U.S. patent application number 17/828643 was filed with the patent office on 2022-09-22 for btk inhibitors with improved dual selectivity.
The applicant listed for this patent is BEIGENE, LTD.. Invention is credited to Yunhang GUO, Zhiwei WANG, Desheng YU.
Application Number | 20220298163 17/828643 |
Document ID | / |
Family ID | 1000006366398 |
Filed Date | 2022-09-22 |
United States Patent
Application |
20220298163 |
Kind Code |
A1 |
WANG; Zhiwei ; et
al. |
September 22, 2022 |
BTK INHIBITORS WITH IMPROVED DUAL SELECTIVITY
Abstract
Disclosed herein is a tri-substituted phenyl Btk inhibitors with
improved dual selectivity, a method and a composition for
inhibiting Btk and treating disease associated with undesirable Btk
activity (Btk-related diseases).
Inventors: |
WANG; Zhiwei; (Beijing,
CN) ; GUO; Yunhang; (Beijing, CN) ; YU;
Desheng; (Beijing, CN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BEIGENE, LTD. |
Camana Bay |
|
KY |
|
|
Family ID: |
1000006366398 |
Appl. No.: |
17/828643 |
Filed: |
May 31, 2022 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16637615 |
Feb 7, 2020 |
11377449 |
|
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PCT/CN2018/100145 |
Aug 12, 2018 |
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17828643 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07D 487/14 20130101;
C07D 487/04 20130101; C07D 471/14 20130101; C07D 519/00
20130101 |
International
Class: |
C07D 487/04 20060101
C07D487/04; C07D 471/14 20060101 C07D471/14; C07D 487/14 20060101
C07D487/14; C07D 519/00 20060101 C07D519/00 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 12, 2017 |
CN |
PCT/CN2017/097291 |
Claims
1-25. (canceled)
26. A method of treating a disease associated with undesirable Btk
activity in a subject comprising administering to the subject, a
compound of formula (I): ##STR00194## or a stereoisomer thereof, or
a pharmaceutically acceptable salt thereof, wherein: L is a bond,
CH.sub.2, NR.sup.12, O, or S; R.sup.1 is halogen, heteroalkyl,
alkyl, alkenyl, cycloalkyl, aryl, saturated or unsaturated
heterocyclyl, heteroaryl, alkynyl, --CN, --NR.sup.13R.sup.14,
--OR.sup.13, --COR.sup.13, --CO.sub.2R.sup.13,
--CONR.sup.13R.sup.14, --C(.dbd.NR.sup.13)NR.sup.14R.sup.15,
--NR.sup.13COR.sup.14, --NR.sup.13CONR.sup.14R.sup.15,
--NR.sup.13CO.sub.2R.sup.14, --SO.sub.2R.sup.13,
--NR.sup.13SO.sub.2NR.sup.14R.sup.15, or
--NR.sup.13SO.sub.2R.sup.4, wherein the alkyl, alkenyl, alkynyl,
cycloalkyl, heteroaryl, aryl, and saturated or unsaturated
heterocyclyl are optionally substituted with at least one
substituent R.sup.16; n is an integer of 2, 3 or 4; each R.sup.2 is
independently halogen, alkyl, --S-alkyl, --CN, --NR.sup.13R.sup.14,
--OR.sup.13, --COR.sup.13, --CO.sub.2R.sup.13,
--CONR.sup.13R.sup.14, --C(.dbd.NR.sup.13)NR.sup.14R,
--NR.sup.13COR.sup.14, --NR.sup.13CONR.sup.14R.sup.15,
--NR.sup.13CO.sub.2R.sup.1, --SO.sub.2R.sup.13,
--NR.sup.13SO.sub.2NR.sup.14R.sup.15, or
--NR.sup.13SO.sub.2R.sup.14, wherein R.sup.2 is, optionally,
partially or fully deuterated; R.sup.4a and R.sup.4b are each
independently hydrogen, halogen, heteroalkyl, alkyl, alkenyl,
cycloalkyl, saturated or unsaturated heterocyclyl, heteroaryl,
alkynyl, --CN, --NR.sup.13R.sup.14, --OR.sup.13, --COR.sup.13,
--CO.sub.2R.sup.13, --CONR.sup.13R.sup.14,
--C(.dbd.NR.sup.13)NR.sup.14R, --NR.sup.13COR.sup.14,
--NR.sup.13CONR.sup.14R.sup.13, --NR.sup.13CO.sub.2R.sup.14,
--SO.sub.2R.sup.13, --NR.sup.13SO.sub.2NR.sup.14R.sup.15, or
--NR.sup.13SO.sub.2R.sup.14, wherein the alkyl, alkenyl, alkynyl,
cycloalkyl, heteroaryl, aryl, and saturated or unsaturated
heterocyclyl are optionally substituted with at least one
substituent R.sup.16; R.sup.5a and R.sup.5b are each independently
hydrogen, halogen, heteroalkyl, alkyl, alkenyl, cycloalkyl, aryl,
saturated or unsaturated heterocyclyl, heteroaryl, alkynyl, --CN,
--NR.sup.13R.sup.14, --OR.sup.13, --COR.sup.13, --CO.sub.2R.sup.13,
--CONR.sup.13R.sup.14, --C(.dbd.NR.sup.13)NR.sup.14R.sup.15,
--NR.sup.13COR.sup.4, --NR.sup.13CONR.sup.4R.sup.1,
--NR.sup.13CO.sub.2R.sup.4, --SO.sub.2R.sup.13,
--NR.sup.13SO.sub.2NR.sup.14R.sup.15, or
--NR.sup.13SO.sub.2R.sup.14, wherein the alkyl, alkenyl, alkynyl,
cycloalkyl, heteroaryl, aryl, and saturated or unsaturated
heterocyclyl are optionally substituted with at least one
substituent R.sup.16; R.sup.6a and R.sup.6b are each independently
hydrogen, halogen, heteroalkyl, alkyl, alkenyl, cycloalkyl, aryl,
saturated or unsaturated heterocyclyl, heteroaryl, alkynyl, --CN,
--NR.sup.13R.sup.14, --OR.sup.13, --COR.sup.13, --CO.sub.2R.sup.13,
--CONR.sup.13R.sup.14, --C(.dbd.NR.sup.13)NR.sup.14R.sup.15,
--NR.sup.13COR.sup.4, --NR.sup.13CONR.sup.4R.sup.1,
--NR.sup.13CO.sub.2R.sup.4, --SO.sub.2R.sup.13,
--NR.sup.13SO.sub.2NR.sup.14R.sup.15, or
--NR.sup.13SO.sub.2R.sup.14, wherein the alkyl, alkenyl, alkynyl,
cycloalkyl, heteroaryl, aryl, and saturated or unsaturated
heterocyclyl are optionally substituted with at least one
substituent R.sup.16; or wherein (R.sup.4a and R.sup.5a), or
(R.sup.4a and R.sup.5b), or (R.sup.4b and R.sup.5a), or (R.sup.4b
and R.sup.5b), or (R.sup.5a and R.sup.6a), or (R.sup.5a and
R.sup.6b), or (R.sup.5b and R.sup.6a), or (R.sup.5b and R.sup.6b),
together with the atoms to which they are attached, may form a
fused ring selected from cycloalkyl, saturated or unsaturated
heterocycle, aryl, and heteroaryl rings, optionally substituted
with at least one substituent R.sup.16; R.sup.12 is H or alkyl;
R.sup.13, R.sup.14 and R.sup.15 are each independently H,
heteroalkyl, alkyl, alkenyl, alkynyl, cycloalkyl, saturated or
unsaturated heterocyclyl, aryl, or heteroaryl; wherein (R.sup.13
and R.sup.14), and/or (R.sup.14 and R.sup.15) together with the
atom(s) to which they are attached, each can form a ring selected
from cycloalkyl, saturated or unsaturated heterocycle, aryl, and
heteroaryl rings optionally substituted with at least one
substituent R.sup.16; and R.sup.16 is halogen, substituted or
unsubstituted alkyl, substituted or unsubstituted alkenyl,
substituted or unsubstituted alkynyl, substituted or unsubstituted
cycloalkyl, substituted or unsubstituted aryl, substituted or
unsubstituted heteroaryl, substituted or unsubstituted
heterocyclyl, oxo, --CN, --OR.sup.a, --NR.sup.aR.sup.b,
--COR.sup.a, --CO.sub.2R.sup.a, --CONR.sup.aR.sup.b,
--C(.dbd.NR.sup.a)NR.sup.bR.sup.c, --NR.sup.aCOR.sup.b,
--NR.sup.aCONR.sup.aR.sup.b, --NR.sup.aCO.sub.2R.sup.b,
--SO.sub.2R.sup.a, --SO.sub.2aryl,
--N.sup.aSO.sub.2NR.sup.bR.sup.c, or --NR.sup.aSO.sub.2R.sup.b,
wherein R.sup.a, R.sup.b, and R.sup.c are independently hydrogen,
halogen, substituted or unsubstituted alkyl, substituted or
unsubstituted alkenyl, substituted or unsubstituted alkynyl,
substituted or unsubstituted cycloalkyl, substituted or
unsubstituted aryl, substituted or unsubstituted heteroaryl,
substituted or unsubstituted heterocyclyl, wherein (R.sup.a and
R.sup.b), and/or (R.sup.a and R.sup.b) together with the atoms to
which they are attached, can form a ring selected from cycloalkyl,
saturated or unsaturated heterocycle, aryl, and heteroaryl
rings.
27. The method of claim 26, wherein L is a bond.
28. The method of claim 26, wherein R.sup.1 is halogen, alkyl,
alkenyl, cycloalkyl, aryl, saturated heterocyclyl, heteroaryl, or
--OR.sup.13, wherein the alkyl, alkenyl, cycloalkyl, and heteroaryl
are optionally substituted with at least one substituent
R.sup.16.
29. The method of claim 26, wherein n is 2.
30. The method of claim 29, wherein one of R.sup.2 is at position 3
of the phenyl ring, and the other R.sup.2 is at position 5 of the
phenyl ring, wherein R.sup.2 is optionally partially or fully
deuterated.
31. The method of claim 30, wherein R.sup.2, at each of its
occurrence, is halogen, alkyl or --O-alkyl, wherein the alkyl group
or moiety is optionally partially or fully deuterated.
32. The method of claim 26, wherein R.sup.4a, R.sup.5a, R.sup.5b,
R.sup.6a and R.sup.6b are hydrogen; and R.sup.4b is alkyl, or
saturated heterocyclyl, wherein the alkyl and saturated
heterocyclyl are optionally substituted with at least one
substituent R.sup.16.
33. The method of claim 32, wherein R.sup.4a, R.sup.5a, R.sup.5b,
R.sup.6a and R.sup.6b are hydrogen; and R.sup.4b is a saturated
heterocyclyl containing a nitrogen atom which is optionally
substituted with acryloyl or propiolyl.
34. The method of claim 33, wherein the saturated heterocyclyl in
the definition of R.sup.4b is a monocyclic ring selected from an
azetidine, pyrrole, piperidine, azapane or azocane ring; or a spiro
or bridged bicyclic ring selected from azabicyclooctanyl,
azaspirononanyl or azaspiroheptanyl.
35. The method of claim 26, wherein (R.sup.4a and R.sup.5a), or
(R.sup.4a and R.sup.5b), or (R.sup.4b and R.sup.5a) or (R.sup.4b
and R.sup.5b), or (R.sup.5a and R.sup.6a), or (R.sup.5a and
R.sup.6b), or (R.sup.5b and R.sup.6a), or (R.sup.5b and R.sup.6b),
together with the atoms to which they are attached, form a fused
ring selected from cycloalkyl, saturated or unsaturated
heterocycle, aryl, and heteroaryl rings, optionally substituted
with at least one substituent R.sup.16.
36. The method of claim 35, wherein the fused saturated or
unsaturated heterocycle ring is an azetidine, pyrrole, piperidine,
azapane or azocane ring optionally substituted with a substituent
R.sup.16 at the nitrogen atom of the fused ring, wherein the
substituent R.sup.16 is acryloyl or propiolyl.
37. A method of treating a disease associated with undesirable Btk
activity in a subject comprising administering to the subject, a
compound of formula (II): ##STR00195## or a stereoisomer thereof,
or a pharmaceutically acceptable salt thereof, wherein: R.sup.x is
halogen, alkyl, or --O-alkyl, wherein the alkyl group or moiety is
optionally partially or fully deuterated; R.sup.y is halogen, alkyl
optionally substituted with at least substituents selected from
hydroxy, halogen, or cycloalkyl; and R.sup.z is halogen, alkyl, or
--O-alkyl, wherein the alkyl group or moiety is optionally
partially or fully deuterated.
38. The method of claim 37, wherein R.sup.x is methoxy, ethoxy,
methyl, ethyl or chloro, wherein the methoxy, ethoxy, methyl, ethyl
is optionally partially or fully deuterated; R.sup.y is
2-hydroxylethyl, trifluoromethyl, isopropyl, cyclopropyl, methyl,
chloro, bromo, or iodo; and R.sup.z is methoxy, methyl, or chloro,
wherein the methoxy or methyl is optionally partially or fully
deuterated.
39. The method of claim 37, wherein R.sup.x is methoxy, ethoxy,
methyl, chloro, CD.sub.3, -CD.sub.2CD.sub.3, OCD.sub.3, or
--O-CD.sub.2CD.sub.3; R.sup.y is 2-hydroxylethyl, CF.sub.3,
isopropyl, cyclopropyl, methyl, chloro, bromo, or iodo; and R.sup.z
is ethoxy, methoxy, methyl, ethyl, chloro, or CD.sub.3,
-CD.sub.2CD.sub.3, OCD.sub.3, or --O-CD.sub.2CD.sub.3.
40. The method of claim 37, wherein R.sup.x is methoxy, ethoxy,
methyl, ethyl, chloro, or OCD.sub.3; R.sup.y is 2-hydroxylethyl,
CF.sub.3, isopropyl, cyclopropyl, methyl, chloro, bromo, or iodo;
and R.sup.z is methoxy, methyl, chloro, or OCD.sub.3.
41. The method of claim 37, wherein the compound is in
(S)-configuration of the following formula: ##STR00196##
42. The method of claim 26, which is selected from: ##STR00197##
##STR00198## ##STR00199## ##STR00200## ##STR00201## ##STR00202##
##STR00203## ##STR00204## ##STR00205## ##STR00206## ##STR00207##
##STR00208## ##STR00209## ##STR00210## ##STR00211## ##STR00212##
##STR00213## ##STR00214## ##STR00215## ##STR00216## ##STR00217##
##STR00218## ##STR00219## ##STR00220## ##STR00221##
pharmaceutically acceptable salt thereof.
43. The method of claim 28, wherein R.sup.1 is alkyl or
cycloalkyl.
44. The method of claim 26, wherein the compound is: ##STR00222##
or a pharmaceutically acceptable salt thereof.
45. The method of claim 26, wherein the compound is: ##STR00223##
or a pharmaceutically acceptable salt thereof.
46. The method of claim 26, wherein the compound is: ##STR00224##
or a stereoisomer thereof, or a pharmaceutically acceptable salt
thereof.
47. The method of claim 26, wherein the compound is: ##STR00225##
or a stereoisomer thereof, or a pharmaceutically acceptable salt
thereof.
48. The method of claim 26, wherein the disease is a B-cell
malignancy.
49. The method of claim 48, wherein the B-cell malignancy is
selected from lymphoma, non-Hodgkin's lymphoma (NHL), diffuse large
B cell lymphoma (DLBCL), mantle cell lymphoma (MCL), follicular
lymphoma (FL), chronic lymphocytic leukemia (CLL), small
lymphocytic lymphoma (SLL), Waldenstrom macroglobulinemia (WM),
marginal zone lymphoma (MZL), Hairy cell leukemia (HCL),
Burkitt's-like leukemia (BL), or a combination of two or more
thereof.
50. The method of claim 37, wherein the disease is a B-cell
malignancy.
51. The method of claim 50, wherein the B-cell malignancy is
selected from lymphoma, non-Hodgkin's lymphoma (NHL), diffuse large
B cell lymphoma (DLBCL), mantle cell lymphoma (MCL), follicular
lymphoma (FL), chronic lymphocytic leukemia (CLL), small
lymphocytic lymphoma (SLL), Waldenstrom macroglobulinemia (WM),
marginal zone lymphoma (MZL), Hairy cell leukemia (HCL),
Burkitt's-like leukemia (BL), or a combination of two or more
thereof.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of International Patent
Application No. PCT/CN2017/097291 filed Aug. 12, 2017, the
disclosures of which are hereby incorporated by reference in its
entirety for all purposes.
FILED OF THE INVENTION
[0002] Disclosed herein is a tri-substituted phenyl Btk inhibitors
with improved dual selectivity, a method and a composition for
inhibiting Btk and treating disease associated with undesirable Btk
activity (Btk-related diseases).
BACKGROUND OF THE INVENTION
[0003] Bruton's tyrosine kinase (Btk) belongs to the Tec family of
cytoplasmic tyrosine kinases, which is the second largest family of
non-receptor kinases in humans (Vetrie et al., Nature 361: 226-233,
1993; Bradshaw, Cell Signal. 22: 1175-84, 2010). It is expressed in
all cell lineages of the hematopoietic system, except for T cells
and is localized in bone marrow, spleen and lymph node tissue
(Smith et al., J. Immunol. 152: 557-565, 1994). Inactivating
mutations in the gene encoding Btk cause X-linked
agammaglobulinemia (XLA) in humans and X-linked immunodeficiency
(XID) in mice (Conley et al., Annu. Rev. Immunol. 27: 199-227,
2009). Both diseases are characterized by dramatic defects in B
cell development and function, suggesting the essential role of Btk
for B cell development and function. In addition, constitutive
activation of Btk in B cells results in the accumulation of
autoreactive plasma cells (Kersseboom et al., Eur J Immunol.
40:2643-2654, 2010). Btk is activated by upstream Src-family
kinases in BCR signaling pathway. Once activated, Btk in turn
phosphorylates phospholipase-C.gamma. (PLC.gamma.), leading to
Ca.sup.2+ mobilization and activation of NF-xB and MAP kinase
pathways. These proximal signaling events promote expression of
genes involved in proliferation and survival (Humphries et al., J.
Biol. Chem. 279: 37651, 2004). In addition to its essential
regulatory role as downstream of BCR, Btk activity also plays a
critical role in FcR signaling. Signaling via FcR.gamma. associated
receptors also promotes Btk-dependent proinflammatory cytokine
production by cells such as macrophages (Di Paolo et al., Nat.
Chem. Biol. 7: 41-50, 2011). Btk has been an important target due
to its proximal location in the BCR and FcR signaling pathways.
