U.S. patent application number 16/579488 was filed with the patent office on 2020-04-23 for substituted tetrahydropyrans as ccr2 modulators.
The applicant listed for this patent is CHEMOCENTRYX, INC.. Invention is credited to Junfa FAN, Jaroslaw Kalisiak, Rebecca M. LUI, Venkat Reddy MALI, Jeffrey P. McMahon, Jay P. Powers, Hiroko Tanaka, Yibin Zeng, Penglie Zhang.
Application Number | 20200121688 16/579488 |
Document ID | / |
Family ID | 57320660 |
Filed Date | 2020-04-23 |
![](/patent/app/20200121688/US20200121688A1-20200423-C00001.png)
![](/patent/app/20200121688/US20200121688A1-20200423-C00002.png)
![](/patent/app/20200121688/US20200121688A1-20200423-C00003.png)
![](/patent/app/20200121688/US20200121688A1-20200423-C00004.png)
![](/patent/app/20200121688/US20200121688A1-20200423-C00005.png)
![](/patent/app/20200121688/US20200121688A1-20200423-C00006.png)
![](/patent/app/20200121688/US20200121688A1-20200423-C00007.png)
![](/patent/app/20200121688/US20200121688A1-20200423-C00008.png)
![](/patent/app/20200121688/US20200121688A1-20200423-C00009.png)
![](/patent/app/20200121688/US20200121688A1-20200423-C00010.png)
![](/patent/app/20200121688/US20200121688A1-20200423-C00011.png)
View All Diagrams
United States Patent
Application |
20200121688 |
Kind Code |
A1 |
FAN; Junfa ; et al. |
April 23, 2020 |
SUBSTITUTED TETRAHYDROPYRANS AS CCR2 MODULATORS
Abstract
Compounds are provided that are modulators of the CCR2 receptor.
The compounds have the general formula (I): ##STR00001## and are
useful in pharmaceutical compositions, methods for the treatment of
diseases and disorders involving the pathologic activation of CCR2
receptors.
Inventors: |
FAN; Junfa; (Palo Alto,
CA) ; Kalisiak; Jaroslaw; (Mountain View, CA)
; LUI; Rebecca M.; (Santa Clara, CA) ; MALI;
Venkat Reddy; (Cupertino, CA) ; McMahon; Jeffrey
P.; (San Francisco, CA) ; Powers; Jay P.;
(Pacifica, CA) ; Tanaka; Hiroko; (Mountain View,
CA) ; Zeng; Yibin; (Foster City, CA) ; Zhang;
Penglie; (Foster City, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
CHEMOCENTRYX, INC. |
Mountain View |
CA |
US |
|
|
Family ID: |
57320660 |
Appl. No.: |
16/579488 |
Filed: |
September 23, 2019 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
15692811 |
Aug 31, 2017 |
10464934 |
|
|
16579488 |
|
|
|
|
15158713 |
May 19, 2016 |
9783540 |
|
|
15692811 |
|
|
|
|
62164957 |
May 21, 2015 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 45/06 20130101;
A61K 31/538 20130101; A61K 31/536 20130101; A61K 31/551 20130101;
C07D 413/14 20130101; C07D 471/04 20130101; A61K 31/4725 20130101;
C07D 405/14 20130101; A61K 31/553 20130101; A61P 37/00 20180101;
A61P 35/00 20180101; A61K 31/453 20130101; C07D 405/04 20130101;
A61K 31/4545 20130101; A61K 31/496 20130101 |
International
Class: |
A61K 31/538 20060101
A61K031/538; A61K 31/453 20060101 A61K031/453; A61K 31/4545
20060101 A61K031/4545; A61K 31/496 20060101 A61K031/496; A61K
31/551 20060101 A61K031/551; A61K 31/553 20060101 A61K031/553; A61P
35/00 20060101 A61P035/00 |
Claims
1.-55. (canceled)
56. A method of treating cancer in a mammal in need thereof,
comprising administering to the mammal an effective amount of a
compound selected from the group consisting of ##STR00064## or a
pharmaceutically acceptable salt thereof.
57. The method of claim 56, wherein said cancer is selected from
the group consisting of Melanoma, Small Cell Lung Carcinoma, Non
Small Cell Lung Carcinoma, Pancreatic Cancer, Breast Cancer,
Bladder Cancer, Renal Cell Carcinoma, Colorectal Cancer,
Hepatocellular Carcinoma, Head and Neck Squamous Cell Carcinoma,
Esophageal Cancer, Ovarian Cancer, Prostate Cancer, Gastric Cancer,
Acute myelogenous leukemia, leukemia.
58. The method of claim 56, wherein said cancer is selected from
the group consisting of Small Cell Lung Carcinoma, Non Small Cell
Lung Carcinoma, Pancreatic Cancer, Breast Cancer, Bladder Cancer,
Renal Cell Carcinoma, Colorectal Cancer, Esophageal Cancer, Ovarian
Cancer, Prostate Cancer, Gastric Cancer.
59. The method of claim 56, wherein said cancer is Colorectal
Cancer.
60. A pharmaceutical composition comprising a compound having the
formula: ##STR00065## or a pharmaceutically acceptable salt,
hydrate, stereoisomer or rotamer thereof; wherein the subscripts m
and n are each independently 0, 1, or 2, wherein the sum of m and n
is less than or equal to 3; R.sup.1 is selected from the group
consisting of aryl, aryl-C.sub.1-4 alkyl, heteroaryl and
heteroaryl-C.sub.1-4 alkyl, wherein the heteroaryl portion has from
1-3 heteroatoms as ring members selected from N, O and S; and
wherein said aryl and heteroaryl groups or portions are optionally
substituted with from 1 to 5 R.sup.x substituents; R.sup.2 is
selected from the group consisting of H, C.sub.1-8 alkyl, C.sub.3-8
cycloalkyl, C.sub.3-8 cycloalkyl-C.sub.1-4 alkyl, aryl,
aryl-C.sub.1-4 alkyl, heteroaryl and heteroaryl-C.sub.1-4 alkyl,
wherein the heteroaryl portion has from 1-3 heteroatoms as ring
members selected from N, O and S; and wherein said aryl and
heteroaryl groups or portions are optionally substituted with from
1 to 4 R.sup.x substituents; or optionally, R.sup.1 and R.sup.2 are
combined with the nitrogen atom to which each is attached to form a
6- to 11-membered monocyclic or fused bicyclic-heterocyclic or
heteroaryl ring, wherein the --NR.sup.1R.sup.2 is optionally
further substituted with from 1 to 4 R.sup.x substituents; R.sup.3
is selected from the group consisting of H, C.sub.1-8 alkyl,
C.sub.3-8 cycloalkyl and C.sub.3-8 cycloalkyl-C.sub.1-4 alkyl, each
of which is optionally substituted with from 1-3 R.sup.y
substituents; R.sup.4 is selected from the group consisting of H,
C.sub.1-8alkyl optionally substituted with 1 to 2 R.sup.y, and
--CO.sub.2H: each R.sup.x is independently selected from the group
consisting of halogen, --CN, --R.sup.c, --CO.sub.2R.sup.a,
--CONR.sup.aR.sup.b, --C(O)R.sup.a, --OC(O)NR.sup.aR.sup.b,
--NR.sup.bC(O)R.sup.a, --NR.sup.bC(O).sub.2R.sup.c,
--NR.sup.a--C(O)NR.sup.aR.sup.b, --NR.sup.aC(O)NR.sup.aR.sup.b,
--NR.sup.aR.sup.b, --OR.sup.a, --O--X.sup.1--OR.sup.a,
--O--X.sup.1--NR.sup.aR.sup.b, --O--X.sup.1--CO.sub.2R.sup.a,
--O--X.sup.1--CONR.sup.aR.sup.b, --X.sup.1--OR.sup.a,
--X.sup.1--NR.sup.aR.sup.b, --X.sup.1--CO.sub.2R.sup.a,
--X.sup.1--CONR.sup.aR.sup.b, --SF.sub.5,
--S(O).sub.2NR.sup.aR.sup.b, and 5- or 6-membered aryl or
heteroaryl, wherein each X.sup.1 is a C.sub.1-4 alkylene; each
R.sup.a and R.sup.b is independently selected from hydrogen,
C.sub.1-8 alkyl, and C.sub.1-8 haloalkyl, or when attached to the
same nitrogen atom can be combined with the nitrogen atom to form a
five or six-membered ring having from 0 to 2 additional heteroatoms
as ring members selected from N, O or S, and optionally substituted
with oxo; each R.sup.c is independently selected from the group
consisting of C.sub.1-8alkyl, C.sub.1-8 haloalkyl and C.sub.3-6
cycloalkyl; and optionally when two R.sup.x substituents are on
adjacent atoms, are combined to form a fused five or six-membered
carbocyclic ring, and wherein the aryl or heteroaryl groups are
optionally substituted with 1-3 members selected from halogen,
hydroxyl, C.sub.1-4 alkyl, C.sub.1-4 alkoxy, C.sub.1-4 haloalkyl,
and C.sub.1-4 haloalkoxy; each R.sup.y is independently selected
from the group consisting of halogen, --CN, --R.sup.f,
--CO.sub.2R.sup.d, --CONR.sup.dR.sup.e, --C(O)R.sup.d,
--OC(O)NR.sup.dR.sup.e, --NR.sup.eC(O)R.sup.d,
--NR.sup.eC(O).sub.2R.sup.f, --NR.sup.dC(O)NR.sup.dR.sup.e,
--NR.sup.dC(O)NR.sup.dR.sup.e, --NR.sup.dR.sup.e, --OR.sup.d, and
--S(O).sub.2NR.sup.dR.sup.e; wherein each R.sup.d and R.sup.e is
independently selected from hydrogen, C.sub.1-8 alkyl, and
C.sub.1-8 haloalkyl, or when attached to the same nitrogen atom can
be combined with the nitrogen atom to form a five or six-membered
ring having from 0 to 2 additional heteroatoms as ring members
selected from N, O or S; each R.sup.f is independently selected
from the group consisting of C.sub.1-8 alkyl, C.sub.1-8 haloalkyl
and C.sub.3-6 cycloalkyl; each R.sup.z is independently selected
from the group consisting of halogen, --CN, --R.sup.1,
--CO.sub.2R.sup.g, --CONR.sup.gR.sup.h, --C(O)R.sup.g,
--OC(O)NR.sup.gR.sup.h, --NR.sup.hC(O)R.sup.g,
--NR.sup.hC(O).sub.2R.sup.i, --NR.sup.gC(O)NR.sup.gR.sup.h,
--NR.sup.gR.sup.h, --OR.sup.g, --S(O).sub.2NR.sup.gR.sup.h,
--X.sup.1--R.sup.j, --X.sup.1--NR.sup.gR.sup.h,
--X.sup.1--CONR.sup.gR.sup.h, --X.sup.1--NR.sup.hC(O)R.sup.g,
--NHR.sup.j, --NHCH.sub.2R.sup.j, and tetrazole; wherein each
R.sup.g and R.sup.h is independently selected from hydrogen,
C.sub.1-8 alkyl, C.sub.3-6 cycloalkyl and C.sub.1-8 haloalkyl, or
when attached to the same nitrogen atom can be combined with the
nitrogen atom to form a five or six-membered ring having from 0 to
2 additional heteroatoms as ring members selected from N, O or S
and is optionally substituted with one or two oxo; each R.sup.i is
independently selected from the group consisting of C.sub.1-8
alkyl, C.sub.1-8 haloalkyl and C.sub.3-6 cycloalkyl; and each
R.sup.j is selected from the group consisting of C.sub.3-6
cycloalkyl, pyrrolinyl, piperidinyl, morpholinyl,
tetrahydrofuranyl, and tetrahydropyranyl; and the subscript q is 1,
2, 3, 4, or 5, and wherein said compound is substantially free of
other stereoisomers.
61. The pharmaceutical composition of claim 60, wherein R.sup.3 is
a member selected from the group consisting of H, methyl, ethyl,
propyl, isopropyl, butyl, isobutyl, sec-butyl, cyclopropyl,
cyclopropylmethyl, cyclobutyl and cyclobutylmethyl.
62. The pharmaceutical composition of claim 60, wherein m and n are
both 0.
63. The pharmaceutical composition of claim 60, wherein m and n are
both 1.
64. The pharmaceutical composition of claim 60, wherein m is 1 and
n is 0.
65. The pharmaceutical composition of claim 60, wherein m is 1 and
n is 2.
66. The pharmaceutical composition of claim 60, wherein
--N(R.sup.1)(R.sup.2) is selected from the group consisting of:
##STR00066## ##STR00067##
67. The pharmaceutical composition of claim 60, wherein the
compound is selected from the group consisting of: ##STR00068##
##STR00069## ##STR00070## ##STR00071## ##STR00072## ##STR00073##
##STR00074## ##STR00075## ##STR00076## ##STR00077## ##STR00078##
##STR00079## ##STR00080## ##STR00081## ##STR00082## ##STR00083##
##STR00084## ##STR00085## ##STR00086## ##STR00087## ##STR00088##
##STR00089## ##STR00090## or a pharmaceutically acceptable salt
thereof.
68. The pharmaceutical composition of claim 60, wherein the
compound is selected from the group consisting of ##STR00091## or a
pharmaceutically acceptable salt thereof.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 15/692,811 filed Aug. 31, 2017, which is a
continuation of U.S. patent application Ser. No. 15/158,713 filed
May 19, 2016 (now U.S. Pat. No. 9,783,540), which application
claims the benefit of priority under 35 U.S.C. .sctn. 119(e) to
U.S. Provisional Application Ser. No. 62/164,957, filed May 21,
2015, each of which is incorporated herein by reference in its
entirety.
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED
RESEARCH AND DEVELOPMENT
[0002] NOT APPLICABLE
REFERENCE TO A "SEQUENCE LISTING," A TABLE, OR A COMPUTER PROGRAM
LISTING APPENDIX SUBMITTED ON A COMPACT DISK
[0003] NOT APPLICABLE
BACKGROUND OF THE INVENTION
[0004] Chemokines, also known as chemotactic cytokines, are a group
of small molecular-weight proteins that are released by a wide
variety of cells and have a variety of biological activities.
Chemokines attract various types of cells of the immune system,
such as macrophages, T cells, eosinophils, basophils and
neutrophils, and cause them to migrate from the blood to various
lymphoid and none-lymphoid tissues. They mediate infiltration of
inflammatory cells to sites of inflammation, and are responsible
for the initiation and perpetuation of many inflammation diseases
(reviewed in Schall, Cytokine, 3:165-183 (1991), Schall et al.,
Curr. Opin. Immunol., 6:865-873 (1994)).
[0005] In addition to stimulating chemotaxis, chemokines can induce
other changes in responsive cells, including changes in cell shape,
granule exocytosis, integrin up-regulation, formation of bioactive
lipids (e.g., leukotrienes), respiratory burst associated with
leukocyte activation, cell proliferation, resistance to induction
of apoptosis and angiogenesis. Thus, chemokines are early triggers
of the inflammatory response, causing inflammatory mediator
release, chemotaxis and extravasation to sites of infection or
inflammation. They are also stimulators of a multitude of cellular
processes that bear important physiological functions as well as
pathological consequences.
[0006] Chemokines exert their effects by activating chemokine
receptors expressed by responsive cells. Chemokine receptors are a
class of G-protein coupled receptors, also known as
seven-transmembrane receptors, found on the surface of a wide
variety of cell types such as leukocytes, endothelial cells, smooth
muscle cells and tumor cells.
[0007] Chemokines and chemokine receptors are expressed by
intrinsic renal cells and infiltrating cells during renal
inflammation (Segerer et al., J. Am. Soc. Nephrol., 11:152-76
(2000); Morii et al., J. Diabetes Complications, 17:11-5 (2003);
Lloyd et al. J. Exp. Med., 185:1371-80 (1997); Gonzalez-Cuadrado et
al. Clin. Exp. Immunol. 106:518-22 (1996); Eddy & Giachelli,
Kidney Int., 47:1546-57 (1995); Diamond et al., Am. J. Physiol.,
266:F926-33 (1994)). In humans, CCR2 and ligand MCP-1 are among the
proteins expressed in renal fibrosis, and are correlated with the
extent of macrophage infiltration into the interstitium (Yang et
al., Zhonghua Yi Xue Za Zhi, 81:73-7 (2001); Stephan et al., J.
Urol., 167:1497-502 (2002); Amann et al., Diabetes Care, 26:2421-5
(2003); Dai et al., Chin. Med. J. (Engl), 114:864-8 (2001)). In
animal models of renal fibrosis, blockade of CCR2 or MCP-1 leads to
a marked reduction in severity of renal inflammation (Kitagawa et
al., Am. J. Pathol., 165:237-46 (2004); Wada et al., Am. J.
Pathol., 165:237-46 (2004); Shimizu et al., J. Am. Soc. Nephrol.,
14:1496-505 (2003)).
[0008] Additionally CCR2 plays a role in the development of
polycystic kidney disease (PKD). The pathology of ADPKD is
characterized by renal cysts that arise from tubular epithelial
cells and enlarge continuously over the patient's lifetime. The
expanding cysts are accompanied by the presence of a large number
of infiltrating macrophages which are recruited to the kidney in
response to injury. Thus, inhibition of immune recruitment to the
kidney constitutes a therapeutic strategy for PKD (J Am Soc
Nephrol. 2011 October; 22(10):1809-14.)
[0009] Rheumatoid arthritis is a chronic disease of the joints
characterized by synovial inflammation that leads to the
destruction of cartilage and bone. Although the underlying causes
of the disease are unknown, it is believed that macrophages and
Th-1 type T cells play a key role in the initiation and
perpetuation of the chronic inflammatory process (Vervoordeldonk et
al., Curr. Rheumatol. Rep., 4:208-17 (2002)).
[0010] MCP-1 is among the several chemokines, including
MIP-1.alpha. and IL-8, identified in rheumatoid synovium (Villiger
et al., J. Immunol., 149:722-7 (1992); Scaife et al., Rheumatology
(Oxford), 43:1346-52 (2004); Shadidi et al., Scand. J Immunol.,
57:192-8 (2003); Taylor et al., Arthritis Rheum., 43:38-47 (2000);
Tucci et al., Biomed. Sci. Instrum., 34:169-74 (1997)). Chemokine
receptors CCR1, CCR2, CCR3 and CCR5 are up-regulated in the joints
from arthritic mice (Plater-Zyberk et al., Immunol. Lett.,
57:117-20 (1997). Blockade of MCP-1 activity using a CCR2
antagonist or an antibody against MCP-1 have been shown efficacious
in reducing joint inflammation in experimental models of rheumatoid
arthritis (Gong et al., J Exp. Med., 186:131-7 (1997); Ogata et
al., J. Pathol., 182:106-14 (1997)).
[0011] Chemokine receptor-mediated infiltration of macrophages in
the fat tissues may also contribute to the complications arising
from obesity, a condition resulting from excessive storage of fat
in the body. Obesity predisposes the affected individuals to many
disorders, such as non-insulin-dependent diabetes, hypertension,
stroke, and coronary artery disease. In obesity, adipose tissues
have altered metabolic and endocrine functions that lead to an
increased release of fatty acids, hormones, and pro-inflammatory
molecules. Adipose tissue macrophages are believed to be a key
source of pro-inflammatory cytokines including TNF-alpha, iNOS and
IL-6 (Weisberg et al., J. Clin. Invest., 112:1796-808 (2003)).
Recruitment of macrophages to the adipose tissue is likely mediated
by MCP-1 produced by adipocytes (Christiansen T, et al., Int J Obes
(Lond). 2005 January; 29(1):146-50; Sartipy et al., Proc. Natl.
Acad. Sci. U.S.A., 100:7265-70 (2003)).
[0012] Elevated MCP-1 may induce adipocyte differentiation and
insulin resistance, and contribute to pathologies associated with
hyper-insulinemia and obesity. MCP-1 is over-expressed in plasma in
obese mice compared to lean controls and white adipose is a major
source. MCP-1 has also been shown to accelerate wound healing, and
has a direct angiogenic effect on epithelial cells, and may play a
direct role in the remodeling of adipose tissue in obesity.
(Sartipy P, Loskutoff D J., Proc. Natl. Acad. Sci. U.S.A., 100:7265
(2003)).
[0013] MCP-1 plasma levels are substantially increased in Diet
Induce Obesity (DIO) mice, and a strong correlation between plasma
MCP-1 levels and body weight has been identified. Furthermore,
elevation of MCP-1 induced by high fat diet causes changes in the
CD11b positive monocyte population in DIO mice. (Takahashi K, et
al., J. Biol. Chem., 46654 (2003)).
[0014] Furthermore, chronic inflammation in fat is thought to play
a crucial role in the development of obesity-related insulin
resistance (Xu H, et al., J Clin Invest. 2003 December;
112(12):1821-30). It has been proposed that obesity related insulin
resistance is, at least in part, a chronic inflammatory disease
initiated in adipose tissue. Many inflammation and macrophage
specific genes are dramatically upregulated in white adipose tissue
in mouse models of genetic and high fat diet-induced obesity (DIO),
and this upregulation precedes a dramatic increase in circulating
insulin.
[0015] Increased expression levels of monocyte CCR2 and monocyte
chemoattractant protein-1 in patients with diabetes mellitus
(Biochemical and Biophysical Research Communications, 344(3):780-5
(2006)) were found in a study involving diabetic patients. Serum
MCP-1 concentrations and surface expression of CCR2 on monocytes in
diabetic patients were significantly higher than in non-diabetics,
and the serum MCP-1 levels correlated with HbA1c, triglycerides,
BMI, hs-CRP. Surface expression levels of CD36 and CD68 on
monocytes were significantly increased in diabetic patients and
more unregulated by MCP-1 in diabetics, augmenting uptake of
ox-LDL, and hence potentially foam cell transformation. Elevated
serum MCP-1 and increased monocyte CCR2, CD36, CD68 expression
correlated with poor blood glucose control and potentially
correlate with increased vessel wall monocyte recruitment.
[0016] MCP-1 is a potential player in negative cross talk between
adipose tissue and skeletal muscle (Bianco J J, et al.,
Endocrinology, 2458 (2006)). MCP-1 can significantly reduce
insulin-stimulated glucose uptake, and is a prominent inducer of
insulin resistance in human skeletal muscle cell. Adipose tissue is
a major secretory and endocrine active organ producing bioactive
proteins regulating energy metabolism and insulin sensitivity.
[0017] CCR2 modulates inflammatory and metabolic effects of
high-fat feeding (Weisberg S P, et al., J. Clin. Invest., 115
(2006)). Genetic deficiency in CCR2 reduced food intake and
attenuated the development of obesity in mice fed a high fat diet.
In obese mice matched for adiposity, CCR2 deficiency reduced
macrophage content and inflammatory profile of adipose tissue,
increased adiponectin expression, and improved glucose homeostatis
and insulin sensitivity. In lean animals, no effect of CCR2
genotype on metabolic trait was found. In high-fat diet mice, CCR2
genotype modulated feeding, the development of obesity and adipose
tissue inflammation. Once established, short term antagonism was
shown to attenuate macrophage accumulation in adipose tissue and
insulin resistance.
[0018] Chemokine and chemokine receptors are the key regulators of
immune cell trafficking. MCP-1 is a potent chemoattractant of
monocytes and T cells; its expression is induced under inflammatory
conditions including proinflammatory cytokine stimulations and
hypoxia. The interaction between MCP-1 and CCR2 mediates migration
of monocytes, macrophage as well as activated T cells and play a
key role in the pathogenesis of many inflammatory diseases.
Inhibition of CCR2 functions using small molecule antagonists
described in this invention represents a new approach for the
treatments of inflammatory disorders.
[0019] Psoriasis is a chronic inflammatory disease characterized by
hyperproliferation of keratinocytes and pronounced leukocyte
infiltration. It is known that keratinocytes from psoriasis lesion
express abundant CCR2 ligand MCP-1, particularly when stimulated by
proinflammatory cytokines such as TNF-.alpha. (Vestergaard et al.,
Acta. Derm. Venereol., 84(5):353-8 (2004); Gillitzer et al., J.
Invest. Dermatol., 101(2):127-31 (1993); Deleuran et al., J.
Dermatol. Sci., 13(3):228-36 (1996)). Since MCP-1 can attract
migration of both macrophages and dendritic cells expressing CCR2
to the skin, this receptor and ligand pair is believed to be
important in regulating the interaction between proliferating
keratinocytes and dermal macrophage during the development of
psoriasis. A small molecule antagonist may thus be useful in the
treatment of psoriasis.
[0020] In addition to inflammatory diseases, chemokines and
chemokine receptors have also been implicated in cancers (Broek et
al., Br. J. Cancer, 88(6):855-62 (2003)). Tumor cells stimulate the
formation of stroma that secretes various mediators pivotal for
tumor growth, including growth factors, cytokines, and proteases.
It is known that the level of MCP-1 is associated significantly
with tumor-associated macrophage accumulation, and prognostic
analysis reveals that high expression of MCP-1 is a significant
indicator of early relapse in breast cancer (Ueno et al., Clin.
Cancer Res., 6(8):3282-9 (2001)). A small molecule antagonist of a
chemokine may thus be able to reduce the release of
growth-stimulating cytokines by blocking accumulation of
macrophages at sites of tumor formation.
[0021] CCR2 and its ligand CCL2 also play a major role in
conditioning the tumor microenvironment, and regulating the influx
of both beneficial and deleterious immune cell populations to the
tumor. As such recent clinical and pre-clinical literature has
demonstrated a role for CCR2 in a variety of solid tumors either
through up-regulation of its expression in transformed cells or in
enhanced chemotaxis of inflammatory monocytes into the tumour that
terminally differentiates into myeloid-derived suppressor cells
(MDSCs) or tumour associated macrophages (TAMs). MDSCs/TAMs are
thought to promote tumorgenesis through the following mechanisms:
(1) contribute to the general immunosuppressive microenvironment,
thereby promoting tumour growth, that abolish the cytotoxic
functionality of infiltrating cytotoxic T cells (Mitchem et al,
Cancer Res 73(3): 1128-1141), (2) enhance angiogenesis through
secretion of vascular endothelial growth factor (VEGF) and other
growth factors (Murdoch et al, Nat Rev Cancer 8: 618-631), (3)
prevent tumour senescence (Di Mitri et al, AOP, Nature, 2014), and
(4) promote tumour metastases into distal organs (Qian et al,
Nature 475: 222-227). Thus, a small molecule antagonist can be
useful in limiting tumourgenesis, enhancing tumour specific immune
activity, and tumour metastases.
[0022] Provided herein are compounds that are CCR2 modulators and
address some of the shortcomings identified with earlier CCR2
modulators.
BRIEF SUMMARY OF THE INVENTION
[0023] In one aspect, the present invention provides compounds
having the formula:
##STR00002##
or a pharmaceutically acceptable salt, hydrate, stereoisomer or
rotamers thereof; wherein the symbols R.sup.1, R.sup.2, R.sup.3,
R.sup.4, A and the subscripts m and n have the meanings provided in
the Detail Description of the Invention.
[0024] In addition to the compounds provided herein, the present
invention further provides pharmaceutical compositions containing
one or more of these compounds, as well as methods for the use of
these compounds in therapeutic methods, primarily to treat diseases
associated CCR2 signalling activity.
[0025] In yet another aspect, the present invention provides
methods of diagnosing disease in an individual. In these methods,
the compounds provided herein are administered in labeled form to a
subject, followed by diagnostic imaging to determine the presence
or absence of CCR2. In a related aspect, a method of diagnosing
disease is carried out by contacting a tissue or blood sample with
a labeled compound as provided herein and determining the presence,
absence, or amount of CCR2 in the sample.
[0026] In still another aspect, the present invention provides a
method for modulating chemokine function, comprising contacting a
chemokine receptor with a therapeutically effective amount of a
compound or composition according to the invention.
[0027] In still another aspect, the present invention provides a
method for treating a chemokine-mediated condition or disease,
comprising administering to a subject a safe and effective amount
of a compound or composition according to the invention.
[0028] In one particular aspect, the present invention relates to a
method of treating a CCR2-mediated condition or disease comprising
administering to a subject a therapeutically effective amount of a
compound or pharmaceutically acceptable salt of a compound of
formula I. In certain embodiments, a CCR2-mediated condition or
disease is atherosclerosis. In certain embodiments, a CCR2-mediated
condition or disease is restenosis. In certain embodiments, a
CCR2-mediated condition or disease is multiple sclerosis. In
certain embodiments, a CCR2-mediated condition or disease is
selected from the group consisting of inflammatory bowel disease,
renal fibrosis, rheumatoid arthritis, obesity and
noninsulin-dependent diabetes. In certain embodiments, a
CCR2-mediated condition or disease is type 2 diabetes. In certain
embodiments, a CCR2-mediated condition or disease is selected from
the group consisting of chronic obstructive pulmonary disease,
idiopathic pulmonary fibrosis and idiopathic pneumonia
syndrome.
[0029] In addition to the compounds provided herein, the present
invention further provides pharmaceutical compositions containing
one or more of these compounds, as well as methods for the use of
these compounds in therapeutic methods, primarily to treat diseases
associated with chemokine signaling activity.
BRIEF DESCRIPTION OF THE DRAWINGS
[0030] FIGS. 1A-1Y provide structures and activity for
representative compounds of the present invention. The compounds
were prepared as described generally below, as well as by methods
provided in the Examples. Activity is provided as follows for the
binding assay as described herein: +, 501 nM.ltoreq.IC50<5000
nM; ++, 101 nM.ltoreq.IC50<500 nM; and +++, 1
nM.ltoreq.IC50.ltoreq.100 nM.
[0031] FIG. 2 provide an ORTEP structure of a component portion of
the compounds described herein and shows the stereochemistry of a
quaternary center (shown as bearing a cyclopropyl group and
identified as having `S` chirality).
DETAILED DESCRIPTION OF THE INVENTION
I. Abbreviation and Definitions
[0032] The term "alkyl", by itself or as part of another
substituent, means, unless otherwise stated, a straight or branched
chain hydrocarbon radical, having the number of carbon atoms
designated (i.e. C.sub.1-8 means one to eight carbons). Examples of
alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl,
t-butyl, isobutyl, sec-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl,
and the like. The term "alkenyl" refers to an unsaturated alkyl
group having one or more double bonds. Similarly, the term
"alkynyl" refers to an unsaturated alkyl group having one or more
triple bonds. Examples of such unsaturated alkyl groups include
vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl),
2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl,
3-butynyl, and the higher homologs and isomers. The term
"cycloalkyl" refers to hydrocarbon rings having the indicated
number of ring atoms (e.g., C.sub.3-6cycloalkyl) and being fully
saturated or having no more than one double bond between ring
vertices. "Cycloalkyl" is also meant to refer to bicyclic and
polycyclic hydrocarbon rings such as, for example,
bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane, etc. The term
"heterocycloalkyl" refers to a cycloalkyl group that contain from
one to five heteroatoms selected from N, O, and S, wherein the
nitrogen and sulfur atoms are optionally oxidized, and the nitrogen
atom(s) are optionally quatemized. The heterocycloalkyl may be a
monocyclic, a bicyclic or a polycylic ring system. Non limiting
examples of heterocycloalkyl groups include pyrrolidine,
imidazolidine, pyrazolidine, butyrolactam, valerolactam,
imidazolidinone, hydantoin, dioxolane, phthalimide, piperidine,
1,4-dioxane, morpholine, thiomorpholine, thiomorpholine-S-oxide,
thiomorpholine-S,S-oxide, piperazine, pyran, pyridone, 3-pyrroline,
thiopyran, pyrone, tetrahydrofuran, tetrhydrothiophene,
quinuclidine, and the like. A heterocycloalkyl group can be
attached to the remainder of the molecule through a ring carbon or
a heteroatom. For terms such as cycloalkylalkyl and
heterocycloalkylalkyl, it is meant that a cycloalkyl or a
heterocycloalkyl group is attached through an alkyl or alkylene
linker to the remainder of the molecule. For example,
cyclobutylmethyl--is a cyclobutyl ring that is attached to a
methylene linker to the remainder of the molecule.
[0033] The term "alkylene" by itself or as part of another
substituent means a divalent radical derived from an alkane, as
exemplified by --CH.sub.2CH.sub.2CH.sub.2CH.sub.2--. Typically, an
alkyl (or alkylene) group will have from 1 to 24 carbon atoms, with
those groups having 10 or fewer carbon atoms being preferred in the
present invention. A "lower alkyl" or "lower alkylene" is a shorter
chain alkyl or alkylene group, generally having four or fewer
carbon atoms. Similarly, "alkenylene" and "alkynylene" refer to the
unsaturated forms of "alkylene" having double or triple bonds,
respectively.
[0034] The term "heteroalkyl," by itself or in combination with
another term, means, unless otherwise stated, a stable straight or
branched chain, or cyclic hydrocarbon radical, or combinations
thereof, consisting of the stated number of carbon atoms and from
one to three heteroatoms selected from the group consisting of O,
N, Si and S, and wherein the nitrogen and sulfur atoms may
optionally be oxidized and the nitrogen heteroatom may optionally
be quatemized. The heteroatom(s) O, N and S may be placed at any
interior position of the heteroalkyl group. The heteroatom Si may
be placed at any position of the heteroalkyl group, including the
position at which the alkyl group is attached to the remainder of
the molecule. Examples include --CH.sub.2--CH.sub.2--O--CH.sub.3,
--CH.sub.2--CH.sub.2--NH--CH.sub.3,
--CH.sub.2--CH.sub.2--N(CH.sub.3)--CH.sub.3,
--CH.sub.2--S--CH.sub.2--CH.sub.3, --CH.sub.2--CH.sub.2,
--S(O)--CH.sub.3, --CH.sub.2--CH.sub.2--S(O).sub.2--CH.sub.3,
--CH.dbd.CH--O--CH.sub.3, --Si(CH.sub.3).sub.3,
--CH.sub.2--CH.dbd.N--OCH.sub.3, and
--CH.dbd.CH--N(CH.sub.3)--CH.sub.3. Up to two heteroatoms may be
consecutive, such as, for example, --CH.sub.2--NH--OCH.sub.3 and
--CH.sub.2--O--Si(CH.sub.3).sub.3. Similarly, the terms
"heteroalkenyl" and "heteroalkynyl" by itself or in combination
with another term, means, unless otherwise stated, an alkenyl group
or alkynyl group, respectively, that contains the stated number of
carbons and having from one to three heteroatoms selected from the
group consisting of O, N, Si and S, and wherein the nitrogen and
sulfur atoms may optionally be oxidized and the nitrogen heteroatom
may optionally be quatemized. The heteroatom(s) O, N and S may be
placed at any interior position of the heteroalkyl group.
[0035] The term "heteroalkylene" by itself or as part of another
substituent means a divalent radical, saturated or unsaturated or
polyunsaturated, derived from heteroalkyl, as exemplified by
--CH.sub.2--CH.sub.2--S--CH.sub.2CH.sub.2-- and
--CH.sub.2--S--CH.sub.2--CH.sub.2--NH--CH.sub.2--,
--O--CH.sub.2--CH.dbd.CH--,
--CH.sub.2--CH.dbd.C(H)CH.sub.2--O--CH.sub.2-- and
--S--CH.sub.2--C.ident.C--. For heteroalkylene groups, heteroatoms
can also occupy either or both of the chain termini (e.g.,
alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, and the
like).
[0036] The terms "alkoxy," "alkylamino" and "alkylthio" (or
thioalkoxy) are used in their conventional sense, and refer to
those alkyl groups attached to the remainder of the molecule via an
oxygen atom, an amino group, or a sulfur atom, respectively.
Additionally, for dialkylamino groups, the alkyl portions can be
the same or different and can also be combined to form a 3-7
membered ring with the nitrogen atom to which each is attached.
Accordingly, a group represented as --NR.sup.aR.sup.b is meant to
include piperidinyl, pyrrolidinyl, morpholinyl, azetidinyl and the
like.
[0037] The terms "halo" or "halogen," by themselves or as part of
another substituent, mean, unless otherwise stated, a fluorine,
chlorine, bromine, or iodine atom. Additionally, terms such as
"haloalkyl," are meant to include monohaloalkyl and polyhaloalkyl.
For example, the term "C.sub.1-4 haloalkyl" is mean to include
trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl,
3-bromopropyl, and the like.
[0038] The term "aryl" means, unless otherwise stated, a
polyunsaturated, typically aromatic, hydrocarbon group which can be
a single ring or multiple rings (up to three rings) which are fused
together or linked covalently. The term "heteroaryl" refers to aryl
groups (or rings) that contain from one to five heteroatoms
selected from N, O, and S, wherein the nitrogen and sulfur atoms
are optionally oxidized, and the nitrogen atom(s) are optionally
quatemized. A heteroaryl group can be attached to the remainder of
the molecule through a heteroatom. Non-limiting examples of aryl
groups include phenyl, naphthyl and biphenyl, while non-limiting
examples of heteroaryl groups include pyridyl, pyridazinyl,
pyrazinyl, pyrimindinyl, triazinyl, quinolinyl, quinoxalinyl,
quinazolinyl, cinnolinyl, phthalaziniyl, benzotriazinyl, purinyl,
benzimidazolyl, benzopyrazolyl, benzotriazolyl, benzisoxazolyl,
isobenzofuryl, isoindolyl, indolizinyl, benzotriazinyl,
thienopyridinyl, thienopyrimidinyl, pyrazolopyrimidinyl,
imidazopyridines, benzothiaxolyl, benzofuranyl, benzothienyl,
indolyl, quinolyl, isoquinolyl, isothiazolyl, pyrazolyl, indazolyl,
pteridinyl, imidazolyl, triazolyl, tetrazolyl, oxazolyl,
isoxazolyl, thiadiazolyl, pyrrolyl, thiazolyl, furyl, thienyl and
the like. Substituents for each of the above noted aryl and
heteroaryl ring systems are selected from the group of acceptable
substituents described below.
[0039] For brevity, the term "aryl" when used in combination with
other terms (e.g., aryloxy, arylthioxy, arylalkyl) includes both
aryl and heteroaryl rings as defined above. Thus, the term
"arylalkyl" is meant to include those radicals in which an aryl
group is attached to an alkyl group that is attached to the
remainder of the molecule (e.g., benzyl, phenethyl, pyridylmethyl
and the like).
