogen in R.sup.8 is optionally substituted
by R.sup.11;
[0015] each R.sup.9 and R.sup.10 is independently hydrogen,
halogen, C.sub.1-12 alkyl, C.sub.3-12 cycloalkyl, C.sub.6-12 aryl,
3-12 membered heteroalicyclic, 5-12 membered heteroaryl,
--S(O).sub.mR.sup.4, --SO.sub.2NR.sup.4R.sup.5,
--S(O).sub.2OR.sup.4, --NO.sub.2, --NR.sup.4R.sup.5,
--(CR.sup.6R.sup.7).sub.nOR.sup.4, --CN, --C(O)R.sup.4,
--OC(O)R.sup.4, --NR.sup.4C(O)R.sup.5,
--(CR.sup.6R.sup.7).sub.nC(O)OR.sup.4,
--(CR.sup.6R.sup.7).sub.nNCR.sup.4R.sup.5,
--NR.sup.4C(O)NR.sup.5R.sup.6, --NR.sup.4S(O).sub.pR.sup.5 or
--C(O)NR.sup.4R.sup.5; R.sup.9 or R.sup.10 may combine with a ring
atom of A or a substituent of A to form a C.sub.3-12 cycloalkyl,
3-12 membered heteroalicyclic, C.sub.6-12 aryl or 5-12 membered
heteroaryl ring fused to A; and each hydrogen in R.sup.9 and
R.sup.10 is optionally substituted by R.sup.3;
[0016] each R.sup.11 is independently halogen, C.sub.1-12 alkyl,
C.sub.1-12 alkoxy, C.sub.3-12 cycloalkyl, C.sub.6-12 aryl, 3-12
membered heteroalicyclic, 5-12 membered heteroaryl, --O--C.sub.1-12
alkyl, --O--(CH.sub.2).sub.nC.sub.3-12 cycloalkyl,
--O--(CH.sub.2).sub.nC.sub.6-12 aryl, --O--(CH.sub.2).sub.n(3-12
membered heteroalicyclic), --O--(CH.sub.2).sub.n(5-12 membered
heteroaryl) or --CN, and each hydrogen in R.sup.11 is optionally
substituted by halogen, --OH, --CN, --C.sub.1-12 alkyl which may be
partially or fully halogenated, --O--C.sub.1-12 alkyl which may be
partially or fully halogenated, --CO, --SO or --SO.sub.2;
[0017] R.sup.12 is hydrogen, halogen, C.sub.1-12 alkyl, C.sub.2-12
alkenyl, C.sub.2-12 alkynyl, C.sub.3-12 cycloalkyl, C.sub.6-12
aryl, 3-12 membered heteroalicyclic, 5-12 membered heteroaryl,
--S(O).sub.mR.sup.4, --SO.sub.2NR.sup.4R.sup.5,
--S(O).sub.2OR.sup.4, --NO.sub.2, --NR.sup.4R.sup.5,
--(CR.sup.6R.sup.7).sub.nOR.sup.4, --CN, --C(O)R.sup.4,
--OC(O)R.sup.4, --O(CR.sup.6R.sup.7).sub.nR.sup.4,
--NR.sup.4C(O)R.sup.5, --(CR.sup.6R.sup.7).sub.nC(O)OR.sup.4,
--(CR.sup.6R.sup.7).sub.nNCR.sup.4R.sup.5,
--C(.dbd.NR.sup.6)NR.sup.4R.sup.5, --NR.sup.4C(O)NR.sup.5R.sup.6,
--NR.sup.4S(O).sub.pR.sup.5 or --C(O)NR.sup.4R.sup.5, and each
hydrogen in R.sup.12 is optionally substituted by R.sup.3;
[0018] each R.sup.13 is independently halogen, C.sub.1-12 alkyl,
C.sub.2-12 alkenyl, C.sub.2-12 alkynyl, C.sub.3-12 cycloalkyl,
C.sub.6-12 aryl, 3-12 membered heteroalicyclic, 5-12 membered
heteroaryl, --S(O).sub.mR.sup.4, --SO.sub.2NR.sup.4R.sup.5,
--S(O).sub.2OR.sup.4, --NO.sub.2, --NR.sup.4R.sup.5,
--(CR.sup.6R.sup.7).sub.nOR.sup.4, --CN, --C(O)R.sup.4,
--OC(O)R.sup.4, --O(CR.sup.6R.sup.7).sub.nR.sup.4,
--NR.sup.4C(O)R.sup.5, --(CR.sup.6R.sup.7).sub.nC(O)OR.sup.4,
--(CR.sup.6R.sup.7).sub.nOR.sup.4,
--(CR.sup.6R.sup.7).sub.nC(O)NR.sup.4R.sup.5,
--(CR.sup.6R.sup.7).sub.nNCR.sup.4R.sup.5,
--C(.dbd.NR.sup.6)NR.sup.4R.sup.5, --NR.sup.4C(O)NR.sup.5R.sup.6,
--NR.sup.4S(O).sub.pR.sup.5, --C(O)NR.sup.4R.sup.5,
--(CR.sup.6R.sup.7).sub.n(3-12 membered heteroalicyclic),
--(CR.sup.6R.sup.7).sub.n(C.sub.3-12 cycloalkyl),
--(CR.sup.6R.sup.7).sub.n(C.sub.6-12 aryl),
--(CR.sup.6R.sup.7).sub.n(5-12 membered heteroaryl),
--(CR.sup.6R.sup.7).sub.nC(O)NR.sup.4R.sup.5, or
--(CR.sup.6R.sup.7).sub.nC(O)R.sup.4, R.sup.13 groups on adjacent
atoms may combine to form a C.sub.6-12 aryl, 5-12 membered
heteroaryl, C.sub.3-12 cycloalkyl or 3-12 membered heteroalicyclic
group, and each hydrogen in R.sup.13 is optionally substituted by
R.sup.3;
[0019] each m is independently 0, 1 or 2;
[0020] each n is independently 0, 1, 2, 3 or 4;
[0021] each p is independently 1 or 2;
or a pharmaceutically acceptable salt, hydrate or solvate
thereof.
[0022] In a particular aspect of this embodiment, R.sup.2 is
hydrogen.
[0023] In another particular aspect of this embodiment, Y is N.
[0024] In another particular aspect of this embodiment, Y is N and
R.sup.2 is hydrogen.
[0025] In another particular aspect of this embodiment, Y is
CR.sup.12.
[0026] In another particular aspect of this embodiment, Y is
CR.sup.12 and R.sup.12 is H.
[0027] In another particular aspect of this embodiment, and in
combination with any other particular aspect not inconsistent,
R.sup.1 is a furan, thiopene, pyrrole, pyrroline, pyrrolidine,
dioxolane, oxazole, thiazole, imidazole, imidazoline,
imidazolidine, pyrazole, pyrazoline, pyrazolidine, isoxazole,
isothiazole, oxadiazole, triazole, thiadiazole, pyran, pyridine,
piperidine, dioxane, morpholine, dithiane, thiomorpholine,
pyridazine, pyrimidine, pyrazine, piperazine, triazine, trithiane
or phenyl group, and each hydrogen in R.sup.1 is optionally
substituted by one or more R.sup.3 groups.
[0028] In another particular aspect of this embodiment, and in
combination with any other particular aspect not inconsistent,
R.sup.1 is a fused ring heteroaryl group, and each hydrogen in
R.sup.1 is optionally substituted by one or more R.sup.3
groups.
[0029] In another particular aspect of this embodiment, and in
combination with any other particular aspect not inconsistent,
R.sup.1 is hydrogen.
[0030] In another particular aspect of this embodiment, and in
combination with any other particular aspect not inconsistent,
R.sup.1 is a halogen.
[0031] In another embodiment, the invention provides an
enantiomerically pure compound of formula 1a
##STR00003##
wherein:
[0032] Y is N or CH;
[0033] R.sup.1 is a furan, thiopene, pyrrole, pyrroline,
pyrrolidine, dioxolane, oxazole, thiazole, imidazole, imidazoline,
imidazolidine, pyrazole, pyrazoline, pyrazolidine, isoxazole,
isothiazole, oxadiazole, triazole, thiadiazole, pyran, pyridine,
piperidine, dioxane, morpholine, dithiane, thiomorpholine,
pyridazine, pyrimidine, pyrazine, piperazine, triazine, trithiane,
azitidine or phenyl group; and each hydrogen in R.sup.1 is
optionally substituted by R.sup.3;
[0034] each R.sup.3 is independently halogen, C.sub.1-12 alkyl,
C.sub.2-12 alkenyl, C.sub.2-12 alkynyl, C.sub.3-12 cycloalkyl,
C.sub.6-12 aryl, 3-12 membered heteroalicyclic, 5-12 membered
heteroaryl, --S(O).sub.mR.sup.4, --SO.sub.2NR.sup.4R.sup.5,
--S(O).sub.2OR.sup.4, --NO.sub.2, --NR.sup.4R.sup.5,
--(CR.sup.6R.sup.7).sub.nOR.sup.4, --CN, --C(O)R.sup.4,
--OC(O)R.sup.4, --O(CR.sup.6R.sup.7).sub.nR.sup.4,
--NR.sup.4C(O)R.sup.5, --(CR.sup.6R.sup.7).sub.nC(O)OR.sup.4,
--(CR.sup.6R.sup.7).sub.nOR.sup.4,
--(CR.sup.6R.sup.7).sub.nC(O)NR.sup.4R.sup.5,
--(CR.sup.6R.sup.7).sub.nNCR.sup.4R.sup.5,
--C(.dbd.NR.sup.6)NR.sup.4R.sup.5, --NR.sup.4C(O)NR.sup.5R.sup.6,
--NR.sup.4S(O).sub.pR.sup.5 or --C(O)NR.sup.4R.sup.5, each hydrogen
in R.sup.3 is optionally substituted by R.sup.8, and R.sup.3 groups
on adjacent atoms may combine to form a C.sub.6-12 aryl, 5-12
membered heteroaryl, C.sub.3-12 cycloalkyl or 3-12 membered
heteroalicyclic group;
[0035] each R.sup.4, R.sup.5, R.sup.6 and R.sup.7 is independently
hydrogen, halogen, C.sub.1-12 alkyl, C.sub.2-12 alkenyl, C.sub.2-12
alkynyl, C.sub.3-12 cycloalkyl, C.sub.6-12 aryl, 3-12 membered
heteroalicyclic, 5-12 membered heteroaryl; or any two of R.sup.4,
R.sup.5, R.sup.6 and R.sup.7 bound to the same nitrogen atom may,
together with the nitrogen to which they are bound, be combined to
form a 3 to 12 membered heteroalicyclic or 5-12 membered heteroaryl
group optionally containing 1 to 3 additional heteroatoms selected
from N, O, and S; or any two of R.sup.4, R.sup.5, R.sup.6 and
R.sup.7 bound to the same carbon atom may be combined to form a
C.sub.3-12 cycloalkyl, C.sub.6-12 aryl, 3-12 membered
heteroalicyclic or 5-12 membered heteroaryl group; and each
hydrogen in R.sup.4, R.sup.5, R.sup.6 and R.sup.7 is optionally
substituted by R.sup.8;
[0036] each R.sup.8 is independently halogen, C.sub.1-12 alkyl,
C.sub.2-12 alkenyl, C.sub.2-12 alkynyl, C.sub.3-12 cycloalkyl,
C.sub.6-12 aryl, 3-12 membered heteroalicyclic, 5-12 membered
heteroaryl, --NH.sub.2, --CN, --OH, --O--C.sub.1-12 alkyl,
--O--(CH.sub.2).sub.nC.sub.3-12 cycloalkyl,
--O--(CH.sub.2).sub.nC.sub.6-12 aryl, --O--(CH.sub.2).sub.n(3-12
membered heteroalicyclic) or --O--(CH.sub.2).sub.n(5-12 membered
heteroaryl); and each hydrogen in R.sup.8 is optionally substituted
by R.sup.11;
[0037] each R.sup.9 and R.sup.10 is independently hydrogen,
halogen, C.sub.1-12 alkyl, C.sub.3-12 cycloalkyl, C.sub.6-12 aryl,
3-12 membered heteroalicyclic, 5-12 membered heteroaryl,
--S(O).sub.mR.sup.4, --SO.sub.2NR.sup.4R.sup.5,
--S(O).sub.2OR.sup.4, --NO.sub.2, --NR.sup.4R.sup.5,
--(CR.sup.6R.sup.7).sub.nOR.sup.4, --CN, --C(O)R.sup.4,
--OC(O)R.sup.4, --NR.sup.4C(O)R.sup.5,
--(CR.sup.6R.sup.7).sub.nC(O)OR.sup.4,
--(CR.sup.6R.sup.7).sub.nNCR.sup.4R.sup.5,
--NR.sup.4C(O)NR.sup.5R.sup.6, --NR.sup.4S(O).sub.pR.sup.5 or
--C(O)NR.sup.4R.sup.5; R.sup.9 or R.sup.10 may combine with a ring
atom of A or a substituent of A to form a C.sub.3-12 cycloalkyl,
3-12 membered heteroalicyclic, C.sub.6-12 aryl or 5-12 membered
heteroaryl ring fused to A; and each hydrogen in R.sup.9 and
R.sup.10 is optionally substituted by R.sup.3;
[0038] each R.sup.11 is independently halogen, C.sub.1-12 alkyl,
C.sub.1-12 alkoxy, C.sub.3-12 cycloalkyl, C.sub.6-12 aryl, 3-12
membered heteroalicyclic, 5-12 membered heteroaryl, --O--C.sub.1-12
alkyl, --O--(CH.sub.2).sub.nC.sub.3-12 cycloalkyl,
--O--(CH.sub.2).sub.nC.sub.6-12 aryl, --O--(CH.sub.2).sub.n(3-12
membered heteroalicyclic), --O--(CH.sub.2).sub.n(5-12 membered
heteroaryl) or --CN, and each hydrogen in R.sup.11 is optionally
substituted by halogen, --OH, --CN, --C.sub.1-12 alkyl which may be
partially or fully halogenated, --O--C.sub.1-12 alkyl which may be
partially or fully halogenated, --CO, --SO or --SO.sub.2;
[0039] each R.sup.13 is independently halogen, C.sub.1-12 alkyl,
C.sub.2-12 alkenyl, C.sub.2-12 alkynyl, C.sub.3-12 cycloalkyl,
C.sub.6-12 aryl, 3-12 membered heteroalicyclic, 5-12 membered
heteroaryl, --S(O).sub.mR.sup.4, --SO.sub.2NR.sup.4R.sup.5,
--S(O).sub.2OR.sup.4, --NO.sub.2, --NR.sup.4R.sup.5,
--(CR.sup.6R.sup.7).sub.nOR.sup.4, --CN, --C(O)R.sup.4,
--OC(O)R.sup.4, --O(CR.sup.6R.sup.7).sub.nR.sup.4,
--NR.sup.4C(O)R.sup.5, --(CR.sup.6R.sup.7).sub.nC(O)OR.sup.4,
--(CR.sup.6R.sup.7).sub.nOR.sup.4,
--(CR.sup.6R.sup.7).sub.nC(O)NR.sup.4R.sup.5,
--(CR.sup.6R.sup.7).sub.nNCR.sup.4R.sup.5,
--C(.dbd.NR.sup.6)NR.sup.4R.sup.5, --NR.sup.4C(O)NR.sup.5R.sup.6,
--NR.sup.4S(O).sub.pR.sup.5, --C(O)NR.sup.4R.sup.5,
--(CR.sup.6R.sup.7).sub.n(3-12 membered heteroalicyclic),
--(CR.sup.6R.sup.7).sub.n(C.sub.3-12 cycloalkyl),
--(CR.sup.6R.sup.7).sub.n(C.sub.6-12 aryl),
--(CR.sup.6R.sup.7).sub.n(5-12 membered heteroaryl),
--(CR.sup.6R.sup.7).sub.nC(O)NR.sup.4R.sup.5, or
--(CR.sup.6R.sup.7).sub.nC(O)R.sup.4, R.sup.13 groups on adjacent
atoms may combine to form a C.sub.6-12 aryl, 5-12 membered
heteroaryl, C.sub.3-12 cycloalkyl or 3-12 membered heteroalicyclic
group, and each hydrogen in R.sup.13 is optionally substituted by
R.sup.3;
[0040] each m is independently 0, 1 or 2;
[0041] each n is independently 0, 1, 2, 3 or 4;
[0042] each p is independently 1 or 2;
or a pharmaceutically acceptable salt, hydrate or solvate
thereof.
[0043] In another embodiment, the invention provides an
enantiomerically pure compound selected from the group consisting
of
5-Bromo-3-[(R)-1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-pyrazin-2-ylamine-
;
5-iodo-3-[(R)-1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-pyridin-2-ylamine-
;
5-bromo-3-[1(R)-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-pyridin-2-ylamine-
;
4-{5-Amino-6-[(R)-1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-pyrazin-2-yl}-
-benzoic acid;
(4-{5-Amino-6-[(R)-1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-pyrazin-2-yl}-
-phenyl)-piperazin-1-yl-methanone;
4-(4-{5-Amino-6-[(R)-1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-pyrazin-2-y-
l}-benzoyl)-piperazine-1-carboxylic acid tert-butyl ester;
3-[(1R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]-5-[4-(piperazin-1-ylcarbon-
yl)phenyl]pyridin-2-amine;
4-{6-amino-5-[(1R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]pyridin-3-yl}-N--
[2-(dimethylamino)ethyl]-N-methylbenzamide;
(4-{6-amino-5-[(1R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]pyridin-3-yl}ph-
enyl)methanol;
4-{6-amino-5-[(1R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]pyridin-3-yl}-N--
[3-(dimethylamino)propyl]-N-methylbenzamide; tert-butyl
4-(4-{6-amino-5-[(1R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]pyridin-3-yl}-
benzoyl)piperazine-1-carboxylate;
3-[(R)-1-(2,6-Dichloro-3-fluoro-phenyl)-ethoxy]-5-[1-(1-methyl-piperidin--
4-yl)-1H-pyrazol-4-yl]-pyridin-2-ylamine;
1-[4-(4-{6-Amino-5-[(R)-1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-pyridin--
3-yl}-pyrazol-1-yl)-piperidin-1-yl]-2-hydroxy-ethanone;
3-[(R)-1-(2,6-Dichloro-3-fluoro-phenyl)-ethoxy]-5-(1-piperidin-4-yl-1H-py-
razol-4-yl)-pyridin-2-ylamine;
3-[(R)-1-(2,6-Dichloro-3-fluoro-phenyl]-ethoxy]-5-(1-piperidin-4-yl-1H-py-
razol-4-yl)-pyridin-2-ylamine;
3-[(R)-1-(2,6-Dichloro-3-fluoro-phenyl)-ethoxy]-5-(1-piperidin-4-yl-1H-py-
razol-4-yl)-pyrazin-2-ylamine;
3-[(R)-1-(2,6-Dichloro-3-fluoro-phenyl)-ethoxy]-5-(1H-pyrazol-4-yl)-pyraz-
in-2-ylamine;
1-[4-(4-{5-Amino-6-[(R)-1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-pyrazin--
2-yl}-pyrazol-1-yl)-piperidin-1-yl]-2-hydroxy-ethanone;
3-[(R)-1-(2,6-Dichloro-3-fluoro-phenyl)-ethoxy]-5-[1-(1-methyl-piperidin--
4-yl)-1H-pyrazol-4-yl]-pyrazin-2-ylamine;
1-[4-(4-{5-Amino-6-[(R)-1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-pyrazin--
2-yl}-pyrazol-1-yl)-piperidin-1-yl]-2-dimethylamino-ethanone;
3-[(R)-1-(2-Chloro-3,6-difluoro-phenyl)-ethoxy]-5-(1-piperidin-4-yl-1H-py-
razol-4-yl)-pyridin-2-ylamine; or a pharmaceutically acceptable
salt, solvate or hydrate thereof.
[0044] In another embodiment, the invention provides a
pharmaceutical composition comprising any of the compounds of the
invention and a pharmaceutically acceptable carrier. Examples of
such compositions are described below.
[0045] Preferred compounds of the invention include those having
c-MET inhibitory activity as defined by any one or more of
IC.sub.50, Ki, or percent inhibition (% I). One skilled in the art
can readily determine if a compound has such activity by carrying
out the appropriate assay, and descriptions of such assays are
shown in the Examples section herein. In one embodiment,
particularly preferred compounds have a c-MET Ki of less than 5
.mu.M or less than 2 .mu.M, or less than 1 .mu.M, or less than 500
nM or less than 200 nM or less than 100 nM. In another embodiment,
particularly preferred compounds have a c-MET inhibition at 1 .mu.M
of at least 10% or at least 20% or at least 30% or at least 40% or
at least 50% or at least 60% or at least 70% or at least 80% or at
least 90%. Methods for measuring c-MET/HGFR activity are described
in the Examples herein.
[0046] In another embodiment, the invention provides a method of
treating abnormal cell growth in a mammal, including a human, the
method comprising administering to the mammal any of the
pharmaceutical compositions of the invention.
[0047] In a specific embodiment of any of the inventive methods
described herein, the abnormal cell growth is cancer, including,
but not limited to, lung cancer, bone cancer, pancreatic cancer,
skin cancer, cancer of the head or neck, cutaneous or intraocular
melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of
the anal region, stomach cancer, colon cancer, breast cancer,
uterine cancer, carcinoma of the fallopian tubes, carcinoma of the
endometrium, carcinoma of the cervix, carcinoma of the vagina,
carcinoma of the vulva, Hodgkin's Disease, cancer of the esophagus,
cancer of the small intestine, cancer of the endocrine system,
cancer of the thyroid gland, cancer of the parathyroid gland,
cancer of the adrenal gland, sarcoma of soft tissue, cancer of the
urethra, cancer of the penis, prostate cancer, chronic or acute
leukemia, lymphocytic lymphomas, cancer of the bladder, cancer of
the kidney or ureter, renal cell carcinoma, carcinoma of the renal
pelvis, neoplasms of the central nervous system (CNS), primary CNS
lymphoma, spinal axis tumors, brain stem glioma, pituitary adenoma,
or a combination of one or more of the foregoing cancers. In
another embodiment of said method, said abnormal cell growth is a
benign proliferative disease, including, but not limited to,
psoriasis, benign prostatic hypertrophy or restinosis.
[0048] In another embodiment, the invention provides a method of
treating an HGFR mediated disorder in a mammal, including a human,
the method comprising administering to the mammal any of the
pharmaceutical compositions of the invention.
[0049] In further specific embodiments of any of the inventive
methods described herein, the method further comprises
administering to the mammal an amount of one or more substances
selected from anti-tumor agents, anti-angiogenesis agents, signal
transduction inhibitors, and antiproliferative agents, which
amounts are together effective in treating said abnormal cell
growth. Such substances include those disclosed in PCT Publication
Nos. WO 00/38715, WO 00/38716, WO 00/38717, WO 00/38718, WO
00/38719, WO 00/38730, WO 00/38665, WO 00/37107 and WO 00/38786,
the disclosures of which are incorporated herein by reference in
their entireties.
[0050] Examples of anti-tumor agents include mitotic inhibitors,
for example vinca alkaloid derivatives such as vinblastine
vinorelbine, vindescine and vincristine; colchines allochochine,
halichondrine, N-benzoyltrimethyl-methyl ether colchicinic acid,
dolastatin 10, maystansine, rhizoxine, taxanes such as taxol
(paclitaxel), docetaxel (Taxotere),
2'-N-[3-(dimethylamino)propyl]glutaramate (taxol derivative),
thiocholchicine, trityl cysteine, teniposide, methotrexate,
azathioprine, fluorouricil, cytocine arabinoside,
2'2'-difluorodeoxycytidine (gemcitabine), adriamycin and mitamycin.
Alkylating agents, for example cis-platin, carboplatin oxiplatin,
iproplatin, Ethyl ester of N-acetyl-DL-sarcosyl-L-leucine (Asaley
or Asalex), 1,4-cyclohexadiene-1,4-dicarbamic acid,
2,5-bis(1-azirdinyl)-3,6-dioxo-, diethyl ester (diaziquone),
1,4-bis(methanesulfonyloxy)butane (bisulfan or leucosulfan)
chlorozotocin, clomesone, cyanomorpholinodoxorubicin, cyclodisone,
dianhydroglactitol, fluorodopan, hepsulfam, mitomycin C,
hycantheonemitomycin C, mitozolamide,
1-(2-chloroethyl)-4-(3-chloropropyl)-piperazine dihydrochloride,
piperazinedione, pipobroman, porfiromycin, spirohydantoin mustard,
teroxirone, tetraplatin, thiotepa, triethylenemelamine, uracil
nitrogen mustard, bis(3-mesyloxypropyl)amine hydrochloride,
mitomycin, nitrosoureas agents such as
cyclohexyl-chloroethylnitrosourea,
methylcyclohexyl-chloroethylnitrosourea
1-(2-chloroethyl)-3-(2,6-dioxo-3-piperidyl)-1-nitroso-urea,
bis(2-chloroethyl)nitrosourea, procarbazine, dacarbazine, nitrogen
mustard-related compounds such as mechloroethamine,
cyclophosphamide, ifosamide, melphalan, chlorambucil, estramustine
sodium phosphate, strptozoin, and temozolamide. DNA
anti-metabolites, for example 5-fluorouracil, cytosine arabinoside,
hydroxyurea,
2-[(3hydroxy-2-pyrinodinyl)methylene]-hydrazinecarbothioamide,
deoxyfluorouridine, 5-hydroxy-2-formyl pyridine thiosemicarbazone,
alpha-2'-deoxy-6-thioguanosine, aphidicolin glycinate,
5-azadeoxycytidine, beta-thioguanine deoxyriboside, cyclocytidine,
guanazole, inosine glycodialdehyde, macbecin II, pyrazolimidazole,
cladribine, pentostatin, thioguanine, mercaptopurine, bleomycin,
2-chlorodeoxyadenosine, inhibitors of thymidylate synthase such as
raltitrexed and pemetrexed disodium, clofarabine, floxuridine and
fludarabine. DNA/RNA antimetabolites, for example, L-alanosine,
5-azacytidine, acivicin, aminopterin and derivatives thereof such
as
N-[2-chloro-5-[[(2,4-diamino-5-methyl-6-quinazolinyl)methyl]amino]benzoyl-
]-L-aspartic acid,
N-[4-[[(2,4-diamino-5-ethyl-6-quinazolinyl)methyl]amino]benzoyl]-L-aspart-
ic acid,
N-[2-chloro-4-[[(2,4-diaminopteridinyl)methyl]amino]benzoyl]-L-as-
partic acid, soluble Baker's antifol, dichloroallyl lawsone,
brequinar, ftoraf, dihydro-5-azacytidine, methotrexate,
N-(phosphonoacetyl)-L-aspartic acid tetrasodium salt, pyrazofuran,
trimetrexate, plicamycin, actinomycin D, cryptophycin, and analogs
such as cryptophycin-52 or, for example, one of the preferred
anti-metabolites disclosed in European Patent Application No.
239362 such as
N-(5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)-N-methylamino]--
2-thenoyl)-L-glutamic acid; growth factor inhibitors; cell cycle
inhibitors; intercalating antibiotics, for example adriamycin and
bleomycin; proteins, for example interferon; and anti-hormones, for
example anti-estrogens such as Nolvadex.TM. (tamoxifen) or, for
example anti-androgens such as Casodex.TM.
(4'-cyano-3-(4-fluorophenylsulphonyl)-2-hydroxy-2-methyl-3'-(trifluoromet-
hyl)propionanilide). Such conjoint treatment may be achieved by way
of the simultaneous, sequential or separate dosing of the
individual components of the treatment.
[0051] Anti-angiogenesis agents include MMP-2
(matrix-metalloprotienase 2) inhibitors, MMP-9
(matrix-metalloprotienase 9) inhibitors, and COX-II (cyclooxygenase
II) inhibitors. Examples of useful COX-II inhibitors include
CELEBREX.TM. (alecoxib), valdecoxib, and rofecoxib. Examples of
useful matrix metalloproteinase inhibitors are described in WO
96/33172 (published Oct. 24, 1996), WO 96/27583 (published Mar. 7,
1996), European Patent Application No. 97304971.1 (filed Jul. 8,
1997), European Patent Application No. 99308617.2 (filed Oct. 29,
1999), WO 98/07697 (published Feb. 26, 1998), WO 98/03516
(published Jan. 29, 1998), WO 98/34918 (published Aug. 13, 1998),
WO 98/34915 (published Aug. 13, 1998), WO 98/33768 (published Aug.
6, 1998), WO 98/30566 (published Jul. 16, 1998), European Patent
Publication 606,046 (published Jul. 13, 1994), European Patent
Publication 931,788 (published Jul. 28, 1999), WO 90/05719
(published May 331, 1990), WO 99/52910 (published Oct. 21, 1999),
WO 99/52889 (published Oct. 21, 1999), WO 99/29667 (published Jun.
17, 1999), PCT International Application No. PCT/IB98/01113 (filed
Jul. 21, 1998), European Patent Application No. 99302232.1 (filed
Mar. 25, 1999), Great Britain patent application number 9912961.1
(filed Jun. 3, 1999), U.S. Provisional Application No. 60/148,464
(filed Aug. 12, 1999), U.S. Pat. No. 5,863,949 (issued Jan. 26,
1999), U.S. Pat. No. 5,861,510 (issued Jan. 19, 1999), and European
Patent Publication 780,386 (published Jun. 25, 1997), all of which
are herein incorporated by reference in their entirety. Preferred
MMP-2 and MMP-9 inhibitors are those that have little or no
activity inhibiting MMP-1. More preferred, are those that
selectively inhibit MMP-2 and/or MMP-9 relative to the other
matrix-metalloproteinases (i.e. MMP-1, MMP-3, MMP-4, MMP-5, MMP-6,
MMP-7, MMP-8, MMP-10, MMP-11, MMP-12, and MMP-13).
[0052] Examples of MMP inhibitors include AG-3340, RO 32-3555, RS
13-0830, and the following compounds:
3-[[4-(4-fluoro-phenoxy)-benzenesulfonyl]-(1-hydroxycarbamoyl-cyclopentyl-
)-amino]-propionic acid;
3-exo-3-[4-(4-fluoro-phenoxy)-benzenesulfonylamino]-8-oxa-bicyclo[3.2.1]o-
ctane-3-carboxylic acid hydroxyamide; (2R, 3R)
1-[4-(2-chloro-4-fluoro-benzyloxy)-benzenesulfonyl]-3-hydroxy-3-methyl-pi-
peridine-2-carboxylic acid hydroxyamide;
4-[4-(4-fluoro-phenoxy)-benzenesulfonylamino]-tetrahydro-pyran-4-carboxyl-
ic acid hydroxyamide;
3-[[4-(4-fluoro-phenoxy)-benzenesulfonyl]-(1-hydroxycarbamoyl-cyclobutyl]-
-amino]-propionic acid;
4-[4-(4-chloro-phenoxy)-benzenesulfonylamino]-tetrahydro-pyran-4-carboxyl-
ic acid hydroxyamide;
3-[4-(4-chloro-phenoxy)-benzenesulfonylamino]-tetrahydro-pyran-3-carboxyl-
ic acid hydroxyamide; (2R,3R)
1-[4-(4-fluoro-2-methyl-benzyloxy)-benzenesulfonyl]-3-hydroxy-3-methyl-pi-
peridine-2-carboxylic acid hydroxyamide;
3-[[4-(4-fluoro-phenoxy)-benzenesulfonyl]-(1-hydroxycarbamoyl-1-methyl-et-
hyl)-amino]-propionic acid;
3-[[4-(4-fluoro-phenoxy)-benzenesulfonyl]-(4-hydroxycarbamoyl-tetrahydro--
pyran-4-yl)-amino]-propionic acid;
3-exo-3-[4-(4-chloro-phenoxy)-benzenesulfonylamino]-8-oxa-bicyclo[3.2.1]o-
ctane-3-carboxylic acid hydroxyamide;
3-endo-3-[4-(4-fluoro-phenoxy)-benzenesulfonylamino]-8-oxa-bicyclo[3.2.1]-
octane-3-carboxylic acid hydroxyamide;
3-[4-(4-fluoro-phenoxy)-benzenesulfonylamino]-tetrahydro-furan-3-carboxyl-
ic acid hydroxyamide; and pharmaceutically acceptable salts,
solvates and hydrates thereof.
[0053] Examples of signal transduction inhibitors include agents
that can inhibit EGFR (epidermal growth factor receptor) responses,
such as EGFR antibodies, EGF antibodies, and molecules that are
EGFR inhibitors; VEGF (vascular endothelial growth factor)
inhibitors; and erbB2 receptor inhibitors, such as organic
molecules or antibodies that bind to the erbB2 receptor, for
example, HERCEPTIN.TM. (Genentech, Inc. of South San Francisco,
Calif., USA).
[0054] EGFR inhibitors are described in, for example in WO 95/19970
(published Jul. 27, 1995), WO 98/14451 (published Apr. 9, 1998), WO
98/02434 (published Jan. 22, 1998), and U.S. Pat. No. 5,747,498
(issued May 5, 1998). EGFR-inhibiting agents include, but are not
limited to, the monoclonal antibodies C225 and anti-EGFR 22Mab
(ImClone Systems Incorporated of New York, N.Y., USA), the
compounds ZD-1839 (AstraZeneca), BIBX-1382 (Boehringer Ingelheim),
MDX-447 (Medarex Inc. of Annandale, N.J., USA), and OLX-103 (Merck
& Co. of Whitehouse Station, N.J., USA), VRCTC-310 (Ventech
Research) and EGF fusion toxin (Seragen Inc. of Hopkinton,
Mass.).
[0055] VEGF inhibitors, for example SU-5416 and SU-6668 (Sugen Inc.
of South San Francisco, Calif., USA), can also be combined or
co-administered with the composition. VEGF inhibitors are described
in, for example in WO 99/24440 (published May 20, 1999), PCT
International Application PCT/IB99/00797 (filed May 3, 1999), in WO
95/21613 (published Aug. 17, 1995), WO 99/61422 (published Dec. 2,
1999), U.S. Pat. No. 5,834,504 (issued Nov. 10, 1998), WO 98/50356
(published Nov. 12, 1998), U.S. Pat. No. 5,883,113 (issued Mar. 16,
1999), U.S. Pat. No. 5,886,020 (issued Mar. 23, 1999), U.S. Pat.
No. 5,792,783 (issued Aug. 11, 1998), WO 99/10349 (published Mar.
4, 1999), WO 97/32856 (published Sep. 12, 1997), WO 97/22596
(published Jun. 26, 1997), WO 98/54093 (published Dec. 3, 1998), WO
98/02438 (published Jan. 22, 1998), WO 99/16755 (published Apr. 8,
1999), and WO 98/02437 (published Jan. 22, 1998), all of which are
herein incorporated by reference in their entirety. Other examples
of some specific VEGF inhibitors are IM862 (Cytran Inc. of
Kirkland, Wash., USA); anti-VEGF monoclonal antibody bevacizumab
(Genentech, Inc. of South San Francisco, Calif.); and angiozyme, a
synthetic ribozyme from Ribozyme (Boulder, Colo.) and Chiron
(Emeryville, Calif.).
[0056] ErbB2 receptor inhibitors, such as GW-282974 (Glaxo Wellcome
plc), and the monoclonal antibodies AR-209 (Aronex Pharmaceuticals
Inc. of The Woodlands, Tex., USA) and 2B-1 (Chiron), may be
administered in combination with the composition. Such erbB2
inhibitors include those described in WO 98/02434 (published Jan.
22, 1998), WO 99/35146 (published Jul. 15, 1999), WO 99/35132
(published Jul. 15, 1999), WO 98/02437 (published Jan. 22, 1998),
WO 97/13760 (published Apr. 17, 1997), WO 95/19970 (published Jul.
27, 1995), U.S. Pat. No. 5,587,458 (issued Dec. 24, 1996), and U.S.
Pat. No. 5,877,305 (issued Mar. 2, 1999), each of which is herein
incorporated by reference in its entirety. ErbB2 receptor
inhibitors useful in the present invention are also described in
U.S. Provisional Application No. 60/117,341, filed Jan. 27, 1999,
and in U.S. Provisional Application No. 60/117,346, filed Jan. 27,
1999, both of which are herein incorporated by reference in their
entirety.
[0057] Other antiproliferative agents that may be used include
inhibitors of the enzyme farnesyl protein transferase and
inhibitors of the receptor tyrosine kinase PDGFr, including the
compounds disclosed and claimed in the following U.S. patent
application Ser. Nos. 09/221,946 (filed Dec. 28, 1998); 09/454,058
(filed Dec. 2, 1999); 09/501,163 (filed Feb. 9, 2000); 09/539,930
(filed Mar. 31, 2000); 09/202,796 (filed May 22, 1997); 09/384,339
(filed Aug. 26, 1999); and 09/383,755 (filed Aug. 26, 1999); and
the compounds disclosed and claimed in the following U.S.
provisional patent applications 60/168,207 (filed Nov. 30, 1999);
60/170,119 (filed Dec. 10, 1999); 60/177,718 (filed Jan. 21, 2000);
60/168,217 (filed Nov. 30, 1999), and 60/200,834 (filed May 1,
2000). Each of the foregoing patent applications and provisional
patent applications is herein incorporated by reference in their
entirety.
[0058] Compositions of the invention can also be used with other
agents useful in treating abnormal cell growth or cancer,
including, but not limited to, agents capable of enhancing
antitumor immune responses, such as CTLA4 (cytotoxic lymphocite
antigen 4) antibodies, and other agents capable of blocking CTLA4;
and anti-proliferative agents such as other farnesyl protein
transferase inhibitors. Specific CTLA4 antibodies that can be used
in the present invention include those described in U.S.
Provisional Application 60/113,647 (filed Dec. 23, 1998) which is
herein incorporated by reference in its entirety.
DEFINITIONS
[0059] Unless otherwise stated, the following terms used in the
specification and claims have the meanings discussed below.
Variables defined in this section, such as R, X, n and the like,
are for reference within this section only, and are not meant to
have the save meaning as may be used outside of this definitions
section. Further, many of the groups defined herein can be
optionally substituted. The listing in this definitions section of
typical substituents is exemplary and is not intended to limit the
substituents defined elsewhere within this specification and
claims.
[0060] "Alkyl" refers to a saturated aliphatic hydrocarbon radical
including straight chain and branched chain groups of 1 to 20
carbon atoms, preferably 1 to 12 carbon atoms, more preferably 1 to
8 carbon atoms, or 1 to 6 carbon atoms, or 1 to 4 carbon atoms.