Preclinical studies show that Btk deficient mice are resistant to
developing collagen-induced arthritis. Moreover, clinical studies
of Rituxan, a CD20 antibody to deplete mature B-cells, reveal the
key role of B-cells in a number of inflammatory diseases such as
rheumatoid arthritis, systemic lupus erythematosus and multiple
sclerosis (Gurcan et al., Int. Immunopharmacol. 9: 10-25, 2009). In
addition, aberrant activating of Btk plays important role in
pathogenesis of B-cell lymphomas indicating that inhibition of Btk
is useful in the treatment of hematological malignancies (Davis et
al., Nature 463: 88-92, 2010).
[0004] The covalent Btk inhibitor, i.e., ibrutinib (Imbruvica.RTM.)
was approved by the US Food and Drug Administration for the
treatment of chronic lymphocytic leukemia (CLL), mantle cell
lymphoma (MCL), Waldenstrom's macroglobulinemia (WM) and chronic
graft versus host disease (cGVHD). Even though excellent efficacy
and general tolerability, there are adverse events like bleeding,
rash and diarrhea reported, which are not typically observed in Btk
deficient patients. From early phase of clinical trials testing
Ibrutinib as monotherapy in MCL, CLL and small lymphocytic lymphoma
(SLL), rash occurred at a frequency of 20-25% and diarrhea occurred
at a frequency of 50-60% (Varinder et al., Ann Hematol
96(7):1175-1184, 2017). These common toxicities are self-limiting
grade 1 or 2 adverse events, and dose interruption or reduction is
typically not required. Bleeding episodes were observed in 44% of
patients in the CLL registration trial (Byrd J C, et al., N Engl J
Med 369: 32-42, 2013) and in up to 61% of patients after a longer
observation period (Byrd J C, et al., Blood 125: 2497-2506, 2015
and Wang M L, N Engl J Med 369: 507-516, 2013). Patients taking
ibrutinib have experienced fatal bleeding events. Grade .gtoreq.3
bleeding, including subdural hematoma, gastrointestinal bleeding,
hematuria, and postprocedural hemorrhage, which have been reported
in up to 6% of patients taking ibrutinib (Lisa A. Raedler, Am
Health Drug Benefits. 9: 89-92, 2016). Bleeding as a common adverse
event (AE) has also been found in other Btk inhibitors such as
ONO-4059, ACP-196, and BGB-3111 (Jun Chen, et al., EHA Learning
Center, meeting abstract 2016).
[0005] These Ibrutinib related adverse effects were thought to be
mostly associated with the off-target effects of ibrutinib that
demonstrated to inhibit EGFR and Tec. Targeting EGFR is well known
to induce dramatic cutaneous toxicity and gastrointestinal adverse
effects because EGFR-signaling cascade is involved in biology of
the skin and gastrointestinal system (Li, et al., Target Oncol
4(2):107-19, 2009 and Melosky, et al., Curr Oncol 19 (Suppl 1):
S59-63, 2012). Both of Btk and Tec are belong to the Tec family
kinases. Platelets express Btk and Tec, which serve as downstream
of Glycoprotein VI (GPVI) signaling. Tec compensates for the
absence of Btk in signaling downstream of GPVI in murine platelets
(Atkinson B T, Blood 102: 3592-3599, 2003). Ibrutinib inhibition of
Tec kinase interferes with platelet aggregation and may contribute
to the observed bleeding (John C. Byrd, et al., N Engl J Med
374:323-332, 2016).
[0006] International application WO2014173289A disclosed a series
of fused heterocyclic compounds, which were proved to be potent Btk
inhibitors with high or comparable affinity to the target kinase.
In WO2014173289, Compound 167 was proved to the most selective
compound against Btk over Tec. The IC50 activity data of Compound
167 against Btk and Tec are reported in Table II of the present
specification (i.e., 167# in Table II). However, Compound 167 was
also proved to have high affinity to EGFR, which is likely to give
rise to adverse effects such as dramatic cutaneous toxicity and
gastrointestinal adverse effects. This compound also showed low
cellular potency, oral bioavailability and pharmacodynamic
activity, which are not acceptable drug like properties.
[0007] Therefore, there is a need to develop a new Btk inhibitor
which exhibits potent inhibitory activity against Btk and, at the
same time, improves the selectivity of Btk over both Tec and EGFR
(i.e., good dual selectivity corresponding to no less than 100 fold
selectivity of Btk over Tec and of Btk over EGFR), which will
reduce the clinical adverse effects significantly.
SUMMARY OF THE INVENTION
[0008] The inventors have unexpectedly found a new Btk inhibitor
which exhibits potent inhibitory activity against Btk and, at the
same time, exhibits improved or good dual selectivity of Btk over
Tec and of Btk over EGFR. The good dual selectivity of Btk over Tec
and of Btk over EGFR may be attributed to a combination of a
tri-substituted phenyl ring and a piperidine ring (particularly
N-substituted acryloyl piperidine ring) in the molecule. Such a
combination may also improve cellular potency, oral bioavailability
and pharmacodynamic activity.
[0009] In a first aspect, disclosed herein is a compound of formula
(I):
##STR00001##
or a stereoisomer thereof, or a pharmaceutically acceptable salt
thereof, wherein:
[0010] L is a bond, CH.sub.2, NR.sup.12, O, or S;
[0011] R.sup.1 is halogen, heteroalkyl, alkyl, alkenyl, cycloalkyl,
aryl, saturated or unsaturated heterocyclyl, heteroaryl, alkynyl,
--CN, --NR.sup.13R.sup.14, --OR.sup.13, --COR.sup.13,
--CO.sub.2R.sup.13, --CONR.sup.13R.sup.14,
--C(.dbd.NR.sup.13)NR.sup.14R.sup.15, --NR.sup.13COR.sup.14,
--NR.sup.3CONR.sup.14R.sup.13, --NR.sup.13CO.sub.2R.sup.4,
--SO.sub.2R.sup.13, --NR.sup.13SO.sub.2NR.sup.14R.sup.15, or
--NR.sup.13SO.sub.2R.sup.14, wherein the alkyl, alkenyl, alkynyl,
cycloalkyl, heteroaryl, aryl, and saturated or unsaturated
heterocyclyl are optionally substituted with at least one
substituent R.sup.16;
[0012] n is an integer of 2, 3 or 4;
[0013] each R.sup.2 is independently halogen, alkyl, --S-alkyl,
--CN, --NR.sup.13R.sup.14, --OR.sup.13, --COR.sup.13,
--CO.sub.2R.sup.13, --CONR.sup.13R.sup.14,
--C(.dbd.NR.sup.13)NR.sup.14R.sup.15, --NR.sup.13COR.sup.14,
--NR.sup.13CONR.sup.14R.sup.13, --NR.sup.13CO.sub.2R.sup.14,
--SO.sub.2R.sup.13, --NR.sup.13SO.sub.2NR.sup.14R, or
--NR.sup.13SO.sub.2R.sup.14;
[0014] R.sup.4a and R.sup.4b are each independently hydrogen,
halogen, heteroalkyl, alkyl, alkenyl, cycloalkyl, saturated or
unsaturated heterocyclyl, heteroaryl, alkynyl, --CN,
--NR.sup.13R.sup.14, --OR.sup.13, --COR.sup.13, --CO.sub.2R.sup.13,
--CONR.sup.3R.sup.4, --C(.dbd.NR.sup.13NR.sup.14R.sup.15,
--NR.sup.3COR.sup.14, --NR.sup.13CON.sup.14R.sup.15,
--NR.sup.13CO.sub.2R.sup.14, --SO.sub.2R.sup.13,
--NR.sup.13SO.sub.2NR.sup.14R.sup.15, or
--NR.sup.13SO.sub.2R.sup.14, wherein the alkyl, alkenyl, alkynyl,
cycloalkyl, heteroaryl, aryl, and saturated or unsaturated
heterocyclyl are optionally substituted with at least one
substituent R.sup.16;
[0015] R.sup.5a and R.sup.5b are each independently hydrogen,
halogen, heteroalkyl, alkyl, alkenyl, cycloalkyl, aryl, saturated
or unsaturated heterocyclyl, heteroaryl, alkynyl, --CN,
--NR.sup.13R.sup.14, --OR.sup.13, --COR.sup.13, --CO.sub.2R.sup.13,
--CONR.sup.13R.sup.14, --C(.dbd.NR.sup.13)NR.sup.14R.sup.15,
--NR.sup.13COR.sup.14, --NR.sup.13CONR.sup.14R.sup.15,
--NR.sup.13CO.sub.2R.sup.13, --SO.sub.2R.sup.13,
--NR.sup.13SO.sub.2NR.sup.14R.sup.15, or
--NR.sup.13SO.sub.2R.sup.14, wherein the alkyl, alkenyl, alkynyl,
cycloalkyl, heteroaryl, aryl, and saturated or unsaturated
heterocyclyl are optionally substituted with at least one
substituent R.sup.16;
[0016] R.sup.6a and R.sup.6b are each independently hydrogen,
halogen, heteroalkyl, alkyl, alkenyl, cycloalkyl, aryl, saturated
or unsaturated heterocyclyl, heteroaryl, alkynyl, --CN,
--NR.sup.13R.sup.14, --OR.sup.13, --COR.sup.13, --CO.sub.2R.sup.13,
--CONR.sup.13R.sup.14, --C(.dbd.NR.sup.13)NR.sup.14R.sup.15,
--NR.sup.13COR.sup.14, --NR.sup.13CONR.sup.14R.sup.13,
--NR.sup.13CO.sub.2R.sup.14, --SO.sub.2R.sup.13,
--NR.sup.13SO.sub.2NR.sup.14R.sup.15, or
--NR.sup.13SO.sub.2R.sup.14, wherein the alkyl, alkenyl, alkynyl,
cycloalkyl, heteroaryl, aryl, and saturated or unsaturated
heterocyclyl are optionally substituted with at least one
substituent R.sup.16; or
[0017] wherein (R.sup.4a and R.sup.5a), or (R.sup.4a and R.sup.5b),
or (R.sup.4b and R.sup.5a), or (R.sup.4b and R.sup.5b), or
(R.sup.5a and R.sup.6a), or (R.sup.5a and R.sup.6b), or (R.sup.5b
and R.sup.6a), or (R.sup.5b and R.sup.6b), together with the atoms
to which they are attached, may form a fused ring selected from
cycloalkyl, saturated or unsaturated heterocycle, aryl, and
heteroaryl rings, optionally substituted with at least one
substituent R.sup.16;
[0018] R.sup.12 is H or alkyl;
[0019] R.sup.13, R.sup.14 and R.sup.15 are each independently H,
heteroalkyl, alkyl, alkenyl, alkynyl, cycloalkyl, saturated or
unsaturated heterocyclyl, aryl, or heteroaryl; wherein (R.sup.13
and R.sup.14), and/or (R.sup.14 and R.sup.15) together with the
atom(s) to which they are attached, each can form a ring selected
from cycloalkyl, saturated or unsaturated heterocycle, aryl, and
heteroaryl rings optionally substituted with at least one
substituent R.sup.16;
[0020] R.sup.16 is halogen, substituted or unsubstituted alkyl,
substituted or unsubstituted alkenyl, substituted or unsubstituted
alkynyl, substituted or unsubstituted cycloalkyl, substituted or
unsubstituted aryl, substituted or unsubstituted heteroaryl,
substituted or unsubstituted heterocyclyl, oxo, --CN, --OR.sup.a,
--NR.sup.aR.sup.b, --COR.sup.a, --CO.sub.2R.sup.a,
--CONR.sup.aR.sup.b, --C(.dbd.NR.sup.a)NR.sup.bR.sup.c,
--NR.sup.aCOR.sup.b, --NR.sup.aCONR.sup.aR.sup.b,
--NR.sup.aCO.sub.2R.sup.b, --SO.sub.2R.sup.a, --SO.sub.2aryl,
--NR.sup.aSO.sub.2NR.sup.bR.sup.c, or --NR.sup.aSO.sub.2R.sup.b,
wherein R.sup.a, R.sup.b, and R.sup.c are independently hydrogen,
halogen, substituted or unsubstituted alkyl, substituted or
unsubstituted alkenyl, substituted or unsubstituted alkynyl,
substituted or unsubstituted cycloalkyl, substituted or
unsubstituted aryl, substituted or unsubstituted heteroaryl,
substituted or unsubstituted heterocyclyl, wherein (R.sup.a and
R.sup.b), and/or (R.sup.a and R.sup.b) together with the atoms to
which they are attached, can form a ring selected from cycloalkyl,
saturated or unsaturated heterocycle, aryl, and heteroaryl
rings.
[0021] In some embodiments of the first aspect, L is a bond.
[0022] In some embodiments of the first aspect, R.sup.1 is halogen,
alkyl, alkenyl, cycloalkyl, aryl, saturated heterocyclyl,
heteroaryl, or --OR', wherein the alkyl, alkenyl, cycloalkyl, and
heteroaryl are optionally substituted with at least one substituent
R.sup.16. In some preferred embodiment, R.sup.1 is alkyl or
cycloalkyl.
[0023] In some embodiments of the first aspect, n is 2.
[0024] In some embodiments of the first aspect, one of R.sup.2 is
at position 3 of the phenyl ring, and the other R.sup.2 is at
position 5 of the phenyl ring.
[0025] In some embodiments of the first aspect, n is 2; and one of
R.sup.2 is at position 3 of the phenyl ring, and the other R.sup.2
is at position 5 of the phenyl ring. In some preferred embodiment,
R.sup.2 is optionally partially or fully deuterated, i.e., one or
more carbon-bound hydrogen(s) in the definition of R.sup.2 are
replaced by one or more deuterium(s). In some more preferred
embodiments, R.sup.2, at each of its occurrence, is halogen, alkyl
or --O-alkyl.
[0026] In some embodiments of the first aspect, R.sup.4a, R.sup.5a,
R.sup.5b, R.sup.6a and R.sup.6b are hydrogen; and R.sup.4b is
alkyl, or saturated heterocyclyl, wherein the alkyl and saturated
heterocyclyl are optionally substituted with at least one
substituent R'.sup.6. In other embodiments, R.sup.4a, R.sup.5a,
R.sup.5b, R.sup.6a and R.sup.6b are hydrogen; and R.sup.4b is a
saturated heterocyclyl containing a nitrogen atom which is
optionally substituted with acryloyl or propiolyl. In further
embodiments, the nitrogen atom of the saturated heterocyclyl in the
definition of R.sup.4b is substituted with acryloyl. In even
further embodiments, the saturated heterocyclyl in the definition
of R.sup.4b is a monocyclic ring selected from a azetidine,
pyrrole, piperidine, azapane and azocane ring; or a Spiro or
bridged bicyclic ring selected from azabicyclooctanyl,
azaspirononanyl and azaspiroheptanyl.
[0027] In alternative embodiments of the first aspect, (R.sup.4a
and R.sup.5a), or (R.sup.4a and R.sup.5b), or (R.sup.4b and
R.sup.5a), or (R.sup.4b and R.sup.5b), or (R.sup.5a and R.sup.6a),
or (R.sup.5a and R.sup.6b), or (R.sup.5b and R.sup.6a), or
(R.sup.5b and R.sup.6b), together with the atoms to which they are
attached, form a fused ring selected from cycloalkyl, saturated or
unsaturated heterocycle, aryl, and heteroaryl rings, optionally
substituted with at least one substituent R.sup.16. In further
embodiments, (R.sup.4a and R.sup.5a), or (R.sup.4a and R.sup.5b),
or (R.sup.4b and R.sup.5a), or (R.sup.4b and R.sup.5b), or
(R.sup.5a and R.sup.6a), or (R.sup.5a and R.sup.6b), or (R.sup.5b
and R.sup.6a), or (R.sup.5b and R.sup.6b), together with the atoms
to which they are attached, form a fused saturated heterocycle ring
optionally substituted with at least one substituent R.sup.16. In
even further embodiments, the fused saturated heterocycle ring is a
azetidine, pyrrole, piperidine, zapane or azocane ring optionally
substituted with a substituent R.sup.16 at the nitrogen atom of the
fused ring, wherein the substituent R.sup.1-6 is acryloyl or
propiolyl.
[0028] In some embodiments of the first aspect, the compounds
disclosed herein are deuterated compounds. Specifically, the
compounds disclosed herein are partially or fully deuterated in the
definition of W. More preferably, the definition of R.sup.2 as
alkyl or O-alkyl is partially or fully deuterated.
[0029] In a second aspect, disclosed herein is a compound of
formula (II):
##STR00002##
or a stereoisomer thereof, or a pharmaceutically acceptable salt
thereof, wherein:
[0030] R.sup.x is halogen, alkyl, or --O-alkyl, wherein the alkyl
group or moiety is optionally partially or fully deuterated;
[0031] R.sup.y is halogen, alkyl optionally substituted with at
least substituents selected from hydroxy or halogen, or cycloalkyl;
and
[0032] R.sup.z is halogen, alkyl, or --O-alkyl, wherein the alkyl
group or moiety is optionally partially or fully deuterated.
[0033] In some embodiments of the second aspect, disclosed herein
is a compound of formula (IIa):
##STR00003##
or a stereoisomer thereof, or a pharmaceutically acceptable salt
thereof, wherein:
[0034] R.sup.x is methoxy, ethoxy, methyl, ethyl or chloro, wherein
the methoxy, ethoxy, methyl, ethyl is optionally partially or fully
deuterated;
[0035] R.sup.y is 2-hydroxylethyl, trifluoromethyl, isopropyl,
cyclopropyl, methyl, chloro, bromo, or iodo; and
[0036] R.sup.z is methoxy, methyl, or chloro, wherein the methoxy
or methyl is optionally partially or fully deuterated.
[0037] In some embodiments of the second aspect, R.sup.x is
methoxy, ethoxy, methyl, chloro, CD.sub.3, -CD.sub.2CD.sub.3, OCD3,
or --O-CD.sub.2CD.sub.3; W is 2-hydroxylethyl, CF.sub.3, isopropyl,
cyclopropyl, Methyl, chloro, bromo, or iodo; and W is ethoxy,
methoxy, methyl, ethyl, chloro, or CD.sub.3, -CD.sub.2CD.sub.3,
OCD.sub.3, or --O-CD.sub.2CD.sub.3.
[0038] In some embodiments of the second aspect, R.sup.x is
methoxy, ethoxy, methyl, ethyl, chloro, or OCD.sub.3; R.sup.y is
2-hydroxylethyl, CF.sub.3, isopropyl, cyclopropyl, methyl, chloro,
bromo, or iodo; and W is methoxy, methyl, chloro, or OCD.sub.3.
[0039] In some embodiments of the second aspect, R.sup.x is methoxy
or OCD.sub.3, R.sup.y is cyclopropyl or methyl; and W is methoxy or
OCD.sub.3.