[0040] The above terms (e.g., "alkyl," "aryl" and "heteroaryl"), in
some embodiments, will include both substituted and unsubstituted
forms of the indicated radical. Preferred substituents for each
type of radical are provided below. For brevity, the terms aryl and
heteroaryl will refer to substituted or unsubstituted versions as
provided below, while the term "alkyl" and related aliphatic
radicals is meant to refer to unsubstituted version, unless
indicated to be substituted.
[0041] Substituents for the alkyl radicals (including those groups
often referred to as alkylene, alkenyl, alkynyl and cycloalkyl) can
be a variety of groups selected from: -halogen, --OR', --NR'R'',
--SR', --SiR'R''R''', --OC(O)R', --C(O)R', --CO.sub.2R',
--CONR'R'', --OC(O)NR'R'', --NR''C(O)R', --NR'--C(O)NR''R''',
--NR''C(O).sub.2R', --NH--C(NH.sub.2).dbd.NH,
--NR'C(NH.sub.2).dbd.NH, --NH--C(NH.sub.2).dbd.NR', --S(O)R',
--S(O).sub.2R', --S(O).sub.2NR'R'', --NR'S(O).sub.2R'', --CN and
--NO.sub.2 in a number ranging from zero to (2 m'+1), where m' is
the total number of carbon atoms in such radical. R', R'' and R'''
each independently refer to hydrogen, unsubstituted C.sub.1-8
alkyl, unsubstituted heteroalkyl, unsubstituted aryl, aryl
substituted with 1-3 halogens, unsubstituted C.sub.1-s alkyl,
C.sub.1-8alkoxy or C.sub.1-8 thioalkoxy groups, or unsubstituted
aryl-C.sub.1-4 alkyl groups. When R' and R'' are attached to the
same nitrogen atom, they can be combined with the nitrogen atom to
form a 3-, 4-, 5-, 6-, or 7-membered ring. For example, --NR'R'' is
meant to include 1-pyrrolidinyl and 4-morpholinyl. The term "acyl"
as used by itself or as part of another group refers to an alkyl
radical wherein two substitutents on the carbon that is closest to
the point of attachment for the radical is replaced with the
substitutent .dbd.O (e.g., --C(O)CH.sub.3,
--C(O)CH.sub.2CH.sub.2OR' and the like).
[0042] Similarly, substituents for the aryl and heteroaryl groups
are varied and are generally selected from: -halogen, --OR',
--OC(O)R', --NR'R'', --SR', --R', --CN, --NO.sub.2, --CO.sub.2R',
--CONR'R'', --C(O)R', --OC(O)NR'R'', --NR''C(O)R',
--NR''C(O).sub.2R', --NR'--C(O)NR''R''', --NH--C(NH.sub.2).dbd.NH,
--NR'C(NH.sub.2).dbd.NH, --NH--C(NH.sub.2).dbd.NR', --S(O)R',
--S(O).sub.2R', --S(O).sub.2NR'R'', --NR'S(O).sub.2R'', --N.sub.3,
perfluoro(C.sub.1-C.sub.4)alkoxy, and
perfluoro(C.sub.1-C.sub.4)alkyl, in a number ranging from zero to
the total number of open valences on the aromatic ring system; and
where R', R'' and R''' are independently selected from hydrogen,
C.sub.1-8 alkyl, C.sub.3-6 cycloalkyl, C.sub.2-8 alkenyl, C.sub.2-8
alkynyl, unsubstituted aryl and heteroaryl, (unsubstituted
aryl)-C.sub.1-4 alkyl, and unsubstituted aryloxy-C.sub.1-4 alkyl.
Other suitable substituents include each of the above aryl
substituents attached to a ring atom by an alkylene tether of from
1-4 carbon atoms.
[0043] Two of the substituents on adjacent atoms of the aryl or
heteroaryl ring may optionally be replaced with a substituent of
the formula -T-C(O)--(CH.sub.2).sub.q--U--, wherein T and U are
independently --NH--, --O--, --CH.sub.2-- or a single bond, and q
is an integer of from 0 to 2. Alternatively, two of the
substituents on adjacent atoms of the aryl or heteroaryl ring may
optionally be replaced with a substituent of the formula
-A-(CH.sub.2).sub.r--B--, wherein A and B are independently
--CH.sub.2--, --O--, --NH--, --S--, --S(O)--, --S(O).sub.2--,
--S(O).sub.2NR'-- or a single bond, and r is an integer of from 1
to 3. One of the single bonds of the new ring so formed may
optionally be replaced with a double bond. Alternatively, two of
the substituents on adjacent atoms of the aryl or heteroaryl ring
may optionally be replaced with a substituent of the formula
--(CH.sub.2).sub.s--X--(CH.sub.2).sub.t--, where s and t are
independently integers of from 0 to 3, and X is --O--, --NR'--,
--S--, --S(O)--, --S(O).sub.2--, or --S(O).sub.2NR'--. The
substituent R' in --NR'-- and --S(O).sub.2NR'-- is selected from
hydrogen or unsubstituted C.sub.1-6 alkyl.
[0044] As used herein, the term "heteroatom" is meant to include
oxygen (O), nitrogen (N), sulfur (S) and silicon (Si).
[0045] For the compounds provided herein, a bond that is drawn from
a substituent (typically an R group) to the center of an aromatic
ring (e.g., benzene, pyridine, and the like) will be understood to
refer to a bond providing a connection at any of the available
vertices of the aromatic ring. In some embodiments, the depiction
will also include connection at a ring which is fused to the
aromatic ring. For example, a bond drawn to the center of the
benzene portion of an indole, will indicate a bond to any available
vertex of the six- or five-membered ring portions of the
indole.
[0046] The term "pharmaceutically acceptable salts" is meant to
include salts of the active compounds which are prepared with
relatively nontoxic acids or bases, depending on the particular
substituents found on the compounds described herein. When
compounds of the present invention contain relatively acidic
functionalities, base addition salts can be obtained by contacting
the neutral form of such compounds with a sufficient amount of the
desired base, either neat or in a suitable inert solvent. Examples
of salts derived from pharmaceutically-acceptable inorganic bases
include aluminum, ammonium, calcium, copper, ferric, ferrous,
lithium, magnesium, manganic, manganous, potassium, sodium, zinc
and the like. Salts derived from pharmaceutically-acceptable
organic bases include salts of primary, secondary and tertiary
amines, including substituted amines, cyclic amines,
naturally-occuring amines and the like, such as arginine, betaine,
caffeine, choline, N,N'-dibenzylethylenediamine, diethylamine,
2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine,
ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine,
glucosamine, histidine, hydrabamine, isopropylamine, lysine,
methylglucamine, morpholine, piperazine, piperadine, polyamine
resins, procaine, purines, theobromine, triethylamine,
trimethylamine, tripropylamine, tromethamine and the like. When
compounds of the present invention contain relatively basic
functionalities, acid addition salts can be obtained by contacting
the neutral form of such compounds with a sufficient amount of the
desired acid, either neat or in a suitable inert solvent. Examples
of pharmaceutically acceptable acid addition salts include those
derived from inorganic acids like hydrochloric, hydrobromic,
nitric, carbonic, monohydrogencarbonic, phosphoric,
monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,
monohydrogensulfuric, hydriodic, or phosphorous acids and the like,
as well as the salts derived from relatively nontoxic organic acids
like acetic, propionic, isobutyric, malonic, benzoic, succinic,
suberic, fumaric, mandelic, phthalic, benzenesulfonic,
p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like.
Also included are salts of amino acids such as arginate and the
like, and salts of organic acids like glucuronic or galactunoric
acids and the like (see, for example, Berge, S. M., et al,
"Pharmaceutical Salts", Journal of Pharmaceutical Science, 1977,
66, 1-19). Certain specific compounds of the present invention
contain both basic and acidic functionalities that allow the
compounds to be converted into either base or acid addition
salts.
[0047] The neutral forms of the compounds may be regenerated by
contacting the salt with a base or acid and isolating the parent
compound in the conventional manner. The parent form of the
compound differs from the various salt forms in certain physical
properties, such as solubility in polar solvents, but otherwise the
salts are equivalent to the parent form of the compound for the
purposes of the present invention.
[0048] In addition to salt forms, the present invention provides
compounds which are in a prodrug form. Prodrugs of the compounds
described herein are those compounds that readily undergo chemical
changes under physiological conditions to provide the compounds of
the present invention. Additionally, prodrugs can be converted to
the compounds of the present invention by chemical or biochemical
methods in an ex vivo environment. For example, prodrugs can be
slowly converted to the compounds of the present invention when
placed in a transdermal patch reservoir with a suitable enzyme or
chemical reagent.
[0049] Certain compounds of the present invention can exist in
unsolvated forms as well as solvated forms, including hydrated
forms. In general, the solvated forms are equivalent to unsolvated
forms and are intended to be encompassed within the scope of the
present invention. Certain compounds of the present invention may
exist in multiple crystalline or amorphous forms. In general, all
physical forms are equivalent for the uses contemplated by the
present invention and are intended to be within the scope of the
present invention.
[0050] Certain compounds of the present invention possess
asymmetric carbon atoms (optical centers) or double bonds; the
racemates, diastereomers, geometric isomers, regioisomers and
individual isomers (e.g., separate enantiomers) are all intended to
be encompassed within the scope of the present invention. When
compounds are provided herein with an identified stereochemistry
(indicated as R or S, or with dashed or wedge bond designations),
those compounds will be understood by one of skill in the art to be
substantially free of other isomers (e.g., at least 80%, 90%, 95%,
98%, 99%, and up to 100% free of the other isomer).
[0051] The compounds of the present invention may also contain
unnatural proportions of atomic isotopes at one or more of the
atoms that constitute such compounds. Unnatural proportions of an
isotope may be defined as ranging from the amount found in nature
to an amount consisting of 100% of the atom in question. For
example, the compounds may incorporate radioactive isotopes, such
as for example tritium (.sup.3H), iodine-125 (.sup.125I) or
carbon-14 (.sup.14C), or non-radioactive isotopes, such as
deuterium (.sup.2H) or carbon-13 (.sup.13C). Such isotopic
variations can provide additional utilities to those described
elsewhere within this application. For instance, isotopic variants
of the compounds of the invention may find additional utility,
including but not limited to, as diagnostic and/or imaging
reagents, or as cytotoxic/radiotoxic therapeutic agents.
Additionally, isotopic variants of the compounds of the invention
can have altered pharmacokinetic and pharmacodynamic
characteristics which can contribute to enhanced safety,
tolerability or efficacy during treatment. All isotopic variations
of the compounds of the present invention, whether radioactive or
not, are intended to be encompassed within the scope of the present
invention.
II. General
[0052] The present invention is directed to compounds and salts
thereof, compositions and methods useful in the modulation of
chemokine receptor function, particularly CCR2 function. Modulation
of chemokine receptor activity, as used herein in its various
forms, is intended to encompass antagonism, agonism, partial
antagonism, inverse agonism and/or partial agonism of the activity
associated with a particular chemokine receptor, preferably the
CCR2 receptor. Accordingly, the compounds of the present invention
are compounds which modulate at least one function or
characteristic of mammalian CCR2, for example, a human CCR2
protein. The ability of a compound to modulate the function of
CCR2, can be demonstrated in a binding assay (e.g., ligand binding
or agonist binding), a migration assay, a signaling assay (e.g.,
activation of a mammalian G protein, induction of rapid and
transient increase in the concentration of cytosolic free calcium),
and/or cellular response assay (e.g., stimulation of chemotaxis,
exocytosis or inflammatory mediator release by leukocytes).
III. Compounds
[0053] In one aspect, the present invention provides compounds
having the formula I:
##STR00003## [0054] or a pharmaceutically acceptable salt, hydrate,
stereoisomer or rotamer thereof; wherein [0055] A is
C(R.sup.5)(R.sup.6) or N(R.sup.5) [0056] the subscripts m and n are
each independently integers of from 0 to 2, and m+n is .ltoreq.3;
[0057] R.sup.1 is selected from the group consisting of aryl,
aryl-C.sub.1-4 alkyl, heteroaryl and heteroaryl-C.sub.1-4 alkyl,
wherein the heteroaryl portion has from 1-3 heteroatoms as ring
members selected from N, O and S; and wherein said aryl and
heteroaryl groups or portions are optionally substituted with from
1 to 5 R.sup.x substituents; [0058] R.sup.2 is selected from the
group consisting of H, C.sub.1-8 alkyl, C.sub.3-8 cycloalkyl,
C.sub.3-8 cycloalkyl-C.sub.1-4 alkyl, aryl, aryl-C.sub.1-4 alkyl,
heteroaryl and heteroaryl-C.sub.1-4 alkyl, wherein the heteroaryl
portion has from 1-3 heteroatoms as ring members selected from N, O
and S; and wherein said aryl and heteroaryl groups or portions are
optionally substituted with from 1 to 4 R.sup.x substituents;
[0059] or optionally, R.sup.1 and R.sup.2 are combined with the
nitrogen atom to which each is attached to form a 6- to 11-membered
monocyclic or fused bicyclic-heterocyclic or heteroaryl ring,
wherein the --NR.sup.1R.sup.2 is optionally further substituted
with from 1 to 4 R.sup.x substituents; [0060] R.sup.3 is selected
from the group consisting of H, C.sub.1-8 alkyl, C.sub.3-8
cycloalkyl and C.sub.3-8 cycloalkyl-C.sub.1-4 alkyl, each of which
is optionally substituted with from 1-3 R.sup.y substituents;
[0061] R.sup.4 is selected from the group consisting of H,
C.sub.1-8alkyl optionally substituted with 1 to 2 R.sup.y, and
--CO.sub.2H: [0062] R.sup.5 is selected from the group consisting
of C.sub.1-8 alkyl, C.sub.1-8 alkoxy, C.sub.3-8 cycloalkyl,
C.sub.3-8 cycloalkyloxy, C.sub.3-8 cycloalkyl-C.sub.1-4 alkyl,
C.sub.1-8 alkylamino, di-C.sub.1-8 alkylamino, aryl, aryloxy,
arylamino, aryl-C.sub.1-4 alkyl, heteroaryl, heteroaryloxy,
heteroarylamino and heteroaryl-C.sub.1-4 alkyl, each of which is
optionally substituted with from 1 to 5 R.sup.z substituents;
[0063] R.sup.6 is selected from the group consisting of H, F, OH,
C.sub.1-8 alkyl and C.sub.1-8alkoxy, wherein the C.sub.1-8 alkyl
and C.sub.1-8 alkoxy groups are optionally substituted with from 1
to 3 R.sup.z substituents; [0064] or optionally, R.sup.5 and
R.sup.6 are joined to form a spirocyclic 5- or 6-membered
cycloalkyl ring which is optionally unsaturated, and has a fused
aryl group which is optionally substituted with from 1 to 4 R.sup.z
substituents; [0065] each R.sup.x is independently selected from
the group consisting of [0066] halogen, --CN, --R.sup.c,
--CO.sub.2R.sup.a, --CONR.sup.aR.sup.b, --C(O)R.sup.a,
--OC(O)NR.sup.aR.sup.b, --NR.sup.bC(O)R.sup.a,
--NR.sup.bC(O).sub.2R.sup.c, --NR.sup.a--C(O)NR.sup.aR.sup.b,
--NR.sup.aC(O)NR.sup.aR.sup.b, --NR.sup.aR.sup.b, --OR.sup.a,
--O--X.sup.1--OR.sup.a, --O--X.sup.1--NR.sup.aR.sup.b,
--O--X.sup.1--CO.sub.2R.sup.a, --O--X.sup.1--CONR.sup.aR.sup.b,
--X.sup.1--OR.sup.a, --X.sup.1--NR.sup.aR.sup.b,
--X.sup.1--CO.sub.2R.sup.a, --X.sup.1--CONR.sup.aR.sup.b,
--SF.sub.5, --S(O).sub.2NR.sup.aR.sup.b, and 5- or 6-membered aryl
or heteroaryl, wherein each X.sup.1 is a C.sub.1-4 alkylene; each
R.sup.a and R.sup.b is independently selected from hydrogen,
C.sub.1-8 alkyl, and C.sub.1-8 haloalkyl, or when attached to the
same nitrogen atom can be combined with the nitrogen atom to form a
five or six-membered ring having from 0 to 2 additional heteroatoms
as ring members selected from N, O or S, and optionally substituted
with oxo; each R.sup.c is independently selected from the group
consisting of C.sub.1-8alkyl, C.sub.1-8 haloalkyl and C.sub.3-6
cycloalkyl; and optionally when two R.sup.x substituents are on
adjacent atoms, are combined to form a fused five or six-membered
carbocyclic ring, and wherein the aryl or heteroaryl groups are
optionally substituted with 1-3 members selected from halogen,
hydroxyl, C.sub.1-4 alkyl, C.sub.1-4 alkoxy, C.sub.1-4 haloalkyl,
and C.sub.1-4 haloalkoxy; [0067] each R.sup.y is independently
selected from the group consisting of halogen, --CN, --R.sup.f,
--CO.sub.2R.sup.d, --CONR.sup.dR.sup.e, --C(O)R.sup.d,
--OC(O)NR.sup.dR.sup.e, --NR.sup.eC(O)R.sup.d,
NR.sup.eC(O).sub.2R.sup.f, --NR.sup.dC(O)NR.sup.dR.sup.e,
--NR.sup.dC(O)NR.sup.dR.sup.e, --NR.sup.dR.sup.e, --OR.sup.d, and
--S(O).sub.2NR.sup.dR.sup.e; wherein each R.sup.d and R.sup.e is
independently selected from hydrogen, C.sub.1-8 alkyl, and
C.sub.1-8 haloalkyl, or when attached to the same nitrogen atom can
be combined with the nitrogen atom to form a five or six-membered
ring having from 0 to 2 additional heteroatoms as ring members
selected from N, O or S; each R.sup.f is independently selected
from the group consisting of C.sub.1-8 alkyl, C.sub.1-8 haloalkyl
and C.sub.3-6 cycloalkyl; [0068] each R.sup.z is independently
selected from the group consisting of halogen, --CN, --R.sup.i,
--CO.sub.2R.sup.g, --CONR.sup.gR.sup.h, --C(O)R.sup.g,
--OC(O)NR.sup.gR.sup.h, --NR.sup.hC(O)R.sup.g,
--NR.sup.hC(O).sub.2R.sup.i, --NR.sup.gC(O)NR.sup.gR.sup.h,
--NR.sup.gR.sup.h, --OR.sup.g, --S(O).sub.2NR.sup.gR.sup.h,
--X.sup.1--R.sup.h, --X.sup.1--NR.sup.gR.sup.h,
--X.sup.1--CONR.sup.gR.sup.h, --X.sup.1--NR.sup.hC(O)R.sup.g,
--NHR.sup.j, --NHCH.sub.2R.sup.j, and tetrazole; wherein each
R.sup.g and R.sup.h is independently selected from hydrogen,
C.sub.1-8 alkyl, C.sub.3-6 cycloalkyl and C.sub.1-8 haloalkyl, or
when attached to the same nitrogen atom can be combined with the
nitrogen atom to form a five or six-membered ring having from 0 to
2 additional heteroatoms as ring members selected from N, O or S
and is optionally substituted with one or two oxo; each R.sup.i is
independently selected from the group consisting of C.sub.1-8
alkyl, C.sub.1-8 haloalkyl and C.sub.3-6 cycloalkyl; and each RR is
selected from the group consisting of C.sub.3-6 cycloalkyl,
pyrrolinyl, piperidinyl, morpholinyl, tetrahydrofuranyl, and
tetrahydropyranyl.
[0069] It shall be understood that when R.sup.1 and R.sup.2 are
combined with the nitrogen atom to which each is attached to form a
6- to 11-membered monocyclic or fused bicyclic-heterocyclic ring,
the 6- to 11-membered monocyclic or fused bicyclic-heterocyclic
ring encompasses monocyclic heterocyclic rings fused with an aryl
or a heteroaryl ring.
[0070] In formula I, the substituent R.sup.3 is, in one embodiment,
selected from the group consisting of H, methyl, ethyl, propyl,
isopropyl, buty, isobutyl, sec-butyl, cyclopropyl,
cyclopropylmethyl, cyclobutyl and cyclobutylmethyl.
[0071] In the descriptions herein, one of skill in the art will
understand that the wavy line intersecting a bond is meant to
identify the point of attachment of a given substituent or group to
the remainder of the molecule.
[0072] As noted above, the subscripts m and n are each integers
selected from 0, 1 and 2, and m+n is .ltoreq.3. When the subscript
is 0, one of skill in the are will understand that a cyclic
structure with ring vertex A is intended, but that adjacent ring
vertices on either side of the parentheses are joined by a bond.
Accordingly, the present invention includes the structures wherein
the ring having A as a vertex is meant to include:
##STR00004##
[0073] In one selected group of embodiments, m and n are both 0. In
another selected group of embodiments, m and n are both 1. In yet
another group of selected embodiments, m is 1 and n is 0. In still
another group of embodiments, m is 1 and n is 2.
[0074] In still other selected embodiments, the ring having vertex
A is represented by a formula selected from:
##STR00005##
[0075] In one subgroup of embodiments, the compounds of formula (I)
are represented by:
##STR00006##
[0076] Within formula (Ia), a number of selected embodiments are
provided as formulae Ia1, Ia2, Ia3, Ia4 and Ia5.
##STR00007##
[0077] In each of formulae Ia, Ia1, Ia2, Ia3, Ia4 and Ia5, the
noted substituents (R.sup.1 through R.sup.6, R.sup.x and R.sup.z)
and subscripts m and n have the meanings provided above with
respect to formula I. The subscripts, p and q, have the following
meanings: for Ia1, Ia4 and Ia5, the subscript q is an integer of
from 0 to 5; for Ia2 and Ia4, the subscript p is an integer of from
0 to 4; and for Ia3 and Ia5, the subscript p is an integer of from
0 to 5.
[0078] In still other selected embodiments, the compounds provided
herein are represented by formulae selected from:
##STR00008##
wherein each compound is substantially free of other stereoisomers,
and wherein the noted substituents (R.sup.1 through R.sup.6,
R.sup.x and R.sup.z) and subscripts m and n have the meanings
provided above with respect to formula I. The subscripts, p and q,
have the following meanings: for Ia1', Ia4' and Ia5', the subscript
q is an integer of from 0 to 5; for Ia2' and Ia4', the subscript p
is an integer of from 0 to 4; and for Ia3' and Ia5', the subscript
p is an integer of from 0 to 5.
[0079] In another group of embodiments of formula I, A is
C(R.sup.5)(R.sup.6), wherein R.sup.5 and R.sup.6 are combined to
form a ring. Selected embodiments are provided as follows:
##STR00009##
[0080] In each of formulae Ib, Ib1 and Ib2, the noted substituents
(R.sup.1 through R.sup.6, R.sup.x and R.sup.z) and subscripts m and
n have the meanings provided above with respect to formula I. The
subscripts, p and q, have the following meanings: for Ib, Ib1 and
Ib2, the subscript q is an integer of from 0 to 5; for Ib1, the
subscript p is an integer of from 0 to 4; and for Ib2, the
subscript p is an integer of from 0 to 5.
[0081] In another group of embodiments of formula I, A is NR.sup.5
(see formula Ic). Selected embodiments are provided as follows:
##STR00010## ##STR00011##
[0082] In each of formulae Ic, Ic1, Ic2, Ic3, Ic4 and Ic5, the
noted substituents (R.sup.1 through R.sup.6, R.sup.x and R.sup.z)
and subscripts m and n have the meanings provided above with
respect to formula I. The subscripts, p and q, have the following
meanings: for Ic1, Ic4 and Ic5, the subscript q is an integer of
from 0 to 5; for Ic2 and Ic4, the subscript p is an integer of from
0 to 4; and for Ic3 and Ic5, the subscript p is an integer of from
0 to 5.
[0083] In still other selected embodiments, the compounds provided
herein are represented by formulae selected from:
##STR00012##
wherein each compound is substantially free of other stereoisomers,
and wherein the noted substituents (R.sup.1 through R.sup.6,
R.sup.x and R.sup.z) and subscripts m and n have the meanings
provided above with respect to formula I. The subscripts, p and q,
have the following meanings: for Ic1', Ic4' and Ic5', the subscript
q is an integer of from 0 to 5; for Ic2' and Ic4', the subscript p
is an integer of from 0 to 4; and for Ic3' and Ic5', the subscript
p is an integer of from 0 to 5.
[0084] Other selected embodiments, compounds are provided in each
of I, Ia, Ia1, Ia1', Ib, Ic, Ic1 and Ic1', described above, wherein
--N(R.sup.1)(R.sup.2) is selected from:
##STR00013## ##STR00014## ##STR00015##
[0085] Still other selected embodiments, are provided in each of I,
Ia, Ia1, Ia1', Ib, Ic, Ic1 and Ic1', described above, wherein
--N(R.sup.1)(R.sup.2) is selected from:
##STR00016## ##STR00017##
[0086] Yet other selected embodiments, are provided in each of I,
Ia, Ia1, Ia1', Ib, Ic, Ic1 and Ic1', described above, wherein
--N(R.sup.1)(R.sup.2) is selected from:
##STR00018##
[0087] In some embodiments, compounds of formulae I, Ia, Ia2, Ia3,
Ia2' and Ia3', are provided wherein A is C(R.sup.5)(R.sup.6), or is
shown in the formula as C(R.sup.5)(R.sup.6), wherein R.sup.5 is
selected from aryl, aryloxy, arylamino, aryl-C.sub.1-4 alkyl,
heteroaryl, heteroaryloxy, heteroarylamino and heteroaryl-C.sub.1-4
alkyl, wherein the aryl or heteroaryl groups or portions are
selected from:
##STR00019## ##STR00020##
[0088] In certain selected embodiments, compounds of formulae I,
Ia, Ia2, Ia3, Ia2' and Ia3', are provided wherein A is
C(R.sup.5)(R.sup.6), or is shown in the formula as
C(R.sup.5)(R.sup.6), wherein R.sup.5 is selected from aryl,
aryloxy, arylamino and aryl-C.sub.1-4 alkyl, wherein the aryl group
or portion is selected from:
##STR00021##
[0089] In still other selected embodiments, compounds of formulae
I, la, Ia2, Ia3, Ia2' and Ia3', are provided wherein A is
C(R.sup.5)(R.sup.6), or is shown in the formula as
C(R.sup.5)(R.sup.6), wherein R.sup.5 is selected from heteroaryl,
heteroaryloxy, heteroarylamino and heteroaryl-C.sub.1-4 alkyl,
wherein the heteroaryl group or portion is selected from:
##STR00022##
[0090] In some embodiments, compounds of formulae I, Ic, Ic2, Ic3,
Ic2' and Ic3', are provided wherein A is N(R.sup.5), or is shown in
the formula as N(R.sup.5), wherein R.sup.5 is selected from aryl,
aryl-C.sub.1-4 alkyl, heteroaryl and heteroaryl-C.sub.1-4 alkyl,
wherein the aryl or heteroaryl groups or portions are selected from
Group 1 above. In certain selected embodiments, compounds of
formulae I, Ic, Ic2, Ic3, Ic2' and Ic3', are provided wherein A is
N(R.sup.5), or is shown in the formula as N(R.sup.5), wherein
R.sup.5 is selected from aryl and aryl-C.sub.1-4 alkyl, wherein the
aryl group or portion is selected from Subgroup 1a, above. In still
other selected embodiments, compounds of formulae I, Ic, Ic2, Ic3,
Ic2' and Ic3', are provided wherein A is N(R.sup.5), or is shown in
the formula as N(R.sup.5), wherein R.sup.5 is selected from
heteroaryl and heteroaryl-C.sub.1-4 alkyl, wherein the heteroaryl
group or portion is selected from Subgroup Ib, above.
Preparation of Compounds
[0091] Those skilled in the art will recognize that there are a
variety of methods available to synthesize molecules represented in
the claims. In general, useful methods for synthesizing compounds
represented in the claims are shown in Scheme 1:
##STR00023##
[0092] Variations on the methods shown above have been used to
prepare compounds of the invention, some of which are described in
the examples.
[0093] A family of specific compounds of particular interest having
formula I consists of compounds, pharmaceutically acceptable salts,
hydrates, stereoisomers and rotamers thereof, as set forth in FIG.
1.
IV. Pharmaceutical Compositions
[0094] In addition to the compounds provided above, compositions
for modulating CCR2 activity in humans and animals will typically
contain a pharmaceutical carrier or diluent.
[0095] The term "composition" as used herein is intended to
encompass a product comprising the specified ingredients in the
specified amounts, as well as any product which results, directly
or indirectly, from combination of the specified ingredients in the
specified amounts. By "pharmaceutically acceptable" it is meant the
carrier, diluent or excipient must be compatible with the other
ingredients of the formulation and not deleterious to the recipient
thereof.
[0096] The pharmaceutical compositions for the administration of
the compounds of this invention may conveniently be presented in
unit dosage form and may be prepared by any of the methods well
known in the art of pharmacy and drug delivery. All methods include
the step of bringing the active ingredient into association with
the carrier which constitutes one or more accessory ingredients. In
general, the pharmaceutical compositions are prepared by uniformly
and intimately bringing the active ingredient into association with
a liquid carrier or a finely divided solid carrier or both, and
then, if necessary, shaping the product into the desired
formulation. In the pharmaceutical composition the active object
compound is included in an amount sufficient to produce the desired
effect upon the process or condition of diseases.
[0097] The pharmaceutical compositions containing the active
ingredient may be in a form suitable for oral use, for example, as
tablets, troches, lozenges, aqueous or oily suspensions,
dispersible powders or granules, emulsions and self emulsifications
as described in U.S. Patent Application 2002-0012680, hard or soft
capsules, syrups, elixirs, solutions, buccal patch, oral gel,
chewing gum, chewable tablets, effervescent powder and effervescent
tablets. Compositions intended for oral use may be prepared
according to any method known to the art for the manufacture of
pharmaceutical compositions and such compositions may contain one
or more agents selected from the group consisting of sweetening
agents, flavoring agents, coloring agents, antioxidants and
preserving agents in order to provide pharmaceutically elegant and
palatable preparations. Tablets contain the active ingredient in
admixture with non-toxic pharmaceutically acceptable excipients
which are suitable for the manufacture of tablets. These excipients
may be for example, inert diluents, such as cellulose, silicon
dioxide, aluminum oxide, calcium carbonate, sodium carbonate,
glucose, mannitol, sorbitol, lactose, calcium phosphate or sodium
phosphate; granulating and disintegrating agents, for example, corn
starch, or alginic acid; binding agents, for example PVP,
cellulose, PEG, starch, gelatin or acacia, and lubricating agents,
for example magnesium stearate, stearic acid or talc. The tablets
may be uncoated or they may be coated, enterically or otherwise, by
known techniques to delay disintegration and absorption in the
gastrointestinal tract and thereby provide a sustained action over
a longer period. For example, a time delay material such as
glyceryl monostearate or glyceryl distearate may be employed. They
may also be coated by the techniques described in the U.S. Pat.
Nos. 4,256,108; 4,166,452; and 4,265,874 to form osmotic
therapeutic tablets for controlled release.
[0098] Formulations for oral use may also be presented as hard
gelatin capsules wherein the active ingredient is mixed with an
inert solid diluent, for example, calcium carbonate, calcium
phosphate or kaolin, or as soft gelatin capsules wherein the active
ingredient is mixed with water or an oil medium, for example peanut
oil, liquid paraffin, or olive oil. Additionally, emulsions can be
prepared with a non-water miscible ingredient such as oils and
stabilized with surfactants such as mono-diglycerides, PEG esters
and the like.
[0099] Aqueous suspensions contain the active materials in
admixture with excipients suitable for the manufacture of aqueous
suspensions. Such excipients are suspending agents, for example
sodium carboxymethylcellulose, methylcellulose,
hydroxy-propylmethylcellulose, sodium alginate,
polyvinyl-pyrrolidone, gum tragacanth and gum acacia; dispersing or
wetting agents may be a naturally-occurring phosphatide, for
example lecithin, or condensation products of an alkylene oxide
with fatty acids, for example polyoxy-ethylene stearate, or
condensation products of ethylene oxide with long chain aliphatic
alcohols, for example heptadecaethyleneoxycetanol, or condensation
products of ethylene oxide with partial esters derived from fatty
acids and a hexitol such as polyoxyethylene sorbitol monooleate, or
condensation products of ethylene oxide with partial esters derived
from fatty acids and hexitol anhydrides, for example polyethylene
sorbitan monooleate. The aqueous suspensions may also contain one
or more preservatives, for example ethyl, or n-propyl,
p-hydroxybenzoate, one or more coloring agents, one or more
flavoring agents, and one or more sweetening agents, such as
sucrose or saccharin.
[0100] Oily suspensions may be formulated by suspending the active
ingredient in a vegetable oil, for example arachis oil, olive oil,
sesame oil or coconut oil, or in a mineral oil such as liquid
paraffin. The oily suspensions may contain a thickening agent, for
example beeswax, hard paraffin or cetyl alcohol. Sweetening agents
such as those set forth above, and flavoring agents may be added to
provide a palatable oral preparation. These compositions may be
preserved by the addition of an anti-oxidant such as ascorbic
acid.
[0101] Dispersible powders and granules suitable for preparation of
an aqueous suspension by the addition of water provide the active
ingredient in admixture with a dispersing or wetting agent,
suspending agent and one or more preservatives. Suitable dispersing
or wetting agents and suspending agents are exemplified by those
already mentioned above. Additional excipients, for example
sweetening, flavoring and coloring agents, may also be present.
[0102] The pharmaceutical compositions of the invention may also be
in the form of oil-in-water emulsions. The oily phase may be a
vegetable oil, for example olive oil or arachis oil, or a mineral
oil, for example liquid paraffin or mixtures of these. Suitable
emulsifying agents may be naturally-occurring gums, for example gum
acacia or gum tragacanth, naturally-occurring phosphatides, for
example soy bean, lecithin, and esters or partial esters derived
from fatty acids and hexitol anhydrides, for example sorbitan
monooleate, and condensation products of the said partial esters
with ethylene oxide, for example polyoxyethylene sorbitan
monooleate. The emulsions may also contain sweetening and flavoring
agents.
[0103] Syrups and elixirs may be formulated with sweetening agents,
for example glycerol, propylene glycol, sorbitol or sucrose. Such
formulations may also contain a demulcent, a preservative,
flavoring and/or coloring agents. Oral solutions can be prepared in
combination with, for example, cyclodextrin, PEG and
surfactants.
[0104] The pharmaceutical compositions may be in the form of a
sterile injectable aqueous or oleagenous suspension. This
suspension may be formulated according to the known art using those
suitable dispersing or wetting agents and suspending agents which
have been mentioned above. The sterile injectable preparation may
also be a sterile injectable solution or suspension in a non-toxic
parenterally-acceptable diluent or solvent, for example as a
solution in 1,3-butane diol. Among the acceptable vehicles and
solvents that may be employed are water, Ringer's solution and
isotonic sodium chloride solution. In addition, sterile, fixed oils
are conventionally employed as a solvent or suspending medium. For
this purpose any bland fixed oil may be employed including
synthetic mono- or diglycerides. In addition, fatty acids such as
oleic acid find use in the preparation of injectables.
[0105] The compounds of the present invention may also be
administered in the form of suppositories for rectal administration
of the drug. These compositions can be prepared by mixing the drug
with a suitable non-irritating excipient which is solid at ordinary
temperatures but liquid at the rectal temperature and will
therefore melt in the rectum to release the drug. Such materials
include cocoa butter and polyethylene glycols.
[0106] Additionally, the compounds can be administered via ocular
delivery by means of solutions or ointments. Still further,
transdermal delivery of the subject compounds can be accomplished
by means of iontophoretic patches and the like. For topical use,
creams, ointments, jellies, solutions or suspensions, etc.,
containing the compounds of the present invention are employed. As
used herein, topical application is also meant to include the use
of mouth washes and gargles.
[0107] The compounds of this invention may also be coupled to a
carrier that can be a suitable polymeric carrier as, for example, a
targetable drug carrier. Such polymers can include
polyvinylpyrrolidone, pyran copolymer,
polyhydroxy-propyl-methacrylamide-phenol,
polyhydroxyethyl-aspartamide-phenol, or
polyethyleneoxide-polylysine substituted with palmitoyl residues.
Furthermore, the compounds of the invention may be coupled to a
carrier that is a class of biodegradable polymers useful in
achieving controlled release of a drug, for example polylactic
acid, polyglycolic acid, copolymers of polylactic and polyglycolic
acid, polyepsilon caprolactone, polyhydroxy butyric acid,
polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates
and cross linked or amphipathic block copolymers of hydrogels.
Polymers and semipermeable polymer matrices may be formed into
shaped articles, such as valves, stents, tubing, prostheses and the
like. In one embodiment of the invention, the compound of the
invention is coupled to a polymer or semipermeable polymer matrix
that is formed as a stent or stent-graft device.