"Lower alkyl" refers specifically to an alkyl group with 1 to 4
carbon atoms. Examples of alkyl groups include methyl, ethyl,
propyl, 2-propyl, n-butyl, iso-butyl, tert-butyl, pentyl, and the
like. Alkyl may be substituted or unsubstituted. Typical
substituent groups include cycloalkyl, aryl, heteroaryl,
heteroalicyclic, hydroxy, alkoxy, aryloxy, mercapto, alkylthio,
arylthio, cyano, halo, carbonyl, thiocarbonyl, O-carbamyl,
N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido,
C-carboxy, O-carboxy, nitro, silyl, amino and --NR.sup.xR.sup.y,
where R.sup.x and R.sup.y are independently selected from the group
consisting of hydrogen, alkyl, cycloalkyl, aryl, carbonyl, acetyl,
sulfonyl, trifluoromethanesulfonyl and, combined, a five- or
six-member heteroalicyclic ring.
[0061] "Cycloalkyl" refers to a 3 to 8 member all-carbon monocyclic
ring, an all-carbon 5-member/6-member or 6-member/6-member fused
bicyclic ring, or a multicyclic fused ring (a "fused" ring system
means that each ring in the system shares an adjacent pair of
carbon atoms with each other ring in the system) group wherein one
or more of the rings may contain one or more double bonds but none
of the rings has a completely conjugated pi-electron system.
Examples, without limitation, of cycloalkyl groups are
cyclopropane, cyclobutane, cyclopentane, cyclopentene, cyclohexane,
cyclohexadiene, adamantane, cycloheptane, cycloheptatriene, and the
like. A cycloalkyl group may be substituted or unsubstituted.
Typical substituent groups include alkyl, aryl, heteroaryl,
heteroalicyclic, hydroxy, alkoxy, aryloxy, mercapto, alkylthio,
arylthio, cyano, halo, carbonyl, thiocarbonyl, C-carboxy,
O-carboxy, O-carbamyl, N-carbamyl, C-amido, N-amido, nitro, amino
and --NR.sup.xR.sup.y, with R.sup.x and R.sup.y as defined above.
Illustrative examples of cycloalkyl are derived from, but not
limited to, the following:
##STR00004##
[0062] "Alkenyl" refers to an alkyl group, as defined herein,
consisting of at least two carbon atoms and at least one
carbon-carbon double bond. Representative examples include, but are
not limited to, ethenyl, 1-propenyl, 2-propenyl, 1-, 2-, or
3-butenyl, and the like.
[0063] "Alkynyl" refers to an alkyl group, as defined herein,
consisting of at least two carbon atoms and at least one
carbon-carbon triple bond. Representative examples include, but are
not limited to, ethynyl, 1-propynyl, 2-propynyl, 1-, 2-, or
3-butynyl, and the like.
[0064] "Aryl" refers to an all-carbon monocyclic or fused-ring
polycyclic groups of 6 to 12 carbon atoms having a completely
conjugated pi-electron system. Examples, without limitation, of
aryl groups are phenyl, naphthalenyl and anthracenyl. The aryl
group may be substituted or unsubstituted. Typical substituents
include halo, trihalomethyl, alkyl, hydroxy, alkoxy, aryloxy,
mercapto, alkylthio, arylthio, cyano, nitro, carbonyl,
thiocarbonyl, C-carboxy, O-carboxy, O-carbamyl, N-carbamyl,
O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, sulfinyl,
sulfonyl, amino and --NR.sup.xR.sup.y, with R.sup.x and R.sup.y as
defined above.
[0065] "Heteroaryl" refers to a monocyclic or fused ring group of 5
to 12 ring atoms containing one, two, three or four ring
heteroatoms selected from N, O, and S, the remaining ring atoms
being C, and, in addition, having a completely conjugated
pi-electron system. Examples, without limitation, of unsubstituted
heteroaryl groups are pyrrole, furan, thiophene, imidazole,
oxazole, thiazole, pyrazole, pyridine, pyrimidine, quinoline,
isoquinoline, purine, tetrazole, triazine, and carbazole. The
heteroaryl group may be substituted or unsubstituted. Typical
substituents include alkyl, cycloalkyl, halo, trihalomethyl,
hydroxy, alkoxy, aryloxy, mercapto, alkylthio, arylthio, cyano,
nitro, carbonyl, thiocarbonyl, sulfonamido, C-carboxy, O-carboxy,
sulfinyl, sulfonyl, O-carbamyl, N-carbamyl, O-thiocarbamyl,
N-thiocarbamyl, C-amido, N-amido, amino and --NR.sup.xR.sup.y with
R.sup.x and R.sup.y as defined above.
[0066] A pharmaceutically acceptable heteroaryl is one that is
sufficiently stable to be attached to a compound of the invention,
formulated into a pharmaceutical composition and subsequently
administered to a patient in need thereof.
[0067] Examples of typical monocyclic heteroaryl groups include,
but are not limited to:
##STR00005##
[0068] Examples of suitable fused ring heteroaryl groups include,
but are not limited to:
##STR00006## ##STR00007## ##STR00008##
[0069] "Heteroalicyclic" or "heterocycle" refers to a monocyclic or
fused ring group having in the ring(s) of 3 to 12 ring atoms, in
which one or two ring atoms are heteroatoms selected from N, O, and
S(O).sub.n (where n is 0, 1 or 2), the remaining ring atoms being
C. The rings may also have one or more double bonds. However, the
rings do not have a completely conjugated pi-electron system.
Examples of suitable saturated heteroalicyclic groups include, but
are not limited to:
##STR00009##
[0070] Examples of suitable partially unsaturated heteroalicyclic
groups include, but are not limited to:
##STR00010##
[0071] The heterocycle group is optionally substituted with one or
two substituents independently selected from halo, lower alkyl,
lower alkyl substituted with carboxy, ester hydroxy, or mono or
dialkylamino.
[0072] "Hydroxy" refers to an --OH group.
[0073] "Alkoxy" refers to both an --O-(alkyl) or an
--O-(unsubstituted cycloalkyl) group. Representative examples
include, but are not limited to, methoxy, ethoxy, propoxy, butoxy,
cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy, and
the like.
[0074] "Haloalkoxy" refers to an --O-(haloalkyl) group.
Representative examples include, but are not limited to,
trifluoromethoxy, tribromomethoxy, and the like.
[0075] "Aryloxy" refers to an --O-aryl or an --O-heteroaryl group,
as defined herein. Representative examples include, but are not
limited to, phenoxy, pyridinyloxy, furanyloxy, thienyloxy,
pyrimidinyloxy, pyrazinyloxy, and the like, and derivatives
thereof.
[0076] "Mercapto" refers to an --SH group.
[0077] "Alkylthio" refers to an --S-(alkyl) or an
--S-(unsubstituted cycloalkyl) group. Representative examples
include, but are not limited to, methylthio, ethylthio, propylthio,
butylthio, cyclopropylthio, cyclobutylthio, cyclopentylthio,
cyclohexylthio, and the like.
[0078] "Arylthio" refers to an --S-aryl or an --S-heteroaryl group,
as defined herein. Representative examples include, but are not
limited to, phenylthio, pyridinylthio, furanylthio, thienylthio,
pyrimidinylthio, and the like and derivatives thereof.
[0079] "Acyl" or "carbonyl" refers to a --C(O)R'' group, where R''
is selected from the group consisting of hydrogen, lower alkyl,
trihalomethyl, unsubstituted cycloalkyl, aryl optionally
substituted with one or more, preferably one, two, or three
substituents selected from the group consisting of lower alkyl,
trihalomethyl, lower alkoxy, halo and --NR.sup.xR.sup.y groups,
heteroaryl (bonded through a ring carbon) optionally substituted
with one or more, preferably one, two, or three substitutents
selected from the group consisting of lower alkyl, trihaloalkyl,
lower alkoxy, halo and --NR.sup.xR.sup.y groups and heteroalicyclic
(bonded through a ring carbon) optionally substituted with one or
more, preferably one, two, or three substituents selected from the
group consisting of lower alkyl, trihaloalkyl, lower alkoxy, halo
and --NR.sup.xR.sup.y groups. Representative acyl groups include,
but are not limited to, acetyl, trifluoroacetyl, benzoyl, and the
like
[0080] "Aldehyde" refers to an acyl group in which R'' is
hydrogen.
[0081] "Thioacyl" or "thiocarbonyl" refers to a --C(S)R'' group,
with R'' as defined above.
[0082] A "thiocarbonyl" group refers to a --C(S)R'' group, with R''
as defined above.
[0083] A "C-carboxy" group refers to a --C(O)OR'' group, with R''
as defined above.
[0084] An "O-carboxy" group refers to a --OC(O)R'' group, with R''
as defined above.
[0085] "Ester" refers to a --C(O)OR'' group with R'' as defined
herein except that R'' cannot be hydrogen.
[0086] "Acetyl" group refers to a --C(O)CH.sub.3 group.
[0087] "Halo" group refers to fluorine, chlorine, bromine or
iodine, preferably fluorine or chlorine.
[0088] "Trihalomethyl" group refers to a methyl group having three
halo substituents, such as a trifluoromethyl group.
[0089] "Cyano" refers to a --C.ident.N group.
[0090] A "sulfinyl" group refers to a --S(O)R'' group wherein, in
addition to being as defined above, R'' may also be a hydroxy
group.
[0091] A "sulfonyl" group refers to a --S(O).sub.2R'' group
wherein, in addition to being as defined above, R'' may also be a
hydroxy group.
[0092] "S-sulfonamido" refers to a --S(O).sub.2NR.sup.xR.sup.y
group, with R.sup.x and R.sup.y as defined above.
[0093] "N-sulfonamido" refers to a --NR.sup.xS(O).sub.2R.sup.y
group, with R.sup.x and R.sup.y as defined above.
[0094] "O-carbamyl" group refers to a --OC(O)NR.sup.xR.sup.y group
with R.sup.x and R.sup.y as defined above.
[0095] "N-carbamyl" refers to an R.sup.yOC(O)NR.sup.x-- group, with
R.sup.x and R.sup.y as defined above.
[0096] "O-thiocarbamyl" refers to a --OC(S)NR.sup.xR.sup.y group
with R.sup.x and R.sup.y as defined above.
[0097] "N-thiocarbamyl" refers to a R.sup.yOC(S)NR.sup.x-- group,
with R.sup.y and R.sup.x as defined above.
[0098] "Amino" refers to an --NR.sup.xR.sup.y group, wherein
R.sup.x and R.sup.y are both hydrogen.
[0099] "C-amido" refers to a --C(O)NR.sup.xR.sup.y group with
R.sup.x and R.sup.y as defined above.
[0100] "N-amido" refers to a R.sup.xC(O)NR.sup.y-- group, with
R.sup.x and R.sup.y as defined above.
[0101] "Nitro" refers to a --NO.sub.2 group.
[0102] "Haloalkyl" means an alkyl, preferably lower alkyl, that is
substituted with one or more same or different halo atoms, e.g.,
--CH.sub.2Cl, --CF.sub.3, --CH.sub.2CF.sub.3, --CH.sub.2CCl.sub.3,
and the like.
[0103] "Hydroxyalkyl" means an alkyl, preferably lower alkyl, that
is substituted with one, two, or three hydroxy groups; e.g.,
hydroxymethyl, 1 or 2-hydroxyethyl, 1,2-, 1,3-, or
2,3-dihydroxypropyl, and the like.
[0104] "Aralkyl" means alkyl, preferably lower alkyl, that is
substituted with an aryl group as defined above; e.g.,
--CH.sub.2-phenyl, --(CH.sub.2).sub.2-phenyl,
--(CH.sub.2).sub.3-phenyl, CH.sub.3CH(CH.sub.3)CH.sub.2-phenyl, and
the like and derivatives thereof.
[0105] "Heteroaralkyl" group means alkyl, preferably lower alkyl,
that is substituted with a heteroaryl group; e.g.,
--CH.sub.2pyridinyl, --(CH.sub.2).sub.2pyrimidinyl,
--(CH.sub.2).sub.3imidazolyl, and the like, and derivatives
thereof.
[0106] "Monoalkylamino" means a radical --NHR where R is an alkyl
or unsubstituted cycloalkyl group; e.g., methylamino,
(1-methylethyl)amino, cyclohexylamino, and the like.
[0107] "Dialkylamino" means a radical --NRR where each R is
independently an alkyl or unsubstituted cycloalkyl group;
dimethylamino, diethylamino, (1-methylethyl)-ethylamino,
cyclohexylmethylamino, cyclopentylmethylamino, and the like.
[0108] "Optional" or "optionally" means that the subsequently
described event or circumstance may but need not occur, and that
the description includes instances where the event or circumstance
occurs and instances in which it does not. For example,
"heterocycle group optionally substituted with an alkyl group"
means that the alkyl may but need not be present, and the
description includes situations where the heterocycle group is
substituted with an alkyl group and situations where the
heterocycle group is not substituted with the alkyl group.
[0109] A "pharmaceutical composition" refers to a mixture of one or
more of the compounds described herein, or
physiologically/pharmaceutically acceptable salts, solvates,
hydrates or prodrugs thereof, with other chemical components, such
as physiologically/pharmaceutically acceptable carriers and
excipients. The purpose of a pharmaceutical composition is to
facilitate administration of a compound to an organism.
[0110] As used herein, a "physiologically/pharmaceutically
acceptable carrier" refers to a carrier or diluent that does not
cause significant irritation to an organism and does not abrogate
the biological activity and properties of the administered
compound.
[0111] A "pharmaceutically acceptable excipient" refers to an inert
substance added to a pharmaceutical composition to further
facilitate administration of a compound. Examples, without
limitation, of excipients include calcium carbonate, calcium
phosphate, various sugars and types of starch, cellulose
derivatives, gelatin, vegetable oils and polyethylene glycols.
[0112] As used herein, the term "pharmaceutically acceptable salt"
refers to those salts which retain the biological effectiveness and
properties of the parent compound. Such salts include:
[0113] (i) acid addition salts, which can be obtained by reaction
of the free base of the parent compound with inorganic acids such
as hydrochloric acid, hydrobromic acid, nitric acid, phosphoric
acid, sulfuric acid, and perchloric acid and the like, or with
organic acids such as acetic acid, oxalic acid, (D) or (L) malic
acid, maleic acid, methanesulfonic acid, ethanesulfonic acid,
p-toluenesulfonic acid, salicylic acid, tartaric acid, citric acid,
succinic acid or malonic acid and the like; or
[0114] (2) salts formed when an acidic proton present in the parent
compound either is replaced by a metal ion, e.g., an alkali metal
ion, an alkaline earth ion, or an aluminum ion; or coordinates with
an organic base such as ethanolamine, diethanolamine,
triethanolamine, tromethamine, N-methylglucamine, and the like.
[0115] "PK" refers to receptor protein tyrosine kinase (RTKs),
non-receptor or "cellular" tyrosine kinase (CTKs) and
serine-threonine kinases (STKs).
[0116] "Modulation" or "modulating" refers to the alteration of the
catalytic activity of RTKs, CTKs and STKs. In particular,
modulating refers to the activation of the catalytic activity of
RTKs, CTKs and STKs, preferably the activation or inhibition of the
catalytic activity of RTKs, CTKs and STKs, depending on the
concentration of the compound or salt to which the RTK, CTK or STK
is exposed or, more preferably, the inhibition of the catalytic
activity of RTKs, CTKs and STKs.
[0117] "Catalytic activity" refers to the rate of phosphorylation
of tyrosine under the influence, direct or indirect, of RTKs and/or
CTKs or the phosphorylation of serine and threonine under the
influence, direct or indirect, of STKs.
[0118] "Contacting" refers to bringing a compound of this invention
and a target PK together in such a manner that the compound can
affect the catalytic activity of the PK, either directly, i.e., by
interacting with the kinase itself, or indirectly, i.e., by
interacting with another molecule on which the catalytic activity
of the kinase is dependent. Such "contacting" can be accomplished
"in vitro," i.e., in a test tube, a petri dish or the like. In a
test tube, contacting may involve only a compound and a PK of
interest or it may involve whole cells. Cells may also be
maintained or grown in cell culture dishes and contacted with a
compound in that environment. In this context, the ability of a
particular compound to affect a PK related disorder, i.e., the
IC.sub.50 of the compound, defined below, can be determined before
use of the compounds in vivo with more complex living organisms is
attempted. For cells outside the organism, multiple methods exist,
and are well-known to those skilled in the art, to get the PKs in
contact with the compounds including, but not limited to, direct
cell microinjection and numerous transmembrane carrier
techniques.
[0119] "In vitro" refers to procedures performed in an artificial
environment such as, e.g., without limitation, in a test tube or
culture medium.
[0120] "In vivo" refers to procedures performed within a living
organism such as, without limitation, a mouse, rat or rabbit.
[0121] "PK related disorder," "PK driven disorder," and "abnormal
PK activity" all refer to a condition characterized by
inappropriate, i.e., under or, more commonly, over, PK catalytic
activity, where the particular PK can be an RTK, a CTK or an STK.
Inappropriate catalytic activity can arise as the result of either:
(1) PK expression in cells which normally do not express PKs, (2)
increased PK expression leading to unwanted cell proliferation,
differentiation and/or growth, or, (3) decreased PK expression
leading to unwanted reductions in cell proliferation,
differentiation and/or growth. Over-activity of a PK refers to
either amplification of the gene encoding a particular PK or
production of a level of PK activity which can correlate with a
cell proliferation, differentiation and/or growth disorder (that
is, as the level of the PK increases, the severity of one or more
of the symptoms of the cellular disorder increases). Under-activity
is, of course, the converse, wherein the severity of one or more
symptoms of a cellular disorder increase as the level of the PK
activity decreases.
[0122] "Treat", "treating" and "treatment" refer to a method of
alleviating or abrogating a PK mediated cellular disorder and/or
its attendant symptoms. With regard particularly to cancer, these
terms simply mean that the life expectancy of an individual
affected with a cancer will be increased or that one or more of the
symptoms of the disease will be reduced.
[0123] "Organism" refers to any living entity comprised of at least
one cell. A living organism can be as simple as, for example, a
single eukariotic cell or as complex as a mammal, including a human
being.
[0124] "Therapeutically effective amount" refers to that amount of
the compound being administered which will relieve to some extent
one or more of the symptoms of the disorder being treated. In
reference to the treatment of cancer, a therapeutically effective
amount refers to that amount which has at least one of the
following effects: [0125] (1) reducing the size of the tumor;
[0126] (2) inhibiting (that is, slowing to some extent, preferably
stopping) tumor metastasis; [0127] (3) inhibiting to some extent
(that is, slowing to some extent, preferably stopping) tumor
growth, and [0128] (4) relieving to some extent (or, preferably,
eliminating) one or more symptoms associated with the cancer.
[0129] "Monitoring" means observing or detecting the effect of
contacting a compound with a cell expressing a particular PK. The
observed or detected effect can be a change in cell phenotype, in
the catalytic activity of a PK or a change in the interaction of a
PK with a natural binding partner. Techniques for observing or
detecting such effects are well-known in the art. The effect is
selected from a change or an absence of change in a cell phenotype,
a change or absence of change in the catalytic activity of said
protein kinase or a change or absence of change in the interaction
of said protein kinase with a natural binding partner in a final
aspect of this invention.
[0130] "Cell phenotype" refers to the outward appearance of a cell
or tissue or the biological function of the cell or tissue.
Examples, without limitation, of a cell phenotype are cell size,
cell growth, cell proliferation, cell differentiation, cell
survival, apoptosis, and nutrient uptake and use. Such phenotypic
characteristics are measurable by techniques well-known in the
art.
[0131] "Natural binding partner" refers to a polypeptide that binds
to a particular PK in a cell. Natural binding partners can play a
role in propagating a signal in a PK-mediated signal transduction
process. A change in the interaction of the natural binding partner
with the PK can manifest itself as an increased or decreased
concentration of the PK/natural binding partner complex and, as a
result, in an observable change in the ability of the PK to mediate
signal transduction.
[0132] As used herein, the terms "optically pure,"
"enantiomerically pure," "pure enantiomer," and "optically pure
enantiomer" mean a composition that comprises one enantiomer of a
compound and is substantially free of the opposite enantiomer of
the compound. A typical optically pure compound comprises greater
than about 80% by weight of one enantiomer of the compound and less
than about 20% by weight of the opposite enantiomer of the
compound, more preferably greater than about 90% by weight of one
enantiomer of the compound and less than about 10% by weight of the
opposite enantiomer of the compound, even more preferably greater
than about 95% by weight of one enantiomer of the compound and less
than about 5% by weight of the opposite enantiomer of the compound,
and most preferably greater than about 97% by weight of one
enantiomer of the compound and less than about 3% by weight of the
opposite enantiomer of the compound.
DETAILED DESCRIPTION
[0133] General schemes for synthesizing the compounds of the
invention can be found in the Examples section herein.
[0134] Some of the general procedures are shown with reference to
synthesis of compounds wherein the
1-(2,6-dichloro-3-fluorophenyl)-ethoxy moiety is the pure
(R)-isomer, and some are shown with reference to compounds wherein
said moiety is a racemic mixture. It should be understood that the
procedures herein can be used to produce racemic compounds or
enantiomerically pure (R) isomers by choosing the corresponding
racemic or enantiomerically pure starting material.
[0135] The procedures shown herein can be used to produce a wide
variety of enantiomerically pure compounds by selection of the
appropriate enantiomerically pure starting material. In addition to
the compounds shown herein, the invention also provides
enantiomerically pure compounds corresponding to the
3-[1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-pyridin-2-ylamine and
3-[1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-pyrazin-2-ylamine
compounds shown in U.S. patent application Ser. No. 10/786,610
(PCT/US2004/005495); in U.S. application Ser. No. to be assigned,
docket number PC 32546, filed Aug. 26, 2004 and entitled,
"Pyrazolo-Substituted Aminoheteroaryl Compounds as Protein Kinase
Inhibitors"; and in U.S. application Ser. No. to be assigned,
docket number PC 32548, filed Aug. 26, 2004 and entitled,
"Aminoheteroaryl Compounds as Protein Kinase Inhibitors". The
disclosures of these documents are incorporated herein by reference
in their entireties.
[0136] Unless indicated otherwise, all references herein to the
inventive compounds include references to salts, solvates, hydrates
and complexes thereof, and to solvates, hydrates and complexes of
salts thereof, including polymorphs, stereoisomers, and
isotopically labeled versions thereof.
[0137] Pharmaceutically acceptable salts include acid addition and
base salts (including disalts).
[0138] Suitable acid addition salts are formed from acids which
form non-toxic salts. Examples include the acetate, aspartate,
benzoate, besylate, bicarbonate/carbonate, bisulphate/sulfate,
borate, camsylate, citrate, edisylate, esylate, formate, fumarate,
gluceptate, gluconate, glucuronate, hexafluorophosphate, hibenzate,
hydrochloride/chloride, hydrobromide/bromide, hydroiodide/iodide,
isethionate, lactate, malate, maleate, malonate, mesylate,
methylsulfate, naphthylate, 2-napsylate, nicotinate, nitrate,
orotate, oxalate, palmitate, pamoate, phosphate/hydrogen
phosphate/dihydrogen phosphate, saccharate, stearate, succinate,
tartrate, tosylate and trifluoroacetate salts.
[0139] Suitable base salts are formed from bases which form
non-toxic salts. Examples include the aluminum, arginine,
benzathine, calcium, choline, diethylamine, diolamine, glycine,
lysine, magnesium, meglumine, olamine, potassium, sodium,
tromethamine and zinc salts.
[0140] For a review on suitable salts, see "Handbook of
Pharmaceutical Salts: Properties, Selection, and Use" by Stahl and
Wermuth (Wiley-VCH, Weinheim, Germany, 2002), the disclosure of
which is incorporated herein by reference in its entirety.
[0141] A pharmaceutically acceptable salt of the inventive
compounds can be readily prepared by mixing together solutions of
the compound and the desired acid or base, as appropriate. The salt
may precipitate from solution and be collected by filtration or may
be recovered by evaporation of the solvent. The degree of
ionization in the salt may vary from completely ionized to almost
non-ionized.
[0142] The compounds of the invention may exist in both unsolvated
and solvated forms. The term `solvate` is used herein to describe a
molecular complex comprising the compound of the invention and one
or more pharmaceutically acceptable solvent molecules, for example,
ethanol. The term `hydrate` is employed when the solvent is water.
Pharmaceutically acceptable solvates in accordance with the
invention include hydrates and solvates wherein the solvent of
crystallization may be isotopically substituted, e.g. D.sub.2O,
d.sub.6-acetone, d.sub.6-DMSO.
[0143] Also included within the scope of the invention are
complexes such as clathrates, drug-host inclusion complexes
wherein, in contrast to the aforementioned solvates, the drug and
host are present in stoichiometric or non-stoichiometric amounts.
Also included are complexes of the drug containing two or more
organic and/or inorganic components which may be in stoichiometric
or non-stoichiometric amounts. The resulting complexes may be
ionized, partially ionized, or non-ionized. For a review of such
complexes, see J Pharm Sci, 64 (8), 1269-1288 by Haleblian (August
1975), the disclosure of which is incorporated herein by reference
in its entirety.
[0144] Also within the scope of the invention are polymorphs,
prodrugs, and isomers (including optical, geometric and tautomeric
isomers) of the inventive compounds
[0145] Derivatives of compounds of the invention which may have
little or no pharmacological activity themselves but can, when
administered to a patient, be converted into the inventive
compounds, for example, by hydrolytic cleavage. Such derivatives
are referred to as `prodrugs`. Further information on the use of
prodrugs may be found in `Pro-drugs as Novel Delivery Systems, Vol.
14, ACS Symposium Series (T Higuchi and W Stella) and
`Bioreversible Carriers in Drug Design`, Pergamon Press, 1987 (ed.
E B Roche, American Pharmaceutical Association), the disclosures of
which are incorporated herein by reference in their entireties.
[0146] Prodrugs in accordance with the invention can, for example,
be produced by replacing appropriate functionalities present in the
inventive compounds with certain moieties known to those skilled in
the art as `pro-moieties` as described, for example, in "Design of
Prodrugs" by H Bundgaard (Elsevier, 1985), the disclosure of which
is incorporated herein by reference in its entirety.
[0147] Some examples of prodrugs in accordance with the invention
include:
[0148] (i) where the compound contains a carboxylic acid
functionality (--COOH), an ester thereof, for example, replacement
of the hydrogen with (C.sub.1-C.sub.8)alkyl;
[0149] (ii) where the compound contains an alcohol functionality
(--OH), an ether thereof, for example, replacement of the hydrogen
with (C.sub.1-C.sub.6)alkanoyloxymethyl; and
[0150] (iii) where the compound contains a primary or secondary
amino functionality (--NH.sub.2 or --NHR where R.noteq.H), an amide
thereof, for example, replacement of one or both hydrogens with
(C.sub.1-C.sub.10)alkanoyl.
[0151] Further examples of replacement groups in accordance with
the foregoing examples and examples of other prodrug types may be
found in the aforementioned references.
[0152] Finally, certain inventive compounds may themselves act as
prodrugs of other of the inventive compounds.
[0153] Compounds of the invention containing one or more asymmetric
carbon atoms can exist as two or more stereoisomers. Where a
compound of the invention contains an alkenyl or alkenylene group,
geometric cis/trans (or Z/E) isomers are possible. Where the
compound contains, for example, a keto or oxime group or an
aromatic moiety, tautomeric isomerism (`tautomerism`) can occur. A
single compound may exhibit more than one type of isomerism.
[0154] Included within the scope of the invention are all
stereoisomers, geometric isomers and tautomeric forms of the
inventive compounds, including compounds exhibiting more than one
type of isomerism, and mixtures of one or more thereof. Also
included are acid addition or base salts wherein the counterion is
optically active, for example, D-lactate or L-lysine, or racemic,
for example, DL-tartrate or DL-arginine.
[0155] Cis/trans isomers may be separated by conventional
techniques well known to those skilled in the art, for example,
chromatography and fractional crystallization.
[0156] Conventional techniques for the preparation/isolation of
individual enantiomers include chiral synthesis from a suitable
optically pure precursor or resolution of the racemate (or the
racemate of a salt or derivative) using, for example, chiral high
pressure liquid chromatography (HPLC).
[0157] Alternatively, the racemate (or a racemic precursor) may be
reacted with a suitable optically active compound, for example, an
alcohol, or, in the case where the compound contains an acidic or
basic moiety, an acid or base such as tartaric acid or
1-phenylethylamine. The resulting diastereomeric mixture may be
separated by chromatography and/or fractional crystallization and
one or both of the diastereoisomers converted to the corresponding
pure enantiomer(s) by means well known to one skilled in the
art.
[0158] Chiral compounds of the invention (and chiral precursors
thereof) may be obtained in enantiomerically-enriched form using
chromatography, typically HPLC, on an asymmetric resin with a
mobile phase consisting of a hydrocarbon, typically heptane or
hexane, containing from 0 to 50% isopropanol, typically from 2 to
20%, and from 0 to 5% of an alkylamine, typically 0.1%
diethylamine. Concentration of the eluate affords the enriched
mixture.
[0159] Stereoisomeric conglomerates may be separated by
conventional techniques known to those skilled in the art; see, for
example, "Stereochemistry of Organic Compounds" by E L Eliel
(Wiley, New York, 1994), the disclosure of which is incorporated
herein by reference in its entirety.
[0160] The invention also includes isotopically-labeled compounds
of the invention, wherein one or more atoms is replaced by an atom
having the same atomic number, but an atomic mass or mass number
different from the atomic mass or mass number usually found in
nature. Examples of isotopes suitable for inclusion in the
compounds of the invention include isotopes of hydrogen, such as
.sup.2H and .sup.3H, carbon, such as .sup.11C, .sup.13C and
.sup.14C, chlorine, such as .sup.36Cl, fluorine, such as .sup.18F,
iodine, such as .sup.123I and .sup.125I, nitrogen, such as .sup.13N
and .sup.15N, oxygen, such as .sup.15O, .sup.17O and .sup.18O,
phosphorus, such as .sup.32P, and sulfur, such as .sup.35S. Certain
isotopically-labeled compounds of the invention, for example, those
incorporating a radioactive isotope, are useful in drug and/or
substrate tissue distribution studies. The radioactive isotopes
tritium, .sup.3H, and carbon-14, .sup.14C, are particularly useful
for this purpose in view of their ease of incorporation and ready
means of detection. Substitution with heavier isotopes such as
deuterium, .sup.2H, may afford certain therapeutic advantages
resulting from greater metabolic stability, for example, increased
in vivo half-life or reduced dosage requirements, and hence may be
preferred in some circumstances. Substitution with positron
emitting isotopes, such as .sup.11C, .sup.18F, .sup.15O and
.sup.13N, can be useful in Positron Emission Topography (PET)
studies for examining substrate receptor occupancy.
[0161] Isotopically-labeled compounds of the invention can
generally be prepared by conventional techniques known to those
skilled in the art or by processes analogous to those described
herein, using an appropriate isotopically-labeled reagent in place
of the non-labeled reagent otherwise employed.
[0162] Pharmaceutically acceptable solvates in accordance with the
invention include those wherein the solvent of crystallization may
be isotopically substituted, e.g. D.sub.2O, d.sub.6-acetone,
d.sub.6-DMSO.
[0163] Compounds of the invention intended for pharmaceutical use
may be administered as crystalline or amorphous products, or
mixtures thereof. They may be obtained, for example, as solid
plugs, powders, or films by methods such as precipitation,
crystallization, freeze drying, spray drying, or evaporative
drying. Microwave or radio frequency drying may be used for this
purpose.
[0164] The compounds can be administered alone or in combination
with one or more other compounds of the invention, or in
combination with one or more other drugs (or as any combination
thereof). Generally, they will be administered as a formulation in
association with one or more pharmaceutically acceptable
excipients. The term "excipient" is used herein to describe any
ingredient other than the compound(s) of the invention. The choice
of excipient will to a large extent depend on factors such as the
particular mode of administration, the effect of the excipient on
solubility and stability, and the nature of the dosage form.
[0165] Pharmaceutical compositions suitable for the delivery of
compounds of the invention and methods for their preparation will
be readily apparent to those skilled in the art. Such compositions
and methods for their preparation can be found, for example, in
`Remington's Pharmaceutical Sciences`, 19th Edition (Mack
Publishing Company, 1995), the disclosure of which is incorporated
herein by reference in its entirety.
Oral Administration
[0166] The compounds of the invention may be administered orally.
Oral administration may involve swallowing, so that the compound
enters the gastrointestinal tract, or buccal or sublingual
administration may be employed by which the compound enters the
blood stream directly from the mouth.
[0167] Formulations suitable for oral administration include solid
formulations such as tablets, capsules containing particulates,
liquids, or powders, lozenges (including liquid-filled), chews,
multi- and nano-particulates, gels, solid solution, liposome, films
(including muco-adhesive), ovules, sprays and liquid
formulations.
[0168] Liquid formulations include suspensions, solutions, syrups
and elixirs. Such formulations may be used as fillers in soft or
hard capsules and typically include a carrier, for example, water,
ethanol, polyethylene glycol, propylene glycol, methylcellulose, or
a suitable oil, and one or more emulsifying agents and/or
suspending agents. Liquid formulations may also be prepared by the
reconstitution of a solid, for example, from a sachet.
[0169] The compounds of the invention may also be used in
fast-dissolving, fast-disintegrating dosage forms such as those
described in Expert Opinion in Therapeutic Patents, 11 (6), 981-986
by Liang and Chen (2001), the disclosure of which is incorporated
herein by reference in its entirety.
[0170] For tablet dosage forms, depending on dose, the drug may
make up from 1 wt % to 80 wt % of the dosage form, more typically
from 5 wt % to 60 wt % of the dosage form. In addition to the drug,
tablets generally contain a disintegrant. Examples of disintegrants
include sodium starch glycolate, sodium carboxymethyl cellulose,
calcium carboxymethyl cellulose, croscarmellose sodium,
crospovidone, polyvinylpyrrolidone, methyl cellulose,
microcrystalline cellulose, lower alkyl-substituted hydroxypropyl
cellulose, starch, pregelatinized starch and sodium alginate.
Generally, the disintegrant will comprise from 1 wt % to 25 wt %,
preferably from 5 wt % to 20 wt % of the dosage form.
[0171] Binders are generally used to impart cohesive qualities to a
tablet formulation. Suitable binders include microcrystalline
cellulose, gelatin, sugars, polyethylene glycol, natural and
synthetic gums, polyvinylpyrrolidone, pregelatinized starch,
hydroxypropyl cellulose and hydroxypropyl methylcellulose. Tablets
may also contain diluents, such as lactose (monohydrate,
spray-dried monohydrate, anhydrous and the like), mannitol,
xylitol, dextrose, sucrose, sorbitol, microcrystalline cellulose,
starch and dibasic calcium phosphate dihydrate.
[0172] Tablets may also optionally include surface active agents,
such as sodium lauryl sulfate and polysorbate 80, and glidants such
as silicon dioxide and talc. When present, surface active agents
are typically in amounts of from 0.2 wt % to 5 wt % of the tablet,
and glidants typically from 0.2 wt % to 1 wt % of the tablet.
[0173] Tablets also generally contain lubricants such as magnesium
stearate, calcium stearate, zinc stearate, sodium stearyl fumarate,
and mixtures of magnesium stearate with sodium lauryl sulphate.
Lubricants generally are present in amounts from 0.25 wt % to 10 wt
%, preferably from 0.5 wt % to 3 wt % of the tablet.
[0174] Other conventional ingredients include anti-oxidants,
colorants, flavoring agents, preservatives and taste-masking
agents.
[0175] Exemplary tablets contain up to about 80 wt % drug, from
about 10 wt % to about 90 wt % binder, from about 0 wt % to about
85 wt % diluent, from about 2 wt % to about 10 wt % disintegrant,
and from about 0.25 wt % to about 10 wt % lubricant.
[0176] Tablet blends may be compressed directly or by roller to
form tablets. Tablet blends or portions of blends may alternatively
be wet-, dry-, or melt-granulated, melt congealed, or extruded
before tableting. The final formulation may include one or more
layers and may be coated or uncoated; or encapsulated.
[0177] The formulation of tablets is discussed in detail in
"Pharmaceutical Dosage Forms: Tablets, Vol. 1", by H. Lieberman and
L. Lachman, Marcel Dekker, N.Y., N.Y., 1980 (ISBN 0-8247-6918-X),
the disclosure of which is incorporated herein by reference in its
entirety.
[0178] Solid formulations for oral administration may be formulated
to be immediate and/or modified release. Modified release
formulations include delayed-, sustained-, pulsed-, controlled-,
targeted and programmed release.
[0179] Suitable modified release formulations are described in U.S.
Pat. No. 6,106,864. Details of other suitable release technologies
such as high energy dispersions and osmotic and coated particles
can be found in Verma et al, Pharmaceutical Technology On-line,
25(2), 1-14 (2001). The use of chewing gum to achieve controlled
release is described in WO 00/35298. The disclosures of these
references are incorporated herein by reference in their
entireties.
Parenteral Administration
[0180] The compounds of the invention may also be administered
directly into the blood stream, into muscle, or into an internal
organ. Suitable means for parenteral administration include
intravenous, intraarterial, intraperitoneal, intrathecal,
intraventricular, intraurethral, intrasternal, intracranial,
intramuscular and subcutaneous. Suitable devices for parenteral
administration include needle (including micro needle) injectors,
needle-free injectors and infusion techniques.
[0181] Parenteral formulations are typically aqueous solutions
which may contain excipients such as salts, carbohydrates and
buffering agents (preferably to a pH of from 3 to 9), but, for some
applications, they may be more suitably formulated as a sterile
non-aqueous solution or as a dried form to be used in conjunction
with a suitable vehicle such as sterile, pyrogen-free water.
[0182] The preparation of parenteral formulations under sterile
conditions, for example, by lyophilization, may readily be
accomplished using standard pharmaceutical techniques well known to
those skilled in the art.
[0183] The solubility of compounds of the invention used in the
preparation of parenteral solutions may be increased by the use of
appropriate formulation techniques, such as the incorporation of
solubility-enhancing agents.
[0184] Formulations for parenteral administration may be formulated
to be immediate and/or modified release. Modified release
formulations include delayed-, sustained-, pulsed-, controlled-,
targeted and programmed release. Thus compounds of the invention
may be formulated as a solid, semi-solid, or thixotropic liquid for
administration as an implanted depot providing modified release of
the active compound. Examples of such formulations include
drug-coated stents and PGLA microspheres.
Topical Administration
[0185] The compounds of the invention may also be administered
topically to the skin or mucosa, that is, dermally or
transdermally. Typical formulations for this purpose include gels,
hydrogels, lotions, solutions, creams, ointments, dusting powders,
dressings, foams, films, skin patches, wafers, implants, sponges,
fibers, bandages and microemulsions. Liposomes may also be used.
Typical carriers include alcohol, water, mineral oil, liquid
petrolatum, white petrolatum, glycerin, polyethylene glycol and
propylene glycol. Penetration enhancers may be incorporated; see,
for example, J Pharm Sci, 88 (10), 955-958 by Finnin and Morgan
(October 1999). Other means of topical administration include
delivery by electroporation, iontophoresis, phonophoresis,
sonophoresis and micro needle or needle-free (e.g. Powderject.TM.,
Bioject.TM., etc.) injection. The disclosures of these references
are incorporated herein by reference in their entireties.