[0040] In some embodiments of the above first and second aspects,
disclosed herein is a compound or a stereoisomer thereof, or a
pharmaceutically acceptable salt thereof, which is selected from:
Example Nos. A1, A1a, A1b, A2, A2a, A2b, A3, A4, A5, A6, A6a, A6b,
A7, A8, A9, A10, A11, A11a, A11b, A12, A13, A14, A15, A16, A17,
A18, A19, A20, A21, A22, A23, A24, A25, A26, A27, A28, A29, A30,
A31, A32, A33, A34, A35, A36, A37, A38, A39, A40, A41, A42, A43,
A44, A45, A46, A47, A48, A49, A50, A51, A52, A52a, A52b, B1, B2,
B3, C1, C2, D1, D2, E1, F1, G1, H1,
##STR00004## ##STR00005## ##STR00006## ##STR00007## ##STR00008##
##STR00009##
[0041] In other embodiments of the above first and second aspects,
disclosed herein is a compound or a stereoisomer thereof, or a
pharmaceutically acceptable salt thereof, which is selected from:
A1a, A2a, A5, A6a, A8, A11, A11b, A18, A20, A22, A33, A34, A36, A46
and C1.
[0042] In a third aspect, disclosed herein is a method of treating
a disease associated with undesirable Btk activity in a subject by
administering to the subject the compound disclosed herein or a
stereoisomer thereof, or a pharmaceutically acceptable salt
thereof.
[0043] In a fourth aspect, disclosed herein is the compound
disclosed herein or a stereoisomer thereof, or a pharmaceutically
acceptable salt thereof for use in treating a disease associated
with undesirable Btk activity.
[0044] In a fifth aspect, disclosed herein is a pharmaceutical
composition comprising the therapeutically effective amount of a
compound disclosed herein or a stereoisomer thereof, or a
pharmaceutically acceptable salt thereof, and at least one
pharmaceutically acceptable carrier.
BRIEF DESCRIPTIONS OF THE DRAWINGS
[0045] FIG. 1A shows in vivo PD result of Compounds A1a, A2a and
A6a compared to Compound 176 in PBMC;
[0046] FIG. 1B shows in vivo PD result of Compounds A1a, A2a and
A6a compared to compound 176 in Spleen;
[0047] FIG. 2A shows in vivo PD result of Compounds 169* and 167*
compared to Compound 176 in PBMC; and
[0048] FIG. 2B shows in vivo PD result of Compounds 169* and 167*
compared to Compound 176 in Spleen.
[0049] FIG. 3 shows the single crystal of compound A1a used to
determine the (S)-configuration.
DETAILED DESCRIPTION OF THE INVENTION
[0050] Unless specifically defined elsewhere in this document, all
other technical and scientific terms used herein have the meaning
commonly understood by one of ordinary skill in the art to which
this invention belongs.
[0051] As used herein, including the appended claims, the singular
forms of words such as "a", "an", and "the", include their
corresponding plural references unless the context clearly
indicates otherwise.
[0052] The term "or" is used to mean, and is used interchangeably
with, the term "and/or" unless the context clearly dictates
otherwise.
[0053] The term "alkyl" refers to a hydrocarbon group selected from
linear and branched saturated hydrocarbon groups of 1-18, or 1-12,
1-6 or 1-4 carbon atoms. Examples of the alkyl group include
methyl, ethyl, 1-propyl or n-propyl ("n-Pr"), 2-propyl or isopropyl
("i-Pr"), 1-butyl or n-butyl ("n-Bu"), 2-methyl-1-propyl or
isobutyl ("i-Bu"), 1-methylpropyl or s-butyl ("s-Bu"), and
1,1-dimethylethyl or t-butyl ("t-Bu"). Other examples of the alkyl
group include 1-pentyl, 2-pentyl, 3-pentyl, 2-methyl-2-butyl,
3-methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl, 1-hexyl,
2-hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl,
4-methyl-2-pentyl, 3-methyl-3-pentyl, 2-methyl-3-pentyl,
2,3-dimethyl-2-butyl and 3,3-dimethyl-2-butyl groups. Lower alkyl
means 1-8, preferably 1-6, more preferably 1-4 carbon atoms; lower
alkenyl or alkynyl means 2-8, 2-6 or 2-4 carbon atoms. The alkyl
group can be optionally partially or fully enriched in deuterium,
e.g., -CD.sub.3, -CD.sub.2CD.sub.3 and the like.
[0054] The term "--O-alkyl" refer to o an alkyl group, as
previously defined, attached to the parent molecular moiety through
an oxygen atom.
[0055] The term "alkenyl" refers to a hydrocarbon group selected
from linear and branched hydrocarbon groups comprising at least one
C.dbd.C double bond and of 2-18, or 2-12, or 2-6 carbon atoms.
Examples of the alkenyl group may be selected from ethenyl or
vinyl, prop-1-enyl, prop-2-enyl, 2-methylprop-1-enyl, but-1-enyl,
but-2-enyl, but-3-enyl, buta-1,3-dienyl, 2-methylbuta-1,3-diene,
hex-1-enyl, hex-2-enyl, hex-3-enyl, hex-4-enyl, and hexa-1,3-dienyl
groups. The alkenyl group can be optionally partially or fully
enriched in deuterium.
[0056] The term "alkynyl" refers to a hydrocarbon group selected
from linear and branched hydrocarbon group, comprising at least one
C.dbd.C triple bond and of 2-18, or 2-12, or 2-6 carbon atoms.
Examples of the alkynyl group include ethynyl, 1-propynyl,
2-propynyl (propargyl), 1-butynyl, 2-butynyl, and 3-butynyl groups.
The alkynyl group can be optionally partially or fully enriched in
deuterium.
[0057] The term "cycloalkyl" refers to a hydrocarbon group selected
from saturated and partially unsaturated cyclic hydrocarbon groups,
comprising monocyclic and polycyclic (e.g., bicyclic and tricyclic)
groups. For example, the cycloalkyl group may be of 3-12, or 3-8,
or 3-6 carbon atoms. Even further for example, the cycloalkyl group
may be a monocyclic group of 3-12, or 3-8, or 3-6 carbon atoms.
Examples of the monocyclic cycloalkyl group include cyclopropyl,
cyclobutyl, cyclopentyl, 1-cyclopent-1-enyl, 1-cyclopent-2-enyl,
1-cyclopent-3-enyl, cyclohexyl, 1-cyclohex-1-enyl,
1-cyclohex-2-enyl, 1-cyclohex-3-enyl, cyclohexadienyl, cycloheptyl,
cyclooctyl, cyclononyl, cyclodecyl, cycloundecyl, and cyclododecyl
groups. Examples of the bicyclic cycloalkyl groups include those
having 7-12 ring atoms arranged as a bicycle ring selected from
[4,4], [4,5], [5,5], [5,6] and [6,6] ring systems, or as a bridged
bicyclic ring selected from bicyclo[2.2.1]heptane,
bicyclo[2.2.2]octane, and bicyclo[3.2.2]nonane. The ring may be
saturated or have at least one double bond (i.e. partially
unsaturated), but is not fully conjugated, and is not aromatic, as
aromatic is defined herein. The cycloalkyl group can be optionally
partially or fully enriched in deuterium.
[0058] The term "deuterated compound" refers to a compound wherein
one or more carbon-bound hydrogen(s) are replaced by one or more
deuterium(s). Similarly, the term "deuterated" is be used herein to
modify a chemical structure or an organic group or radical, wherein
one or more carbon-bound hydrogen(s) are replaced by one or more
deuterium(s), e.g., "deuterated-alkyl", "deuterated-cycloalkyl",
"deuterated-heterocycloalkyl", "deuterated-aryl",
"deuterated-morpholinyl", and the like. For example, the term
"deuterated-alkyl" defined above refers to an alkyl group as
defined herein, wherein at least one hydrogen atom bound to carbon
is replaced by a deuterium. In a deuterated alkyl group, at least
one carbon atom is bound to a deuterium; and it is possible for a
carbon atom to be bound to more than one deuterium; it is also
possible that more than one carbon atom in the alkyl group is bound
to a deuterium.
[0059] The term "aryl" herein refers to a group selected from: 5-
and 6-membered carbocyclic aromatic rings, for example, phenyl;
bicyclic ring systems such as 7-12 membered bicyclic ring systems
wherein at least one ring is carbocyclic and aromatic, selected,
for example, from naphthalene, and indane; and tricyclic ring
systems such as 10-15 membered tricyclic ring systems wherein at
least one ring is carbocyclic and aromatic, for example, fluorene.
For example, the aryl group is selected from 5- and 6-membered
carbocyclic aromatic rings fused to a 5- to 7-membered cycloalkyl
or heterocyclic ring optionally comprising at least one heteroatom
selected from N, O, and S, provided that the point of attachment is
at the carbocyclic aromatic ring when the carbocyclic aromatic ring
is fused with a heterocyclic ring, and the point of attachment can
be at the carbocyclic aromatic ring or at the cycloalkyl group when
the carbocyclic aromatic ring is fused with a cycloalkyl group.
Bivalent radicals formed from substituted benzene derivatives and
having the free valences at ring atoms are named as substituted
phenylene radicals. Bivalent radicals derived from univalent
polycyclic hydrocarbon radicals whose names end in "-yl" by removal
of one hydrogen atom from the carbon atom with the free valence are
named by adding "-idene" to the name of the corresponding univalent
radical, e.g., a naphthyl group with two points of attachment is
termed naphthylidene. Aryl, however, does not encompass or overlap
with heteroaryl, separately defined below. Hence, if one or more
carbocyclic aromatic rings are fused with a heterocyclic aromatic
ring, the resulting ring system is heteroaryl, not aryl, as defined
herein.
[0060] The term "halogen" or "halo" refers to F, Cl, Br or I.
[0061] The term "heteroalkyl" refers to alkyl comprising at least
one heteroatom.
[0062] The term "heteroaryl" refers to a group selected from: 5- to
7-membered aromatic, monocyclic rings comprising 1, 2, 3 or 4
heteroatoms selected from N, O, and S, with the remaining ring
atoms being carbon; 8- to 12-membered bicyclic rings comprising 1,
2, 3 or 4 heteroatoms, selected from N, O, and S, with the
remaining ring atoms being carbon and wherein at least one ring is
aromatic and at least one heteroatom is present in the aromatic
ring; and 11- to 14-membered tricyclic rings comprising 1, 2, 3 or
4 heteroatoms, selected from N, O, and S, with the remaining ring
atoms being carbon and wherein at least one ring is aromatic and at
least one heteroatom is present in an aromatic ring. For example,
the heteroaryl group includes a 5- to 7-membered heterocyclic
aromatic ring fused to a 5- to 7-membered cycloalkyl ring. For such
fused, bicyclic heteroaryl ring systems wherein only one of the
rings comprises at least one heteroatom, the point of attachment
may be at the heteroaromatic ring or at the cycloalkyl ring. When
the total number of S and O atoms in the heteroaryl group exceeds
1, those heteroatoms are not adjacent to one another. In some
embodiments, the total number of S and O atoms in the heteroaryl
group is not more than 2. In some embodiments, the total number of
S and O atoms in the aromatic heterocycle is not more than 1.
Examples of the heteroaryl group include, but are not limited to,
(as numbered from the linkage position assigned priority 1) pyridyl
(such as 2-pyridyl, 3-pyridyl, or 4-pyridyl), cinnolinyl,
pyrazinyl, 2,4-pyrimidinyl, 3,5-pyrimidinyl, 2,4-imidazolyl,
imidazopyridinyl, isoxazolyl, oxazolyl, thiazolyl, isothiazolyl,
thiadiazolyl, tetrazolyl, thienyl, triazinyl, benzothienyl, furyl,
benzofuryl, benzoimidazolyl, indolyl, isoindolyl, indolinyl,
phthalazinyl, pyrazinyl, pyridazinyl, pyrrolyl, triazolyl,
quinolinyl, isoquinolinyl, pyrazolyl, pyrrolopyridinyl (such as
1H-pyrrolo[2,3-b]pyridin-5-yl), pyrazolopyridinyl (such as
1H-pyrazolo[3,4-b]pyridin-5-yl), benzoxazolyl (such as
benzo[d]oxazol-6-yl), pteridinyl, purinyl, 1-oxa-2,3-diazolyl,
1-oxa-2,4-diazolyl, 1-oxa-2,5-diazolyl, 1-oxa-3,4-diazolyl,
1-thia-2,3-diazolyl, 1-thia-2,4-diazolyl, 1-thia-2,5-diazolyl,
1-thia-3,4-diazolyl, furazanyl, benzofurazanyl, benzothiophenyl,
benzothiazolyl, benzoxazolyl, quinazolinyl, quinoxalinyl,
naphthyridinyl, furopyridinyl, benzothiazolyl (such as
benzo[d]thiazol-6-yl), indazolyl (such as 1H-indazol-5-yl) and
5,6,7,8-tetrahydroisoquinoline.
[0063] The term "heterocyclic" or "heterocycle" or "heterocyclyl"
refers to a ring selected from 4- to 12-membered monocyclic,
bicyclic and tricyclic, saturated and partially unsaturated rings
comprising at least one carbon atoms in addition to 1, 2, 3 or 4
heteroatoms, selected from oxygen, sulfur, and nitrogen.
"Heterocycle" also refers to a 5- to 8-membered heterocyclic ring
comprising at least one heteroatom selected from N, O, and S fused
with 5-, 6-, 7-, and/or 8-membered cycloalkyl, carbocyclic aromatic
or heteroaromatic ring, provided that the point of attachment is at
the heterocyclic ring when the heterocyclic ring is fused with a
carbocyclic aromatic or a heteroaromatic ring, and that the point
of attachment can be at the cycloalkyl or heterocyclic ring when
the heterocyclic ring is fused with cycloalkyl.
[0064] The "heterocycle" also refers to an aliphatic spirocyclic
ring comprising at least one heteroatom selected from N, O, and S,
provided that the point of attachment is at the heterocyclic ring.
The rings may be saturated or have at least one double bond (i.e.
partially unsaturated). The heterocycle may be substituted with
oxo. The point of the attachment may be carbon or heteroatom in the
heterocyclic ring. A heterocyle is not a heteroaryl as defined
herein. Examples of the heterocycle include, but not limited to,
(as numbered from the linkage position assigned priority 1)
1-pyrrolidinyl, 2-pyrrolidinyl, 2,4-imidazolidinyl,
2,3-pyrazolidinyl, 1-piperidinyl, 2-piperidinyl, 3-piperidinyl,
4-piperidinyl, 2,5-piperazinyl, pyranyl, 2-morpholinyl,
3-moholinyl, oxiranyl, aziridinyl, thiiranyl, azetidinyl, oxetanyl,
thietanyl, 1,2-dithietanyl, 1,3-difhietanyl, dihydropyridinyl,
tetrahydropyridinyl, thiomorpholinyl, thioxanyl, piperazinyl,
homopiperazinyl, homopiperidinyl, azepanyl, oxepanyl, thiepanyl,
1,4-oxathianyl, 1,4-dioxepanyl, 1,4-oxafhiepanyl, 1,4-oxaazepanyl,
1,4-difhiepanyl, 1,4-fhiazepanyl and 1,4-diazepane 1,4-dithianyl,
1,4-azathianyl, oxazepinyl, diazepinyl, thiazepinyl,
dihydrofhienyl, dihydropyranyl, dihydrofuranyl, tetrahydrofuranyl,
tetrahydrothienyl, tetrahydropyranyl, tetrahydrothiopyranyl,
1-pyrrolinyl, 2-pyrrolinyl, 3-pyrrolinyl, indolinyl, 2H-pyranyl,
4H-pyranyl, 1,4-dioxanyl, 1,3-dioxolanyl, pyrazolinyl,
pyrazolidinyl, dithianyl, difhiolanyl, pyrazolidinylimidazolinyl,
pyrimidinonyl, 1,1-dioxo-thiomoholinyl, 3-azabicyco[3.1.0]hexanyl,
3-azabicyclo[4.1.0]heptanyl and azabicyclo[2.2.2]hexanyl.
Substituted heterocycle also includes ring systems substituted with
one or more oxo moieties, such as piperidinyl N-oxide,
morpholinyl-N-oxide, 1-oxo-1-thiomorpholinyl and
1,1-dioxo-1-thiomorpholinyl. 100481 The term "fused ring" herein
refers to a polycyclic ring system, e.g., a bicyclic or tricyclic
ring system, in which two rings share only two ring atoms and one
bond in common. Examples of fused rings may comprise a fused
bicyclic cycloalkyl ring such as those having from 7 to 12 ring
atoms arranged as a bicyclic ring selected from [4,4], [4,5],
[5,5], [5,6] and [6,6] ring systems as mentioned above; a fused
bicyclic aryl ring such as 7 to 12 membered bicyclic aryl ring
systems as mentioned above, a fused tricyclic aryl ring such as 10
to 15 membered tricyclic aryl ring systems mentioned above; a fused
bicyclic heteroaryl ring such as 8- to 12-membered bicyclic
heteroaryl rings as mentioned above, a fused tricyclic heteroaryl
ring such as 11- to 14-membered tricyclic heteroaryl rings as
mentioned above; and a fused bicyclic or tricyclic heterocyclyl
ring as mentioned above.
[0065] When compounds contain olefin double bonds, unless specified
otherwise, such double bonds are meant to include both E and Z
geometric isomers.
[0066] Some of the compounds may exist with different points of
attachment of hydrogen, referred to as tautomers. For example,
compounds including carbonyl --CH.sub.2C(O)-- groups (keto forms)
may undergo tautomerism to form hydroxyl --CH.dbd.C(OH)-- groups
(enol forms). Both keto and enol forms, individually as well as
mixtures thereof, are also intended to be included where
applicable.
[0067] "Pharmaceutically acceptable salt" refers to conventional
acid-addition salts or base-addition salts that retain the
biological effectiveness and properties of the compounds of the
present invention and are formed from suitable non-toxic organic or
inorganic acids or organic or inorganic bases. In addition, if a
compound is obtained as an acid addition salt, the free base can be
obtained by basifying a solution of the acid salt. Conversely, if
the product is a free base, an addition salt, such as a
pharmaceutically acceptable addition salt, may be produced by
dissolving the free base in a suitable organic solvent and treating
the solution with an acid, in accordance with conventional
procedures for preparing acid addition salts from base compounds.
Those skilled in the art will recognize various synthetic
methodologies that may be used without undue experimentation to
prepare non-toxic pharmaceutically acceptable addition salts.
[0068] The terms "administration", "administering", "treating" and
"treatment" herein, when applied to an animal, human, experimental
subject, cell, tissue, organ, or biological fluid, mean contact of
an exogenous pharmaceutical, therapeutic, diagnostic agent, or
composition to the animal, human, subject, cell, tissue, organ, or
biological fluid. Treatment of a cell encompasses contact of a
reagent to the cell, as well as contact of a reagent to a fluid,
where the fluid is in contact with the cell. The term
"administration" and "treatment" also means in vitro and ex vivo
treatments, e.g., of a cell, by a reagent, diagnostic, binding
compound, or by another cell. The term "subject" herein includes
any organism, preferably an animal, more preferably a mammal (e.g.,
rat, mouse, dog, cat, rabbit) and most preferably a human.