[0108] In some embodiments, a pharmaceutical composition comprising
the compounds of the invention and further comprising one or more
additional therapeutic compound is provided. In some embodiments,
the one or more additional therapeutic compound is selected from
one or more of a Btk tyrosine kinase inhibitor, an Erbb2 tyrosine
kinase receptor inhibitor; an Erbb4 tyrosine kinase receptor
inhibitor, an mTOR inhibitor, a Thymidylate synthase inhibitor, an
EGFR tyrosine kinase receptor inhibitor, an Epidermal growth factor
antagonist, a Fyn tyrosine kinase inhibitor, a Kit tyrosine kinase
inhibitor, a Lyn tyrosine kinase inhibitor, a NK cell receptor
modulator, a PDGF receptor antagonist, a PARP inhibitor, a Poly ADP
ribose polymerase inhibitor, a Poly ADP ribose polymerase 1
inhibitor, a Poly ADP ribose polymerase 2 inhibitor, a Poly ADP
ribose polymerase 3 inhibitor, a Galactosyltransferase modulator, a
Dihydropyrimidine dehydrogenase inhibitor, an Orotate
phosphoribosyltransferase inhibitor, a Telomerase modulator, a
Mucin 1 inhibitor, a Mucin inhibitor, a Secretin agonist, a TNF
related apoptosis inducing ligand modulator, an IL17 gene
stimulator, an Interleukin 17E ligand, a Neurokinin receptor
agonist, a Cyclin G1 inhibitor, a checkpoint inhibitor, a PD-1
inhibitor, a PD-L1 inhibitor, a CTLA4 inhibitor, a Topoisomerase I
inhibitor, an Alk-5 protein kinase inhibitor, a Connective tissue
growth factor ligand inhibitor, a Notch-2 receptor antagonist, a
Notch-3 receptor antagonist, a Hyaluronidase stimulator, a MEK-1
protein kinase inhibitor; MEK-2 protein kinase inhibitor, a GM-CSF
receptor modulator; TNF alpha ligand modulator, a Mesothelin
modulator, an Asparaginase stimulator, a Caspase-3 stimulator;
Caspase-9 stimulator, a PKN3 gene inhibitor, a Hedgehog protein
inhibitor; Smoothened receptor antagonist, an AKT1 gene inhibitor,
a DHFR inhibitor, a Thymidine kinase stimulator, a CD29 modulator,
a Fibronectin modulator, an Interleukin-2 ligand, a Serine protease
inhibitor, a D40LG gene stimulator; TNFSF9 gene stimulator, a 2
oxoglutarate dehydrogenase inhibitor, a TGF-beta type II receptor
antagonist, an Erbb3 tyrosine kinase receptor inhibitor, a
Cholecystokinin CCK2 receptor antagonist, a Wilms tumor protein
modulator, a Ras GTPase modulator, an Histone deacetylase
inhibitor, a Cyclin-dependent kinase 4 inhibitor A modulator, an
Estrogen receptor beta modulator, a 4-1BB inhibitor, a 4-1BBL
inhibitor, a PD-L2 inhibitor, a B7-H3 inhibitor, a B7-H4 inhibitor,
a BTLA inhibitor, a HVEM inhibitor, aTIM3 inhibitor, a GAL9
inhibitor, a LAG3 inhibitor, a VISTA inhibitor, a KIR inhibitor, a
2B4 inhibitor, a CD160 inhibitor, a CD66e modulator, an Angiotensin
II receptor antagonist, a Connective tissue growth factor ligand
inhibitor, a Jak1 tyrosine kinase inhibitor, a Jak2 tyrosine kinase
inhibitor, a dual Jak1/Jak2 tyrosine kinase inhibitor, an
Angiotensin converting enzyme 2 stimulator, a Growth hormone
receptor antagonist, a Galectin-3 inhibitor, a Sodium glucose
transporter-2 inhibitor, a Endothelin ET-A antagonist, a
Mineralocorticoid receptor antagonist, an Endothelin ET-B
antagonist, an Advanced glycosylation product receptor antagonist,
an Adrenocorticotrophic hormone ligand, a Famesoid X receptor
agonist, a G-protein coupled bile acid receptor 1 agonist, an
Aldose reductase inhibitor, a Xanthine oxidase inhibitor, a PPAR
gamma agonist, a Prostanoid receptor antagonist, a FGF receptor
antagonist, a PDGF receptor antagonist, a TGF beta antagonist, a
p38 MAP kinase inhibitor, a VEGF-1 receptor antagonist, a Protein
tyrosine phosphatase beta inhibitor, a Tek tyrosine kinase receptor
stimulator, a PDE 5 inhibitor, a Mineralocorticoid receptor
antagonist, an ACE inhibitor, a I-kappa B kinase inhibitor, a
NFE2L2 gene stimulator, a Nuclear factor kappa B inhibitor, a STAT3
gene inhibitor, a NADPH oxidase 1 inhibitor, a NADPH oxidase 4
inhibitor, a PDE 4 inhibitor, a Renin inhibitor, a MEKK-5 protein
kinase inhibitor, a Membrane copper amine oxidase inhibitor, an
Integrin alpha-V/beta-3 antagonist, an Insulin sensitizer, a
Kallikrein 1 modulator, a Cyclooxygenase 1 inhibitor and a
Phenylalanine hydroxylase stimulator.
[0109] In some embodiments, the one or more additional therapeutic
compound is selected from one or more of bavituximab, IMM-101,
CAP1-6D, Rexin-G, genistein, CVac, MM-D37K, PCI-27483, TG-01,
mocetinostat, LOAd-703, CPI-613, upamostat, CRS-207, NovaCaps,
trametinib, Atu-027, sonidegib, GRASPA, trabedersen, nastorazepide,
Vaccell, oregovomab, istiratumab, refametinib, regorafenib,
lapatinib, selumetinib, rucaparib, pelareorep, tarextumab,
PEGylated hyaluronidase, varlitinib, aglatimagene besadenovec,
GBS-01, GI-4000, WF-10, galunisertib, afatinib, RX-0201, FG-3019,
pertuzumab, DCVax-Direct, selinexor, glufosfamide, virulizin,
yttrium (90Y) clivatuzumab tetraxetan, brivudine, nimotuzumab,
algenpantucel-L, tegafur+gimeracil+oteracil potassium+calcium
folinate, olaparib, ibrutinib, pirarubicin, Rh-Apo2L, tertomotide,
tegafur+gimeracil+oteracil potassium, tegafur+gimeracil+oteracil
potassium, masitinib, Rexin-G, mitomycin, erlotinib, adriamycin,
dexamethasone, vincristine, cyclophosphamide, fluorouracil,
topotecan, taxol, interferons, platinum derivatives, taxane,
paclitaxel, vinca alkaloids, vinblastine, anthracyclines,
doxorubicin, epipodophyllotoxins, etoposide, cisplatin, rapamycin,
methotrexate, actinomycin D, dolastatin 10, colchicine, emetine,
trimetrexate, metoprine, cyclosporine, daunorubicin, teniposide,
amphotericin, alkylating agents, chlorambucil, 5-fluorouracil,
campthothecin, cisplatin, metronidazole, Gleevec, Avastin,
Vectibix, abarelix, aldesleukin, alemtuzumab, alitretinoin,
allopurinol, altretamine, amifostine, anastrozole, arsenic
trioxide, asparaginase, azacitidine, AZD9291, BCG Live,
bevacuzimab, fluorouracil, bexarotene, bleomycin, bortezomib,
busulfan, calusterone, capecitabine, camptothecin, carboplatin,
carmustine, celecoxib, cetuximab, chlorambucil, cladribine,
clofarabine, cyclophosphamide, cytarabine, dactinomycin,
darbepoetin alfa, daunorubicin, denileukin, dexrazoxane, docetaxel,
doxorubicin (neutral), doxorubicin hydrochloride, dromostanolone
propionate, epirubicin, epoetin alfa, estramustine, etoposide
phosphate, etoposide, exemestane, filgrastim, floxuridine
fludarabine, fulvestrant, gefitinib, gemcitabine, gemtuzumab,
goserelin acetate, histrelin acetate, hydroxyurea, ibritumomab,
idarubicin, ifosfamide, imatinib mesylate, interferon alfa-2a,
interferon alfa-2b, irinotecan, lenalidomide, letrozole,
leucovorin, leuprolide acetate, levamisole, lomustine, megestrol
acetate, melphalan, mercaptopurine, 6-MP, mesna, methotrexate,
methoxsalen, mitomycin C, mitotane, mitoxantrone, nandrolone,
nelarabine, nofetumomab, oprelvekin, oxaliplatin, nab-paclitaxel,
palifermin, pamidronate, pegademase, pegaspargase, pegfilgrastim,
pemetrexed disodium, pentostatin, pipobroman, plicamycin, porfimer
sodium, procarbazine, quinacrine, rasburicase, rituximab,
rociletinib, sargramostim, sorafenib, streptozocin, sunitinib
maleate, talc, tamoxifen, temozolomide, teniposide, VM-26,
testolactone, thioguanine, 6-TG, thiotepa, topotecan, toremifene,
tositumomab, trastuzumab, tretinoin, ATRA, uracil mustard,
valrubicin, vinblastine, vincristine, vinorelbine, zoledronate,
zoledronic acid, pembrolizumab, nivolumab, IBI-308, mDX--400,
BGB-108, MEDI-0680, SHR-1210, PF-06801591, PDR-001, GB-226,
STI-1110, durvalumab, atezolizumab, avelumab, BMS-936559, ALN-PDL,
TSR-042, KD-033, CA-170, STI-1014, FOLFIRINOX, KY-1003, olmesartan
medoxomil, candesartan, PBI-4050, baricitinib, GSK-2586881,
losartan, dapagliflozin propanediol, pegvisomant, GR-MD-02,
canagliflozin, irbesartan, FG-3019, atrasentan, finerenone,
sparsentan, bosentan, defibrotide, fimasartan, azeliragon,
pyridoxamine, corticotropin, INT-767, epalrestat, topiroxostat,
SER-150-DN, pirfenidone, VEGFR-1 mAb, AKB-9778, PF-489791, SHP-627,
CS-3150, imidapril, perindopril, captopril, enalapril, lisinopril,
Zofenopril, Lisinopril, Quinapril, Benazepril, Trandolapril,
Cilazapril, Fosinopril, Ramipril, bardoxolone methyl,
irbesartan+propagermanium, GKT-831, MT-3995, TAK-648, TAK-272,
GS-4997, DW-1029M, ASP-8232, VPI-2690B, DM-199, rhein, PHN-033,
GLY-230, and sapropterin, sulodexide.
[0110] In some embodiments, the one or more additional therapeutic
compound is an angiotensin converting enzyme (ACE) inhibitor or an
angiotensin receptor II blocker (ARB). In some embodiments, the one
or more additional therapeutic compound is an angiotensin
converting enzyme (ACE) inhibitor. In some embodiments, the one or
more additional therapeutic compound is an angiotensin receptor II
blocker (ARB). In some embodiments, the one or more additional
therapeutic compound is olmesartan medoxomil, candesartan,
losartan, irbesartan, sparsentan, fimasartan, GSK-2586881,
imidapril, perindopril, captopril, enalapril, lisinopril,
Zofenopril, Lisinopril, Quinapril, Benazepril, Trandolapril,
Cilazapril, Fosinopril or Ramipril.
[0111] In some embodiments, the one or more additional therapeutic
compound is FOLFIRINOX. In some embodiments, the one or more
additional therapeutic compound is gemcitabine and paclitaxel. In
some embodiments, the one or more additional therapeutic compound
is gemcitabine and nab-paclitaxel.
Compounds that Modulate CCR2 Activity
[0112] The present invention provides compounds that modulate at
least one CCR2 activity. Chemokine receptors are integral membrane
proteins which interact with an extracellular ligand, such as a
chemokine, and mediate a cellular response to the ligand, e.g.,
chemotaxis, increased intracellular calcium ion concentration, etc.
Therefore, modulation of a chemokine receptor function, e.g.,
interference with a chemokine receptor ligand interaction, will
modulate a chemokine receptor mediated response, and treat or
prevent a chemokine receptor mediated condition or disease.
Modulation of a chemokine receptor function includes both
inducement and inhibition of the function. The type of modulation
accomplished will depend on the characteristics of the compound,
i.e., antagonist or full, partial or inverse agonist.
[0113] Without intending to be bound by any particular theory, it
is believed that the compounds provided herein interfere with the
interaction between a chemokine receptor and one or more cognate
ligands. In particular, it is believed that the compounds interfere
with the interaction between CCR2 and a CCR2 ligand, such as MCP-1.
Compounds contemplated by the invention include, but are not
limited to, the exemplary compounds provided herein and salts
thereof.
[0114] For example, compounds of this invention act as potent CCR2
antagonists, and this antagonistic activity has been further
confirmed in animal testing for inflammation, one of the hallmark
disease states for CCR2. Accordingly, the compounds provided herein
are useful in pharmaceutical compositions, methods for the
treatment of CCR2-mediated diseases, and as controls in assays for
the identification of competitive CCR2 antagonists.
Methods of Treatment
[0115] Modulation of CCR2 Receptor Function
[0116] The compounds of the invention may be used as agonists,
(preferably) antagonists, partial agonists, inverse agonists, of
CCR2 receptors in a variety of contexts, both in vitro and in vivo.
In one embodiment, the compounds of the invention are CCR2
antagonists that can be used to inhibit the binding of CCR2
receptor ligand to the CCR2 receptor in vitro or in vivo. In
general, such methods comprise the step of contacting a CCR2
receptor with a sufficient amount of one or more CCR2 receptor
modulators as provided herein, in the presence of CCR2 receptor
ligand in aqueous solution and under conditions otherwise suitable
for binding of the ligand to CCR2 receptor. The CCR2 receptor may
be present in suspension (e.g., in an isolated membrane or cell
preparation), in a cultured or isolated cell, or in a tissue or
organ.
[0117] Preferably, the amount of CCR2 receptor modulator contacted
with the receptor should be sufficient to inhibit ligand binding to
the CCR2 receptor in vitro as measured, for example, using a
radioligand binding assay, calcium mobilization assay, or
chemotaxis assay as described herein.
[0118] In one embodiment of the invention, the CCR2 modulators of
the invention are used to modulate, preferably inhibit, the
signal-transducing activity of a CCR2 receptor, for example, by
contacting one or more compound(s) of the invention with a CCR2
receptor (either in vitro or in vivo) under conditions suitable for
binding of the modulator(s) to the receptor. The receptor may be
present in solution or suspension, in a cultured or isolated cell
preparation or within a patient. Any modulation of the signal
transducing activity may be assessed by detecting an effect on
calcium ion calcium mobilization or by detecting an effect on CCR2
receptor-mediated cellular chemotaxis. In general, an effective
amount of CCR2 modulator(s) is an amount sufficient to modulate
CCR2 receptor signal transducing activity in vitro within a calcium
mobilization assay or CCR2 receptor-mediated cellular chemotaxis
within a migration assay.
[0119] When compounds of the invention are used to inhibit CCR2
receptor-mediated cellular chemotaxis, preferably leukocyte
chemotaxis, in an in vitro chemotaxis assay, such methods comprise
contacting white blood cells (particularly primate white blood
cells, especially human white blood cells) with one or more
compounds of the invention. Preferably the concentration is
sufficient to inhibit chemotaxis of white blood cells in an in
vitro chemotaxis assay, so that the levels of chemotaxis observed
in a control assay are significantly higher, as described above,
than the levels observed in an assay to which a compound of the
invention has been added.
[0120] In another embodiment, the compounds of the present
invention further can be used for treating patients suffering from
conditions that are responsive to CCR2 receptor modulation. As used
herein, the term "treating" or "treatment" encompasses both
disease-modifying treatment and symptomatic treatment, either of
which may be prophylactic (i.e., before the onset of symptoms, in
order to prevent, delay or reduce the severity of symptoms) or
therapeutic (i.e., after the onset of symptoms, in order to reduce
the severity and/or duration of symptoms). As used herein, a
condition is considered "responsive to CCR2 receptor modulation" if
modulation of CCR2 receptor activity results in the reduction of
inappropriate activity of a CCR2 receptor. As used herein, the term
"patients" include primates (especially humans), domesticated
companion animals (such as dogs, cats, horses, and the like) and
livestock (such as cattle, pigs, sheep, and the like), with dosages
as described herein.
[0121] In some embodiments, the compounds of the present invention
further can be used for treating patients suffering from an
inflammatory disease or disorder, a cardiovascular or
cerebrovascular disorder, an autoimmune disorder, a cancer or a
solid tumor. In some embodiments, the compounds of the present
invention can be used for treating patients suffering from a
disease or disorder selected from the group consisting of diabtetic
nephropathy, nephropathy, neutropenia, neutrophilia, Albuminuria,
Diabetic retinopathy, Focal segmental glomerulosclerosis,
glomerulosclerosis, Allergy, fibrosis, NASH (Nonalcoholic
Steatohepatitis), Hemolytic uremic syndrome, atypical hemolytic
uremic syndrome (aHUS), C3-glomerulopathy, C3-glomerulonephritis,
dense deposit disease, membranoproliferative glomerulonephritis,
sepsis, septic shock, Alzheimer's disease, multiple sclerosis,
stroke, inflammatory bowel disease, chronic obstructive pulmonary
disorder, inflammation associated with burns, lung injury,
osteoarthritis, atopic dermatitis, chronic urticaria,
ischemia-reperfusion injury, acute respiratory distress syndrome,
systemic inflammatory response syndrome, multiple organ dysfunction
syndrome, tissue graft rejection, Graft versus host disease,
hyperacute rejection of transplanted organs, myocardial infarction,
coronary thrombosis, vascular occlusion, post-surgical vascular
reocclusion, artherosclerosis, traumatic central nervous system
injury, ischemic heart disease, rheumatoid arthritis, systemic
lupus erythematosus, Guillain-Barre syndrome, pancreatitis, lupus
nephritis, lupus glomerulonephritis, psoriasis, Crohn's disease,
vasculitis, Anca-vasculitis, irritable bowel syndrome,
dermatomyositis, multiple sclerosis, bronchial asthma, pemphigus,
pemphigoid, scleroderma, myasthenia gravis, autoimmune hemolytic
and thrombocytopenic states, Goodpasture's syndrome,
immunovasculitis, tissue graft rejection, hyperacute rejection of
transplanted organs, Melanoma, Small Cell Lung Carcinoma, Non Small
Cell Lung Carcinoma, Pancreatic Cancer, Breast Cancer, Bladder
Cancer, Renal Cell Carcinoma, Colorectal Cancer, Hepatocellular
Carcinoma, Head and Neck Squamous Cell Carcinoma, Esophageal
Cancer, Ovarian Cancer, Prostate Cancer, Gastric Cancer, Acute
myelogenous leukemia, leukemia, a pathologic sequelae associated
with the group consisting of insulin-dependent diabetes, mellitus,
lupus nephropathy, Heyman nephritis, membranous nephritis,
glomerulonephritis, contact sensitivity responses, and inflammation
resulting from contact of blood with artificial surfaces.
[0122] In some embodiments, the compounds of the present invention
can be used for treating patients suffering from a disease or
disorder and are administered with one or more additional
therapeutic compound. In some embodiments, the one or more
additional therapeutic compound is selected from one or more of a
Btk tyrosine kinase inhibitor, an Erbb2 tyrosine kinase receptor
inhibitor; an Erbb4 tyrosine kinase receptor inhibitor, an mTOR
inhibitor, a Thymidylate synthase inhibitor, an EGFR tyrosine
kinase receptor inhibitor, an Epidermal growth factor antagonist, a
Fyn tyrosine kinase inhibitor, a Kit tyrosine kinase inhibitor, a
Lyn tyrosine kinase inhibitor, a NK cell receptor modulator, a PDGF
receptor antagonist, a PARP inhibitor, a Poly ADP ribose polymerase
inhibitor, a Poly ADP ribose polymerase 1 inhibitor, a Poly ADP
ribose polymerase 2 inhibitor, a Poly ADP ribose polymerase 3
inhibitor, a Galactosyltransferase modulator, a Dihydropyrimidine
dehydrogenase inhibitor, an Orotate phosphoribosyltransferase
inhibitor, a Telomerase modulator, a Mucin 1 inhibitor, a Mucin
inhibitor, a Secretin agonist, a TNF related apoptosis inducing
ligand modulator, an IL17 gene stimulator, an Interleukin 17E
ligand, a Neurokinin receptor agonist, a Cyclin G1 inhibitor, a
checkpoint inhibitor, a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA4
inhibitor, a Topoisomerase I inhibitor, an Alk-5 protein kinase
inhibitor, a Connective tissue growth factor ligand inhibitor, a
Notch-2 receptor antagonist, a Notch-3 receptor antagonist, a
Hyaluronidase stimulator, a MEK-1 protein kinase inhibitor; MEK-2
protein kinase inhibitor, a GM-CSF receptor modulator; TNF alpha
ligand modulator, a Mesothelin modulator, an Asparaginase
stimulator, a Caspase-3 stimulator; Caspase-9 stimulator, a PKN3
gene inhibitor, a Hedgehog protein inhibitor; Smoothened receptor
antagonist, an AKT1 gene inhibitor, a DHFR inhibitor, a Thymidine
kinase stimulator, a CD29 modulator, a Fibronectin modulator, an
Interleukin-2 ligand, a Serine protease inhibitor, a D40LG gene
stimulator; TNFSF9 gene stimulator, a 2 oxoglutarate dehydrogenase
inhibitor, a TGF-beta type II receptor antagonist, an Erbb3
tyrosine kinase receptor inhibitor, a Cholecystokinin CCK2 receptor
antagonist, a Wilms tumor protein modulator, a Ras GTPase
modulator, an Histone deacetylase inhibitor, a Cyclin-dependent
kinase 4 inhibitor A modulator, an Estrogen receptor beta
modulator, a 4-1BB inhibitor, a 4-1BBL inhibitor, a PD-L2
inhibitor, a B7-H3 inhibitor, a B7-H4 inhibitor, a BTLA inhibitor,
a HVEM inhibitor, aTIM3 inhibitor, a GAL9 inhibitor, a LAG3
inhibitor, a VISTA inhibitor, a KIR inhibitor, a 2B4 inhibitor, a
CD160 inhibitor, a CD66e modulator, an Angiotensin II receptor
antagonist, a Connective tissue growth factor ligand inhibitor, a
Jak1 tyrosine kinase inhibitor, a Jak2 tyrosine kinase inhibitor, a
dual Jak1/Jak2 tyrosine kinase inhibitor, an Angiotensin converting
enzyme 2 stimulator, a Growth hormone receptor antagonist, a
Galectin-3 inhibitor, a Sodium glucose transporter-2 inhibitor, a
Endothelin ET-A antagonist, a Mineralocorticoid receptor
antagonist, an Endothelin ET-B antagonist, an Advanced
glycosylation product receptor antagonist, an Adrenocorticotrophic
hormone ligand, a Famesoid X receptor agonist, a G-protein coupled
bile acid receptor 1 agonist, an Aldose reductase inhibitor, a
Xanthine oxidase inhibitor, a PPAR gamma agonist, a Prostanoid
receptor antagonist, a FGF receptor antagonist, a PDGF receptor
antagonist, a TGF beta antagonist, a p38 MAP kinase inhibitor, a
VEGF-1 receptor antagonist, a Protein tyrosine phosphatase beta
inhibitor, a Tek tyrosine kinase receptor stimulator, a PDE 5
inhibitor, a Mineralocorticoid receptor antagonist, an ACE
inhibitor, a I-kappa B kinase inhibitor, a NFE2L2 gene stimulator,
a Nuclear factor kappa B inhibitor, a STAT3 gene inhibitor, a NADPH
oxidase 1 inhibitor, a NADPH oxidase 4 inhibitor, a PDE 4
inhibitor, a Renin inhibitor, a MEKK-5 protein kinase inhibitor, a
Membrane copper amine oxidase inhibitor, an Integrin alpha-V/beta-3
antagonist, an Insulin sensitizer, a Kallikrein 1 modulator, a
Cyclooxygenase 1 inhibitor and a Phenylalanine hydroxylase
stimulator. In some embodiments, the one or more additional
therapeutic compound is selected from one or more of bavituximab,
IMM-101, CAP1-6D, Rexin-G, genistein, CVac, MM-D37K, PCI-27483,
TG-01, mocetinostat, LOAd-703, CPI-613, upamostat, CRS-207,
NovaCaps, trametinib, Atu-027, sonidegib, GRASPA, trabedersen,
nastorazepide, Vaccell, oregovomab, istiratumab, refametinib,
regorafenib, lapatinib, selumetinib, rucaparib, pelareorep,
tarextumab, PEGylated hyaluronidase, varlitinib, aglatimagene
besadenovec, GBS-01, GI-4000, WF-10, galunisertib, afatinib,
RX-0201, FG-3019, pertuzumab, DCVax-Direct, selinexor,
glufosfamide, virulizin, yttrium (90Y) clivatuzumab tetraxetan,
brivudine, nimotuzumab, algenpantucel-L, tegafur+gimeracil+oteracil
potassium+calcium folinate, olaparib, ibrutinib, pirarubicin,
Rh-Apo2L, tertomotide, tegafur+gimeracil+oteracil potassium,
tegafur+gimeracil+oteracil potassium, masitinib, Rexin-G,
mitomycin, erlotinib, adriamycin, dexamethasone, vincristine,
cyclophosphamide, fluorouracil, topotecan, taxol, interferons,
platinum derivatives, taxane, paclitaxel, vinca alkaloids,
vinblastine, anthracyclines, doxorubicin, epipodophyllotoxins,
etoposide, cisplatin, rapamycin, methotrexate, actinomycin D,
dolastatin 10, colchicine, emetine, trimetrexate, metoprine,
cyclosporine, daunorubicin, teniposide, amphotericin, alkylating
agents, chlorambucil, 5-fluorouracil, campthothecin, cisplatin,
metronidazole, Gleevec, Avastin, Vectibix, abarelix, aldesleukin,
alemtuzumab, alitretinoin, allopurinol, altretamine, amifostine,
anastrozole, arsenic trioxide, asparaginase, azacitidine, AZD9291,
BCG Live, bevacuzimab, fluorouracil, bexarotene, bleomycin,
bortezomib, busulfan, calusterone, capecitabine, camptothecin,
carboplatin, carmustine, celecoxib, cetuximab, chlorambucil,
cladribine, clofarabine, cyclophosphamide, cytarabine,
dactinomycin, darbepoetin alfa, daunorubicin, denileukin,
dexrazoxane, docetaxel, doxorubicin (neutral), doxorubicin
hydrochloride, dromostanolone propionate, epirubicin, epoetin alfa,
estramustine, etoposide phosphate, etoposide, exemestane,
filgrastim, floxuridine fludarabine, fulvestrant, gefitinib,
gemcitabine, gemtuzumab, goserelin acetate, histrelin acetate,
hydroxyurea, ibritumomab, idarubicin, ifosfamide, imatinib
mesylate, interferon alfa-2a, interferon alfa-2b, irinotecan,
lenalidomide, letrozole, leucovorin, leuprolide acetate,
levamisole, lomustine, megestrol acetate, melphalan,
mercaptopurine, 6-MP, mesna, methotrexate, methoxsalen, mitomycin
C, mitotane, mitoxantrone, nandrolone, nelarabine, nofetumomab,
oprelvekin, oxaliplatin, nab-paclitaxel, palifermin, pamidronate,
pegademase, pegaspargase, pegfilgrastim, pemetrexed disodium,
pentostatin, pipobroman, plicamycin, porfimer sodium, procarbazine,
quinacrine, rasburicase, rituximab, rociletinib, sargramostim,
sorafenib, streptozocin, sunitinib maleate, talc, tamoxifen,
temozolomide, teniposide, VM-26, testolactone, thioguanine, 6-TG,
thiotepa, topotecan, toremifene, tositumomab, trastuzumab,
tretinoin, ATRA, uracil mustard, valrubicin, vinblastine,
vincristine, vinorelbine, zoledronate, zoledronic acid,
pembrolizumab, nivolumab, IBI-308, mDX--400, BGB-108, MEDI-0680,
SHR-1210, PF-06801591, PDR-001, GB-226, STI-1110, durvalumab,
atezolizumab, avelumab, BMS-936559, ALN-PDL, TSR-042, KD-033,
CA-170, STI-1014, FOLFIRINOX, KY-1003, olmesartan medoxomil,
candesartan, PBI-4050, baricitinib, GSK-2586881, losartan,
dapagliflozin propanediol, pegvisomant, GR-MD-02, canagliflozin,
irbesartan, FG-3019, atrasentan, finerenone, sparsentan, bosentan,
defibrotide, fimasartan, azeliragon, pyridoxamine, corticotropin,
INT-767, epalrestat, topiroxostat, SER-150-DN, pirfenidone, VEGFR-1
mAb, AKB-9778, PF-489791, SHP-627, CS-3150, imidapril, perindopril,
captopril, enalapril, lisinopril, Zofenopril, Lisinopril,
Quinapril, Benazepril, Trandolapril, Cilazapril, Fosinopril,
Ramipril, bardoxolone methyl, irbesartan+propagermanium, GKT-831,
MT-3995, TAK-648, TAK-272, GS-4997, DW-1029M, ASP-8232, VPI-2690B,
DM-199, rhein, PHN-033, GLY-230, and sapropterin, sulodexide.
[0123] In some embodiments, the one or more additional therapeutic
compound is an angiotensin converting enzyme (ACE) inhibitor or an
angiotensin receptor II blocker (ARB). In some embodiments, the one
or more additional therapeutic compound is an angiotensin
converting enzyme (ACE) inhibitor. In some embodiments, the one or
more additional therapeutic compound is an angiotensin receptor II
blocker (ARB). In some embodiments, the one or more additional
therapeutic compound is olmesartan medoxomil, candesartan,
losartan, irbesartan, sparsentan, fimasartan, GSK-2586881,
imidapril, perindopril, captopril, enalapril, lisinopril,
Zofenopril, Lisinopril, Quinapril, Benazepril, Trandolapril,
Cilazapril, Fosinopril or Ramipril.
[0124] In some embodiments, the one or more additional therapeutic
compound is FOLFIRINOX. In some embodiments, the one or more
additional therapeutic compound is gemcitabine and paclitaxel. In
some embodiments, the one or more additional therapeutic compound
is gemcitabine and nab-paclitaxel. Conditions that can be treated
by CCR2 modulation:
[0125] Autoimmune disorders--e.g., Rheumatoid arthritis, systemic
lupus erythematosus, Guillain-Barre syndrome, pancreatitis, lupus
nephritis, lupus glomerulonephritis, psoriasis, Crohn's disease,
vasculitis, irritable bowel syndrome, dermatomyositis, multiple
sclerosis, bronchial asthma, pemphigus, pemphigoid, scleroderma,
myasthenia gravis, autoimmune hemolytic and thrombocytopenic
states, Goodpasture's syndrome (and associated glomerulonephritis
and pulmonary hemorrhage), immunovasculitis, tissue graft
rejection, hyperacute rejection of transplanted organs; and the
like.
[0126] Inflammatory disorders and related conditions--e.g.,
Neutropenia, sepsis, septic shock, Alzheimer's disease, multiple
sclerosis, stroke, inflammatory bowel disease (IBD), inflammation
associated with severe burns, lung injury, and ischemia-reperfusion
injury, osteoarthritis, as well as acute (adult) respiratory
distress syndrome (ARDS), chronic pulmonary obstructive disorder
(COPD), systemic inflammatory response syndrome (SIRS), atopic
dermatitis, psoriasis, chronic urticaria and multiple organ
dysfunction syndrome (MODS). Also included are pathologic sequellae
associated with insulin-dependent diabetes mellitus (including
diabetic retinopathy), lupus nephropathy, Heyman nephritis,
membranous nephritis and other forms of glomerulonephritis, contact
sensitivity responses, and inflammation resulting from contact of
blood with artificial surfaces that can cause complement
activation, as occurs, for example, during extracorporeal
circulation of blood (e.g., during hemodialysis or via a heart-lung
machine, for example, in association with vascular surgery such as
coronary artery bypass grafting or heart valve replacement), or in
association with contact with other artificial vessel or container
surfaces (e.g., ventricular assist devices, artificial heart
machines, transfusion tubing, blood storage bags, plasmapheresis,
plateletpheresis, and the like). Also included are diseases related
to ischemia/reperfusion injury, such as those resulting from
transplants, including solid organ transplant, and syndromes such
as ischemic reperfusion injury, ischemic colitis and cardiac
ischemia. Compounds of the instant invention may also be useful in
the treatment of age-related macular degeneration (Hageman et al,
P.N.A.S. 102: 7227-7232, 2005).
[0127] Cardiovascular and Cerebrovascular Disorders--e.g.,
myocardial infarction, coronary thrombosis, vascular occlusion,
post-surgical vascular reocclusion, atherosclerosis, traumatic
central nervous system injury, and ischemic heart disease. In one
embodiment, an effective amount of a compound of the invention may
be administered to a patient at risk for myocardial infarction or
thrombosis (i.e., a patient who has one or more recognized risk
factor for myocardial infarction or thrombosis, such as, but not
limited to, obesity, smoking, high blood pressure,
hypercholesterolemia, previous or genetic history of myocardial
infarction or thrombosis) in order reduce the risk of myocardial
infarction or thrombosis.
[0128] Diseases of Vasculitis--Vasculitic diseases are
characterized by inflammation of the vessels. Infiltration of
leukocytes leads to destruction of the vessel walls, and the
complement pathway is believed to play a major role in initiating
leukocyte migration as well as the resultant damage manifested at
the site of inflammation (Vasculitis, Second Edition, Edited by
Ball and Bridges, Oxford University Press, pp 47-53, 2008). The
compounds provided in the present invention can be used to treat
leukoclastic vasculitis, Wegener's granulomatosis, microscopic
polyangiitis, Churg-Strauss syndrome, Henoch-Schonlein purpura,
polyateritis nodosa, Rapidly Progressive Glomerulonephritis (RPGN),
cryoglobulinaemia, giant cell arteritis (GCA), Behcet's disease and
Takayasu's arteritis (TAK).
[0129] Cancer--CCR2 and its ligand CCL2 also play a major role in
conditioning the tumor microenvironment, and regulating the influx
of both beneficial and deleterious immune cell populations to the
tumor. As such recent clinical and pre-clinical literature has
demonstrated a role for CCR2 in a variety of solid tumors including
Melanoma, Small Cell Lung Carcinoma, Non Small Cell Lung Carcinoma,
Pancreatic Cancer, Breast Cancer, Bladder Cancer, Renal Cell
Carcinoma, Colorectal Cancer, Hepatocellular Carcinoma, Head and
Neck Squamous Cell Carcinoma, Esophageal Cancer, Ovarian Cancer,
Prostate Cancer, and Gastric Cancer.
[0130] HIV infection, AIDS and Viral Infections--CCR2 receptor
modulators provided herein may be used to inhibit HIV infection,
delay AIDS progression or decrease the severity of symptoms or HIV
infection and AIDS, as well as in the treatment of hepatitis C.
[0131] Neurodegenerative disorders and related diseases--Within
further aspects, CCR2 antagonists provided herein may be used to
treat Alzheimer's disease, multiple sclerosis, and cognitive
function decline associated with cardiopulmonary bypass surgery and
related procedures.
[0132] In one embodiment of the invention, the compounds of the
invention can be used for the treatment of diseases selected from
the group consisting of sepsis (and associated disorders), COPD,
rheumatoid arthritis, lupus nephritis and multiple sclerosis.
[0133] Treatment methods provided herein include, in general,
administration to a patient an effective amount of one or more
compounds provided herein. Suitable patients include those patients
suffering from or susceptible to (i.e., prophylactic treatment) a
disorder or disease identified herein. Typical patients for
treatment as described herein include mammals, particularly
primates, especially humans. Other suitable patients include
domesticated companion animals such as a dog, cat, horse, and the
like, or a livestock animal such as cattle, pig, sheep and the
like.
[0134] In general, treatment methods provided herein comprise
administering to a patient an effective amount of a compound one or
more compounds provided herein. In a preferred embodiment, the
compound(s) of the invention are preferably administered to a
patient (e.g., a human) orally or topically. The effective amount
may be an amount sufficient to modulate CCR2 receptor activity
and/or an amount sufficient to reduce or alleviate the symptoms
presented by the patient. Preferably, the amount administered is
sufficient to yield a plasma concentration of the compound (or its
active metabolite, if the compound is a pro-drug) high enough to
detectably inhibit white blood cell chemotaxis in vitro. Treatment
regimens may vary depending on the compound used and the particular
condition to be treated; for treatment of most disorders, a
frequency of administration of 4 times daily or less is preferred.
In general, a dosage regimen of 2 times daily is more preferred,
with once a day dosing particularly preferred. It will be
understood, however, that the specific dose level and treatment
regimen for any particular patient will depend upon a variety of
factors including the activity of the specific compound employed,
the age, body weight, general health, sex, diet, time of
administration, route of administration, rate of excretion, drug
combination (i.e., other drugs being administered to the patient)
and the severity of the particular disease undergoing therapy, as
well as the judgment of the prescribing medical practitioner. In
general, the use of the minimum dose sufficient to provide
effective therapy is preferred. Patients may generally be monitored
for therapeutic effectiveness using medical or veterinary criteria
suitable for the condition being treated or prevented.
[0135] Depending on the disease to be treated and the subject's
condition, the compounds and compositions of the present invention
may be administered by oral, parenteral (e.g., intramuscular,
intraperitoneal, intravenous, ICV, intracisternal injection or
infusion, subcutaneous injection, or implant), inhalation, nasal,
vaginal, rectal, sublingual, or topical routes of administration
and may be formulated, alone or together, in suitable dosage unit
formulations containing conventional non toxic pharmaceutically
acceptable carriers, adjuvants and vehicles appropriate for each
rouse of administration. The present invention also contemplates
administration of the compounds and compositions of the present
invention in a depot formulation.
[0136] Dosage levels of the order of from about 0.1 mg to about 140
mg per kilogram of body weight per day are useful in the treatment
or preventions of conditions involving pathogenic CCR2 activity
(about 0.5 mg to about 7 g per human patient per day). The amount
of active ingredient that may be combined with the carrier
materials to produce a single dosage form will vary depending upon
the host treated and the particular mode of administration. Dosage
unit forms will generally contain between from about 1 mg to about
500 mg of an active ingredient. For compounds administered orally,
transdermally, intravaneously, or subcutaneously, it is preferred
that sufficient amount of the compound be administered to achieve a
serum concentration of 5 ng (nanograms)/mL-10 .mu.g (micrograms)/mL
serum, more preferably sufficient compound to achieve a serum
concentration of 20 ng-1 .mu.g/ml serum should be administered,
most preferably sufficient compound to achieve a serum
concentration of 50 ng/ml-200 ng/ml serum should be administered.
For direct injection into the synovium (for the treatment of
arthritis) sufficient compounds should be administered to achieve a
local concentration of approximately 1 micromolar.
[0137] Frequency of dosage may also vary depending on the compound
used and the particular disease treated. However, for treatment of
most disorders, a dosage regimen of 4 times daily, three times
daily, or less is preferred, with a dosage regimen of once daily or
2 times daily being particularly preferred. It will be understood,
however, that the specific dose level for any particular patient
will depend upon a variety of factors including the activity of the
specific compound employed, the age, body weight, general health,
sex, diet, time of administration, route of administration, and
rate of excretion, drug combination (i.e., other drugs being
administered to the patient), the severity of the particular
disease undergoing therapy, and other factors, including the
judgment of the prescribing medical practitioner.