[0186] Formulations for topical administration may be formulated to
be immediate and/or modified release. Modified release formulations
include delayed-, sustained-, pulsed-, controlled-, targeted and
programmed release.
Inhaled/Intranasal Administration
[0187] The compounds of the invention can also be administered
intranasally or by inhalation, typically in the form of a dry
powder (either alone, as a mixture, for example, in a dry blend
with lactose, or as a mixed component particle, for example, mixed
with phospholipids, such as phosphatidylcholine) from a dry powder
inhaler or as an aerosol spray from a pressurized container, pump,
spray, atomizer (preferably an atomizer using electrohydrodynamics
to produce a fine mist), or nebulizer, with or without the use of a
suitable propellant, such as 1,1,1,2-tetrafluoroethane or
1,1,1,2,3,3,3-heptafluoropropane. For intranasal use, the powder
may include a bioadhesive agent, for example, chitosan or
cyclodextrin.
[0188] The pressurized container, pump, spray, atomizer, or
nebulizer contains a solution or suspension of the compound(s) of
the invention comprising, for example, ethanol, aqueous ethanol, or
a suitable alternative agent for dispersing, solubilizing, or
extending release of the active, a propellant(s) as solvent and an
optional surfactant, such as sorbitan trioleate, oleic acid, or an
oligolactic acid.
[0189] Prior to use in a dry powder or suspension formulation, the
drug product is micronized to a size suitable for delivery by
inhalation (typically less than 5 microns). This may be achieved by
any appropriate comminuting method, such as spiral jet milling,
fluid bed jet milling, supercritical fluid processing to form
nanoparticles, high pressure homogenization, or spray drying.
[0190] Capsules (made, for example, from gelatin or HPMC), blisters
and cartridges for use in an inhaler or insufflator may be
formulated to contain a powder mix of the compound of the
invention, a suitable powder base such as lactose or starch and a
performance modifier such as l-leucine, mannitol, or magnesium
stearate. The lactose may be anhydrous or in the form of the
monohydrate, preferably the latter. Other suitable excipients
include dextran, glucose, maltose, sorbitol, xylitol, fructose,
sucrose and trehalose.
[0191] A suitable solution formulation for use in an atomizer using
electrohydrodynamics to produce a fine mist may contain from 1
.mu.g to 20 mg of the compound of the invention per actuation and
the actuation volume may vary from 1 .mu.L to 100 .mu.L. A typical
formulation includes a compound of the invention, propylene glycol,
sterile water, ethanol and sodium chloride. Alternative solvents
which may be used instead of propylene glycol include glycerol and
polyethylene glycol.
[0192] Suitable flavors, such as menthol and levomenthol, or
sweeteners, such as saccharin or saccharin sodium, may be added to
those formulations of the invention intended for inhaled/intranasal
administration.
[0193] Formulations for inhaled/intranasal administration may be
formulated to be immediate and/or modified release using, for
example, poly(DL-lactic-coglycolic acid (PGLA). Modified release
formulations include delayed-, sustained-, pulsed-, controlled-,
targeted and programmed release.
[0194] In the case of dry powder inhalers and aerosols, the dosage
unit is determined by means of a valve which delivers a metered
amount. Units in accordance with the invention are typically
arranged to administer a metered dose or "puff" containing a
desired mount of the compound of the invention. The overall daily
dose may be administered in a single dose or, more usually, as
divided doses throughout the day.
Rectal/Intravaginal Administration
[0195] Compounds of the invention may be administered rectally or
vaginally, for example, in the form of a suppository, pessary, or
enema. Cocoa butter is a traditional suppository base, but various
alternatives may be used as appropriate.
[0196] Formulations for rectal/vaginal administration may be
formulated to be immediate and/or modified release. Modified
release formulations include delayed-, sustained-, pulsed-,
controlled-, targeted and programmed release.
Ocular Administration
[0197] Compounds of the invention may also be administered directly
to the eye or ear, typically in the form of drops of a micronized
suspension or solution in isotonic, pH-adjusted, sterile saline.
Other formulations suitable for ocular and aural administration
include ointments, biodegradable (e.g. absorbable gel sponges,
collagen) and non-biodegradable (e.g. silicone) implants, wafers,
lenses and particulate or vesicular systems, such as niosomes or
liposomes. A polymer such as crossed-linked polyacrylic acid,
polyvinylalcohol, hyaluronic acid, a cellulosic polymer, for
example, hydroxypropylmethylcellulose, hydroxyethylcellulose, or
methyl cellulose, or a heteropolysaccharide polymer, for example,
gelan gum, may be incorporated together with a preservative, such
as benzalkonium chloride. Such formulations may also be delivered
by iontophoresis.
[0198] Formulations for ocular/aural administration may be
formulated to be immediate and/or modified release. Modified
release formulations include delayed-, sustained-, pulsed-,
controlled-, targeted, or programmed release.
Other Technologies
[0199] Compounds of the invention may be combined with soluble
macromolecular entities, such as cyclodextrin and suitable
derivatives thereof or polyethylene glycol-containing polymers, in
order to improve their solubility, dissolution rate, taste-masking,
bioavailability and/or stability for use in any of the
aforementioned modes of administration.
[0200] Drug-cyclodextrin complexes, for example, are found to be
generally useful for most dosage forms and administration routes.
Both inclusion and non-inclusion complexes may be used. As an
alternative to direct complexation with the drug, the cyclodextrin
may be used as an auxiliary additive, i.e. as a carrier, diluent,
or solubilizer. Most commonly used for these purposes are alpha-,
beta- and gamma-cyclodextrins, examples of which may be found in
PCT Publication Nos. WO 91/11172, WO 94/02518 and WO 98/55148, the
disclosures of which are incorporated herein by reference in their
entireties.
Dosage
[0201] The amount of the active compound administered will be
dependent on the subject being treated, the severity of the
disorder or condition, the rate of administration, the disposition
of the compound and the discretion of the prescribing physician.
However, an effective dosage is typically in the range of about
0.001 to about 100 mg per kg body weight per day, preferably about
0.01 to about 35 mg/kg/day, in single or divided doses. For a 70 kg
human, this would amount to about 0.07 to about 7000 mg/day,
preferably about 0.7 to about 2500 mg/day. In some instances,
dosage levels below the lower limit of the aforesaid range may be
more than adequate, while in other cases still larger doses may be
used without causing any harmful side effect, with such larger
doses typically divided into several smaller doses for
administration throughout the day.
Kit-of-Parts
[0202] Inasmuch as it may desirable to administer a combination of
active compounds, for example, for the purpose of treating a
particular disease or condition, it is within the scope of the
present invention that two or more pharmaceutical compositions, at
least one of which contains a compound in accordance with the
invention, may conveniently be combined in the form of a kit
suitable for coadministration of the compositions. Thus the kit of
the invention includes two or more separate pharmaceutical
compositions, at least one of which contains a compound of the
invention, and means for separately retaining said compositions,
such as a container, divided bottle, or divided foil packet. An
example of such a kit is the familiar blister pack used for the
packaging of tablets, capsules and the like.
[0203] The kit of the invention is particularly suitable for
administering different dosage forms, for example, oral and
parenteral, for administering the separate compositions at
different dosage intervals, or for titrating the separate
compositions against one another. To assist compliance, the kit
typically includes directions for administration and may be
provided with a memory aid.
EXAMPLES
[0204] In the following examples, "Et" means ethyl, "Ac" means
acetyl, "Me" means methyl, "Ms" means methanesulfonyl
(CH.sub.3SO.sub.2), "iPr" means isopropyl, "HATU" means
2-(7-Aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate, "Ph" means phenyl, "Boc" means
tert-butoxycarbonyl, "EtOAc" means ethyl acetate, "HOAc" means
acetic acid, "NEt.sub.3" or "Et.sub.3N" means triethylamine, "THF"
means tetrahydrofuran, "DIC" means diisopropylcarbodiimide, "HOBt"
means hydroxy benzotriazole, "MeOH" means methanol, "i-PrOAc" means
isopropyl acetate, "KOAc" means potassium acetate, "DMSO" means
dimethylsulfoxide, "AcCl" means acetyl chloride, "CDCl.sub.3" means
deuterated chloroform, "MTBE" means methyl t-butyl ether, "DMF"
means dimethyl formamide, "Ac.sub.2O" means acetic anhydride,
"Me3SOI" means trimethylsulfoxonium iodide, "DMAP" means
4-dimethylaminopyridine, "dppf" means diphenylphosphino ferrocene,
"DME" means ethylene glycol dimethyl ether, HOBT means
1-hydroxybenzotriazole, EDC means
1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide.
[0205] The following examples are given to illustrate the present
invention. It should be understood, however, that the invention is
not to be limited to the specific conditions or details described
in these examples.
[0206] Reagents can be synthesized as shown herein, or are
available from commercial sources (e.g., Aldrich, Milwaukee, Wis.;
Acros, Morris Plains, N.J.; Biosynth International, Naperville,
Ill.; Frontier Scientific, Logan, Utah; TCI America, Portland,
Oreg.; Combi-Blocks, San Diego, Calif.; Matrix Scientific,
Columbia, S.C.; Acros, Morris Plains, N.J.; Alfa Aesar, Ward Hill,
Mass.; Apollo Scientific, UK; etc.) or can be synthesized by
procedures known in the art.
[0207] The synthesis of several specific reagents is shown in U.S.
patent application Ser. No. 10/786,610, entitled "Aminoheteroaryl
Compounds as Protein Kinase Inhibitors", filed Feb. 26, 2004, and
corresponding international application PCT/US2004/005495 of the
same title, filed Feb. 26, 2004. Other reagents can be synthesized
by adapting the procedures therein, and one skilled in the art can
readily adapt those procedures to produce the desired compounds.
Further, these references contain general procedures and specific
examples for the preparation of a large number of heteroarylamino
compounds, and one skilled in the art can readily adapt such
procedures and examples to the preparation of compounds of the
present invention. The disclosures of these references are
incorporated herein by reference in their entireties.
[0208] When a general or exemplary synthetic procedure is referred
to, one skilled in the art can readily determine the appropriate
reagents, if not indicated, extrapolating from the general or
exemplary procedures. Some of the general procedures are given as
examples for preparing specific compounds. One skilled in the art
can readily adapt such procedures to the synthesis of other
compounds. It should be understood that R groups shown in the
general procedures are meant to be generic and non-limiting, and do
not correspond to definitions of R groups elsewhere in this
document. Each such R group represents one or multiple chemical
moieties that can be the same or different from other chemical
moieties also represented by the same R symbol. One skilled in the
art can readily appreciate the range of R groups suitable in the
exemplary syntheses. Moreover, representation of an unsubstituted
position in structures shown or referred to in the general
procedures is for convenience and does not preclude substitution as
described elsewhere herein. For specific groups that can be
present, either as R groups in the general procedures or as
optional substituents not shown, refer to the descriptions in the
remainder of this document, including the claims, summary and
detailed description.
[0209] Some of the general procedures are shown with reference to
synthesis of compounds wherein the
1-(2,6-dichloro-3-fluorophenyl)-ethoxy moiety is the pure
(R)-isomer, and some are shown with reference to compounds wherein
said moiety is a racemic mixture. It should be understood that the
procedures herein can be used to produce racemic compounds or
enantiomerically pure (R) isomers by choosing the corresponding
racemic or enantiomerically pure starting material.
[0210] The procedures shown herein can be used to produce a wide
variety of enantiomerically pure compounds by selection of the
appropriate enantiomerically pure starting material. In addition to
the compounds shown herein, the invention also provides
enantiomerically pure compounds corresponding to the
3-[1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-pyridin-2-ylamine and
3-[1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-pyrazin-2-ylamine
compounds shown in U.S. patent application Ser. No. 10/786,610
(PCT/US2004/005495); in U.S. application Ser. No. to be assigned,
docket number PC 32546, filed Aug. 26, 2004 and entitled,
"Pyrazolo-Substituted Aminoheteroaryl Compounds as Protein Kinase
Inhibitors"; and in U.S. application Ser. No. to be assigned,
docket number PC 32548, filed Aug. 26, 2004 and entitled,
"Aminoheteroaryl Compounds as Protein Kinase Inhibitors". The
disclosures of these documents are incorporated herein by reference
in their entireties.
Select Starting Materials
5-bromo-3-[1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-pyridin-2-ylamine
(racemate)
##STR00011##
[0212] 1. 2,6-Dichloro-3-fluoroacetophenone (15 g, 0.072 mol) was
stirred in THF (150 mL, 0.5M) at 0.degree. C. using an ice bath for
10 min. Lithium aluminum hydride (2.75 g, 0.072 mol) was slowly
added. The reaction was stirred at ambient temperature for 3 hr.
The reaction was cooled in ice bath, and water (3 mL) was added
drop wisely followed by adding 15% NaOH (3 mL) slowly. The mixture
was stirred at ambient temperature for 30 min. 15% NaOH (9 mL),
MgSO.sub.4 were added and the mixture filtered to remove solids.
The solids were washed with THF (50 mL) and the filtrate was
concentrated to give 1-(2,6-dichloro-3-fluoro-phenyl)-ethanol (14.8
gm, 95% yield) as a yellow oil. .sup.1H NMR (400 MHz, DMSO-d.sub.6)
.delta. 1.45 (d, 3H), 5.42 (m, 2H), 7.32 (m, 1H), 7.42 (m, 1H).
[0213] 2. To a stirred solution of triphenyl phosphine (8.2 g, 0.03
mol) and DEAD (13.65 mL of a 40% solution in toluene) in THF (200
mL) at 0.degree. C. was added a solution of
1-(2,6-dichloro-3-fluoro-phenyl)-ethanol (4.55 g, 0.021 mol) and
3-hydroxy-nitropyridine (3.35 g, 0.023 mol) in THF (200 mL). The
resulting bright orange solution was stirred under a nitrogen
atmosphere at ambient temperature for 4 hours at which point all
starting materials had been consumed. The solvent was removed, and
the crude material was dry loaded onto silica gel, and eluted with
ethyl acetate-hexanes (20:80) to yield
3-(2,6-dichloro-3-fluoro-benzyloxy)-2-nitro-pyridine (6.21 g, 0.021
mol, 98%) as a pink solid. .sup.1H NMR (CDCl.sub.3, 300 MHz)
.delta.1.8-1.85 (d, 3H), 6.0-6.15 (q, 1H), 7.0-7.1 (t, 1H),
7.2-7.21 (d, 1H), 7.25-7.5 (m, 2H), 8.0-8.05 (d, 1H).
[0214] 3. To a stirred mixture of AcOH (650 mL) and EtOH (500 mL)
was suspended 3-(2,6-dichloro-3-fluoro-benzyloxy)-2-nitro-pyridine
(9.43 g, 0.028 mol) and iron chips (15.7 g, 0.28 mol). The reaction
was heated slowly to reflux and allowed to stir for 1 hr. The
reaction was cooled to room temperature then diethyl ether (500 mL)
and water (500 mL) was added. The solution was carefully
neutralized by the addition of sodium carbonate. The combined
organic extracts were washed with sat'd NaHCO.sub.3 (2.times.100
mL), H.sub.2O (2.times.100 mL) and brine (1.times.100 mL) then
dried (Na.sub.2SO.sub.4), filtered and concentrated to dryness
under vacuum to yield
3-(2,6-dichloro-3-fluoro-benzyloxy)-pyridin-2-ylamine (9.04 g,
0.027 mol, 99%) as a light pink solid. .sup.1H NMR (CDCl.sub.3, 300
MHz) .delta.1.8-1.85 (d, 3H), 4.9-5.2 (brs, 2H), 6.7-6.84 (q, 1H),
7.0-7.1 (m, 1H), 7.2-7.3 (m, 1H), 7.6-7.7 (m, 1H).
[0215] 4. A stirring solution of
3-(2,6-dichloro-3-fluoro-benzyloxy)-pyridin-2-ylamine (9.07 g, 0.03
mol) in acetonitrile was cooled to 0.degree. C. using an ice bath.
To this solution was added N-bromosuccinimide (NBS) (5.33 g, 0.03
mol) portionwise. The reaction was stirred at 0.degree. C. for 15
min. The reaction was concentrated to dryness under vacuum. The
resulting dark oil was dissolved in EtOAc (500 mL), and purified
via silica gel chromatography. The solvents were then removed in
vacuo to yield
5-bromo-3-(2,6-dichloro-3-fluoro-benzyloxy)-pyridin-2-ylamine (5.8
g, 0.015 mol, 51%) as a white crystalline solid. .sup.1H NMR
(CDCl.sub.3, 300 MHz) .delta.1.85-1.95 (d, 3H), 4.7-5.0 (brs, 2H),
5.9-6.01 (q, 1H), 6.8-6.95 (d, 1H), 7.01-7.2 (t, 1H), 7.4-7.45 (m,
1H), 7.8-7.85 (d, 1H).
5-iodo-3-[1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-pyridin-2-ylamine
(racemate)
##STR00012##
[0217] To a solution of
3-[1-(2,6-Dichloro-3-fluoro-phenyl)ethoxy]-pyridin-2-ylamine (10.0
g, 33.2 mmol) in acetonitrile (600 mL) and acetic acid (120 mL) was
added N-iodosuccinimide (11.2 g, 49.8 mmol). The mixture was
stirred at room temperature for 4 h and the reaction was quenched
with Na.sub.2S.sub.2O.sub.5 solution. After evaporation, the
residue was partitioned between ethyl acetate and water. The
organic layer was washed with 2N NaOH solution, brine, and dried
over Na.sub.2SO.sub.4. The crude product was purified on a silica
gel column to provide
5-iodo-3-[1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-pyridin-2-ylamine
(7.1 g, 50% yield). MS m/z 427 [M+1]. .sup.1H NMR (400 MHz,
DMSO-D6) .delta. ppm 1.74 (d, J=6.57 Hz, 3H) 5.91-5.99 (m, 3H) 6.82
(d, J=1.26 Hz, 1H) 7.46 (t, J=8.72 Hz, 1H) 7.56 (dd, J=8.97, 4.93
Hz, 1H) 7.62 (d, J=1.52 Hz, 1H).
5-bromo-3-[1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-pyrazin-2-ylamine
(racemate)
##STR00013##
[0219] 1. 2,6-Dichloro-3-fluoroacetophenone (15 g, 0.072 mol) was
stirred in THF (150 mL, 0.5M) at 0.degree. C. using an ice bath for
10 min. Lithium aluminum hydride (from Aldrich, 2.75 g, 0.072 mol)
was slowly added. The reaction was stirred at ambient temperature
for 3 h. The reaction was cooled in ice bath, and water (3 mL) was
added drop wisely followed by adding 15% NaOH (3 mL) slowly. The
mixture was stirred at ambient temperature for 30 min. 15% NaOH (9
mL), MgSO.sub.4 were added and the mixture filtered to remove
solids. The solids were washed with THF (50 mL) and the filtrate
was concentrated to give 1-(2,6-dichloro-3-fluoro-phenyl)-ethanol
(14.8 gm, 95% yield) as a yellow oil. .sup.1H NMR (400 MHz,
DMSO-d.sub.6) .delta. 1.45 (d, 3H), 5.42 (m, 2H), 7.32 (m, 1H),
7.42 (m, 1H).
[0220] 2.
5-Bromo-3-[1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-pyrazin-2-yl-
amine was prepared following procedure 2 below, from
1-(2,6-dichloro-3-fluoro-phenyl)-ethanol and
3,5-dibromo-pyrazin-2-ylamine. .sup.1H NMR (400 MHz, DMSO-d.sub.6)
.delta. 1.74 (d, 3H), 6.40 (m, 1H), 6.52 (br s, 2H), 7.30 (m, 1H),
7.48 (m, 1H), 7.56 (s, 1H); MS m/z 382 (M+1).
Enantiomerically Pure Starting Materials
[0221] PLE is an enzyme produced by Roche and sold through
Biocatalytics Inc. as a crude esterase preparation from pig liver,
commonly known as PLE-AS (purchased from Biocatalytics as ICR-123,
sold as an ammonium sulfate suspension). The enzyme is classified
in the CAS registry as a "carboxylic-ester hydrolase, CAS no.
9016-18-6". The corresponding enzyme classification number is EC
3.1.1.1. The enzyme is known to have broad substrate specificity
towards the hydrolysis of a wide range of esters. The lipase
activity is determined using a method based on hydrolysis of
ethylbutyrate in a pH titrator. 1 LU (lipase unit) is the amount of
enzyme which liberates 1 .mu.mol titratable butyric acid per minute
at 22.degree. C., pH 8.2. The preparation reported herein (PLE-AS,
as a suspension) is usually shipped as an opaque brown-green liquid
with a declared activity of >45 LU/mg (protein content around 40
mg/mL).
(1S)-1-(2,6-dichloro-3-fluorophenyl)ethanol
[0222] (1S)-1-(2,6-dichloro-3-fluorophenyl)ethanol, shown as
compound (S-1) in the schemes below, was prepared by a combination
of enzymatic hydrolysis of racemic
1-(2,6-dichloro-3-fluorophenyl)ethyl acetate, esterification and
chemical hydrolysis with inversion according to Scheme B. Racemic
1-(2,6-dichloro-3-fluorophenyl)ethyl acetate (compound A2) was
prepared according to Scheme A.
##STR00014##
[0223] 1-(2,6-dichloro-3-fluorophenyl)ethanol (A1): Sodium
borohydride (90 mg, 2.4 mmol) was added to a solution of
2',6'-dichloro-3'-fluoro-acetophenone (Aldrich, catalog #52, 294-5)
(207 mg, 1 mmol) in 2 mL of anhydrous CH.sub.3OH. The reaction
mixture was stirred at room temperature for 1 h then was evaporated
to give a colorless oil residue. The residue was purified by flash
chromatography (eluting with 0.fwdarw.10% EtOAc in hexanes) to give
compound A1 as a colorless oil (180 mg; 0.88 mmol; 86.5% yield); MS
(APCI) (M-H).sup.- 208; .sup.1H NMR (400 MHz, chloroform-D) .delta.
ppm 1.64 (d, J=6.82 Hz, 3H) 3.02 (d, J=9.85 Hz, 1H) 6.97-7.07 (m,
1H) 7.19-7.33 (m, 1H).
[0224] 1-(2,6-dichloro-3-fluorophenyl)ethyl acetate (A2): Acetic
anhydride (1.42 mL, 15 mmol) and pyridine (1.7 mL, 21 mmol) were
added sequentially to a solution of compound A1 (2.2 g, 10.5 mmol)
in 20 mL of CH.sub.2Cl.sub.2. The reaction mixture was stirred at
room temperature for 12 h and then evaporated to give a yellowish
oil residue. The residue was purified by flash chromatography
(eluting with 7.fwdarw.9% EtOAc in hexanes) to give compound A2 as
a colorless oil (2.26 g; 9.0 mmol; 85.6% yield); .sup.1H NMR (400
MHz, chloroform-D) .delta. ppm 1.88 (d, J=6.82 Hz, 3H) 2.31 (s, 3H)
6.62 (q, J=6.82 Hz, 1H) 7.25 (t, J=8.46 Hz, 1H) 7.49 (dd, J=8.84,
5.05 Hz, 1H).
##STR00015##
[0225] To a 50 mL jacketed flask equipped with a pH electrode, an
overhead stirrer and a base addition line (1M NaOH), was added 1.2
mL of 100 mM potassium phosphate buffer pH 7.0 and 0.13 mL of PLE
AS suspension. Then, compound A2 (0.13 g, 0.5 mmol, 1.00 eq) was
added dropwise and the resulting mixture was stirred at room
temperature for 20 h, maintaining the pH of the reaction constant
at 7.0 using 1 M NaOH. Both the conversion and ee's of the reaction
were monitored by RP-HPLC, and stopped after 50% starting material
was consumed (approximately 17 hours under these conditions). The
mixture was then extracted three times with 10 mL of ethyl acetate
to recover both ester and alcohol as a mixture of R-1 and S-2.
[0226] Methanesulfonyl chloride (0.06 mL, 0.6 mmol) was added to a
solution of a mixture of R-1 and S-2 (0.48 mmol) in 4 mL of
pyridine under nitrogen atmosphere. The reaction mixture was
stirred at room temperature for 3 h then evaporated to obtain an
oil. Water (20 mL) was added to the mixture and then EtOAc (20
mL.times.2) was added to extract the aqueous solution. The organic
layers were combined, dried, filtered, and evaporated to give a
mixture of R-3 and S-2. This mixture was used in the next step
reaction without further purification. .sup.1H NMR (400 MHz,
chloroform-D) .delta. ppm 1.66 (d, J=7.1 Hz, 3H) 1.84 (d, J=7.1 Hz,
3H) 2.09 (s, 3H) 2.92 (s, 3H) 6.39 (q, J=7.0 Hz, 1H) 6.46 (q, J=6.8
Hz, 1H) 6.98-7.07 (m, 1H) 7.07-7.17 (m, 1H) 7.23-7.30 (m, 1H) 7.34
(dd, J=8.8, 4.80 Hz, 1H).
[0227] Potassium acetate (0.027 g, 0.26 mmol) was added to a
mixture of R-3 and S-2 (0.48 mmol) in 4 mL of DMF under nitrogen
atmosphere. The reaction mixture was heated to 100.degree. C. for
12 h. Water (20 mL) was added to the reaction mixture and EtOAc (20
mL.times.2) was added to extract the aqueous solution. The combined
organic layer was dried, filtered, and evaporated to give an oil of
S-2 (72 mg, 61% yield in two steps). Chirality ee: 97.6%. .sup.1H
NMR (400 MHz, chloroform-D) .delta. ppm 1.66 (d, J=7.1 Hz, 3H) 2.09
(s, 3H) 6.39 (q, J=6.8 Hz, 1H) 7.02 (t, J=8.5 Hz, 1H) 7.22-7.30 (m,
1H).
[0228] Sodium methoxide (19 mmol; 0.5 M in methanol) was added
slowly to compound S-2 (4.64 g, 18.8 mmol) under a nitrogen
atmosphere at 0.degree. C. The resulting mixture was stirred at
room temperature for 4 hours. The solvent was evaporated and
H.sub.2O (100 mL) was added. The cooled reaction mixture was
neutralized with sodium acetate-acetic acid buffer solution to pH
7. Ethyl acetate (100 mL.times.2) was added to extract the aqueous
solution. The combined organic layers were dried over
Na.sub.2SO.sub.4, filtered, and evaporated to obtain a white solid
(4.36 g, 94.9% yield); SFC-MS: 97% ee. .sup.1H NMR (400 MHz,
chloroform-D) .delta. ppm 1.65 (d, J=6.8 Hz, 3H) 5.58 (q, J=6.9 Hz,
1H) 6.96-7.10 (m, 1H) 7.22-7.36 (m, 1H).
3-[(1R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]-2-nitropyridine
##STR00016##
[0230] 3-Hydroxy-2-nitropyridine (175 mg, 1.21 mmol) and
triphenylphosphine (440 mg, 1.65 mmol) were added sequentially to a
stirred solution of (1S)-1-(2,6-dichloro-3-fluorophenyl)ethanol
(229.8 mg, 1.1 mmol) in THF (10 mL) under a nitrogen atmosphere.
The reaction mixture was maintained at room temperature for 1 h and
then diisopropyl azo-dicarboxylate (0.34 mL, 1.65 mmol) was added
at 0.degree. C. The mixture was stirred for an additional 12 h. The
reaction mixture was evaporated under vacuum to give an oil. The
residue was purified by flash chromatography (eluting with
20.fwdarw.25% EtOAc in hexanes) to give the title compound as a
white solid (321.5 mg; 0.97 mmol; 88.3% yield); MS (APCI)
(M+H).sup.+ 331; SFC-MS: 99.5% ee. .sup.1H NMR (400 MHz,
chloroform-D) .delta. ppm 1.85 (d, J=6.6 Hz, 3H) 6.10 (q, J=6.6 Hz,
1H) 7.04-7.13 (m, 1H) 7.21 (dd, J=8.5, 1.14 Hz, 1H) 7.30 (dd,
J=9.0, 4.9 Hz, 1H) 7.37 (dd, J=8.6, 4.6 Hz, 1H) 8.04 (dd, J=4.6,
1.3 Hz, 1H).
3-[(1R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]pyridin-2-amine
##STR00017##
[0232] Iron (365 mg) was added to a stirred solution of
3-[(1R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]-2-nitropyridine (321
mg, 0.97 mmol) in a mixture of EtOH (2 mL) and 2M HCl (0.2 mL) at
0.degree. C. The resulting solution was heated to 85.degree. C. for
2 h. Celite (0.5 g) was added to the cooled reaction mixture. This
mixture was filtered over a bed of celite and evaporated to give
the title compound as a dark oil. MS (APCI) (M+H).sup.+ 301.
5-bromo-3-[1(R)-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-pyridin-2-ylamine
##STR00018##
[0234] The enantiomerically pure R isomer was prepared as described
above for the racemate, but using the enantiomerically pure
starting materials described above. .sup.1H NMR (400 MHz,
DMSO-d.sub.6) .delta. 1.74 (d, 3H), 6.40 (m, 1H), 6.52 (br s, 2H),
7.30 (m, 1H), 7.48 (m, 1H), 7.56 (s, 1H); MS m/z 382 (M+1).
5-iodo-3-[(R)1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-pyridin-2-ylamine
##STR00019##
[0236] Periodic acid (60 mg, 0.24 mmol), iodine (130 mg, 0.5 mmol),
and sulfuric acid (0.03 mL) were added sequentially to a stirred
solution of
3-[(1R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]pyridin-2-amine (0.97
mmol) in a mixture of acetic acid (3 mL) and H.sub.2O (0.5 mL). The
resulting solution was heated to 80.degree. C. for 5 h. The cooled
reaction mixture was quenched with Na.sub.2SO.sub.3 (80 mg) and
basicified with saturated Na.sub.2CO.sub.3 (2.times.100 mL) to pH
7. CH.sub.2Cl.sub.2 (2.times.50 mL) was added to extract the
aqueous solution. The combined organic layers were dried over
Na.sub.2SO.sub.4 then filtered and concentrated under vacuum. The
residue was purified by flash chromatography (eluting with
35.fwdarw.40% EtOAc in hexanes) to give the title compound as a
yellow oil (254 mg; 0.6 mmol; 61.6% yield); MS (APCI) (M+H).sup.+
426. .sup.1H NMR (400 MHz, chloroform-D) .delta. ppm 1.81 (d, J=6.8
Hz, 3H) 4.86 (s, 2H) 5.98 (q, J=6.57 Hz, 1H) 6.96 (d, J=1.5 Hz, 1H)
7.08 (dd, J=9.0, 8.0 Hz, 1H) 7.31 (dd, J=8.8, 4.8 Hz, 1H) 7.78 (d,
J=1.8 Hz, 1H).
5-bromo-3-[(R)-1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-pyrazin-2-ylamine
##STR00020##
[0238] The title compound was prepared according to procedure 2,
from (1S)-1-(2,6-dichloro-3-fluorophenyl)ethanol. .sup.1H NMR (400
MHz, DMSO-d6) .delta. 7.53 (s, 1H), 7.48 (m, 1H), 7.39 (t, 1H),
6.48 (s, 2H), 6.41 (q, 1H), 1.74 (d, 3H); LCMS: 381 [M+1]; c-Met
Ki: 0.796 .mu.M.
##STR00021##
General Procedure 1 for the Synthesis of
5-Bromo-3-(Substituted-Benzyloxy)-Pyridin-2-ylamine (5)
[0239] 1. Preparation of 3-(substituted-benzyloxy)-2-nitro-pyridine
(3): To a stirred solution of Cs.sub.2CO.sub.3 (1.0 molar
equivalent)) in DMF (0.2 M) under a N.sub.2 atmosphere containing
3-hydroxy-4-nitro-pyridine (Aldrich, 1.0 molar equivalent) is added
substituted benzyl bromide (1.0 molar equivalent). The mixture is
stirred for 6 h at ambient temperature. The reaction is then
diluted with EtOAc, and partitioned with H.sub.2O. The aqueous
layer is extracted with EtOAc twice. The organic layers are then
combined, washed with H.sub.2O and brine, dried over
Na.sub.2SO.sub.4, filtered, and concentrated to dryness under
vacuum to yield 3-(substituted-benzyloxy)-2-nitro-pyridine (3) as a
solid.
[0240] 2. Preparation of
3-(substituted-benzyloxy)-pyridin-2-ylamine (4): To a stirred
mixture of AcOH and EtOH (1.3:1) is suspended
3-(substituted-benzyloxy-2-nitro-pyridine (1.0 molar equivalent, 1
M) and iron chips (1.0 molar equivalent). The reaction is heated
slowly to reflux and allowed to stir for 1 hr. The reaction is
cooled to room temperature then filtered through a pad of celite.
The resulting filtrate is neutralized with conc. NH.sub.4OH, and
then extracted with EtOAc for three times. The combined organic
extracts are washed with saturated NaHCO.sub.3, H.sub.2O, and
brine, dried over Na.sub.2SO.sub.4, filtered and concentrated to
dryness under vacuum to yield
3-(substituted-benzyloxy)-pyridin-2-ylamine (4) as a solid.
[0241] 3. Preparation of 5-bromo-3-(substituted
benzyloxy)-pyridin-2-ylamine (5): A stirring solution of
3-(substituted-benzyloxy)-pyridin-2-ylamine (4) (1.0 molar
equivalent) in acetonitrile is cooled to 0.degree. C. using an ice
bath. To this solution is added N-bromosuccinimide (Aldrich, 1.0
molar equivalent) portionwise. The reaction is stirred at 0.degree.
C. for 15 min. The reaction is concentrated to dryness under
vacuum. The resulting dark oil is dissolved in EtOAc and
partitioned with H.sub.2O. The organic is then washed with
saturated NaHCO.sub.3 twice and brine once. Activated charcoal is
added to the organic layer and warmed to reflux. The solution is
then cooled to room temperature and filtered through a pad of
celite. The organic is then concentrated to dryness under vacuum to
one third the original volume. The solids are then filtered off to
yield 5-bromo-3-(substituted benzyloxy)-pyridin-2-ylamine (5) as a
solid.
##STR00022##
General Procedure 2 for the Synthesis of
5-Bromo-3-(Substituted-Benzyloxy)-Pyrazin-2-ylamine
##STR00023##
[0243] To an ice cooled solution of substituted benzyl alcohol (1.0
molar equivalent) and anhydrous tetrahydrofuran (0.14 M) was added
sodium hydride (1.0 molar equivalent) slowly under nitrogen
atmosphere. After stirring for 30 minutes,
3,5-dibromopyrazin-2-ylamine (1.0 molar equivalent) in
tetrahydrofuran (0.56 M) was added via an addition funnel at a fast
dropwise rate. Once the addition was complete the ice bath was
removed and the reaction was refluxed under nitrogen and monitored
by reversed phase HPLC. After 18 hr HPLC showed that the majority
of the starting 3,5-dibromopyrazin-2-ylamine had been consumed and
the reaction was allowed to cool to room temperature. The reaction
mixture was concentrated, diluted with ethyl acetate, and washed
with brine. The organic layer was dried over anhydrous magnesium
sulfate and concentrated in vacuum. The crude product was purified
using a silica gel eluting with 1:1 ethyl acetate/dichloromethane
to yield the 5-bromo-3-(substituted-benzyloxy)-pyrazin-2-ylamine as
a white solid in 60-90% yield.
General Procedure 3 for the Synthesis of
5-Aryl-3-(Substituted-Benzyloxy)-Pyridin-2-ylamine and
5-Aryl-3-(Substituted-Benzyloxy)-Pyrazin-2-ylamine
##STR00024##
[0245] A mixture of
5-bromo-3-(substituted-benzyloxy)-pyridin-2-ylamine or
5-bromo-3-(substituted-benzyloxy)-pyrazin-2-ylamine (1 molar
equivalent), aryl boronic acid or ester (1.2 molar equivalent),
bis(triphenylphosphine) palladium II chloride (0.03 molar
equivalent) and sodium carbonate (3.0 molar equivalent.) in
ethylene glycol dimethyl ether and water (10:0.5, 0.03 M) is
de-gassed and charged with nitrogen for three times, and then
heated to reflux under nitrogen for overnight. The reaction is
cooled to ambient temperature and diluted with ethyl acetate. The
mixture is washed with water, brine, dried over Na.sub.2SO.sub.4,
and purified on a silica gel column to afford
5-aryl-3-(substituted-benzyloxy)-pyridin-2-ylamine, or
5-aryl-3-(substituted-benzyloxy)-pyrazin-2-ylamine.
General Procedure 4 for Amidation Reaction of
6-amino-5-(substituted-benzyloxy)-pyridin-3-yl]-benzoic acid
##STR00025##
[0247] To a solution of
6-amino-5-(substituted-benzyloxy)-pyridin-3-yl]-benzoic acid (1
molar equivalent), 1-hydroxybenzotriazole hydrate (HOBT, 1.2 molar
equivalent), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride (EDC, 1.2 molar equivalent) in DMF (0.2 M) is added
amine (1.2 molar equivalent). The reaction solution is stirred at
room temperature for overnight, then diluted with EtOAc, and
partitioned with H.sub.2O. The organic is separated and the aqueous
is extracted with EtOAc. The organic layers are combined, washed
with saturated NaHCO.sub.3, and concentrated to dryness under
vacuum. The material is purified using column chromatography
(silica gel, 99:1 to 95:5 CH.sub.2Cl.sub.2/MeOH). The fractions
containing product are concentrated under vacuum to yield the amide
product.
General procedure 5 for the preparation of
3-(substituted-benzyloxy)-5-(3-dialkylaminomethyl-1H-indol-5-yl)-pyridin--
2-ylamine
##STR00026##
[0249] To a solution of benzotriazole (1.0 molar equivalent) in
dichloromethane (0.2 M) Is added amine (1.0 molar equivalent). The
reaction Is stirred for 5 minutes at room temperature after which
formaldehyde (37% by wt, 1.0 molar equivalent) Is added and the
reaction capped and stirred at room temperature for 3 h. Once TLC
(10% ethyl acetate:dichloromethane) shows the consumption of
starting benzotriazole the reaction is dried with anhydrous
magnesium sulfate (10 g), filtered and concentrated in vacuo. The
crude product is purified with a silica gel column eluting with 1:1
ethyl acetate:dichloromethane to yield the desired product as a
white solid.
[0250] To a solution of the aminomethylbenzotriazole intermediate
(1.0 molar equivalent) in dichloromethane (0.43 M) is added
aluminum chloride (2.0 molar equivalent) followed by
3-(2,6-dichloro-benzyloxy)-5-(1H-indol-5-yl)-pyridine-2-ylamin (1.1
molar equivalent). The reaction is capped and heated with stirring
to 40.degree. C. for 3-4 h. The reaction is then removed from the
heat and allowed to cool to room temperature. The reaction mixture
is diluted with sodium hydroxide (0.2 M) and chloroform, recapped
and vigorously stirred at room temperature to dissolve the residue
in the vial. The chloroform is extracted away from the aqueous,
dried over anhydrous sodium sulfate and concentrated in vacuo. The
crude product is purified with a silica gel column, first eluting
with 1:1, ethyl acetate:dichloromethane, to elute the less polar
impurities and then eluting the product with 90:9:1,
chloroform:methanol:ammonium hydroxide. (Yields 10-67%.)