[0069] An "effective amount" refers to an amount of at least one
compound and/or at least one stereoisomer thereof, and/or at least
one pharmaceutically acceptable salt thereof effective to "treat" a
disease or disorder in a subject, and that will elicit, to some
significant extent, the biological or medical response of a tissue,
system, animal or human that is being sought, such as when
administered, is sufficient to prevent development of, or alleviate
to some extent, one or more of the symptoms of the condition or
disorder being treated. The therapeutically effective amount will
vary depending on the compound, the disease and its severity and
the age, weight, etc., of the mammal to be treated.
[0070] The term "at least one substituent" includes, for example,
from 1 to 4, such as from 1 to 3, further as 1 or 2, substituents,
depending on the available valence. For example, "at least one
substituent R.sup.16" herein includes from 1 to 4, such as from 1
to 3, further as 1 or 2, substituents selected from the list of
R.sup.1'' as disclosed herein.
[0071] The compound disclosed herein or a stereoisomer thereof, or
a pharmaceutically acceptable salt thereof can be used to treat a
disease selected from an allergic disease, an autoimmune disease,
an inflammatory disease, a cancer, or a combination of two or more
thereof. The compound disclosed herein or a stereoisomer thereof,
or a pharmaceutically acceptable salt thereof can also be used to
treat a B-cell proliferative disease. Specifically, the B-cell
proliferative disease is B-cell malignancies, selected from
lymphoma, non-Hodgkin's lymphoma (NHL), diffuse large B cell
lymphoma (DLBCL), mantle cell lymphoma (MCL), follicular lymphoma
(FL), chronic lymphocytic leukemia (CLL), small lymphocytic
lymphoma (SLL), waldenstrom macroglobulinemia (WM), marginal zone
lymphoma (MZL), Hairy cell leukemia (HCL), Burkitt's-like leukemia
(BL), or a combination of two or more thereof.
EXAMPLES
[0072] The examples below are intended to be purely exemplary and
should not be considered to be limiting in any way. Efforts have
been made to ensure accuracy with respect to numbers used (for
example, amounts, temperature, etc.), but some experimental errors
and deviations should be accounted for. Unless indicated otherwise,
temperature is in degrees Centigrade. Reagents were purchased from
commercial suppliers such as Sigma-Aldrich, Alfa Aesar, or TCI, and
were used without further purification unless indicated
otherwise.
[0073] Unless indicated otherwise, the reactions set forth below
were performed under a positive pressure of nitrogen or argon or
with a drying tube in anhydrous solvents; the reaction flasks were
fitted with rubber septa for the introduction of substrates and
reagents via syringe; and glassware was oven dried and/or heat
dried.
[0074] .sup.1H NMR spectra were recorded on a Agilent instrument
operating at 400 MHz. .sup.1HNMR spectra were obtained using
CDCl.sub.3, CD.sub.2Cl.sub.2, CD.sub.3OD, D.sub.2O, d.sub.6-DMSO,
d.sub.6-acetone or (CD.sub.3).sub.2CO as solvent and
tetramethylsilane (0.00 ppm) or residual solvent (CDCl.sub.3: 7.25
ppm; CD.sub.3OD: 3.31 ppm; D.sub.2O: 4.79 ppm; d.sub.6-DMSO: 2.50
ppm; d.sub.6-acetone: 2.05; (CD.sub.3).sub.2CO: 2.05) as the
reference standard. When peak multiplicities are reported, the
following abbreviations are used: s (singlet), d (doublet), t
(triplet), q (quartet), qn (quintuplet), sx (sextuplet), m
(multiplet), br (broadened), dd (doublet of doublets), dt (doublet
of triplets). Coupling constants, when given, are reported in Hertz
(Hz).
[0075] LC-MS spectrometer (Agilent 1260) Detector: MWD (190-400
nm), Mass detector: 6120 SQ
[0076] Mobile phase: A: acetonitrile with 0.1% Formic acid, B:
water with 0.1% Formic acid
[0077] Column: Poroshell 120 EC-C18, 4.6.times.50 mm, 2.7 .mu.m
[0078] Gradient method: Flow: 1.8 mL/min
TABLE-US-00001 Time A B (min) (%) (%) 0.00 5 95 1.5 95 5 2.0 95 5
2.1 5 95 3.0 5 95
[0079] Preparative HPLC was conducted on a column (150.times.21.2
mm ID, 5 .mu.m, Gemini NX-C18) at a flow rate of 20 mL/min,
injection volume 2 ml, at room temperature and UV Detection at 214
nm and 254 nm.
[0080] In the following examples, the abbreviations below are used:
[0081] AcOH or HOAc Acetic acid [0082] aq. aqueous [0083] Brine
Saturated aqueous sodium chloride solution [0084] DCM
Dichloromethane [0085] C2H5I Iodoethane [0086] DMF
N,N-Dimethylformamide [0087] DMAC Dimethylacetamide [0088] DMSO
Dimethyl sulfoxide [0089] EA Ethyl acetate [0090] EDCI
1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride [0091]
EtOH ethyl alcohol [0092] h hour [0093] H.sub.2O.sub.2 Hydrogen
peroxide [0094] HOBt Hydroxybenzotriazole [0095] MeOH Methanol
[0096] MsOH Methanesulfonic acid [0097] n-BuLi n-Butyllithium
[0098] NCS N-Chlorosuccinimide [0099] NHaCI Ammonium chloride
[0100] Pd/C Palladium on carbon powder [0101] Pd(dppf)Cl.sub.2
[1,1'-Bis(diphenylphosphino)ferrocene]dichloropalladium(II) [0102]
Pd(OAc).sub.2 Palladium acetate [0103] Pd(OH).sub.2/C Palladium
hydroxide on carbon powder [0104] PE Petroleum ether [0105] pH
-lg(hydrogen ion concentration) [0106] Pre-TLC Prepared thin layer
chromatography [0107] RT room temperature [0108] sat. Saturated
[0109] t-Bu.sub.3P Tritert-butylphosphane [0110] THF
Tetrahydrofuran [0111] TEA Triethylamine [0112] TFA Trifluoroacetic
acid
Examples of a Series
Example A1:
7-(1-acryloylpiperidin-4-yl)-2-(3,5-dimethoxy-4-methylphenyl)-4,5,6,7-tet-
rahydropyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00010##
[0113] Step 1:
2-((3,5-dimethoxy-4-methylphenyl)(hydroxy)methylene)malononitrile
##STR00011##
[0115] To a mixture of 3,5-dimethoxy-4-methylbenzoic acid (5.0 g,
25 mmol), malononitrile (1.65 g, 25 mmol), HOBT (3.4 g, 25 mmol)
and EDCI (5.37 g, 25 mmol) in EA (200 mL) was added TEA (7.5 g, 75
mmol). The reaction was stirred at RT for 10 h. The mixture was
filtered and the filtrate was washed by sat. NaHCO.sub.3 (50 mL)
and H.sub.2O (50 mL), then stirred with 1.5 N HCl (50 mL) for 30
min, washed with NaCl (50 mL) and evaporated to give the product as
a yellow solid (5.1 g, 80%). .sup.1H NMR (400 MHz, DMSO-d.sub.6)
.delta. 6.87 (s, 2H), 3.77 (s, 6H), 1.99 (s, 3H). MS (ESI, m/e)
[M+1].sup.+ 244.9.
Step 2:
2-((3,5-dimethoxy-4-methylphenyl)(methoxy)methylene)malononitrile
##STR00012##
[0117] A solution of
2-((3,5-dimethoxy-4-methylphenyl)(hydroxy)methylene)malononitrile
(5.0 g, 20 mmol) in trimethoxymethane (50 mL) was stirred at
100.degree. C. for 10 h, then evaporated to give the crude product
as a brown oil (5.5 g). MS (ESI, m/e) [M+1].sup.+ 259.1.
Step 3:
5-amino-3-(3,5-dimethoxy-4-methylphenyl)-1H-pyrazole-4-carbonitril-
e
##STR00013##
[0119] To a solution of
2-((3,5-dimethoxy-4-methylphenyl)(methoxy)methylene) malononitrile
(5.0 g, 19 mmol) in EtOH (50 mL) was added hydrazine hydrate (2.0
mL) at 0.degree. C. Then the reaction was allowed to warm to RT and
stirred for 2 h. The mixture was evaporated to give the crude
product as a brown oil (6.0 g). MS (ESI, m/e) [M+1].sup.+
259.1.
Step 4: tert-butyl
4-(3-cyano-2-(3,5-dimethoxy-4-methylphenyl)pyrazolo[1,5-a]pyrimidin-7-yl)-
piperidine-1-carboxylate
##STR00014##
[0121] To a solution of
5-amino-3-(3,5-dimethoxy-4-methylphenyl)-1H-pyrazole-4-carbonitrile
(3.0 g, 11.6 mmol) in toluene (50 mL) and AcOH (10 mL) was added
tert-butyl
(E)-4-(3-(dimethylamino)acryloyl)piperidine-1-carboxylate (ref:
PCT/CN2016/095510, 3.27 g, 11.6 mmol). The reaction was stirred at
110.degree. C. for 16 h. The reaction was cooled to RT and
evaporated under reduced pressure. EA (30 mL) was added to the
residue. The solid was collected by filtration and dried to give
the product as a yellow solid (2.0 g, 36%). MS (ESI, m/e)
[M+1].sup.+ 477.8.
Step 5: tert-butyl
4-(3-cyano-2-(3,5-dimethoxy-4-methylphenyl)-4,5,6,7-tetrahydropyrazolo[1,-
5-a]pyrimidin-7-yl)piperidine-1-carboxylate
##STR00015##
[0123] To a suspension of tert-butyl
4-(3-cyano-2-(3,5-dimethoxy-4-methylphenyl)pyrazolo[1,5-a]pyrimidin-7-yl)-
piperidine-1-carboxylate (2.0 g, 4.1 mmol) in MeOH (500 mL) was
added NaBH.sub.4 (465 mg, 12.5 mmol) in portions over 30 min. The
reaction was stirred at RT for 2 h. When the system turned clear,
the starting material was consumed, the mixture was evaporated, the
residue was partitioned between DCM (500 mL) and H.sub.2O (300 mL).
The organic phase was evaporated to give the crude product as a
light yellow solid (2.3 g). MS (ESI, m/e) [M+1].sup.+ 481.8.
Step 6:
2-(3,5-dimethoxy-4-methylphenyl)-7-(piperidin-4-yl)-4,5,6,7-tetrah-
ydropyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00016##
[0125] A solution of tert-butyl
4-(3-cyano-2-(3,5-dimethoxy-4-methylphenyl)-4,5,6,7-tetrahydropyrazolo[1,-
5-a]pyrimidin-7-yl)piperidine-1-carboxylate (2.3 g, 4.78 mmol) in
methanesulfonic acid (5 mL) was heated at 100.degree. C. for 2 h.
The reaction was then cooled to RT and added dropwise to a system
of sat. NaHCO.sub.3 (100 mL) and DCM (50 mL). The organic phase was
washed with sat. NaCl (50 mL), evaporated to give the desired
product as a light yellow solid (1.5 g, 78.6%). MS (ESI, m/e)
[M+1].sup.+ 399.8.
Step 7:
7-(1-acryloylpiperidin-4-yl)-2-(3,5-dimethoxy-4-methylphenyl)-4,5,-
6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00017##
[0127] To a mixture of
2-(3,5-dimethoxy-4-methylphenyl)-7-(piperidin-4-yl)-4,5,6,7-tetrahydropyr-
azolo[1,5-a]pyrimidine-3-carboxamide (1.5 g, 3.9 mmol) in sat.
NaHCO.sub.3 (20 mL) and DCM (50 mL) was added acryloyl chloride
(354 mg, 3.9 mmol) dropwise over 3 min at 0.degree. C. The reaction
was allowed to warm to RT and stirred for 2 h. Then the organic
phase was washed with sat. NH.sub.4Cl (30 mL) and sat. NaCl (30
mL), evaporated and purified by flash column chromatography (eluted
with DCM:MeOH=50:1) to yield the compound (400 mg, 23.4%).
Example A1a and A1b: (S or
R)-7-(1-acryloylpiperidin-4-yl)-2-(3,5-dimethoxy-4-methylphenyl)-4,5,6,7--
tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide and (R or
S)-7-(1-acryloylpiperidin-4-yl)-2-(3,5-dimethoxy-4-methylphenyl)-4,5,6,7--
tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00018##
[0129] Two enantiomers A1a (faster isomer) and A1b (slower isomer)
were separated by chiral preparative HPLC. The chiral separation
conditions are shown below. The faster enantiomer was eluted at
retention time of 6.4 min to give 185 mg of product. The slower
enantiomer was eluted at retention time of 8.2 min to give 181 mg
of product. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 6.79 (dd,
J=16.8, 10.4 Hz, 1H), 6.71 (s, 2H), 6.07 (dd, J=16.8, 2.4, Hz, 1H),
5.64 (dd, J=10.4, 2.4 Hz, 1H), 4.54-4.36 (m, 1H), 4.17-3.97 (m,
2H), 3.78 (s, 6H), 3.36-3.22 (m, 2H), 3.07-2.88 (m, 1H), 2.65-2.50
(m, 1H), 2.32-2.15 (m, 1H), 2.10-1.96 (m, 1H), 2.03 (s, 3H),
1.96-1.52 (m, 3H), 1.37-1.13 (m, 2H). MS (EST, m/e) [M+1].sup.+
453.8.
TABLE-US-00002 Column CHIRAL ART Cellulose-SB Column size 2 cm
.times. 25 cm, 5 um Injection 0.5 mL Mobile phase Hex:EtOH = 50:50
Flow rate 20 ml/min Wave length UV 220 nm Temperature 25.degree. C.
Sample solution 45.6 mg/ml in MeOH:DCM = 1:1 Prep-HPLC equipment
Prep-Gilson-HPLC
[0130] The absolute stereochemistry of the more potent compound A1a
was confirmed to be (S)-configuration on the chiral carbon atom by
X-ray single crystal diffraction as shown in FIG. 3. Data
collection were performed on a Bruker D8 VENTURE (Cu/K.alpha.
radiation, .lamda.=1.54178 .ANG.) diffractometer and analyzed with
the APEX3 software package. The Flack parameter was refined to 0.00
(4).
##STR00019##
[0131] Accordingly the absolute stereochemistry of compound A1b was
assigned to be (R)-configuration.
##STR00020##
Example A2:
7-(1-acryloylpiperidin-4-yl)-2-(4-cyclopropyl-3,5-dimethoxyphenyl)-4,5,6,-
7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00021##
[0132] Step 1: methyl 4-bromo-3,5-dimethoxybenzoate
##STR00022##
[0134] A mixture of methyl 4-bromo-3,5-dihydroxybenzoate (10 g,
40.5 mmol), CH.sub.3I (14.2 g, 101.2 mmol), K.sub.2CO.sub.3 (16.8
g, 121.5 mmol) in 100 mL of DMF was heated at 50.degree. C. for 16
h. After cooling down to RT the mixture was poured into 200 mL of
water, extracted with EA (100 mL.times.3). The combined organic
layers were washed with brine, dried over Na.sub.2SO.sub.4,
concentrated to give the product as an off-white solid (10.5 g,
94.6%). MS (ESI) m/e [M+1].sup.+274.7 and 276.7.
Step 2: 4-bromo-3,5-dimethoxybenzoic acid
##STR00023##
[0136] A mixture of methyl 4-bromo-3,5-dimethoxybenzoate (10.5 g,
38.2 mmol), LiOH.H.sub.2O (4.8 g, 114.5 mmol) in THF (80 mL) and
H.sub.2O (80 mL) was heated to reflux for 16 h. The mixture was
concentrated to remove the organic solvent under reduced pressure,
the residue was adjusted to pH=1-2 with 6 HCl, extracted with EA
(10 mL.times.3). The combined organic layers were washed with
brine, dried over Na.sub.2SO.sub.4, filtered and concentrated to
give the product as yellow solid (8.2 g, 82.2%). MS (ESI) m/e
[M+1].sup.+ 260.7 and 262.7.
Step 3:
2-((4-bromo-3,5-dimethoxyphenyl)(hydroxy)methylene)malononitrile
##STR00024##
[0138] A mixture of 4-bromo-3,5-dimethoxybenzoic acid (7.5 g, 28.7
mmol), propanedinitrile (1.89 g, 28.7 mmol), EDCI (5.51 g, 28.7
mmol), HOBT (3.87 g, 28.7 mmol) and TEA (5.80 g, 28.7 mmol) in EA
(100 mL) was stirred at RT for 16 h. The mixture was then washed
with 6 N HCl (100 mL). The organic layer was dried over
Na.sub.2SO.sub.4, filtered and concentrated to give the product as
a yellow solid (8.0 g, 90.3%). MS (ESI, m/e) [M+1].sup.+ 308.7 and
310.7.
Step 4:
2-((4-bromo-3,5-dimethoxyphenyl)(methoxy)methylene)malononitrile
##STR00025##
[0140] A solution of
2-((4-bromo-3,5-dimethoxyphenyl)(hydroxy)methylene)malononitrile
(8.0 g, 25.9 mmol) in CH(OMe).sub.3 (100 mL) was heated to reflux
for 4 h. Then the mixture was concentrated to give the crude
product as brown oil (8.2 g, 98.1%). MS (ESI) m/e [M+1].sup.+ 312.7
and 314.7.
Step 5:
5-amino-3-(4-bromo-3,5-dimethoxyphenyl)-1H-pyrazole-4-carbonitrile
##STR00026##
[0142] A mixture of
2-((4-bromo-3,5-dimethoxyphenyl)(methoxy)methylene)malononitrile
(8.2 g, 25.4 mmol) and hydrazine hydrate (5.0 mL) in EtOH (50 mL)
was stirred at RT for 2 h. Then the mixture was filtrated to give
the product as a yellow solid (5.5 g, 67.1%).
Step 6: tert-butyl
4-(2-(4-bromo-3,5-dimethoxyphenyl)-3-cyanopyrazolo[1,5-a]pyrimidin-7-yl)p-
iperidine-1-carboxylate
##STR00027##
[0144] A mixture of
5-amino-3-(4-bromo-3,5-dimethoxyphenyl)-1H-pyrazole-4-carbonitrile
(5.5 g, 17.0 mmol) and tert-butyl 4-(3-(dimethylamino)acryloyl)
piperidine-1-carboxylate (ref: PCT/CN2016/095510, 4.8 g, 17.0 mmol)
in HOAc (10 mL) and toluene (50 mL) was heated to 100.degree. C.
for 16 h. Then the mixture was filtered to give the product as a
yellow solid (6.5 g, 70.3%). MS (ESI) m/e [M+1].sup.+ 542.2 and
544.1.