[0138] In some embodiments, the treatment or prevention of
conditions which require CCR2 receptor modulation, an appropriate
dosage level will generally be about 0.001 to 100 mg per kg patient
body weight per day which can be administered in single or multiple
doses. Preferably, the dosage level will be about 0.01 to about 25
mg/kg per day; more preferably about 0.05 to about 10 mg/kg per
day. A suitable dosage level may be about 0.01 to 25 mg/kg per day,
about 0.05 to 10 mg/kg per day, or about 0.1 to 5 mg/kg per day.
Within this range the dosage may be 0.005 to 0.05, 0.05 to 0.5, 0.5
to 5.0, or 5.0 to 50 mg/kg per day. For oral administration, the
compositions are preferably provided in the form of tablets
containing 1.0 to 1000 milligrams of the active ingredient,
particularly 1.0, 5.0, 10.0, 15.0, 20.0, 25.0, 50.0, 75.0, 100.0,
150.0, 200.0, 250.0, 300.0, 400.0, 500.0, 600.0, 750.0, 800.0,
900.0, and 1000.0 milligrams of the active ingredient for the
symptomatic adjustment of the dosage to the patient to be treated.
The compounds may be administered on a regimen of 1 to 4 times per
day, preferably once or twice per day.
[0139] It will be understood, however, that the specific dose level
and frequency of dosage for any particular patient may be varied
and will depend upon a variety of factors including the activity of
the specific compound employed, the metabolic stability and length
of action of that compound, the age, body weight, hereditary
characteristics, general health, sex, diet, mode and time of
administration, rate of excretion, drug combination, the severity
of the particular condition, and the host undergoing therapy.
[0140] Combination Treatments--Pharmacologics to be Used in
Conjunction with CCR2 Compounds
[0141] Pharmacological agents that can be used in conjunction with
the CCR2 antagonists of the current invention include those used
for the treatments of atherosclerosis, restenosis, multiple
sclerosis, pulmonary fibrosis, inflammatory bowel disease,
rheumatoid arthritis, graft-versus-host disease, renal fibrosis,
psoriasis, transplantation rejection, obesity, diabetes,
hypercholesterolemia and cancer.
[0142] In the treatment of cancer, immune modulators such as those
of the invention may be used in conjunction with other compounds
and reagents which regulate the immune response or tumor immune
environment. This is termed "immuno-oncology", and a wide variety
of reagants can be utilized either alone or in combinations,
including small molecules, protein and peptides, antibodies,
speigelmers, viruses, oligoribonucleotides and other methods of
genetic information delivery and other therapeutics known in the
art. As such compositions of the invention can be utilized in the
formulation of immunoimmune-oncologic medicaments.
[0143] In still other embodiments, the present methods are directed
to the treatment of allergic diseases, wherein a compound or
composition of the invention is administered either alone or in
combination with a second therapeutic agent, wherein said second
therapeutic agent is an antihistamine. When used in combination,
the practitioner can administer a combination of the compound or
composition of the present invention and a second therapeutic
agent. Also, the compound or composition and the second therapeutic
agent can be administered sequentially, in any order.
[0144] The compounds and compositions of the present invention can
be combined with other compounds and compositions having related
utilities to prevent and treat the condition or disease of
interest, such as inflammatory conditions and diseases, including
inflammatory bowel disease, allergic diseases, psoriasis, atopic
dermatitis and asthma, and those pathologies noted above. Selection
of the appropriate agents for use in combination therapies can be
made one of ordinary skill in the art. The combination of
therapeutic agents may act synergistically to effect the treatment
or prevention of the various disorders. Using this approach, one
may be able to achieve therapeutic efficacy with lower dosages of
each agent, thus reducing the potential for adverse side
effects.
[0145] In treating, preventing, ameliorating, controlling or
reducing solid tumour growth and metastases, the compounds of the
present invention may be used in conjuction with the following: (1)
cancer vaccination strategies, (2) immune-checkpoint modulators
such as antagonistic antibodies against immune-checkpoint
inhibitors (anti-PD1, anti-PD-L1, anti-CTLA4, anti-Tim3,
anti-VISTA, anti-KIR) or agonistic antibodies against
immune-accelators (anti-Lag3, anti-OX40, anti-ICOS, anti-4-1BB, (3)
blocking or depleting antibodies against cell surface proteins
commonly up-regulated in transformed cells (CEACAM1, Syndecan-2,
GRP78), (4) anti-angiogenic therapies (anti-VEGF, anti-VEGFR, VEGFR
small molecule inhibitors), (5) anti-lymphangiogenesis (blocking
antibodies or inhibitors against VEGF, FDF2, PDGF as well as its
respective receptors), (6) standard chemotherapeutic therapies
(Gemcitabine, Paclitaxel, FOLFORINOX), (7) irradiation therapy, (8)
other chemokine antagonists (CCR1, CCR4, CCR6, CXCR4, CXCR2, CXCR7
small molecule inhibitors, blocking antibodies, or depleting
antibodies), (9) depleting antibodies against chemokines that
activate the aforementioned chemokine receptors, (10) inhibitors
targeting common somatic mutations in cancer such as those
specifically targeting the following genes (BRAF, KRAS, NRAS, EGFR,
CTNNB1, NOTCH1, PIK3CA, PTEN, APC, FLT3, IDH1, IDH2, KIT, TP53,
JAK2).
[0146] In treating, preventing, ameliorating, controlling or
reducing the risk of inflammation, the compounds of the present
invention may be used in conjunction with an anti-inflammatory or
analgesic agent such as an opiate agonist, a lipoxygenase
inhibitor, such as an inhibitor of 5-lipoxygenase, a cyclooxygenase
inhibitor, such as a cyclooxygenase-2 inhibitor, an interleukin
inhibitor, such as an interleukin-1 inhibitor, an NMDA antagonist,
an inhibitor of nitric oxide or an inhibitor of the synthesis of
nitric oxide, a non-steroidal antiinflammatory agent, or a
cytokine-suppressing anti-inflammatory agent, for example with a
compound such as acetaminophen, aspirin, codeine, biological TNF
sequestrants, fentanyl, ibuprofen, indomethacin, ketorolac,
morphine, naproxen, phenacetin, piroxicam, a steroidal analgesic,
sufentanyl, sunlindac, tenidap, and the like.
[0147] Similarly, the compounds of the present invention may be
administered with a pain reliever; a potentiator such as caffeine,
an H2-antagonist, simethicone, aluminum or magnesium hydroxide; a
decongestant such as pseudophedrine; an antitussive such as
codeine; a diuretic; a sedating or non-sedating antihistamine; a
very late antigen (VLA-4) antagonist; an immunosuppressant such as
cyclosporin, tacrolimus, rapamycin, EDG receptor agonists, or other
FK-506 type immunosuppressants; a steroid; a non-steroidal
anti-asthmatic agent such as a 32-agonist, leukotriene antagonist,
or leukotriene biosynthesis inhibitor; an inhibitor of
phosphodiesterase type IV (PDE-IV); a cholesterol lowering agent
such as a HMG-CoA reductase inhibitor, sequestrant, or cholesterol
absorption inhibitor; and an anti-diabetic agent such as insulin,
.alpha.-glucosidase inhibitors or glitazones.
[0148] The weight ratio of the compound of the present invention to
the second active ingredient may be varied and will depend upon the
effective dose of each ingredient. Generally, an effective dose of
each will be used. Thus, for example, when a compound of the
present invention is combined with an NSAID the weight ratio of the
compound of the present invention to the NSAID will generally range
from about 1000:1 to about 1:1000, preferably about 200:1 to about
1:200. Combinations of a compound of the present invention and
other active ingredients will generally also be within the
aforementioned range, but in each case, an effective dose of each
active ingredient should be used.
[0149] In view of the above, a number of selected embodiments are
provided herein.
[0150] In one embodiment, the present invention provides a method
of treating a CCR2-mediated condition or disease involving
administering to a subject a safe and effective amount of the
compound or composition of the invention, where the CCR2-mediated
condition or disease is atherosclerosis.
[0151] In one embodiment, the present invention provides a method
of treating a CCR2-mediated condition or disease involving
administering to a subject a safe and effective amount of the
compound or composition of the invention, where the CCR2-mediated
condition or disease is restenosis.
[0152] In one embodiment, the present invention provides a method
of treating a CCR2-mediated condition or disease involving
administering to a subject a safe and effective amount of the
compound or composition of the invention, where the CCR2-mediated
condition or disease is multiple sclerosis.
[0153] In one embodiment, the present invention provides a method
of treating a CCR2-mediated condition or disease involving
administering to a subject a safe and effective amount of the
compound or composition of the invention, where the CCR2-mediated
condition or disease is selected from the group consisting of
inflammatory bowel disease, renal fibrosis, rheumatoid arthritis,
obesity and noninsulin-dependent diabetes.
[0154] In one embodiment, the present invention provides a method
of treating a CCR2-mediated condition or disease involving
administering to a subject a safe and effective amount of the
compound or composition of the invention, where the CCR2-mediated
condition or disease is type 2 diabetes.
[0155] In one embodiment, the present invention provides a method
of treating a CCR2-mediated condition or disease involving
administering to a subject a safe and effective amount of the
compound or composition of the invention, where the CCR2-mediated
condition or disease is diabetic nephropathy.
[0156] In one embodiment, the present invention provides a method
of treating a CCR2-mediated condition or disease involving
administering to a subject a safe and effective amount of the
compound or composition of the invention, where the CCR2-mediated
condition or disease is pancreatic cancer
[0157] In one embodiment, the present invention provides a method
of treating a CCR2-mediated condition or disease involving
administering to a subject a safe and effective amount of the
compound or composition of the invention, where the CCR2-mediated
condition or disease is a cancer or immunoimmune-oncology related
indication.
[0158] In one embodiment, the present invention provides a method
of treating a CCR2-mediated condition or disease involving
administering to a subject a safe and effective amount of the
compound or composition of the invention, where the CCR2-mediated
condition or disease is selected from the group consisting of
chronic obstructive pulmonary disease, idiopathic pulmonary
fibrosis and idiopathic pneumonia syndrome.
[0159] In one embodiment, the present invention provides a method
of treating a CCR2-mediated condition or disease involving
administering to a subject a safe and effective amount of the
compound or composition of the invention, where the administering
is oral, parenteral, rectal, transdermal, sublingual, nasal or
topical.
[0160] In one embodiment, the present invention provides a method
of treating a CCR2-mediated condition or disease involving
administering to a subject a safe and effective amount of the
compound or composition of the invention, where the compound is
administered in combination with an anti-inflammatory or analgesic
agent.
[0161] In one embodiment, the present invention provides a method
of treating a CCR2-mediated condition or disease involving
administering to a subject a safe and effective amount of the
compound or composition of the invention, where an
anti-inflammatory or analgesic agent is also administered.
[0162] In one embodiment, the present invention provides a method
of modulating CCR2 function in a cell, where the CCR2 function in
the cell is modulated by contacting the cell with a CCR2 modulating
amount of the compound of the present invention.
[0163] In one embodiment, the present invention provides a method
of treating a CCR2-mediated condition or disease involving
administering to a subject a safe and effective amount of the
compound or composition of the invention, where the disease is
selected from the group consisting of pulmonary fibrosis,
transplantation rejection, graft-versus-host disease and
cancer.
[0164] In yet other embodiments, the present methods are directed
to the treatment of psoriasis wherein a compound or composition of
the invention is used alone or in combination with a second
therapeutic agent such as a corticosteroid, a lubricant, a
keratolytic agent, a vitamin D.sub.3 derivative, PUVA and
anthralin.
[0165] In other embodiments, the present methods are directed to
the treatment of atopic dermatitis using a compound or composition
of the invention either alone or in combination with a second
therapeutic agent such as a lubricant and a corticosteroid.
[0166] In further embodiments, the present methods are directed to
the treatment of asthma using a compound or composition of the
invention either alone or in combination with a second therapeutic
agent such as a P2-agonist and a corticosteroid.
[0167] In yet another aspect of the invention, the compounds of the
invention can be used in a variety of non-pharmaceutical in vitro
and in vivo application. For example, the compounds of the
invention may be labeled and used as probes for the detection and
localization of CCR2 receptor (cell preparations or tissue sections
samples). The compounds of the invention may also be used as
positive controls in assays for CCR2 receptor activity, i.e., as
standards for determining the ability of a candidate agent to bind
to CCR2 receptor, or as radiotracers for positron emission
tomography (PET) imaging or for single photon emission computerized
tomography (SPECT). Such methods can be used to characterize CCR2
receptors in living subjects. For example, a CCR2 receptor
modulator may be labeled using any of a variety of well known
techniques (e.g., radiolabeled with a radionuclide such as
tritium), and incubated with a sample for a suitable incubation
time (e.g., determined by first assaying a time course of binding).
Following incubation, unbound compound is removed (e.g., by
washing), and bound compound detected using any method suitable for
the label employed (e.g., autoradiography or scintillation counting
for radiolabeled compounds; spectroscopic methods may be used to
detect luminescent groups and fluorescent groups). As a control, a
matched sample containing labeled compound and a greater (e.g.,
10-fold greater) amount of unlabeled compound may be processed in
the same manner. A greater amount of detectable label remaining in
the test sample than in the control indicates the presence of CCR2
receptor in the sample. Detection assays, including receptor
autoradiography (receptor mapping) of CCR2 receptor in cultured
cells or tissue samples may be performed as described by Kuhar in
sections 8.1.1 to 8.1.9 of Current Protocols in Pharmacology (1998)
John Wiley & Sons, New York.
[0168] The compounds provided herein may also be used within a
variety of well known cell separation methods. For example,
modulators may be linked to the interior surface of a tissue
culture plate or other support, for use as affinity ligands for
immobilizing and thereby isolating, CCR2 receptors (e.g., isolating
receptor-expressing cells) in vitro. In one preferred application,
a modulator linked to a fluorescent marker, such as fluorescein, is
contacted with the cells, which are then analyzed (or isolated) by
fluorescence activated cell sorting (FACS).
[0169] In FIG. 1, structures and activity are provided for
representative compounds described herein. Activity is provided as
follows for the binding assay as described herein: +, 501
nM.ltoreq.IC50<5000 nM; ++, 101 nM.ltoreq.IC50<500 nM; and
+++, 1 nM.ltoreq.IC50.ltoreq.100 nM.
V. Examples
[0170] The following examples are offered to illustrate, but not to
limit the claimed invention.
[0171] Reagents and solvents used below can be obtained from
commercial sources such as Aldrich Chemical Co. (Milwaukee, Wis.,
USA). .sup.1H-NMR spectra were recorded on a Varian Mercury 400 MHz
NMR spectrometer. Significant peaks are provided relative to TMS
and are tabulated in the order: multiplicity (s, singlet; d,
doublet; t, triplet; q, quartet; m, multiplet) and number of
protons. Mass spectrometry results are reported as the ratio of
mass over charge, followed by the relative abundance of each ion
(in parenthesis). In the examples, a single m/e value is reported
for the M+H (or, as noted, M-H) ion containing the most common
atomic isotopes. Isotope patterns correspond to the expected
formula in all cases. Electrospray ionization (ESI) mass
spectrometry analysis was conducted on a Hewlett-Packard MSD
electrospray mass spectrometer using the HP1100 HPLC for sample
delivery. Normally the analyte was dissolved in methanol at 0.1
mg/mL and 1 microlitre was infused with the delivery solvent into
the mass spectrometer, which scanned from 100 to 1500 daltons. All
compounds could be analyzed in the positive ESI mode, using
acetonitrile/water with 1% formic acid as the delivery solvent. The
compounds provided below could also be analyzed in the negative ESI
mode, using 2 mM NH.sub.4OAc in acetonitrile/water as delivery
system.
[0172] The following abbreviations are used in the Examples and
throughout the description of the invention:
aq: aqueous; BBr.sub.3: boron tribromide; CH.sub.2Cl.sub.2 or DCM:
dichloromethane; CH.sub.3CN: acetonitrile; CH.sub.3OH or MeOH:
methanol; DAST, (Diethylamino)sulfur trifluoride; DavePhos,
2-Dicyclohexylphosphino-2'-(N,N-dimethylamino)biphenyl; 1,2-DCE,
1,2-dichloroethane; DIEA: N,N-diisopropylethylamine; DMF: dimethyl
formamide; DMP, Dess-Martin periodinane; DMSO: dimethyl sulfoxide;
equiv. or eq.: equivalents; Et.sub.3N: triethylamine; Et.sub.2O:
diethyl ether; EtOH: ethanol; EtONa, sodium ethoxide; h: hour(s);
HATU, O-(7-Azabenzotriazol-1-yl)-N,N,N,N'-tetramethyluronium
hexafluorophosphate; HCl: hydrogen chloride; H.sub.2O: water;
K.sub.2CO.sub.3: potassium carbonate; KHSO.sub.4: potassium
bisulfate; LAH, lithium aluminum hydride; LDA, lithium
diisopropylamide; MgSO.sub.4: magnesium sulfate; mL: milliliter;
NaCl: sodium chloride; NaH: sodium hydride; NaHCO.sub.3: sodium
bicarbonate; NaOEt: sodium ethoxide; NaOH: sodium hydroxide; NaOMe:
sodium methoxide; Na.sub.2SO.sub.4: sodium sulfate; NH.sub.4Cl:
ammonium chloride; NMP: N-methyl pyrrolidinone; pH: -log [H.sup.+];
POCl.sub.3, phosphoryl trichloride; PPTS, pyridinium
p-toluenesulfonate; RP-HPLC, reversed phase high pressure liquid
chromatography; RT, room temperature; TEA, triethylamine; TFA:
trifluoroacetic acid; TFAA, trifluoroacetic anhydride; THF:
tetrahydrofuran; TLC: thin layer chromatography
[0173] Compounds within the scope of this invention can be
synthesized as described below, using a variety of reactions known
to the skilled artisan. One skilled in the art will also recognize
that alternative methods may be employed to synthesize the target
compounds of this invention, and that the approaches described
within the body of this document are not exhaustive, but do provide
broadly applicable and practical routes to compounds of
interest.
[0174] Certain molecules claimed in this patent can exist in
different enantiomeric and diastereomeric forms and all such
variants of these compounds are claimed.
[0175] The detailed description of the experimental procedures used
to synthesize key compounds in this text lead to molecules that are
described by the physical data identifying them as well as by the
structural depictions associated with them.
[0176] Those skilled in the art will also recognize that during
standard work up procedures in organic chemistry, acids and bases
are frequently used. Salts of the parent compounds are sometimes
produced, if they possess the necessary intrinsic acidity or
basicity, during the experimental procedures described within this
patent.
EXAMPLES
Example 1
Synthesis of
[(2S,5S)-5-[4-(4-fluorophenyl)-1-piperidyl]-2-methyl-tetrahydropyran-2-yl-
]-[3-(trifluoromethyl)-7,8-dihydro-5H-1,6-naphthyridin-6-yl]methanone
and
[(2R,5R)-5-[4-(4-fluorophenyl)-1-piperidyl]-2-methyl-tetrahydropyran-2-yl-
]-[3-(trifluoromethyl)-7,8-dihydro-5H-1,6-naphthyridin-6-yl]methanone
##STR00024##
[0178] Step a:
[0179] A microwave vial was charged with freshly distilled acrolein
(40.0 g, 48.0 mL, 710 mmol) and hydroquinone (50 mg). The mixture
was heated for 70 min at 140.degree. C., then for 30 min at
150.degree. C. Afterwards, the contents of the vial were filtered,
washed with CH.sub.2Cl.sub.2 and concentrated under reduced
pressure. The resulting 3,4-dihydro-2H-pyran-2-carboxaldehyde (20.0
g, 50%) was pure enough to be immediately used in the next
step.
[0180] Step b:
[0181] A solution of AgNO.sub.3 (65 g, 383 mmol) in water (400 mL)
was added to a stirred solution of
3,4-dihydro-2H-pyran-2-carbaldehyde (13 g, 116 mmol) in ethanol
(300 mL), followed by the slow addition of a solution of KOH (43 g,
766 mmol) in water (300 mL) over 1 hour. The mixture was filtered
and evaporated. The residue was extracted with ether. The aqueous
layer was adjusted to pH=3 with 6 N HCl and extracted with MTBE
(2.times.200 mL). The organic layer was evaporated and the residue
was treated with 1 N NaOH until the pH=12. The mixture was
co-evaporated with methanol to dryness to give
3,4-dihydro-2H-pyran-2-carboxalicacid (8 g).
[0182] Step c:
[0183] To a stirred suspension of the carboxylic acid attained
above, (8.0 g, 53.0 mmol) in DMF (70 mL) at rt was added
Na.sub.2CO.sub.3 (5.0 g, 47.2 mmol) and ethyl iodide (9.9 g, 63.6
mmol). The reaction mixture was stirred for 6 h at 110.degree. C.
then allowed to cool to room temperature and diluted with H.sub.2O
(100 mL). The crude product was extracted with Et.sub.2O
(4.times.100 mL) and the combined organic layers were washed with
brine, dried over Na.sub.2SO.sub.4, filtered and concentrated under
reduced pressure to afford ethyl 3,4-dihydro-2H-pyran-2-carboxylate
(5 g, 60%) as a yellow oil.
[0184] Step d:
[0185] To a cold (-78.degree. C.) solution of ethyl
3,4-dihydro-2H-pyran-2-carboxylate (10.4 g, 66.6 mmol) in anhydrous
THF (150 mL) was added a solution of LDA in THF (2.0 M, 40 mL, 80.0
mmol). The reaction mixture was stirred at -78.degree. C. for 1
hour, treated with methyl iodide (16.6 mL, 266.4 mmol), stirred for
1 h at -78.degree. C. and allowed to warm to room temperature. The
reaction mixture was quenched with saturated aqueous ammonium
chloride and extracted with ether (2.times.100 mL). The organic
extract was washed with brine, dried over Na.sub.2SO.sub.4,
filtered, and concentrated under vacuum to afford
ethyl-3,4-dihydro-2-methyl-2H-pyran-2-carboxylate (5.1 g, 45%) as a
colorless oil. .sup.1H-NMR (400 MHz, CDCl.sub.3): .delta. 6.39 (dt,
J=1.9 Hz, 1H), 4.73-4.69 (m, 1H), 4.21 (q, J=7.5 Hz, 2H), 2.23 (dt,
J=1.9 Hz, 1H), 2.0-1.95 (m, 2H), 1.79-1.71 (m, 1H), 1.48 (s, 3H),
1.28 (t, J=7.1 Hz, 3H).
[0186] Step e:
[0187] To a stirred solution of
ethyl-3,4-dihydro-2-methyl-2H-pyran-2-carboxylate (5.1 g, 30.0
mmol) in THF (50 mL) at 0.degree. C. was added slowly BH.sub.3 in
THF (60 mL, 60.0 mmol, 1M solution). The reaction mixture was
stirred at 0.degree. C. for 4 h and the mixture was kept at
0.degree. C. for 16 h. The mixture was then treated with NaOAc
(7.98 g, 60.0 mmol) followed by H.sub.2O.sub.2 (14.0 mL, 70.0 mmol,
30% solution). The reaction mixture was stirred at 0.degree. C. for
2 h, then allowed to warm to room temperature and quenched with
saturated Na.sub.2SO.sub.3 solution. The mixture was extracted with
EtOAc (2.times.100 mL) and the combined organic layers were washed
with brine, dried over Na.sub.2SO.sub.4, filtered and concentrated
under reduced pressure. The crude product was purified by column
chromatography on silica gel, eluting with ethyl acetate-hexanes
10% to 60% ethyl acetate/hexane gradient to afford ethyl
tetrahydro-5-hydroxy-2-methyl-2H-pyran-2-carboxylate (4.43 g, 79%)
as a colorless viscous liquid.
[0188] Step f:
[0189] To a stirred solution of
ethyl-tetrahydro-5-hydroxy-2-methyl-2H-pyran-2-carboxylate (4.4 g,
23.6 mmol) in CH.sub.2Cl.sub.2 (150 mL) at 0.degree. C. was added
Dess-Martin periodinane (15.0 g, 35.4 mmol). The reaction mixture
was stirred at rt for 16 h. After completion of the reaction, the
mixture was quenched with saturated Na.sub.2S.sub.2O.sub.4
solution, then stirred for 30 min and the resulting mixture was
extracted with CH.sub.2Cl.sub.2 (2.times.150 mL) and washed with
aqueous NaHCO.sub.3 solution and brine, dried over
Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure.
The crude residue was triturated with hexanes and filtered to
afford ethyl tetrahydro-2-methyl-5-oxo-2H-pyran-2-carboxylate (3.45
g).
[0190] Step g:
[0191] To a mixed solution of ethyl
tetrahydro-2-methyl-5-oxo-2H-pyran-2-carboxylate (0.75 g, 4.07
mmol), 4-(4-fluorophenyl)piperidine (963 mg, 4.48 mmol), in
CH.sub.2Cl.sub.2 (15 mL) was added Na(OAc).sub.3BH (27.8 g, 124.05
mmol) at rt. The reaction mixture was heated at 50.degree. C. for
16 h. After completion of the reaction, the mixture was quenched
with saturated NaHCO.sub.3 solution. The resulting mixture was
extracted with CH.sub.2Cl.sub.2 (2.times.150 mL) and washed with
aqueous NaHCO.sub.3 solution and brine, dried over
Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure.
The crude product was purified by column chromatography on silica
gel, eluting with ethyl acetate-hexanes 10% to 60% ethyl
acetate/hexane gradient to afford ethyl-5-(4-(4-fluorophenyl)
piperidin-1-yl)-tetrahydro-2-methyl-2H-pyran-2-carboxylate (1.2 g,
47%). MS: (ES) m/z calculated for C.sub.20H.sub.29FNO.sub.3
[M+H].sup.+ 350.2, found 350.2.
[0192] Step h:
[0193] To a stirred solution of ethyl-5-(4-(4-fluorophenyl)
piperidin-1-yl)-tetrahydro-2-methyl-2H-pyran-2-carboxylate (1.2 g,
14.9 mmol) in MeOH (5 mL) at rt was added 4M NaOH (3.0 mL). The
reaction mixture was heated at 80.degree. C. for 16 h. After
completion of the reaction, this was allowed to cool to rt and
quenched with 2N HCl (3 mL), after which the pH=5. The aqueous
solution was extracted with CHCl.sub.3: iPrOH 3:1 (3.times.50 mL)
and the combined organic layers were washed with brine, dried over
Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure.
The crude compound (1.05 g) was recovered as a brown solid and
carried to the next step without purification. MS: (ES) m/z
calculated for C.sub.18H.sub.24FNO.sub.3 [M+H].sup.+ 321.2, found
321.2.
[0194] Step i:
[0195]
5-(4-(4-Fluorophenyl)piperidin-1-yl)-tetrahydro-2-methyl-2H-pyran-2-
-carboxylic acid (150 mg, 0.467 mmol),
3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,6-naphthyridine HCl (143
mg, 0.70 mmol), HATU (266 mg, 0.70 mmol) and diisopropylethylamine
(300 mg, 2.33 mmol) were mixed in DMF (2.5 mL). The reaction
mixture was stirred for 16 h. After completion of the reaction, the
mixture was diluted with H.sub.2O and the aqueous solution was
extracted with EtOAc (2.times.25 mL). The combined organic layers
were washed with brine, dried over Na.sub.2SO.sub.4, filtered and
concentrated under reduced pressure. The crude product was purified
by column chromatography on silica gel, eluting with ethyl
acetate-hexanes (20-100%) to give a mixture of diastereomers, which
was further purified on a preparative TLC plate (8:2 ethyl
acetate:hexanes) to separate the diastereomers. The racemic cis
isomer,
[(2S,5S)-5-[4-(4-fluorophenyl)-1-piperidyl)]-2-methyl-tetrahydrop-
yran-2-yl]-[3-(trifluoromethyl)-7,8-dihydro-5H-1,6-napthyridin-6-yl]-metha-
none and
[(2R,5R)-5-[4-(4-fluorophenyl)-1-piperidyl]-2-methyl-tetrahydropy-
ran-2-yl]-[3-(trifluoromethyl)-7,8-dihydro-5H-1,6-naphthyridin-6-yl]methan-
one, (20 mg, 10%) was isolated as a white fluffy solid: .sup.1H NMR
(400 MHz, MeOH-d.sub.4) .delta. 8.71 (s, 1H), 8.04 (s, 1H), 7.23
(dd, J=7.4, 5.5 Hz, 2H), 7.04 (dd, J=8.6, 5.5 Hz, 2H), 5.35-5.25
(m, 1H), 5.05 (dd, J=18.4, Hz, 1H), 4.72-4.62 (m, 1H), 4.40-4.35
(m, 1H), 4.30-4.22 (m, 1H), 4.15-3.90 (m, 1H), 3.70-3.55 (m, 2H),
3.50-3.40 (m, 2H), 3.28-3.05 (m, 4H), 2.95-2.80 (m, 1H), 2.75-2.60
(m, 1H), 2.28-2.20 (m, 1H), 2.15-2.05 (m, 2H), 1.95-1.75 (m, 3H),
1.53 (s, 3H). MS: (ES) m/z calculated for
C.sub.27H.sub.32F.sub.4N.sub.3O.sub.2 [M+H].sup.+ 506.2 found
506.2.
Synthesis of (2S)-2-cyclopropyl-3,4-dihydropyran-2-carboxylic
acid
##STR00025##
[0197] Step a:
[0198] A 5 L flask fitted with a magnetic stirrer was charged with
Mg turnings (106 g, 4.34 mol, 1.58 equiv) and anhydrous THF (2.00
L) and purged with N.sub.2. Mg was activated by addition of
Br.sub.2 (1 mL, 19.5 mmol) and stirring at rt for 15 min. A
solution of cyclopropyl bromide (500 g, 4.13 mol, 1.5 equiv) in
anhydrous THF (1.9 L) was added slowly over 2 h. The reaction was
kept on ice during the addition and the rate controlled so the
internal temperature was kept below 35.degree. C. After addition
the mixture was stirred at rt for 1.5 h then cooled on ice
overnight. The next day the reaction was gently warmed to
22.degree. C. to redissolve most of the precipitate and used in the
following step.
[0199] Step b:
[0200] In a 12 L flask fitted with a mechanical stirrer (rinsed
1.times. with dry THF), diethyl oxalate (373 mL, 2.75 mol, 1 equiv)
was dissolved in THF (1.00 L) and this solution was cooled in a
reagent alcohol/dry ice bath. The Grignard solution from the first
step was transferred via teflon cannula to the oxalate solution
over the course of 3.5 h, keeping the internal temperature below
-60.degree. C. The Grignard flask was rinsed with THF (100 mL) and
this was also transferred via Teflon cannula to the reaction
mixture. After addition, the reaction was stirred for an additional
1.5 h, then quenched by adding an aqueous solution of
H.sub.2SO.sub.4 (3M, 740 mL) until the pH .about.2. The reaction
was removed from the bath and allowed to warm to room temperature
and stirred overnight. To the mixture was added brine (600 mL),
H.sub.2O (500 mL), and hexanes (1.5 L). The mixture was stirred
vigorously and the layers were separated. The aqueous layer was
extracted with Et.sub.2O (2.times.400 mL), dried over MgSO.sub.4,
filtered and concentrated to give a brown oil. The oil was
distilled under vacuum (.about.20 mmHg) to give a yellow oil (318
g, 88% pure by .sup.1H NMR) which was used in the next step.
[0201] Step c:
[0202] A 12 L, 3-neck flask equipped with a mechanical stirrer and
internal thermometer was rinsed with anhydrous THF (3.times.) and
charged with [3aR-[2(3'aR*,8'aS*),
3'a.beta.,8'a.beta.]]-(+)-2,2'-methylenebis[3a,8a-dihydro-8H-indeno[1,2-d-
]oxazole] (49.56 g, 0.15 mol, 0.055 equiv) and anhydrous THF (6 L)
and 3 A molecular sieves (500 g). The mixture was purged with
N.sub.2 for 5 min, then Cu(OTf).sub.2 was added. This mixture was
stirred for 1 h then cooled in a reagent alcohol/dry ice bath. When
the internal temperature reached -50.degree. C. the ketone (467 g,
84% pure, 2.76 mol, 1 equiv) was added in THF (200 mL) over 15 min.
After addition, the reaction mixture was stirred for 45 min, during
which time the internal temperature reached -75.degree. C. The
diene (510 g, 2.96 mol, 1.07 equiv) in THF (200 mL) was added over
60 min, keeping the internal temperature below -67.degree. C. The
reaction was then stirred for 18 h in the -78.degree. C. bath. The
reaction was then removed from the bath and 1 M HCl in brine (3 L,
3.00 mol) was added and the reaction was allowed to stir for 2 h.
Hexanes (1 L) was added and the layers were then separated. The
aqueous layer was extracted with MTBE (750 mL) and the combined
organic layers were washed with brine (750 mL), dried over
MgSO.sub.4 and filtered through celite. The filtrate was
concentrated to give a dark brown oil (867 g). The oil was
dissolved in acetone (1 L) and then MTBE (2 L) was added at which
point a precipitate formed. This mixture was filtered through
celite and concentrated. The product was then dissolved in MeCN (1
L) and washed with hexanes (3.times.400 mL). The combined hexanes
were extracted with MeCN (200 mL) and the combined MeCN was then
concentrated to give the product as a dark brown oil (715 g, 86%
ee). The enantiopurity was established by HPCL using a Pirkle
Covalent (R,R) Whelk-01 5/100 Kromasil chiral column (30:70
iPrOH:hexanes as eluent at 1 mL/min, t.sub.R(major).dbd.8.4 min and
t.sub.R (minor).dbd.10.6 min).
[0203] Step d:
[0204] A 12 L 3-neck flask equipped with a mechanical stirrer and
internal thermometer was charged with CeCl.sub.3.7H.sub.2O (514 g,
1.38 mol, 0.5 equiv) and MeOH (2.2 L). Once all solids dissolved,
the mixture was cooled to 0.degree. C. and crude intermediate 1
(714 g, 2.76 mol, 1 equiv)) in CH.sub.2Cl.sub.2 (2.2 L) was added.
When the mixture reached an internal temperature of 5.degree. C.,
solid NaBH.sub.4 (124 g, 3.28 mol, 1.2 equiv) was added portionwise
(.about.10 g per portion) keeping the internal temperature below
16.degree. C. After addition, the bath was removed. H.sub.2O (3 L)
was added slowly, then 1 M citric acid (2.2 L). The layers were
separated and the aqueous was extracted with CH.sub.2Cl.sub.2
(3.times.500 mL). The combined organic was washed with
1:1H.sub.2O:brine (600 mL), then dried over MgSO.sub.4, filtered
and concentrated. The product was recovered as a dark oil (598 g)
containing 15 mol % CH.sub.2Cl.sub.2 and was kept in the freezer
overnight. This was used in the next reaction the following day
without further purification.
[0205] Step e:
[0206] In a 12 L, 3-necked flask equipped with mechanical stirrer
and an addition funnel, the product of the previous step (598 g,
92% pure, 2.59 mol) was dissolved in CH.sub.2Cl.sub.2 (4 L) and
cooled in a reagent alcohol/dry ice bath. When the internal
temperature reached -75.degree. C., Et.sub.3SiH (497 mL, 3.11 mol,
1.2 equiv.) was added and stirred for 5 min. Then
BF.sub.3.Et.sub.2O (384 mL, 3.11 mol, 1.2 equiv.) in
CH.sub.2Cl.sub.2 (768 mL) was added dropwise over 1.25 hour,
keeping the internal temperature below -68.degree. C. Cooling bath
was removed and the reaction was stirred for 1.5 h, after which
time the internal temperature reached 2.degree. C. The reaction was
diluted with H.sub.2O (1 L) and carefully quenched with saturated
NaHCO.sub.3 (2.5 L). The reaction was stirred at rt for 20 min then
the organic layer was separated, and the aqueous layer was
extracted with CH.sub.2Cl.sub.2 (500 mL). The combined organics
were dried over MgSO.sub.4, filtered and concentrated to give crude
product (558 g, >100%), which was used as is in the next
step.
[0207] Step f:
[0208] In a 5 L, 1-necked flask equipped with magnetic stirrer,
crude starting material (2.59 mol) was dissolved in toluene (2.59
L) and N.sub.2 was bubbled through the solution for 20 min.
Et.sub.3SiH (414 mL, 2.59 mol) was added followed by the
Wilkinson's catalyst (43.1 g, 47 mmol, 0.018 equiv). N.sub.2 was
bubbled through the reaction for 5 more min, then the mixture was
stirred at rt overnight. The reaction was evaporated. 4M NaOH (1.13
L, 4.53 mol, 1.75 equiv.) in MeOH:H.sub.2O (1:1) was added and the
mixture was stirred at 70.degree. C. for 1 h. The reaction was
concentrated in vacuo to .about.50% of the initial volume and
neutralized to pH=1-2 using 2M NaHSO.sub.4. The dark brown mixture
was extracted with EtOAc (3.times.1.0 L). The combined organics
were dried over MgSO.sub.4 and filtered. The solution was diluted
with EtOAc to 9.0 L total volume. The solution was stirred
vigorously while (S)-1-(1-naphthyl)ethyl amine (314 g, 1.83 mol,
0.7 equiv) in EtOH (150 mL) was added. Immediately a white solid
precipitates and the mixture was stirred for 2 h then filtered,
rinsing the solid with EtOAc (500 mL). This was dried under vacuum
to give the product as a tan solid (456 g, 88% ee). To the solid
was added 10 L MeCN, and this was refluxed for 1 h. The reaction
was filtered to give the product as an off-white solid (428 g, 91%
ee). To the solid was added MeCN (6.8 L) and H.sub.2O (270 mL) and
the mixture was refluxed for 9 h. This was allowed to cool to room
temperature. Filtered the solid, rinsing with MeCN. This was dried
in a vacuum oven to give the product as an off-white solid (352 g,
>98% ee).