General Procedure 6 for the synthesis of
3-(Substituted-benzyloxy)-5-phenyl-pyridin-2-ylamine using
3-(3-methoxy-benzyloxy)-5-phenyl-pyridin-2-ylamine
##STR00027##
[0252] To a solution of 3-benzyloxy-5-phenyl-pyridin-2-ylamine
(Example I-87, 3.27 g, 11.8 mmol) in methanol (30 mL) was added
Pd(OH).sub.2 (2.5 g, 2.37 mmol). The mixture was degassed and
charged with hydrogen three times, and then stirred under hydrogen
balloon for 5 h. The reaction was filtered through a celite pad,
washed with methanol, and condensed. After high vacuum dry,
2-amino-5-phenyl-pyridin-3-ol was obtained (2.04 g, 93% yield). MS
m/z 187 [M+1].
[0253] To a solution of 2-amino-5-phenyl-pyridin-3-ol (2.04 g,
10.95 mmol) in THF (anhydrous, 30 mL) was added NaH (1.31 g, 32.85
mmol) slowly. The mixture was stirred under nitrogen for 20
minutes, and then trityl chloride (3.66 g, 13.14 mmol) was added.
The reaction was stirred at room temperature for over night under
nitrogen. The solvent was evaporated, and the residue was dissolved
in dichloromethane, washed with water, and dried over
Na.sub.2SO.sub.4. After filtration and condensation, the crude
product was purified on a silica gel column eluting with
EtOAc-Hexane (1:10) to provide
5-phenyl-2-(trityl-amino)-pyridin-3-ol (1.09 g, 23% yield). MS m/z
427 [M+1].
[0254] To a solution of 5-phenyl-2-(trityl-amino)-pyridin-3-ol (100
mg, 0.24 mmol) in THF (3 mL) was added Cs.sub.2CO.sub.3 (79 mg,
0.24 mmol). The mixture was stirred at room temperature for 20
minutes, and then 3-methoxybenzylbromide (0.037 mL, 0.26 mmol) was
added. The reaction was stirred at room temperature overnight,
diluted with dichloromethane (5 mL), and filtered to remove the
salts. The solvents were evaporated, and the residue was dissolved
in 10% trifluoroacetic acid in dichloromethane (2 mL). The reaction
was stirred for 2 hr, and evaporated. The residue was dissolved in
dichloromethane, washed by sat. NaHCO.sub.3, and dried over
Na.sub.2SO.sub.4. After filtration and concentration, the crude
product was purified on a silica gel column eluting with
methanol-dichloromethane (from 3% to 15% gradient) to provide
3-(3-methoxy-benzyloxy)-5-phenyl-pyridin-2-ylamine as a white solid
(43.5 mg, 60% yield).
General Procedure 7 for the Synthesis of
3-(Substituted-benzyloxy)-5-Aryl-pyridin-2-ylamine using
5-[4-(2-morpholin-4-yl-ethoxy)-phenyl]-3-(3-nitro-benzyloxy)-pyridin-2-yl-
amine
##STR00028##
[0256] To a solution of
2-amino-5-[4-(2-morpholin-4-yl-ethoxy)-phenyl]-pyridin-3-ol
(prepared according to the procedures for
2-amino-5-phenyl-pyridin-3-ol in Example I-88 of U.S. patent
application Ser. No. 10/786,610 (PCT/US2004/005495) (45.5 mg, 0.14
mmol) in DMF (3 mL) at 0.degree. C. was added NaH (60% in oil) (5.6
mg, 0.14 mmol) and the mixture was stirred at 0.degree. C. for 20
min. Then 1-bromomethyl-3-nitro-benzene was added and the mixture
was stirred at 0.degree. C. for 1 hr and at room temperature for 2
hr. Cold 1 N aqueous HCl (0.1 mL) was added and the solvent was
removed under reduced pressure. The residue was purified with
silica gel chromatography
(CH.sub.2Cl.sub.2:MeOH:NH.sub.4OH=100:3:0.3) to give
5-[4-(2-morpholin-4-yl-ethoxy)-phenyl]-3-(3-nitro-benzyloxy)-pyridin-2-yl-
amine as yellow solid (44 mg, 68%).
General Procedure 8 for the Synthesis of
{4-[6-Amino-5-(substituted-benzyloxy)-pyridin-3-yl]-phenyl}-[(2R)-2-pyrro-
lidin-1-ylmethyl-pyrrolidin-1-yl]-methanone using
{4-[6-amino-5-(4-fluoro-2-trifluoromethyl-benzyloxy)-pyridin-3-yl]-phenyl-
}-[(2R)-2-pyrrolidin-1-ylmethyl-pyrrolidin-1-yl]-methanone:
##STR00029## ##STR00030##
[0258] 1. 6-Amino-5-benzyloxy-nicotinic acid was prepared according
to procedure 3 from 3-benzyloxy-5-bromo-pyridin-2-ylamine and
4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-benzoic acid. MS
m/z 321 (M+1).
[0259] 2.
[4-(6-amino-5-benzyloxy-pyridin-3-yl)-phenyl]-[(2R)-2-pyrrolidin-
-1-ylmethyl-pyrrolidin-1-yl]-methanone was prepared following
procedure 4 using 6-amino-5-benzyloxy-nicotinic acid and
(2R)-pyrrolidin-1-ylmethyl-pyrrolidine (prepared in Example I-39 of
U.S. patent application Ser. No. 10/786,610 (PCT/US2004/005495)).
MS m/z 457 (M+1).
[0260] 3. To a solution of
[4-(6-amino-5-benzyloxy-pyridin-3-yl)-phenyl]-[(2R)-pyrrolidin-1-ylmethyl-
-pyrrolidin-1-yl]-methanone (2.28 g, 5.00 mmol) in methanol (25 mL)
was added 10% Pd/C (100 mg). The mixture was degassed and charged
with hydrogen for three times, and then stirred under hydrogen
balloon overnight. The reaction was filtered through a celite pad,
washed with methanol, and condensed. After high vacuum dry,
[4-(6-amino-5-hydroxy-pyridin-3-yl)-phenyl]-[(2R)-2-pyrrolidin-1-ylmethyl-
-pyrrolidin-1-yl)-methanone was obtained (1.74 g, 95% yield).
.sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 7.79 (s, 1H), 7.54 (m,
3H), 7.46 (m, 2H), 7.14 (s, 1H), 5.68 (s, 2H), 4.22 (m, 1H), 3.45
(m, 2H), 2.66 (m, 1H), 2.52 (m, 4H), 1.96 (m, 2H), 1.84 (m, 3H),
1.64 (m, 4H); MS m/z 367 (M+1).
[0261] 4. To a stirred solution of
[4-(6-amino-5-hydroxy-pyridin-3-yl)-phenyl]-[(2R)-2-pyrrolidin-1-ylmethyl-
-pyrrolidin-1-yl]-methanone (100 mg, 0.27 mmol) in anhydrous DMF
(15 mL) under a N.sub.2 atmosphere containing, at 0.degree. C.,
sodium hydride (60% dispersion in mineral oil, 11 mg, 0.49 mmol)
was added. The mixture was allowed to stir at 0.degree. C. for 30
min. 1-(Bromomethyl)-4-fluoro-2-(trifluoromethyl)benzene (0.046 mL,
0.27 mmol) was added. The mixture was stirred at room temperature
for 2 hr. The reaction was diluted with EtOAc, and partitioned with
H.sub.2O. The aqueous layer was extracted with EtOAc (2.times.25
mL). The organic layers were combined, washed with H.sub.2O
(1.times.15 mL), brine (1.times.15 mL), dried over MgSO.sub.4,
filtered, concentrated, and purified on a silica gel column to
yield
{4-[6-amino-5-(4-fluoro-2-trifluoromethyl-benzyloxy)-pyridin-3-yl]-phenyl-
}-[(2R)-2-pyrrolidin-1-ylmethyl-pyrrolidin-1-yl]-methanone as
off-white crystals.
General Procedure 9 for the Synthesis 2-Dialkylamino-ethanesulfonic
acid [6-amino-5-(substituted-benzyloxy)-pyridin-3-yl]-phenyl-amide
using 2-diethylamino-ethanesulfonic acid
{4-[6-amino-5-(2-chloro-3,6-difluoro-benzyloxy)-pyridin-3-yl]-phenyl}-ami-
de
##STR00031##
[0263] 1. To a solution of
4-(4,4,5,5-Tetramethyl-[1,3,2]dioxaborolan-2-yl)phenylamine (5 g,
22.8 mmol) in dichloromethane (120 mL) was added N-methyl
morpholine (7.5 mL, 68.4 mmol). This mixture was cooled to
0.degree. C. under nitrogen atmosphere. 2-Chloroethanesulfonyl
chloride (2.5 mL, 23.9 mmol) in dichloromethane (60 mL) was then
added drop wise with stirring. Once the addition was complete the
flask was stirred at 0.degree. C. for 1 hr and then at room
temperature while monitoring by TLC (1:1 ethyl acetate:hexanes) and
staining with ninhydrin. After 4 h stirring some starting boronic
ester still remained and an additional 0.2 equivalents (0.5 mL) of
2-chloroethanesulfonyl chloride in dichloromethane (25 mL) was
added drop wise at room temperature. After 1 hr the boronic ester
had been consumed as shown by TLC and the total reaction volume was
reduced by one-half via rotary evaporation. The contents were
diluted with ethyl acetate (200 mL), washed with 50% brine
(2.times.100 mL), dried over anhydrous sodium sulfate and
concentrated in vacuum. The crude product was purified using silica
gel (120 g) and eluting with 10% ethyl acetate, dichloromethane to
yield ethenesulfonic acid
[4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenyl]-amidea as
a white solid (6.2 g, 20.2 mmol, 89% yield). .sup.1H NMR
(CDCl.sub.3, 300 MHz), .delta. 7.76 (d, J=8.4, 2H), 7.12 (d,
J=8.45, 2H) 6.65 (s, 1H), 6.55 (dd, J=9.77, 6.7, 1H), 6.31 (d,
J=16.54, 1H), 5.96 (d, J=9.8, 1H), 1.33 (s, 12H).
[0264] 2. To a solution of ethenesulfonic acid
[4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenyl]-amide
(0.500 g, 1.6 mmol) in methanol (5 mL) was added diethylamine
(0.707 g, 4.0 mmol) in methanol (5 mL), and the reaction was
stirred at room temperature and monitored by TLC (1:1 Ethyl
acetate:hexanes). After 2 hr the reaction was concentrated in
vacuum and the residue partitioned between ethyl acetate (50 mL)
and water (50 mL). The ethyl acetate was then washed with 50% brine
(1.times.50 mL), dried over anhydrous sodium sulfate, filtered and
concentrated in vacuum. Crude product was purified using a 10 g
prepacked silica gel column, eluting with 1:1 ethyl
acetate:dichloromethane to provide 2-diethylamino-ethanesulfonic
acid
[4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenyl]-amide as
a white solid (0.346 g, 0.90 mmol, 56%). .sup.1H NMR (CDCl.sub.3,
300 MHz) .delta. 7.78 (d, J=6.65, 2H) 7.15 (d, J=6.66, 2H), 3.20
(m, 2H), 3.0 (m, 2H), 2.55 (q, J=7.15, 7.16 4H), 1.34 (s, 12H),
1.05 (t, J=7.19, 6H).
[0265] 3. 2-diethylamino-ethanesulfonic acid
{4-[6-amino-5-(2-chloro-3,6-difluoro-benzyloxy)-pyridin-3-yl]-phenyl}-ami-
de was prepared following the general Suzuki coupling procedure 3
from 5-bromo-3-(2-chloro-3,6-difluoro-benzyloxy)-pyridin-2-ylamine
and 2-diethylamino-ethanesulfonic acid
[4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenyl]-amide
prepared in part 2 as a white solid in 60% yield.
General Procedure 10
[0266] 1: 4-(4,4,5,5-tetramethyl 1,3,2 dioxaboralan-2-yl) aniline
(3 g, 0.013 mol) was dissolved in dichloromethane (350 mL) to which
pyridine (1.02 g, 0.013 mol) and 4-nitrophenyl chloroformate was
added. The reaction was stirred for 13 hr where TLC analysis showed
consumption of all starting materials. The solution was washed with
saturated NaHCO.sub.3 (3.times.50 mL), water (3.times.50 mL) and
brine (3.times.50 mL). The organic layer was dried over
Na.sub.2SO.sub.4 and solvent removed to yield a white crystalline
solid
[4-(4,4,5,5-Tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenyl]-carbamic
acid phenyl ester, 4.45 g, 91%. .sup.1H NMR (CDCl.sub.3 300 MHz)
.delta. 1.4 (s, 12H), 7.1 (brs, 1H), 7.3 (d, 2H), 7.5 (d, 2H), 7.8
(d, 2H), 8.3 (d, 2H).
##STR00032##
[0267] 2:
[4-(4,4,5,5-Tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenyl]-carbam-
ic acid phenyl ester (500 mg, 1.3 mmol) was dissolved in anhydrous
dichloromethane (0.5 mL) and triethylamine (0.187 mL, 1.3 mmol). To
this stirred solution was added 1-methyl piperazine (or any other
amine) (0.144 mL, 1.3 mmol). The solution turned yellow instantly,
and tlc analysis showed consumption of all starting material. The
reaction was washed with water (3.times.500 mL), saturated sodium
bicarbonate (2.times.200 mL) and dried prior to removal of solvents
in vacuo. The boronic esters were used without purification.
[0268] 3: To a mixture of 2.1 mL of DME and 2.8 mL of 2N
Na.sub.2CO.sub.3 was added 100 mg of the bromide scaffold, 1
equivalent of the boronic acid, and 5 mol % of Pd(PPh.sub.3).sub.4.
The reaction was stirred and heated at 80.degree. C. overnight in a
two dram vial. The crude mixture was filtered through ceolite and
extracted with EtOAc (2.times.100 mL). The combined extracts were
washed with NaHCO.sub.3 (1.times.100 mL), followed by water
(1.times.100 mL), and then saturated brine (1.times.100 mL). The
resulting mixture was concentrated in vacuum. The residue was
dissolved in hexane and purified via column chromatography.
General Procedure 11
##STR00033## ##STR00034##
[0270] 1: To a solution of
3-[1-(2,6-Dichloro-3-fluoro-phenyl)ethoxy]-pyridin-2-ylamine (10.0
g, 33.2 mmol) in acetonitrile (600 mL) and acetic acid (120 mL) was
added N-iodosuccinimide (11.2 g, 49.8 mmol). The mixture was
stirred at room temperature for 4 hr and the reaction was quenched
with Na.sub.2S.sub.2O.sub.5 solution. After evaporation, the
residue was partitioned between ethyl acetate and water. The
organic layer was washed with 2N NaOH solution, brine, and dried
over Na.sub.2SO.sub.4. The crude product was purified on a silica
gel column to provide
3-[1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-5-iodo-pyridin-2-ylamine
(7.1 g, 50% yield). MS m/z 427 [M+1]
[0271] 2: To a solution of
3-[1-(2,6-Dichloro-3-fluoro-phenyl)-ethoxy]-5-iodo-pyridin-2-ylamine
(7.1 g, 16.6 mmol) and prop-2-ynyl-carbamic acid tert-butyl ester
(3.1 g, 20.0 mmol) in THF (60 mL) and Et.sub.3N (60 mL) was added
CuI (63 mg, 0.3 mmol) and Pd(PPh.sub.3).sub.4 (384 mg, 0.3 mmol).
The mixture was stirred under nitrogen and monitored by TLC until
the reaction was complete. The mixture was extracted with EtOAc and
washed by water. The crude product was purified on a silica gel
column eluting with 20-40% EtOAc in hexanes to provide
(3-{6-Amino-5-[1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-pyridin-3-yl}-pro-
p-2-ynyl)-carbamic acid tert-butyl ester (2.2 g, 29% yield).
[0272] 3: The solution of
(3-{6-Amino-5-[1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-pyridin-3-yl}-pro-
p-2-ynyl)-carbamic acid tert-butyl ester in 25% TFA in
dichloromethane was stirred for 2 hr, then washed by 2N NaOH, water
twice, brine, dried over Na.sub.2SO.sub.4. After filtration and
evaporation,
5-(3-amino-prop-1-ynyl)-3-[1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-pyrid-
in-2-ylamine was obtained in 93% yield.
[0273] 4: To a solution of
5-(3-amino-prop-1-ynyl)-3-[1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-pyrid-
in-2-ylamine (0.282 mmol, 1 eq) and 4-nitrophenyl chloroformate (1
eq) in anhydrous dichloromethane (10 mL) was added pyridine (1 eq).
The reaction was stirred for 4 hr under nitrogen, and then the
selected amine (1 eq) and triethylamine (1 eq) were added. The
mixture was refluxed for 5 minutes and cooled to room temperature.
The reaction mixture was washed with water. The organic layer was
evaporated and purified on a silica gel column eluting with 0-20%
methanol in dichloromethane on prepacked silica columns. Final
yields varied between 24% and 71%.
General Procedure 12
##STR00035##
[0275] 1: To a solution of
5-(3-amino-prop-1-ynyl)-3-[1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-pyrid-
in-2-ylamine (prepared in procedure 11) (400 mg, 1.1 mmol) in
dichloromethane (17 mL) was added chloroacetyl chloride (153 mg,
1.4 mmol). The reaction was stirred at room temperature with TLC
monitor of the completion of the reaction. After the completion,
the solvent was evaporated to get the crude product.
[0276] 2: To a solution of
N-(3-{6-Amino-5-[1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-pyridin-3-yl}-p-
rop-2-ynyl)-2-chloro-acetamide (1 eq) in acetonitrile (5 eq) was
added the individual amine (5 eq). The mixture was refluxing under
nitrogen overnight. After evaporation of solvent, the residue was
purified on a silica gel column eluting with 1-10% methanol in
dichloromethane to provide the product with yields varied between
47% to 97%.
General Procedure 13
##STR00036## ##STR00037##
[0278] 1. To a stirred solution of 2-amino-3-benzyloxypyridine
(42.0 g, 0.21 mol) in CH.sub.3CN (600 mL) at 0.degree. C. was added
N-bromosuccinimide (37.1 g, 0.21 mol) over 30 minutes. The mixture
was stirred for 0.5 hr, after which the reaction was then diluted
with EtOAc (900 mL) and partitioned with H.sub.2O (900 mL). The
organic layer was washed with brine and dried (Na.sub.2SO.sub.4),
filtered and concentrated to dryness under vacuum to yield
3-benzyloxy-5-bromo-pyridin-2-ylamine (31.0 g, 0.11 mol, 53%).
.sup.1H NMR (CDCl.sub.3, 300 MHz) .delta. 4.63-4.78 (brs, 2H), 5.04
(s, 2H), 7.07 (d, 1H, J, 1.8 Hz), 7.33-7.42 (m, 5H), 7.73 (d, 1H,
J, 1.8 Hz).
[0279] 2. To a stirred mixture of
3-benzyloxy-5-bromo-pyridin-2-ylamine (31.0 g, 0.11 mol) in a
mixture of DME (600 mL) and H.sub.2O (600 mL) was added
4-carboxymethylboronic acid (29.9 g, 0.11 mol), Pd(PPh.sub.3).sub.4
(6.4 g, 5.55 mmol), and Na.sub.2CO.sub.3 (82.0 g, 0.78 mol). The
reaction was heated slowly to reflux and allowed to stir for 3 hr.
The reaction was cooled to room temperature, then diluted with
CH.sub.2Cl.sub.2 (1.5 L) and partitioned with H.sub.2O (700 mL).
The organic layer was washed with saturated NaHCO.sub.3 (700 mL),
dried (Na.sub.2SO.sub.4), filtered, and concentrated in vacuo. The
crude material was purified by column chromatography (silica gel,
1:1 to 4:1 EtOAc:hexanes) and the fractions containing product were
combined and concentrated in vacuo to yield
4-(6-amino-5-benzyloxy-pyridin-3-yl)-benzoic acid methyl ester
(29.4 g, 0.086 mol, 79%). .sup.1H NMR (CDCl.sub.3, 300 MHz) .delta.
3.92 (s, 3H), 4.82-4.94 (brs, 2H), 5.15 (s, 2H), 7.22 (d, 1H, J,
1.8 Hz), 7.33-7.42 (m, 5H), 7.54 (d, 2H, J, 8.6), 7.98 (d, 1H, J,
1.8 Hz), 8.06 (d, 2H, J, 8.6 Hz).
[0280] 3. To a stirring solution of
4-(6-amino-5-benzyloxy-pyridin-3-yl)-benzoic acid methyl ester
(10.0 g, 0.03 mol) in EtOH:H.sub.2O (95:5, 600 mL) was added Pd/C
(15.9 g, 0.015 mol) (the reaction was de-gassed under vacuum). The
solution was allowed to stir under an H.sub.2 atmosphere for 22 hr.
The solution was filtered through wet celite and the celite washed
with EtOH. The filtrate was concentrated under vacuum to yield
4-(6-Amino-5-hydroxy-pyridin-3-yl)-benzoic acid methyl ester (2.3
g, 9.3 mmol, 31%). .sup.1H NMR (MeOD, 300 MHz) .delta. 3.90 (s,
3H), 7.21 (d, 1H, J, 1.9 Hz), 7.62 (d, 2H, J, 8.5 Hz), 7.76 (d, 1H,
J, 1.9 Hz), 8.04 (d, 2H, J, 8.5 Hz).
[0281] 4. To a stirring solution of
4-(6-amino-5-hydroxy-pyridin-3-yl)-benzoic acid methyl ester (2.3
g, 9.3 mmol) in CH.sub.2Cl.sub.2 (180 mL) was added
N,N-diisopropylethylamine (3.2 mL, 0.019 mol),
4-methyl-benzenesulfonyl chloride (2.66 g, 0.014 mol), and PS-DMAP
(catalytic amount). The reaction was stirred at ambient temperature
for 6 hr then filtered to remove the resin. The resin was washed
with CH.sub.2Cl.sub.2 (3.times.20 mL), and the combined fractions
were washed with 10% citric acid (100 mL), saturated NaCl (100 mL),
dried (Na.sub.2SO.sub.4) and filtered and concentrated in vacuo.
The resulting crude material was purified by column chromatography
(silica gel, 100% CH.sub.2Cl.sub.2 to 95:5 CH.sub.2Cl.sub.2:MeOH)
and the fractions containing the desired product were combined and
concentrated in vacuo to yield
4-[6-Amino-5-(toluene-4-sulfonyloxy)-pyridin-3-yl]-benzoic acid
methyl ester (3.3 g, 8.2 mmol, 88%). .sup.1H NMR (CDCl.sub.3, 300
MHz) .delta. 2.47 (s, 3H), 3.93 (s, 3H), 4.81-4.88 (brs, 2H),
7.36-7.44 (m, 5H), 7.81 (d, 2H, J, 8.3 Hz), 8.05 (d, 2H, J, 8.4
Hz), 8.19-8.27 (brs, 1H).
[0282] 5. To a stirred solution of
1-(3-fluoro-2-trifluoromethyl-phenyl)-ethanol (2.0 g, 9.6 mmol) in
anhydrous DMF (500 mL) at 0.degree. C. under a N.sub.2 atmosphere
was added NaH (0.38 g, 9.6 mmol). The reaction was allowed to stir
for 0.5 hr. A solution of
4-[6-Amino-5-(toluene-4-sulfonyloxy)-pyridin-3-yl]-benzoic acid
methyl ester (3.8 g, 9.6 mmol) in anhydrous DMF (30 mL) was added
to the reaction mixture which was allowed to come to ambient
temperature slowly and stirred for 21 hr at this temperature. The
reaction was diluted with EtOAc (500 mL) and H.sub.2O (100 mL). The
organic layer was separated off and the aqueous was further
extracted with EtOAc (1.times.200 mL). The organic layers were
combined and washed with brine (1.times.100 mL), dried with
Na.sub.2SO.sub.4 and concentrated to dryness under vacuum. The
crude mixture was purified by column chromatography (silica gel,
40:60 to 70:30 EtOAc:hexanes) and the fractions containing product
were combined and concentrated in vacuo to yield
4-{6-amino-5-[1-(3-fluoro-2-trifluoromethyl-phenyl)-ethoxy]-pyridin-3-yl}-
-benzoic acid methyl ester (1.4 g, 3.2 mmol, 34%). .sup.1H NMR
(CDCl.sub.3, 300 MHz) .delta. 1.73 (d, 3H, J, 6.2 Hz), 3.91 (s,
3H), 4.87-4.64 (brs, 2H), 5.81 (q, 1H, J, 6.1, 6.3 Hz), 6.92 (d,
1H, J, 1.8 Hz), 7.38 (d, 2H, J, 8.5 Hz), 7.46-7.66 (m, 3H), 7.93
(d, 1H, J, 1.8 Hz), 8.02 (d, 2H, J, 8.5 Hz).
[0283] 6. To a stirred solution of
4-{6-amino-5-[1-(3-fluoro-2-trifluoromethyl-phenyl)-ethoxy]-pyridin-3-yl}-
-benzoic acid methyl ester (1.4 g, 3.2 mmol) in warm IPA (72 mL)
was added H.sub.2O (38 mL) containing LiOH (0.68 g, 16.2 mmol). The
reaction was heated to reflux for 3.5 hr. The reaction was
neutralized and diluted with EtOAc (200 mL) and extracted upon
cooling. The organic layer was washed with brine (50 mL), dried
over Na.sub.2SO.sub.4 and concentrated under vacuum to yield
4-{6-Amino-5-[1-(3-fluoro-2-trifluoromethyl-phenyl)-ethoxy]-pyridin-3-yl}-
-benzoic acid (1.2 g, 2.8 mmol, 88%). .sup.1H NMR (MeOD, 300 MHz)
.delta. 1.75 (d, 3H, J, 6.2 Hz), 4.88-4.93 (m, 1H), 7.01 (d, 1H, J,
1.8 Hz), 7.39 (d, 2H, J, 8.3 Hz), 7.52-7.67 (m, 3H), 7.80 (d, 1H,
J, 1.8 Hz), 7.97 (d, 2H, J, 8.3 Hz).
[0284] 7. Preparation of amide compounds: A stirring solution of
4-{6-Amino-5-[1-(3-fluoro-2-trifluoromethyl-phenyl)-ethoxy]-pyridin-3-yl}-
-benzoic acid (50 mg, 0.12 mmol), EDC (27.0 mg, 0.13 mmol) and HOBt
(18.0 mg, 0.13 mmol) in DMF (2 mL) was added to a two dram vial
containing NHR.sub.1R.sub.2 (0.12 mmol). The reaction was stirred
at room temperature for 18 hr. The reaction was then diluted with
CH.sub.2Cl.sub.2 (3 mL) and partitioned with H.sub.2O. The organic
was separated washed with saturated NaCl (1.times.2 mL) and
saturated NaHCO.sub.3 (1.times.2 mL). The organic was concentrated
to dryness under vacuum. The material was purified using column
chromatography (silica gel, 99:1 to 95:5 CH.sub.2Cl.sub.2/MeOH).
The fractions containing product were concentrated under vacuum to
yield amide compounds.
General Procedure 14
##STR00038##
[0286] 1: To a mixture of 1-(2-chloroethyl)pyrrolidine
hydrochloride (200 mg, 1.18 mmol) and
4-[4-(4,4,5,5-Tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenyl]-1H-pyrazole
(229 mg, 1.19 mmol) in DMF (6 mL) was added Cs.sub.2CO.sub.3. The
mixture was stirred at room temperature overnight. Water (10 mL)
was then added to the mixture. The product was extracted with EtOAc
(3.times.10 mL). The combined extracts were then washed with brine
(5.times.10 mL) to remove the DMF, then dried over
Na.sub.2SO.sub.4, and concentrated (142 mg, 41% yield).
[0287] 2: To a mixture of
3-[1-(2,6-Dichloro-3-fluoro-phenyl)-ethoxy]-5-iodo-pyridin-2-ylamine
(200 mg, 0.468 mmol), pinacol boronic ester (1.2 eq),
Na.sub.2CO.sub.3 (149 mg, 1.41 mmol) in water (1.25 mL), and
dimethyl ethyl glycol (3.75 mL, 0.1M) was added
Pd(PPh.sub.3).sub.2Cl.sub.2 (16 mg, 0.020 mmol) in a microwave
reaction vessel. The system was degassed and charged with nitrogen.
The mixture was stirred at 160.degree. C. in a microwave apparatus
for 15 minutes. The mixture was cooled to room temperature followed
by the addition of water (10 mL). The product was extracted with
EtOAc (3.times.20 mL), dried over Na.sub.2SO.sub.4, and
concentrated. The crude product was purified by reverse phase HPLC
with 0.1% TFA in water and acetonitrile.
General Procedure 15
##STR00039##
[0289] 1: To a solution of 3H-oxazolo[4,5-b]pyridin-2-one (13.6 g,
100 mmol) in acetonitrile (600 mL) and acetic acid (120 mL) was
added N-bromosuccinimide (21.4 g, 120 mmol). The mixture was
stirred at room temperature for 4 hr and the reaction was quenched
with Na.sub.2S.sub.2O.sub.5 solution. After evaporation, the
residue was partitioned between ethyl acetate and water. The
organic layer was washed with 2N NaOH solution, brine, and dried
over Na.sub.2SO.sub.4. The crude product was purified on a silica
gel column to provide 6-bromo-3H-oxazolo[4,5-b]pyridin-2-one (11.5
g, 55% yield).
[0290] 2: 6-Bromo-3H-oxazolo[4,5-b]pyridin-2-one (21.5 g, 100 mmol)
was suspended in NaOH solution (2N, 250 mL, 500 mmol). The mixture
was refluxed overnight and a clear solution was obtained. After
cooling to room temperature, the reaction solution was neutralized
to pH .about.7. A lot of CO.sub.2 was released and also precipitate
was observed. The product was filtered, washed with water, and
dried under high vacuum to provide 2-amino-5-bromo-pyridin-3-ol as
an off-white solid (17.8 g, 98% yield).
[0291] 3: To a solution of 2-amino-5-bromo-pyridin-3-ol (358 mg,
1.89 mmol) in DMF (8 mL) was added Cs.sub.2CO.sub.3 (620 mg, 1.89
mmol). The mixture was stirred at room temperature under nitrogen
for 1 hr. To the reaction mixture was added bromo-compound (0.9 eq)
in DMF (5 mL) slowly. The reaction solution was stirred under
nitrogen for five hr, and then partitioned between water and ethyl
acetate. The organic layer was washed with brine for three times,
dried over MgSO.sub.4. The crude product was purified on a silica
gel column eluting with hexane-ethyl acetate (4:1) to provide the
product with 70%-80% yield.
General Procedure 16 using Example I-488 of U.S. patent application
Ser. No. 10/786,610 (PCT/US2004/005495)
##STR00040##
[0293] 1. To a solution of 3-benzyloxy-5-bromo-pyridin-2-ylamine (1
g, 3.58 mmol) in dimethylsulfoxide (7 mL) was added sequentially
bis(pinacolato)diborane (1.0 g, 3.94 mmol), potassium acetate (1.05
g, 10.7 mmol)
[1,1'-bis(diphenylphosphino)ferrocine]dichloropalladium (II),
complex with dichloromethane (1:1) (146 mg, 0.18 mmol). The mixture
was heated to 80.degree. C. for 16 hr and then cooled to room
temperature. The reaction mixture was diluted with ethyl acetate
(50 mL) and filtered. The filtrate was washed with water
(2.times.50 mL) and dried over magnesium sulfate. Concentration in
vacuo yielded the crude boronate as a brown solid (1.13 g, 97%).
.sup.1H NMR (CDCl.sub.3) .delta. 1.32 (s, 12H), 5.08 (s, 2H), 5.44
(br s, 2H), 7.33-7.42 (m, 6H), 8.03 (s, 1H).
[0294] 2. An 18 mL reaction vessel was charged with the crude
3-benzyloxy-5-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-pyridin-2-yl-
amine (161 mg, 0.49 mmol), dimethoxyethane (3 mL) and
2-bromopyridine (117 mg, 0.74 mmol). To this solution was added
[1,1'-bis(diphenylphosphino)ferrocine]dichloropalladium (II),
complex with dichloromethane (1:1) (20 mg, 0.05 mmol) and a 2 M
solution of cesium carbonate in water (0.75 mL, 1.5 mmol). The
reactor was warmed to 80.degree. C. for 66 hr under a nitrogen
atmosphere, then cooled to room temperature. The reaction mixture
was partitioned between ethyl acetate (5 mL) and water (5 mL). The
organic layer was washed with additional water (5 mL) and diluted
with dimethylformamide (5 mL). Polymer-bound sulfonic acid (0.5 g,
2.1 mmol) was added to the organic solution, and the resulting
mixture was gently agitated for 2 hr. The resin was filtered and
washed with dimethylformamide, methanol and methylene chloride
(3.times.5 mL each solvent). Then the polymer was reacted with 2 M
ammonia in methanol for 1 hr. The resin was filtered and washed
with additional 2 M ammonia in methanol (2.times.5 mL), and the
combined filtrates were concentrated in vacuo. Purification of the
crude product by flash column chromatography yielded 52.2 mg of
product as a tan solid (38% yield).
General Procedure 17
##STR00041##
[0296] 1. To the solution of
3-(2-Chloro-3,6-difluoro-benzyloxy)-5-(4,4,5,5-tetramethyl-[1,3,2]dioxabo-
rolan-2-yl)-pyridin-2-ylamine (procedure 16) (10.0 g, 24.3 mmol) in
t-butyl alcohol (50 mL) was added boc anhydride (5.83 g, 26.7 mmol)
and reaction stirred at room temperature overnight. Additional boc
anhydride (2.25 g, 10.3 mmol) was added and reaction stirred
overnight again. Material was concentrated to a viscous black oil
and used as-is.
[0297] 2. The crude boronic ester (24.3 mmol theoretical) in THF
(150 mL) was added to a solution of sodium bicarbonate (16.3 g, 194
mmol) in water (150 mL) and acetone (23 mL). The mixture was cooled
to 2.degree. C. and oxone (13.5 g, 21.9 mmol) added slowly, keeping
temperature below 8.degree. C. Upon completion of addition,
reaction was stirred for 5 minutes then quenched with sodium
bisulfite (14.2 g) in water (28 mL). Ethyl acetate was added (200
mL) and layers separated. Aqueous layer was neutralized with 6N HCl
and extracted with ethyl acetate (2.times.200 mL). Combined
organics were washed with water (250 mL) and brine (250 mL), dried
(Na.sub.2SO.sub.r) and concentrated to a crude black oil. Silica
gel chromatography (ethyl acetate/hexane) gave the product as a
light brown foam (4.78 g, 49.0%). .sup.1H NMR (CDCl.sub.3) .delta.
1.48 (s, 9H), 1.74 (d, 3H), 5.75 (q, 1H), 6.61 (d, 1H), 76.89 (dt,
1H), 6.94-7.04 (m, 2H), 7.26 (d, 1H), 8.19 (bs, 1H). MS m/z 401
(M+H).sup.+.
[0298] 3. To cesium carbonate in a 2 dram vial was added
[3-(2-Chloro-3,6-difluoro-benzyloxy)-5-hydroxy-pyridin-2-yl]-carbamic
acid tert-butyl ester (100 mg, 0.25 mmol) in anhydrous DMF (1 mL)
followed by benzyl bromide (89.2 .mu.L, 0.75 mmol). The vial was
capped and stirred at 90.degree. C. overnight. Reaction was
filtered through a 5 mL Chem-Elut tube pre-wetted with water (3.5
mL) and eluted with 1:1 ethyl acetate:methylene chloride. After
partial concentration, 4N HCl in dioxane (1-2 mL) was added and
solution concentrated. Reverse phase chromatography
(water:acetonitrile, 0.05% TFA) followed by lyophilization, gave
the desired product as an off white amorphous solid (25.3 mg,
20.0%) and the bis-addition product as a tan amorphous solid (35.2
mg, 23.7%).
General Procedure 18
##STR00042##
[0300] Sodium borohydride (1.5 molar equivalent) is added to
solution of ketone (3.89 mmol) in 10 mL of ethanol under a nitrogen
atmosphere. The resulting mixture is stirred at room temperature
for 12 hr. The mixture is then put in an ice bath and quenched with
dilute aqueous HCl. The ethanol is evaporated and EtOAc is added to
extract the aqueous solution. The EtOAc layer is dried over
Na.sub.2SO.sub.4. The Na.sub.2SO.sub.4 is filtered off and the
filtrate evaporated to give a oil residue, compound A5. The residue
is used without further purification.
[0301] 3-Hydroxy-2-nitropyridine (1.1 molar equivalent) and
triphenylphosphine (1.5 molar equivalent) are added to a solution
of compound A5 (1.1 mmol) in 10 mL of THF. The reaction mixture is
then put in an ice bath and diisopropyl azodicarboxylate (1.5 molar
equivalent) is added. The ice bath is removed and the mixture
stirred at room temperature for 12 hr. The solvent is evaporated to
give a yellow oil residue. The residue is purified by silica gel
chromatography (eluting EtOAc in hexanes) to give compound A1.
[0302] 2 M HCl (0.2 mL) is added to solution of compound A1 (0.97
mmol) in 2 mL of ethanol. The mixture is then put in an ice bath
and Fe powder (365 mg) is added slowly. The reaction is heated to
85.degree. C. for 1 hr and cooled to room temperature. Celite (0.5
g) is added to stir and the resulting mixture is filtered through a
bed of celite and rinsed with ethanol. The filtrated is evaporated
to give a brown oil residue, compound A2. The residue is used
without further purification.
[0303] Periodic acid (0.25 molar equivalent), iodine (0.5 molar
equivalent), H.sub.2O (0.5 mL), and concentrate sulfuric acid (0.03
mL) are added to a solution of compound A2 in 3 mL of acetic acid.
The reaction mixture is heated to 85.degree. C. for 5 hr. The
reaction mixture is then cooled in an ice bath and basified with
saturated aq. Na.sub.2CO.sub.3 to a pH of 3-4. Ethyl acetate is
added to extract the aqueous solution. Dry EtOAc layer over
Na.sub.2SO.sub.4. The Na.sub.2SO.sub.4 is filtered off and the
filtrated evaporated to give a brown oil residue. The residue is
purified by silica gel chromatography (eluting with EtOAc and
hexanes) to give desired product, compound A3.