Step 7: tert-butyl
4-(3-cyano-2-(4-cyclopropyl-3,5-dimethoxyphenyl)pyrazolo[1,5-a]pyrimidin--
7-yl)piperidine-1-carboxylate
##STR00028##
[0146] A mixture of tert-butyl
4-(2-(4-bromo-3,5-dimethoxyphenyl)-3-cyanopyrazolo[1,5-a]pyrimidin-7-yl)p-
iperidine-1-carboxylate (500 mg, 0.90 mmol), cyclopropylboronic
acid (150 mg, 1.80 mmol), Pd(dppf)Cl.sub.2 (100 mg, 0.90 mmol) and
K.sub.3PO.sub.4 (570 mg, 2.70 mmol) in dioxane (50 mL) was heated
to 100.degree. C. for 16 h under N.sub.2 atmosphere. The mixture
was filtered, concentrated and purified by column chromatograph on
silica gel using EA/PE (1/1) as eluent to afford the product as a
yellow solid (150 mg, 33.0%). MS (ESI) m/e [M+1].sup.+504.3.
Step 8: tert-butyl
4-(3-cyano-2-(4-cyclopropyl-3,5-dimethoxyphenyl)-4,5,6,7-tetrahydropyrazo-
lo[1,5-a]pyrimidin-7-yl)piperidine-1-carboxylate
##STR00029##
[0148] A mixture of tert-butyl
4-(3-cyano-2-(4-cyclopropyl-3,5-dimethoxyphenyl)pyrazolo
[1,5-a]pyrimidin-7-yl)piperidine-1-carboxylate (150 mg, 0.30 mmol),
10% w/w Pd/C (50 mg) in MeOH (20 mL) and DCM (20 mL) was stirred
for 16 h under a balloon of H.sub.2. The mixture was filtered,
concentrated to give the product as a yellow solid (120 mg, 80%).
MS (ESI, m/e) [M+1].sup.+ 508.3.
Step 9: tert-butyl
4-(3-carbamoyl-2-(4-cyclopropyl-3,5-dimethoxyphenyl)-4,5,6,7-tetrahydropy-
razolo[1,5-a]pyrimidin-7-yl)piperidine-1-carboxylate
##STR00030##
[0150] To a mixture of tert-butyl
4-(3-cyano-2-(4-cyclopropyl-3,5-dimethoxyphenyl)-4,5,6,7-tetrahydropyrazo-
lo[1,5-a]pyrimidin-7-yl)piperidine-1-carboxylate (120 mg, 0.24
mmol), DMSO (2 mL), aqueous NaOH (3N, 2 mL) in EtOH (20 mL) was
slowly added H.sub.2O.sub.2 (2 mL, 30% solution) at 65.degree. C.
The reaction mixture was stirred at 65.degree. C. for 1 h, cooled
to RT, concentrated to remove EtOH. The residue was partitioned
between water (50 mL) and EA (50 mL). The aqueous phase was
extracted with EA (50 mL.times.2). The combined organic layers were
washed with brine, dried over Na.sub.2SO.sub.4 and concentrated to
give the product as a yellow solid (80 mg, 63.5%). MS (ESI, m/e)
[M+1].sup.+ 526.3.
Step 10:
2-(4-cyclopropyl-3,5-dimethoxyphenyl)-7-(piperidin-4-yl)-4,5,6,7--
tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00031##
[0152] A solution of tert-butyl
4-(3-carbamoyl-2-(4-cyclopropyl-3,5-dimethoxyphenyl)-4,5,6,7-tetrahydropy-
razolo[1,5-a]pyrimidin-7-yl)piperidine-1-carboxylate (80 mg, 0.15
mmol) in TFA/DCM (5 mL/15 mL) was stirred at RT for 16 h. The
mixture was concentrated to remove the solvent and the residue was
partitioned between aqueous NaHCO.sub.3 solution (50 mL) and DCM
(50 mL). The organic layer was dried over Na.sub.2SO.sub.4,
concentrated to get the product as a yellow solid (55 mg, 85.9%).
MS (ESI, m/e) [M+1].sup.+ 426.3.
Step 11:
7-(1-acryloylpiperidin-4-yl)-2-(4-cyclopropyl-3,5-dimethoxyphenyl-
)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00032##
[0154] To a mixture of
2-(4-cyclopropyl-3,5-dimethoxyphenyl)-7-(piperidin-4-yl)-4,5,6,7-tetrahyd-
ropyrazolo[1,5-a]pyrimidine-3-carboxamide (55 mg, 0.13 mmol),
NaHCO.sub.3 (56 mg, 0.65 mmol) in DCM (10 mL) and H.sub.2O (10 mL)
was added acryloyl chloride (14 mg, 0.16 mmol) at RT. After the
reaction was completed, the organic layer was dried over
Na.sub.2SO.sub.4, concentrated and further purified by Prep-TLC
(DCM:MeOH=20:1) to afford the product (40 mg, 64.5%). .sup.1H NMR
(400 MHz, DMSO-d.sub.6) .delta. 6.79 (dd, J=16.8, 10.8 Hz, 1H),
6.73 (br s, 1H), 6.68 (s, 2H), 6.07 (dd, J=16.8, 2.2 Hz, 1H), 5.64
(d, J=10.8 Hz, 1H), 4.54-4.40 (m, 1H), 4.15-3.97 (m, 2H), 3.75 (s,
6H), 3.50-3.20 (m, 3H), 3.05-2.90 (m, 1H), 2.63-2.52 (m, 1H),
2.30-2.15 (m, 1H), 2.10-1.81 (m, 3H), 1.77-1.10 (m, 3H), 1.02-0.90
(m, 2H), 0.82-0.68 (m, 2H). MS (ESI, m/e) [M+1].sup.+ 480.2.
Example A2a and A2b: (S or
R)-7-(1-acryloylpiperidin-4-yl)-2-(4-cyclopropyl-3,5-dimethoxyphenyl)-4,5-
,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide and (R or
5)-7-(1-acryloylpiperidin-4-yl)-2-(4-cyclopropyl-3,5-dimethoxyphenyl)-4,5-
,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00033##
[0156] Two enantiomers A2a (faster isomer) and A2b (slower isomer)
were separated by chiral preparative HPLC. The chiral separation
conditions are shown below. The faster enantiomer was eluted at
retention time of 7.2 min to give 231 mg. The slower enantiomer was
eluted at retention time of 10.0 min to give 236 mg.
TABLE-US-00003 Column CHIRAL ART Cellulose-SB Column size 2 cm
.times. 25 cm, 5 um Injection 0.6 mL Mobile phase CO.sub.2:MeOH =
50:50 Flow rate 40 ml/min Wave length UV 220 nm Temperature
25.degree. C. Sample solution 24.3 mg/ml in MeOH Prep-HPLC
equipment Prep-SFC80
[0157] A2a was assigned to (S)-configuration and A2b was assigned
to (R)-configuration:
##STR00034##
Example A3:
7-(1-acryloylpiperidin-4-yl)-2-(4-ethyl-3,5-dimethoxyphenyl)-4,5,6,7-tetr-
ahydropyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00035##
[0159] Example A3 was synthesized from tert-butyl
4-(2-(4-bromo-3,5-dimethoxyphenyl)-3-cyanopyrazolo[1,5-a]pyrimidin-7-yl)p-
iperidine-1-carboxylate and
4,4,5,5-tetramethyl-2-vinyl-1,3,2-dioxaborolane following the
procedures similar to those in Example A2. .sup.1H NMR (400 MHz,
DMSO-d.sub.6) .delta. 6.76 (dd, J=16.8, 10.2 Hz, 1H), 6.67 (s, 2H),
6.03 (dd, J=16.8, 2.2 Hz, 1H), 5.60 (d, J=10.2 Hz, 1H), 4.50-4.39
(m, 1H), 4.12-3.94 (m, 2H), 3.75 (s, 6H), 3.30-3.22 (m, 2H),
3.02-2.89 (m, 1H), 2.56 (q, J=7.4 Hz, 2H), 2.54-2.50 (m, 1H),
2.27-1.47 (m, 5H), 1.32-1.09 (m, 2H), 0.99 (t, J=7.4 Hz, 31-1). MS
(ESI, m/e) [M+1].sup.+ 468.2.
Example A4:
7-(1-acryloylpiperidin-4-yl)-2-(3,5-dimethoxy-4-propylphenyl)-4,5,6,7-tet-
rahydropyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00036##
[0161] Example A4 was a byproduct when preparing A3 due to the
impurity
((E)-4,4,5,5-tetramethyl-2-(prop-1-en-1-yl)-1,3,2-dioxaborolane) in
4,4,5,5-tetramethyl-2-vinyl-1,3,2-dioxaborolane. .sup.1H NMR (400
MHz, DMSO-d.sub.6) .delta. 6.76 (dd, J=16.8, 10.2 Hz, 1H), 6.67 (s,
2H), 6.03 (d, J=16.8 Hz, 1H), 5.60 (d, J=10.2 Hz, 1H), 4.52-4.36
(m, 1H), 4.12-3.94 (m, 2H), 3.74 (s, 6H), 3.34-3.20 (m, 2H),
3.04-2.85 (m, 1H), 2.60-2.50 (m, 3H), 2.28-1.48 (m, 5H), 1.47-1.34
(m, 2H), 1.32-1.08 (m, 2H), 0.85 (t, J=7.3 Hz, 3H). MS (ESI, m/e)
[M+1].sup.+ 482.3.
Example A5:
7-(1-acryloylpiperidin-4-yl)-2-(4-isopropyl-3,5-dimethoxyphenyl)-4,5,6,7--
tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00037##
[0163] Example A5 was synthesized from tert-butyl
4-(2-(4-bromo-3,5-dimethoxyphenyl)-3-cyanopyrazolo[1,5-a]pyrimidin-7-yl)p-
iperidine-1-carboxylate and
4,4,5,5-tetramethyl-2-(prop-1-en-2-yl)-1,3,2-dioxaborolane
following the procedures similar to those in Example A2. .sup.1H
NMR (400 MHz, DMSO-d.sub.6) .delta. 6.79 (dd, J=16.8, 10.4 Hz, 1H),
6.73 (s, 1H), 6.70 (s, 2H), 6.07 (dd, J=16.8, 2.0 Hz, 1H), 5.64 (d,
J=10.4 Hz, 1H), 4.52-4.42 (m, 1H), 4.15-3.95 (m, 2H), 3.77 (s, 6H),
3.62-3.49 (m, 1H), 3.35-3.25 (m, 2H), 3.07-2.89 (m, 1H), 2.58-2.52
(m, 1H), 2.30-1.50 (m, 5H), 1.24 (d, J=6.8 Hz, 6H), 1.35-1.13 (m,
2H). MS (ESI, m/e) [M+1].sup.+ 482.2.
Example A6:
7-(1-acryloylpiperidin-4-yl)-2-(4-bromo-3,5-dimethoxyphenyl)-4,5,6,7-tetr-
ahydropyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00038##
[0164] Step 1: Tert-butyl
4-(2-(4-bromo-3,5-dimethoxyphenyl)-3-cyano-4,5,6,7-tetrahydropyrazolo[1,5-
-a]pyrimidin-7-yl)piperidine-1-carboxylate
##STR00039##
[0166] To a mixture solution of tert-butyl
4-(2-(4-bromo-3,5-dimethoxyphenyl)-3-cyanopyrazolo[1,5-a]pyrimidin-7-yl)p-
iperidine-1-carboxylate (2.0 g, 3.70 mmol) in MeOH (100 mL) and DCM
(100 mL) was added NaBH.sub.4 (1.4 g, 37.0 mmol) in portions, then
the mixture was stirred for 2 h at RT. The reaction was quenched
with H.sub.2O (100 mL), concentrated to remove organic solvent. The
residue was extracted with EA (50 mL.times.3). The combined organic
layers were washed with brine, dried over Na.sub.2SO.sub.4,
concentrated to afford the product as a yellow solid (1.65 g,
81.7%). MS (ESI, m/e) [M+1].sup.+ 546.1 and 548.1.
Step 2: tert-butyl
4-(2-(4-bromo-3,5-dimethoxyphenyl)-3-carbamoyl-4,5,6,7-tetrahydropyrazolo-
[1,5-a]pyrimidin-7-yl)piperidine-1-carboxylate
##STR00040##
[0168] To a solution of tert-butyl
4-(2-(4-bromo-3,5-dimethoxyphenyl)-3-cyano-4,5,6,7-tetrahydropyrazolo[1,5-
-a]pyrimidin-7-yl)piperidine-1-carboxylate (1.65 g, 3.02 mmol),
DMSO (10 mL) and aq. NaOH (3 N, 10 mL) in EtOH (50 mL) was slowly
added H.sub.2O.sub.2 (10 mL, 30% solution) at 65.degree. C. The
reaction mixture was stirred at 65.degree. C. for 1 h. Then the
mixture was concentrated to remove EtOH. The residue was
partitioned between water (50 mL) and EA (50 mL), the aqueous phase
was extracted with EA (50 mL.times.3). The combined organic layers
were washed with brine, dried over Na.sub.2SO.sub.4 and
concentrated to afford the crude product as a yellow solid (1.05 g,
61.8%). MS (ESI, m/e) [M+1].sup.+ 564.1 and 566.1.
Step 3:
2-(4-bromo-3,5-dimethoxyphenyl)-7-(piperidin-4-yl)-4,5,6,7-tetrahy-
dropyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00041##
[0170] A solution of tert-butyl
4-(2-(4-bromo-3,5-dimethoxyphenyl)-3-carbamoyl-4,5,6,7-tetrahydropyrazolo-
[1,5-a]pyrimidin-7-yl)piperidine-1-carboxylate (1.05 g, 1.86 mmol)
in TFA/DCM (5 mL/50 mL) was stirred at RT for 16 h. The mixture was
concentrated to remove solvent and the residue was partitioned
between sat. NaHCO.sub.3 (50 mL) and DCM (50 mL). The organic layer
was dried over Na.sub.2SO.sub.4, filtered and concentrated to give
the crude product as a yellow solid (680 mg, 82.6%). MS (ESI, m/e)
[M+1].sup.+ 464.1 and 466.1.
Step 4:
7-(1-acryloylpiperidin-4-yl)-2-(4-bromo-3,5-dimethoxyphenyl)-4,5,6-
,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00042##
[0172] To a solution of
2-(4-bromo-3,5-dimethoxyphenyl)-7-(piperidin-4-yl)-4,5,6,7-tetrahydropyra-
zolo[1,5-a]pyrimidine-3-carboxamide (680 mg, 1.47 mmol) in sat.
NaHCO.sub.3 (100 mL) and DCM (100 mL) was added acryloyl chloride
(160 mg, 1.76 mmol) at ambient temperature, the reaction was
stirred at ambient temperature for about 1 h. The aqueous phase was
extracted with DCM (50 mL.times.3), the combined organic phases
were washed with sat. NaCl (50 mL), dried over Na.sub.2SO.sub.4,
concentrated and purified by column chromatograph on silica gel
(100-200 mesh, eluent: MeOH:DCM=1:50) to afford the product (550
mg, 72.3%). .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 6.83 (s,
2H), 6.77 (dd, J=16.8, 10.4 Hz, 1H), 6.73 (s, 1H), 6.07 (dd,
J=16.8, 2.2 Hz, 1H), 5.64 (dd, J=10.4, 2.2 Hz, 1H), 4.53-4.41 (m,
1H), 4.16-3.98 (m, 2H), 3.85 (s, 6H), 3.35-3.25 (m, 2H), 3.05-2.95
(m, 1H), 2.62-2.50 (m, 1H), 2.32-2.18 (m, 1H), 2.10-1.95 (m, 1H),
1.99-1.92 (m, 1H), 1.78-1.72 (m, 1H), 1.62-1.55 (m, 1H), 1.37-1.13
(m, 2H). MS (ESI, m/e) [M+1].sup.+ 518.1 and 520.1.
Example A6a and A6b: (S or
R)-7-(1-acryloylpiperidin-4-yl)-2-(4-bromo-3,5-dimethoxyphenyl)-4,5,6,7-t-
etrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide and (R or
S)-7-(1-acryloylpiperidin-4-yl)-2-(4-bromo-3,5-dimethoxyphenyl)-4,5,6,7-t-
etrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00043##
[0174] Two enantiomers A6a (faster isomer) and A6b (slower isomer)
were separated by chiral preparative HPLC. The chiral separation
conditions are shown below. The faster enantiomer was eluted at
retention time of 3.2 min to give 294 mg. The slower enantiomer was
eluted at retention time of 4.0 min to give 310 mg.
TABLE-US-00004 Column CHIRAL ART Cellulose-SB Column size 2 cm
.times. 25 cm, 5 um Injection 1.0 mL Mobile phase CO.sub.2:MeOH =
50:50 Flow rate 45 ml/min Wave length UV 254 nm Temperature
25.degree. C. Sample solution 24.5 mg/ml in MeOH Prep-HPLC
equipment Prep-SFC80
[0175] A6a was assigned to (S)-configuration and A6b was assigned
to (R)-configuration:
##STR00044##
Example A7:
7-(1-acryloylpiperidin-4-yl)-2-(3,5-dichloro-4-methylphenyl)-4,5,6,7-tetr-
ahydropyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00045##
[0177] Example A7 was synthesized from 3,5-dichloro-4-methylbenzoic
acid following the procedures similar to those in Example A2
without the Suzuki reaction step. NaBH.sub.4 was used in the
reduction step. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 7.56
(s, 2H), 6.86-6.72 (m, 1H), 6.60 (s, 1H), 6.07 (dd, J=16.8, 2.2 Hz,
1H), 5.64 (dd, J=10.5, 2.2 Hz, 1H), 4.56-4.40 (m, 1H), 4.20-3.95
(m, 2H), 3.33-3.22 (m, 2H), 3.07-2.89 (m, 1H), 2.69-2.54 (m, 1H),
2.44 (s, 3H), 2.35-2.12 (m, 1H), 2.10-1.82 (m, 2H), 1.78-1.45 (m,
2H), 1.37-1.10 (m, 2H). MS (ESI, m/e) [M+1].sup.+ 462.1.
Example A8:
7-(1-acryloylpiperidin-4-yl)-2-(4-iodo-3,5-dimethoxyphenyl)-4,5,
tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00046##
[0179] Example A8 was synthesized from 4-iodo-3,5-dimethoxybenzoic
acid following the procedures similar to those in Example A2
without the Suzuki reaction step. NaBH.sub.4 was used in the
reduction step. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 6.79
(dd, J=16.6, 10.4 Hz, 1H), 6.74 (s, 2H), 6.07 (dd, J=16.6, 2.2 Hz,
1H), 5.64 (dd, J=10.4, 2.2 Hz, 1H), 4.55-4.42 (m, 1H), 4.15-3.95
(m, 2H), 3.83 (s, 6H), 3.33-3.26 (m, 1H), 3.05-2.90 (m, 2H),
2.63-2.52 (m, 1H), 2.31-1.12 (m, 7H). MS (ESI, m/e) [M+1].sup.+
565.0.