[0209] Step g:
[0210] To the salt obtained above (46.0 g, 136 mmol, 1.0 equiv) was
added Et.sub.2O (500 mL) and 0.5 M aqueous HCl (326 mL, 163 mmol,
1.2 equiv). The mixture was stirred until the acid had completely
dissolved and the layers were then separated. The aqueous was
extracted with Et.sub.2O (2.times.350 mL). The combined organic was
then dried over MgSO.sub.4, filtered, and concentrated to give the
free acid which was used without purification.
Synthesis of benzyl
(2S)-2-cyclopropyl-5-oxo-tetrahydropyran-2-carboxylate
##STR00026##
[0212] Step a:
[0213] To (2S)-2-cyclopropyl-3,4-dihydro-2H-pyran-2-carboxylic acid
(5.4 g, 32.1 mmol, 1 equiv) dissolved in DMF (70 mL) was added
Na.sub.2CO.sub.3 (5.1 g, 48.1 mmol, 1.5 equiv). This was stirred at
50.degree. C. for 5 min, then benzyl bromide (4.6 mL, 38.5 mmol,
1.2 equiv) was added and the reaction was stirred at 90.degree. C.
for 3 h. The reaction was allowed to cool to room temperature, then
diluted with H.sub.2O (150 mL) and extracted with Et.sub.2O (100 mL
then 2.times.50 mL). The combined organic was dried over
MgSO.sub.4, filtered and concentrated to give the product as a
yellow oil (8.3 g).
[0214] Step b:
[0215] (2S)-2-Cyclopropyl-3,4-dihydro-2H-pyran-2-carboxylic acid
benzyl ester (8.3 g, 32 mmol, 1 equiv) was dissolved in THF (100
mL) and cooled in a brine/ice bath. BH.sub.3.THF (1M in THF, 32 mL,
32 mmol, 1 equiv) was added dropwise over 5 min. The reaction was
kept in an 8.degree. C. refrigerator overnight, then returned to a
brine/ice bath. A solution of NaOAc (5.3 g, 64 mmol, 2 equiv) in
H.sub.2O (20 mL) was then slowly added and the reaction was stirred
for 10 min. H.sub.2O.sub.2 (9.7 mL, 96 mmol, 3 equiv) was then
added in one portion, the cooling bath was removed, and the
reaction was stirred for 3 h. The reaction was diluted with brine
(50 mL) and the organic layer was separated. The aqueous layer was
extracted with Et.sub.2O (100 mL), and the combined organic was
dried over MgSO.sub.4, filtered, and concentrated. The residue was
redissolved in Et.sub.2O and a white precipitate was removed by
filtration through celite. The filtrate was concentrated to give a
yellow oil (8.8 g).
[0216] Step c:
[0217] The oil from the previous step (99 g, 359 mmol, 1 equiv) was
dissolved in CH.sub.2Cl.sub.2 (100 mL) and Dess-Martin periodinane
(82.5 g, 430 mmol, 1.5 equiv) was added in portions. After
addition, the reaction was warmed to reflux and stirred for 5.5 h.
Dess-Martin periodinane (15.2 g, 35.9 mmol, 0.1 equiv) was added
and the mixture was stirred for 1.5 h at reflux. Reaction was
allowed to cool to room temperature and stirred overnight.
Saturated aqueous NaHCO.sub.3 (3 L) was carefully added to the
reaction, then saturated aqueous Na.sub.2S.sub.2O.sub.3 (100 mL)
was added and the mixture was stirred for 1.5 h. This was then
filtered through celite, and the organic layer was separated. The
aqueous layer was extracted with CH.sub.2Cl.sub.2 (2.times.300 mL)
and the combined organic was dried over MgSO.sub.4, filtered, and
concentrated to one third of the total volume. Cyclohexane (500 mL)
was added and evaporated to one third the volume. Cyclohexane (1 L)
and celite (35 g) were added and the mixture was stirred at
40.degree. C. for 5 min. This was then filtered through celite,
washed with cyclohexane and evaporated to give a dark yellow oil
(93.5 g). A portion of this material (31.5 g) was purified by
silica gel chromatography using 8:2 hexanes:EtOAc as eluent to give
the pure product (21 g, 67%).
Example 2
Synthesis of
5-[1-[(3R,6S)-6-cyclopropyl-6-[[3-fluoro-5-(trifluoromethyl)phenyl]methyl-
carbamoyl]tetrahydropyran-3-yl]-4-piperidyl]-2-fluorobenzoic
acid
##STR00027##
[0219] Step a:
[0220] To a mixture of the
(2S)-2-cycloproyl-3,4-dihydro-2H-2-carboxylic acid (730 mg, 4.3
mmol), 3-fluoro-5-trfluoromethyl-benzylamine (1.0 g, 5.2 mmol),
diethylisopropyl amine (650 mg, 5 mmol) in DMF (10 mL) was added
HATU (2.2 g, 5.8 mmol). The resulting solution was stirred at room
temperature for 3 h. The reaction mixture was quenched with water,
extracted with ethyl acetate and washed with brine. The organic
layer was concentrated in vacuo and the residue purified by flash
chromatography (2 to 25% ethyl acetate in hexane as eluent) to give
1.2 g of
(2S)-2-cyclopropyl-N-(3-fluoro-5-(trifluoromethyl)benzyl)-3,4-dihydro-2H--
pyran-2-carboxamide (84% yield) as a light yellow syrup and was
used directly in the next step. MS: (ES) m/z calculated for
C.sub.17H.sub.18F.sub.4NO.sub.2 [M+H].sup.+ 344.1, found 344.1.
[0221] Step b:
[0222] The amide prepared above (1.2 g, 3.5 mmol) was dissolved in
THF (10 mL), cooled in an ice-bath under N.sub.2 and treated with
borane in THF (1M, 10 mL, 10 mmol). The resulting mixture was
stirred at 0.degree. C. for 4 h (monitored by LC-MS) and quenched
slowly with NaOAc trihydrate (2 g, 15 mmol) in water (6 mL),
followed by 30% H.sub.2O.sub.2 (2.5 mL). The resulting mixture was
allowed to warm up to room temperature and stirred for 1 h. The
mixture was diluted with EtOAc, washed with NaHCO.sub.3 and brine,
and dried over MgSO.sub.4, filtered and concentrated in vacuo. The
residue was dissolved in CH.sub.2Cl.sub.2 (50 mL) and treated with
Dess-Martin periodinane (3.2 g, 7.5 mmol). The mixture was stirred
at room temperature overnight and quenched with sat
Na.sub.2S.sub.2O.sub.3 solution and concentrated under reduced
pressure. The residue was diluted with EtOAc, washed with
NaHCO.sub.3, 1N HCl, brine, and dried over MgSO.sub.4, filtered and
concentrated under reduced pressure. The residue was purified by
flash chromatography (20 to 45% EtOAc in hexane as eluent) to give
0.97 g of
(2S)-2-cyclopropyl-N-(3-fluoro-5-(trifluoromethyl)benzyl)-5-oxotetrahydro-
-2H-pyran-2-carboxamide (60% yield) as a light yellow syrup and was
used directly in the next step. MS: (ES) m/z calculated for
C.sub.17H.sub.18F.sub.4NO.sub.3 [M+H].sup.+ 360.1, found 360.1.
[0223] Step c:
[0224] The ketone prepared above (140 mg, 0.39 mmol) was added to a
mixture of methyl 2-fluoro-5-(piperidin-4-yl)benzoate hydrochloride
(200 mg, 0.73 mmol) and N,N-diisopropylethylamine (650 mg, 5 mmol)
in CH.sub.2Cl.sub.2 (10 mL), followed by addition of
NaBH(OAc).sub.3 (160 mg, 0.76 mmol). The resulting mixture was
stirred at room temperature for 1 h and then at 45.degree. C. bath
overnight. The reaction mixture was cooled to room temperature,
quenched with sat NaHCO.sub.3 solution, extracted with 10% MeOH in
CH.sub.2Cl.sub.2 and dried over MgSO.sub.4. After filtration and
concentration in vacuo, the residue was purified by flash
chromatography (2 to 5% MeOH in CH.sub.2Cl.sub.2 as eluent) and
followed by preparative TLC (75% EtOAc in hexane as eluent) to give
30 mg of methyl
5-(1-((3R,6S)-6-cyclopropyl-6-(3-fluoro-5-(trifluoromethyl)benz-
ylcarbamoyl)tetrahydro-2H-pyran-3-yl)piperidin-4-yl)-2-fluorobenzoate
as a yellow foam and was used directly in the next step. MS: (ES)
m/z calculated for C.sub.30H.sub.34F.sub.5N.sub.2O.sub.4
[M+H].sup.+ 581.2, found 581.3.
[0225] Step d:
[0226] The ester prepared above (30 mg, 0.05 mmol) was dissolved in
MeOH (3 mL) and water (1 mL) and treated with LiOH monohydrate (100
mg, 2.38 mmol). The resulting mixture was stirred at at room
temperature overnight, diluted with 2 N HCl and extracted with 10%
MeOH in CH.sub.2Cl.sub.2 and dried over MgSO.sub.4. After
filtration and concentration in vacuo, the residue was purified by
preparative TLC (75% EtOAc in hexane as eluent) and followed by
reverse phase HPLC (C18 column, acetonitrile-H.sub.2O with 0.1% TFA
as eluent) to give 25 mg of title compound as a white solid.
.sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 8.84 (br, 1H), 7.83 (dd,
J=2.5, 6.9 Hz, 1H), 7.49 (dt, J=2.1, 7.9 Hz, 2H), 7.36 (dd, J=7.9,
9.3 Hz, 2H), 7.19 (dd, J=8.5, 10.6 Hz, 1H), 4.68 (dd, J=7.1, 15.4
Hz, 1H), 4.36 (dd, J=5.4, 15.4 Hz, 1H), 4.22-4.18 (m, 1H),
3.66-3.62 (m, 3H), 3.40-3.18 (m, 4H), 2.98-2.93 (m, 1H), 2.69-2.63
(m, 1H), 2.27-2.10 (m, 3H), 1.97-1.93 (m, 2H), 1.75-1.57 (m, 2H),
1.17-1.06 (m, 1H), 0.70-0.65 (m, 1H), 0.60-0.36 (m, 3H). MS: (ES)
m/z calculated for C.sub.29H.sub.32F.sub.5N.sub.2O.sub.4
[M+H].sup.+ 567.2, found 567.3.
Example 3
Synthesis of
3-[(3S,4R)-1-[(3R,6S)-6-cyclopropyl-6-[[3-fluoro-5-(trifluoromethyl)pheny-
l]methylcarbamoyl]tetrahydropyran-3-yl]-3-methyl-4-piperidyl]benzoic
acid and
3-[(3R,4S)-1-[(3R,6S)-6-cyclopropyl-6-[[3-fluoro-5-(trifluoromethyl)p-
henyl]methylcarbamoyl]tetrahydropyran-3-yl]-3-methyl-4-piperidyl]benzoic
acid
##STR00028##
[0228] Step a:
[0229] To a mixture of 3-(ethoxycarbonyl)phenylboronic acid (4.0 g,
20.6 mmol), tert-butyl
4-(trifluoromethylsulfonyloxy)-5,6-dihydropyridine-1(2H)-carboxylate
(4.0 g, 12 mmol), K.sub.2CO.sub.3 (4.2 g, 30 mmol), and LiCl (4.3
g, 100 mmol) in p-dioxane (75 mL) and water (15 mL) was added
Pd(PPh.sub.3).sub.4 (600 mg, 0.5 mmol). The resulting mixture was
purged with N.sub.2 and then heated to 100.degree. C. under N.sub.2
for 3 h. After cooling to room temperature, the mixture was diluted
with EtOAc and washed with brine. The organic layer was
concentrated in vacuo and the residue was purified by flash
chromatography (2 to 25% ethyl acetate in hexane as eluent) to give
2.4 g of tert-butyl
4-(3-(ethoxycarbonyl)phenyl)-5,6-dihydropyridine-1(2H)-carboxylate
(60% yield) as a light yellow syrup. This was dissolved in EtOH (40
mL), charged with 10% Pd/C (400 mg), and hydrogenated overnight
under a H.sub.2-balloon at room temperature. The mixture was
filtered through celite and the filtrate was concentrated in vacuo
to give 2.4 g of tert-butyl
4-(3-(ethoxycarbonyl)phenyl)piperidine-1-carboxylate as a light
yellow syrup, which was used directly in the next step. MS: (ES)
m/z calculated for C.sub.19H.sub.28NO.sub.4 [M+H].sup.+ 334.2,
found 334.2.
[0230] Step b:
[0231] The Boc-piperidine prepared above (1.4 g, 4.2 mmol) was
dissolved in EtOAc (70 mL) and added slowly (over 30 min) to a
solution of RuO.sub.4 (200 mg, 1.21 mmol) and NaIO.sub.4 (5 g, 23.4
mmol) in water (20 mL). The resulting dark mixture was stirred at
room temperature for 3 h. After quenching the reaction with 10%
Na.sub.2S.sub.2O.sub.3 solution (100 mL), the mixture was extracted
with EtOAc and washed with NaHCO.sub.3, brine, and dried over
MgSO.sub.4. After filtration and concentration in vacuo, the
residue was purified by flash chromatography (5 to 25% EtOAc in
hexane as eluent) to give tert-butyl
4-(3-(ethoxycarbonyl)phenyl)-2-oxopiperidine-1-carboxylate (900 mg)
as a light yellow syrup, which was used directly in the next step.
MS: (ES) m/z calculated for C.sub.19H.sub.25NNaO.sub.5
[M+Na].sup.+370.2, found 370.1.
[0232] Step c:
[0233] The Boc-piperidone prepared above (900 mg, 2.6 mmol) was
dissolved in THF (5 mL) and added dropwise to a solution of LiHMDS
(1M, 3 mL, 3 mmol) in THF under N.sub.2 at -78.degree. C. The
resulting solution was stirred at -78.degree. C. for 30 min,
followed by addition of MeI (710 mg, 5 mmol) and then the
temperature was allowed to slowly warm up to -20.degree. C. over 3
h. After quenching the reaction with saturated aqueous NH.sub.4Cl,
the mixture was extracted with EtOAc and washed with brine. The
organic layer was concentrated in vacuo and the residue was
purified by flash chromatography (5 to 25% EtOAc in hexane as
eluent) to give (.+-.)-(3S,4R)-tert-butyl
4-(3-(ethoxycarbonyl)phenyl)-3-methyl-2-oxopiperidine-1-carboxylate
(230 mg) as a light yellow syrup, which was used directly in the
next step. MS: (ES) m/z calculated for C.sub.20H.sub.27NNaO.sub.5
[M+Na].sup.+384.2, found 384.1.
[0234] Step d:
[0235] The Boc-methylpiperidone prepared above (230 mg, 0.63 mmol)
was dissolved in CH.sub.2Cl.sub.2 (5 mL) and treated with 4N HCl in
dioxane (5 mL, 20 mmol). After stirring at room temperature for 2
h, the solvent was concentrated in vacuo and the residue was
dissolved in THF (5 mL). To this solution cooled in an ice-bath was
added slowly BH.sub.3.Me.sub.2S (2M, 1 mL). The resulting mixture
was stirred at 0.degree. C. for 6 h, followed by standing at
4.degree. C. for 72 h. The solvent was concentrated in vacuo and
the residue dissolved in EtOH (5 mL) with conc. HCl (0.1 mL, 12
mmol). The mixture was stirred at 50.degree. C. for 30 min. The
organic layer was concentrated in vacuo and the residue was
purified by flash chromatography (2 to 20% MeOH in CH.sub.2Cl.sub.2
with 2% NH.sub.4OH as eluent) to give 110 mg of (.+-.)-ethyl
3-((3S,4R)-3-methylpiperidin-4-yl)benzoate as a light yellow foam,
which was used directly in the next step. MS: (ES) m/z calculated
for C.sub.15H.sub.22NO.sub.2 [M+H].sup.+ 248.2, found 248.2.
[0236] Step e:
[0237] The amine prepared above (110 mg, 0.44 mmol) was dissolved
in CH.sub.2Cl.sub.2 (10 mL) and charged with
N,N-diisopropylethylamine (390 mg, 3 mmol),
(2S)-2-cyclopropyl-N-(3-fluoro-5-(trifluoromethyl)benzyl)-5-oxotetrahydro-
-2H-pyran-2-carboxamide (120 mg, 0.33 mmol) and followed by
addition of NaBH(OAc).sub.3 (110 mg, 0.52 mmol). The resulting
mixture was stirred at room temperature for 1 h and then at
45.degree. C. overnight. The reaction mixture was cooled to room
temperature, quenched with saturated aqueous NaHCO.sub.3, extracted
with 10% MeOH in CH.sub.2Cl.sub.2 and dried over MgSO.sub.4. After
filtration and concentration in vacuo, the residue was purified by
flash chromatography (2 to 5% MeOH in CH.sub.2Cl.sub.2 as eluent)
and by preparative TLC (60% EtOAc in hexane as eluent) to give a
mixture (75 mg) of ethyl
3-[(3S,4R)-1-[(3R,6S)-6-cyclopropyl-6-[[3-fluoro-5-(trifluoromethyl)pheny-
l]methylcarbamoyl]tetrahydropyran-3-yl]-3-methyl-4-piperidyl]benzoate
and ethyl
3-[(3R,4S)-1-[(3R,6S)-6-cyclopropyl-6-[[3-fluoro-5-(trifluoromethyl-
)phenyl]methylcarbamoyl]tetrahydropyran-3-yl]-3-methyl-4-piperidyl]benzoat-
e as a yellow foam and was used directly in the next step. MS: (ES)
m/z calculated for C.sub.32H.sub.39F.sub.4N.sub.2O.sub.4
[M+H].sup.+ 591.3, found 591.2.
[0238] Step f:
[0239] To a solution of the ester prepared above (75 mg, 0.13 mmol)
in MeOH (5 mL) and water (2 mL) was added LiOH monohydrate (210 mg,
5 mmol). The resulting mixture was stirred at 80.degree. C. for 2
h, diluted with 2 N HCl and extracted with 10% MeOH in
CH.sub.2Cl.sub.2 and dried over MgSO.sub.4. After filtration and
concentration in vacuo, the residue was purified by preparative TLC
(20% MeOH in EtOAc as eluent) and then by reverse phase HPLC (C18
column, acetonitrile-H.sub.2O with 0.1% TFA as eluent) to give 30
mg of title compound (mixture of diastereomers) as an off-white
solid. .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 8.87 (br, 1H),
7.85-7.91 (m, 2H), 7.27-7.51 (m, 5H), 4.68 (ddd, J=4.0, 7.0, 15.6
Hz, 1H), 4.37 (dt, J=4.6, 15.4 Hz, 1H), 4.26-4.20 (m, 1H),
3.75-3.55 (m, 3H), 3.34-3.15 (m, 3H), 2.90 (q, J=11.9 Hz, 1H),
2.70-2.63 (m, 1H), 2.57-2.52 (m, 1H), 2.33-2.20 (m, 1H), 2.10-1.96
(m, 2H), 1.72-1.55 (m, 3H), 1.15-1.06 (m, 1H), 0.77 (d, J=8.0 Hz,
3H), 0.76-0.67 (m, 1H), 0.61-0.38 (m, 3H). MS: (ES) m/z calculated
for C.sub.30H.sub.35F.sub.4N.sub.2O.sub.4 [M+H]+ 563.3, found
563.3.
Example 4
Synthesis of
5-[(2S)-4-[(3R,6S)-6-cyclopropyl-6-[[3-fluoro-5-(trifluoromethyl)phenyl]m-
ethylcarbamoyl]tetrahydropyran-3-yl]-2-methyl-piperazin-1-yl]-2-fluoro-ben-
zoic acid
##STR00029##
[0241] Step a:
[0242] To a mixture of (S)-methyl
2-fluoro-5-(2-methylpiperazin-1-yl)benzoate di-HCl salt (150 mg,
0.46) and N,N-diisopropylethylamine (390 mg, 3 mmol) in
CH.sub.2C.sub.2(10 mL) were added
(2S)-2-cyclopropyl-N-(3-fluoro-5-(trifluoromethyl)benzyl)-5-oxotetrahydro-
-2H-pyran-2-carboxamide (150 mg, 0.42 mmol) and NaBH(OAc).sub.3
(211 mg, 1 mmol). The resulting mixture was stirred at room
temperature for 1 h and then at 45.degree. C. overnight. The
reaction mixture was cooled to room temperature, quenched with
saturated aqueous NaHCO.sub.3, extracted with 10% MeOH in
CH.sub.2Cl.sub.2 and dried over MgSO.sub.4. After filtration and
concentration in vacuo, the residue was purified by flash
chromatography (2 to 5% MeOH in CH.sub.2Cl.sub.2 as eluent) and
then by preparative TLC (50% EtOAc in hexane as eluent) to give
methyl
5-[(2S)-4-[(3R,6S)-6-cyclopropyl-6-[[3-fluoro-5-(trifluoromethyl)phenyl]m-
ethylcarbamoyl]tetrahydropyran-3-yl]-2-methyl-piperazin-1-yl]-2-fluoro-ben-
zoate (60 mg) as a yellow foam that was used directly in the next
step. MS: (ES) m/z calculated for
C.sub.30H.sub.35F.sub.5N.sub.3O.sub.4 [M+H].sup.+ 596.3, found
596.4.
[0243] Step b:
[0244] To a solution of the ester prepared above (60 mg, 0.10 mmol)
in MeOH (5 mL) and water (2 mL) was added LiOH monohydrate (210 mg,
5 mmol). The resulting mixture was stirred at 80.degree. C. for 2
h, diluted with 2 N HCl and extracted with 10% MeOH in
CH.sub.2Cl.sub.2 and dried over MgSO.sub.4. After filtration and
concentration in vacuo, the residue was purified by preparative TLC
(EtOAc as eluent) and then by reverse phase HPLC (C18 column,
acetonitrile-H.sub.2O with 0.1% TFA as eluent) to give 30 mg of the
title compound as an off-white solid. .sup.1H NMR (400 MHz,
CD.sub.3OD) .delta. 8.67 (br, 1H), 7.51-7.29 (m, 4H), 7.16-6.99 (m,
2H), 4.59 (dd, J=6.8, 15.4 Hz, 1H), 4.43 (dd, J=5.6, 15.4 Hz, 1H),
3.99 (ddd, J=2.2, 4.3, 11.4 Hz, 1H), 3.54 (dt, J=3.3, 6.3 Hz, 1H),
3.44-3.32 (m, 1H), 3.10-3.02 (m, 2H), 2.86-2.82 (m, 2H), 2.75-2.67
(m, 1H), 2.67-2.47 (m, 3H), 2.05-1.97 (m, 1H), 1.55 (dt, J=3.6,
13.4 Hz, 1H), 1.44-1.26 (m, 1H), 1.15-1.02 (m, 1H), 0.94 (d, J=6
Hz, 3H), 0.94-0.83 (m, 1H), 0.77-0.66 (m, 1H), 0.61-0.36 (m, 3H).
MS: (ES) m/z calculated for C.sub.29H.sub.33F.sub.5N.sub.3O.sub.4
[M+H].sup.+ 582.2, found 582.2.
Example 5
Synthesis of
5-[(2S)-4-[(3R,6S)-6-cyclopropyl-6-[4-[3-(trifluoromethyl)phenyl]piperazi-
ne-1-carbonyl]tetrahydropyran-3-yl]-2-methyl-piperazin-1-yl]-2-fluoro-benz-
oic acid
##STR00030##
[0246] Step a:
[0247] To a mixture of
(2S,5R)-2-cyclopropyl-5-[(3S)-4-(4-fluoro-3-methoxycarbonyl-phenyl)-3-met-
hyl-piperazin-1-yl]tetrahydropyran-2-carboxylic acid (70 mg, 0.17
mmol), 1-(3-(trifluoromethyl)phenyl)piperazine (70 mg, 0.3 mmol),
N,N-diisopropylethylamine (130 mg, 1 mmol) in DMF (5 mL) was added
HATU (110 mg, 0.29 mmol). The resulting solution was stirred at
room temperature for 1 h. The reaction mixture was quenched with
water, extracted with ethyl acetate and washed with brine. The
organic layer was concentrated in vacuo and the residue was
purified by flash chromatography (10 to 35% ethyl acetate in hexane
as eluent) to give 85 mg of the intermediate ester. This was
dissolved in MeOH (5 mL) and water (2 mL) and treated with LiOH
monohydrate (210 mg, 5 mmol). The resulting mixture was stirred at
room temperature for 2 h, diluted with 2 N HCl and extracted with
10% MeOH in CH.sub.2Cl.sub.12 and dried over MgSO.sub.4. After
filtration and concentration in vacuo, the residue was purified by
preparative TLC (EtOAc as eluent) and then by reverse phase HPLC
(C18 column, acetonitrile-H.sub.2O with 0.1% TFA as eluent) to give
50 mg of the title compound as an off-white solid. .sup.1H NMR (400
MHz, CD.sub.3OD) .delta. 7.18-7.80 (m, 7H), 4.20-4.40 (m, 4H),
3.20-3.98 (m, 14H), 3.01-2.88 (m, 1H), 2.63 (d, J=13.7 Hz, 1H),
2.35-2.25 (m, 1H), 1.85-1.78 (m, 1H), 1.56-1.48 (m, 1H), 1.30-1.20
(m, 1H), 1.04-0.94 (m, 3H), 0.92-0.83 (m, 1H), 0.74-0.60 (m, 2H),
0.49-0.40 (m, 1H). MS: (ES) m/z calculated for
C.sub.32H.sub.39F.sub.4N.sub.4O.sub.4 [M+H].sup.+ 619.3, found
619.2.
Synthesis of
(2S,5R)-2-cyclopropyl-5-[(3S,4R)-4-(4-fluorophenyl)-3-methoxycarbonyl-1-p-
iperidyl]tetrahydropyran-2-carboxylic acid
##STR00031##
[0249] Step a:
[0250] [(3S,4R)-4-(4-Fluoro-phenyl)-piperidin-3-yl]-methanol (3.00
g, 14.3 mmol, 1 equiv) was suspended in CH.sub.2Cl.sub.2 (10 mL)
and Boc anhydride (3.23 g, 14.8 mmol, 1.03 equiv) was added. This
was stirred for 2 h then concentrated to give the crude, which was
used in the next reaction without further purification.
[0251] Step b:
[0252] To the crude from step a (298 mg, 0.965 mmol, 1 equiv) was
added CH.sub.2Cl.sub.2 (2 mL) and Dess-Martin periodinane (449 mg,
1.06 mmol, 1.1 equiv). The reaction was stirred for 1.5 h at room
temperature. This mixture was then purified by silica gel
chromatography (hexanes:EtOAc as eluent) to give
(3S,4R)-4-(4-fluoro-phenyl)-3-hydroxymethyl-piperidine-1-carboxylic
acid tert-butyl ester (167 mg, 56%).
[0253] Step c:
[0254] To
(3S,4R)-4-(4-Fluoro-phenyl)-3-hydroxymethyl-piperidine-1-carboxy-
lic acid tert-butyl ester (167 mg, 0.543 mmol, 1 equiv) was added
t-BuOH (3.5 mL) and 2-methyl-2-butene in THF (2M, 2 mL, 4 mmol, 7
equiv). This was cooled in an ice bath and a mixture of NaClO.sub.2
(80%, 329 mg, 2.9 mmol, 5 equiv) and NaH.sub.2PO.sub.4 (535 mg,
3.88 mmol, 7 equiv) in H.sub.2O (1.8 mL) was added. The mixture was
stirred for 30 min then concentrated. The residue was partitioned
between EtOAc and H.sub.2O, separated and extracted with EtOAc
(3.times.). The organic was dried over Na.sub.2SO.sub.4, filtered,
and concentrated to give the crude (176 mg).
[0255] Step d:
[0256] To the crude from the previous step (4.17 g, 11 mmol, 1
equiv) was added MeOH (15 mL) and the solution was cooled in an ice
bath. Trimethylsilyldiazomethane in hexanes (2M, 10 mL, 20 mmol,
1.8 equiv) was added until the solution remains yellow. This was
then concentrated and purified by silica gel chromatography
(hexanes:EtOAc as eluent) to give
(3S,4R)-4-(4-Fluoro-phenyl)-piperidine-1,3-dicarboxylic acid
1-tert-butyl ester 3-methyl ester (3.69 g).
[0257] Step e:
[0258] To (3S,4R)-4-(4-Fluoro-phenyl)-piperidine-1,3-dicarboxylic
acid 1-tert-butyl ester 3-methyl ester (3.67 g, 10.9 mmol, 1 equiv)
was added CH.sub.2Cl.sub.2 (10 mL) and HCl in dioxane (4M, 10 mL,
40 mmol, 3.7 equiv). This was stirred for 1.5 h, then concentrated.
The residue was diluted with CH.sub.2Cl.sub.2 and washed with
saturated aqueous NaHCO.sub.3. The aqueous was extracted with
CH.sub.2Cl.sub.2 (2.times.), then the organic was dried over
Na.sub.2SO.sub.4, filtered and concentrated to give
(3S,4R)-4-(4-Fluoro-phenyl)-piperidine-3-carboxylic acid methyl
ester (2.55 g).
[0259] Step f:
[0260] To (3S,4R)-4-(4-fluoro-phenyl)-piperidine-3-carboxylic acid
methyl ester (2.55 g, 10.8 mmol, 1.1 equiv) was added
(2S)-2-Cyclopropyl-5-oxo-tetrahydro-pyran-2-carboxylic acid benzyl
ester (2.73 g, 9.95 mmol, 1 equiv) in dichloroethane (30 mL) and
the mixture was cooled in an ice bath. NaBH(OAc).sub.3 (3.28 g,
15.5 mmol, 1.5 equiv) was added and the reaction was then stirred
overnight at room temperature. The reaction was diluted with
CH.sub.2Cl.sub.2 and washed with saturated aqueous NaHCO.sub.3. The
aqueous was extracted with CH.sub.2Cl.sub.2(2.times.) and the
organic was then dried over Na.sub.2SO.sub.4, filtered and
concentrated. The crude was purified by silica gel chromatography
(hexanes:EtOAc as eluent) to give the product (3.74 g) as a
cis/trans mixture.
[0261] Step g:
[0262] To the mixture from step f (3.74 g, 7.54 mmol) was added
MeOH (25 mL) followed by 10% wet Degussa type Pd/C (304 mg). The
reaction was stirred under H.sub.2 for 3 h. The reaction was then
filtered through celite, rinsing with MeOH and CH.sub.2Cl.sub.2.
The solution was then concentrated and purified by silica gel
chromatography (CH.sub.2Cl.sub.2:MeOH as eluent) to give
(2S,5R)-2-cyclopropyl-5-[(3S,4R)-4-(4-fluorophenyl)-3-methoxycarbonyl-1-p-
iperidyl]tetrahydropyran-2-carboxylic acid (1.318 g, 43%) as the
pure isomer.
Example 6
Synthesis of
(3S,4R)-1-[(3R,6S)-6-cyclopropyl-6-[8-fluoro-6-(trifluoromethyl)-2,4-dihy-
dro-1,3-benzoxazine-3-carbonyl]tetrahydropyran-3-yl]-4-(4-fluorophenyl)pip-
eridine-3-carboxylic acid
##STR00032##
[0264] Step a:
[0265] To a mixture of
(2S,5R)-2-cyclopropyl-5-[(3S,4R)-4-(4-fluorophenyl)-3-methoxycarbonyl-1-p-
iperidyl]tetrahydropyran-2-carboxylic acid (100 mg, 0.25 mmol),
2-(aminomethyl)-6-fluoro-4-(trifluoromethyl)phenol (80 mg, 0.38
mmol), and N,N-diisopropylethylamine (390 mg, 3 mmol) in DMF (6 mL)
was added HATU (150 mg, 0.39 mmol). The resulting solution was
stirred at room temperature for 1 h. The reaction mixture was
quenched with water, extracted with ethyl acetate and washed with
brine. The organic layer was concentrated in vacuo and the residue
was dissolved in toluene (10 mL) and treated with paraformaldehyde
(600 mg, 20 mmol) and p-TsOH (150 mg, 0.87 mmol). The resulting
mixture was stirred at 100.degree. C. for 6 h. After cooling to
room temperature, the mixture was diluted with EtOAc and washed
with sat NaHCO.sub.3 solution, brine and dried over MgSO.sub.4.
After filtration and concentration in vacuo, the residue was
purified by flash chromatography (10% to 30% EtOAc in hexane as
eluent) to give methyl
(3S,4R)-1-[(3R,6S)-6-cyclopropyl-6-[8-fluoro-6-(trifluoromethyl)-2-
,4-dihydro-1,3-benzoxazine-3-carbonyl]tetrahydropyran-3-yl]-4-(4-fluorophe-
nyl)piperidine-3-carboxylate (80 mg) as a yellow foam, which was
used directly in the next step. MS: (ES) m/z calculated for
C.sub.31H.sub.34F.sub.5N.sub.2O.sub.5 [M+H].sup.+ 609.2, found
609.3.
[0266] Step b:
[0267] The ester prepared above (80 mg, 0.13 mmol) was dissolved in
MeOH (5 mL) and water (2 mL) and then treated with LiOH monohydrate
(210 mg, 5 mmol). The resulting mixture was stirred at 60.degree.
C. for 1 h, diluted with 2 N HCl and extracted with 10% MeOH in
CH.sub.2Cl.sub.2 and dried over MgSO.sub.4. After filtration and
concentration in vacuo, the residue was purified by preparative TLC
(EtOAc as eluent) and then by reverse phase HPLC (C18 column,
acetonitrile-H.sub.2O with 0.1% TFA as eluent) to give 50 mg of the
title compound as an off-white solid. .sup.1H NMR (400 MHz,
CD.sub.3OD) .delta. 7.40-7.36 (m, 2H), 7.32-7.20 (m, 2H), 7.04 (td,
J=2.4, 9.1 Hz, 1H), 6.20 (br, 1H), 5.75 (br, 1H), 4.98-4.88 (m,
1H), 4.45-4.36 (m, 1H), 3.77-3.40 (m, 4H), 3.35-2.98 (m, 8H), 2.65
(dt, J=3.6, 13.7 Hz, 1H), 2.33-2.23 (m, 1H), 2.09-1.95 (m, 2H),
1.84-1.72 (m, 1H), 1.65-1.58 (m, 1H), 1.25-1.15 (m, 1H), 0.65-0.46
(m, 3H). MS: (ES) m/z calculated for
C.sub.30H.sub.32F.sub.5N.sub.2O.sub.5 [M+H]+ 595.2, found
595.3.
Synthesis of ethyl 3-[(3R,4S)-3-methyl-4-piperidyl]benzoate
##STR00033##
[0269] Step a:
[0270] 3-Carbethoxyphenylboronic acid (6.0 g, 31 mmol, 2.2 equiv)
was dissolved in dioxane (50 mL) and cooled in an ice bath.
K.sub.2CO.sub.3 (4.6 g, 33 mmol, 2.4 equiv) was added followed by
H.sub.2O (5 mL). N.sub.2 was bubbled through the solution for 2
min, then (R.sup.h(cod)C.sub.1).sub.2 (600 mg, 1.2 mmol, 0.08
equiv) and (S)-BINAP (1.68 g, 2.56 mmol, 0.18 equiv) was added.
N.sub.2 was bubbled through the solution for 5 more min, then
6-oxo-3,6-dihydro-2H-pyridine-1-carboxylic acid tert-butyl ester
(2.70 g, 14 mmol, 1 equiv) was added slowly. N.sub.2 was bubbled
through the solution for 5 more min, then the reaction was allowed
to warm to room temperature over 1 h. The reaction was heated to
45.degree. C. for 1 h, then was allowed to cool to room temperature
and stirred overnight. The reaction was diluted with EtOAc, washed
with H.sub.2O, then brine and then concentrated. The residue was
purified by silica gel chromatography (5% to 20% EtOAc in hexanes)
to give
(S)-4-(3-ethoxycarbonyl-phenyl)-2-oxo-piperidine-1-carboxylic acid
tert-butyl ester (3.5 g, 74%).
[0271] Step b:
[0272] LiHMDS (1M, 13.4 mL, 13.4 mmol, 1.3 equiv) was added to THF
(20 mL) and this solution was cooled to -60.degree. C.
(S)-4-(3-Ethoxycarbonyl-phenyl)-2-oxo-piperidine-1-carboxylic acid
tert-butyl ester (3.5 g, 10 mmol, 1 equiv) in THF was then added
over 10 min while keeping the internal temperature below
-45.degree. C. The reaction was stirred for 45 min in the
-78.degree. C. bath, then MeI (2 mL) was added. The reaction was
stirred in the bath for 3 h, then quenched by adding NH.sub.4OH and
warmed to room temperature. The mixture was extracted with EtOAc
(150 mL) and concentrated. The resulting residue was purified by
silica gel chromatography (5% to 15% EtOAc in hexanes) to give
(3R,4S)-4-(3-Ethoxycarbonyl-phenyl)-3-methyl-2-oxo-piperidine-1-carb-
oxylic acid tert-butyl ester (3.0 g, 83%).
[0273] Step c:
[0274]
(3R,4S)-4-(3-Ethoxycarbonyl-phenyl)-3-methyl-2-oxo-piperidine-1-car-
boxylic acid tert-butyl ester (3.0 g, 8.3 mmol) was dissolved in
CH.sub.2Cl.sub.2 (20 mL) and HCl in dioxane (4N, 20 mL, 80 mmol)
was added. This mixture was allowed to stir at room temperature for
4 h then concentrated to give the product, which was used directly
in the next step.
[0275] Step d:
[0276] 3-((3R,4S)-3-Methyl-2-oxo-piperidin-4-yl)-benzoic acid ethyl
ester (8.3 mmol, 1 equiv) was dissolved in THF (35 mL) and
BH.sub.3.SMe.sub.2 in THF (2M, 12 mL, 24 mmol, 3 equiv) was slowly
added. The reaction was allowed to sit in a 4.degree. C.
refrigerator for 3 days, then the solution was concentrated. The
residue was dissolved in EtOH (35 mL) and concentrated HCl (1.8 mL,
22 mmol) was added. This solution was stirred at 60.degree. C. for
2 h then concentrated. The residue was purified by silica gel
chromatography (5% to 15% MeOH in CH.sub.2Cl.sub.2) to give ethyl
3-[(3R,4S)-3-methyl-4-piperidyl]benzoate (1.2 g, 59%).