General Procedure 19
##STR00043##
[0305] Boronic ester or boronic acid (1.3 molar equivalent) is
added to a solution of compound A3 (0.47 mmol) in 5 mL of DME. The
mixture was purged with nitrogen several times and then
dichlorobis(triphenylphosphino) palladium (II) (0.05 molar
equivalent) is added. Sodium carbonate (3 molar equivalent) in 1 mL
of H.sub.2O is added to the reaction mixture and the resulting
solution heated to 85.degree. C. for 12 hr. Water is added to the
reaction mixture to quench the reaction. EtOAc is then added to
extract the aqueous solution. Dry EtOAc layer over
Na.sub.2SO.sub.4. The Na.sub.2SO.sub.4 is filtered off and the
filtrated evaporated to give a dark brown oil residue. The residue
is purified by silica gel chromatography (eluting with CH.sub.3OH,
CH.sub.2Cl.sub.2, EtOAc, and hexanes) to give desired product,
compound A4.
General Procedure 20
##STR00044##
[0307] Compound A6 was prepared using general procedure 19.
O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium phosphorus
pentafluoride (HATU) (1.1 molar equivalent), diisopropylethyl amine
(5 molar equivalent) and amine (1.3 molar equivalent) are added to
a solution of compound A6 (0.17 mmol) in 3 mL of DMF under a
nitrogen atmosphere. The reaction is allowed to stir at room
temperature for 12 hr. Saturated NaHCO.sub.3 is added to the
reaction mixture to quench the reaction. EtOAc is then added to
extract the aqueous solution. Dry EtOAc layer over
Na.sub.2SO.sub.4. The Na.sub.2SO.sub.4 is filtered off and the
filtrate is evaporated to give a brown oil residue. The residue is
purified by silica gel chromatography (eluting with EtOAc and
hexanes) to give desired amide product, compound A7, as a yellow
oil.
General Procedure 21
##STR00045##
[0309] Acid (16 molar equivalent or less) is added to compound A7
(0.13 mmol) at room temperature. The resulting solution is stirred
at room temperature or heated to 60.degree. C. for 12 hr. The
reaction mixture is evaporated and the residue is purified by
silica gel chromatography (eluting with CH.sub.3OH, EtOAc and
CH.sub.2Cl.sub.2) to give desired amide product, compound A8, as a
yellowish to white solid.
General Procedure 22
##STR00046##
[0311] Compound A9 is prepared using general procedure 19.
Di-tert-butyl dicarbonate (3 molar equivalent) and
4-(dimethylamino)pyridine (0.14 molar equivalent) are added to a
solution of compound A9 (3 mmol) in 20 mL of DMF. The reaction
mixture is stirred at room temperature for 12 hr. Water is added to
the reaction mixture to quench the reaction. EtOAc is then added to
extract the aqueous solution. Dry EtOAc layer over
Na.sub.2SO.sub.4. The Na.sub.2SO.sub.4 is filtered off and the
filtrated evaporated to give a brown yellow oil residue. The
residue is purified by silica gel chromatography (eluting with
25.fwdarw.30% EtOAc in hexanes) to give desired product, compound
A10 as a yellowish oil (87.8% yield). Ozone is bubbled through a
solution of compound A10 in 50 mL of CH.sub.2Cl.sub.2 at
-78.degree. C. and dimethyl sulfide is added to quench the
reaction. Saturated sodium chloride is added to the reaction
mixture and EtOAc is added to extract the aqueous solution.
Combined EtOAc layer is dried over Na.sub.2SO.sub.4. The
Na.sub.2SO.sub.4 is filtered off and the filtrated is evaporated to
give a yellow oil residue. The residue is purified by silica gel
chromatography (eluting with 35.fwdarw.40% EtOAc in hexanes) to
give desired product, compound A11 as a yellowish oil (58.4%
yield).
General Procedure 23: Reductive Amination
##STR00047##
[0313] Amine hydrochloride salt (1.2 molar equivalent), sodium
acetate (2 molar equivalent to the amine hydrochloride salt) are
added to a solution of compound A11 (0.45 mmol) in 4 mL of
CH.sub.3OH under a nitrogen atmosphere. Molecular sieve (0.5 g) is
added to the reaction mixture and then sodium cyanoborohydride (2
molar equivalent) is added. The resulting mixture is stirred at
room temperature for 12 hr under a nitrogen atmosphere. The
reaction mixture is filtered through a bed of celite and the
filtrate is evaporated and purified by silica gel chromatography
(eluting CH.sub.3OH, EtOAc, and CH.sub.2CL.sub.2) to give desired
product, compound A12 as an oil (52.6% yield). Acid (16 molar
equivalent or less) is added to compound A12 (0.17 mmol) at room
temperature. The resulting solution is stirred at room temperature
or heated to 60.degree. C. for 12 hr. The reaction mixture is
evaporated and the residue was purified by silica gel
chromatography (eluting with CH.sub.3OH, EtOAc and
CH.sub.2Cl.sub.2) to give desired product, compound A13.
General Procedure 24
##STR00048##
[0315] O-phenyldiamines (1.2 molar equivalent) and sodium bisulfite
(2.1 molar equivalent) are added to a solution of compound A11
(0.41 mmol) in 5 mL of DMA. The resulting solution is heated to
110.degree. C. for 12 hr. Water is added to the reaction mixture to
quench the reaction. EtOAc is then added to extract the aqueous
solution. Dry EtOAc layer over Na.sub.2SO.sub.4. The
Na.sub.2SO.sub.4 is filtered off and the filtrated is evaporated to
give a brown yellow oil residue. The residue is purified by silica
gel chromatography (eluting with EtOAc in hexanes) to give desired
product, compound A14. Acid (16 molar equivalent or less) is added
to compound A14 (0.16 mmol) at room temperature. The resulting
solution is stirred at room temperature or heated to 60.degree. C.
for 12 hr. The reaction mixture is evaporated and the residue is
purified by silica gel chromatography (eluting with CH.sub.3OH,
EtOAc and CH.sub.2Cl.sub.2) to give desired amide product, compound
A15.
General Procedure 25
##STR00049##
[0317] Di-tert-butyl dicarbonate (3 molar equivalent),
4-(dimethylamino)pyridine (0.14 molar equivalent) are added to a
solution of compound A3b (2 mmol) in 10 mL of DMF. The reaction
mixture is stirred at room temperature for 12 hr. Water is added to
the reaction mixture to quench the reaction. EtOAc is then added to
extract the aqueous solution. Dry EtOAc layer over
Na.sub.2SO.sub.4. The Na.sub.2SO.sub.4 is filtered off and the
filtrated is evaporated to give a brown yellow oil residue
(compound a16). The residue is used without further
purification.
[0318] Bis(pinacolato)diboron (1.2 molar equivalent) and potassium
acetate (3.4 molar equivalent) are added to a solution of compound
a16 in 4 mL of DMSO. The mixture is purged with nitrogen several
times and then dichlorobis(triphenylphosphino) palladium (II) (0.05
molar equivalent) is added. The resulting solution is heated to
80.degree. C. for 12 hr. Water is added to the reaction mixture to
quench the reaction. EtOAc is then added to extract the aqueous
solution. Dry EtOAc layer over Na.sub.2SO.sub.4. The
Na.sub.2SO.sub.4 is filtered off and the filtrated is evaporated to
give a dark brown oil residue. The residue is purified by silica
gel chromatography (eluting with 30% EtOAc in hexanes) to give
desired product, compound A17 (76% yield). HCl (5 molar equivalent)
is added to a solution of compound A17 (0.43 mmol) in 4 mL of
CH.sub.2Cl.sub.2. The resulting mixture is heated to 50.degree. C.
for 12 hr. Saturated NaHCO.sub.3 is added to the reaction mixture
to neutralize the reaction. EtOAc is then added to extract the
aqueous solution. Dry EtOAc layer over Na.sub.2SO.sub.4. The
Na.sub.2SO.sub.4 is filtered off and the filtrated is evaporated to
give the desired product (compound A18) as a yellow solid (75%
yield).
General Procedure 26
##STR00050##
[0320] Compound A17 (1.3 molar equivalent) is added to a solution
of aryl halide (0.36 mmol) in 3 mL of DME. The mixture is purged
with nitrogen several times and then
dichlorobis(triphenylphosphino) palladium (II) (0.05 molar
equivalent) is added. Sodium carbonate (3 molar equivalent) in 0.8
mL of H.sub.2O is added to the reaction mixture and the resulting
solution is heated to 85.degree. C. for 12 hr. Water is added to
the reaction mixture to quench the reaction. EtOAc is then added to
extract the aqueous solution. Dry EtOAc layer over
Na.sub.2SO.sub.4. The Na.sub.2SO.sub.4 is filtered off and the
filtrated is evaporated to give a dark brown oil residue. The
residue is purified by silica gel chromatography (eluting with
EtOAc in hexanes) to give desired product, compound A19 (74.4%
yield). HCl (5 molar equivalent) is added to a solution of compound
A19 (0.26 mmol) in 10 mL of isopropyl alcohol. The resulting
mixture is heated to 50.degree. C. for 12 hr. The solvent is
evaporated to give the desired product, compound A20.
General Procedure 27
##STR00051##
[0322] Compound A18 (1.3 molar equivalent) is added to a solution
of aryl halide (0.21 mmol) in 3 mL of DME. The mixture is purged
with nitrogen several times and then
dichlorobis(triphenylphosphino) palladium (II) (0.05 molar
equivalent) is added. Sodium carbonate (3 molar equivalent) in 0.6
mL of H.sub.2O is added to the reaction mixture and the resulting
solution is heated to 85.degree. C. for 12 hr. Water is added to
the reaction mixture to quench the reaction. EtOAc is then added to
extract the aqueous solution. Dry EtOAc layer over
Na.sub.2SO.sub.4. The Na.sub.2SO.sub.4 is filtered off and the
filtrated is evaporated to give a dark brown oil residue. The
residue is purified by silica gel chromatography (eluting with
CH.sub.3OH, CH.sub.2Cl.sub.2, EtOAc, and hexanes) to give desired
product, compound A21.
General Procedure 28
##STR00052##
[0324] Amine (1.5 molar equivalent) and K.sub.2CO.sub.3 (1.5 molar
equivalent) are added to a solution of 4-halobenzyl halide (1.0
molar equivalent) in 2 mL of toluene. The resulting mixture is
microwaved using Smithsynthesizer (150.degree. C., 1 hr). Water is
added to the reaction mixture to quench the reaction. EtOAc is then
added to extract the aqueous solution. Dry EtOAc layer over
Na.sub.2SO.sub.4. The Na.sub.2SO.sub.4 is filtered off and the
filtrated is evaporated to give the desired product, compound A23.
The residue is used in procedure 11 without further purification to
synthesize compound A22.
General Procedure 29
##STR00053##
[0326] Amine (1.2 molar equivalent) and diisopropylamine (5 molar
equivalent) are added to a solution of 4-bromobenzenesulfonyl
chloride (0.77 mmol) in 5 mL of CHCl.sub.3 under a nitrogen
atmosphere. The resulting mixture is stirred at room temperature
for 4 hr. Water is added to the reaction mixture to quench the
reaction. EtOAc is then added to extract the aqueous solution. Dry
EtOAc layer over Na.sub.2SO.sub.4. The Na.sub.2SO.sub.4 is filtered
off and the filtrated is evaporated to give the desired product,
compound A25. The residue is used in procedure 11 without further
purification to synthesize compound A24.
General Procedure 30
##STR00054##
[0328] Boronic ester or boronic acid (1.2 molar equivalent) is
added to a solution of 1-chloro-4-iodobenzene (0.84 mmol) in 10 mL
of (DME) under a nitrogen atmosphere. The mixture is purged with
nitrogen several times and then dichlorobis(triphenylphosphino)
palladium (II) (0.05 molar equivalent) is added. Sodium carbonate
(3 molar equivalent) in 1.8 mL of H.sub.2O is added to the reaction
mixture and the resulting solution is heated to 85.degree. C. for
12 hr. Water is added to the reaction mixture to quench the
reaction. EtOAc is then added to extract the aqueous solution. Dry
EtOAc layer over Na.sub.2SO.sub.4. The Na.sub.2SO.sub.4 is filtered
off and the filtrated is evaporated to give a dark brown oil
residue. The residue is purified by silica gel chromatography
(eluting with CH.sub.3OH, CH.sub.2Cl.sub.2, EtOAc, and hexanes) to
give desired product, compound A27. Compound A27 is used in
procedure 11 to synthesize compound A26.
General Procedure 31 for Chiral Separation of Racemates
[0329] The racemic sample is purified using preparative
supercritical fluid chromatography SFC-MS. Exemplary purification
conditions: column--Chiralpak AD-H, 250.times.21 mm, 5 micron, 100A
column (Column #: ADH0CJ-C1003); column temperature 35.degree. C.;
mobile phase 35% methanol (with 0.1% isopropylamine)-modified
CO.sub.2; preparative flow rate 52 mL/min; isobaric pressure at 120
bar.
General Procedure 32: using
(4-{6-Amino-5-[1-(3-trifluoromethyl-phenyl)-ethoxy]-pyridin-3-yl}-phenyl)-
-(3,5-dimethyl-piperazin-1-yl)-methanone
##STR00055##
[0331] To a mixture of
4-[4-(6-Amino-5-hydroxy-pyridin-3-yl)-benzoyl]-2,6-dimethyl-piperazine-1--
carboxylic acid tert-butyl ester (100 mg, 0.23 mmol) and
1-(1-bromo-ethyl)-3-trifluoromethyl-benzene (64 mg, 0.25 mmol) in
DMF (2 ml) was added NaH (12 mg, 0.47 mmol) at 0.degree. C. The
mixture was stirred overnight. LCMS showed that the reaction was
completed, DMF and water were removed. TFA (2 mL) was added to the
residue and stirred at room temperature for 3 hr. TFA was removed
followed by addition of methanol. The residue was purified by
prep-HPLC to afford
(4-{6-Amino-5-[1-(3-trifluoromethyl-phenyl)-ethoxy]-pyridin-3-yl}-phenyl)-
-(3,5-dimethyl-piperazin-1-yl)-methanone (30 mg, yield 25.7%).
General Procedure 33: using
(4-{6-amino-5-[1-(2-trifluoromethyl-phenyl)-ethoxy]-pyridin-3-yl}-phenyl)-
-(3,5-dimethyl-piperazin-1-yl)-methanone
##STR00056##
[0333] To a mixture of
4-[4-(6-Amino-5-hydroxy-pyridin-3-yl)-benzoyl]-2,6-dimethyl-piperazine-1--
carboxylic acid tert-butyl ester (50 mg, 0.12 mmol) and
1-(1-bromo-ethyl)-2-trifluoromethyl-benzene (32 mg, 0.12 mmol) in
DMF (2 ml) was added 2 M Cs.sub.2CO.sub.3 (0.18 mL, 0.35 mmol),
followed by water (0.5 mL), the mixture was stirred overnight then
heated at 70.degree. C. for 8 hr, LCMS showed that the reaction was
completed. The DMF and water were removed. TFA (2 mL was added to
the residue and stirred at room temperature for 3 hr. The TFA was
removed, followed by addition of methanol. The residue was purified
by prep-HPLC to afford
(4-{6-amino-5-[1-(2-trifluoromethyl-phenyl)-ethoxy]-pyridin-3-yl}-phenyl)-
-(3,5-dimethyl-piperazin-1-yl)-methanone (20 mg, yield 34.2%).
General Procedure 34: using
{4-[6-Amino-5-(2-methyl-benzyloxy)-pyridin-3-yl]-phenyl}-(3,5-dimethyl-pi-
perazin-1-yl)-methanone
##STR00057##
[0335] To a mixture of
(2R,6S)-4-[4-(6-Amino-5-hydroxy-pyridin-3-yl)-benzoyl]-2,6-dimethyl-piper-
azine-1-carboxylic acid tert-butyl ester (100 mg, 0.23 mmol) and
1-bromomethyl-2-methyl-benzene (47 mg, 0.25 mmol) in DMF (2 mL) was
added 2 M Cs.sub.2CO.sub.3 (0.35 mL, 0.7 mmol) followed by water
(0.5 mL). The mixture was stirred at room temperature overnight.
LCMS showed the reaction was completed, DMF was removed, followed
by addition of 4 N HCl in dioxane (2 mL) and the reaction was
stirred at room temperature for 3 hr. The volatiles were removed
followed by addition of methanol. This solution was purified by
prep-HPLC to afford
{4-[6-Amino-5-(2-methyl-benzyloxy)-pyridin-3-yl]-phenyl}-(3,5-dimethyl-pi-
perazin-1-yl)-methanone (47 mg, yield 46.6%).
General Procedure 35: using
(6-amino-3-aza-bicyclo[3.1.0]hex-3-yl)-(4-{6-amino-5-[1-(2,6-dichloro-3-f-
luoro-phenyl)-ethoxy]-pyridin-3-yl}-phenyl)-methanone
##STR00058##
[0337] To a mixture of
[3-(4-iodo-benzoyl)-3-aza-bicyclo[3.1.0]hex-6-yl]-carbamic acid
tert-butyl ester (100 mg, 0.234 mmol) and
3-[1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-5-(4,4,5,5-tetramethyl-[1,3,2-
]dioxaborolan-2-yl)-pyridin-2-ylamine (100 mg, 0.234 mmol) in DME
(2 mL) was added Pd(dppf).sub.2Cl.sub.2.CH.sub.2Cl.sub.2 (10 mg,
0.012 mmol) and Cs.sub.2CO.sub.3 (351 mg, 0.702 mmol). The mixture
was bubbled with nitrogen for 10 min then microwaved at 150.degree.
C. for 30 min. LCMS checked that the reaction was completed. The
crude reaction mixture was diluted with ethyl acetate followed by
washings with water and brine. The solution was dried over
MgSO.sub.4. Purification by prep-HPLC afforded a solid. The solid
was stirred with 4 N HCl/dioxane (3 mL) for 3 hr at room
temperature. Removal of the volatiles led to a residue that was
purified by prep-HPLC to afford
(6-amino-3-aza-bicyclo[3.1.0]hex-3-yl)-(4-{6-amino-5-[1-(2,6-dichloro-3-f-
luoro-phenyl)-ethoxy]-pyridin-3-yl}-phenyl)-methanone (30 mg, yield
26%).
General Procedure 36: using
5-[1-(2,6-Dichloro-3-fluoro-phenyl)-ethoxy]-6'-(2-morpholin-4-yl-ethoxy)--
[3,3']bipyridinyl-6-ylamine
##STR00059##
[0339] To a mixture of
6'-amino-5'-[1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-[3,3']bipyridinyl-6-
-ol (78 mg, 0.20 mmol), triphenylphosphine (63 mg, 0.24 mmol) and
2-morpholin-4-yl-ethanol (0.026 mL, 0.22 mmol) was added DEAD
(0.034 mL, 0.22 mmol). After stirring overnight more PPh.sub.3 (63
mg, 0.24 mmol) and more DEAD (0.034 mL, 0.22 mmol) were added.
After several hours, more alcohol (0.026 mL, 0.22 mmol) was added.
After several more hours, more PPh.sub.3 (63 mg, 0.24 mmol) and
more DEAD (0.034 mL, 0.22 mmol) were added. After stirring
overnight, the mixture was partitioned between dichloromethane and
half-saturated brine. The phases were separated and the aqueous
phase was extracted with dichloromethane. The combined organic
phases were dried over Na.sub.2SO.sub.4 and concentrated by rotary
evaporation. The residue was purified by silica gel chromatography
using gradient elution of dichloromethane, methanol to afford
5-[1-(2,6-Dichloro-3-fluoro-phenyl)-ethoxy]-6'-(2-morpholin-4-yl-ethoxy)--
[3,3']bipyridinyl-6-ylamine (53 mg, 53%).
General Procedure 37: using Example I-650 of U.S. patent
application Ser. No. 10/786,610 (PCT/US2004/005495)
##STR00060##
[0341]
3-(2,6-Dichloro-3-fluoro-benzyloxy)-5-thiazol-2-yl-pyridin-2-ylamin-
e: To a microwave tube equipped with a stir bar was added the
iodo-pyridyl starting material (300 mg, 0.702 mmol),
tetrakis(triphenylphosphine) palladium (0) (40 mg, 5 mol %) and
tetrahydrofuran (anhydrous, 6 mL). The vial was capped and purged
with nitrogen for 5 minutes. 2-Thiazolylzinc bromide (0.5 M in THF,
1.4 mmol, 2.8 mL) was then added via syringe. The vial was heated
to 120.degree. C. in the microwave for 10 minutes. TLC (1:1 ethyl
acetetate:methylene chloride) showed a large amount of starting
material remaining. Additional 2-thiazolylzinc bromide (0.5 M in
THF, 500 .mu.L) was added and the vial was heated to 120.degree. C.
in the microwave for 20 minutes. TLC (1:1 ethyl actetate:methylene
chloride) showed a large amount of starting material still
remaining. Additional 2-thiazolylzinc bromide (0.5 M in THF, 500
.mu.L) was added and the vial was heated to 120.degree. C. in the
microwave for 60 minutes. TLC (1:1 ethyl actetate:methylene
chloride) still showed a large amount of starting material still
remaining but also had become very messy. The vial contents were
poured into a sat. NH.sub.4Cl solution (10 mL) and this solution
extracted with ethyl acetate (2.times.30 mL). The combined ethyl
acetate layers were dried over Na.sub.2SO.sub.4, filtered and
concentrated in vacuo. The crude product was loaded onto a 10 g
prepacked silica gel column and 1:1 ethyl acetate:methylene
chloride used to elute the desired product. (40 mg, 15%).
General Procedure 38: using Example I-652 of U.S. patent
application Ser. No. 10/786,610 (PCT/US2004/005495)
##STR00061##
[0343]
3-[1-(2,6-Dichloro-3-fluoro-phenyl)-ethoxy]-5-(1-methyl-1H-imidazol-
-2-yl)-pyridin-2-ylamine: N-methyl imidazole (92 mg, 1.1 mmol) was
dissolved in tetrahydrofuran (anhydrous, 4 mL) in a 50 mL round
bottom flask. The flask was cooled with a dry-ice/acetone bath
under nitrogen atmosphere. N-butyl lithium (2.5 M, 562 .mu.L, 1.4
mmol) was added via syringe in 100 .mu.L portions over 5 minutes.
The reaction was stirred at -70.degree. C. for 30 minutes. Solid
zinc chloride (anhydrous, 383 mg, 2.8 mmol) was added and the
reaction stirred for 15 minutes. The ice bath was then removed and
the reaction allowed to warm to room temperature. Once all of the
zinc chloride was in solution and the reaction at room temperature,
iodo scaffold (400 mg, 0.936 mmol) was added in tetrahydrofuran
(anhydrous, 4 mL), followed by tetrakis(triphenylphosphine)
palladium (0) (108 mg, 10 mol %) and the reaction heated to reflux.
The reaction was monitored by LC/MS until all of the starting iodo
scaffold was consumed. The reaction was allowed to cool and then
diluted with a sat. NH.sub.4Cl solution (20 mL). This solution was
extracted with ethyl acetate (2.times.50 mL). The combined ethyl
acetate layers were dried over Na.sub.2SO.sub.4, filtered and
concentrated in vacuo. The crude product was loaded onto a 10 g
prepacked silica gel column and 10% methanol:ethyl acetate was used
to elute the desired product (25 mg, 7%).
General Procedure 39: using Example I-657 of U.S. patent
application Ser. No. 10/786,610 (PCT/US2004/005495)
##STR00062##
[0345] To
6-Amino-5-[1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-nicotinonitr-
ile (400 mg, 1.23 mmol) in 70 mL dry methanol at 0.degree. C. was
bubbled HCl gas for 3 minutes. Stirred overnight at 3.degree. C.
Removed volatiles and washed the solids with diethyl ether to yield
quantitatively the imidate. To 200 mg of the imidate in 4 mL
methanol at 0.degree. C. was added 2N methylamine in THF (837
.mu.L). Let stir at O.degree. C. for about 1 hr then let warm to rt
overnight. The volatiles were removed and the residue was
chromatographed with 10-20% methanol/dichloromethane to yield 70 mg
of product.
General Procedure 40
##STR00063##
[0347] 1. 6-Nitro-5-hydroxynicotinic acid (B2): To a solution of
5-hydroxynicotinic acid (B1) (7.0 g, 50 mmol) in concentrated
H.sub.2SO.sub.4 was added 9 mL of fuming HNO.sub.3 (90%) (9 mL).
The reaction mixture was stirred at 55-60.degree. C. in a sealed
tube for four days. The mixture was then poured into ice and the pH
was adjusted to 3 with 50% NaOH. MgSO.sub.4 was added to saturate
the aqueous mixture, which was then extracted with isopropyl
alcohol (4.times.45 mL). After the removal of isopropyl alcohol
under reduced pressure, 5.93 g (64% yield) of B2 was obtained as a
yellow solid. MS (APCI), (M+H).sup.+ 185. .sup.1HNMR (DMSO-d6)
.delta. 8.01 (d, 1H, Ar--H), 8.41 (d, 1H, Ar--H).
[0348] 2.
2,6-Dichlorobenzyl-6-nitro-5-[(2,6-dichlorobenzyl)oxy]nicotinate
(B3): 6-nitro-5-hydroxynicotinic acid (B2) (3.4 g, 18.5 mmol),
2,6-dichlorobenzyl bromide (8.88 g, 37 mmol), DIPEA (5.5 g, 42.5
mmol) were dissolved in DMF (25 mL) in a 250 mL round bottomed
flask and the reaction was stirred at room temperature for 4.5 hr
and then concentrated under reduced pressure. The resulting mixture
was poured into ice and the filtered. The solid collected was dried
under reduced pressure to give 4.25 g (46% yield) of B3. MS (APCI)
(M+H).sup.+ 503. .sup.1HNMR (DMSO-d6) .delta. 5.47 (s, 2H,
ArCH.sub.2O), 5.71 (s, 2H, ArCH.sub.2O), 7.24-7.43 (m, 6H, Ar--H),
8.26 (d, 1H, Ar--H), 8.66 (d, 1H, Ar--H).
[0349] 3.
2,6-Dichlorobenzyl-6-amino-5-[(2,6-dichlorobenzyl)oxy]nicotinate
(B4): A mixture of
2,6-dichlorobenzyl-6-nitro-5-[(2,6-dichlorobenzyl)oxy]nicotinate
(B3) (5.5 g, 10.96 mmol), iron powder (0.92 g, 16.43 mmol), glacial
acetic acid (20 mL) and methanol (17 mL) was stirred at 85.degree.
C. for three hr. The reaction mixture was concentrated to near
dryness, and ammonium hydroxide (30%) was added to neutralize the
mixture. Minimum amount of DMF was added to dissolve the reaction
mixture, which was purified by flash column chromatograph (eluent:
EtOAc-EtOH, 9:1) to give 4.5 g (87%) of B4 as a pale yellow solid.
MS (APCI) (M+H).sup.+ 473.
[0350] 4. 6-Amino-5-[(2,6-dichlorobenzyl)oxy]nicotinic acid (B5): A
mixture of
2,6-dichlorobenzyl-6-amino-5-[(2,6-dichlorobenzyl)oxy]nicotinate
(B4) (3.5 g, 7.4 mmol), lithium hydroxide (0.41 g, 17 mmol), water
(22 mL) and methanol (30 mL) was stirred and reflux at 85.degree.
C. for 5 hr. The mixture was concentrated to dryness under reduced
pressure. The resulting residue was dissolved in water, extracted
with a mixture of Et.sub.2O/hexane (1:1, 4.times.25 mL),
neutralized with 1N HCl to form white precipitation, which was
filtered and dried under reduced pressure to provide 1.83 grams
(79%) of B5 as a white solid. MS (APCI) (M+H).sup.+ 313. .sup.1HNMR
(DMSO-d6) .delta. 5.26 (s, 2H, ArCH.sub.2O), 6.37 (s, 2H,
NH.sub.2), 7.43-7.48 (t, 1H, Ar--H), 7.54 (s, 2H, Ar--H), 7.56 (s,
1H, Ar--H), 8.18 (s, 1H, Ar--H).
##STR00064##
[0351] To an array of 400 .mu.L of 0.2 M solution of different
amines in DMF in a 96-well plate was added 400 .mu.L (0.2 M in DMF)
of
4-[6-amino-5-(2,6-dichloro-3-fluoro-benzyloxy)-pyridin-3-yl]-benzoic
acid, 80 .mu.L of triethylamine (1 M in DMF) and 160 .mu.L of HATU
(0.5 M in DMF) and the reactions were stirred at 70.degree. C. for
2 hr. The solvent was removed using the SpeedVac apparatus and the
crude reaction mixtures were redissolved in DMSO and transferred
using a liquid handler to a 1 mL 96-well plate to give a final
theoretical concentration of .about.10 mM. The reactions were
analyzed and positive product identification was made using LC/MS.
The mother stock solution was diluted to 50 nM and assayed for
percent inhibition of c-MET at 50 nM.
General Procedure 41
##STR00065##
[0353] To an array of 400 .mu.L of 0.2 M solution of different
amines in DMF in a 96-well plate was added 400 .mu.L (0.2 M in DMF)
of 6-Amino-5-[(2,6-dichlorobenzyl)oxy]nicotinic acid, 80 .mu.L of
triethylamine (1M in DMF) and 160 .mu.L of HATU (0.5 M in DMF) and
the reactions were stirred at 70.degree. C. for 2 hr. The solvent
was removed using the SpeedVac apparatus and the crude reaction
mixtures were redissolved in DMSO and transferred using a liquid
handler to a 1 mL 96-well plate to give a final theoretical
concentration of .about.10 mM. The reactions were analyzed and
positive product identification was made using LC/MS. The mother
stock solution was diluted to 1 .mu.M and assayed
General Procedure 42 using
2-(4-{6-amino-5-[1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-pyridin-3-yl}-p-
yrazol-1-yl)-N-(3-dimethylamino-propyl)-isobutyramide
##STR00066## ##STR00067##
[0355] To a solution of
4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-1H-pyrazole (5 g,
25.77 mmol) and 2-bromo-2-methyl-propionic acid methyl ester (12.6
g, 27.06 mmol) in DMF (85 mL), was added Cs.sub.2CO.sub.3 (12.6 g,
38.65 mmol). The reaction mixture was heated to 90.degree. C. in an
oil bath overnight. The reaction solution was cooled to room
temperature, and partitioned between water and ethyl acetate. The
combined ethyl acetate solution was washed with water five times,
dried over Na.sub.2SO.sub.4, and concentrated to give the product
2-methyl-2-[4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-pyrazol-1-yl-
]propionic acid methyl ester (4.776 g, 63% yield).
[0356] To a solution of
3-[1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-5-iodo-pyridin-2-ylamine
(6.363 g, 14.901 mmol) and
2-methyl-2-[4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-pyrazol-1-yl-
]propionic acid methyl ester (4.6 g, 15.64 mmol) in DME (27 mL) was
added a solution of CsF (6.79 g, 44.7 mmol) in water (9.3 mL). The
reaction mixture was degassed 3 times with N.sub.2.
Pd(dppf)CH.sub.2Cl.sub.2 was added and the reaction mixture was
degassed 3 times with N.sub.2. The reaction was heated to
120.degree. C. in the microwave (subsequent Pd was added in
intervals of 30 minutes until the reaction was complete). Water was
added and the reaction was extracted with EtOAc, dried over
Na.sub.2SO.sub.4, and concentrated to give
2-(4-{6-amino-5-[1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-pyridin-3-yl}-p-
yrazol-1-yl)-2-methyl-propionic acid methyl ester. The crude
product was purified by a silica gel column chromatography with a
gradient of 25%-50% EtOAc/hexanes to provide
2-(4-{6-Amino-5-[1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-pyridin-3-yl}-p-
yrazol-1-yl)-2-methyl-propionic acid methyl ester (1.46 g, 21%
yield) with a R.sub.f 0.11 (50% EtOAc/hexanes).
[0357] To a solution of the methyl ester (2.92 g, 6.25 mmol) in
MeOH (31 mL) was added a solution of LiOH (450 mg, 18.76 mmol) in
water (6.25 mL). The reaction was heated to 60.degree. C. until
LCMS showed complete hydrolysis (about 45 minutes). The MeOH was
removed in vacuo and MeOH (2.5 mL) and water (1 mL) was added. The
pH was adjusted to pH 5 with 1N HCl, in which the product
precipitated out. The
2-(4-{6-amino-5-[1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-pyridin-3-yl}-p-
yrazol-1-yl)-2-methyl-propionic acid product was obtained after
filtration (2.825 g, quant.).
[0358] To a solution of
2-(4-{6-amino-5-[1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-pyridin-3-yl}-p-
yrazol-1-yl)-2-methyl-propionic acid (1.00 g, 2.20 mmol) in DMF
(5.5 mL) were added HOBT (300 mg, 2.20 mmol), EDC (633 mg, 3.30
mmol), and N,N-dimethyl-propane-1,3-diamine (225 mg, 2.20 mmol).
The reaction was stirred overnight at room temperature. The
reaction was then purified by reversed phase C-18 prep HPLC eluting
with acetonitrile/water with 0.1% acetic acid to afford
2-(4-{6-amino-5-[1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-pyridin-3-yl}-p-
yrazol-1-yl)-N-(3-dimethylamino-propyl)-isobutyramide (170 mg, 14%
yield).
General Procedure 43 using
3-[1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-5-(3-methyl-pyrazol-1-yl)-pyr-
idin-2-ylamine
##STR00068##
[0360] To a stirred solution of
3-[1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-5-iodo-pyridin-2-ylamine
(100 mg, 0.23 mmol) and 3-methyl-1H-pyrazole (59 mg, 0.70 mmol) in
DMSO (1 mL was added K.sub.3PO.sub.4 (101 mg, 0.47 mmol), dodecane
(0.015 mL, 0.05 mmol), cyclohexanediamine (0.009 mL, 0.07 mmol) and
copper iodide (CuI) (14 mg, 0.07 mmol). The solution was bubbled
with nitrogen for 5 minutes, then radiated with microwave at
150.degree. C. for 2 hours, LCMS checked that the reaction was
completed, the mixture was purified by prep-HPLC to leave
3-[1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-5-(3-methyl-pyrazol-1-y-
l)-pyridin-2-ylamine (30 mg), yield 34.2%
General Procedure 44
##STR00069##
[0362] 2,5-dibromopyridine (1 molar eq.) was dissolved in anhydrous
toluene (0.085 M) and cooled to -78.degree. C. n-BuLi (1.2 molar
eq.) was slowly added over 5 minutes and then the resulting mixture
allowed to stir at -78.degree. C. After 2 h, R.sub.1COR.sub.2 (1.3
molar eq.) was added and the solution kept at -78.degree. C. After
1 h, saturated aqueous NH.sub.4Cl was added and the solution was
warmed to room temperature. The product was extracted with EtOAc
(3.times.) and the organic extracts were combined, dried
(Na.sub.2SO.sub.4), concentrated, and purified by column
chromatography (10% EtOAc/Hexanes-100% EtOAc) to afford crude
product. It was used directly in General Procedure 27 to afford
25.
General Procedure 45
##STR00070##
[0364] To a solution of
3-[1-(2,6-dichloro-3-fluoro-phenyl)ethoxy]-pyridin-2-ylamine (1.8
g, 6.04 mmol), zinc cyanide, 98% (2.07 g, 12.07 mmol) and
1,1'-bis(diphenylphosphino)-ferrocene, 97% (0.4 g, 0.712 mmol) in
DMF (48 mL) was added
[1,1'-bis(diphenylphosphino)-ferrocene]dichloropalladium(II)
complex with dichloromethane (1:1) (0.25 g, 0.30 mmol). The
reaction mixture was heated to 150.degree. C. for overnight under
nitrogen atmosphere. The reaction was diluted with EtOAc (50 mL),
washed with 4:1:4 saturated NH.sub.4Cl/28% NH.sub.4OH/H.sub.2O
(2.times.28 mL), dried over Na.sub.2SO.sub.4. The crude mixture was
purified with a silica gel column eluting with a linear gradient of
25%-50% (EtOAc/hexanes) to provide
2-[1-(2-amino-pyridin-3-yloxy)-ethyl]-3-chloro-4-dimethylamino-benzonitri-
le as a yellow solid (37% yield) and
2-[1-(2-amino-pyridin-3-yloxy)-ethyl]-4-dimethylamino-isophthalonitrile
as a dark brown solid (33% yield).
General Procedure 46
##STR00071##
[0366] To a mixture of 4-bromo-imidazole (995 mg, 6.77 mmol),
potassium hydroxide (380 mg, 6.77 mmol), potassium carbonate (936
mg, 6.77 mmol) and tetra-n-butyl ammonium bromide (109 mg, 0.339
mmol) in dichloromethane (7 mL) was added tert-butyl bromo acetate
(0.50 mL, 3.4 mmol). After stirring overnight the reaction was
filtered. The filtrate was dried over sodium sulphate, filtered and
concentrated by rotary evaporation. The residue was purified by
silica gel chromatography using gradient elution of
dichloromethane, ethyl acetate to afford
(4-Bromo-imidazol-1-yl)-acetic acid tert-butyl ester (696 mg,
79%).
General Procedure 47
##STR00072##
[0368] A 4 M solution of hydrochloric acid in dioxane (0.22 mL,
0.89 mmol) was added to a solution of
(4-{6-Amino-5-[1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-pyridin-3-yl}-imi-
dazol-1-yl)-acetic acid tert-butyl ester (86 mg, 0.18 mmol) in
dichloromethane (2 mL). After stirring for two days the reaction
was concentrated by rotary evaporation and the residue was
dissolved in a minimum amount of methanol. This solution was added
dropwise to ether and the resulting mixture allowed to stand
overnight. The mixture was filtered and the precipitate was washed
with ether and air dried to give
(4-{6-Amino-5-[1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-pyridin-3-yl}-imi-
dazol-1-yl)-acetic acid (83 mg, 93%).
General Procedure 48
##STR00073##
[0370] A mixture of 4-bromo-imidazole (217 mg, 1.48 mmol) and
cesium carbonate (875 mg, 2.69 mmol) in dimethylformamide (5 mL)
was stirred for 30 minutes. 4-(2-Chloro-ethyl)-morpholine
hydrochloride (250 mg, 1.34 mmol) was added and the mixture was
heated to 50.degree. C. After heating overnight the reaction was
concentrated by rotary evaporation. The residue was suspended in a
mixture of dichloromethane and methanol and filtered. The filtrate
was concentrated by rotary evaporation. The residue was purified by
silica gel chromatography using gradient elution of
dichloromethane, methanol to afford
4-[2-(4-Bromo-imidazol-1-yl)ethyl]-morpholine (148 mg, 42%).
General Procedure 49
##STR00074##
[0372] Isoxazole (0.64 mL, 10 mmol) was added to a solution of
N-iodosuccinimide (2.3 g, 10 mmol) in trifluoroacetic acid (20 mL).
After stirring overnight, water (50 mL), hexanes (50 mL) and sodium
bisulfite were added to the reaction. The phases were separated and
the organic phase was dried over Na.sub.2SO.sub.4, filtered and
concentrated by rotary evaporation to give 4-iodo-isoxazole (218
mg, 11%).