Example A9:
7-(1-acryloylpiperidin-4-yl)-2-(3,5-dimethoxyphenyl)-4,5,6,7-tetrahydropy-
razolo[1,5-a]pyrimidine-3-carboxamide
##STR00047##
[0181] Example A9 is a byproduct when preparing Example A8. .sup.1H
NMR (400 MHz, DMSO-d.sub.6) .delta. 6.85-6.74 (m, 1H), 6.72 (br s,
1H), 6.63-6.58 (m, 2H), 6.57-6.54 (m, 1H), 6.06 (dd, J=16.6, 2.2
Hz, 1H), 5.64 (d, J=10.6 Hz, 1H), 4.52-4.42 (m, 1H), 4.17-3.95 (m,
2H), 3.76 (s, 6H), 3.33-3.26 (m, 2H), 3.07-2.87 (m, 1H), 2.64-2.54
(m, 1H), 2.35-2.15 (m, 1H), 2.10-1.84 (m, 2H), 1.78-1.66 (m, 1H),
1.63-1.48 (m, 1H), 1.35-1.12 (m, 2H). MS (ESI, m/e) [M+1].sup.+
439.4.
Example A10:
7-(1-acryloylpiperidin-4-yl)-2-(2,6-dimethoxy-[1,1'-biphenyl]-4-yl)-4,5,6-
,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00048##
[0183] Example A10 was synthesized from tert-butyl
4-(2-(4-bromo-3,5-dimethoxyphenyl)-3-cyanopyrazolo[1,5-a]pyrimidin-7-yl)p-
iperidine-1-carboxylate and phenylboronic acid following the
procedures similar to those in Example A2. NaBH.sub.4 was used in
the reduction step. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta.
7.39-7.24 (m, 5H), 6.85 (s, 2H), 6.82-6.71 (m, 2H), 6.07 (d, J=16.8
Hz, 1H), 5.64 (d, J=10.4 Hz, 1H), 4.52-4.45 (m, 1H), 4.15-4.00 (m,
2H), 3.71 (s, 6H), 3.05-2.95 (m, 2H), 2.65-2.55 (m, 1H), 2.32-2.25
(m, 1H), 2.08-1.90 (m, 2H), 1.80-1.70 (m, 1H), 1.62-1.55 (m, 1H),
1.35-1.23 (m, 2H). MS (ESI, m/e) [M+1].sup.+ 516.2.
Example A11:
7-(1-acryloylpiperidin-4-yl)-2-(3,5-bis(methoxy-d3)-4-methylphenyl)-4,5,6-
,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00049##
[0184] Step 1: Methyl 3,5-dihydroxy-4-methylbenzoate
##STR00050##
[0186] A mixture solution of 3,5-dihydroxy-4-methylbenzoic acid
(4.7 g, 27.9 mmol) and H.sub.2SO.sub.4 (1.0 mL) in methanol (50 mL)
was stirred at 60.degree. C. for overnight. Then the mixture
solution was concentrated and the residue was dissolved in EA (100
mL), washed with saturated NaHCO.sub.3 solution (100 mL.times.3),
dried over Na.sub.2SO.sub.4, filtered and concentrated to give the
product (4.93 g, 96.8%). MS (ESI, m/e) [M+1].sup.+ 183.1.
Step 2: Methyl 3,5-bis(methoxy-d3)-4-methylbenzoate
##STR00051##
[0188] A mixture solution of methyl 3,5-dihydroxy-4-methylbenzoate
(5.27 g, 28.9 mmol), CD.sub.3I (9.24 g, 63.7 mmol) and
K.sub.2CO.sub.3 (9.97 g, 72.3 mmol) in DMF (50 mL) was stirred at
RT for 16 h. Then the mixture solution was concentrated and further
purified by chromatography on silica gel eluting with EA/PE=1/5 to
give the product (5.3 g, 84.8%) as a white solid. MS (ESI, m/e)
[M+1].sup.+ 217.1.
Step 3: 3,5-bis(methoxy-d3)-4-methylbenzoic acid
##STR00052##
[0190] A mixture solution of methyl
3,5-bis(methoxy-d3)-4-methylbenzoate (5.3 g, 24.5 mmol) and NaOH
solution (17 mL, 3N) in THF (17 mL) was stirred at 60.degree. C.
for 2 h. Then the mixture solution was concentrated. And the
residue was adjusted to the pH value of 4-5, extracted with EA (20
mL.times.3). The combined organic layers were dried over
Na.sub.2SO.sub.4, filtered and concentrated to give the product
(4.9 g, 98.4%) as a yellow solid. MS (ESI, m/e)
[M+1].sup.+203.1.
Step 4:
7-(1-acryloylpiperidin-4-yl)-2-(3,5-bis(methoxy-d3)-4-methylphenyl-
)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00053##
[0192] Example A11 was then synthesized from
3,5-bis(methoxy-d3)-4-methylbenzoic acid following the procedures
similar to those in Example A2 without the Suzuki reaction step.
.sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 6.86-6.72 (m, 2H), 6.70
(s, 2H), 6.06 (d, J=16.8 Hz, 1H), 5.64 (d, J=10.4 Hz, 1H),
4.55-4.40 (m, 1H), 4.18-3.96 (m, 2H), 3.33-3.22 (m, 2H), 3.07-2.89
(m, 1H), 2.70-2.54 (m, 1H), 2.32-2.16 (m, 1H), 2.10-1.96 (m, 1H),
2.03 (s, 3H), 1.95-1.50 (m, 3H), 1.35-1.14 (m, 2H).
Example A11a and A11b: (S or
R)-7-(1-acryloylpiperidin-4-yl)-2-(3,5-bis(methoxy-d3)-4-methylphenyl)-4,-
5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide and (R or
S)-7-(1-acryloylpiperidin-4-yl)-2-(3,5-bis(methoxy-d3)-4-methylphenyl)-4,-
5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00054##
[0194] Two enantiomers A11a (faster isomer) and A11b (slower
isomer) were separated by chiral preparative HPLC. The chiral
separation conditions are shown below. The faster enantiomer was
eluted at retention time of around 2.0 min. The slower enantiomer
was eluted at retention time of around 2.6 min.
TABLE-US-00005 Column CHIRALPAK ID Column size 2 cm .times. 5 cm, 5
um Injection 1 mL Mobile phase (Hex:DCM = 3:1):IPA = 50:50 Flow
rate 16 ml/min Wave length UV 220 nm Temperature 25.degree. C.
Sample solution 26.25 mg/ml in EtOH:DCM = 3:l Prep-HPLC equipment
Prep-Gilson-HPLC
[0195] A11a was assigned to (R)-configuration and A11b was assigned
to (S)-configuration:
##STR00055##
Example A12:
7-(1-acryloylpiperidin-4-yl)-2-(3-ethoxy-5-methoxy-4-methylphenyl)-4,5,6,-
7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00056##
[0196] Step 1: methyl 3-hydroxy-5-methoxy-4-methylbenzoate
##STR00057##
[0198] To a mixture solution of methyl
3,5-dihydroxy-4-methylbenzoate (3.0 g, 16.4 mmol) and
K.sub.2CO.sub.3 (4.52 g, 32.8 mmol) in DMF (30 mL) was added
CH.sub.3I (2.45 g, 17.3 mmol). The mixture was then stirred at RT
for 72 h, concentrated under reduced pressure. To the residue was
added EA (100 mL), washed with water (100 mL.times.3), dried over
Na.sub.2SO.sub.4, filtered, concentrated and purified by column
chromatography on silica gel (200-300 mesh, eluent: PE:EA=5:1) to
give the product as a white solid (1.1 g, 34%). MS (ESI, m/e)
[M+1].sup.+ 197.1.
Step 2: methyl 3-ethoxy-5-methoxy-4-methylbenzoate
##STR00058##
[0200] To a mixture solution of methyl
3-hydroxy-5-methoxy-4-methylbenzoate (1.1 g, 5.6 mmol) and
K.sub.2CO.sub.3 (1.05 g, 6.7 mmol) in DMF (30 mL) was added
C.sub.2H.sub.5I (1.55 g, 11.2 mmol). Then the mixture was stirred
at RT for 16 h, concentrated under reduced pressure. To the residue
was added EA (100 mL), washed with water (20 mL.times.3), dried
over Na.sub.2SO.sub.4, filtered and concentrated to give the
product (1.2 g, 95.2%). MS (ESI, m/e) [M+1].sup.+ 225.1.
Step 3: 3-ethoxy-5-methoxy-4-methylbenzoic acid
##STR00059##
[0202] A mixture solution of methyl
3-ethoxy-5-methoxy-4-methylbenzoate (1.2 g, 5.4 mmol) and NaOH (3.6
mL, 3N) in THF (5 mL) and H.sub.2O (1 mL) was stirred at RT for 16
h, and then heated to 60.degree. C. for 2 h. Then the mixture
solution was acidified with 6N HCl and extracted with EA (20
mL.times.3). The organic layers were combined, dried over
Na.sub.2SO.sub.4, filtered and concentrated to give the product
(1.1 g, 97.8%) as a yellow solid. MS (ESI, m/e) [M+1].sup.+
211.1.
Step 4:
7-(1-acryloylpiperidin-4-yl)-2-(3-ethoxy-5-methoxy-4-methylphenyl)-
-4,5,6,7 tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00060##
[0204] Example A12 was then synthesized from
3-ethoxy-5-methoxy-4-methylbenzoic acid following the procedures
similar to those in Example A2 without the Suzuki reaction step.
.sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 6.79 (dd, J=16.8, 10.6
Hz, 1H), 6.69 (s, 2H), 6.07 (d, J=16.8 Hz, 1H), 5.64 (d, J=10.6 Hz,
1H), 4.59-4.39 (m, 1H), 4.19-3.91 (m, 4H), 3.78 (s, 3H), 3.36-3.23
(m, 2H), 3.10-2.90 (m, 1H), 2.70-2.52 (m, 1H), 2.35-2.15 (m, 1H),
2.03 (s, 3H), 2.12-1.96 (m, 1H), 1.97-1.52 (m, 3H), 1.34 (t, J=6.8
Hz, 3H) 1.36-1.12 (m, 2H). MS (ESI, m/e) [M+1].sup.+ 468.2.
Example A13:
7-(1-acryloylpiperidin-4-yl)-2-(3,5-diethoxy-4-methylphenyl)-4,5,6,7-tetr-
ahydropyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00061##
[0206] Example A13 was synthesized from methyl
3,5-dihydroxy-4-methylbenzoate following the procedures similar to
those in Example A11. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta.
6.79 (dd, J=16.6, 10.4 Hz, 1H), 6.72 (s, 1H), 6.66 (s, 2H), 6.07
(dd, J=16.6, 2.2 Hz, 1H), 5.64 (dd, J=10.4, 2.2 Hz, 1H), 4.55-4.39
(m, 1H), 4.15-3.96 (m, 6H), 3.33-3.25 (m, 2H), 3.08-2.90 (m, 1H),
2.54-2.52 (m, 1H), 2.31-2.15 (m, 1H), 2.04 (s, 3H), 2.10-1.96 (m,
1H), 1.96-1.51 (m, 3H), 1.36 (t, J=6.8 Hz, 6H), 1.41-1.09 (m,
2H).
Example A14:
7-(1-acryloylpiperidin-4-yl)-2-(3,4,5-trimethoxyphenyl)-4,5,6,7-tetrahydr-
opyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00062##
[0208] Example A15 was synthesized from 3,4,5-trimethoxybenzoic
acid following the procedures similar to those in Example A2
without the Suzuki reaction step. NaBH.sub.4 was used in the
reduction step. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 6.79
(dd, J=16.8, 11.2 Hz, 1H), 6.76 (s, 2H), 6.73 (br s, 1H), 6.07 (dd,
J=16.8, 2.4 Hz, 1H), 5.64 (dd, J=11.2, 2.4 Hz, 1H), 4.54-4.42 (m,
1H), 4.15-3.97 (m, 2H), 3.79 (s, 6H), 3.70 (s, 3H), 3.33-3.26 (m,
2H), 3.06-2.92 (m, 1H), 2.62-2.50 (m, 1H), 2.31-2.16 (m, 1H),
2.08-1.96 (m, 1H), 1.95-1.84 (m, 1H), 1.77-1.67 (m, 1H), 1.61-1.51
(m, 1H), 1.34-1.16 (m, 2H). MS (ESI) m/e [M+1].sup.+ 470.2.
Example A15:
7-(1-acryloylpiperidin-4-yl)-2-(3,5-dichloro-4-methoxyphenyl)-4,5,6,7-tet-
rahydropyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00063##
[0210] Example A15 was synthesized from
3,5-dichloro-4-methoxybenzoic acid following the procedures similar
to those in Example A2 without the Suzuki reaction step. NaBH.sub.4
was used in the reduction step. .sup.1H NMR (400 MHz, CDCl.sub.3)
.delta. 7.51 (s, 2H), 6.59 (dd, J=16.8, 10.6 Hz, 1H), 6.27 (dd,
J=16.8, 1.8 Hz, 1H), 5.68 (dd, J=10.6, 1.8 Hz, 1H), 5.45 (br s,
2H), 4.88-4.64 (m, 1H), 4.14-4.04 (m, 2H), 3.94 (s, 3H), 3.50-3.40
(m, 2H), 3.17-2.92 (m, 1H), 2.75-2.26 (m, 2H), 2.25-2.00 (m, 2H),
1.85-1.73 (m, 1H), 1.72-1.56 (m, 1H), 1.54-1.41 (m, 1H), 1.40-1.24
(m, 1H). MS (EST) m/e [M+1].sup.+ 478.1.
Example A16:
7-(1-acryloylpiperidin-4-yl)-2-(3,5-dibromo-4-methylphenyl)-4,5,6,7-tetra-
hydropyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00064##
[0212] Example A16 was synthesized from 3,5-dibromo-4-methylbenzoic
acid following the procedures similar to those in Example A1.
.sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 7.75 (s, 2H), 6.85-6.73
(m, 1H), 6.43 (br s, 2H), 6.07 (dd, J=16.8, 2.2 Hz, 1H), 5.64 (dd,
J=10.4, 2.2 Hz, 1H), 4.54-4.41 (m, 1H), 4.15-3.93 (m, 2H),
3.33-3.23 (m, 2H), 3.07-2.92 (m, 1H), 2.64-2.54 (m, 1H), 2.53 (s,
3H), 2.34-2.17 (m, 1H), 2.08-1.96 (m, 1H), 1.95-1.83 (m, 1H),
1.75-1.63 (m, 1H), 1.60-1.47 (m, 1H), 1.35-1.10 (m, 2H). MS (ESI)
m/e [M+1].sup.+ 552.0.
Example A17:
7-(1-acryloylpiperidin-4-yl)-2-(3,4,5-trichlorophenyl)-4,5,6,7-tetrahydro-
pyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00065##
[0214] Example A17 was synthesized from 3,4,5-trichlorobenzoic acid
following the procedures similar to those in Example A1. .sup.1H
NMR (400 MHz, DMSO-d.sub.6) .delta. 7.73 (s, 2H), 6.87-6.73 (m,
1H), 6.62 (br s, 2H), 6.55 (br s, 1H), 6.08 (dd, J=16.6, 1.8 Hz,
1H), 5.64 (dd, J=10.4, 1.8 Hz, 1H), 4.55-4.41 (m, 1H), 4.7-3.96 (m,
2H), 3.35-3.20 (m, 2H), 3.08-2.92 (m, 1H), 2.69-2.50 (m, 1H),
2.34-2.17 (m, 1H), 2.10-1.96 (m, 1H), 1.96-1.82 (m, 1H), 1.77-1.64
(m, 1H), 1.62-1.49 (m, 1H), 1.36-1.12 (m, 2H). MS (ESI) m/e
[M+1].sup.+ 482.1.
Example A18:
7-(1-acryloylpiperidin-4-yl)-2-(4-bromo-3,5-dichlorophenyl)-4,5,6,7-tetra-
hydropyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00066##
[0216] Example A1b was synthesized from 4-bromo-3,5-dichlorobenzoic
acid following the procedures similar to those in Example A1.
.sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 7.70 (s, 2H), 6.96-6.73
(m, 1H), 6.62 (br s, 2H), 6.54 (br s, 1H), 6.07 (dd, J=16.8, 2.0
Hz, 1H), 5.64 (dd, J=10.4, 2.0 Hz, 1H), 4.55-4.42 (m, 1H),
4.16-3.98 (m, 2H), 3.33-3.23 (m, 2H), 3.07-2.92 (m, 1H), 2.69-2.52
(m, 1H), 2.35-2.16 (m, 1H), 2.10-1.96 (m, 1H), 1.96-1.83 (m, 1H),
1.77-1.64 (m, 1H), 1.62-1.47 (m, 1H), 1.35-1.10 (m, 2H). MS (ESI)
m/e [M+1].sup.+ 526.4.
Example A19:
7-(1-acryloylpiperidin-4-yl)-2-(4-(furan-2-yl)-3,5-dimethoxyphenyl)-4,5,6-
,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00067##
[0218] Example A19 was synthesized from tert-butyl
4-(2-(4-bromo-3,5-dimethoxyphenyl)-3-cyanopyrazolo[1,5-a]pyrimidin-7-yl)p-
iperidine-1-carboxylate and furan-2-ylboronic acid following the
procedures similar to those in Example A2. NaBH.sub.4 was used in
the reduction step. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta.
7.70 (s, 1H), 6.87-6.84 (m, 1H), 6.85 (s, 2H), 6.80 (dd, J=16.8,
10.4 Hz, 1H), 6.56-6.53 (m, 1H), 6.50 (d, J=3.2 Hz, 1H), 6.07 (dd,
J=16.8, 2.0 Hz, 1H), 5.64 (dd, J=10.4, 2.0 Hz, 1H), 4.92 (br s,
3H), 4.57-4.42 (m, 1H), 4.16-4.01 (m, 2H), 3.76 (s, 6H), 3.35-3.27
(m, 2H), 3.07-2.92 (m, 1H), 2.65-2.52 (m, 1H), 2.34-2.17 (m, 1H),
2.11-1.98 (m, 1H), 1.98-1.84 (m, 1H), 1.79-1.68 (m, 1H), 1.64-1.53
(m, 1H), 1.36-1.15 (m, 2H). MS (ESI) m/e [M+1].sup.+ 506.2.
Example A20:
7-(1-acryloylpiperidin-4-yl)-2-(4-bromo-3,5-dimethylphenyl)-4,5,6,7-tetra-
hydropyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00068##
[0220] Example A20 was synthesized from 4-bromo-3,5-dimethylbenzoic
acid following the procedures similar to those in Example A1.
.sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 7.30 (s, 2H), 6.79 (dd,
J=16.2, 10.6 Hz, 1H), 6.07 (dd, J=16.2, 1.6 Hz, 1H), 5.64 (dd,
J=10.6, 1.6 Hz, 1H), 4.55-4.41 (m, 1H), 4.16-3.95 (m, 2H),
3.38-3.22 (m, 2H), 3.06-2.90 (m, 1H), 2.64-2.50 (m, 1H), 2.40 (s,
6H), 2.32-2.15 (m, 1H), 2.10-1.96 (m, 1H), 1.96-1.84 (m, 1H),
1.79-1.64 (m, 1H), 1.63-1.47 (m, 1H), 1.36-1.10 (m, 2H). MS (ESI)
m/e [M+1]+486.2.
Example A21:
7-(1-acryloylpiperidin-4-yl)-2-(3,5-dimethoxy-4-(1-methylcyclopropyl)phen-
yl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00069##
[0221] Step 1: methyl 3,5-dimethoxy-4-(prop-1-en-2-yl)benzoate
##STR00070##
[0223] To a solution of methyl 4-bromo-3,5-dimethoxybenzoate (3.1
g, 11.3 mmol) in dioxane (50 mL) and sat. K.sub.2CO.sub.3 (50 mL)
was added
4,4,5,5-tetramethyl-2-(prop-1-en-2-yl)-1,3,2-dioxaborolane (2.3 g,
13.5 mmol) and Pd(dppf)Cl.sub.2 (830 mg, 1.13 mmol), the reaction
atmosphere was exchanged with N.sub.2 for three times, warmed to
90.degree. C., stirred for about 16 h. Cooled to ambient
temperature, the mixture was extracted with EA (30 mL.times.3), the
combined organic phases were washed with sat. NaCl (20 mL), dried
over anhydrous Na.sub.2SO.sub.4, filtered and concentrated to give
the crude product (4.0 g) as a black oil which was purified by
column chromatograph on silica gel (100-200 mesh, eluent:
PE:EA=10:1) to afford the product as a white solid (2.6 g, 97.7%).
MS (ESI, m/e) [M+1].sup.+ 237.1.
Step 2: methyl 3,5-dimethoxy-4-(1-methylcyclopropyl)benzoate
##STR00071##
[0225] To a solution of methyl
3,5-dimethoxy-4-(prop-1-en-2-yl)benzoate (2.3 g, 9.75 mmol) in
toluene (50 mL) was added CH.sub.2I.sub.2 (13.1 g, 48.7 mmol), the
reaction was cooled to 0-5.degree. C., stirred for about 15 min,
ZnEt.sub.2 (97.5 mL, 97.5 mmol, 1 M in n-hexane) was dropwisely
added to the reaction, the reaction was slowly warmed to ambient
temperature, stirred for about 16 h. CH.sub.2I.sub.2 (6.0 g, 22.4
mmol) was added to the reaction, the reaction was stirred at
ambient temperature for about another 16 h. The reaction was
concentrated under reduced pressure to remove solvent, the residue
was portioned between EA (30 mL) and sat. NH.sub.4Cl (30 mL), the
aqueous phase was extracted EA (30 mL.times.3), the combined
organic phases were washed with sat. NaCl (10 mL), dried over
anhydrous Na.sub.2SO.sub.4, filtered, concentrated and purified by
column chromatography of silica gel (200-300 mesh, eluent:
PE:EA=10:1) to give the product as a yellow oil (1.8 g, 65.7%). MS
(ESI, m/e) [M+1].sup.+ 251.1.
Step 3: 3,5-dimethoxy-4-(1-methylcyclopropyl)benzoic acid
##STR00072##
[0227] To a solution of methyl
3,5-dimethoxy-4-(1-methylcyclopropyl)benzoate (1.8 g, 7.20 mmol) in
THF (30 mL) and H.sub.2O (10 mL) was added LiOH.H.sub.2O (1.52 g,
36.0 mmol), the reaction was warmed to 60.degree. C. for about 6 h.
The reaction was cooled to ambient temperature, concentrated under
reduced pressure to remove THF, the residue was adjusted to the pH
value of 2-3 with 3 N HCl, the aqueous phase was extracted with EA
(30 mL.times.3), the combined organic phases were washed with sat.
NaCl (20 mL), dried over anhydrous Na.sub.2SO.sub.4, filtered and
concentrated to give the crude product as a yellow solid (1.5 g,
88.3%). MS (ESI, m/e) [M+1].sup.+ 237.1.
Step 4:
7-(1-acryloylpiperidin-4-yl)-2-(3,5-dimethoxy-4-(1-methylcycloprop-
yl)phenyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00073##
[0229] Example A21 was then synthesized from
3,5-dimethoxy-4-(1-methylcyclopropyl)benzoic acid following the
procedures similar to those in Example A2 without the Suzuki
reaction step. NaBH.sub.4 was used in the reduction step. .sup.1H
NMR (400 MHz, DMSO-d.sub.6) .delta. 6.78 (dd, J=16.4, 10.4 Hz, 1H),
6.73 (br s, 1H), 6.69 (s, 2H), 6.06 (d, J=16.4 Hz, 1H), 5.63 (d,
J=10.4 Hz, 1H), 4.53-4.42 (m, 1H), 4.15-3.97 (m, 2H), 3.79 (s, 6H),
3.32-3.25 (m, 2H), 3.05-2.90 (m, 1H), 2.64-2.50 (m, 1H), 2.34-2.15
(m, 1H), 2.10-1.96 (m, 1H), 1.96-1.83 (m, 1H), 177-1.66 (m, 1H),
1.61-1.50 (m, 1H), 1.35-1.19 (m, 1H), 1.18 (s, 3H), 0.68-0.59 (m,
4H). MS (ESI) m/e [M+1].sup.+ 494.2.
Example 22:
7-(1-acryloylpiperidin-4-yl)-2-(4-chloro-3,5-dimethoxyphenyl)-4,5,
tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00074##
[0230] Step 1: 4-chloro-3,5-dimethoxyaniline
##STR00075##
[0232] To a solution of 3,5-dimethoxyaniline (20.0 g, 130 mmol) in
AcOH (50 mL) was added NCS (17.0 g, 130 mmol) in portions over 10
min. The mixture was stirred at RT for 16 h. The reaction was
poured into DCM (200 mL) and H.sub.2O (200 mL), the organic phase
was washed with Sat. NaCl (100 mL), dried over anhydrous
Na.sub.2SO.sub.4, filtered and evaporated to give a crude product
which was purified by column chromatography on silica gel
(PE:EA=12:1) to give desired product as a white solid (3.2 g,
13.1%). .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 5.96 (s, 2H),
5.28 (s, 2H), 3.71 (s, 6H), MS (ESI, m/e) [M+1].sup.+ 188.1.
Step 2: 2-chloro-5-iodo-1,3-dimethoxybenzene
##STR00076##
[0234] To a solution of 4-chloro-3,5-dimethoxyaniline (2.0 g, 10.70
mmol) in H.sub.2O (30 mL) and con. H.sub.2SO.sub.4 (3.0 mL) was
added the solution of NaNO.sub.2 (1.11 g, 16.04 mmol) in water (6.0
mL) at 0-5.degree. C., the reaction was stirred at 0-5.degree. C.
for about 30 min, the solution was added to the pre-warmed mixture
of KI (7.11 g, 42.80 mmol) and 12 (1.36 g, 5.35 mmol) in H.sub.2O
(30 mL) and con. H.sub.2SO.sub.4 (3.0 mL) at 80.degree. C., the
resulting mixture was stirred at 80.degree. C. for about 30 min.
The reaction was allowed to cool to ambient temperature, sat.
Na.sub.2S.sub.2O.sub.3 (100 mL) was added to quench the reaction.
The mixture was extracted with EA (30 mL.times.3), the combined
organic phases were washed with sat. NaCl (20 mL), dried over
anhydrous Na.sub.2SO.sub.4, filtered, concentrated and purified by
column chromatography on silica gel (200-300 mesh, eluent:
PE:EA=10:1) to give the product as a white solid (2.1 g, 65.9%).
.sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 7.09 (s, 2H), 3.84 (s,
6H). MS (ESI) m/e [M+1].sup.+ 298.9.
Step 3: 4-chloro-3,5-dimethoxybenzoic acid
##STR00077##
[0236] To a solution of 2-chloro-5-iodo-1,3-dimethoxybenzene (2.2
g, 7.38 mmol) in THF (50 mL) was added n-BuLi (5.5 mL, 8.86 mmol,
1.6 M in hexane) at -78.degree. C., the reaction was stirred at
-78.degree. C. for about 30 min, CO.sub.2 was bubbled into the
reaction after drying over with anhydrous CaCl.sub.2). The reaction
was stirred at -78.degree. C. for about 30 min, then warmed to
ambient temperature, concentrated under reduced pressure to remove
solvent, the residue was portioned between EA (50 mL) and water (20
mL), the aqueous phase was adjusted to the pH value of 2-3 with 3 N
HCl and extracted with EA (30 mL.times.3). The combined organic
phases were washed with sat. NaCl (15 mL), dried over anhydrous
Na.sub.2SO.sub.4, filtered and concentrated to give the crude
product as a pale yellow solid (2.0 g, 99.0%). MS (ESI) m/e
[M+1].sup.+ 217.1.
Step 4:
7-(1-acryloylpiperidin-4-yl)-2-(4-chloro-3,5-dimethoxyphenyl)-4,5,-
6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00078##
[0238] Example A22 was then synthesized from
4-chloro-3,5-dimethoxybenzoic acid following the procedures similar
to those in Example A2 without the Suzuki reaction step. NaBH.sub.4
was used in the reduction step. .sup.1H NMR (400 MHz, DMSO-d.sub.6)
.delta. 6.87 (s, 2H), 6.79 (dd, J=16.4, 10.4 Hz, 1H), 6.73 (br s,
1H), 6.07 (dd, J=16.4, 2.0 Hz, 1H), 5.64 (d, J=10.4 Hz, 1H),
4.54-4.42 (m, 1H), 4.15-3.99 (m, 2H), 3.85 (s, 6H), 3.34-3.25 (m,
2H), 3.06-2.92 (m, 1H), 2.64-2.50 (m, 1H), 2.32-2.16 (m, 1H),
2.11-1.97 (m, 1H), 1.96-1.83 (m, 1H), 1.78-1.67 (m, 1H), 1.63-1.51
(m, 1H), 1.36-1.14 (m, 2H). MS (ESI) m/e [M+1].sup.+ 474.1.
Example A23:
7-(1-acryloylpiperidin-4-yl)-2-(4-bromo-3-chloro-5-methoxyphenyl)-4,5,6,7-
-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00079##
[0239] Step 1: Methyl 4-amino-3-chloro-5-methoxybenzoate
##STR00080##
[0241] A mixture solution of methyl 4-amino-3-methoxybenzoate (9.06
g, 50 mmol) and NCS (7.34 g, 55 mmol) in CH.sub.3CN (50 mL) was
stirred at RT for 16 h. The mixture solution was washed with sat.
NaHCO.sub.3 (50 mL) and sat. NaCl (50 mL.times.2), dried over
anhydrous Na.sub.2SO.sub.4, filtered and concentrated to give the
crude product as a yellow solid (10.2 g, 94.4%). MS (ESI, m/e)
[M+1].sup.+ 216.0.
Step 2: Methyl 4-bromo-3-chloro-5-methoxybenzoate
##STR00081##
[0243] To a solution of methyl 4-amino-3-chloro-5-methoxybenzoate
(2.16 g, 10.0 mmol) in CH.sub.3CN (120 mL) and water (12 mL) was
added CuBr.sub.2 (3.04 g, 13.6 mmol) and tert-butyl nitrite (2.0
mL, 17.0 mmol). The resulting mixture solution was warmed to
60.degree. C., stirred for about 1 h. The reaction mixture was
cooled to ambient temperature and partitioned between EA (150 mL)
and water (30 mL), the organic layer was washed with brine (60 mL),
dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated to
give a brown oil which was purified by column chromatography on
silica gel (200-300 mesh, eluting: EA:PE=0-30%) to give the product
as a white solid (5.3 g, 84.8%). MS (ESI, m/e) [M-F1].sup.+278.9
and 280.9.
Step 2: 4-bromo-3-chloro-5-methoxybenzoic acid
##STR00082##
[0245] To a solution of Methyl 4-bromo-3-chloro-5-methoxybenzoate
(2.5 g, 8.9 mmol) in EtOH (20 mL) was added NaOH (6.0 mL, 3 N), the
reaction was stirred as reflux for about 2 h. The reaction was
concentrated under reduced pressure to remove EtOH, the residue was
adjusted to the pH value of 1-2 with 3 N HCl, the aqueous phase was
extracted with EA (50 mL.times.3), the combined organic layers were
dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated to
give the crude product as white solid (2.16 g, 91.3%). MS (ESI,
m/e) [M+1].sup.+264.9 and 266.9.
Step 3:
7-(1-acryloylpiperidin-4-yl)-2-(4-bromo-3-chloro-5-methoxyphenyl)--
4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00083##
[0247] Example 23 was then synthesized from
4-bromo-3-chloro-5-methoxybenzoic acid following the procedures
similar to those in Example A2 without the Suzuki reaction step.
NaBH.sub.4 was used in the reduction step. .sup.1H NMR (400 MHz,
DMSO-d.sub.6) .delta. 7.31 (d, J=1.2 Hz, 1H), 7.19 (d, J=1.2 Hz,
1H), 6.85-6.74 (m, 1H), 6.36 (br s, 1H), 6.07 (dd, J=16.4, 1.8 Hz,
1H), 5.64 (dd, J=10.4, 1.8 Hz, 1H), 5.08 (br s, 2H), 4.54-4.42 (m,
1H), 4.16-3.98 (m, 2H), 3.89 (s, 3H), 3.34-3.24 (m, 2H), 3.06-2.92
(m, 1H), 2.64-2.51 (m, 1H), 2.35-2.16 (m, 1H), 2.10-1.96 (m, 1H),
1.96-1.82 (m, 1H), 1.78-1.65 (m, 1H), 1.62-1.50 (m, 1H), 1.37-1.12
(m, 2H). MS (ESI, m/e) [M+1].sup.+ 522.1 and 524.0.
Example A24:
7-(1-acryloylpiperidin-4-yl)-2-(3,5-dichloro-4-cyclopropylphenyl)-4,5,6,7-
-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00084##
[0249] Example A24 was synthesized from tert-butyl
4-(2-(4-bromo-3,5-dichlorophenyl)-3-cyanopyrazolo[1,5-a]pyrimidin-7-yl)pi-
peridine-1-carboxylate (one intermediate from A18) and
cyclopropylboronic acid following the procedures similar to those
in Example A2. NaBH.sub.4 was used in the reduction step. .sup.1H
NMR (400 MHz, DMSO-d.sub.6) .delta. 7.53 (s, 2H), 6.85-6.73 (m,
1H), 6.59 (br s, 1H), 6.07 (dd, J=16.6, 2.2 Hz, 1H), 5.64 (dd,
J=10.6, 2.2 Hz, 1H), 4.54-4.42 (m, 1H), 4.15-3.97 (m, 2H),
3.33-3.26 (m, 2H), 3.06-2.92 (m, 1H), 2.64-2.50 (m, 1H), 2.34-2.16
(m, 1H), 2.08-1.96 (m, 1H), 1.95-1.84 (m, 1H), 1.84-1.79 (m, 1H),
1.75-1.64 (m, 1H), 1.59-1.48 (m, 1H), 1.32-1.18 (m, 2H), 1.19-1.10
(m, 2H), 0.76-0.69 (m, 2H). MS (ESI) m/e [M+1].sup.+ 488.1.
Example A25:
7-(1-acryloylpiperidin-4-yl)-2-(3,4,5-trimethylphenyl)-4,5,6,7-tetrahydro-
pyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00085##
[0250] Step 1: 4-bromo-3,5-dimethylbenzoic acid
##STR00086##
[0252] To a solution of NaOH (40 mL, 25% in H.sub.2O) was added
4-bromo-3,5-dimethylbenzonitrile (2.0 g, 9.52 mmol), the reaction
was warmed to 95.degree. C., stirred for about 17 h. Cooled to
ambient temperature, the solution was adjusted to the pH value of
1-2 with 12 N HCl, the solid was collected by filtration to give
the crude product as a white solid (2.3 g, 99.0%). MS (ESI) m/e
[M+1].sup.+ 228.3 and 230.3.
Step 2:
7-(1-acryloylpiperidin-4-yl)-2-(3,4,5-trimethylphenyl)-4,5,6,7-tet-
rahydropyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00087##
[0254] Example A25 was then synthesized from tert-butyl
4-(2-(4-bromo-3,5-dimethylphenyl)-3-cyanopyrazolo[1,5-a]pyrimidin-7-yl)pi-
peridine-1-carboxylate (one intermediate from A20) and
methylboronic acid following the procedures similar to those in
Example A2. NaBH.sub.4 was used in the reduction step. MsOH was
used in the cyano hydrolysis step. .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta. 7.15 (s, 2H), 6.66-6.49 (m, 2H), 6.25 (d,
J=16.4 Hz, 1H), 5.66 (d, J=10.8 Hz, 1H), 5.55-5.00 (m, 2H),
4.84-4.67 (m, 1H), 4.19-3.96 (m, 2H), 3.47-3.38 (m, 2H), 3.14-2.94
(m, 1H), 2.65-2.50 (m, 1H), 2.31 (s, 6H), 2.20 (s, 3H), 2.12-2.02
(m, 1H), 1.90-1.75 (m, 2H), 1.73-1.55 (m, 1H), 1.54-1.40 (m, 1H),
1.35-1.22 (m, 2H). MS (ESI) m/e [M+1].sup.+ 422.2.
Example A26:
7-(1-acryloylpiperidin-4-yl)-2-(4-bromo-3-chloro-5-(trifluoromethoxy)phen-
yl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00088##
[0255] Step 1: methyl 4-amino-3-(trifluoromethoxy)benzoate
##STR00089##
[0257] To a solution of 4-amino-3-(trifluoromethoxy)benzoic acid
(10.0 g, 45.3 mmol) in MeOH (120 mL) was added SOCl.sub.2 (10 mL)
for dropwise, the reaction was stirred at 60.degree. C. for about
16 h. The reaction was cooled to ambient temperature, concentrated
under reduced pressure to remove solvent, the residue was
partitioned between EA (80 mL) and H.sub.2O (20 mL), the organic
phase was washed with sat. NaHCO.sub.3 (20 mL.times.2) and brine
(20 mL), dried over anhydrous Na.sub.2SO.sub.4, filtered and
concentrated to give the crude product as a brown solid (9.8 g,
92.2%). MS (ESI) m/e [M+1].+-.236.0.
Step 2: methyl 4-amino-3-chloro-5-(trifluoromethoxy)benzoate
##STR00090##
[0259] To a solution of methyl 4-amino-3-(trifluoromethoxy)benzoate
(4.7 g, 20.0 mmol) in CH.sub.3CN (100 mL) was added DMAC (10 mL)
and NCS (2.94 g, 22.0 mmol), the reaction was warmed to 70.degree.