Example 7
Synthesis of
3-[(3R,4S)-1-[(3R,6S)-6-cyclopropyl-6-[8-fluoro-6-(trifluoromethyl)-2,4-d-
ihydro-1,3-benzoxazine-3-carbonyl]tetrahydropyran-3-yl]-3-methyl-4-piperid-
yl]benzoic acid
##STR00034##
[0278] Step a:
[0279] To a mixture of
(2S,5R)-2-cyclopropyl-5-[(3R,4S)-4-(3-ethoxycarbonylphenyl)-3-methyl-1-pi-
peridyl]tetrahydropyran-2-carboxylic acid (85 mg, 0.21 mmol),
2-(aminomethyl)-6-fluoro-4-(trifluoromethyl)phenol (60 mg, 0.29
mmol), N,N-diisopropylethylamine amine (390 mg, 3 mmol) in DMF (6
mL) was added HATU (120 mg, 0.32 mmol). The resulting solution was
stirred at room temperature for 1 h. The reaction mixture was
quenched with water (25 mL), extracted with ethyl acetate (50 mL)
and washed with brine (25 mL). The organic layer was concentrated
in vacuo and the residue was dissolved in toluene (10 mL) and
treated with paraformaldehyde (600 mg, 20 mmol) and p-TsOH (150 mg,
0.87 mmol) at 100.degree. C. for 6 h. After cooling to room
temperature, the mixture was diluted with EtOAc and washed with
saturated aqueous NaHCO.sub.3(20 mL), brine (20 mL) and dried over
MgSO.sub.4. After filtration and concentration in vacuo, the
residue was purified by preparative TLC (35% EtOAc in hexane as
eluent) to give ethyl
3-[(3R,4S)-1-[(3R,6S)-6-cyclopropyl-6-[8-fluoro-6-(trifluoromethyl)-2,4-d-
ihydro-1,3-benzoxazine-3-carbonyl]tetrahydropyran-3-yl]-3-methyl-4-piperid-
yl]benzoate (15 mg) as a yellow foam and this was used directly in
the next step. MS: (ES) m/z calculated for
C.sub.33H.sub.39F.sub.4N.sub.2O.sub.5 [M+H].sup.+619.3, found
619.3.
[0280] Step b:
[0281] The ester prepared above (15 mg, 0.024 mmol) was dissolved
in MeOH (5 mL) and water (2 mL) and then treated with LiOH
monohydrate (100 mg, 2.5 mmol) at 60.degree. C. for 3 h. The
reaction was diluted with 2 N HCl, extracted with 10% MeOH in
CH.sub.2Cl.sub.2 and dried over MgSO.sub.4. After filtration and
concentration in vacuo, the residue was purified by preparative TLC
(EtOAc as eluent) and followed by reverse phase HPLC (C18 column,
acetonitrile-H.sub.2O with 0.1% TFA as eluent) to give 7 mg of the
title compound as an off-white solid. .sup.1H NMR (400 MHz,
CD.sub.3OD) .delta. 7.94-7.82 (m, 2H), 7.48-7.37 (m, 4H), 6.20 (br,
1H), 5.75 (br, 1H), 4.98-4.90 (m, 1H), 4.45-4.35 (m, 1H), 3.70-3.38
(m, 4H), 3.35-3.02 (m, 4H), 2.90-2.80 (m, 1H), 2.68-2.62 (m, 1H),
2.60-2.53 (m, 1H), 2.32-1.92 (m, 3H), 1.85-1.70 (m, 1H), 1.65-1.55
(m, 1H), 1.32-1.20 (m, 2H), 0.75 (d, J=6.7 Hz, 3H), 0.65-0.46 (m,
3H). MS: (ES) m/z calculated for
C.sub.31H.sub.35F.sub.4N.sub.2O.sub.5 [M+H]+ 591.2, found
591.3.
Synthesis of 2-(aminomethyl)-6-chloro-4-(trifluoromethyl)phenol
##STR00035##
[0283] Step a:
[0284] A solution of 2-fluoro-5-(trifluoromethyl)benzonitrile (50.0
g, 265 mmol, 1 equiv) in THF (1.0 L) was treated with NaOtBu (30.5
g, 317 mmol, 1.2 equiv) for 10 min on ice and then at room
temperature overnight. The mixture was then concentrated and the
residue was diluted with H.sub.2O (400 mL) and extracted with
hexanes (2.times.300 mL). The combined organic was dried over
MgSO.sub.4, filtered and concentrated to give a yellow oil (61.7 g,
96%) that solidified upon standing. This was used in step b.
[0285] Step b:
[0286] 2-tert-Butoxy-5-trifluoromethyl-benzonitrile (20.0 g, 82.3
mmol) was dissolved in EtOH (100 mL). NH.sub.4OH (10 mL) was added,
followed by Raney Nickel slurry in water (5 mL). The reaction was
shaken under H.sub.2 (50 psi) overnight. The mixture was filtered
through celite and concentrated. The residue was dissolved in
hexanes and dried over MgSO.sub.4, filtered and concentrated to
give a yellow oil (19.8 g, 97%).
[0287] Step c:
[0288] Trifluoroacetic acid (2 mL) was added to
2-tert-butoxy-5-trifluoromethyl-benzylamine (2.5 g, 10 mmol, 1
equiv) with stirring. Triflic acid (10 mL) was carefully added to
the solution, followed by portionwise addition of
N-chlorosuccinamide (2.7 g, 20 mmol, 2 equiv). The reaction was
stirred at room temperature for 2 h, then poured into H.sub.2O and
neutralized with saturated aqueous NaHCO.sub.3. The mixture was
extracted with EtOAc (2.times.25 mL), then the combined organic was
dried over MgSO.sub.4, filtered and concentrated. The resulting
brown residue was dissolved in minimal MeOH and a gray precipitate
formed. The solid was filtered and washed with minimal MeOH to give
the product as a gray solid (850 mg, 37%).
Example 8
Synthesis of
3-[(3R,4S)-1-[(3R,6S)-6-[8-chloro-6-(trifluoromethyl)-2,4-dihydro-1,3-ben-
zoxazine-3-carbonyl]-6-cyclopropyl-tetrahydropyran-3-yl]-3-methyl-4-piperi-
dyl]benzoic acid
##STR00036##
[0290] Step a:
[0291] To a mixture of the
(2S,5R)-2-cyclopropyl-5-[(3R,4S)-4-(3-ethoxycarbonylphenyl)-3-methyl-1-pi-
peridyl]tetrahydropyran-2-carboxylic acid (80 mg, 0.19 mmol),
2-(aminomethyl)-6-fluoro-4-(trifluoromethyl)phenol (50 mg, 0.22
mmol), N,N-diisopropylethylamine (260 mg, 2 mmol) in DMF (6 mL) was
added HATU (100 mg, 0.26 mmol). The resulting solution was stirred
at room temperature for 1 h. The reaction mixture was quenched with
water, extracted with ethyl acetate and washed with brine. The
organic layer was concentrated in vacuo and the residue was
dissolved in toluene (10 mL) and treated with paraformaldehyde (600
mg, 20 mmol) and p-TsOH (150 mg, 0.87 mmol). The resulting mixture
was stirred at 100.degree. C. for 6 h. After cooling to room
temperature, the mixture was diluted with EtOAc (25 mL) and washed
with saturated aqueous NaHCO.sub.3 solution (15 mL), brine (15 mL)
and dried over MgSO.sub.4. After filtration and concentration in
vacuo, the residue was purified by preparative TLC (35% EtOAc in
hexane as eluent) give 50 mg of ethyl
3-[(3R,4S)-1-[(3R,6S)-6-[8-chloro-6-(trifluoromethyl)-2,4-dihydro-1,3-ben-
zoxazine-3-carbonyl]-6-cyclopropyl-tetrahydropyran-3-yl]-3-methyl-4-piperi-
dyl]benzoate as a yellow foam that was used directly in the next
step. MS: (ES) m/z calculated for
C.sub.33H.sub.39ClF.sub.3N.sub.2O.sub.5 [M+H].sup.+635.2, found
635.2.
[0292] Step b:
[0293] The ester prepared above (50 mg, 0.079 mmol) in MeOH (5 mL)
and water (2 mL) was treated with LiOH monohydrate (210 mg, 5 mmol)
at 60.degree. C. for 3 h. This mixture was diluted with 2 N HCl and
extracted with 10% MeOH in CH.sub.2Cl.sub.2 and dried over
MgSO.sub.4. After filtration and concentration in vacuo, the
residue was purified by reverse phase HPLC (C18 column,
acetonitrile-H.sub.2O with 0.1% TFA as eluent) to give 40 mg of the
title compound as an off-white solid. .sup.1H NMR (400 MHz,
CD.sub.3OD) .delta. 7.96-7.84 (m, 2H), 7.61-7.53 (m, 2H), 7.45 (d,
J=4.7 Hz, 2H), 6.17 (br, 1H), 5.88 (br, 1H), 4.98-4.90 (m, 1H),
4.45-4.35 (m, 1H), 3.70-3.37 (m, 4H), 3.35-3.05 (m, 4H), 2.85 (t,
J=12.2 Hz, 1H), 2.65 (td, J=3.7, 13.9 Hz, 1H), 2.54 (dt, J=4.8,
11.2 Hz, 1H), 2.34-2.26 (m, 1H), 2.16-1.92 (m, 2H), 1.85-1.70 (m,
1H), 1.65-1.55 (m, 1H), 1.32-1.20 (m, 2H), 0.75 (d, J=6.7 Hz, 3H),
0.65-0.46 (m, 3H). MS: (ES) m/z calculated for
C.sub.31H.sub.35ClF.sub.3N.sub.2O.sub.5 [M+H].sup.+ 607.2, found
607.4.
Example 9
Synthesis of
5-[(3R,4S)-1-[(3R,6S)-6-cyclopropyl-6-[[3-fluoro-5-(trifluoromethyl)pheny-
l]methylcarbamoyl]tetrahydropyran-3-yl]-3-methyl-4-piperidyl]-2-fluoro-ben-
zoic acid
##STR00037##
[0295] To a mixture of
(2S,5R)-2-cyclopropyl-5-[(3R,4S)-4-(4-fluoro-3-methoxycarbonyl-phenyl)-3--
methyl-1-piperidyl]tetrahydropyran-2-carboxylic acid (35 mg, 0.083
mmol), 3-fluoro-5-trfluoromethyl-benzylamine (40 mg, 0.2 mmol), and
N,N-diisopropylethylamine (130 mg, 1 mmol) in DMF (5 mL) was added
HATU (60 mg, 0.15 mmol). The resulting solution was stirred at room
temperature for 1 h. The reaction mixture was quenched with water
(5 mL), extracted with ethyl acetate (25 mL) and washed with brine
(25 mL). The organic layer was concentrated in vacuo and the
residue purified by preparative TLC (75% EtOAc in hexane as eluent)
to give 50 mg of the intermediate ester. This was dissolved in MeOH
(5 mL) and water (2 mL) and then treated with LiOH monohydrate (210
mg, 5 mmol). The resulting mixture was stirred at 60.degree. C. for
3 h, diluted with 2 N HCl and extracted with 10% MeOH in
CH.sub.2Cl.sub.2 and dried over MgSO.sub.4. After filtration and
concentration in vacuo, the residue was purified by reverse phase
HPLC (C18 column, acetonitrile-H.sub.2O with 0.1% TFA as eluent) to
give 50 mg of the title compound as an off-white solid. .sup.1H NMR
(400 MHz, CD.sub.3OD) .delta. 8.87 (t, J=6.3 Hz, 1H), 7.79 (dd,
J=2.4, 6.9 Hz, 1H), 7.53-7.32 (m, 3H), 7.20 (dd, J=8.5, 10.6 Hz,
1H), 4.98-4.90 (m, 1H), 4.66 (dd, J=7.0, 15.6 Hz, 1H), 4.36 (dd,
J=5.4, 15.6 Hz, 1H), 4.23-4.15 (m, 1H), 3.69-3.52 (m, 3H),
3.44-3.22 (m, 3H), 3.22-3.10 (m, 1H), 2.87 (t, J=12.0 Hz, 1H),
2.70-2.60 (m, 1H), 2.54 (dt, J=3.9, 11.8 Hz, 1H), 2.30-2.20 (m,
1H), 2.10-1.86 (m, 3H), 1.75-1.57 (m, 2H), 1.17-1.05 (m, 1H), 0.78
(d, J=6.5 Hz, 3H), 0.70-0.46 (m, 3H). MS: (ES) m/z calculated for
C.sub.30H.sub.34F.sub.5N.sub.2O.sub.4 [M+H].sup.+581.2, found
581.4.
Example 10
Synthesis of
3-[(3R,4S)-1-[(3R,6S)-6-cyclopropyl-6-[4-[3-fluoro-5-(trifluoromethyl)phe-
nyl]piperazine-1-carbonyl]tetrahydropyran-3-yl]-3-methyl-4-piperidyl]benzo-
ic acid
##STR00038##
[0297] Step a:
[0298] To a mixture of 1-bromo-3-fluoro-5-(trifluoromethyl)benzene
(1.0 g, 4.1 mmol), tert-butyl piperazine-1-carboxylate (1.0 g, 5.4
mmol), and Cs.sub.2CO.sub.3 (2.4 g, 7.4 mmol) in p-dioxane (10 mL)
was added DavePhos (120 mg, 0.3 mmol). The resulting mixture was
purged with nitrogen gas and then heated to 100.degree. C. for 13
h. After cooling to room temperature, the reaction mixture was
diluted with EtOAc and filtered through celite. The filtrate was
concentrated in vacuo and the residue was purified by flash
chromatography (2 to 25% ethyl acetate in hexane as eluent) to give
1.2 g of the intermediate Boc-piperazine. This was dissolved in
CH.sub.2Cl.sub.2 (5 mL) and treated with 4N HCl in dioxane (6 mL,
24 mmol). After stirring at room temperature for 3 h, the solvent
was concentrated in vacuo to give 1.2 g of
1-(3-fluoro-5-(trifluoromethyl)phenyl)piperazine dihydrochloride
salt as a yellow solid, which was used directly in the next step.
MS: (ES) m/z calculated for C.sub.11H.sub.13F.sub.4N.sub.2
[M+H].sup.+249.1, found 249.1.
[0299] Step b:
[0300] To a mixture of the piperazine di-HCl salt prepared above
(60 mg, 0.19 mmol),
(2S,5R)-2-cyclopropyl-5-[(3R,4S)-4-(3-ethoxycarbonylphenyl)-3-methyl-1-pi-
peridyl]tetrahydropyran-2-carboxylic acid (75 mg, 0.18 mmol), and
N,N-diisopropylethylamine (130 mg, 1 mmol) in DMF (5 mL) was added
HATU (110 mg, 0.29 mmol). The resulting solution was stirred at
room temperature for 1 h. The reaction was quenched with water,
extracted with ethyl acetate and washed with brine. The organic
layer was concentrated in vacuo and the residue purified by
preparative TLC (75% EtOAc in hexane as eluent) to give 15 mg of
the intermediate ester. This was dissolved in MeOH (5 mL) and water
(2 mL) and then treated with LiOH monohydrate (210 mg, 5 mmol) at
room temperature for 2 h, diluted with 2 N HCl, extracted with 10%
MeOH in CH.sub.2Cl.sub.2 and dried over MgSO.sub.4. After
filtration and concentration in vacuo, the residue was purified by
reverse phase HPLC (C18 column, acetonitrile-H.sub.2O with 0.1% TFA
as eluent) to give 12 mg of the title compound as an off-white
solid. .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 7.97-7.85 (m, 2H),
7.51-7.42 (m, 2H), 7.08-6.94 (m, 2H), 6.82 (d, J=13.7 Hz, 1H),
4.38-4.30 (m, 2H), 4.30-4.14 (m, 4H), 3.90-3.55 (m, 5H), 3.48-3.05
(m, 4H), 2.92-2.82 (m, 1H), 2.66-2.49 (m, 2H), 2.27-1.98 (m, 4H),
1.85-1.72 (m, 1H), 1.60-1.50 (m, 1H), 1.25-1.18 (m, 1H), 0.95-0.82
(m, 1H), 0.78 (d, J=6.5 Hz, 3H), 0.74-0.60 (m, 2H), 0.50-0.42 (m,
1H). MS: (ES) m/z calculated for
C.sub.33H.sub.40F.sub.4N.sub.3O.sub.4 [M+H].sup.+ 618.3, found
618.3.
Example 11
Synthesis of
(2S,5R)-2-cyclopropyl-N-[[3-fluoro-5-(trifluoromethyl)phenyl]methyl]-5-[4-
-hydroxy-4-(3-pyridyl)-1-piperidyl]tetrahydropyran-2-carboxamide
##STR00039##
[0302] Step a:
[0303] To a -78.degree. C. solution of 3-bromopyridine (1.58 g, 10
mmol) in 20 mL of THF was added BuLi (1.6M, 6.25 mL, 10 mmol). The
resulting mixture was stirred at -78.degree. C. for 20 min. A
solution of 1-Boc-4-piperidone (1.99 g, 10 mmol) in THF (10 mL) was
added dropwise. The reaction mixture was slowly warmed to room
temperature overnight. The reaction was quenched with aqueous
NH.sub.4Cl (10 mL) and extracted with ether (100 mL). The combined
organic was washed with brine and dried over MgSO.sub.4. After
concentration, the crude was purified by silica gel chromatography
(20% EtOA in hexane) to gave a colorless oil (1.5 g, 50%), which
was dissolved in CH.sub.2Cl.sub.2 (30 mL) and cooled to 0.degree.
C. 2.0 mL of TFA was added and the resulting mixture was stirred at
room temperature for 4 hours before it was concentrated under
reduced pressure to give a sticky oil (2.1 g, 95%), which was used
in the next step without further purification.
[0304] Step b:
[0305] The TFA salt of the amine (202 mg, 0.5 mmol) was dissolved
in 1,2-dichloroethane (2 mL) at room temperature followed by
addition of iPr.sub.2NEt (130 mg, 1 mmol). The resulting mixture
was stirred at room temperature for 30 min. A solution of the
ketone (104 mg, 0.3 mmol) in 1,2-dichloroethane (1.0 mL) was added
followed by addition of NaBH(OAc).sub.3 (318 mg, 1.5 mmol). The
resulting mixture was stirred at room temperature overnight. The
reaction was quenched by addition of 5 mL of aqueous NaHCO.sub.3
and extracted with 30 mL of CH.sub.2Cl.sub.2. The combined organic
was washed with brine and dried over MgSO.sub.4. After
concentration, the crude was purified by reverse phase preparative
HPLC to give the desired product (12 mg). .sup.1H NMR (400 MHz,
Chloroform-d) .delta. 8.75 (s, 1H), 7.37 (s, 1H), 7.22 (d, J=8.7
Hz, 2H), 7.00-6.84 (m, 2H), 4.69 (dd, J=15.6, 7.1 Hz, 1H), 4.42
(dd, J=15.6, 5.6 Hz, 1H), 3.91 (ddd, J=10.9, 4.4, 2.2 Hz, 1H), 3.35
(t, J=10.7 Hz, 1H), 3.08-2.68 (m, 4H), 2.69-2.56 (m, 1H), 2.50-1.96
(m, 6H), 1.98-1.88 (m, 1H), 1.75-1.69 (m, 2H), 1.55-1.35 (m, 2H),
1.12 (tt, J=8.5, 5.5 Hz, 1H), 0.61-0.35 (m, 4H). MS: (ES) m/z
calculated for C.sub.27H.sub.32F.sub.4N.sub.3O.sub.3 [M+1]522.2,
found 522.2.
Example 12
Synthesis of
2-[3-[[[(2S,5R)-2-cyclopropyl-5-[4-(4-fluorophenyl)-1-piperidyl]tetrahydr-
opyran-2-carbonyl]amino]methyl]-5-(trifluoromethyl)phenoxy]acetic
acid
##STR00040##
[0307] Step a:
[0308] To a solution of 3-bromo-5-trifluoromethylphenol (7.2 g, 30
mmol) in DMF (100 mL) was added Zn(CN).sub.2 (3.51 g, 30 mmol) and
Pd(PPh.sub.3).sub.4(3.5 g, 6 mmol). The resulting mixture was
stirred at 100.degree. C. under nitrogen atmosphere for 4 hours.
EtOAc (250 mL) was added and the mixture was washed with water
(2.times.50 mL) and brine (100 mL) before it was dried over
MgSO.sub.4 and concentrated under reduced pressure. The residue was
purified by silica gel chromatography (20% EtOA in hexane) to give
a colorless oil (3.36 g, 60%).
[0309] Step b:
[0310] To a solution of 3-cyano-5-trifluoromethylphenol (1.87 g, 10
mmol) in DMF (20 mL) was added ethyl, 2-bromoacetate (2.5 g, 15
mmol), Cs.sub.2CO.sub.3 (6.5 g, 20 mmol) and NaI (1.5 g, 10 mmol).
The resulting mixture was stirred at 100.degree. C. overnight
before it was cooled down to room temperature. EtOAc (250 mL) was
added and the mixture was washed with water (2.times.50 mL) and
brine (100 mL) before it was dried over MgSO.sub.4 and concentrated
under reduced pressure. The residue was purified by silica gel
chromatography (10% EtOA in hexane) to give a colorless oil (2.18
g, 80%).
[0311] Step c:
[0312] To a solution of the cyanide (1.35 g, 5 mmol) in EtOH (20
mL) was added Raney-nickel slurry (1 mL) and NH.sub.4OH (2 ml). The
resulting suspension was stirred at room temperature under 45 psi
of hydrogen for 4 hours. The reaction mixture was filtered through
a pad of Celite and concentrated under reduced pressure to give a
light blue oil (1.1 g) that was used in the next step without
further purification.
[0313] Step d:
[0314] To a solution of
(2S,5R)-2-cyclopropyl-5-[4-(4-fluorophenyl)-1-piperidyl]tetrahydropyran-2-
-carboxylic acid acid intermediate (52 mg, 0.149 mmol) in DMF (2
mL) was added the amine from step c (100 mg), triethylamine (0.5
ml) and HATU (190 mg). The resulting mixture was stirred at room
temperature overnight. EtOAc (20 mL) was added and the mixture was
washed with saturated aqueous NaHCO.sub.3 (2.times.5 mL) and brine
(5 mL). The combined organic was dried over MgSO.sub.4 and
concentrated under reduced pressure. The residue was purified by
preparative TLC (100% EtOAc) to give the desired product (35
mg).
[0315] Step e:
[0316] A mixture of the ester (35 mg) in THF (1 mL) and 1N aqueous
LiOH (1 mL) was stirred at room temperature overnight. 1N aqueous
HCl was slowly added to adjust the pH of the mixture to 7.0 and
then the mixture was extracted with 2:1 CHCl.sub.3:iPrOH (20 mL).
The organic was dried over MgSO.sub.4 and concentrated under
reduced pressure. The residue was purified using reverse phase HPLC
to give the desired product (15 mg). .sup.1H NMR (400 MHz,
CD.sub.3OD) .delta. 7.51 (brs, 1H), 7.40-7.30 (m, 2H), 7.24-7.18
(m, 2H), 7.02-6.95 (m, 2H), 4.55, 4.44 (ABq, J=15.4 Hz, 2H), 3.94
(ddd, J=11.2, 4.4, 2.2 Hz, 1H), 3.39 (t, J=11.0 Hz, 1H), 3.00-2.90
(m, 4H), 2.58-2.41 (m, 3H), 2.39-2.24 (m, 2H), 2.00-1.92 (m, 1H),
1.82-1.60 (m, 4H), 1.52 (td, J=13.5, 3.7 Hz, 1H), 1.40-1.26 (m,
1H), 1.10-1.02 (m, 1H), 0.70-0.64 (m, 1H), 0.57-0.49 (m, 1H),
0.46-0.30 (m, 3H). MS: (ES) m/z calculated for
C.sub.30H.sub.35F.sub.4N.sub.2O.sub.5 [M+H].sup.+ 579.2, found
579.6.
Synthesis of
(2S,5R)-2-cyclopropyl-5-[4-(4-fluoro-3-methoxycarbonyl-phenyl)-1-piperidy-
l]tetrahydropyran-2-carboxylic acid
##STR00041##
[0318] Step a:
[0319] 2-Fluoro-5-piperidin-4-yl-benzoic acid methyl ester
hydrochloride (5.0 g, 18 mmol, 1 equiv) and diisopropylethylamine
(3.2 mL, 18.2 mmol, 1 equiv) in 1,2-dichloroethane (100 mL) was
stirred at reflux until homogeneous. The solution was allowed to
cool to room temperature and
(2S)-2-Cyclopropyl-5-oxo-tetrahydro-pyran-2-carboxylic acid benzyl
ester (5.0 g, 18.2 mmol, 1 equiv) was added followed by
NaBH(OAc).sub.3 (7.7 g, 36.4 mmol, 2 equiv). This was stirred at
room temperature for 2 days then neutralized with saturated aqueous
NaHCO.sub.3. The organic layer was separated, dried over
MgSO.sub.4, filtered and concentrated to give the crude.
[0320] Step b:
[0321] To the crude from step a (18.2 mmol) was added MeOH (100 mL)
and 10% Pd/C (50% wet, 7.7 g, 3.6 mmol, 0.2 equiv). This was
stirred under a balloon filled with H.sub.2. When complete, the
reaction was filtered through celite and the filter cake was rinsed
with 1:1 MeOH:AcOH. The solution was then concentrated. The residue
was diluted with acetone. The resulting white precipitate is
filtered to give
(2S,5R)-2-cyclopropyl-5-[4-(4-fluoro-3-methoxycarbonyl-phenyl)-1-piperidy-
l]tetrahydropyran-2-carboxylic acid (1.1 g, 15%) as the pure cis
diastereomer.
Synthesis of
5-chloro-7-(trifluoromethyl)-1,2,3,4-tetrahydroisoquinoline
##STR00042##
[0323] Step a:
[0324] To a 0.degree. C. solution of
2-chloro-4-trifluoromethylbenzaldehyde (1.8 g, 9 mmol) in EtOH (20
mL) was added CH.sub.3NO.sub.2 (610 mg, 10 mmol). Aqueous NaOH
(10M, 1 mL) was added dropwise and the resulting mixture was
stirred at room temperature for one hour. The reaction was quenched
with 1N HCl (10 mL) and extracted with EtOAc (200 mL). The combined
organic was washed with aq. NaHCO.sub.3 and brine before it was
dried over MgSO.sub.4 and concentrated under reduced pressure to
give a colorless oil, which was used in the next step without
further purification.
[0325] Step b:
[0326] The oil obtained from the previous step was dissolved in
CH.sub.2Cl.sub.2 (20 mL) and the solution was cooled to 0.degree.
C. followed by addition of triethylamine (3.3 g, 30 mmol). MsCl
(3.42 g, 30 mmol) was added dropwise and the resulting mixture was
stirred at room temperature for 30 minutes before it was diluted
with CH.sub.2Cl.sub.2 (200 mL). The combined organic was washed
with aqueous NaHCO.sub.3 and brine before it was dried over
MgSO.sub.4 and concentrated under reduced pressure. The residue was
purified by silica gel chromatography (5% EtOAc in hexane) to give
1.35 g of the desired product as a yellow solid.
[0327] Step c:
[0328] To a 0.degree. C. solution of product from the previous step
(625 mg, 2.5 mmol) in THF (20 mL) was added dropwise a solution of
LiAlH.sub.4 in THF (1M, 8 mL). The resulting mixture was warmed to
50.degree. C. for five hours. The mixture was cooled to 0.degree.
C. and slowly quenched with water followed by filtration through a
pad of celite and washed with MTBE. The filtrate was dried and
concentrated to give a colorless oil, which was used in the next
step.
[0329] Step d:
[0330] The oil from step c was dissolved in CH.sub.2Cl.sub.2 (20
mL) and the solution was cooled to 0.degree. C. Trifluoroacetic
anhydride (630 mg, 3 mmol) was added dropwise followed by stirring
at room temperature for one hour. The mixture was diluted with of
CH.sub.2Cl.sub.2 (100 mL) and washed with aqueous NaHCO.sub.3 and
brine before it was dried over MgSO.sub.4 and concentrated under
reduced pressure. The residue was purified by silica gel
chromatography (10% EtOAc in hexane) to give 600 mg of the desired
product as a white solid.
[0331] Step e:
[0332] To a solution of TFA-protected amine from the previous step
(1.0 g, 3.13 mmol) in a mixture of H.sub.2SO.sub.4 (4 mL) and HOAc
(8 mL) was added paraformaldehyde (290 mg). The resulting mixture
was stirred at room temperature for 72 hours before it was diluted
with EtOAc (200 mL). The mixture was washed with water and brine
and dried over MgSO.sub.4 followed by concentration under reduced
pressure. The residue was purified by silica gel chromatography
(10% EtOAc in hexane) to give 300 mg of the desired product as a
white solid.
[0333] Step f:
[0334] The solid from step e was dissolved in a mixture of MeOH (6
mL) and water (2 mL) followed by addition of K.sub.2CO.sub.3 (300
mg). The resulting mixture was stirred at room temperature
overnight. 200 mL of CHCl.sub.3: iPrOH (2:1) was added and the
organic was separated and dried over MgSO.sub.4 followed by
concentration under reduced pressure. The residue was purified by
silica gel chromatography (50% EtOAc in hexane) to give 190 mg of
the desired product as a colorless oil.
Example 13
Synthesis of
5-[(3R,4S)-1-[(3R,6S)-6-[5-chloro-7-(trifluoromethyl)-3,4-dihydro-1H-isoq-
uinoline-2-carbonyl]-6-cyclopropyl-tetrahydropyran-3-yl]-3-methyl-4-piperi-
dyl]-2-fluoro-benzoic acid
##STR00043##
[0336] Step a:
[0337] To a solution of the acid intermediate (52 mg, 0.149 mmol)
in DMF (2 mL) was added the amine (100 mg), triethylamine (0.5 ml)
and HATU (190 mg). The resulting mixture was stirred at room
temperature overnight. EtOAc (20 mL) was added and the mixture was
washed with saturated aqueous NaHCO.sub.3(2.times.5 mL) and brine
(5 mL). The combined organic was dried over MgSO.sub.4 and
concentrated under reduced pressure. The residue was purified by
preparative TLC (100% EtOAc) to give 25 mg of the desired
product.
[0338] Step b:
[0339] The product from step a was treated with aqueous LiOH in THF
as previously described to give the final product (12 mg). .sup.1H
NMR (400 MHz, CD.sub.3OD) .delta. 7.43-7.40 (m, 1H), 7.34-7.26 (m,
1H), 7.20-7.14 (m, 1H), 7.04-6.96 (m, 2H), 5.49-5.16 (m, 1H),
4.85-4.52 (m, 1H), 4.25-3.96 (m, 2H), 3.30-3.18 (m, 1H), 3.12-2.85
(m, 4H), 2.59-2.38 (m, 3H), 2.36-2.19 (m, 2H), 2.09-1.97 (m, 1H),
1.85-1.74 (m, 2H), 1.73-1.58 (m, 2H), 1.56-1.38 (m, 1H), 1.34-1.27
(m, 1H), 1.23-0.97 (m, 1H), 0.78 (d, J=6.5 Hz, 3H), 0.76-0.20 (m,
4H). MS: (ES) m/z calculated for
C.sub.32H.sub.36F.sub.4ClN.sub.2O.sub.4 [M+H]+ 623.2, found
623.1.
Example 14
Synthesis of 3-[(3R,
4S)-1-[(3R,6S)-6-[5-chloro-7-(trifluoromethyl)-3,4-dihydro-1H-isoquinolin-
e-2-carbonyl]-6-cyclopropyl-tetrahydropyran-3-yl]-4-piperidyl]benzoic
acid
##STR00044##
[0341] Step a:
[0342] To a solution of the acid intermediate (52 mg, 0.149 mmol)
in DMF (2 mL) was added the amine (100 mg), triethylamine (0.5 ml)
and HATU (190 mg). The resulting mixture was stirred at room
temperature overnight. EtOAc (20 mL) was added and the mixture was
washed with saturated aqueous NaHCO.sub.3 (2.times.5 mL) and brine
(5 mL). The combined organic was dried over MgSO.sub.4 and
concentrated under reduced pressure. The residue was purified by
preparative TLC (100% EtOAc) to give 25 mg of the desired
product.
[0343] Step b:
[0344] The product from step a was treated with aqueous LiOH in THF
as previously described to give the final product (12 mg). .sup.1H
NMR .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 7.41-7.38 (m, 2H),
7.34-7.26 (m, 1H), 7.20-7.14 (m, 1H), 7.04-6.96 (m, 2H), 5.49-5.16
(m, 1H), 4.85-4.52 (m, 1H), 4.25-3.96 (m, 2H), 3.30-3.18 (m, 1H),
3.12-2.85 (m, 4H), 2.59-2.38 (m, 3H), 2.36-2.19 (m, 2H), 2.09-1.97
(m, 1H), 1.85-1.74 (m, 2H), 1.73-1.58 (m, 2H), 1.56-1.38 (m, 1H),
1.34-1.27 (m, 1H), 1.23-0.97 (m, 1H), 0.78 (d, J=6.5 Hz, 3H),
0.76-0.20 (m, 4H). MS: (ES) m/z calculated for
C.sub.32H.sub.37F.sub.3ClN.sub.2O.sub.4 [M+H].sup.+ 605.2, found
605.1.
Example 15
Synthesis of
N-[[3,5-bis(trifluoromethyl)phenyl]methyl]-5-spiro[indene-1,4'-piperidine-
]-1'-yl-tetrahydropyran-2-carboxamide
##STR00045##
[0346] Step a:
[0347] Ethyl 5-hydroxytetrahydropyran-2-carboxylate (2.0 g, 11.5
mmol) was dissolved in anhydrous CH.sub.2Cl.sub.2 (80 mL) and
Dess-Martin periodinane (7.3 g, 17.2 mmol) was added. The reaction
mixture was stirred at rt overnight, then 10%
Na.sub.2S.sub.2O.sub.3 solution (50 mL) and saturated NaHCO.sub.3
(50 mL) were added. The mixture was stirred for 15 min and the
organic layer was separated, dried over MgSO.sub.4, filtered and
evaporated. The residue was diluted with Et.sub.2O, white solid was
filtered off and the filtrate was evaporated to give the crude
product as a yellow oil (1.5 g, 76%).
[0348] Step b:
[0349] To a suspension of 4-spiroindene-piperidine hydrochloride
(554 mg, 2.5 mmol) in anhydrous 1,2-dichloroethane (10 mL),
triethylamine (0.35 mL, 2.5 mmol) was added and the mixture was
stirred at rt for 15 min. The crude ketone from step a (400 mg, 2.3
mmol) was added followed by solid NaBH(OAc).sub.3 (0.97 g, 4.6
mmol). The reaction mixture was stirred at rt overnight, then
H.sub.2O (10 mL) and 2M K.sub.2CO.sub.3 (10 mL) were added. The
organic layer was separated and the aqueous phase was further
extracted with CH.sub.2Cl.sub.2 (2.times.15 mL). The combined
organics were dried over MgSO.sub.4, filtered and evaporated. The
residue was purified by column chromatography (silica gel,
CH.sub.2Cl.sub.2.fwdarw.10% MeOH in CH.sub.2Cl.sub.2) to give the
product as a mixture of diastereomers (460 mg, 59%). MS: (ES) m/z
calculated for C.sub.21H.sub.28NO.sub.3 [M+H].sup.+ 342.2, found
342.1.
[0350] Step c:
[0351] To a suspension of
3,5-bis(trifluoromethyl)phenyl)-methanamine hydrochloride (195.7
mg, 0.7 mmol) in anhydrous toluene (1 mL),
N,N-diisopropylethylamine (0.12 mL, 0.7 mmol) was added and the
mixture was stirred at rt for 15 min. 2M AlMe.sub.3 (0.7 mL, 1.4
mmol) was added dropwise and after 10 min., ester from step b (120
mg, 0.35 mmol) in anhydrous toluene (2 mL) was added. The mixture
was stirred at rt overnight then H.sub.2O (5 mL) and potassium
tartrate (500 mg) were added. The organic layer was separated and
the aqueous phase was further extracted with Et.sub.2O. The
combined organics were dried over MgSO.sub.4, filtered and
evaporated. The residue was purified by column chromatography
(silica gel, CH.sub.2Cl.sub.2.fwdarw.1:1 CH.sub.2Cl.sub.2:EtOAc)
and then by C18 HPLC to give the product as a mixture of
diastereomers (75 mg, 33%). .sup.1H NMR (400 MHz, CDCl.sub.3)
.delta. 7.83-7.71 (m, 3H), 7.41-7.27 (m, 4H), 7.05 (t, J=6.3 Hz,
1H), 6.94-6.87 (m, 1H), 6.74-6.66 (m, 1H), 4.80-4.32 (m, 3H),
4.21-3.92 (m, 1H), 3.90-3.60 (m, 2H), 3.27-2.89 (m, 6H), 2.86-2.63
(m, 1H), 2.58-2.38 (m, 1H), 2.24-1.94 (m, 2H), 1.72-1.47 (m, 2H).
MS: (ES) m/z calculated for C.sub.28H.sub.29F.sub.6N.sub.2O.sub.2
[M+H].sup.+ 539.2, found 539.2.
Example 16
Synthesis of
(2S,5R)-5-[4-(4-fluorophenyl)-1-piperidyl]-2-isopropyl-N-[[5-(trifluorome-
thyl)-3-pyridyl]methyl]tetrahydropyran-2-carboxamide and
(2R,5S)-5-[4-(4-fluorophenyl)-1-piperidyl]-2-isopropyl-N-[[5-(trifluorome-
thyl)-3-pyridyl]methyl]tetrahydropyran-2-carboxamide
##STR00046##
[0353] Step a:
[0354] 2-Isopropyl-3,4-dihydro-2H-pyran-2-carboxylic acid ethyl
ester (7.0 g, 35.0 mmol) was dissolved in anhydrous THF (100 mL)
and cooled to -10.degree. C., then 1M BH.sub.3 in THF (75 mL, 75.0
mmol) was added dropwise. The reaction was kept at 4.degree. C.
overnight, then cooled to -10.degree. C. and a solution of NaOAc
(5.7 g, 70 mmol) in H.sub.2O (15 mL) was slowly added. After 10
min, 35% H.sub.2O.sub.2(10.2 mL, 105 mmol) was added and the
reaction mixture was stirred at rt for 3 h. The reaction was
diluted with brine (50 mL) and the organic layer was separated,
dried over MgSO.sub.4, filtered and evaporated. The residue was
diluted with Et.sub.2O (150 mL), white solid was filtered off and
the filtrate was evaporated to give crude product as oil (7.5 g,
98%).