General Procedure 50
##STR00075##
[0374] Trifluoroacetic acid (5 mL) was added to a solution of
6'-bromo-5-[1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-[3,3']bipyridinyl-6--
yl-bis-(tert-butoxycarbonyl)-amine (1.3 g, 2.0 mmol) in
dichloromethane (15 mL). After 3 hours, equal portions of water and
saturated aqueous sodium bicarbonate were added. The phases were
separated and the aqueous phase was extracted with dichloromethane.
The combined organic phases were dried over Na.sub.2SO.sub.4 and
concentrated by rotary evaporation to give
6'-bromo-5-[1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-[3,3']bipyri-
dinyl-6-ylamine (968 mg, 106%).
[0375] A tube was charged with
6'-bromo-5-[1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-[3,3']bipyridinyl-6--
ylamine (92 mg, 0.20 mmol), 4-pyrrolidin-1-yl-piperidine (0.62 g,
4.0 mmol) and N-methylpyrrolidinone (0.8 mL). The tube was sealed
and the mixture was heated at 80.degree. C. overnight. The
temperature was increased to 100.degree. C. for 5.5 hours and then
heating was ceased. The reaction was partitioned between ethyl
acetate and water. The phases were separated and the aqueous phase
was extracted with ethyl acetate. The combined organic phases were
dried over MgSO.sub.4 and concentrated by rotary evaporation. The
residue was purified by silica gel chromatography using gradient
elution of dichloromethane, methanol, ammonium hydroxide to afford
5''-[1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-4-pyrrolidin-1-yl-3,4,5,6-t-
etrahydro-2H-[1,2';5',3'']terpyridin-6''-ylamine (53 mg, 50%).
General Procedure 51
##STR00076##
[0377] Sodium hydride (56 mg, 2.3 mmol) was added to a solution of
piperidin-4-ol (214 mg, 2.11 mmol) in DMSO (8 mL). After stirring
for 30 minutes, 2,5-dibromopyridine was added. After stirring for
24 hours, sodium hydride (56 mg, 2.3 mmol) was added. After
stirring for another 24 hours the reaction was partitioned between
ethyl acetate and water. The phases were separated and the aqueous
phase was extracted with ethyl acetate. The combined organic phases
were dried over MgSO.sub.4 and concentrated by rotary evaporation.
The residue was purified by silica gel chromatography using
gradient elution of dichloromethane, methanol, ammonium hydroxide
to afford 5-bromo-2-(piperidin-4-yloxy)-pyridine (316 mg, 58%).
General Procedure 52
##STR00077##
[0379] A tube was charged with 2,5-dibromopyridine (0.24 g, 1.0
mmol), 4-Amino-piperidine-1-carboxylic acid tert-butyl ester (0.22
g, 1.1 mmol), di-isopropylethylamine (0.19 mL, 1.1 mmol) and
N-methylpyrrolidinone (1.0 mL). The tube was sealed and the mixture
was heated at 80.degree. C. overnight. The temperature was
increased to 120.degree. C. and heated overnight. The reaction was
partitioned between ethyl acetate and water. The phases were
separated and the aqueous phase was extracted with ethyl acetate.
The combined organic phases were dried over MgSO.sub.4 and
concentrated by rotary evaporation. The residue was purified by
silica gel chromatography using gradient elution of ethyl acetate
and hexanes to afford
4-(5-bromo-pyridin-2-ylamino)-piperidine-1-carboxylic acid
tert-butyl ester (36 mg, 10%).
General Procedure 53
##STR00078##
[0381]
4-(4-{6-Amino-5-[1-(2,6-dichloro-3-ethoxy-phenyl)-ethoxy]-pyridin-3-
-yl}-benzoyl)-piperazine-1-carboxylic acid tert-butyl ester: To 4
mL of DMSO was added 0.124 ml ethanol followed by 32 mg NaH. After
stirring for 30 minutes 250 mg of 250 mg
4-(4-{6-Amino-5-[1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-pyridin-3-yl}-b-
enzoyl)-piperazine-1-carboxylic acid tert-butyl ester was added and
the reaction was heated to 40.degree. C. After three hours the
reaction was cooled and poured into water to precipitate. After
neutralization to pH 6, 200 mg of a tan solid was isolated,
77%.
General Procedure 54
##STR00079##
[0383]
(4-{6-Amino-5-[1-(2,6-dichloro-3-hydroxy-phenyl)-ethoxy]-pyridin-3--
yl}-phenyl)-piperazin-1-yl-methanone: To 140 mg
4-[4-(6-Amino-5-{1-[2,6-dichloro-3-(2,4,6-trimethoxy-benzyloxy)-phenyl]-e-
thoxy}-pyridin-3-yl)-benzoyl]-piperazine-1-carboxylic acid
tert-butyl ester (from general procedure 53) was added 1 mL TFA,
the solution turned reddish immediately followed by addition of 100
.mu.L triethyl silane 3 seconds later. The solution turned to
yellow. After stirring for four hours 5 mL of toluene were added
and the solvent was removed in vacuo. Chromatography with 10%
MeOH/CH.sub.2Cl.sub.2 to 0.5% to 1% NH.sub.4OH/9.5 to 9% MeOH/90%
CH.sub.2Cl.sub.2 led to 55 mg of a white solid, 62% yield.
General Procedure 55
##STR00080##
[0385] 2-(4-bromo-2-methoxyphenoxy)ethanol (8a): Potassium
carbonate (1.4 g, 10 mmol) was added to a solution of ethylene
carbonate (1.8 g, 20 mmol) and 4-bromo-2-methoxyphenol (1.05 g, 5
mmol) in 5 mL of toluene under an inert atmosphere. The reaction
was heated at 115.degree. C. for 12 h. Water (50 mL) and ethyl
acetate (2.times.100 mL) were added to the reaction mixture to
stir. The organic layers were combined, dried, filtered, and
evaporated to get a yellow oil residue. The residue was purified by
flash chromatography (eluting with 40.fwdarw.45% EtOAc in hexanes)
to give compound 8a as a light brown yellow oil (1 g; 4.13 mmol;
82.6% yield); MS (APCI) (M+H).sup.+ 246. .sup.1H NMR (400 MHz,
chloroform-D) .delta. ppm 2.83 (t, J=6.3 Hz, 1H) 3.84 (s, 3H)
3.89-4.01 (m, 2H) 4.03-4.13 (m, 2H) 6.78 (d, J=8.3 Hz, 1H) 6.99 (d,
1H) 7.02 (d, 1H).
[0386] 4-bromo-1-(2-chloroethoxy)-2-methoxybenzene (8b): Thionyl
chloride (0.3 mL) was added to solution of compound 1 in 1 mL of
pyridine in an ice bath. The reaction was stirred in the ice bath
for 10 minutes then heated to 100.degree. C. for 2 h. The reaction
was cooled to room temperature and neutralized with dilute HCl (1
M). CH.sub.2Cl.sub.2 (2.times.100 mL) was added to extract the
aqueous solution. The combined organic layers were dried over
Na.sub.2SO.sub.4 then concentrated under vacuum. The residue was
purified by flash chromatography (eluting with 10.fwdarw.15% EtOAc
in hexanes) to give compound 8b as a colorless oil (485 mg; 1.84
mmol; 50.3% yield); MS (APCI) (M+H).sup.+ 264. .sup.1H NMR (400
MHz, chloroform-D) .delta. ppm 3.81 (t, J=6.2 Hz, 2H) 3.85 (s, 3H)
4.23 (t, J=6.2 Hz, 2H) 6.78 (d, J=8.6 Hz, 1H).
[0387] Compound 9: Compounds of formula 9 can be formed by the
following exemplary procedure: Compound A18 (1.3 molar equivalent)
is added to a solution of aryl halide (0.51 mmol) in 7 mL of DME.
The mixture is purged with nitrogen several times and then
dichlorobis(triphenylphsophino) palladium (II) (0.05 molar
equivalent) is added. Sodium carbonate (3 molar equivalent) in 1.5
mL of H.sub.2O is added to the reaction mixture and the resulting
solution is heated to 85.degree. C. for 12 h. Water (20 mL) is
added to the reaction mixture to quench the reaction. EtOAc (50
mL.times.2) is then added to extract the aqueous solution. Dry
EtOAc layer over Na.sub.2SO.sub.4. The Na.sub.2SO.sub.4 is filtered
off and the filtrated is evaporated to give a dark brown oil
residue. The residue is purified by silica gel chromatography
(eluting with CH.sub.3OH, CH.sub.2Cl.sub.2, EtOAc, and hexanes) to
give desired product, compound 9.
[0388] Compound 10: Compounds of formula 10 can be formed by the
following exemplary procedure: Amine (7 molar equivalent) is added
to a solution of compound 9 (0.17 mmol) in 3 mL of
2-methoxyethanol. The resulting solution is heated to 85.degree. C.
for 12 h. Water (20 mL) is added to the reaction mixture to quench
the reaction. EtOAc (50 mL.times.2) is then added to extract the
aqueous solution. The EtOAc layer is dried over Na.sub.2SO.sub.4.
The Na.sub.2SO.sub.4 is filtered off and the filtrated is
evaporated to give a light brown oil residue. The residue is
purified by silica gel chromatography (eluting with CH.sub.3OH,
CH.sub.2Cl.sub.2, EtOAc, and hexanes) to give desired product,
compound 10.
General Procedure 56
##STR00081##
[0390] Compound 14: Compounds of formula 14 can be formed by the
following exemplary procedure: Lithium hexamethyldisilazide (1.2
molar equivalent; 1M in THF) is added to a solution of alcohol (1
mmol) in 2 mL of THF. The mixture is stirred at room temperature
under a nitrogen atmosphere for 30 min and then
5-bromo-2-chloropyrimidine (1 molar equivalent) is added. The
resulting solution is heated to 75.degree. C. for 12 h. Water (20
mL) is added to the reaction mixture to quench the reaction. EtOAc
(50 mL.times.2) is then added to extract the aqueous solution. Dry
EtOAc layer over Na.sub.2SO.sub.4. The Na.sub.2SO.sub.4 is filtered
off and the filtrated is evaporated to give an oil residue. The
residue is purified by silica gel chromatography (eluting with
EtOAc in hexanes) to give desired product, compound 14.
[0391] Compound 11: Compound A18 (1.3 molar equivalent) is added to
a solution of 5-bromo-2-chloropyrimidine or compound 14 (1 mmol) in
24 mL of DME. The mixture is purged with nitrogen several times and
then dichlorobis(triphenylphosphino) palladium (II) (0.05 molar
equivalent) is added. Sodium carbonate (3 molar equivalent) in 3 mL
of H.sub.2O is added to the reaction mixture and the resulting
solution is heated to 85.degree. C. for 12 h. Water (50 mL) is
added to the reaction mixture to quench the reaction. EtOAc (100
mL.times.2) is then added to extract the aqueous solution. Dry
EtOAc layer over Na.sub.2SO.sub.4. The Na.sub.2SO.sub.4 is filtered
off and the filtrated is evaporated to give a dark brown oil
residue. The residue is purified by flash chromatography (eluting
with 40.fwdarw.55% EtOAc in hexanes) to give compound 11.
[0392] Compound 12: Amine (2 molar equivalent) is added to a
solution of compound 11 in 3 mL of n-butanol. The reaction mixture
is irradiated in microwave at 120.degree. C. for 30 min. The
resulting mixture is poured into a mixture of H.sub.2O and EtOAc
(100 mL; v:v: 1:1). The organic layer is dried, filtered, and
evaporated to give a light brown oil residue. The residue is
purified by silica gel chromatography (eluting with CH.sub.3OH,
CH.sub.2Cl.sub.2, EtOAc, and hexanes) to give desired product,
compound 12.
[0393] Compound 13: Acid (16 molar equivalent or less) is added to
compound 12 (0.14 mmol) at room temperature. The resulting solution
is stirred at room temperature or heated to 60.degree. C. for 12 h.
The reaction mixture is evaporated and the residue is purified by
silica gel chromatography (eluting with CH.sub.3OH, EtOAc and
CH.sub.2Cl.sub.2) to give desired amide product, compound 13, as a
yellowish to white solid.
General Procedure 57
##STR00082##
[0395] Compound 15: Sodium hydride (1.3 molar equivalent) and RX
(1.1 molar equivalent) were added to a solution of
2-amino-5-bromopyridine (0.84 mmol) in 3 mL of DMF. The reaction
mixture is irradiated in microwave at 100.degree. C. for 20 min.
The resulting mixture is poured into a mixture of H.sub.2O and
EtOAc (100 mL; v:v: 1:1). The organic layer is dried, filtered, and
evaporated to give a light brown oil residue. The residue is
purified by silica gel chromatography (eluting with CH.sub.3OH,
CH.sub.2Cl.sub.2, EtOAc, and hexanes) to give desired product,
compound 15.
[0396] Compound 16: Compound A18 (1.3 molar equivalent) is added to
a solution of compound 15 (0.25 mmol) in 5 mL of DME. The mixture
is purged with nitrogen several times and then
dichlorobis(triphenylphosphino) palladium (II) (0.05 molar
equivalent) is added. Sodium carbonate (3 molar equivalent) in 0.8
mL of H.sub.2O is added to the reaction mixture and the resulting
solution is heated to 85.degree. C. for 12 h. Water (50 mL) is
added to the reaction mixture to quench the reaction. EtOAc (100
mL.times.2) is then added to extract the aqueous solution. Dry
EtOAc layer over Na.sub.2SO.sub.4. The Na.sub.2SO.sub.4 is filtered
off and the filtrated is evaporated to give a dark brown oil
residue. The residue is purified by flash chromatography (eluting
with CH.sub.3OH, CH.sub.2Cl.sub.2, EtOAc, and hexanes) to give
desired product, compound 16.
[0397] Compound 17: Acid (16 molar equivalent or less) is added to
compound 16 (0.114 mmol) at room temperature. The resulting
solution is stirred at room temperature or heated to 60.degree. C.
for 12 h. The reaction mixture is evaporated and the residue is
purified by silica gel chromatography (eluting with CH.sub.3OH,
EtOAc and CH.sub.2Cl.sub.2) to give desired amide product, compound
17, as a yellowish to white solid.
General Procedure 58
##STR00083## ##STR00084##
[0399] 1-(t-butoxycarbonyl)azetidine-3-carboxylic acid
(1-1)(AXL016917, 1000 mg, 4.97 mmol) was dissolved in MeOH (5
mL)/Toluene (20 mL) and then cooled to 0.degree. C. TMSCHNN
(trimethylsilyldiazomethane) (7.45 mmol) was then added drop-wise
over 15 minutes with some bubbling observed. The color started
clear and slowly turned yellow. The solution was stirred for 10
minutes at 0.degree. C. and then warmed to room temperature over 30
minutes. The solution was then concentrated and pumped on to remove
toluene to afford 1.055 g of 1-t-butyl 3-methyl
azetidine-1,3-dicarboxylate (1-2) that was used directly in the
next step without being purified (99% crude yield).
[0400] 1-tert-butyl 3-methyl azetidine-1,3-dicarboxylate (1055 mg,
4.90 mmol) was dissolved in THF (17 mL) and then cooled to
0.degree. C. MeOH (0.397 mL, 9.80 mmol) and LiBH.sub.4 (14.7 mmol)
were added sequentially. The reaction was warmed to room
temperature over 3 h. Then 10% aqueous potassium sodium tartrate
tetrahydrate (Rochelle's Salt) (30 mL) and EtOAc (30 mL) were added
and the solution stirred at room temperature over 30 minutes. The
organic layer was separated and then dried (Na.sub.2SO.sub.4) and
concentrated to afford 674 mg of t-butyl
3-(hydroxymethyl)azetidine-1-carboxylate (1-3) as a crude product
(clear oil). The product was used directly in the next step without
purification.
[0401] t-butyl 3-(hydroxymethyl)azetidine-1-carboxylate (674 mg,
3.60 mmol) was dissolved in CH.sub.2Cl.sub.2 (13 mL, 0.25M) and
then Et.sub.3N (1.0 mL, 7.20 mmol), DMAP (44 mg, 0.360 mmol), and
methanesulfonyl chloride (0.31 mL, 3.96 mmol) were added
sequentially at 0.degree. C. with the MsCl addition being done
slowly. The solution was warmed to rt over 1 h. After 15 h,
saturated aqueous NaHCO.sub.3 (50 mL) was added and then the
product was extracted with CH.sub.2Cl.sub.2 (2.times.50 mL) and the
combined organic extracts were washed with brine (50 mL), dried
(Na.sub.2SO.sub.4), concentrated, and purified by flash
chromatography (Biotage Horizon--10% EtOAc/hexanes--100% EtOAc) to
afford 962 mg of (1-4) as an oil (quantitative).
[0402] NaH (95%, 96 mg, 3.99 mmol) was combined in DMF (10 mL)
under N.sub.2 at rt. 4-Bromopyrazole (533 mg, 3.63 mmol) was then
added and the mixture stirred at rt. After 30 minutes (1-4) was
added and the solution heated to 95.degree. C. After 2 h, saturated
aqueous NH.sub.4Cl (50 mL) was added and then EtOAc (50 mL). The
organic extract was dried (Na.sub.2SO.sub.4) and concentrated, and
then run through a short pad of silica gel with 50% EtOAc/Hexanes
to afford 846 mg of crude (1-5) that was used directly in the next
step (74% crude yield).
[0403] (1-5) (846 mg, 2.68 mmol), (1-6) (815 mg, 3.21 mmol),
[1,1'-bis(diphenylphosphino)-ferrocene)dichloropalladium (108 mg,
0.133 mmol), and KOAc (893 mg, 9.10 mmol) were combined in DMSO (10
mL, purged with N.sub.2 for 10 minutes) and then the solution was
warmed to 80.degree. C. After 16 h, the solution was filtered
through Celite and then H.sub.2O (50 mL) and EtOAc (50 mL) was
added. The organic phase was extracted and dried
(Na.sub.2SO.sub.4), concentrated, and then passed through a silica
plug with 50% EtOAc/Hexane. The solvent was concentrated to afford
1.22 g of crude (1-7) used directly in the next step.
[0404] The boronic ester (1-7) (4144 mg, 11.4 mmol), (1-8) (2890
mg, 7.60 mmol), dichlorobis(triphenylphosphine)palladium(II) (534
mg, 0.760 mmol), DME (40 mL, De-gassed for 30 minutes with
N.sub.2), and 1N Na.sub.2CO.sub.3 (40 mL, De-gassed for 30 minutes
with N.sub.2) were combined and heated to 80.degree. C. After 16 h,
the reaction was cooled to rt and EtOAc (80 mL) was added. The
solution was filtered through celite and then water (80 mL) was
added. The organic layer was separated, dried (Na.sub.2SO.sub.4),
and concentrated. The product was purified by flash chromatography
to afford 1486 mg of (1-9) as a tan solid (36%).
[0405] 1 gram of DOWEX 50WX2-400 ion-exchange resin was prepared by
washing it with H.sub.2O (500 mL), 1:1 H.sub.2O/MeOH, MeOH
(5.times.250 mL), CH.sub.2Cl.sub.2 (500 mL), and hexanes (500 mL).
The DOWEX was then dried in a vacuum oven at 40.degree. C. for 1
day. (1-9) was dissolved in MeOH and then DOWEX (588 mg, 1.096
mmol) was added. The solution was stirred at rt for 2 h. The
solution was then filtered and the resin was washed with MeOH
(3.times.200 mL) and the wash was discarded. The resin was then
washed with 3.5M NH.sub.3/MeOH and collected. The solution was then
concentrated to afford 374 mg of (1-10) as a gummy solid (78%).
[0406] To form compounds of formula (1-11), the following exemplary
procedure can be followed. 1 molar equivalent of (1-10) is
dissolved in DMF or CH.sub.2Cl.sub.2 and then base (3 molar
equivalents) and/or coupling reagent (1.5 molar equivalents) is
added. To the solution is added X--R (1.1 molar equivalent), where
X is, for example, Cl, Br, I, OMs, COCl, CO, COOH, ethylene or
carbonate and R is a desired group such as those shown in the
examples herein or similar groups. The resultant solution is
stirred at rt for 4 h. H.sub.2O and EtOAc are added and the organic
phase extracted, dried (Na.sub.2SO.sub.4), and concentrated. The
crude product can purified by purified by preparative HPLC or other
methods well known in the art to afford the product (1-11).
General Procedure 59
##STR00085## ##STR00086##
[0408] 3-Azetidinol (2-2): A reaction mixture of
N-benzhydrylazetidin-3-ol HCl salt (2.76 g, 10.0 mmol) with
palladium hydroxide, 20% Pd (dry base) on C (400 mg) in 50 mL of
MeOH was hydrogenated at 55 psi for 48 h. The reaction mixture was
filtered through Celite pad and washed well with MeOH. The filtrate
was concentrated under vacuum at room temperature water bath. The
residue was treated with ether (3.times.30 ml) and the solvent is
decanted. The solid was air dried to give 571 mg of HCl salt
product (2-2) as white solid (52% yield). .sup.1H NMR (400 MHz,
DMSO-D6) .delta. ppm 3.33 (s, 1H) 3.63-3.80 (m, 2H) 3.93-4.09 (m,
2H) 4.40-4.58 (m, 1H) 6.18 (d, J=6.32 Hz, 1H).
[0409] 3-Hydroxy-azetidine-1-carboxlic acid tert-butyl ester (3-3):
To a cold (0.degree. C. bath) stirred solution of compound (2-2)
(570 mg, 5.20 mmol) in 10 mL of EtOH was added Et.sub.3N (1.8 mL,
13.0 mmol) and di-tert-butyldicarbonate (1.702 g, 7.38 mmol). The
resulting mixture of clear solution was stirred at room temperature
overnight. The reaction mixture was concentrated by vacuum. The
residue was portioned between EtOAc (200 mL) and 0.5N citric acid
solution (30 mL; brine (30 mL). The organic layer was dried
(Na.sub.2SO.sub.4), then concentrated by vacuum to give 899 mg
(2-3) as clear oil (52%). .sup.1H NMR (400 MHz, chloroform-D)
.delta. ppm 1.42 (s, 9H) 3.78 (dd, J=9.47, 4.42 Hz, 2H) 4.13 (dd,
J=9.35, 6.57 Hz, 2H) 4.49-4.63 (m, 1H).
[0410] 3-Methanesulfonyloxy-azetidine-1-carboxylic acid tert-butyl
ester (2-4): To a solution of compound (2-3) (466 mg; 2.69 mmol)
with Et.sub.3N (0.75 mL; 5.38 mmol) and 4-(dimethylamino)-pyridine
(33 mg, 0.269 mmol) in 10 mL of CH.sub.2Cl.sub.2 at 0.degree. C.
was added methanesulfonyl chloride (0.25 mL 3.23 mmol). The
resulting mixture of brown color solution was stirred at 0.degree.
C. to room temperature for overnight. The reaction mixture was
quenched with NaHCO.sub.3, then partitioned between
CH.sub.2Cl.sub.2 (200 mL) and saturated NaHCO.sub.3 solution (50
mL). The organic layer was dried (Na.sub.2SO.sub.4), then filtered
through silica gel pad, eluted with hexane: EtOAc/1:1; the filtrate
was concentrated by vacuum to give 614 mg (2-4) as yellow oil (91%
yield). .sup.1H NMR (400 MHz, chloroform-D) .delta. ppm 1.43 (s,
9H) 3.05 (s, 3H) 4.08 (dd, J=10.36, 4.29 Hz, 2H) 4.26 (dd, J=10.36,
6.82 Hz, 2H) 5.11-5.26 (m, 1H).
[0411] 1-(3-Azetidine-1-carboxylic acid tert-butyl
ester)-4-bromoprazole (2-6): A 5 mL microwave tube was charge with
compound (2-4) (304 mg, 1.21 mmol); 4-bromopyrazole (2-5, 178 mg,
1.21 mmol) and NaH 60% in mineral oil (73 mg, 1.82 mmol.) with 2 mL
of DMF. The resulting mixture was microwaved at 110.degree. C. for
30 minutes. The reaction mixture was partitioned between EtOAc (200
mL) and saturated NaHCO.sub.3 solution (2.times.50 mL); brine (50
mL). The organic layer was dried (Na.sub.2SO.sub.4), then
concentrated by vacuum to afford 360 mg of (2-6) as yellow oil
(98%). .sup.1H NMR (400 MHz, DMSO-D6) .delta. ppm 1.36-1.43 (m, 9H)
4.08 (s, 2H) 4.18-4.31 (m, 2H) 5.12-5.22 (m, 1H) 7.67 (s, 1H) 8.14
(s, 1H).
[0412] tert-Butyl
3-[4-(4,4,5,5-tetramethyl-1,3-dioxoborolan-2-yl)-1H-pyrazol-1-yl]azetidin-
e-1-carboxylate (2-8): A reaction mixture of compound (2-6) (225
mg, 0.74 mmol) and bis(pinacolate)diboron (2-7, 227 mg, 0.89 mmol)
with KOAc (247 mg, 2.52 mmol) in 3 mL of DMSO was purged with
N.sub.2 for 15 minutes, then PdCl.sub.2(dppf).sub.2CH.sub.2Cl.sub.2
(30 mg, 2.52 mmol) was added. The resulting mixture was stirred at
80.degree. C. under N.sub.2 for overnight. After it cooled down to
room temperature, the mixture was filtered through Celite pad and
washed well with EtOAc. The filtrate was extracted with H.sub.2O
(2.times.50 mL), brine (50 mL). The organic layer was dried
(Na.sub.2SO.sub.4), then concentrated by vacuum. The residue was
then filtered through silica gel pad, eluted with hexane:EtOAc/3:2.
The filtrate was concentrated by vacuum to give 250 mg of (2-8) as
a clear oil (97% yield). .sup.1H NMR (400 MHz, chloroform-D)
.delta. ppm 1.18-1.27 (m, 9H) 1.28-1.34 (m, 6H) 1.41-1.49 (m, 6H)
4.22-4.33 (m, 2H) 4.36 (t, J=8.59 Hz, 2H) 4.98-5.13 (m, 1H) 7.83
(s, 2H).
[0413] tert-Butyl
3-(4-{6-amino-5-[1-(2,6-dichloro-3-fluorophenyl)ethoxy]pyridin-3-yl}-1H-p-
yrazol-1-yl)azetidine-1-carboxylate (2-10): A reaction mixture of
compound (2-8) (459 mg; 1.31 mmol) and
3-[1-(2,6-dichloro-3-fluorophenyl)ethoxy]-5-iodopyridin-2-amine
(2-9) (374 mg; 0.88 mmol) in 13 mL of ethylene glycol
dimethylether, anhydrous (DME) was purged with N.sub.2 for 15
minutes, then Pd(II)(PPh.sub.3).sub.2Cl.sub.2 (46 mg, 0.07 mmol)
was added and continued to purge with N.sub.2 for another 15
minutes. Another 1.0 N Na.sub.2CO.sub.3 solution (3.9 mL; 3.9 mmol)
was added after purging with N.sub.2 for 15 minutes. The resulting
mixture was stirred at 85.degree. C. under N.sub.2 for overnight.
The reaction mixture was filtered through Celite pad and washed
well with MeOH. The filtrate was concentrated by vacuum. The
residue was partitioned between EtOAc (200 mL) and saturated
NaHCO.sub.3 solution (2.times.50 mL); brine (50 mL). The organic
layer was dried (Na.sub.2SO.sub.4), then concentrated by vacuum.
The residue was purified by Biotage system (25 M, 100%
CH.sub.2Cl.sub.2; 100% CH.sub.2Cl.sub.2 to 90% CH.sub.2Cl.sub.2
with 10% MeOH) to collect the desired fraction to afford 421 mg of
(2-10) as a brown color grease (92% yield). .sup.1H NMR (400 MHz,
chloroform-D) .delta. ppm 1.17-1.26 (m, 9H) 1.80-1.87 (m, 3H)
4.04-4.18 (m, 2H) 4.20-4.33 (m, 2H) 4.34-4.41 (m, 1H) 4.79 (s, 2H)
5.02 (d, J=7.58 Hz, 1H) 7.04 (t, J=8.46 Hz, 1H) 7.33-7.41 (m, 1H)
7.44-7.52 (m, 1H) 7.53-7.58 (m, 1H) 7.59-7.65 (m, 1H) 7.72-7.78 (m,
1H); LCMS calcd for C.sub.24H.sub.26Cl.sub.2FN.sub.5O.sub.3 (M+H)
523. Found 523.
[0414]
5-(1-Azetidin-3-yl-1H-pyrazol-4-yl)-3-[1-(2,6-dichloro-3-fluorophen-
yl)ethoxy]yridin-2-amine (2-11): A reaction mixture of compound
(2-10) (421 mg; 0.81 mmol) with 4.0 M HCl in dioxane (2.0 mL; 8.1
mmol) in 5 mL of CH.sub.2Cl.sub.2 was stirred at room temperature
for 2.0 hours. The reaction mixture was concentrated by vacuum. The
residue was treated with EtOAc. The precipitated solid was filtered
off and washed well with EtOAc, hexane, then dried under vacuum to
give 275 mg of (2-11) as a sand color solid of HCl salt (81%
yield). .sup.1H NMR (400 MHz, DMSO-D6) .delta. ppm 1.79-1.89 (m,
3H) 3.56 (s, 1H) 4.35 (s, 4H) 5.40 (s, 1H) 6.23 (d, J=6.57 Hz, 2H)
7.09 (s, 1H) 7.40-7.54 (m, 1H) 7.59 (dd, J=8.84, 5.05 Hz, 1H)
7.73-7.83 (m, 1H) 7.86 (s, 1H) 8.12 (s, 1H) 9.20 (s, 1H). LCMS
calcd for C.sub.19H.sub.18Cl.sub.2FN.sub.5O (M+H) 423. Found
423.
[0415] Compounds of formula 2-12 can be prepared by the following
exemplary procedure: To a reaction mixture of compound (2-11) (1.0
eq.) with Et.sub.3N (2.0 eq.) in 2.0 mL of DMF at room temperature
is added alkyl bromide (1.1 eq.). The resulting mixture is stirred
under N.sub.2 at room temperature for overnight. The reaction
mixture is partitioned between EtOAc (200 mL) and saturated
NaHCO.sub.3 solution (2.times.50 mL); brine (50 mL). The organic
layer is dried (Na.sub.2SO.sub.4), then concentrated by vacuum. The
residue is purified by Dionex system (5% to 95% MeCN:H.sub.2O w
0.1% HOAc buffer) to collect the desired fraction to afford
(2-12).
[0416] Alternatively, compounds of formula 2-12 can be prepared by
the following exemplary procedure: To a reaction solution of alkyl
amine (1.0 eq.) with iPr.sub.2EtN (diisopropylethylamine) (3.0 eq.)
in 2.0 mL of DMF is added HATU (1.5 eq.). After stirring for 30
minutes, compound (2-11) (1.0 eq.) is added. The resulting mixture
is stirred at room temperature for overnight. The reaction mixture
is partitioned between EtOAc (200 mL) and saturated NaHCO.sub.3
solution (2.times.50 mL) and brine (50 mL). The organic layer is
dried (Na.sub.2SO.sub.4) and concentrated by vacuum. The residue is
purified by Dionex System (5% to 95% McCN:H.sub.2O w 0.1% HOAc) to
collect the desired product to afford (2-12).
General Procedure 60
##STR00087##
[0418] tert-Butyl 1-oxa-6-azaspiro[2.5]octane-6-carboxylate (3-2):
A solution of dimethylsulfoxonium methylide was prepared under
N.sub.2 from NaH 60% dispersion in mineral oil (440 mg; 11.0 mmol)
and trimethylsulfoxonium iodide (2.421 g; 11.0 mmol) in 5 ml of
anhydrous DMSO. Another solution of
1-Boc-4-oxo-1-piperidincarboxylate (3-1, 1.993 g; 10.0 mmol) in 5
mL of DMSO was added dropwise. The resulting mixture was stirred at
55.degree. C. for 6 hours. The cooled reaction mixture was poured
into ice-H.sub.2O and extracted with EtOAc (2.times.200 mL). The
combined organic layers were washed with H.sub.2O (50 mL); brine (5
0 mL) and then dried (Na.sub.2SO.sub.4), then concentrated by
vacuum to give 1.4791 g of (3-2) as a yellow oil (69% yield).
.sup.1H NMR (400 MHz, chloroform-D) .delta. ppm 1.37-1.52 (m, 11H)
1.71-1.84 (m, 2H) 2.63-2.72 (m, 2H) 3.35-3.49 (m, 2H) 3.62-3.78 (m,
2H).
[0419] tert-Butyl
4-hydroxy-4-{[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazol--
1-yl]methyl}piperidine-1-carboxylate (3-4): A reaction mixture of
compound (3-2) (214 mg; 1.0 mmol) and
4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (3-3,
194 mg; 1.0 mmol) with NaH 60% dispersion in mineral oil (60 mg;
1.5 mmol) in 3 mL of DMF was stirred at 90.degree. C. for 3 hours.
The reaction mixture was partitioned between EtOAc (200 mL) and
saturated NaHCO.sub.3 solution (50 mL) and brine (50 mL). The
organic layer was dried (Na.sub.2SO.sub.4) and concentrated by
vacuum to give 361 mg of (3-4) as a yellow grease (89% yield).
.sup.1H NMR (400 MHz, chloroform-D) .delta. ppm 1.21-1.34 (m, 12H)
1.39-1.50 (m, 9H) 1.56-1.78 (m, 4H) 3.14 (s, 2H) 3.72-3.91 (m,
J=32.34 Hz, 2H) 4.05 (s, 2H) 7.65 (s, 1H) 7.80 (s, 1H) 8.00 (s,
1H). LCMS calcd for C.sub.20H.sub.34BN.sub.3O.sub.5 (M+H) 408.
Found 408. HPLC purity 85%.
[0420] tert-Butyl
4-[(4-{6-amino-5-[1-(2,6-dichloro-3-fluorophenyl)ethoxy]pyridin-3-yl}-1H--
pyrazol-1-yl)methyl]-4-hydroxypiperidine-1-carboxylate (3-6): A
reaction mixture of compound (3-4) (361 mg; 0.89 mmol) and
3-[1-(2,6-dichloro-3-fluorophenyl)ethoxy]-5-iodopyridin-2-amine
(3-5) (378 mg; 0.89 mmol) in 9.0 mL of ethylene glycol
dimethylether, anhydrous (DME) was purged with N.sub.2 for 15
minutes, then Pd(II)(PPh.sub.3).sub.2Cl.sub.2 (32 mg, 0.05 mmol)
was added and continued to purge with N.sub.2 for another 15
minutes. Another 1.0 N Na.sub.2CO.sub.3 solution (3.9 mL; 3.9 mmol)
was added after purging with N.sub.2 for 15 minutes. The resulting
mixture was stirred at 85.degree. C. under N.sub.2 for overnight.
The reaction mixture was filtered through Celite pad and washed
well with MeOH. The filtrate was concentrated by vacuum. The
residue was partitioned between EtOAc (200 mL) and saturated
NaHCO.sub.3 solution (2.times.50 mL); brine (50 mL). The organic
layer was dried (Na.sub.2SO.sub.4), then concentrated by vacuum.
The residue was purified by Dionex system (25% to 95% MeCN:H.sub.2O
w 0.1% HOAc buffer) to collect the desired fraction to afford 147
mg of (3-6) as a white solid (28% yield). .sup.1H NMR (400 MHz,
DMSO-D6) .delta. ppm 1.34-1.39 (m, 9H) 1.70-1.77 (m, 2H) 1.79 (d,
J=6.57 Hz, 3H) 3.06 (d, J=12.63 Hz, 2H) 3.62 (s, 2H) 4.03 (s, 2H)
4.79 (s, 1H) 5.66 (s, 2H) 6.08 (d, J=6.82 Hz, 1H) 6.86 (d, J=1.52
Hz, 1H) 7.44 (t, J=8.72 Hz, 1H) 7.51-7.58 (m, 2H) 7.58-7.65 (m, 2H)
7.73 (d, J=1.52 Hz, 1H) 7.78 (s, 1H). LCMS calcd for
C.sub.27H.sub.32Cl.sub.2FN.sub.5O.sub.4 (M+H) 581. Found 581. HPLC
purity 87%.
[0421]
4-[(4-{6-amino-5-[1-(2,6-dichloro-3-fluorophenyl)ethoxy]pyridin-3-y-
l}-1H-pyrazol-1-yl)methyl]piperidin-4-ol (3-7): A reaction mixture
of compound (3-6) (145 mg; 0.25 mmol) with 4.0 M HCl in dioxane
(2.0 mL; 8.1 mmol) in 5 mL of CH.sub.2Cl.sub.2 was stirred at room
temperature for 2.0 hours. The reaction mixture was concentrated by
vacuum. The residue was purified by Dionex system (5% to 95%
MeCN:H.sub.2O w 0.1% HOAc buffer) to collect the desired fraction
to afford 76 mg of (3-7) as a yellow grease (63% yield). .sup.1H
NMR (400 MHz, DMSO-D6) .delta. ppm 1.41-1.55 (m, 2H) 1.59-1.71 (m,
2H) 1.81 (d, J=6.57 Hz, 3H) 2.88-3.00 (m, 2H) 3.02-3.14 (m, 2H)
4.08 (s, 2H) 5.17 (s, 2H) 6.14-6.27 (m, J=6.57 Hz, 1H) 7.05 (s, 1H)
7.40-7.49 (m, J=8.72, 8.72 Hz, 1H) 7.51-7.60 (m, J=9.09, 4.80 Hz,
1H) 7.63 (s, 1H) 7.76 (s, 1H) 7.91 (s, 1H) 8.51 (s, 1H) 8.81 (s,
1H). LCMS calcd for C.sub.22H.sub.24Cl.sub.2FN.sub.5O.sub.2 (M+H)
481. Found 481. HPLC purity 98%. Anal.
(C.sub.22H.sub.24Cl.sub.2FN.sub.5O.sub.2.times.2.2HOAc.times.2.3H.sub.2O)-
C, H, N.
General Procedure 61
##STR00088##
[0423] Ethyl
2-[(4-bromo-1H-pyrazol-1-yl)methyl]cyclopropanecarboxylate (4-3):
To a reaction solution of ethyl
2-(hydroxymethyl)cyclopropanecarboxylate (4-1) (577 mg; 4.0 mmol)
with Et.sub.3N (1.1 mL; 8.0 mmol) and DMAP (49 mg; 0.4 mmol) in 12
mL of CH.sub.2Cl.sub.2 at 0.degree. C. was added methanesulfonyl
chloride (0.4 mL; 4.8 mmol). The resulting mixture of brown color
suspension was stirred at 0.degree. C. to room temperature under
N.sub.2 for overnight. The reaction mixture was quenched with
NaHCO.sub.3, then partitioned between CH.sub.2Cl.sub.2 (200 mL) and
saturated NaHCO.sub.3 solution (50 mL); brine (50 mL). The organic
layer was dried (Na.sub.2SO.sub.4), then filtered through silica
gel pad, eluted with hexane:EtOAc/1:1. The filtrate was
concentrated by vacuum to give 880 mg of ethyl
2-{[(methylsulfonyl)oxy]methyl}cyclopropanecarboxylate as a yellow
oil (99% yield). .sup.1H NMR (400 MHz, chloroform-D) .delta. ppm
0.91-1.02 (m, 1H) 1.26 (q, J=6.99 Hz, 3H) 1.29-1.36 (m, 1H)
1.63-1.74 (m, 1H) 1.79-1.92 (m, 1H) 3.02 (s, 3H) 3.99-4.24 (m,
4H).