[0355] Step b:
[0356] The oxidation step was carried out analogously to example 15
to give a yellow oil, 96%.
[0357] Step c:
[0358] The reductive amination step was carried out analogously to
example 15 to give the product as a mixture of diastereomers (52%).
MS: (ES) m/z calculated for C.sub.22H.sub.33FNO.sub.3 [M+H].sup.+
378.2, found 378.1.
[0359] Step d:
[0360] The product from Step c (4.6 g, 12.2 mmol) was dissolved in
DMSO (25 mL) and KOtBu (3.4 g, 30.5 mmol) was added. The mixture
was stirred at 100.degree. C. for 20 min, then cooled down and
diluted with H.sub.2O (100 mL), AcOH (10 mL) and extracted with
EtOAc (3.times.100 mL). The combined organics were dried over
MgSO.sub.4, filtered and evaporated. The residue was diluted with
Et.sub.2O (50 mL), white solid of the product ((rac)-cis
diastereomer) was filtered off and dried under vacuum (600 mg,
14%). MS: (ES) m/z calculated for C.sub.20H.sub.29FNO.sub.3
[M+H].sup.+ 350.2, found 350.1.
[0361] Step e:
[0362] To the mixture of the acid from Step d (40 mg, 0.115 mmol),
HATU (87 mg, 0.23 mmol) in anhydrous DMF (1 mL),
diisopropylethylamine (0.1 mL, 0.575 mmol) was added. The mixture
was stirred at rt for 15 min, then
5-trifluoromethyl-pyridin-3-yl)-methylamine dihydrochloride (29 mg,
0.116 mmol) was added. The reaction was stirred at rt overnight,
then diluted with H.sub.2O (8 mL) and extracted with EtOAc
(2.times.5 mL). The combined organics were dried over MgSO.sub.4,
filtered and evaporated. The residue was purified by preparative
TLC (silica gel, 2:8 hexanes:EtOAc) to give the product as a
(rac)-cis diastereomer (10 mg, 17%). .sup.1H NMR (400 MHz,
CD.sub.3OD) .delta. 8.85-8.76 (m, 2H), 8.14 (brs, 1H), 7.28-7.13
(m, 2H), 7.06-6.90 (m, 2H), 4.54, 4.49 (ABq, J=15.1 Hz, 2H), 3.97
(ddd, J=11.3, 4.6, 2.4 Hz, 1H), 3.35 (t, J=10.9 Hz, 1H), 3.00-2.83
(m, 2H), 2.57-2.21 (m, 5H), 2.01-1.92 (m, 1H), 1.88-1.58 (m, 5H),
1.44 (td, J=13.2, 3.6 Hz, 1H), 1.36-1.23 (m, 1H), 0.91 (d, J=6.9
Hz, 3H), 0.83 (d, J=6.9 Hz, 3H). MS: (ES) m/z calculated for
C.sub.27H.sub.34F.sub.4N.sub.3O.sub.2 [M+H].sup.+ 508.3, found
508.0.
Example 17
Synthesis of
(2S,5R)-2-cyclopropyl-5-[4-(4-fluorophenyl)-1-piperidyl]-N-[[3-fluoro-5-(-
trifluoromethyl)phenyl]methyl]tetrahydropyran-2-carboxamide
##STR00047##
[0364] Step a:
[0365] To a solution of 4-(4-fluorophenyl)piperidine (3.3 g, 13.9
mmol) and benzyl
(2S)-2-cyclopropyl-5-oxo-tetrahydropyran-2-carboxylate (4.0 g, 14.6
mmol) in anhydrous 1,2-dichloroethane (100 mL), solid
NaBH(OAc).sub.3 (5.9 g, 27.8 mmol) was added. The reaction mixture
was stirred at rt overnight, then H.sub.2O (100 mL) and 2M
K.sub.2CO.sub.3 (10 mL) were added. The organic layer was separated
and the aqueous phase was further extracted with CH.sub.2Cl.sub.2
(2.times.50 mL). The combined organics were dried over MgSO.sub.4,
filtered and evaporated. The residue was re-dissolved in MeOH (100
mL) and 10% Pd/C (50% wet) (3 g, 1.4 mmol) was added under N.sub.2.
The reaction mixture was vigorously stirred under H.sub.2
atmosphere (balloon) overnight, then filtered through celite and
evaporated. The residue was diluted with acetone (15 mL) and
Et.sub.2O (15 mL). The product as white solid was filtered off,
washed with Et.sub.2O (15 mL) and dried under vacuum (850 mg, 15%).
MS: (ES) m/z calculated for C.sub.20H.sub.27FNO.sub.3 [M+H]+ 348.2,
found 348.4.
[0366] Step b:
[0367] The amide coupling step was carried out analogously to
example 16 to give a yellowish solid, 52%. .sup.1H NMR (400 MHz,
CD.sub.3OD) .delta. 7.51 (brs, 1H), 7.40-7.30 (m, 2H), 7.24-7.18
(m, 2H), 7.02-6.95 (m, 2H), 4.55, 4.44 (ABq, J=15.4 Hz, 2H), 3.94
(ddd, J=11.2, 4.4, 2.2 Hz, 1H), 3.39 (t, J=11.0 Hz, 1H), 3.00-2.90
(m, 2H), 2.58-2.41 (m, 3H), 2.39-2.24 (m, 2H), 2.00-1.92 (m, 1H),
1.82-1.60 (m, 4H), 1.52 (td, J=13.5, 3.7 Hz, 1H), 1.40-1.26 (m,
1H), 1.10-1.02 (m, 1H), 0.70-0.64 (m, 1H), 0.57-0.49 (m, 1H),
0.46-0.30 (m, 2H). MS: (ES) m/z calculated for
C.sub.28H.sub.32F.sub.5N.sub.2O.sub.2 [M+H].sup.+ 523.2, found
523.0.
Example 18
Synthesis of
[(2S,5R)-2-cyclopropyl-5-[4-(4-fluorophenyl)-1-piperidyl]tetrahydropyran--
2-yl]-[5-fluoro-7-(trifluoromethyl)-3,4-dihydro-1H-isoquinolin-2-yl]methan-
one
##STR00048##
[0369] Step a:
[0370] The title compound was obtained by an amide coupling
analogous to example 16 (18%). .sup.1H NMR (400 MHz, CD.sub.3OD)
.delta. 7.38 (brs, 1H), 7.34-7.26 (m, 1H), 7.25-7.14 (m, 2H),
7.04-6.91 (m, 2H), 5.49 (brd, J=17.4 Hz, 0.5H), 5.16 (brd, J=17.3
Hz, 0.5H), 4.85-4.52 (m, 1H), 4.25-3.96 (m, 2H), 3.30-3.18 (m, 1H),
3.12-2.85 (m, 4H), 2.59-2.38 (m, 3H), 2.36-2.19 (m, 2H), 2.09-1.97
(m, 1H), 1.85-1.74 (m, 2H), 1.73-1.58 (m, 2H), 1.56-1.38 (m, 2H),
1.34-1.27 (m, 1H), 1.23-0.97 (m, 1H), 0.76-0.20 (m, 4H). MS: (ES)
m/z calculated for C.sub.30H.sub.34F.sub.5N.sub.2O.sub.2
[M+H].sup.+ 549.3, found 549.0.
Example 19
Synthesis of
5-[(2S)-4-[(3R,6S)-6-cyclopropyl-6-[6-(trifluoromethyl)-2,4-dihydro-1,3-b-
enzoxazine-3-carbonyl]tetrahydropyran-3-yl]-2-methyl-piperazin-1-yl]-2-flu-
oro-benzoic acid
##STR00049##
[0372] Step a:
[0373] To a solution of t-butyl
(3S)-3-methylpiperazine-1-carboxylate (10.0 g, 50.0 mmol) and
methyl 5-bromo-2-fluoro-benzoate (11.6 g, 50 mmol) in anhydrous,
degassed 1,4-dioxane (100 mL), anhydrous Cs.sub.2CO.sub.3 (24.4 g,
75.0 mmol) was added, followed by DavePhos (1.2 g, 3.0 mmol) and
Pd.sub.2(dba).sub.3 (2.3 g, 2.5 mmol). The reaction mixture was
stirred under N.sub.2, at 100.degree. C. overnight, then cooled
down to rt, filtered thought celite and evaporated. The residue was
re-dissolved in EtOAc (200 mL), washed with 0.5M AcOH (2.times.100
mL), dried over MgSO.sub.4, filtered and evaporated. Crude t-butyl
(3S)-4-(4-fluoro-3-methoxycarbonyl-phenyl)-3-methyl-piperazine-1-carboxyl-
ate was dissolved in 1,4-dioxane (40 mL) and 4M HCl in 1,4-dioxane
(40 mL). The reaction mixture was stirred at rt for overnight, then
evaporated. The residue was diluted with saturated NaHCO.sub.3 (100
mL) and extracted with EtOAc (2.times.100 mL). The combined
organics were dried over MgSO.sub.4, filtered and evaporated. The
crude product (5 g, 40%) was used without further purification.
[0374] Step b:
[0375] The reductive amination step and the acid synthesis were
carried out analogously to example 17. The product was purified by
column chromatography (silica gel, CH.sub.2Cl.sub.2.fwdarw.9:1
CH.sub.2Cl.sub.2:MeOH) to give yellow solid (32%). MS: (ES) m/z
calculated for C.sub.22H.sub.30FN.sub.2O.sub.5 [M+H].sup.+ 421.2,
found 421.0.
[0376] Step c:
[0377] To the mixture of the acid from Step b (150 mg, 0.36 mmol)
and HATU (205 mg, 0.54 mmol) in anhydrous DMF (3 mL),
diisopropylethylamine (0.19 mL, 1.1 mmol) was added. The mixture
was stirred at rt for 15 min, then
2-t-butoxy-5-(trifluoromethyl)phenyl]methanamine (99 mg, 0.4 mmol)
was added. The reaction was stirred at rt for overnight, then
diluted with H.sub.2O (8 mL) and extracted with EtOAc (2.times.5
mL). The combined organics were dried over MgSO.sub.4, filtered and
evaporated. The residue was dissolved in 1,4-dioxane (2 mL) and 4M
HCl in 1,4-dioxane (2 mL) and stirred at rt for 1 h, then
evaporated. The residue was diluted with saturated NaHCO.sub.3 (10
mL) and extracted with EtOAc (3.times.5 mL). The combined organics
were dried over MgSO.sub.4, filtered and evaporated. The crude
product (170 mg, 80%) was used without further purification.
[0378] Step d:
[0379] The mixture of the crude phenol from Step c (170 mg, 0.29
mmol), paraformaldehyde (900 mg) and p-TsOH.H.sub.2O (300 mg) in
toluene (10 mL) was stirred under reflux (Dean Stark apparatus) for
3 h, and then evaporated. The residue was diluted with saturated
NaHCO.sub.3 (10 mL) and extracted with EtOAc (3.times.5 mL). The
combined organics were dried over MgSO.sub.4, filtered and
evaporated. The crude ester was dissolved in MeOH (2 mL) and 4M
NaOH solution (0.5 mL, 1:1 MeOH:H.sub.2O) was added. The mixture
was stirred at rt for 2 h, and then AcOH (0.5 mL) was added. The
reaction mixture was purified by C18 HPLC to give the product (52
mg, 22%). .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 7.65 (s, 1H),
7.54 (s, 1H), 7.51-7.44 (m, 1H), 7.35 (s, 1H), 7.23-7.13 (m, 1H),
7.04 (d, J=8.6 Hz, 1H), 6.27-6.09 (m, 1H), 5.73-5.57 (m, 1H),
4.87-4.76 (m, 1H), 4.48-4.33 (m, 1H), 3.71-3.34 (m, 4H), 3.28-2.85
(m, 5H), 2.64 (dt, J=14.0, 3.7 Hz, 1H), 2.37-2.24 (m, 1H),
1.82-1.67 (m, 1H), 1.58 (td, J=13.2, 3.6 Hz, 1H), 1.35-1.14 (m,
2H), 1.05-0.85 (m, 3H), 0.72-0.36 (m, 4H). MS: (ES) m/z calculated
for C.sub.30H.sub.34F.sub.4N.sub.3O.sub.5 [M+H].sup.+ 592.2, found
592.0.
Example 20
Synthesis of
5-[(2S)-4-[(3R,6S)-6-cyclopropyl-6-[8-fluoro-6-(trifluoromethyl)-2,4-dihy-
dro-1,3-benzoxazine-3-carbonyl]tetrahydropyran-3-yl]-2-methyl-piperazin-1--
yl]-2-fluoro-benzoic acid
##STR00050##
[0381] Step a:
[0382] To 20% fuming H.sub.2SO.sub.4 (100 mL) cooled to 0.degree.
C., 1,2-difluoro-4-(trifluoromethyl)benzene (52.0 g, 285.7 mmol)
was added, followed by solid NIS (70.7 g, 314.3 mmol). The dark
brown, thick mixture was stirred at 0.degree. C. for 5 min., then
slowly warmed up to rt, and stirred for 1 h. The reaction mixture
was poured into ice and extracted with hexanes (2.times.300 mL).
The combined organics were washed with 10% Na.sub.2SO.sub.3
solution, saturated NaHCO.sub.3, and finally with brine. The
organic layer was dried over MgSO.sub.4, filtered and evaporated to
give the crude product as a clear oil (74 g, 84%).
[0383] Step b:
[0384] To 1,2-difluoro-3-iodo-5-(trifluoromethyl)benzene from Step
a (60.0 g, 194.8 mmol) in anhydrous DMF (200 mL) cooled to
0.degree. C., solid KOtBu (24.0 g, 214.3 mmol) was added. The
reaction was stirred at 0.degree. C. for 30 min. then diluted with
H.sub.2O (1 L) and extracted with hexanes (3.times.300 mL).
Combined organics were dried over MgSO.sub.4, filtered and
evaporated. The residue was purified by filtration through short
silica-gel pad and washing with 10% EtOAc in hexanes to give a
yellow oil (63 g, 89%).
[0385] Step c:
[0386] 2-t-Butoxy-1-fluoro-3-iodo-5-(trifluoromethyl)benzene from
Step b (30.0 g, 82.9 mmol) was dissolved in anhydrous, degassed DMF
(200 mL), then Zn(CN).sub.2 (9.7 g, 82.9 mmol) was added followed
by Pd(PPh.sub.3).sub.4(4.7 g, 4.1 mmol). The reaction mixture was
stirred under N.sub.2 at 100.degree. C. for overnight, then diluted
with H.sub.2O (1 L) and extracted with Et.sub.2O (2.times.300 mL).
The combined organics were washed with 1M NaOH (2.times.80 mL) and
then the aqueous layer was neutralized with 2M HCl and extracted
using Et.sub.2O (2.times.300 mL). The combined organics were dried
over MgSO.sub.4, filtered and evaporated to give the crude product
as yellow oil (11 g, 65%) that was used without further
purification.
[0387] Step d:
[0388] 3-Fluoro-2-hydroxy-5-(trifluoromethyl)benzonitrile from Step
c (11.0 g, 53.6 mmol) was dissolved in EtOH (50 mL) and 28%
NH.sub.4OH (5 mL) and Raney-nickel 2800 slurry in H.sub.2O (3 mL)
was added. The reaction mixture was shaken in a Parr apparatus
under H.sub.2 (50 psi) overnight and then filtered through celite
and evaporated. The crude product was washed with Et.sub.2O to give
the product as a yellow solid (4.6 g, 41%). MS: (ES) m/z calculated
for C.sub.8H.sub.8F.sub.4NO [M+H].sup.+ 210.1, found 210.2.
[0389] Step e:
[0390] The title compound was obtained analogously to the previous
examples (27%). .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 7.69-7.60
(m, 1H), 7.46-7.28 (m, 3H), 7.18 (t, J=9.5 Hz, 1H), 6.40-6.14 (m,
1H), 5.85-5.63 (m, 1H), 5.11-4.92 (m, 1H), 4.51-4.31 (m, 1H),
3.71-3.36 (m, 4H), 3.28-2.74 (m, 6H), 2.65 (dt, J=13.4, 3.4 Hz,
1H), 2.40-2.24 (m, 1H), 1.83-1.67 (m, 1H), 1.59 (td, J=13.5, 3.8
Hz, 1H), 1.32-1.11 (m, 1H), 1.08-0.84 (m, 3H), 0.77-0.27 (m, 4H).
MS: (ES) m/z calculated for C.sub.30H.sub.33F.sub.5N.sub.3O.sub.5
[M+H].sup.+ 610.2, found 610.0.
Example 21
Synthesis of
5-[1-[(3R,6S)-6-cyclopropyl-6-[8-fluoro-6-(trifluoromethyl)-2,4-dihydro-1-
,3-benzoxazine-3-carbonyl]tetrahydropyran-3-yl]-4-piperidyl]-2-fluoro-benz-
oic acid
##STR00051##
[0392] Step a:
[0393] The title compound was obtained analogously to the previous
examples (42%). .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 7.80 (dd,
J=6.9, 2.5 Hz, 1H), 7.53-7.43 (m, 1H), 7.44-7.34 (m, 2H), 7.17 (dd,
J=10.6, 8.5 Hz, 1H), 6.25 (brs, 1H), 5.72 (brs, 1H), 5.12-4.91 (m,
2H), 4.51-4.21 (m, 1H), 3.75-3.51 (m, 3H), 3.46-3.35 (m, 1H),
3.23-3.08 (m, 2H), 2.99-2.86 (m, 1H), 2.70-2.56 (m, 1H), 2.33-2.20
(m, 1H), 2.20-2.06 (m, 2H), 1.95-1.67 (m, 3H), 1.68-1.53 (m, 1H),
1.32-1.11 (m, 1H), 0.79-0.22 (m, 4H). MS: (ES) m/z calculated for
C.sub.30H.sub.32F.sub.5N.sub.2O.sub.5 [M+H]+ 595.2, found
595.0.
Example 22
Synthesis of
5-[(2S)-4-[(3R,6S)-6-cyclopropyl-6-[4-fluoro-7-(trifluoromethyl)-3,4-dihy-
dro-1H-isoquinoline-2-carbonyl]tetrahydropyran-3-yl]-2-methyl-piperazin-1--
yl]-2-fluoro-benzoic acid
##STR00052##
[0395] Step a:
[0396] 2-Benzyl-7-(trifluoromethyl)-1,3-dihydroisoquinolin-4-one
(3.0 g, 8.8 mmol) was dissolved in AcOH (40 mL) and 10% Pd/C (50%
wet) (1.9 g, 0.9 mmol) was added under N.sub.2. The reaction
mixture was vigorously stirred under H.sub.2 atmosphere (balloon)
overnight, then filtered through celite and evaporated. The residue
was washed with CH.sub.3CN and Et.sub.2O to give a greenish solid
(2 g, 90%). MS: (ES) m/z calculated for C.sub.10H.sub.11F.sub.3NO
[M+H].sup.+ 218.1, found 218.0.
[0397] Step b:
[0398] The amide coupling step was carried out analogously to the
previous examples (53%). MS: (ES) m/z calculated for
C.sub.32H.sub.38F.sub.4N.sub.3O.sub.5 [M+H].sup.+ 620.3, found
620.0.
[0399] Step c:
[0400] To the crude alcohol from Step b (111 mg, 0.18 mmol) in
anhydrous CH.sub.2Cl.sub.2 (5 mL) cooled to -78.degree. C.,
(diethylamino)sulfur trifluoride (94 .mu.L, 0.72 mmol) was added
and the reaction was stirred at -78.degree. C. for 1 h. MeOH (1 mL)
was added and the solvent was evaporated. The residue was dissolved
in MeOH (1 mL) and 4M NaOH solution (1 mL, 1:1 MeOH:H.sub.2O) was
added. The mixture was stirred at rt for 1 h, and then AcOH (0.5
mL) was added. The reaction mixture was purified by C18 HPLC to
give the product (15 mg, 10%). .sup.1H NMR (400 MHz, CD.sub.3OD)
.delta. 7.73-7.63 (m, 4H), 7.42-7.29 (m, 1H), 7.22-7.12 (m, 1H),
5.94-5.58 (m, 2H), 5.52-5.25 (m, 1H), 4.50-4.22 (m, 1H), 3.80-3.36
(m, 3H), 3.26-2.80 (m, 7H), 2.68-2.56 (m, 2H), 2.35-2.20 (m, 1H),
1.86-1.46 (m, 2H), 1.35-0.30 (m, 8H). MS: (ES) m/z calculated for
C.sub.31H.sub.35F.sub.5N.sub.3O.sub.4 [M+H].sup.+ 608.3, found
608.0.
Synthesis of methyl
2-fluoro-5-[[(3R)-pyrrolidin-3-yl]amino]benzoate
##STR00053##
[0402] Step a:
[0403] To 5-bromo-2-fluoro-benzoic acid (6.38 g, 29.1 mmol, 1
equiv) was added thionyl chloride (10 mL, 290 mmol, 10 equiv). The
mixture was stirred at 80.degree. C. until complete, then
concentrated. K.sub.2CO.sub.3 (10.87 g, 78.6 mmol, 2.7 equiv) was
added, followed by MeOH (30 mL). This solution was stirred at room
temperature. The reaction was filtered and the solid was rinsed
with CH.sub.2Cl.sub.2. The organic was then washed with H.sub.2O,
dried over MgSO.sub.4, filtered and concentrated to give the
product (3.29 g, 14.1 mmol, 49%).
[0404] Step b:
[0405] To 5-bromo-2-fluoro-benzoic acid methyl ester (652 mg, 2.80
mmol, 1 equiv) was added Cs.sub.2CO.sub.3 (2.34 g, 7.18 mmol, 2.5
equiv) followed by
2-dicyclohexylphosphino-2'-(N,N-dimethylamino)biphenyl (132 mg,
0.335 mmol, 0.12 equiv), dioxane (6 mL), and
(R)-3-Amino-pyrrolidine-1-carboxylic acid tert-butyl ester (0.695
mL, 3.37 mmol, 1.2 equiv). N.sub.2 was bubbled through the mixture
for 5 min, then Pd.sub.2(dba).sub.3 was added to the reaction and
N.sub.2 was again bubbled through the mixture for 5 min. The
reaction was then heated to 90.degree. C. for 2 days. The reaction
was allowed to cool to room temperature, then diluted with
CH.sub.2Cl.sub.2, washed with H.sub.2O (3.times.20 mL) then brine
and dried over MgSO.sub.4, filtered and concentrated to give the
crude. This was purified by silica gel chromatography
(hexanes/EtOAc) to give the product (328 mg, 0.969 mmol, 35%).
[0406] Step c:
[0407] To
(R)-3-(4-Fluoro-3-methoxycarbonyl-phenylamino)-pyrrolidine-1-car-
boxylic acid tert-butyl ester (328 mg, 0.969 mmol) was added
CH.sub.2Cl.sub.2 (1 mL) followed by 4 N HCl in dioxane (1 mL, 4
mmol). This was stirred overnight and then concentrated. The
residue was partitioned between saturated NaHCO.sub.3 and EtOAc.
The layers were separated, then the aqueous layer was extracted
with 2:1 CHCl.sub.3:iPrOH (5.times.). This was then dried over
Na.sub.2SO.sub.4, filtered and concentrated to give
(R)-2-fluoro-5-(pyrrolidin-3-ylamino)-benzoic acid methyl ester
(216 mg).
Example 23
Synthesis of
5-[[(3R)-1-[(3R,6S)-6-cyclopropyl-6-[6-(trifluoromethyl)-2,4-dihydro-1,3--
benzoxazine-3-carbonyl]tetrahydropyran-3-yl]pyrrolidin-3-yl]amino]-2-fluor-
o-benzoic acid
##STR00054## ##STR00055##
[0409] Step a:
[0410] To the mixture of
(2S)-2-cyclopropyl-3,4-dihydropyran-2-carboxylic acid (6.5 g, 38.9
mmol) and HATU (16.3 g, 42.8 mmol) in anhydrous DMF (80 mL),
diisopropylethylamine (13.5 mL, 77.8 mmol) was added and the
mixture was stirred at rt for 15 min. Then a solution of the
2-t-butoxy-5-(trifluoromethyl)phenyl]methanamine (10.5 g, 42.8
mmol) in anhydrous DMF (20 mL) was added and the reaction was
stirred at rt for 1 day. This was diluted with H.sub.2O (600 mL)
and extracted using EtOAc (3.times.200 mL). The combined organic
layers were washed with brine (4.times.50 mL), dried over
MgSO.sub.4, filtered and evaporated. The crude product was purified
by column chromatography (silica gel, hexanes.fwdarw.9:1
hexanes:EtOAc) to give a white solid (10.4 g, 67%).
[0411] Step b:
[0412] The product from step a (10.0 g, 25.2 mmol) was dissolved in
anhydrous THF (100 mL) and cooled to -10.degree. C., then 1M
BH.sub.3 in THF (25.2 mL, 25.2 mmol) was added dropwise. The
reaction was kept at 4.degree. C. overnight, then cooled to
-10.degree. C. and a solution of NaOAc (4.1 g, 50.4 mmol) in
H.sub.2O (15 mL) was slowly added. After 10 min, 35%
H.sub.2O.sub.2(7.6 mL, 75.6 mmol) was added and the reaction
mixture was stirred at rt for 3 h. This was diluted with brine (50
mL) and the organic layer was separated, dried over MgSO.sub.4,
filtered and evaporated. The residue was diluted with Et.sub.2O
(150 mL), a white solid was filtered off and the filtrate was
evaporated to give crude product as oil (10.5 g, quant).
[0413] Step c:
[0414] To the product from Step b (10.4 g, 25.2 mmol) was added
triethylamine (5.3 mL, 37.8 mmol) and DMAP (0.3 g, 2.5 mmol) in
anhydrous CH.sub.2Cl.sub.2 (150 mL). Acetic anhydride (3.6 mL, 37.8
mmol) was added dropwise at rt and the mixture was stirred for 1
day. The reaction was washed with H.sub.2O (100 mL) then a
saturated solution of NaHCO.sub.3 (100 mL), and the organic layer
was dried over MgSO.sub.4, filtered and evaporated. Crude product
was purified by column chromatography (silica gel,
hexanes.fwdarw.7:3 hexanes:EtOAc) to give a yellow thick oil (8.0
g, 70%).
[0415] Step d:
[0416] The product from Step c (8.0 g, 17.5 mmol) was dissolved in
anhydrous 1,4-dioxane (10 mL) and 4M HCl in 1,4-dioxane (30 mL);
stirred at rt for 1 h then evaporated. The residue was dissolved in
toluene (10 mL), then paraformaldehyde (20 g) and p-TsOH.H.sub.2O
(332 mg) were added and the reaction was stirred under reflux (Dean
Stark apparatus) for 2 h, and then evaporated. The residue was
purified by column chromatography (silica gel, hexanes.fwdarw.7:3
hexanes:EtOAc) to give a yellow thick oil (6.2 g, 86%).
[0417] Step e:
[0418] The product from Step d (6.2 g, 15.0 mmol) was dissolved in
MeOH (70 mL) and solution of LiOH.H.sub.2O (3.1 g, 75 mmol) in
H.sub.2O (35 mL) was added. The reaction mixture was stirred at rt
for 2 h, then neutralized to pH=7 with 2M AcOH and evaporated. The
residue was diluted with brine (100 mL), extracted using
CH.sub.2Cl.sub.2 (3.times.50 mL) and combined organic layer were
dried over MgSO.sub.4, filtered and evaporated to give white solid
(5.3 g, 95%).
[0419] Step f:
[0420] The oxidation step was carried out analogously to the
previous examples to give a yellow oil (quant).
[0421] Step g:
[0422] To the product from Step f (150 mg, 0.4 mmol) and methyl
2-fluoro-5-[[(3R)-pyrrolidin-3-yl]amino]benzoate (98 mg, 0.4 mmol)
in anhydrous 1,2-dichloroethane (5 mL), NaBH(OAc).sub.3 (174 mg,
0.8 mmol) was added. The reaction mixture was stirred at rt for
overnight then washed with saturated solution of NaHCO.sub.3 (10
mL). The organic layer was separated, dried over MgSO.sub.4,
filtered and evaporated. The residue was dissolved in MeOH (1 mL)
and 4M NaOH solution (1 mL, 1:1 MeOH:H.sub.2O) was added. The
mixture was stirred at rt for 1 h, and then AcOH (0.5 mL) was
added. The reaction mixture was purified by C18 HPLC to give the
product (39 mg, 12%). .sup.1H NMR (400 MHz, CD.sub.3OD) .delta.
7.57-7.40 (m, 2H), 7.14-6.95 (m, 3H), 6.86-6.77 (m, 1H), 6.27-6.07
(m, 1H), 5.69-5.54 (m, 1H), 4.38-4.10 (m, 2H), 3.57-3.34 (m, 2H),
2.63-2.51 (m, 2H), 2.29-2.16 (m, 2H), 2.15-1.90 (m, 2H), 1.80-1.62
(m, 2H), 1.55 (td, J=13.3, 3.8 Hz, 2H), 1.30-1.09 (m, 2H),
0.72-0.36 (m, 5H). MS: (ES) m/z calculated for
C.sub.29H.sub.32F.sub.4N.sub.3O.sub.5 [M+H].sup.+ 578.2, found
577.9.
Example 24
Synthesis of
3-[(2S)-4-[(3R,6S)-6-cyclopropyl-6-[6-(trifluoromethyl)-2,4-dihydro-1,3-b-
enzoxazine-3-carbonyl]tetrahydropyran-3-yl]-2-methyl-piperazin-1-yl]propan-
oic acid
##STR00056##
[0424] Step a:
[0425] The title compound was obtained analogously to the previous
examples but using ethyl 3-[(2S)-2-methylpiperazin-1-yl]propanoate
(7.5%). .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 7.56-7.43 (m,
2H), 7.05-6.97 (m, 1H), 6.22-6.06 (m, 1H), 5.80-5.61 (m, 1H),
4.16-3.98 (m, 1H), 3.60-3.39 (m, 2H), 3.27-2.87 (m, 8H), 2.79-2.57
(m, 4H), 2.57-2.41 (m, 2H), 2.05-1.91 (m, 1H), 1.54-1.39 (m, 2H),
1.32 (d, J=6.4 Hz, 3H), 1.22-1.06 (m, 1H), 0.70-0.33 (m, 4H). MS:
(ES) m/z calculated for C.sub.26H.sub.35F.sub.3N.sub.3O.sub.5
[M+H].sup.+ 526.3, found 526.0.
Example 25
Synthesis of
5-[(2S)-4-[(3R,6S)-6-cyclopropyl-6-[8-ethoxy-6-(trifluoromethyl)-2,4-dihy-
dro-1,3-benzoxazine-3-carbonyl]tetrahydropyran-3-yl]-2-methyl-piperazin-1--
yl]-2-fluoro-benzoic acid
##STR00057##
[0427] Step a:
[0428] To a solution of
2-tert-butoxy-5-(trifluoromethyl)phenyl]methanamine (4.0 g, 16.2
mmol) in TFA (20 mL), N-iodosuccinamide (4.4 g, 19.4 mmol) was
added and the reaction was stirred at rt for 30 min. This was
evaporated, then the residue was dissolved in EtOAc (250 mL) and
washed with saturated solution of NaHCO.sub.3 (50 mL) then 10%
Na.sub.2SO.sub.3 (50 mL). The organic layer was dried over
MgSO.sub.4, filtered and evaporated to give a yellow solid (4.8 g,
93%).
[0429] Step b:
[0430] Step b was carried out analogously to the previous examples
to give a yellow solid (90%). MS: (ES) m/z calculated for
C.sub.31H.sub.35F.sub.41N.sub.3O.sub.5 [M+H].sup.+ 732.2, found
732.2.
[0431] Step c:
[0432] The product from Step b (100 mg, 0.14 mmol),
1,10-phenanthroline (13 mg, 0.07 mmol), CuI (13 mg, 0.07 mmol) and
Cs.sub.2CO.sub.3 (91 mg, 0.28 mmol) were placed in a microwave vial
and diluted with anhydrous EtOH (3 mL). The reaction was carried
out in a microwave reactor at 110.degree. C. for 1 h, then AcOH (1
mL) was added and reaction mixture was purified by C18 HPLC to give
the product (12 mg, 10%). .sup.1H NMR (400 MHz, CD.sub.3OD) .delta.
7.78-7.59 (m, 1H), 7.45-7.27 (m, 1H), 7.25-7.03 (m, 3H), 6.32-6.15
(m, 1H), 5.69-5.51 (m, 1H), 4.83-4.72 (m, 1H), 4.51-4.34 (m, 1H),
4.10 (q, J=7.0 Hz, 2H), 3.70-3.36 (m, 4H), 3.27-2.82 (m, 5H), 2.64
(dt, J=13.7, 3.6 Hz, 1H), 2.37-2.25 (m, 1H), 1.83-1.66 (m, 1H),
1.59 (td, J=13.4, 3.7 Hz, 1H), 1.42 (t, J=7.0 Hz, 3H), 1.39-1.14
(m, 2H), 1.07-0.85 (m, 3H), 0.73-0.37 (m, 4H). MS: (ES) m/z
calculated for C.sub.32H.sub.38F.sub.4N.sub.3O.sub.6 [M+H].sup.+
636.3, found 636.0.
Synthesis of methyl 3-[(3R,4S)-3-methyl-4-piperidyl]benzoate
##STR00058##
[0434] Step a:
[0435] EDCI (191.7 g, 1.0 mol) was added to an ice bath cooled
solution of 3-(tert-butoxycarbonylamino)propanoic acid (189.2 g,
1.0 mol), meldrum's acid (144.1 g, 1.0 mol) and DMAP (135.0 g, 1.1
mol) in DMF (400 mL). The resulting reaction was allowed to 15 stir
at room temperature overnight. The reaction was quenched with 200
mL of H.sub.2O. The resulting mixture was added dropwise to
H.sub.2O (2.0 L) while being mechanically stirred. The resulting
solid was filtered and washed thoroughly with water until
pH.about.7 to give the product (283 g, 90% yield). .sup.1H NMR (300
MHz, CDCl.sub.3): .delta. 4.9 (bs, 1H), 3.55 (q, 2H), 3.45-3.25 (t,
3H), 1.75 (s, 6H), 1.4 (s, 9H).
[0436] Step b:
[0437] A solution of
tert-butyl-3-(2,2-dimethyl-4,6-dioxo-1,3-dioxan-5-yl)-3-oxopropylcarbamat-
e (100 g, 317.46 mmol) in toluene (1500 mL) was heated at
100.degree. C. for 4 hours. The reaction was cooled to room
temperature and concentrated under reduced pressure to afford the
crude product. Purification by MTBE-trituration gave the product
(56 g, 83% yield). .sup.1H NMR (300 MHz, CDCl.sub.3): .delta. 4.1
(t, 2H), 3.5 (s, 2H), 2.6 (t, 2H), 1.55 (s, 9H).
[0438] Step c:
[0439] tert-Butyl 2,4-dioxopiperidine-1-carboxylate (106.5 g, 500
mmol) was dissolved in CH.sub.2Cl.sub.2 (1.0 L) and HOAc (60 mL).
The resulting solution was cooled to 0.degree. C., then
NaBH.sub.3CN (37.8 g, 600 mmol) was added in portions. After
stirring at room temperature overnight, the reaction mixture was
cooled to 0.degree. C. and aqueous NH.sub.4OH was slowly added to
adjust the pH to 9. The organic phase was separated and washed with
brine (2.times.400 mL) then dried over MgSO.sub.4, filtered and
concentrated to give the product as a sticky oil.
[0440] Step d:
[0441] The product from step c was dissolved in CH.sub.2Cl.sub.2
(1.0 L) and the solution was cooled to 0.degree. C. Triethylamine
(151.5 g, 1.5 mol) was added and the resulting mixture was stirred
at 0.degree. C. for 30 minutes. Acetic anhydride (53.5 g, 525 mmol)
was added dropwise. After stirring at room temperature overnight,
water (400 ml) was added and the organic layer was separated and
washed with a 5% KHSO.sub.4 solution (3.times.300 mL). The organic
phase was dried over MgSO.sub.4 and then concentrated under reduced
pressure. The residue was passed through a short pad of SiO.sub.2
(500 g) and washed with 20% of EtOA in hexanes. The combined
organic was concentrated to give the product (69 g) as a colorless
oil which was directly used in the next step without further
purification.
[0442] Step e:
[0443] Chloro(1,5-cyclooctadien)rhodium(I) dimer (493 mg, 1 mmol)
and (S)-BINAP (1.56 g, 2.5 mmol) were added to a mixture of
3-methoxycarbonylphenylboronic acid (9.0 g, 50 mmol) in dioxane (65
mL). The resulting mixture was stirred at room temperature under
nitrogen atmosphere for 1 hour. The mixture was cooled to 0.degree.
C. and H.sub.2O (10 mL) was added, followed by
6-oxo-3,6-dihydro-2H-pyridine-1-carboxylic acid tert-butyl ester
(9.3 g, 47.5 mmol) in dioxane (5 mL) and triethylamine (4.8 g, 47.5
mmol). The resulting mixture was stirred at room temperature
overnight under nitrogen atmosphere. The mixture was diluted with
heptane (65 mL), MTBE (17.5 mL), and H.sub.2O (50 mL). The organic
phase was separated and washed with H.sub.2O (50 mL). The aqueous
phase was combined and extracted with MTBE/heptane (1:2, 50 mL).
The combined organics were washed with brine and dried over
MgSO.sub.4. Concentration under reduced pressure gave a pale solid
which was washed with 10% EtOAc in hexane (100 mL) to give 10 g of
a white solid (Compound 5, 60% yield, 99% ee). The % ee was
measured by HPLC using a chiral column (Regiscell 25 cm.times.4.6
mm, 5 micron spherical) with 30% iPrOH in hexanes as eluent.
[0444] Step f:
[0445] 4-(3-Methoxycarbonyl-phenyl)-2-oxo-piperidine-1-carboxylic
acid tert-butyl ester (40 g, 120 mmol) was dissolved in anhydrous
THF (120 mL) and the solution was cooled to -78.degree. C. LiHMDS
in THF (1M, 122 mL, 122 mmol) was added dropwise and the resulting
mixture was stirred at -78.degree. C. for 3 hours before MeI (33.84
g, 240 mmol) was added dropwise at the same temperature. The
mixture was slowly warmed to room temperature overnight. Water (100
mL) was added and the organic layer was separated and washed with
brine and dried. Concentration under reduced pressure gave a brown
solid which was recrystallized with 10% EtOAc in hexane (400 mL) to
give the product (35 g, 84%) as a pale solid.
[0446] Step g:
[0447]
4-(3-Methoxycarbonyl-phenyl)-3-methyl-2-oxo-piperidine-1-carboxylic
acid tert-butyl ester (10.4 g, 30 mmol) was added portionwise to
HCl in dioxane (4M, 20 mL, 80 mmol). The resulting mixture was
stirred at room temperature for two hours before it was
concentrated under reduced pressure to give a white solid.
[0448] Step h:
[0449] The product from step g was dissolved in 30 mL of anhydrous
THF. The resulting solution was cooled to -78.degree. C. and a
solution of BH.sub.3.SMe.sub.2 in THF (2M, 45 mL, 90 mmol) was
added dropwise. After addition, the mixture was stirred at room
temperature for 48 hours before it was cooled to -78.degree. C.
MeOH (10 mL) was cautiously added to quench the reaction.
Concentrated HCl (8 mL) was added and the mixture was stirred at
60.degree. C. for two hours. MeOH was removed under reduced
pressure and the residue was diluted with water (20 mL). The
resulting aqueous mixture was extracted with MTBE:heptane (1:2, 50
mL). The separated aqueous layer was adjusted to pH 9 by adding
NH.sub.4OH aqueous solution followed by extraction with
iPrOH:CHCl.sub.3 (1:2, 3.times.100 mL). The combined organic was
washed with brine and dried over MgSO.sub.4 before it was
concentrated under reduced pressure. The residue was purified by
silica gel chromatography (10% MeOH in CH.sub.2Cl.sub.2, plus 1%
NH.sub.40H) to give the desired product as a colorless oil.
Synthesis of 2-(aminomethyl)-4,6-bis(trifluoromethyl)phenol
##STR00059##
[0451] Step a:
[0452] A mixture of H.sub.2O (30 mL) and H.sub.2SO.sub.4 (30 mL)
was cooled in an ice bath. 2-Chloro-3,5-bis-trifluoromethyl aniline
(5.00 g, 19.0 mmol, 1 equiv) was added in MeCN (30 mL) over 5 min.
This was stirred for 10 min then NaNO.sub.2 (2.36 g, 34.2 mmol, 1.8
equiv) in H.sub.2O (17 mL) was added dropwise over 5 min, during
which the internal temperature reached 10.degree. C. This was
stirred 10 min, then poured into an ice cooled solution of KI (11.0
g, 66.5 mmol, 3.5 equiv) in H.sub.2O (30 mL). This was stirred for
2 h then diluted with CHCl.sub.3 (60 mL). The layers were separated
and the aqueous was extracted with CHCl.sub.3. The combined organic
was washed with saturated aqueous NaHCO.sub.3 (2.times.) then
saturated aqueous Na.sub.2S.sub.2O.sub.3. The organic was then
dried over MgSO.sub.4, filtered and concentrated to give the
product (6.58 g, 93%) as a light brown oil.
[0453] Step b:
[0454] 2-Chloro-1-iodo-3,5-bis-trifluoromethyl-benzene (11.8 g,
31.5 mmol, 1 equiv) was dissolved in N-methylpyrrolidone (32 mL)
and CuCN (3.4 g, 38.2 mmol, 1.2 equiv) was added. The reaction was
heated to 120.degree. C. for 6 h. The reaction was allowed to cool
to room temperature, then diluted with H.sub.2O (100 mL) and
heptane (100 mL). This was then filtered through celite and the
layers were separated. The organic layer was filtered through
silica gel (10 g) using heptane (50 mL) to elute. The solution was
then concentrated to give the product (6.6 g, 76%).
[0455] Step c:
[0456] 2-Chloro-3,5-bis-trifluoromethyl-benzonitrile (57.1 g, 209
mmol, 1 equiv) was dissolved in THF (417 mL) and cooled in an ice
bath. NaOtBu (24.0 g, 250 mmol, 1.2 equiv) was added portionwise
over 10 min, keeping the internal temperature below 8.degree. C.
After 30 min the reaction was allowed to warm to room temperature,
then stirred for 3 h. Aqueous HCl (4N, 627 mL, 2.51 mol, 12 equiv)
was added and this was stirred at room temperature overnight. The
reaction was then heated to 50.degree. C. for 2 h. The solution was
concentrated, then heptane (400 mL) was added. The layers were
separated and the organic was dried over MgSO.sub.4, filtered and
concentrated. The resulting oil was diluted with heptane to a total
weight of 160 g, then cooled to -20.degree. C. This was filtered
and the solid was washed with cold heptane (60 mL) to give the
product (23.6 g, 44%).
[0457] Step d:
[0458] 2-Hydroxy-3,5-bis-trifluoromethyl-benzonitrile (2.79 g, 10.9
mmol) was dissolved in EtOH (50 mL) and NH.sub.4OH (5 ml). Raney
nickel slurry in water (2.00 mL) was added and the reaction was
shaken under H.sub.2 (40 psi) for 3 days. The reaction was
filtered, the filter cake was rinsed with MeOH, and the combined
solution was then concentrated. The resulting residue was taken up
in MeOH and HCl in Et.sub.2O (2M, 6 mL, 12 mmol) was added. The
mixture was concentrated, then taken up in EtOAc. This was washed
with NH.sub.4OH then concentrated. The resulting solid was
triturated with Et.sub.2O to give
2-aminomethyl-4,6-bis-trifluoromethyl-phenol (1.55 g, 55%).
Example 26
Synthesis of
3-[(3R,4S)-1-[(3R,6S)-6-[6,8-bis(trifluoromethyl)-2,4-dihydro-1,3-benzoxa-
zine-3-carbonyl]-6-cyclopropyl-tetrahydropyran-3-yl]-3-methyl-4-piperidyl]-
benzoic acid
##STR00060##
[0460] Step a:
[0461] To a mixture of 3-[(3R,4S)-3-methyl-4-piperidyl]benzoate
(4.5 g, 19.3 mmol), benzyl
(2S)-2-cyclopropyl-5-oxo-tetrahydropyran-2-carboxylate (5.8 mmol,
21.2 mmol) and triethylamine (5.4 mL) in anhydrous
1,2-dichloroethane (100 mL), Na(OAc).sub.3BH (8.2 g, 38.6 mmol) was
added. The reaction mixture was stirred at rt overnight, then
diluted with H.sub.2O (100 mL) and the organic layer was separated.
The aqueous layer was extracted with CH.sub.2Cl.sub.2 (50 mL), and
the combined organics were dried over MgSO.sub.4, filtered and
evaporated. The residue was dissolved in MeOH (100 mL) and 10% Pd/C
(50% wet) (2.1 g, 1.0 mmol) was added under N.sub.2. The reaction
mixture was vigorously shaken in a Parr shaker apparatus at 55 psi
H.sub.2 overnight, then filtered through celite and evaporated. The
residue was diluted with acetone (45 mL) and the product was
filtered off, washed with acetone (5 mL) and Et.sub.2O (15 mL) and
dried under vacuum (2.4 g, 31%).
[0462] Step b:
[0463] To the mixture of the acid from Step a (2.2 g, 5.5 mmol),
HATU (2.2 g, 5.8 mmol) in anhydrous DMF (30 mL),
diisopropylethylamine (1.9 mL, 11.0 mmol) was added. The mixture
was stirred at rt for 15 min, then
2-(aminomethyl)-4,6-bis(trifluoromethyl)phenol (1.5 g, 5.8 mmol) in
anhydrous DMF (10 mL) was added. The reaction was stirred at rt
overnight, then diluted with H.sub.2O (300 mL) and extracted with
EtOAc (3.times.100 mL). The combined organics were washed with
brine (4.times.50 mL) then dried over MgSO.sub.4, filtered and
evaporated. The residue was dissolved in toluene (300 mL) and
paraformaldehyde (20 g), p-TsOH.H.sub.2O (2.1 g, 11 mmol) and MsOH
(0.7 mL, 11 mmol) were added. The reaction was stirred at reflux
(Dean Stark apparatus) for 5 h, then cooled to rt, diluted with
saturated NaHCO.sub.3 (200 mL) and extracted with EtOAc
(2.times.100 mL). The combined organics were dried over MgSO.sub.4,
filtered and evaporated. The crude ester was purified by column
chromatography (silica gel, hexanes.fwdarw.1:1 hexanes:EtOAc) to
give a white solid (2.5 g, 70%). MS: (ES) m/z calculated for
C.sub.33H.sub.37F.sub.6N.sub.2O.sub.5 [M+H].sup.+ 655.3, found
655.5.
[0464] Step c:
[0465] The product from Step b (2.5 g, 3.8 mmol) was diluted with
1M NaOH (40 mL, 95:5; MeOH:H.sub.2O) and stirred at 40.degree. C.
for 5 h. AcOH (2.3 mL, 40 mmol) was added and the mixture was
evaporated. The residue was diluted with H.sub.2O (200 mL) and
extracted using CH.sub.2Cl.sub.2 (2.times.100 mL). The combined
organics were dried over MgSO.sub.4, filtered and 2M HCl in
Et.sub.2O (5.7 mL, 11.4 mmol) was added. The mixture was evaporated
and the residue was washed with Et.sub.2O (50 mL) to give a
yellowish solid that was the product as the HCl salt (2.15 g, 83%).
.sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 7.97-7.83 (m, 3H),
7.81-7.73 (m, 1H), 7.51-7.41 (m, 2H), 6.21-5.89 (m, 2H), 5.19-4.92
(m, 2H), 4.45-4.32 (m, 1H), 3.71-3.52 (m, 3H), 3.48-3.35 (m, 1H),
3.24-3.09 (m, 1H), 2.85 (t, J=12.0 Hz, 1H), 2.71-2.60 (m, 1H),
2.60-2.48 (m, 1H), 2.37-2.24 (m, 1H), 2.23-2.08 (m, 1H), 2.08-1.94
(m, 2H), 1.89-1.70 (m, 1H), 1.70-1.55 (m, 1H), 1.24-1.13 (m, 1H),
0.75 (d, J=6.6 Hz, 3H), 0.72-0.28 (m, 4H). MS: (ES) m/z calculated
for C.sub.32H.sub.35F.sub.6N.sub.2O.sub.5 [M+H].sup.+ 641.2, found
641.5.
Example 27
Synthesis of
5-[(2S)-4-[(3R,6S)-6-[[3-chloro-5-(trifluoromethyl)phenyl]methylcarbamoyl-
]-6-cyclopropyl-tetrahydropyran-3-yl]-2-methyl-piperazin-1-yl]-2-fluoro-be-
nzoic acid
##STR00061##
[0467] Step a:
[0468] In a 20 mL vial,
(2S,5R)-2-cyclopropyl-5-[(3S)-4-(4-fluoro-3-methoxycarbonyl-phenyl)-3-met-
hyl-piperazin-1-yl]tetrahydropyran-2-carboxylic acid (39.7 mg,
0.094 mmol) was added followed by HATU (38.2 mg, 0.100 mmol) and
DMF (0.5 mL) at ambient temperature. After addition of iPr.sub.2NEt
(25 .mu.L, 0.144 mmol), the reaction was stirred for 1 minute
before 3-chloro-5-trifluoromethylbenzylamine (18 .mu.L, 0.116 mmol)
was added. The reaction mixture was stirred overnight. The reaction
was diluted with ethyl acetate (20 mL) and water (10 mL). The
layers were separated and the aqueous phase was extracted with
ethyl acetate (2.times.20 mL). After drying with anhydrous sodium
sulfate, the crude reaction mixture was concentrated under reduced
pressure. The product was purified using flash silica gel
chromatography using a gradient of 15% to 33% ethyl acetate in
hexanes. Methyl
5-[(2S)-4-[(3R,6S)-6-[[3-chloro-5-(trifluoromethyl)phenyl]methylcarbamoyl-
]-6-cyclopropyl-tetrahydropyran-3-yl]-2-methyl-piperazin-1-yl]-2-fluoro-be-
nzoate was obtained in 59% yield (34.2 mg).
[0469] Step b:
[0470] Methyl
5-[(2S)-4-[(3R,6S)-6-[[3-chloro-5-(trifluoromethyl)phenyl]methylcarbamoyl-
]-6-cyclopropyl-tetrahydropyran-3-yl]-2-methyl-piperazin-1-yl]-2-fluoro-be-
nzoate (34.2 mg, 0.0559 mmol) was dissolved in THF (0.25 mL) at
ambient temperature. 1.5 N-lithium hydroxide solution (60 .mu.L,
0.090 mmol) was added followed by methanol (60 .mu.L). The reaction
mixture was stirred overnight. The crude product was purified using
reverse phase HPLC using a gradient of acetonitrile in water with
0.01% trifluoromethylacetic acid (20%-95%). Upon removal of the
solvent,
5-[(2S)-4-[(3R,6S)-6-[[3-chloro-5-(trifluoromethyl)phenyl]methylcarbamoyl-
]-6-cyclopropyl-tetrahydropyran-3-yl]-2-methyl-piperazin-1-yl]-2-fluoro-be-
nzoic acid was obtained as a bis TFA salt (39.0 mg, 84% yield).
.sup.1H NMR (400 MHz, Methanol-d.sub.4) .delta. 8.85 (t, J=6.3 Hz,
1H), 7.71 (br, 1H), 7.63 (d, J=16 Hz, 1H), 7.61 (s, 1H), 7.40 (br,
1H), 7.22-7.17 (m, 1H), 4.84-4.90 (m, 1H), 4.70 (dd, J=15.5, 7.1
Hz, 1H), 4.32 (dd, J=15.6, 5.3 Hz, 1H), 4.24 (d, J=11.3 Hz, 1H),
3.62 (t, J=10.8 Hz, 1H), 3.00-3.62 (m, 8H), 2.74-2.58 (m, 1H),
2.32-2.27 (m, 1H), 1.79-1.52 (m, 2H), 1.22-1.06 (m, 1H), 0.99 (br,
3H), 0.71 (dt, J=9.5, 4.6 Hz, 1H), 0.55 (dt, J=9.8, 5.4 Hz, 1H),
0.47 (ddq, J=13.9, 9.0, 4.4 Hz, 1H). MS: (ES) m/z calculated for
C.sub.29H.sub.33ClF.sub.4N.sub.3O.sub.4 [M+H].sup.+ 598.2, found
598.1.
Example 28
Synthesis of
(3S,4R)-1-[(3R,6S)-6-cyclopropyl-6-[8-ethoxy-6-(trifluoromethyl)-2,4-dihy-
dro-1,3-benzoxazine-3-carbonyl]tetrahydropyran-3-yl]-4-(4-fluorophenyl)pip-
eridine-3-carboxylic acid
##STR00062##
[0472] Step a:
[0473] A 20 mL vial was charged with
(2S,5R)-2-cyclopropyl-5-[(3S,4R)-4-(4-fluorophenyl)-3-methoxycarbonyl-1-p-
iperidyl]tetrahydropyran-2-carboxylic acid (360 mg, 0.887 mmol),
HATU (356 mg, 0.936 mmol) and DMF (3.5 mL) at ambient temperature.
iPr.sub.2NEt (234 .mu.L, 1.34 mL) was added and the reaction
mixture was stirred for 15 minutes. Part of reaction mixture (1.56
mL) was removed for use in a different reaction. To the remaining
reaction mixture was added
2-hydroxy-3-iodo-5-trifluoromethylbenzylamine (188 mg, 0.593 mmol)
and the reaction was stirred for 5 hours. Additional
2-hydroxy-3-iodo-5-trifluoromethylbenzylamine (34.1 mg, 0.108 mmol)
was added to drive the reaction to completion. When all starting
material was consumed, the reaction was diluted with water (10 mL)
and ethyl acetate (25 mL). The layers were separated and the
aqueous layer was extracted with ethyl acetate (2.times.20 mL). The
combined organic layer was dried over anhydrous sodium sulfate
followed by removal of solvent under reduced pressure. The obtained
crude product was used directly in the next step.
[0474] Step b:
[0475] Crude product from step a (.about.0.5 mmol) was dissolved in
toluene (5 mL). p-Toluene sulfonic acid monohydrate (195 mg, 1.03
mmol) was added followed by paraformaldehyde (.about.170 mg, 5.71
mmol). The reaction mixture was stirred at 100.degree. C. The
sublimed paraformaldehyde was scraped off from the wall into the
reaction mixture, at the same time, more paraformaldehyde (one
scoop every 10 to 20 minutes) was added. This process was continued
while heating at 100.degree. C. until the reaction conversion
reached more than 60% (about 5 hours). The reaction mixture was
cooled and diluted with ethyl acetate (20 mL) and saturated aqueous
sodium bicarbonate (10 mL). The organic layer was separated and
extracted with ethyl acetate (2.times.20 mL). The combined organic
layer was dried over anhydrous sodium sulfate. After removal of
solvent under reduced pressure, the product was purified using
silica gel chromatography. The compound was eluted using a gradient
of 17% to 50% ethyl acetate in hexanes. The product was obtained in
69% yield (247 mg).
[0476] Step c:
[0477] The product from the previous step (97.5 mg, 0.136 mmol) was
added to a microwave vessel followed by cesium carbonate (89.5 mg,
0.275 mmol), 1,10-phenanthroline (12.0 mg, 0.0666 mmol), copper (I)
iodide (12.9 mg, 0.0677 mmol) and ethanol (1 mL). The reaction
mixture was irradiated under microwave for 45 minutes at
100.degree. C. Water (0.1 mL) was added and it was further
irradiated for 10 minutes at 100.degree. C. After addition of
1N-NaOH (0.1 mL), the reaction was diluted with dichloromethane
followed by filtration of the insoluble material. The filtrate was
concentrated and purified using preparative TLC using 17% methanol
in ethyl acetate as the eluent. The obtained product was further
purified using reverse phase HPLC to afford
(3S,4R)-1-[(3R,6S)-6-cyclopropyl-6-[8-ethoxy-6-(trifluoromethyl)-2,4-dihy-
dro-1,3-benzoxazine-3-carbonyl]tetrahydropyran-3-yl]-4-(4-fluorophenyl)pip-
eridine-3-carboxylic acid (13.0 mg, 16% yield) as a TFA salt.
.sup.1H NMR (400 MHz, Methanol-d.sub.4) .delta. 7.23-7.28 (m, 2H),
7.02-7.15 (m, 4H), 6.24-6.21 (m, 1H), 5.63-5.59 (m, 1H), 4.94-4.89
(m, 1H), 4.43-4.35 (m, 1H), 4.10 (q, J=7.0 Hz, 2H), 3.73-3.78 (m,
1H), 3.62-3.67 (m, 1H), 3.53-3.60 (m, 1H), 3.46-3.49 (m, 1H),
3.12-3.37 (m, 3H), 3.04-3.10 (m, 1H), 2.92-2.99 (m, 1H), 2.67-2.61
(m, 1H), 2.30-2.24 (m, 1H), 2.11-2.04 (m, 1H), 1.90-1.97 (m, 1H),
1.69-1.79 (m, 1H), 1.56-1.65 (m, 1H), 1.42 (t, J=7.0 Hz, 3H),
1.20-1.26 (m, 1H), 0.59-0.70 (m, 3H), 0.44-0.49 (m, 1H). MS: (ES)
m/z calculated for C.sub.32H.sub.37F.sub.4N.sub.2O.sub.6
[M+H].sup.+ 621.3, found 621.4.
Example 29
Synthesis of
5-[(3R,4S)-1-[(3R,6S)-6-[6,8-bis(trifluoromethyl)-2,4-dihydro-1,3-benzoxa-
zine-3-carbonyl]-6-cyclopropyl-tetrahydropyran-3-yl]-3-methyl-4-piperidyl]-
-2-fluorobenzoic acid
##STR00063##
[0479] Step a:
[0480] To a mixture of the acid (2.2 g, 5.5 mmol) and HATU (2.2 g,
5.8 mmol) in anhydrous DMF (30 mL), diisopropylethylamine (1.9 mL,
11.0 mmol) was added. The mixture was stirred at rt for 15 min,
then 2-(aminomethyl)-4,6-bis(trifluoromethyl)phenol (1.5 g, 5.8
mmol) in anhydrous DMF (10 mL) was added. The reaction was stirred
at rt overnight, then diluted with H.sub.2O (300 mL) and extracted
with EtOAc (3.times.100 mL). The combined organics were washed with
brine (4.times.50 mL) then dried over MgSO.sub.4, filtered and
evaporated. The residue was dissolved in toluene (300 mL) and
paraformaldehyde (20 g), p-TsOH.times.H.sub.2O (2.1 g, 11 mmol) and
MsOH (0.7 mL, 11 mmol) were added. The reaction was stirred at
reflux (Dean Stark apparatus) for 5 h, then cooled to rt, diluted
with saturated NaHCO.sub.3(200 mL) and extracted with EtOAc
(2.times.100 mL). The combined organics were dried over MgSO.sub.4,
filtered and evaporated. The crude ester was purified by column
chromatography (silica gel, hexanes.fwdarw.1:1 hexanes:EtOAc) to
give a white solid (2.5 g, 70%).
[0481] Step b:
[0482] The product from Step a (2.5 g, 3.8 mmol) was diluted with
1N NaOH (40 mL, 95:5; MeOH:H.sub.2O) and stirred at 40.degree. C.
for 5 h. AcOH (2.3 mL, 40 mmol) was added and the mixture was
evaporated. The residue was diluted with H.sub.2O (200 mL) and
extracted using CH.sub.2Cl.sub.2 (2.times.100 mL). The combined
organics were dried over MgSO.sub.4, filtered and 2M HCl in
Et.sub.2O (5.7 mL, 11.4 mmol) was added. The mixture was evaporated
and the residue was washed with Et.sub.2O (50 mL) to give the HCl
salt of the product as a yellowish solid (2.15 g, 83%). .sup.1H NMR
(400 MHz, CD.sub.3OD) .delta. 7.97-7.83 (m, 2H), 7.79-7.70 (m, 1H),
7.51-7.41 (m, 2H), 6.21-5.81 (m, 2H), 5.19-4.92 (m, 2H), 4.45-4.32
(m, 1H), 3.71-3.52 (m, 3H), 3.48-3.35 (m, 1H), 3.24-3.09 (m, 1H),
2.85 (t, J=12.0 Hz, 1H), 2.71-2.60 (m, 1H), 2.60-2.48 (m, 1H),
2.37-2.24 (m, 1H), 2.23-2.08 (m, 1H), 2.08-1.94 (m, 2H), 1.89-1.70
(m, 1H), 1.70-1.55 (m, 1H), 1.24-1.13 (m, 1H), 0.75 (d, J=6.6 Hz,
3H), 0.72-0.28 (m, 4H). MS: (ES) m/z calculated for
C.sub.32H.sub.34F.sub.7N.sub.2O.sub.5 [M+H].sup.+ 659.2, Found
658.7.
Biological Examples
[0483] In Vitro Assays
[0484] Reagents
[0485] THP-1 cells were obtained from the American Type Culture
Collection (Manassas, Va.) and cultured in RPMI tissue culture
medium supplemented with 10% fetal calf serum (FCS) in a humidified
5% CO.sub.2 incubator at 37.degree. C. Recombinant human chemokine
proteins MCP-1 were obtained from R&D Systems (Minneapolis,
Minn.). .sup.125-labeled MCP-1 protein was obtained from Amersham
(Piscataway, N.J.). ChemoTX.RTM. chemotaxis microchambers were
purchased from Neuro Probe (Gaithersburg, Md.). CyQUANT.RTM. cell
proliferation kits were purchased from Molecular Probes (Eugene,
Oreg.). Calcium indicator dye Fluo-4 AM was purchased from
Molecular Devices (Mountain View, Calif.).
[0486] Ligand Binding Assay
[0487] Ligand binding assay can be used to determine the ability of
potential CCR2 antagonists to block the interaction between CCR2
and its ligand MCP-1. CCR2 expressing THP-1 cells are centrifuged
and resuspended in assay buffer (20 mM HEPES pH 7.1, 140 mM NaCl, 1
mM CaCl.sub.2, 5 mM MgCl.sub.2, and with 0.2% bovine serum albumin)
to a concentration of 2.2.times.105 cells/mL. Binding assays are
set up as follows. First, 0.09 mL of cells (1.times.105 THP-1
cells/well) was added to the assay plates containing the compounds,
giving a final concentration of .about.2-10 uM each compound for
screening (or part of a dose response for compound IC50
determinations). Then 0.09 mL of 125I labeled MCP-1 (obtained from
Amersham; Piscataway, N.J.) diluted in assay buffer to a final
concentration of .about.50 .mu.M, yielding .about.30,000 cpm per
well, is added, the plates sealed and incubated for approximately 3
hours at 4.degree. C. on a shaker platform. Reactions are aspirated
onto GF/B glass filters pre-soaked in 0.3% polyethyleneimine (PEI)
solution, on a vacuum cell harvester (Packard Instruments; Meriden,
Conn.). Scintillation fluid (50 uL; Microscint 20, Packard
Instruments) is added to each well, the plates are sealed and
radioactivity measured in a Top Count scintillation counter
(Packard Instruments). Control wells containing either diluent only
(for total counts) or excess MCP-1 (1 ug/mL, for non-specific
binding) are used to calculate the percent of total inhibition for
compound. The computer program Prism from GraphPad, Inc. (San
Diego, Ca) is used to calculate IC50 values. IC50 values are those
concentrations required to reduce the binding of labeled MCP-1 to
the receptor by 50%.
[0488] Calcium Flux Assay
[0489] Calcium flux assay measures an increase in intracellular
calcium following ligand-induced receptor activationand may be
employed as a secondary assay following primary screening. Such an
assay may be carried out, for instance, on a FLIPR machine
(Molecular Devices, Mountain View, Calif.). To begin an assay,
chemokine expressing cells (such as THP-1 cells for CCR2 assay) are
harvested by centrifugation of cell suspension, and resuspended to
1.5.times.10.sup.6 cells/mL in HBSS (with 1% fetal calf serum).
Cells are then labeled with a calcium indicator dye Fluo-4 AM for
45 minutes at 37.degree. C. with gentle shaking. Following
incubation, cells are pelleted, washed once with HBSS and
resuspended in the same buffer at a density of 1.6.times.10.sup.6
cells/mL. One hundred microliters of labeled cells are mixed with
10 uL of test compound at the appropriate concentrations on an
assay plate. Chemokine protein (MCP-1 at a final concentration of
0.1 nM for CCR2 assay) is added to activate the receptor. The
degree of inhibition is determined by comparing calcium signals
between compound-treated and untreated cells. IC50 calculations are
further performed by non-linear squares regression analysis using
Graphpad Prism (Graphpad Software, San Diego, Calif.).
[0490] Conventional Migration Assay
[0491] Conventional migration assay was used to determine the
efficacy of potential receptor antagonists in blocking migration
mediated through chemokines (such as CCR2). This assay was
routinely performed using the ChemoTX.RTM. microchamber system with
a 5-.mu.m pore-sized polycarbonate membrane. To begin such an
assay, chemokine expressing cells (such as THP-1 cells for CCR2
assay) were harvested by centrifugation of cell suspension at 1000
RPM on a GS-6R Beckman centrifuge. The cell pellet was resuspended
in chemotaxis buffer (HBSS with 0.1% BSA) at 10.times.10.sup.6
cells/mL for the CCR2 assay. Test compounds at desired
concentrations were prepared from 10 mM stock solutions by serial
dilutions in chemotaxis buffer. An equal volume of cells and
compounds were mixed and incubated at room temperature for 15
minutes. Afterwards, 20 .mu.L of the mixture was transferred onto
the porous membrane of a migration microchamber, with 29 .mu.L of
chemokine ligand (0.1 nM chemokine MCP-1 protein for CCR2 assay)
placed at the lower chamber. Following an incubation at 37.degree.
C. (90-minute for CCR2), during which cells migrated against the
chemokine gradient, the assay was terminated by removing the cell
drops from atop the filter. To quantify cells migrated across the
membrane, 5 .mu.L of 7.times.CyQUANT.RTM. solution was added to
each well in the lower chamber, and the fluorescence signal
measured on a Spectrafluor Plus fluorescence plate reader (TECAN,
Durham, N.C.). The degree of inhibition was determined by comparing
migration signals between compound-treated and untreated cells.
IC.sub.50 calculation was further performed by non-linear squares
regression analysis using Graphpad Prism (Graphpad Software, San
Diego, Calif.).
[0492] In Vivo Efficacy
[0493] Evaluation of Compounds in a Rat Model of Collagen-Induced
Arthritis
[0494] A 17-day study of type II collagen-induced arthritis can be
conducted to evaluate the effects of compounds of the invention on
arthritis-induced clinical ankle swelling. Rat collagen-induced
arthritis is an experimental model of polyarthritis that has been
widely used for preclinical testing of numerous anti-arthritic
agents (see Trentham et al., J. Exp. Med. 146(3):857-868 (1977),
Bendele et al., Toxicologic Pathol. 27:134-142 (1999), Bendele et
al., Arthritis Rheum. 42:498-506 (1999)). The hallmarks of this
model are reliable onset and progression of robust, easily
measurable polyarticular inflammation, marked cartilage destruction
in association with pannus formation and mild to moderate bone
resorption and periosteal bone proliferation. Female Lewis rats
(approximately 0.2 kilograms) are anesthetized with isoflurane and
injected with Freund's Incomplete Adjuvant containing 2 mg/mL
bovine type II collagen at the base of the tail and two sites on
the back on days 0 and 6 of this 17-day study. Compound is dosed
daily by sub-cutaneous injection from day 9 to day 17 at a dose of,
for instance, 100 mg/kg and a volume of 1 mL/kg in an appropriate
vehicle. Caliper measurements of the ankle joint diameter are taken
daily, and reducing joint swelling is taken as a measure of
efficacy.
[0495] Evaluation of Compounds in Animal Models of Human Ulcerative
Colitis
[0496] A murine model described by Panwala and coworkers (Panwala,
et al., J Immunol., 161(10):5733-44 (1998)) involves genetic
deletion of the murine multi-drug resistant gene (MDR). MDR
knockout mice (MDR-/-) are susceptible to developing a severe,
spontaneous intestinal inflammation when maintained under specific
pathogen-free facility conditions. The intestinal inflammation seen
in MDR-/- mice has a pathology similar to that of human
inflammatory bowel disease (IBD) and is defined by Th1 type T cells
infiltration into the lamina propria of the large intestine.
[0497] Another murine model for IBD has been described by Powrie et
al., Int Immunol., 5(11):1461-71 (1993), in which a subset of CD4+
T cells (called CD45RB(high)) from immunocompetent mice are
purified and adoptively transferred into immunodeficient mice (such
as C.B-17 scid mice). The animal restored with the CD45RBhighCD4+ T
cell population developed a lethal wasting disease with severe
mononuclear cell infiltrates in the colon, pathologically similar
with human IBD.
[0498] Evaluation of Compounds in Animal Models of Human Crohn's
Disease
[0499] The TNF ARE(-/-) model. The role of TNF in Crohn's disease
in human has been demonstrated more recently by success of
treatment using anti-TNF alpha antibody by Targan et al., N Engl J
Med., 337(15):1029-35 (1997). Mice with aberrant production of
TNF-alpha due to genetic alteration in the TNF gene (ARE-/-)
develop Crohn's-like inflammatory bowel diseases (see Kontoyiannis
et al., Immunity, 10(3):387-98 (1999)).
[0500] The SAMP/yit model. This model is described by Kosiewicz et
al., J Clin Invest., 107(6):695-702 (2001). The mouse strain,
SAMP/Yit, spontaneously develops a chronic inflammation localized
to the terminal ileum. The resulting ileitis is characterized by
massive infiltration of activated T lymphocytes into the lamina
propria, and bears a remarkable resemblance to human Crohn's
disease.
[0501] Evaluation of Compounds in a Mouse Model of
Thioglycollate-Induced Peritoneal Inflammation
[0502] A 2-day study of thioglycollate-induced inflammation was
conducted to evaluate the effects of the test compound, Compound 53
(FIG. 1). The hallmarks of this model are reliable onset and
progression of robust, easily measurable inflammatory cellular
infiltrate. For the induction of inflammatory peritonitis in Lewis
rats, Brewer-Thioglycollate (1.0 mL, 4% solution in distilled
water) was injected intra peritoneal (i.p.). Before this injection,
the treatment group received test compound,
4-chloro-3-trifluoromethyl-N-[5-chloro-2-(2-methanesulfonyl-benzoyl)-pyri-
din-3-yl]-benzenesulfonamide, or vehicle and the control group
received the same volume of PBS as i.p. injection. After 2 days, a
peritoneal lavage was performed with ice-cold PBS containing 1 mM
EDTA. The recovered cells were counted with a cell counter (Coulter
Counter; Coulter Pharmaceutical, Palo Alto, Calif.) and
monocytes/macrophages were identified by flow cytometry using
light-scatter properties.
[0503] The test compound significantly and specifically inhibited
the number of inflammatory macrophages elicited following
tioglycollate injection.
[0504] Evaluation of Compounds in a Mouse Model of Bacterial
Infection
[0505] A 1-day study of Streptococcus pneumoniae infection can be
conducted to evaluate the effects of the test compound. The model
measures bacterial infection and spread in an animal following
pulmonary infection with live bacterial cultures, measured by
inflammatory cellular infiltrate, and assessment of bacterial
burden. C57/B6 mice are inoculated intra nasally with LD50 400 CFU
at day 0. Groups are either compound or vehicle control treated 1
day prior to bacterial inoculation and twice daily throughout the
study. Bacterial burden is measured at 24 hours by plating serial
dilutions of homogenized lung tissue on agar plates and counting
colonies.
[0506] Evaluation of Compounds in a Mouse Model of Lung
Carcinoma
[0507] Many tumor models in animals are known in the art, and may
be employed to evaluate a compound of instance. For instance, in a
lung carcinoma xenograft study, A549 tumor fragments (30-40 mg) are
implanted into the sub cutaneous space in nude mice. Tumors are
permitted to grow until approximately 150 mg in size (between 100
and 200 mg) at which point mice are enrolled in the study and
treatment begins. Mice are treated with a compound of interest or
the vehicle control. Melphalan may be included as a positive
control (9 mpk/dose, ip administration, Q4D.times.3). Tumors are
measured twice weekly with a caliper in two dimensions and
converted to tumor mass using the formula for a prolate ellipsoid
(a.times.b2/2), where a is the longer dimension and b is the
shorter dimension, and assuming unit density (1 mm3=1 mg). Body
weights may also be measured twice weekly to assess any adverse
effects of compound dosing. Antitumor activity is assessed by the
delay in tumor growth of the treated group in comparison to the
vehicle-treated control group.
[0508] Evaluation of Compounds in a Mouse Model of Glioblastoma
[0509] Many tumor models in animals are known in the art, and may
be employed to evaluate a compound of instance. For instance, in a
murine glioblastoma model, 1.times.106 U251MG cells are implanted
by stereotactic injection into the into the brains of nude mice.
After 20 days tumors are irradiated with between 1-15 Gy of
radiation. Following irradiation mice are treated (eg via
subcutaneous, intraperitoneal, oral, parenteral or other route)
with compound or vehicle control and tumors are allowed to
progress. Tumor growth and/or mortality are monitored for the
remainder of the study. Tumors are measured twice weekly with a
caliper in two dimensions and converted to tumor mass using the
formula for a prolate ellipsoid (a.times.b2/2), where a is the
longer dimension and b is the shorter dimension, and assuming unit
density (1 mm3=1 mg). Body weights may also be measured twice
weekly to assess any adverse effects of compound dosing. Antitumor
activity is assessed by the delay in tumor growth of the treated
group in comparison to the vehicle-treated control group.
[0510] Evaluation of Compounds in a Mouse Model of Pancreatic
Cancer
[0511] Many tumor models in animals are known in the art, and may
be employed to evaluate a compound of instance. For instance, in a
murine pancreatic cancer model, panco2 cells (NCI) were implanted
orthotopically in the pancreas of C57bl/6 mice. Tumors were allowed
to progress for three weeks, then animals were treated with either
Compound 53 (FIG. 1) or vehicle control by subcutaneous injection
once daily for three weeks. Compound efficacy was assessed by
analysis of tumor growth. Animals treated with compound Compound 53
(FIG. 1) displayed significantly smaller tumors than vehicle
treated animals.
[0512] Evaluation of Compounds in a Mouse Model of Polycystic
Kidney Disease
[0513] In a mouse model of polycystic kidney disease
Pkd1flox/flox:Pkhd1-Cre mice (Jackson Labs, ME) these mice
spontaneously develop polycystic kidney disease due to a
kidneyselective genetic deficiency of the mouse ortholog of the
human PKD1 gene, mutations in which are responsible for the most
common and most severe form of ADPKD. Disease is allowed to
develop, and at day 10 animals are treated with compound o the
invention or the vehicle control by subcutaneous injectin once
daily until day 26. At this time animals are sacrificed and kidney
mass, blood urea and kidney cystic counts are performed. Efficacy
is measured as an improvement of these measures when treated with
compound Compound 53 (FIG. 1).
[0514] It is understood that the examples and embodiments described
herein are for illustrative purposes only and that various
modifications or changes in light thereof will be suggested to
persons skilled in the art and are to be included within the spirit
and purview of this application and scope of the appended
claims.
[0515] All publications, patents, and patent applications cited
herein are hereby incorporated by reference in their entirety for
all purposes.
* * * * *