[0424] A reaction mixture of ethyl
2-{[(methylsulfonyl)oxy]methyl}cyclopropanecarboxylate (880 mg; 4.0
mmol), 4-bromopyrazole (4-2, 588 mg, 4.0 mmol) and NaH 60% in
mineral oil (240 mg, 6.0 mmol) with 3.0 mL of DMF was formed. The
resulting mixture was stirred at 90.degree. C. under N.sub.2 for
four hours. The reaction mixture was partitioned between EtOAc (200
mL) and saturated NaHCO.sub.3 solution (2.times.50 mL); brine (50
mL). The organic layer was dried (Na.sub.2SO.sub.4), then
concentrated by vacuum to afford 812 mg of (4-3) as a yellow oil
(74%). .sup.1H NMR (400 MHz, chloroform-D) .delta. ppm 0.85 (dd,
J=7.96, 3.16 Hz, 1H) 0.88-0.98 (m, 1H) 1.18-1.29 (m, 3H) 1.56-1.71
(m, 1H) 1.79-1.94 (m, 1H) 3.96-4.08 (m, 2H) 4.07-4.17 (m, 2H) 7.45
(d, J=3.79 Hz, 2H). LCMS calcd for C.sub.10H.sub.13BrN.sub.2O.sub.2
(M+H) 274. Found 274. HPLC purity 95%.
[0425] Ethyl
2-{[4-(4,4,5,5-tetramethyl-1,3-dioxoborolan-2-yl)-1H-pyrazol-1-yl]methyl}-
cyclopropanecarboxylate (4-4): A reaction mixture of compound (4-3)
(812 mg, 2.97 mmol) and bis(pinacolate)diboron (906 mg, 3.57 mmol)
with KOAc (991 mg, 10.10 mmol) in 10.0 mL of DMSO was purged with
N.sub.2 for 15 minutes, then PdCl.sub.2(dppf).sub.2CH.sub.2Cl.sub.2
(122 mg, 0.15 mmol) was added. The resulting mixture was stirred at
80.degree. C. under N.sub.2 for overnight. After cooling down to
room temperature, the mixture was filtered through Celite pad and
washed well with EtOAc. The filtrate was extracted with H.sub.2O
(2.times.50 mL), brine (50 mL). The organic layer was dried
(Na.sub.2SO.sub.4), then concentrated by vacuum. The residue was
then filtered through silica gel pad, and eluted with
hexane:EtOAc/3:1. The filtrate was concentrated by vacuum to give
945 mg of (4-4) as a yellow oil (98% yield). .sup.1H NMR (400 MHz,
chloroform-D) .delta. ppm 0.85 (dd, J=7.83, 3.03 Hz, 1H) 0.90-0.96
(m, 1H) 1.20-1.24 (m, 3H) 1.29-1.34 (m, 12H) 1.62-1.71 (m, 1H)
1.84-1.97 (m, 1H) 3.96-4.07 (m, 1H) 4.06-4.14 (m, 2H) 4.15-4.23 (m,
J=14.27, 6.44 Hz, 1H) 7.73 (s, 1H) 7.77 (s, 1H).
[0426] Ethyl
2-[(4-{6-amino-5-[1-(2,6-dichloro-3-fluorophenyl)ethoxy]pyridin-3-yl}-1H--
pyrazol-1-yl)methyl]cyclopropanecarboxylate (4-6): A reaction
mixture of compound (4-4) (643 mg; 2.01 mmol) and
3-[1-(2,6-dichloro-3-fluorophenyl)ethoxy]-5-iodopyridin-2-amine
(4-5) (572 mg; 1.34 mmol) in 20.0 mL of ethylene glycol
dimethylether, anhydrous (DME) was purged with N.sub.2 for 15
minutes, then Pd(II)(PPh.sub.3).sub.2Cl.sub.2 (71 mg, 0.1 mmol) was
added and continued to purge with N.sub.2 for another 15 minutes.
Another 1.0 N Na.sub.2CO.sub.3 solution (6.0 mL; 6.0 mmol) was
added after purging with N.sub.2 for 15 minutes. The resulting
mixture was stirred at 85.degree. C. under N.sub.2 for overnight.
The reaction mixture was filtered through Celite pad and washed
well with MeOH. The filtrate was concentrated by vacuum. The
residue was partitioned between EtOAc (200 mL) and saturated
NaHCO.sub.3 solution (2.times.50 mL); brine (50 mL). The organic
layer was dried (Na.sub.2SO.sub.4), then concentrated by vacuum.
The residue was purified by Biotage system (25 M CH.sub.2Cl.sub.2
100%; CH.sub.2Cl.sub.2 100% to 90% CH.sub.2Cl.sub.2: 10% MeOH) to
collect the desired fraction to afford 600 mg of (4-6) as a brown
color grease (91% yield). .sup.1H NMR (400 MHz, DMSO-D6) .delta.
ppm 0.96-1.10 (m, 2H) 1.15 (t, J=7.07 Hz, 2H) 1.74 (s, 3H) 1.79 (d,
J=6.57 Hz, 3H) 3.95-4.14 (m, 4H) 5.66 (s, 2H) 6.08 (d, J=6.57 Hz,
1H) 6.88 (s, 1H) 7.43 (t, J=8.72 Hz, 1H) 7.49-7.62 (m, 2H) 7.73 (s,
1H) 7.88 (s, 1H). LCMS calcd for
C.sub.23H.sub.23Cl.sub.2FN.sub.4O.sub.3 (M+H) 494. Found 494. HPLC
purity 95%.
[0427]
2-[(4-{6-amino-5-[1-(2,6-dichloro-3-fluorophenyl)ethoxy]pyridin-3-y-
l}-1H-pyrazol-1-yl)methyl]cyclopropanecarboxylic acid (4-7): To a
reaction solution of compound (4-6) (377 mg, 0.76 mmol) in 5.0 mL
of MeOH at room temperature under N.sub.2 was added another
solution of 2.0 N NaOH (2) (1.5 mL, 3.04 mmol). The resulting
mixture was stirred at 80.degree. C. for 3 hours. The reaction
mixture was concentrated by vacuum to remove most of the MeOH and
acidified by 2 M HCl to pH 4.0. The mixture was extracted with
CH.sub.2Cl.sub.2 (2.times.200 mL); the organic layers were washed
with brine (50 mL), and dried (Na.sub.2SO.sub.4) and concentrated
by vacuum to give 324 mg of (4-7) as a yellow solid. (92% yield).
.sup.1H NMR (400 MHz, DMSO-D6) .delta. ppm 0.92-1.04 (m, 2H)
1.57-1.72 (m, 2H) 1.76-1.90 (m, 3H) 3.98-4.18 (m, 2H) 6.46 (s, 2H)
6.89-7.02 (m, 1H) 7.29-7.52 (m, 2H) 7.52-7.63 (m, 2H) 7.73 (d,
J=1.52 Hz, 1H) 7.94 (s, 1H) 12.19 (s, 1H). LCMS calcd for
C.sub.21H.sub.19Cl.sub.2FN.sub.4O.sub.3 (M-H) 463. Found 463. HPLC
purity 87%.
[0428]
2-[(4-{6-amino-5-[1-(2,6-dichloro-3-fluorophenyl)ethoxy]pyridin-3-y-
l}-1H-pyrazol-1-yl)methyl]-N-methylcyclopropanecarboxamide (4-8)
(R=Me, R'.dbd.H): To a reaction solution of (4-7) (1.0 eq.) with
iPr.sub.2EtN (2.0 eq.) in 1.0 mL of DMF was added HATU (1.5 eq.).
After stirring for 30 minutes, alkylamine (1.1 eq.) was added. The
resulting mixture was stirred at room temperature for overnight.
The reaction mixture was partitioned between EtOAc (200 mL) and
saturated NaHCO.sub.3 solution (2.times.50 mL) and brine (50 mL).
The organic layer was dried (Na.sub.2SO.sub.4) and concentrated by
vacuum. The sample was free based by partitioning between EtOAc
(200 mL) and saturated NaHCO.sub.3 solution (50 mL) and brine (50
mL). The organic layer was dried (Na.sub.2SO.sub.4) and
concentrated by vacuum. The residue was treated with 1.0 mL of
H.sub.2O and lyophilized to afford (4-8).
General Procedure 62
##STR00089## ##STR00090##
[0430] To a solution of
5-bromo-3-[(R)-1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-pyridin-2-ylamine
(12.83 g, 33.76 mmol) in anhydrous DMF (100 mL) was added
di-tert-butyl dicarbonate (21.25 g, 97.35 mmol) and
4-dimethylaminopyridine (0.793 g, 6.49 mmol). The reaction was
stirred at ambient temperature for 18 hours under nitrogen. To the
mixture was added saturated NaHCO.sub.3 solution (300 mL), and
extracted with EtOAc (3.times.250 mL). The combined extracts were
washed with water (5.times.100 mL), sat. NaHCO.sub.3, and brine,
then dried over Na.sub.2SO.sub.4. After filtration, evaporation,
and high vacuum drying, di-boc protected
5-bromo-3-[(R)-1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-pyridin-2-ylamine
was obtained as an off-white foam solid (19.59 g, 100% yield).
.sup.1H NMR (DMSO-d.sub.6, 400 MHz) .delta. 8.18 (d, 1H), 7.83 (d,
1H), 7.59 (dd, 1H), 7.48 (t, 1H), 6.25 (q, 1H), 1.75 (d, 3H), 1.39
(s, 9H), 1.19 (s, 9H).
[0431] To a solution of the di-boc protected
5-bromo-3-[(R)-1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-pyridin-2-ylamine
(19.58 g, 33.76 mmol) in DMSO (68 mL) was added potassium acetate
(11.26 g, 114.78 mmol) and bis(pinacolato)diboron (10.29 g, 40.51
mmol). The mixture was degassed and charged with nitrogen three
times, then Pd(dppf)Cl.sub.2.CH.sub.2Cl.sub.2 (1.38 g, 1.69 mmol)
was added. The reaction mixture was degassed and charged with
nitrogen three times, and then stirred at 80.degree. C. oil bath
under nitrogen for 12 hours. The reaction was cooled to ambient
temperature, diluted with ethyl acetate (100 mL), and filtered
through a celite pad which was washed with ethyl acetate. The
combined ethyl acetate solution (700 mL) was washed with water
(5.times.100 mL), brine (100 mL), and dried over Na.sub.2SO.sub.4.
After filtration and concentration, the residue was purified on a
silica gel column eluting with EtOAc/Hexane (0%-50%) to provide
di-boc protected
3-[(R)-1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-5-(4,4,5,5-tetramethyl-[1-
,3,2]dioxaborolan-2-yl)-pyridin-2-ylamine as a foam sold (20.59 g,
97% yield). .sup.1H NMR (DMSO-d.sub.6, 400 MHz) .delta. 8.20 (d,
1H), 7.70 (d, 1H), 7.63 (dd, 1H), 7.47 (t, 1H), 6.20 (q, 1H), 1.73
(d, 3H), 1.50-1.13 (m, 30H).
[0432] To a solution of di-boc protected
3-[(R)-1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-5-(4,4,5,5-tetramethyl-[1-
,3,2]dioxaborolan-2-yl)-pyridin-2-ylamine (20.34 g, 32.42 mmol) in
CH.sub.2Cl.sub.2 (80 mL) was added a solution of dry HCl in dioxane
(4N, 40.5 mL, 162 mmol). The reaction solution was stirred at
40.degree. C. oil bath under nitrogen for 12 hours. The reaction
mixture was cooled to ambient temperature, diluted with EtOAc (400
mL), then washed carefully but quickly with saturated NaHCO.sub.3
until the water layer was basic (pH>8). The organic layer was
washed with brine, and dried over Na.sub.2SO.sub.4. After
filtration, evaporation, and high vacuum drying,
3-[(R)-1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-5-(4,4,5,5-tetramethyl-[1-
,3,2]dioxaborolan-2-yl)-pyridin-2-ylamine was obtained as an
off-white foam solid (13.48 g, 97% yield). .sup.1H NMR
(DMSO-d.sub.6, 400 MHz) .delta. 8.01 (d, 1H), 7.27 (dd, 1H), 7.17
(d, 1H), 7.03 (t, 1H), 6.12 (q, 1H), 5.08 (bs, 2H), 1.81 (d, 3H),
1.30 (s, 6H), 1.28 (s, 6H).
[0433] To a stirred solution of
3-[(R)-1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-5-(4,4,5,5-tetramethyl-[1-
,3,2]dioxaborolan-2-yl)-pyridin-2-ylamine (4.2711 g, 10.0 mmol) and
4-(4-bromo-pyrazol-1-yl)-piperidine-1-carboxylic acid tert-butyl
ester (see procedure 11) (3.9628 g, 12.0 mmol) in DME (40 mL) was
added a solution of Na.sub.2CO.sub.3 (3.1787 g, 30.0 mmol) in water
(10 mL). The solution was degassed and charged with nitrogen three
times. To the solution was added Pd(PPh.sub.3).sub.2Cl.sub.2 (351
mg, 0.50 mmol). The reaction solution was degassed and charged with
nitrogen again three times. The reaction solution was stirred at
87.degree. C. oil bath for about 16 hours (or until consumption of
the borane pinacol ester), cooled to ambient temperature and
diluted with EtOAc (200 mL). The reaction mixture was filtered
through a pad of celite and washed with EtOAc. The EtOAc solution
was washed with brine, dried over Na.sub.2SO.sub.4, and
concentrated. The crude product was purified on a silica gel column
eluting with EtOAc/hexane system (0% EtOAc to 100% EtOAc) to afford
4-(4-{6-amino-5-[(R)-1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-pyridin-3-y-
l}-pyrazol-1-yl)-piperidine-1-carboxylic acid tert-butyl ester
(3.4167 g, 65% yield, .about.95% purity) with a Rf of 0.15 (50%
EtOAc/Hexanes). MS m/e 550 (M+1).sup.+.
[0434] To a solution of
4-(4-{6-amino-5-[(R)-1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-pyridin-3-y-
l}-pyrazol-1-yl)-piperidine-1-carboxylic acid tert-butyl ester
(566.7 mg, 1.03 mmol) in methanol (5 mL) or dichloromethane (30 mL)
was added 4N HCl/dioxane (15 mL). The solution was stirred for
about 1 hour or until the de-protection was complete. The solvents
were evaporated and the residue was dissolved in methanol and
purified on a reversed phase C-18 preparative HPLC eluting with
acetonitrile/water with 0.1% acetic acid from 5% to 30% with a
linear gradient. After lyophilization,
3-[(R)-1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-5-(1-piperidin-4-yl-1H-py-
razol-4-yl)-pyridin-2-ylamine acetate was obtained as a white solid
(410 mg, 78% yield, 100% HPLC purity, 96.4% ee). .sup.1H NMR
(DMSO-d.sub.6, 400 MHz) .delta. 7.84 (s, 1H), 7.68 (d, 1H), 7.50
(dd, 1H), 7.46 (s, 1H), 7.37 (t, 1H), 6.83 (d, 1H), 6.02 (q, 1H),
5.57 (bs, 2H), 4.09 (m, 1H), 2.98 (m, 2H), 2.53 (m, 2H), 1.88 (m,
2H), 1.82 (s, 3H), 1.73 (d, 3H), 1.70 (m, 2H). MS m/e 450
(M+1).sup.+.
General Procedure 63
##STR00091##
[0436] To a suspension of
3-[1-(2,6-Dichloro-3-fluoro-phenyl)-ethoxy]-5-(1-piperidin-4-yl-1H-pyrazo-
l-4-yl)-pyridin-2-ylamine as the HCl salt (procedure 6) (150 mg,
0.288 mmol) in CH.sub.2Cl.sub.2 (2 mL) was added NEt.sub.3 (0.121
mL, 0.863 mmol) and stirred for 30 minutes at room temperature. The
reaction was cooled to 0.degree. C. and acetic acid
chlorocarbonylmethyl ester was added and stirred for 1 hour at room
temperature. The reaction was monitored by LC-MS and after complete
conversion to the desired product, water (2 mL) was added. The
reaction was extracted with EtOAc (4.times.10 mL), dried over
Na.sub.2SO.sub.4, and concentrated to give quantitative yield of
acetic acid
2-[4-(4-{6-amino-5-[1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-pyridin-3-yl-
}-pyrazol-1-yl)-piperidin-1-yl]-2-oxo-ethyl ester (164 mg,
quant).
[0437] To solution of acetic acid
2-[4-(4-{6-amino-5-[1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-pyridin-3-yl-
}-pyrazol-1-yl)-piperidin-1-yl]-2-oxo-ethyl ester (164 mg, 0.298
mmol) in MeOH (4 mL) was added LiOH (7 mg, 0.298 mmol) dissolved in
1 mL of water. The reaction was stirred for 30 minutes at room
temperature in which LC-MS showed complete conversion to the
1-[4-(4-{6-amino-5-[1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-pyridin-3-yl-
}-pyrazol-1-yl)-piperidin-1-yl]-2-hydroxy-ethanone. The product was
purified on a reversed phase C-18 preparative HPLC eluting with
acetonitrile/water having 0.1% acetic acid from 10% to 40%.
General Procedure 64
##STR00092##
[0439] A 100 mL of flask with a stir bar was dried in an oven and
cooled in a dry nitrogen atmosphere. The flask was equipped with a
rubber syringe cap. The flask was immersed in an ice-water bath
under nitrogen, and 1.6 mL (1.6 mmol) of 1.0 M borane solution in
THF was introduced. Then
2-(4-{5-Amino-6-[1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-pyrazin-2--
yl}-pyrazol-1-yl)-2-methyl-propionic acid (procedure 5) (0.1 g,
0.221 mmol) in anhydrous THF (1.0 mL) was introduced. The resulting
mixture was stirred at ambient temperature under nitrogen for 5
hours, and 6 N HCl (1.1 mL) was added slowly, and then H.sub.2O
(1.1 mL) and MeOH (7.4 mL) were introduced. The reaction mixture
was stirred continually overnight. Most of solvents were evaporated
in vacuo, and then a 1 N NaOH solution was used to adjust pH to 11.
Water was added, and the solution was extracted with EtOAc
(3.times.30 mL) and dried over Na.sub.2SO.sub.4. After filtration
and concentration, the crude product was purified with a reverse
phase preparative HPLC eluting with acetonitrile/water containing
0.1% acetic acid from 10% to 60%. After lyophilization of the pure
fractions,
2-(4-{6-amino-5-[1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-pyridin-3-yl}-p-
yrazol-1-yl)-2-methyl-propan-1-ol acetate was obtained as a white
solid (21 mg, 22% yield).
General Procedure 65
##STR00093##
[0441] To a stirred solution of 4-hydroxy-piperidine-1-carboxylic
acid tert-butyl ester (7.94 g, 39.45 mmol) in CH.sub.2Cl.sub.2 (100
mL), cooled to 0.degree. C., was slowly added NEt.sub.3 (5.54 mL,
39.45 mmol) followed by methane sulfonyl chloride (3.06 mL, 39.45
mmol) and DMAP (48 mg, 0.39 mmol). The mixture was stirred at room
temperature overnight. To the mixture was added water (30 mL).
Extraction with CH.sub.2Cl.sub.2 (3.times.30 mL) followed by drying
(Na.sub.2SO.sub.4) and removal of the solvent in vacuo afforded
4-methanesulfonyloxy-piperidine-1-carboxylic acid tert-butyl ester
as a white solid (11.00 g, >99% yield). .sup.1H NMR (CDCl.sub.3,
400 MHz) .delta. 4.89 (m, 1H), 3.69 (m, 2H), 3.31 (m, 2H), 3.04 (s,
3H), 1.95 (m, 2H), 1.83 (m, 2H), 1.46 (s, 9H).
[0442] To a stirred solution of 4-bromo-pyrazole (10.44 g, 71.03
mmol) in anhydrous DMF (96 mL), cooled to 0.degree. C., was slowly
added NaH (60% in mineral oil) (3.13 g, 78.133 mmol). The solution
was stirred for 1 hour at 0.degree. C.
4-Methanesulfonyloxy-piperidine-1-carboxylic acid tert-butyl ester
(19.82 g, 71.03 mmol) was added slowly and the reaction was heated
to 100.degree. C. overnight or until consumption of the pyrazole by
NMR. The reaction was cooled to room temperature and water added
(20 mL) followed by extraction with EtOAc. The combined extracts
were washed with saturated aqueous NaCl (4.times.20 mL), dried with
Na.sub.2SO.sub.4 and concentrated to afford
4-(4-bromo-pyrazol-1-yl)-piperidine-1-carboxylic acid tert-butyl
ester as an orange oil. The oil was purified using silica gel
chromatography eluting with 10% EtOAc/hexanes to 25% EtOAc/hexanes
to provide 4-(4-bromo-pyrazol-1-yl)-piperidine-1-carboxylic acid
tert-butyl ester as a white solid (10.55 g, 45% yield) with a
R.sub.f=0.4 (25% EtOAc/hexanes, using iodine as the stain). .sup.1H
NMR (CDCl.sub.3, 400 MHz) .delta. 7.46 (s, 1H), 7.43 (s, 1H), 4.23
(m, 3H), 2.88 (m, 2H), 2.10 (m, 2H), 1.88 (m, 2H), 1.47 (s,
9H).
[0443] To a solution of
4-(4-bromo-pyrazol-1-yl)-piperidine-1-carboxylic acid tert-butyl
ester (500 mg, 1.515 mmol) in CH.sub.2Cl.sub.2 (3 mL) was added TFA
(3 mL). The reaction was stirred at room temperature until LCMS
indicated completion of the reaction. The solvents were removed in
vacuo, and the residue was dissolved in MeOH (15 mL). The pH of the
solution was adjusted to 9 with hydroxide resin to afford
4-(4-bromo-pyrazol-1-yl)-piperidine.
[0444] To a solution of 4-(4-bromo-pyrazol-1-yl)-piperidine (375
mg, 1.63 mmol) in DMF (3.26 mL) was added NEt.sub.3 (230 .mu.L,
1.63 mmol) and stirred for 5 minutes. Methyliodide (Mel) (1.63 mL,
1M Mel in DMF, freshly made) was added and the reaction was stirred
overnight at room temperature. Water was added and the solution was
extracted with EtOAc (4.times.10 mL). The organic solution was
washed with brine, dried with Na.sub.2SO.sub.4, concentrated, and
dried in vacuo to afford
4-(4-bromo-pyrazol-1-yl)-1-methyl-piperidine (251 mg, 63%
yield).
General Procedure 66
##STR00094##
[0446] To a solution of
3-[(R)-1-(2,6-dichloro-3-fluoro-phenyl)ethoxy]-5-(1H-pyrazol-4-yl)-pyrazi-
n-2-ylamine (295 mg, 0.80 mmol) in anhydrous DMF (4 mL) was added
NaH (60% in mineral oil, 30.7 mg, 0.80 mmol). The mixture was
stirred at ambient temperature under nitrogen for 0.5 h, and then
4-methanesulfonyloxy-piperidine-1-carboxylic acid tert-butyl ester
(223.5 mg, 0.80 mmol) was introduced. The reaction mixture was
heated to 90.degree. C. oil bath for 0.5 h under nitrogen, and
cooled to ambient temperature. Water was added slowly to the
mixture, which was extracted with EtOAc, washed with brine, and
dried over Na.sub.2SO.sub.4. The crude product was purified on a
silica gel column to provide
4-(4-{5-amino-6-[(R)-1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-pyrazin-2-y-
l}-pyrazol-1-yl)-piperidine-1-carboxylic acid tert-butyl ester as a
white solid (265 mg, 59% yield).
[0447] To a solution of
4-(4-{5-amino-6-[1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-pyrazin-2-yl}-p-
yrazol-1-yl)-piperidine-1-carboxylic acid tert-butyl ester (265 mg,
0.48 mmol) in CH.sub.2Cl.sub.2 was added 4N HCl/dioxane (4 mL). The
mixture was stirred at ambient temperature for one hour. After
evaporation, the residue was dissolved in methanol (2.5 mL), and
was purified on a reverse phase C-18 reparative HPLC eluting with
acetonitrile/water containing 0.1% acetic acid with a linear
gradient of 10%-40%. After lyophilization,
3-[(R)-1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-5-(1-piperidin-4-yl-1H-py-
razol-4-yl)-pyrazin-2-ylamine acetate was obtained as a white solid
(125 mg, 51% yield).
General Procedure 67
##STR00095##
[0449] O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
phosphorus pentafluoride (HATU) (66 mg, 0.17 mmol) was added to a
solution of
2-(4-{6-Amino-5-[1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-pyridin-3-yl}-p-
yrazol-1-yl)-propionic acid (69 mg, 0.16 mmol), triethylamine
(0.024 mL, 0.17 mmol) and 3-dimethylamino-propylamine (0.022 mL,
0.17 mmol) in 1.6 mL of DMF. After stirring for 3 hours, the
reaction was concentrated by rotary evaporation. The residue was
purified by silica gel chromatography using gradient elution of
dichloromethane, methanol, ammonium hydroxide to afford
2-(4-{6-Amino-5-[1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-pyrid-
in-3-yl}-pyrazol-1-yl)-N-(3-dimethylamino-propyl)-propionamide. (41
mg, 50%).
General Procedure 68
##STR00096##
[0451] Diethylazodicarboxylate (0.48 mL, 3.1 mmol) was added to a
0.degree. C. solution of triphenylphosphine (0.80 g, 3.1 mmol) in
THF (20 mL). After stirring for 5 minutes, 4-bromo-pyrazole (0.30
mg, 2.0 mmol) was added. After another 5 minutes of stirring,
(2-hydroxyethyl)-methyl-carbamic acid tert-butyl ester (0.45 g, 2.6
mmol) was added. The reaction was allowed to warm to room
temperature and stir overnight. The reaction was cooled to
0.degree. C. and filtered. The filtrate was concentrated by rotary
evaporation. The residue was purified by silica gel chromatography
using gradient elution of dichloromethane, ethyl acetate to afford
[2-(4-bromo-pyrazol-1-yl)-ethyl]-methyl-carbamic acid tert-butyl
ester (541 mg, 87%).
General Procedure 69
##STR00097##
[0453] Sodium hydride (0.12 g, 4.9 mmol) was added to a solution of
4-bromo-4H-pyrazole (0.60 g, 4.1 mmol) in DMF (10 mL). After
stirring for 10 minutes, a solution of 2-chloro-propionic acid
methyl ester in DMF (4 mL) was added. After stirring for 4 hours,
the reaction was partitioned between ethyl acetate and water. The
phases were separated and the aqueous phase was extracted with
ethyl acetate. The combined organic phases were dried over
MgSO.sub.4 and concentrated by rotary evaporation. The residue was
purified by silica gel chromatography using gradient elution of
ethyl acetate and hexanes to afford
2-(4-bromo-pyrazol-1-yl)-propionic acid methyl ester (733 mg,
77%).
General Procedure 70
##STR00098##
[0455] A solution of LiOH (34 mg, 1.4 mmol) in water (0.4 mL) was
added to a solution of
2-(4-{6-Amino-5-[1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-pyridin-3-yl}-p-
yrazol-1-yl)-propionic acid methyl ester (70 mg, 0.15 mmol) in a
mixture THF (1.5 mL) and MeOH (0.4 mL). After stirring overnight,
the reaction was partitioned between dichloromethane and
half-saturated brine. A small amount of ethanol was added and the
pH was adjusted to 7 with 1 M HCl. The phases were separated and
the aqueous phase was extracted with dichloromethane. The combined
organic phases were dried over Na.sub.2SO.sub.4, filtered and
concentrated by rotary evaporation to give
2-(4-{6-amino-5-[1-(2,6-dichloro-3-fluoro-phenyl)ethoxy]-pyridin-3-yl}-py-
razol-1-yl)-propionic acid (69 mg, 100%).
General Procedure 71
##STR00099##
[0457] To a stirred solution of
4-(3-{6-Amino-5-[1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-pyridin-3-yl}-p-
yrazol-1-yl)-pyrrolidine-2-carboxylic acid methyl ester (105 mg,
0.21 mmol) in THF (5 mL) was added 2 M CH.sub.3NH.sub.2 in THF
(1.06 mL, 2.12 mmol), the mixture was stirred and heated at
55.degree. C. for 18 hours, LCMS checked that the reaction was
completed, remove THF, the residue was purified by prep-HPLC to
leave
4-(4-{6-amino-5-[1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-pyridin-3-yl}-p-
yrazol-1-yl)-pyrrolidine-2-carboxylic acid methylamide (30 mg),
yield 28.6%.
General Procedure 72
##STR00100##
[0459] tert-butyl
4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole-1-carboxylate
(21-1): Di-tert-butyl dicarbonate (7.2 molar equivalent),
4-(dimethylamino)pyridine (0.84 molar equivalent) were added to a
solution of
4,4,5,5-tetramethyl-2-(1H-pyrazole-4-yl)-1,3,2-dioxaborolane (6
mmol) in 40 mL of DMF. The reaction mixture was stirred at room
temperature for 12 h. Water was added to the reaction mixture to
quench the reaction. EtOAc was then added to extract the aqueous
solution. Dry EtOAc layer over Na.sub.2SO.sub.4. The
Na.sub.2SO.sub.4 was filtered off and the filtrate was evaporated
to give a brown yellow oil residue as compound 21-1 (1.32 g; 4.56
mmol; 76%). .sup.1H NMR (400 MHz, chloroform-D) .delta. ppm 1.32
(s, 12H) 1.63 (s, 9H) 7.91 (s, 1H) 8.37 (s, 1H). The residue was
used for the next step reaction without further purification.
[0460] Compound 21-3, shown with the specific example of
3-[1-(2,6-dichloro-3-fluorophenyl)ethoxy]-5-(1H-pyrazol-4-yl)pyridin-2-am-
ine (21-3a):
##STR00101##
[0461] Compound 21-1 (1.0 molar equivalent) was added to a solution
of compound 21-2a (Compound 21-2, with R substituents to give
2,6-dichloro-3-fluorophenyl) (1.92 mmol) in 20 mL of DME. The
mixture was stirred at room temperature under a nitrogen atmosphere
for 30 minutes and then dichlorobis(triphenylphosphino) palladium
(II) (0.05 molar equivalent) was added. Sodium carbonate (3 molar
equivalent) in 4 mL of H.sub.2O was added to the reaction mixture
and the resulting solution was heated to 85.degree. C. for 12 h.
Alternative bases used were CsF and Cs.sub.2CO.sub.3 in with 1 or 2
equivalents of boronic ester, and at room temperature (CsF) or
80.degree. C. (all). Water was added to the reaction mixture to
quench the reaction. EtOAc (150 mL.times.2) was then added to
extract the aqueous solution. Dry EtOAc layer over
Na.sub.2SO.sub.4. The Na.sub.2SO.sub.4 was filtered off and the
filtrated was evaporated to give a dark brown oil residue. The
residue was purified by silica gel chromatography (eluting with
eluting with 0.fwdarw.10% MeOH in ethyl acetate) to give the
desired product, compound 21-3a (2.05 g, 53.6% yield). .sup.1H NMR
(400 MHz, chloroform-D) .delta. ppm 1.60 (s, 1H) 1.84 (d, J=6.57
Hz, 3H) 5.07 (s, 2H) 6.06 (q, J=6.57 Hz, 1H) 6.89 (d, J=1.77 Hz,
1H) 6.96-7.06 (m, 1H) 7.22-7.33 (m, 1H) 7.67 (s, 2H) 7.80 (d,
J=1.52 Hz, 1H).
[0462] To make compounds of formula 21-4, the following exemplary
procedure can be used: sodium hydride (1.2 molar equivalent) is
added to a solution of compound 21-3 (0.87 mmol) in 10 mL of DMF.
The mixture is stirred at room temperature under a nitrogen
atmosphere for 30 min and then compound 21-6 (1 molar equivalent)
is added. The resulting solution is heated to 85-90.degree. C. for
12 h. Water (20 mL) is added to the reaction mixture to quench the
reaction. EtOAc (50 mL.times.2) is then added to extract the
aqueous solution. Dry EtOAc layer over Na.sub.2SO.sub.4. The
Na.sub.2SO.sub.4 is filtered off and the filtrate is evaporated.
The residue is purified by silica gel chromatography (eluting with
EtOAc in hexanes) to give the desired product, compound 21-4
(20-50% yield).
General Procedure 73
##STR00102##
[0464] Compounds of formula 22-3 can be prepared by the following
exemplary procedure: Compound 22-2 (1.2 molar equivalent) is added
to a solution of compound 22-1 (0.24 mmol) and base (3-5 molar
equivalent) and/or coupling reagent (1 molar equivalent) in 5 mL of
DMF. The mixture is stirred under a nitrogen atmosphere for 12 h.
Water (20 mL) is added to the reaction mixture to quench the
reaction. EtOAc (50 mL.times.2) is then added to extract the
aqueous solution. Dry EtOAc layer over Na.sub.2SO.sub.4. The
Na.sub.2SO.sub.4 is filtered off and the filtrate evaporated. The
residue is purified by silica gel chromatography (eluting with
CH.sub.3OH, CH.sub.2Cl.sub.2, EtOAc, and hexanes) to give the
desired product, compound 22-3.
General Procedure 74
[0465] The following procedure can be used to prepare
piperidine-pyrazole-2-aminopyridine derivatives.
##STR00103## ##STR00104##
tert-butyl 4-(4-iodo-1H-pyrazol-1-yl)piperidine-1-carboxylate
(23-1a)
[0466] NaH (1.2 eq., 0.68 mmol) was added portionwise to a stirred
solution of 4-iodopyrazole (0.57 mmol) in DMF (2 L) at 4.degree. C.
The resulting mixture was stirred for 1 hour at 4.degree. C. and
compound 23-4 (1.1 eq., 0.63 mmol) was then added. The resulting
mixture was heated to 100.degree. C. for 12 h. The reaction was
quenched with H.sub.2O and extracted with EtOAc several times. The
combined organic layers were dried, filtered, and concentrated to
afford an orange oil. The residue was purified by silica gel
chromatography (eluting with 5% EtOAc in pentane) to give compound
23-1a as a white solid (140 g, 66%).
tert-butyl-4-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazol-1-
-yl]piperidine-1-carboxylate (23-1b)
[0467] Bis(pinacolato)diboron (1.4 eq., 134 g, 0.52 mol) and
potassium acetate (4 eq., 145 g, 1.48 mol) were added sequentially
to a solution of compound 23-1a (140 g, 0.37 mol) in 1.5 L of DMSO.
The mixture was purged with nitrogen several times and
dichlorobis(triphenylphosphino) palladium (II) (0.05 eq., 12.9 g,
0.018 mol) was then added. The resulting mixture was heated at
80.degree. C. for 2 h. The reaction mixture was cooled to room
temperature and filtered through a bed of celite and washed with
EtOAc. The filtrate was washed with saturated NaCl (500
mL.times.2), dried over Na.sub.2SO.sub.4, filtered and
concentrated. The residue was purified by silica gel chromatography
(eluting with 5% EtOAc in hexanes) to give compound 23-1b as a
white solid (55 g, 40%).
[0468] Compound 23-2 (1.0 molar equivalent) was added to a solution
of compound 23-1b (1.3 molar equivalent) in 15 mL of DME. The
mixture was purged with nitrogen several times and then
dichlorobis(triphenylphosphino) palladium (II) (0.05 molar
equivalent) was added. Cesium carbonate (3 molar equivalent) in 4
mL of H.sub.2O was added to the reaction mixture and the resulting
solution was heated to 85.degree. C. for 12 h. Water (10 mL) was
added to the reaction mixture to quench the reaction. EtOAc (150
mL.times.2) was then added to extract the aqueous solution. Dry
EtOAc layer over Na.sub.2SO.sub.4. The Na.sub.2SO.sub.4 was
filtered off and the filtrated was evaporated to give a dark brown
oil residue. The residue was purified by silica gel chromatography
(eluting with eluting with 75.fwdarw.100% EtOAc in hexanes) to give
compound 23-3a (61% yield).
[0469] Hydrochloride (19 eq., 12 mmol) was added to a solution of
compound 23-3a (0.63 mmol) in MeOH (4 mL). The mixture was stirred
at room temperature for 12 h. The solvent was evaporated and
H.sub.2O (10 mL) was added. Saturated NaHCO.sub.3(aq) was added to
neutralize the solution to pH 7. Ethyl acetate (100 mL.times.2) was
added to extract the aqueous solution. The combined organic layer
was dried over Na.sub.2SO.sub.4, filtered, and evaporated to give
compound 23-5a as a solid reside (0.6 mmol, 95% yield).
[0470] Compounds of formula 23-7 can be formed according to the
following general procedure: Compound 23-8 (1.2 molar equivalent)
is added to a solution of compound 23-5a (0.24 mmol) and base (3-5
molar equivalent) and/or coupling reagent (1 molar equivalent) in 5
mL of DMF. The mixture is stirred under a nitrogen atmosphere for
12 h. Water (20 mL) is added to the reaction mixture to quench the
reaction. EtOAc (50 mL.times.2) is then added to extract the
aqueous solution. Dry EtOAc layer over Na.sub.2SO.sub.4. The
Na.sub.2SO.sub.4 is filtered off and the filtrated is evaporated to
give an oil residue. The residue is purified by silica gel
chromatography (eluting with CH.sub.3OH, CH.sub.2Cl.sub.2, EtOAc,
and hexanes) to give the desired product, compound 23-7a.
General Procedure 75
##STR00105##
[0472] 3-methoxy compounds can be prepared from the corresponding
3-fluoro compounds by the following general procedure. To 4 mL of
DMSO is added 0.124 mL ethanol followed by 32 mg NaH. After
stirring for 30 minutes 250 mg of 24-1 is added and the reaction
heated to 40.degree. C. After three hours the reaction is cooled
and poured into water to precipitate. After neutralization to pH 6,
the product 24-2 is isolated.
General Procedure 76
##STR00106##
[0474] To a stirred solution of
3-[1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-5-[1-(2,2-dimethyl-[1,3]dioxo-
lan-4-ylmethyl)-1H-pyrazol-4-yl]-pyridin-2-ylamine (150 mg, 0.31
mmol) in THF (3 mL) and H.sub.2O (2 mL) was added TFA (2 mL) at
0.degree. C., the mixture was stirred and warmed to room
temperature, then heated at 50.degree. C. for 5 hours, LCMS checked
that the reaction was completed, remove THF, the residue was
purified by prep-HPLC to leave
3-(4-{6-amino-5-[1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-pyridin-3-yl}-p-
yrazol-1-yl)-propane-1,2-diol (102 mg), yield 74.2%.
General Procedure 77
##STR00107##
[0476] To a stirred solution of 4-bromo-1H-pyrazole in DMF was
added sodium hydride at room temperature. The mixture was stirred
for 30 minutes, [1,3]dioxolan-2-one was added, the mixture was
stirred and slowly warmed to room temperature. The reaction was
monitored by TLC. After the reaction was done, EtOAc was added,
washed with saturated NaHCO.sub.3, water and brine, dried with
Na.sub.2SO.sub.4, filtered and concentrated. The residue was
purified by silica gel, eluants EtOAc and DCM 10%, to give
2-(4-Bromo-pyrazol-1-yl)-ethanol 0.22 g, yield 34%. .sup.1H NMR
(400 MHz, chloroform-D) .delta. ppm 7.49 (s, 1H) 7.46 (s, 1H)
4.18-4.23 (m, 2H) 3.93-3.98 (m, 2H) 3.09 (s, 1H).
Example 1
5-Bromo-3-[(R)-1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-pyrazin-2-ylamine
##STR00108##
[0478] The title compound was prepared according to procedure 2,
from (1S)-1-(2,6-dichloro-3-fluorophenyl)ethanol. .sup.1H NMR (400
MHz, DMSO-d6) .delta. 7.53 (s, 1H), 7.48 (m, 1H), 7.39 (t, 1H),
6.48 (s, 2H), 6.41 (q, 1H), 1.74 (d, 3H); LCMS: 381 [M+1]; c-Met
Ki: 0.796 .mu.M.
Example 2
4-{5-Amino-6-[(R)-1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-pyrazin-2-yl}-b-
enzoic acid
##STR00109##
[0480] The title compound was prepared according to procedure 3.
.sup.1H NMR (400 MHz, DMSO-d6) .delta. 8.16 (s, 1H), 7.84 (d, 2H),
7.77 (d, 2H), 7.53 (m, 1H), 7.37 (t, 1H), 6.64 (s, 2H), 6.53 (q,
1H), 1.78 (d, 3H); LCMS: 422 [M+1]; c-Met Ki: 0.154 .mu.M.
Example 3
(4-{5-Amino-6-[(R)-1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-pyrazin-2-yl}--
phenyl)-piperazin-1-yl-methanone
##STR00110##
[0482] The title compound was prepared according to procedure 4.
.sup.1H NMR (400 MHz, DMSO-d6) .delta. 8.11 (s, 1H), 7.73 (d, 2H),
7.53 (m, 1H), 7.37 (t, 1H), 7.31 (d, 2H), 6.55 (m, 3H), 3.51 (br,
2H), 3.32 (br, 2H), 2.67 (br, 4H), 1.77 (d, 3H); LCMS: 490 [M+1];
c-Met Ki: 0.027 .mu.M.
Example 4
4-(4-{5-Amino-6-[(R)-1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-pyrazin-2-yl-
}-benzoyl)-piperazine-1-carboxylic acid tert-butyl ester
##STR00111##
[0484] The title compound was prepared according to procedure 16
followed by 20. .sup.1H NMR (400 MHz, DMSO-d6) .delta. 8.12 (s,
1H), 7.72 (d, 2H), 7.50 (m, 1H), 7.33 (t, 3H), 6.55 (m, 3H), 3.51
(br, 2H), 3.39 (m, 3H), 3.32 (br, 3H), 1.77 (d, 3H), 1.40 (s, 9H);
LCMS: 590 [M+1]; c-Met Ki: 0.335 .mu.M.
Example 5
3-[(1R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]-5-[4-(piperazin-1-ylcarbony-
l)phenyl]pyridin-2-amine
##STR00112##
[0486] The title compound was prepared according to procedure 20
followed by 21 as a racemic mixture with the corresponding S
enantiomer of Example 119, followed by separation by chiral
chromatography. The title compound was also prepared as an
enantiomerically pure compound starting from the chiral starting
material .sup.1H NMR (400 MHz, DMSO-D6) .delta. ppm 1.83 (d, J=6.57
Hz, 3H) 3.35 (s, 4H) 3.69 (s, 4H) 6.24 (q, J=6.57 Hz, 1H) 6.91-7.08
(m, 2H) 7.10 (d, J=1.26 Hz, 1H) 7.46 (t, J=8.72 Hz, 1H) 7.50 (s,
4H) 7.58 (dd, J=8.97, 4.93 Hz, 1H) 7.91 (d, J=1.77 Hz, 1H) 9.35 (s,
2H); LCMS: 490 [M+1]; c-Met Ki: 0.01 .mu.M.
Example 6
4-{6-amino-5-[(1R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]pyridin-3-yl}-N-[-
2-(dimethylamino)ethyl]-N-methylbenzamide
##STR00113##
[0488] The title compound was prepared according to procedure 20.
.sup.1H NMR (400 MHz, DMSO-D6) .delta. ppm 1.80 (d, J=6.82 Hz, 3H)
1.97 (s, 3H) 2.19 (s, 3H) 2.30-2.42 (m, J=1.77 Hz, 2H) 2.93 (s, 3H)
3.22-3.29 (m, 1H) 3.44-3.61 (m, 1H) 5.95 (s, 2H) 6.14 (q, J=6.57
Hz, 1H) 6.98 (d, J=1.01 Hz, 1H) 7.30-7.39 (m, 2H) 7.40-7.47 (m, 3H)
7.51-7.62 (m, 1H) 7.87 (d, J=1.77 Hz, 1H); LCMS: 506 [M+1]; c-Met
Ki: 0.01 .mu.M.
Example 7
(4-{6-amino-5-[(1R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]pyridin-3-yl}phe-
nyl)methanol
##STR00114##
[0490] The title compound was prepared according to procedure 27.
.sup.1H NMR (400 MHz, DMSO-D6) .delta. ppm 1.84 (d, J=6.57 Hz, 3H)
4.49 (d, J=5.81 Hz, 2H) 5.20 (t, J=5.81 Hz, 1H) 6.25 (q, J=6.57 Hz,
1H) 6.46-6.88 (m, 2H) 7.04 (d, J=1.52 Hz, 1H) 7.34 (s, 4H) 7.46 (t,
J=8.72 Hz, 1H) 7.59 (dd, J=8.97, 4.93 Hz, 1H) 7.76 (d, J=1.52 Hz,
1H); LCMS: 408 [M+1]; c-Met Ki: 0.051 .mu.M.
Example 8
4-{6-amino-5-[(1R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]pyridin-3-yl}-N-[-
3-(dimethylamino)propyl]-N-methylbenzamide
##STR00115##
[0492] The title compound was prepared according to procedure 27.
.sup.1H NMR (400 MHz, DMSO-D6) .delta. ppm 1.60-1.73 (m, 2H) 1.80
(d, J=6.57 Hz, 3H) 1.94 (s, 3H) 2.13 (s, 3H) 2.20-2.29 (m, 2H) 2.92
(s, 3H) 3.36-3.50 (m, 2H) 5.96 (s, 2H) 6.14 (q, J=6.57 Hz, 1H) 6.98
(s, 1H) 7.37 (s, 2H) 7.40-7.51 (m, 3H) 7.55 (dd, J=8.84, 4.80 Hz,
1H) 7.86 (d, J=1.77 Hz, 1H); LCMS: 520 [M+1]; c-Met Ki: 0.01
.mu.M.
Example 9
tert-butyl
4-(4-{6-amino-5-[(1R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]pyr-
idin-3-yl}benzoyl)piperazine-1-carboxylate
##STR00116##
[0494] The title compound was prepared according to procedure 20.
.sup.1H NMR (400 MHz, chloroform-D) .delta. ppm 1.46 (s, 9H) 1.86
(d, J=6.82 Hz, 3H) 3.30-3.89 (m, 8H) 4.90 (s, 2H) 6.11 (q, J=6.57
Hz, 1H) 6.98 (d, J=1.52 Hz, 1H) 7.01-7.10 (m, 1H) 7.30 (dd, J=8.97,
4.93 Hz, 1H) 7.35-7.43 (m, 4H) 7.88 (d, J=1.77 Hz, 1H); LCMS: 590
[M+1]; c-Met Ki: 0.03 .mu.M.
Example 10
3-[(R)-1-(2,6-Dichloro-3-fluoro-phenyl)-ethoxy]-5-[1-(1-methyl-piperidin-4-
-yl)-1H-pyrazol-4-yl]-pyridin-2-ylamine
##STR00117##
[0496] The title compound was prepared according to procedure 62
using
3-[(R)-1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-5-(4,4,5,5-tetramethyl-[1-
,3,2]dioxaborolan-2-yl)-pyridin-2-ylamine and
4-(4-bromo-pyrazol-1-yl)-1-methyl-piperidine (prepared according to
general procedure 11. .sup.1H NMR (400 MHz, CDCl.sub.3) .delta.
7.65 (s, 1H), 7.55 (s, 1H), 7.50 (s, 1H), 7.31 (m, 1H), 7.06 (m,
1H), 6.87 (s, 1H), 6.08 (m, 1H), 5.50 (bs, 2H), 4.18 (m, 1H), 3.11
(m, 2H), 2.40 (s, 3H), 2.30 (m, 2H), 2.20 (m, 4H), 2.07 (s, 3H),
1.86 (d, J=8 Hz, 3H); LCMS: 464 [M+1]; c-Met Ki: 0.01 .mu.M.
Example 11
1-[4-(4-{6-Amino-5-[(R)-1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-pyridin-3-
-yl}-pyrazol-1-yl)-piperidin-1-yl]-2-hydroxy-ethanone
##STR00118##
[0498] The title compound was prepared according to procedure 63.
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.72 (s, 1H), 7.57 (s,
1H), 7.47 (s, 1H), 7.31 (m, 1H), 7.06 (m, 1H), 6.86 (s, 1H), 6.08
(m, 1H), 5.00 (bs, 2H), 4.70 (m, 1H), 4.36 (m, 1H), 4.21 (s, 1H),
3.70 (m, 1H), 3.18 (m, 1H), 3.00 (m, 1H), 2.223 (m, 2H), 2.01 (m,
2H), 1.86 (d, J=8 Hz, 3H); LCMS: 508 [M+1]; c-Met Ki: 0.004
.mu.M.
Example 12
3-[(R)-1-(2,6-Dichloro-3-fluoro-phenyl)-ethoxy]-5-(1-piperidin-4-yl-1H-pyr-
azol-4-yl)-pyridin-2-ylamine
##STR00119##
[0500] The title compound was prepared according to procedure 62
using
3-[(R)-1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-5-(4,4,5,5-tetramethyl-[1-
,3,2]dioxaborolan-2-yl)-pyridin-2-ylamine and
4-(4-bromo-pyrazol-1-yl)-1-cyclopentyl-piperidine (prepared
according to general procedure 11 using bromocyclopentane as
alkylation reagent). .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.73
(s, 1H), 7.55 (s, 1H), 7.48 (s, 1H), 7.31 (m, 1H), 7.07 (m, 1H),
6.88 (s, 1H), 6.08 (m, 1H), 4.64 (m, 1H), 2.04 (m, 2H), 1.98 (m,
2H), 1.86 (d, J=8 Hz, 3H), 1.73 (m, 2H); LCMS: 435 [M+1]; c-Met Ki:
0.02 .mu.M.
Example 13
3-[(R)-1-(2,6-Dichloro-3-fluoro-phenyl)-ethoxy]-5-(1-piperidin-4-yl-1H-pyr-
azol-4-yl)-pyridin-2-ylamine
##STR00120##
[0502] The title compound was prepared according to procedure 62.
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.69 (s, 1H), 7.56 (s,
1H), 7.50 (s, 1H), 7.32 (m, 1H), 7.07 (m, 1H), 6.87 (m, 1H), 6.07
(m, 1H), 5.25 (bs, 2H), 4.30 (m, 1H), 3.41 (m, 2H), 2.96 (m, 2H),
2.26 (m, 2H), 2.12 (m, 2H), 1.86 (d, J=8 Hz, 3H); LCMS: 450 [M+1];
c-Met Ki: 0.003 .mu.M.
Example 14
3-[(R)-1-(2,6-Dichloro-3-fluoro-phenyl)-ethoxy]-5-(1-piperidin-4-yl-1H-pyr-
azol-4-yl)-pyrazin-2-ylamine
##STR00121##
[0504] The title compound was prepared according to procedure 66.
.sup.1H NMR (400 MHz, DMSO-d6) .delta. 7.86 (s, 1H), 7.76 (s, 1H),
7.63 (m, 2H), 7.54 (m, 1H), 7.37 (t, 1H), 6.46 (q, 1H), 6.15 (s,
1H), 4.10 (m, 1H), 3.01 (m, 2H), 1.95 (m, 2H), 1.85 (s, 2H), 1.75
(d, 3H), 1.67 (dd, 1H); LCMS: 451 [M+1]; c-Met Ki: 0.010 .mu.M.
Example 15
3-[(R)-1-(2,6-Dichloro-3-fluoro-phenyl)-ethoxy]-5-(1H-pyrazol-4-yl)-pyrazi-
n-2-ylamine
##STR00122##
[0506] The title compound was prepared according to procedure 3
using
5-bromo-3-[(R)-1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-pyrazin-2-ylamine
and
4-(4,4,5,5-Tetramethyl-[1,3,2]dioxaborolan-2-yl)-pyrazole-1-carboxyli-
c acid tert-butyl ester. .sup.1H NMR (400 MHz, DMSO-d6) .delta.
12.81 (s, 1H), 7.79 (s, 1H), 7.48 (m, 1H), 7.36 (t, 1H), 6.48 (q,
1H), 6.12 (s, 2H), 1.75 (d, 3H); LCMS: 368 [M+1]; c-Met Ki: 0.065
.mu.M.
Example 16
1-[4-(4-{5-Amino-6-[(R)-1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-pyrazin-2-
-yl}-pyrazol-1-yl)-piperidin-1-yl]-2-hydroxy-ethanone
##STR00123##
[0508] The title compound was prepared according to procedures 62
and 63, using
5-bromo-3-[(R)-1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-pyrazin-2-y-
lamine as the starting material. .sup.1H NMR (400 MHz, DMSO-d6)
.delta. 7.91 (s, 1H), 7.76 (s, 1H), 7.64 (s, 1H), 7.49 (m, 1H),
7.36 (t, 1H), 6.46 (q, 1H), 6.15 (s, 2H), 4.57 (br, 1H), 4.40 (m,
2H), 4.12 (br, 2H), 3.77 (m, 1H), 3.35 (m, 2H), 3.43 (m, 1H), 3.16
(m, 2H), 1.75 (d, 3H); LCMS: 509 [M+1]; c-Met Ki: 0.015 .mu.M.
Example 17
3-[(R)-1-(2,6-Dichloro-3-fluoro-phenyl)-ethoxy]-5-[1-(1-methyl-piperidin-4-
-yl)-1H-pyrazol-4-yl]-pyrazin-2-ylamine
##STR00124##
[0510] The title compound was prepared according to procedure 62
using
5-bromo-3-[(R)-1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-pyrazin-2-ylamine
and 4-(4-bromo-pyrazol-1-yl)-1-methyl-piperidine (prepared
according to general procedure 11). .sup.1H NMR (400 MHz, DMSO-d6)
.delta. 7.88 (s, 1H), 7.76 (s, 1H), 7.64 (s, 1H), 7.49 (m, 1H),
7.36 (t, 1H), 6.46 (q, 1H), 6.15 (s, 2H), 4.02 (m, 1H), 2.84 (m,
2H), 2.19 (s, 3H), 2.00 (m, 4H), 1.85 (m, 3H), 1.75 (d, 3H); LCMS:
465 [M+1]; c-Met Ki: 0.03 .mu.M.
Example 18
1-[4-(4-{5-Amino-6-[(R)-1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-pyrazin-2-
-yl}-pyrazol-1-yl)-piperidin-1-yl]-2-dimethylamino-ethanone
##STR00125##
[0512] The title compound was prepared according to procedure 63
using
3-[(R)-1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-5-(1-piperidin-4-yl-1H-py-
razol-4-yl)-pyrazin-2-ylamine coupled with dimethylamino-acetic
acid in the presence of HOBt/EDC/triethylamine in DMF as described
in procedure 5 using
5-bromo-3-[(R)-1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-pyrazin-2-y-
lamine as the starting material. .sup.1H NMR (400 MHz, DMSO-d6)
.delta. 7.90 (s, 1H), 7.76 (s, 1H), 7.65 (s, 1H), 7.49 (m, 1H),
7.36 (t, 1H), 6.47 (q, 1H), 6.15 (s, 2H), 4.39 (m, 1H), 4.16 (m,
1H), 3.16 (m, 2H), 3.02 (m, 1H), 2.75 (m, 1H), 2.19 (s, 6H), 2.01
(m, 2H), 1.88 (s, 1H), 1.75 (d, 3H); LCMS: 536 [M+1]; c-Met Ki:
0.015 .mu.M.
Example 19
3-[(R)-1-(2-Chloro-3,6-difluoro-phenyl)-ethoxy]-5-(1-piperidin-4-yl-1H-pyr-
azol-4-yl)-pyridin-2-ylamine
##STR00126##
[0514] The title compound was prepared according to procedure 62
using
5-bromo-3-[(R)-1-(2-chloro-3,6-difluoro-phenyl)-ethoxy]-pyridin-2-ylamine
as starting material (according to the methods for the synthesis of
5-bromo-3-[1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-pyridin-2-ylamine
from (S)-1-(2-chloro-3,6-difluoro-phenyl)ethanol, obtained from
SynChem, Inc.). .sup.1H NMR (400 MHz, DMSO-d6) .delta. 7.88 (s,
1H), 7.70 (s, 1H), 7.50 (s, 1H), 7.38 (m, 1H), 7.25 (m, 1H), 6.99
(s, 1H), 5.88 (m, 1H), 5.48 (bs, 2H), 4.08 (m, 1H), 2.96 (m, 2H),
2.53 (m, 1H), 2.45 (m, 1H), 1.89 (m, 1H), 1.80 (m, 4H), 1.67 (m,
4H); LCMS: 434 [M+1]; c-Met Ki: 0.09 .mu.M.
Biological Examples
[0515] It will be appreciated that, in any given series of
compounds, a range of biological activities will be observed. In
its presently preferred aspects, this invention relates to novel
compounds capable of modulating, regulating and/or inhibiting
protein kinase activity. The following assays may be employed to
select those compounds demonstrating the optimal degree of the
desired activity.
Assay Procedures
[0516] The following in vitro assay may be used to determine the
level of activity and effect of the different compounds of the
present invention on one or more of the PKs. Similar assays can be
designed along the same lines for any PK using techniques well
known in the art. A literature reference is provided
(Technikova-Dobrova Z, Sardanelli A M, Papa S FEBS Lett. 1991 Nov.
4; 292: 69-72).
[0517] The general procedure is as follows: compounds and kinase
assay reagents are introduced into test wells. The assay is
initiated by addition of the kinase enzyme. Enzyme inhibitors
reduce the measured activity of the enzyme.
[0518] In the continuous-coupled spectrophotometric assay the
time-dependent production of ADP by the kinase is determined by
analysis of the rate of consumption of NADH by measurement of the
decrease in absorbance at 340 nm. As the PK produces ADP it is
re-converted to ATP by reaction with phosphoenol pyruvate and
pyruvate kinase. Pyruvate is also produced in this reaction.
Pyruvate is subsequently converted to lactate by reaction with
lactate dehydrogenase, which simultaneously converts NADH to NAD.
NADH has a measurable absorbance at 340 nm whereas NAD does
not.
[0519] The presently preferred protocol for conducting the
continuous-coupled spectrophotometric experiments for specific PKs
is provided below. However, adaptation of this protocol for
determining the activity of compounds against other RTKs, as well
as for CTKs and STKs, is well within the scope of knowledge of
those skilled in the art.
HGFR Continuous-Coupled Spectrophotometric Assay
[0520] This assay analyzes the tyrosine kinase activity of HGFR on
the Met-2 substrate peptide, a peptide derived from the activation
loop of the HGFR.
Materials and Reagents:
[0521] 1. HGFR enzyme from Upstate (Met, active) Cat. #14-526
[0522] 2. Met-2 Peptide (HGFR Activation Loop) Ac-ARDMYDKEYYSVHNK
(MW=1960). Dissolve up in 200 mM HEPES, pH 7.5 at 10 mM stock.
[0523] 3. 1 M PEP (phospho-enol-pyruvate) in 200 mM HEPES, pH 7.5
[0524] 4. 100 mM NADH (B-Nicotinamide Adenine Dinucleotide, Reduced
Form) in 200 mM HEPES, pH 7.5 [0525] 5. 4 M MgCl.sub.2 (Magnesium
Chloride) in ddH.sub.2O [0526] 6. 1 M DTT (Dithiothreitol) in 200
mM HEPES, pH 7.5 [0527] 7. 15 Units/mL LDH (Lactic Dehydrogenase)
[0528] 8. 15 Units/mL PK (Pyruvate Kinase) [0529] 9. 5M NaCl
dissolved in ddH.sub.2O [0530] 10. Tween-20 (Protein Grade) 10%
Solution [0531] 11. 1 M HEPES buffer:
(N-[2-Hydroxethyl]piperazine-N-[2-ethanesulfonic acid]) Sodium
Salt. Dissolve in ddH2O, adjust pH to 7.5, bring volume to 1 L.
Filter at 0.1 .mu.m. [0532] 12. HPLC Grade Water; Burdick and
Jackson #365-4, 1.times.4 liters (or equivalent) [0533] 13. 100%
DMSO (SIGMA) [0534] 14. Costar #3880--black clear flat bottom half
area plates for K.sub.i determination and % inhibition [0535] 15.
Costar #3359--96 well polypropylene plates, round bottom for serial
dilutions [0536] 16. Costar #3635--UV-plate clear flat bottom
plates for % inhibition [0537] 17. Beckman DU-650 w/micro cell
holders [0538] 18. Beckman 4-position micro cell cuvette
Procedure:
[0539] Prep Dilution Buffer (DB) for Enzyme (For 30 mL prep) [0540]
1. DB final concentration is 2 mM DTT, 25 mM NaCl.sub.2, 5 mM
MgCl.sub.2, 0.01% Tween-20, and 50 mM HEPES buffer, pH 7.5. [0541]
2. Make up 50 mM HEPES by adding 1.5 mL 1 M HEPES into 28.1 mL of
ddH2O. Add rest of the reagents. Into 50 mL conical vial, add 60
.mu.L of 1M DTT, 150 .mu.L 5M NaCl.sub.2, 150 .mu.L 1M MgCl.sub.2,
and 30 .mu.L of 10% Tween-20 to give total volume of 30 mL. [0542]
3. Vortex for 5-10 seconds. [0543] 4. Aliquot out DB at 1 mL/tube
and label tubes as "DB HGFR" [0544] 5. Note: This can be prepared
and stored ahead of time. [0545] 6. Freeze un-used aliquots in
microcentrifuge tubes at -20.degree. C. freezer.
[0546] Prep Compounds [0547] 1. For compound dilution plate, add 4
.mu.L of 10 mM stock into column 1 of plate, and bring volume to
100 .mu.L with 100% DMSO. [0548] 2. Set up the Precision 2000
dilution method. A final concentration of 200 .mu.M compound in 50%
DMSO, 100 mM HEPES (1:2 serial dilution).
[0549] Prep Coupled Enzymatic Buffer: [0550] 1. Final concentration
in assay:
TABLE-US-00001 [0550] Reagent (Stock Conc.) Final Conc. In Assay a.
PEP (1 M) 1 mM b. NADH (100 mM) 300 .mu.M c. MgCl.sub.2 (4 M) 20 mM
d. DTT (1 M) 2 mM e. ATP (500 mM) 300 .mu.M f. HEPES 200 mM (pH
7.5) 100 mM g. Pyruvate Kinase (PK) 15 units/mL h. Lactic
Dehydrogenase (LDH) 15 units/mL i. Met-2 peptide (10 mM) 0.500 mM
j. HGFR 50 nM
[0551] 2. For a 10 mL reaction buffer add 10 .mu.L of 1M PEP, 33
.mu.L of 100 mM NADH, 50 .mu.L of 4M MgCl.sub.2, 20 .mu.L of 1M
DTT, 6 .mu.L of 500 mM ATP, and 500 .mu.L of 10 mM Met-2 peptide
into 100 mM HEPES buffer pH 7.5 and vortex/mix. [0552] 3. Add
coupling enzymes, LDH and PK, into reaction mix. Mix by gentle
inversion.
[0553] Running samples [0554] 1. Spectrophotometer settings:
TABLE-US-00002 [0554] i. Absorbance wavelength (.lamda.): 340 nm
ii. Incubation time: 10 min iii. Run time: 10 min iv. Temperature:
37.degree. C.
[0555] 2. Add 85 .mu.L of CE reaction mix into each well of assay
plate. [0556] 3. Add 5 .mu.L of diluted compound into a well of the
assay plate. [0557] 4. Add 5 .mu.L of 50% DMSO for negative control
into last column of assay plate. [0558] 5. Mix with multi-channel
pipettor or orbital shaker. [0559] 6. Pre-incubate for 10 minutes
at 37.degree. C. [0560] 7. Add 10 .mu.L of 500 nM HGFR to each well
of assay plate; the final HGFR concentration is 50 nM in a total
final volume of 100 .mu.L. [0561] 8. Measure activity for 10
minutes at .lamda.=340 nm and 37.degree. C.
[0562] The following in vitro assays may be used to determine the
level of activity and effect of the different compounds of the
present invention on one or more of the PKs. Similar assays can be
designed along the same lines for any PK using techniques well
known in the art.
[0563] Several of the assays described herein are performed in an
ELISA (Enzyme-Linked Immunosorbent Sandwich Assay) format (Voller,
et al., 1980, "Enzyme-Linked Immunosorbent Assay," Manual of
Clinical Immunology, 2d ed., Rose and Friedman, Am. Soc. Of
Microbiology, Washington, D.C., pp. 359-371). General procedure is
as follows: a compound is introduced to cells expressing the test
kinase, either naturally or recombinantly, for a selected period of
time after which, if the test kinase is a receptor, a ligand known
to activate the receptor is added. The cells are lysed and the
lysate is transferred to the wells of an ELISA plate previously
coated with a specific antibody recognizing the substrate of the
enzymatic phosphorylation reaction. Non-substrate components of the
cell lysate are washed away and the amount of phosphorylation on
the substrate is detected with an antibody specifically recognizing
phosphotyrosine compared with control cells that were not contacted
with a test compound.
[0564] The presently preferred protocols for conducting the ELISA
experiments for specific PKs is provided below. However, adaptation
of these protocols for determining the activity of compounds
against other RTKs, as well as for CTKs and STKs, is well within
the scope of knowledge of those skilled in the art.
[0565] Other assays described herein measure the amount of DNA made
in response to activation of a test kinase, which is a general
measure of a proliferative response. General procedure for this
assay is as follows: a compound is introduced to cells expressing
the test kinase, either naturally or recombinantly, for a selected
period of time after which, if the test kinase is a receptor, a
ligand known to activate the receptor is added. After incubation at
least overnight, a DNA labeling reagent such as 5-bromodeoxyuridine
(BrdU) or H.sup.3-thymidine is added. The amount of labeled DNA is
detected with either an anti-BrdU antibody or by measuring
radioactivity and is compared to control cells not contacted with a
test compound.
MET Transphosphorylation Assay
[0566] This assay is used to measure phosphotyrosine levels on a
poly(glutamic acid:tyrosine, 4:1) substrate as a means for
identifying agonists/antagonists of met transphosphorylation of the
substrate.
Materials and Reagents:
[0567] 1. Corning 96-well ELISA plates, Corning Catalog #25805-96.
2. Poly(glu-tyr), 4:1, Sigma, Cat. No; P 0275.
3. PBS, Gibco Catalog #450-1300EB
4. 50 mM HEPES
[0568] 5. Blocking Buffer: Dissolve 25 g Bovine Serum Albumin,
Sigma Cat. No A-7888, in 500 mL PBS, filter through a 4 .mu.m
filter. 6. Purified GST fusion protein containing the Met kinase
domain, SUGEN, Inc.
7. TBST Buffer.
[0569] 8. 10% aqueous (MilliQue H.sub.2O) DMSO. 9. 10 mM aqueous
(dH.sub.2O) Adenosine-5'-triphosphate, Sigma Cat. No. A-5394. 10.
2.times. Kinase Dilution Buffer: for 100 mL, mix 10 mL 1M HEPES at
pH 7.5 with 0.4 mL 5% BSA/PBS, 0.2 mL 0.1 M sodium orthovanadate
and 1 mL 5M sodium chloride in 88.4 mL dH.sub.2O. 11. 4.times. ATP
Reaction Mixture: for 10 mL, mix 0.4 mL 1 M manganese chloride and
0.02 mL 0.1 M ATP in 9.56 mL dH.sub.2O. 12. 4.times. Negative
Controls Mixture: for 10 mL, mix 0.4 mL 1 M manganese chloride in
9.6 mL dH.sub.2O. 13. NUNC 96-well V bottom polypropylene plates,
Applied Scientific Catalog #S-72092
14. 500 mM EDTA.
[0570] 15. Antibody Dilution Buffer: for 100 mL, mix 10 mL 5%
BSA/PBS, 0.5 mL 5% Carnation.RTM. Instant Milk in PBS and 0.1 mL
0.1 M sodium orthovanadate in 88.4 mL TBST. 16. Rabbit polyclonal
antophosphotyrosine antibody, SUGEN, Inc. 17. Goat anti-rabbit
horseradish peroxidase conjugated antibody, Biosource, Inc. 18.
ABTS Solution: for 1 L, mix 19.21 g citric acid, 35.49 g
Na.sub.2HPO.sub.4 and 500 mg ABTS with sufficient dH.sub.2O to make
1 L. 19. ABTS/H.sub.2O.sub.2: mix 15 mL ABST solution with
24H.sub.2O.sub.2 five minutes before use.
20. 0.2 M HCl
Procedure:
[0571] 1. Coat ELISA plates with 2 .mu.g Poly(Glu-Tyr) in 100 .mu.L
PBS, hold overnight at 4.degree. C. 2. Block plate with 150 .mu.L
of 5% BSA/PBS for 60 min. 3. Wash plate twice with PBS then once
with 50 mM Hepes buffer pH 7.4. 4. Add 50 .mu.L of the diluted
kinase to all wells. (Purified kinase is diluted with Kinase
Dilution Buffer. Final concentration should be 10 ng/well.) 5. Add
25 .mu.L of the test compound (in 4% DMSO) or DMSO alone (4% in
dH.sub.2O) for controls to plate. 6. Incubate the kinase/compound
mixture for 15 minutes. 7. Add 25 .mu.L of 40 mM MnCl.sub.2 to the
negative control wells. 8. Add 25 .mu.L ATP/MnCl.sub.2 mixture to
the all other wells (except the negative controls). Incubate for 5
min. 9. Add 25 .mu.L 500 mM EDTA to stop reaction. 10. Wash plate
3.times. with TBST. 11. Add 100 .mu.L rabbit polyclonal anti-Ptyr
diluted 1:10,000 in Antibody Dilution Buffer to each well.
Incubate, with shaking, at room temperature for one hour. 12. Wash
plate 3.times. with TBST. 13. Dilute Biosource HRP conjugated
anti-rabbit antibody 1:6,000 in Antibody Dilution buffer. Add 100
.mu.L per well and incubate at room temperature, with shaking, for
one hour. 14. Wash plate 1.times. with PBS. 15. Add 100 .mu.l of
ABTS/H.sub.2O.sub.2 solution to each well. 16. If necessary, stop
the development reaction with the addition of 100 .mu.L of 0.2M HCl
per well. 17. Read plate on Dynatech MR7000ELISA reader with the
test filter at 410 nM and the reference filter at 630 nM.
BrdU Incorporation Assays
[0572] The following assays use cells engineered to express a
selected receptor and then evaluate the effect of a compound of
interest on the activity of ligand-induced DNA synthesis by
determining BrdU incorporation into the DNA.
[0573] The following materials, reagents and procedure are general
to each of the following BrdU incorporation assays. Variances in
specific assays are noted.
General Materials and Reagents:
[0574] 1. The appropriate ligand. 2. The appropriate engineered
cells.
3. BrdU Labeling Reagent: 10 mM, in PBS, pH7.4 (Roche Molecular
Biochemicals, Indianapolis, Ind.).
[0575] 4. FixDenat: fixation solution (Roche Molecular
Biochemicals, Indianapolis, Ind.). 5. Anti-BrdU-POD: mouse
monoclonal antibody conjugated with peroxidase (Chemicon, Temecula,
Calif.). 6. TMB Substrate Solution: tetramethylbenzidine (TMB,
ready to use, Roche Molecular Biochemicals, Indianapolis,
Ind.).
7. PBS Washing Solution: 1.times.PBS, pH 7.4.
[0576] 8. Albumin, Bovine (BSA), fraction V powder (Sigma Chemical
Co., USA).
General Procedure:
[0577] 1. Cells are seeded at 8000 cells/well in 10% CS, 2 mM Gln
in DMEM, in a 96 well plate. Cells are incubated overnight at
37.degree. C. in 5% CO.sub.2. 2. After 24 hours, the cells are
washed with PBS, and then are serum-starved in serum free medium
(0% CS DMEM with 0.1% BSA) for 24 hours. 3. On day 3, the
appropriate ligand and the test compound are added to the cells
simultaneously. The negative control wells receive serum free DMEM
with 0.1% BSA only; the positive control cells receive the ligand
but no test compound. Test compounds are prepared in serum free
DMEM with ligand in a 96 well plate, and serially diluted for 7
test concentrations. 4. After 18 hours of ligand activation,
diluted BrdU labeling reagent (1:100 in DMEM, 0.1% BSA) is added
and the cells are incubated with BrdU (final concentration is 10
.mu.M) for 1.5 hours. 5. After incubation with labeling reagent,
the medium is removed by decanting and tapping the inverted plate
on a paper towel. FixDenat solution is added (50 .mu.l/well) and
the plates are incubated at room temperature for 45 minutes on a
plate shaker. 6. The FixDenat solution is removed by decanting and
tapping the inverted plate on a paper towel. Milk is added (5%
dehydrated milk in PBS, 200 .mu.L/well) as a blocking solution and
the plate is incubated for 30 minutes at room temperature on a
plate shaker. 7. The blocking solution is removed by decanting and
the wells are washed once with PBS. Anti-BrdU-POD solution is added
(1:200 dilution in PBS, 1% BSA, 50 .mu.L/well) and the plate is
incubated for 90 minutes at room temperature on a plate shaker. 8.
The antibody conjugate is removed by decanting and rinsing the
wells 5 times with PBS, and the plate is dried by inverting and
tapping on a paper towel. 9. TMB substrate solution is added (100
.mu.l/well) and incubated for 20 minutes at room temperature on a
plate shaker until color development is sufficient for photometric
detection. 10. The absorbance of the samples are measured at 410 nm
(in "dual wavelength" mode with a filter reading at 490 nm, as a
reference wavelength) on a Dynatech ELISA plate reader.
HGF-Induced BrdU Incorporation Assay
Materials and Reagents:
[0578] 1. Recombinant human HGF (Cat. No. 249-HG, R&D Systems,
Inc. USA). 2. BxPC-3 cells (ATCC CRL-1687). Remaining Materials and
Reagents, as above.
Procedure:
[0579] 1. Cells are seeded at 9000 cells/well in RPMI 10% FBS in a
96 well plate. Cells are incubated overnight at 37.degree. C. in 5%
CO.sub.2. 2. After 24 hours, the cells are washed with PBS, and
then are serum starved in 100 .mu.L serum-free medium (RPMI with
0.1% BSA) for 24 hours. 3. On day 3, 25 .mu.L containing ligand
(prepared at 1 .mu.g/mL in RPMI with 0.1% BSA; final HGF conc. is
200 ng/mL) and test compounds are added to the cells. The negative
control wells receive 25 .mu.L serum-free RPMI with 0.1% BSA only;
the positive control cells receive the ligand (HGF) but no test
compound. Test compounds are prepared at 5 times their final
concentration in serum-free RPMI with ligand in a 96 well plate,
and serially diluted to give 7 test concentrations. Typically, the
highest final concentration of test compound is 100 .mu.M, and 1:3
dilutions are used (i.e. final test compound concentration range is
0.137-100 .mu.M). 4. After 18 hours of ligand activation, 12.5
.mu.L of diluted BrdU labeling reagent (1:100 in RPMI, 0.1% BSA) is
added to each well and the cells are incubated with BrdU (final
concentration is 10 .mu.M) for 1 hour.
5. Same as General Procedure.
6. Same as General Procedure.
[0580] 7. The blocking solution is removed by decanting and the
wells are washed once with PBS. Anti-BrdU-POD solution (1:100
dilution in PBS, 1% BSA) is added (100 .mu.L/well) and the plate is
incubated for 90 minutes at room temperature on a plate shaker.
8. Same as General Procedure.
9. Same as General Procedure.
10. Same as General Procedure.
Cellular HGFR Autophosphorylation Assay
[0581] A549 cells (ATCC) were used in this assay. Cells were seeded
in the growth media (RPMI+10% FBS) into 96 well plates and cultured
overnight at 37.degree. C. for attachment. Cells were exposed to
the starvation media (RPMI+0.05% BSA). Dilutions of the inhibitors
were added to the plates and incubated at 37.degree. C. for 1 hour.
Cells were then stimulated by adding 40 ng/mL HGF for 15 minutes.
Cells were washed once with 1 mM Na.sub.3VO.sub.4 in HBSS and then
lysed. The lysates were diluted with 1 mM Na.sub.3VO.sub.4 in HBSS
and transferred to a 96 well goat ant-rabbit coated plate (Pierce)
which was pre-coated with anti-HGFR antibody (Zymed Laboratories).
The plates were incubated overnight at 4.degree. C. and washed with
1% Tween 20 in PBS for seven times. HRP-PY20 (Santa Cruz) was
diluted and added to the plates for 30 minutes incubation. Plates
were then washed again and TMB peroxidase substrate (Kirkegaard
& Perry) was added and incubated for 10 minutes. The reaction
was then stopped by adding 0.09NH.sub.2SO.sub.4. Plates were
measured at OD-450 nm using a spectrophotometer. IC.sub.50 values
were calculated by curve fitting using a four-parameter
analysis.
[0582] Compounds of the invention were measured for HGFR inhibition
activity; the data are shown in each Example. Ki data were obtained
using the HGFR Continuous-Coupled Spectrophotometric Assay, and
IC.sub.50 data were obtained using the Cellular HGFR
Autophosphorylation Assay, both of which are described above.
[0583] While the invention has been illustrated by reference to
specific and preferred embodiments, those skilled in the art will
recognize that variations and modifications may be made through
routine experimentation and practice of the invention. Thus, the
invention is intended not to be limited by the foregoing
description, but to be defined by the appended claims and their
equivalents.
[0584] All references cited herein, including any priority
documents, are hereby incorporated by reference in their
entireties.
* * * * *