C., stirred for about 2 h. The reaction was cooled to ambient
temperature, concentrated under reduced pressure to remove
CH.sub.3CN, the residue was diluted with EA (80 mL), the solution
was washed with H.sub.2O (20 mL.times.5) and brine (20 mL), dried
over anhydrous Na.sub.2SO.sub.4, filtered, concentrated and
purified by column chromatography on silica gel (200-300 mesh,
eluent PE:EA=4:1) to give the product as a yellow solid (4.8 g,
89.0%). MS (ESI) m/e [M+1].sup.+ 270.0.
Step 3: methyl 4-bromo-3-chloro-5-(trifluoromethoxy)benzoate
##STR00091##
[0261] To a solution of methyl
4-amino-3-chloro-5-(trifluoromethoxy)benzoate (2.4 g, 8.92 mmol) in
CH.sub.3CN (50 mL) and H.sub.2O (5 mL) was added CuBr.sub.2 (2.79
g, 12.49 mmol), then tert-butyl nitrite (1.56 g, 15.2 mmol) was
added to the reaction for dropwise at RT, the reaction was warmed
to 60.degree. C., stirred for about 2 h. The reaction was cooled to
RT, concentrated under reduced pressure to remove most of the
solvent, the residue mixture was extracted with DCM (20
mL.times.2), the combined organic layers were concentrated and
purified by column chromatography on silica gel (100-200 mesh,
eluent: EA:DCM=1:4) to give the product as yellow solid (2.5 g,
84.5%).
Step 4: 4-bromo-3-chloro-5-(trifluoromethoxy)benzoic acid
##STR00092##
[0263] To a solution of methyl
4-bromo-3-chloro-5-(trifluoromethoxy)benzoate (2.5 g, 8.9 mmol) in
MeOH (30 mL) and THF (20 mL) was added NaOH (20 mL, 3 N in water),
the reaction was stirred at RT for about 1 h. The reaction was
concentrated under reduced pressure to remove organic solvent, the
resulting solution was cooled to 0-5.degree. C., adjusted to the pH
value of 1-2 with 3N HCl, the solid was collected by filtration to
give the product as a light pink solid (1.6 g, 62.6%. MS (ESI) m/e
[M-1].sup.- 316.8 and 318.8.
Step 5:
7-(1-acryloylpiperidin-4-yl)-2-(4-bromo-3-chloro-5-(trifluorometho-
xy)phenyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00093##
[0265] Example A26 was then synthesized from
4-bromo-3-chloro-5-(trifluoromethoxy)benzoic acid following the
procedures similar to those in Example A1. .sup.1H NMR (400 MHz,
DMSO-d.sub.6) .delta. 7.81 (d, J=1.6 Hz, 1H), 7.62 (s, 1H),
6.84-6.74 (m, 1H), 6.60 (br s, 3H), 6.07 (dd, J=16.7, 2.2 Hz, 1H),
5.64 (dd, J=10.5, 2.2 Hz, 1H), 4.55-4.40 (m, 1H), 4.15-3.97 (m,
2H), 3.30-3.25 (m, 2H), 3.06-2.92 (m, 1H), 2.68-2.52 (m, 1H),
2.34-2.16 (m, 1H), 2.10-1.97 (m, 1H), 1.97-1.83 (m, 1H), 1.76-1.64
(m, 1H), 1.62-1.50 (m, 1H), 1.35-1.15 (m, 2H). MS (ESI) m/e
[M+1].sup.+ 576.1 and 578.1.
Example A27:
7-(1-acryloylpiperidin-4-yl)-2-(3,5-dimethoxy-4-(1H-pyrazol-3-yl)phenyl)--
4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00094##
[0267] Example A27 was synthesized from tert-butyl
4-(2-(4-bromo-3,5-dimethoxyphenyl)-3-cyanopyrazolo[1,5-a]pyrimidin-7-yl)p-
iperidine-1-carboxylate and
1-(tetrahydro-2H-pyran-2-yl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2--
yl)-1H-pyrazole following the procedures similar to those in
Example A2. NaBH.sub.4 was used in the reduction step. MsOH was
used in the cyano hydrolysis step. .sup.1H NMR (400 MHz,
DMSO-d.sub.6) .delta. 7.53 (s, 1H), 6.87 (s, 2H), 6.80 (dd, J=17.0,
10.4 Hz, 1H), 6.75 (br s, 1H), 6.52 (s, 1H), 6.07 (dd, J=17.0, 2.4
Hz, 1H), 5.64 (d, J=10.4 Hz, 1H), 4.54-4.43 (m, 1H), 4.18-4.00 (m,
2H), 3.80 (s, 6H), 3.35-3.20 (m, 2H), 3.10-2.91 (m, 1H), 2.67-2.53
(m, 1H), 2.35-2.17 (m, 1H), 2.12-1.98 (m, 1H), 1.98-1.84 (m, 1H),
1.80-1.69 (m, 1H), 1.64-1.53 (m, 1H), 1.38-1.26 (m, 2H). MS (ESI)
m/e [M+1].sup.+ 506.2.
Example A28:
7-(1-acryloylpiperidin-4-yl)-2-(3,5-dimethoxy-4-(thiophen-3-yl)phenyl)-4,-
5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00095##
[0269] Example A28 was synthesized from tert-butyl
4-(2-(4-bromo-3,5-dimethoxyphenyl)-3-cyanopyrazolo[1,5-a]pyrimidin-7-yl)p-
iperidine-1-carboxylate and thiophen-3-ylboronic acid following the
procedures similar to those in Example A2. NaBH.sub.4 was used in
the reduction step. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta.
7.51-7.44 (m, 2H), 7.19 (dd, J=4.8, 1.2 Hz, 1H), 6.84 (s, 2H),
6.83-6.73 (m, 2H), 6.07 (dd, J=16.8, 2.4 Hz, 1H), 5.64 (d, J=11.6
Hz, 1H), 4.57-4.42 (m, 1H), 4.18-4.01 (m, 2H), 3.73 (s, 6H),
3.33-3.27 (m, 2H), 3.08-2.92 (m, 1H), 2.65-2.52 (m, 1H), 2.35-2.17
(m, 1H), 2.10-1.86 (m, 2H), 1.82-1.68 (m, 1H), 1.64-1.53 (m, 1H),
1.36-1.25 (m, 2H). MS (ESI) m/e [M+1].sup.+ 522.2.
Example A29:
7-(1-acryloylpiperidin-4-yl)-2-(4-cyclopropyl-3,5-dimethylphenyl)-4,5,6,7-
-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00096##
[0271] Example 29 was synthesized from tert-butyl
4-(2-(4-bromo-3,5-dimethylphenyl)-3-cyanopyrazolo[1,5-a]pyrimidin-7-yl)pi-
peridine-1-carboxylate (one intermediate from A20) and
cyclopropylboronic acid following the procedures similar to those
in Example A2. NaBH.sub.4 was used in the reduction step. MsOH was
used in the cyano hydrolysis step. .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta. 7.22-7.10 (m, 2H), 6.56 (dd, J=16.8, 10.8 Hz,
1H), 6.38-6.20 (m, 2H), 5.93-5.59 (m, 3H), 4.84-4.68 (m, 1H),
4.20-3.96 (m, 2H), 3.60-3.32 (m, 2H), 3.14-2.94 (m, 1H), 2.90-2.42
(m, 3H), 2.35-2.20 (m, 6H), 2.17-2.00 (m, 2H), 1.93 (d, J=6.6 Hz,
2H), 1.87-1.57 (m, 2H), 1.46 (d, J=6.6 Hz, 2H), 1.38-1.23 (m, 2H).
MS (ESI) m/e [M+1].sup.+ 448.2.
Example A30:
7-(1-acryloylpiperidin-4-yl)-2-(3,5-dimethylphenyl)-4,5,6,7-tetrahydropyr-
azolo[1,5-a]pyrimidine-3-carboxamide
##STR00097##
[0273] Example A30 is a byproduct when preparing Example A29.
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 8.46 (br s, 2H),
7.20-7.00 (s, 4H), 6.56 (dd, J=16.8, 10.8 Hz, 1H), 6.26 (d, J=16.8
Hz, 1H), 5.83-5.58 (m, 2H), 4.88-4.65 (m, 1H), 4.22-3.96 (m, 2H),
3.56-3.38 (m, 2H), 3.14-2.99 (m, 1H), 2.74-2.58 (m, 1H), 2.36 (s,
6H), 2.35-2.24 (m, 1H), 2.22-1.99 (m, 2H), 1.91-1.59 (m, 2H),
1.57-1.42 (m, 1H), 1.41-1.22 (m, 1H). MS (ESI) m/e
[M-F1].sup.+408.2.
Example A31:
7-(1-acryloylpiperidin-4-yl)-2-(4-(3,5-dimethylisoxazol-4-yl)-3-dimethoxy-
phenyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00098##
[0275] Example A31 was synthesized from tert-butyl
4-(2-(4-bromo-3,5-dimethoxyphenyl)-3-cyanopyrazolo[1,5-a]pyrimidin-7-yl)p-
iperidine-1-carboxylate and (3,5-dimethylisoxazol-4-yl)boronic acid
following the procedures similar to those in Example A2. NaBH.sub.4
was used in the reduction step. .sup.1H NMR (400 MHz, CDCl.sub.3)
.delta. 6.80 (s, 2H), 6.69 (br s, 1H), 6.57 (dd, J=16.8, 10.6 Hz,
1H), 6.27 (d, J=16.8 Hz, 1H), 5.68 (d, J=10.6 Hz, 1H), 5.35 (br s,
2H), 4.87-4.70 (m, 1H), 4.21-4.02 (m, 2H), 3.80 (s, 6H), 3.55-3.40
(m, 2H), 3.17-2.98 (m, 1H), 2.72-2.56 (m, 1H), 2.56-2.33 (m, 1H),
2.22 (s, 3H), 2.20-2.02 (m, 1H), 2.10 (s, 3H), 1.90-1.60 (m, 2H),
1.57-1.45 (m, 1H), 1.44-1.32 (m, 1H), 1.32-1.20 (m, 1H). MS (ESI)
m/e [M+1].sup.+ 535.2.
Example A32:
7-(1-acryloylpiperidin-4-yl)-2-(3,5-dimethoxy-4-(tetrahydro-2H-pyran-4-yl-
)phenyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00099##
[0276] Step 1: tert-butyl
4-(3-carbamoyl-2-(3,5-dimethoxy-4-(tetrahydro-2H-pyran-4-yl)phenyl)-4,5,6-
,7-tetrahydropyrazolo[1,5-a]pyrimidin-7-yl)piperidine-1-carboxylate
##STR00100##
[0278] To a solution of tert-butyl
4-(3-carbamoyl-2-(4-(3,6-dihydro-2H-pyran-4-yl)-3,5-dimethoxyphenyl)-4,5,-
6,7-tetrahydropyrazolo[1,5-a]pyrimidin-7-yl)piperidine-1-carboxylate
(100 mg, 0.18 mmol, prepared from tert-butyl
4-(2-(4-bromo-3,5-dimethoxyphenyl)-3-cyanopyrazolo[1,5-a]pyrimidin-7-yl)p-
iperidine-1-carboxylate and
2-(3,6-dihydro-2H-pyran-4-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane
following the procedures similar to those in Example A2. NaBH.sub.4
was used in the reduction step) in MeOH (10 mL) was added
Pd(OH).sub.2/C (50 mg), the mixture was stirred RT under H.sub.2
atmosphere for about 3 h. The reaction was then heated to
50.degree. C., stirred for about 50 h. The solid was filtered and
the filtrate was concentrated under reduced pressure to give the
crude product as a brown solid (100 mg, 99.0%). MS (ESI) m/e
[M+1].sup.+ 570.2.
[0279] Example A32 was then synthesized from tert-butyl
4-(3-carbamoyl-2-(3,5-dimethoxy-4-(tetrahydro-2H-pyran-4-yl)phenyl)-4,5,6-
,7-tetrahydropyrazolo[1,5-a]pyrimidin-7-yl)piperidine-1-carboxylate
following the procedures similar to those in Example A2. .sup.1H
NMR (400 MHz, CDCl.sub.3) .delta. 6.68 (s, 2H), 6.65 (br s, 1H),
6.56 (dd, J=16.6, 10.6 Hz, 1H), 6.25 (d, J=16.6 Hz, 1H), 5.66 (d,
J=10.6 Hz, 1H), 5.34 (br s, 2H), 4.85-4.65 (m, 1H), 4.20-3.97 (m,
4H), 3.82 (s, 6H), 3.60-3.33 (m, 5H), 3.14-2.95 (m, 1H), 2.67-2.30
(m, 3H), 2.19-2.00 (m, 2H), 1.97-1.75 (m, 2H), 1.73-1.59 (m, 1H),
1.53-1.20 (m, 4H). MS (ESI) m/e [M+1].sup.+ 524.2.
Example A33:
7-(1-acryloylpiperidin-4-yl)-2-(4-cyclopropyl-3-ethoxy-5-methoxyphenyl)-4-
,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00101##
[0280] Step 1: methyl 4-bromo-3,5-dihydroxybenzoate
##STR00102##
[0282] To a solution of 4-bromo-3,5-dihydroxybenzoic acid (50.0 g,
214.6 mmol) in methanol (500 mL) was added SOCl.sub.2 (20 mL), the
reaction mixture was stirred at 60.degree. C. for about 3 h. The
reaction mixture was concentrated under reduced pressure. The
residue was poured into methanol (100 mL) and H.sub.2O (100 mL),
the solution was stirred at RT for about 1 h, the precipitate was
filtered, collected and dried in vacuum to afford the product as a
white solid (55.0 g, 99.0%). MS (ESI, m/e) [M+1].sup.+ 246.9 and
248.9.
Step 2: methyl 4-bromo-3-ethoxy-5-hydroxybenzoate
##STR00103##
[0284] To a solution of methyl 4-bromo-3,5-dihydroxybenzoate (10.0
g, 40.5 mmol) in DMF (110 mL) was added K.sub.2CO.sub.3 (11.2 g,
81.0 mmol) and iodoethane (3.55 mL, 44.5 mmol), the reaction
mixture was stirred at ambient temperature for about 16 h. EA (300
mL) was added, the mixture was washed with H.sub.2O (100
mL.times.2) and sat. NaCl (100 mL), concentrated and purified by
column chromatograph on silica gel (200-300 mesh, eluent:
PE:EA=10:1) to afford the product as a white solid (2.3 g, 20.6%).
MS (ESI, m/e) [M+1].sup.+ 275.0 and 277.0.
Step 3: methyl 4-bromo-3-ethoxy-5-methoxybenzoate
##STR00104##
[0286] To a solution of methyl 4-bromo-3-ethoxy-5-hydroxybenzoate
(2.3 g, 8.36 mmol) in DMF (40 mL) was added K.sub.2CO.sub.3 (2.3 g,
16.73 mmol) and iodomethane (1.78 g, 12.55 mmol), the reaction was
stirred at ambient temperature for about 60 h. EA (50 mL) was
added, the mixture was washed with H.sub.2O (30 mL.times.3) and
sat. NaCl (20 mL), dried over anhydrous Na.sub.2SO.sub.4, filtered
and concentrated to afford the product as a white solid (2.4 g,
96.0%).
Step 4: methyl 4-cyclopropyl-3-ethoxy-5-methoxybenzoate
##STR00105##
[0288] To a solution of methyl 4-bromo-3-ethoxy-5-methoxybenzoate
(2.3 g, 7.96 mmol) in 1,4-dioxane (30 mL) and H.sub.2O (10 mL) was
added cyclopropylboronic acid (2.15 g, 23.88 mmol), K.sub.2CO.sub.3
(4.4 g, 31.83 mmol) and Pd(dppf)Cl.sub.2 (1.16 g, 1.59 mmol), the
reaction atmosphere was exchanged with N.sub.2 for three times,
warmed to 80.degree. C. and stirred for about 20 h. Cooled to
ambient temperature, the mixture was filtered through a Celite pad
and the filtrate was concentrated under reduced pressure to remove
1,4-dioxane. The resulting mixture was extracted with EA (30
mL.times.2). The combined organic phases were washed with H.sub.2O
(20 mL), concentrated and purified by column chromatograph on
silica gel (100-200 mesh, eluent: PE:EA=2:1) to afford the product
as a white solid (1.8 g, 90.5%). MS (EST, m/e)
[M+1].sup.+251.1.
Step 5: 4-cyclopropyl-3-ethoxy-5-methoxybenzoic acid
##STR00106##
[0290] To a solution of methyl
4-cyclopropyl-3-ethoxy-5-methoxybenzoate (1.8 g, 7.20 mmol) in
methanol (20 mL) was added 3N NaOH (10 mL), the mixture was stirred
at ambient temperature for about 3 h. The mixture was concentrated
under reduced pressure to remove methanol. The residue was adjusted
to the pH value of 2-3 with 3 N HCl. The precipitate was collected
by filtration and dried in vacuum to give the product as a white
solid (1.0 g, 58.6%). MS (EST, m/e) [M+1].sup.+ 237.1.
Step 6:
7-(1-acryloylpiperidin-4-yl)-2-(4-cyclopropyl-3-ethoxy-5-methoxyph-
enyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00107##
[0292] Example A33 was then synthesized from
4-cyclopropyl-3-ethoxy-5-methoxybenzoic acid following the
procedures similar to those in Example A2 without the Suzuki
reaction step. NaBH.sub.4 was used in the reduction step. .sup.1H
NMR (400 MHz, DMSO-d.sub.6) .delta. 6.78 (dd, J=16.8, 10.4 Hz, 1H),
6.72 (br s, 1H), 6.66 (s, 1H), 6.65 (s, 1H), 6.06 (dd, J=2.4, 16.8
Hz, 1H), 5.63 (dd, J=2.4, 10.4 Hz, 1H), 4.53-4.42 (m, 1H),
4.14-3.91 (m, 4H), 3.75 (s, 3H), 3.33-3.25 (m, 2H), 3.06-2.91 (m,
1H), 2.64-2.50 (m, 1H), 2.31-2.15 (m, 1H), 2.09-1.96 (m, 1H),
1.96-1.83 (m, 2H), 1.78-1.66 (m, 1H), 1.62-1.50 (m, 1H), 1.32 (t,
J=6.8 Hz, 3H), 1.30-1.14 (m, 2H), 1.07-1.01 (m, 2H), 0.79-0.72 (m,
2H). MS (ESI) m/e [M+1].sup.+ 494.2.
Example A34:
7-(1-acryloylpiperidin-4-yl)-2-(3-methoxy-4,5-dimethylphenyl)-4,5,6,7-tet-
rahydropyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00108##
[0293] Step 1: Methyl
3-bromo-5-methoxy-4-(((trifluoromethyl)sulfonyl)oxy)benzoate
##STR00109##
[0295] A mixture solution of methyl
3-bromo-4-hydroxy-5-methoxybenzoate (13.0 g, 50 mmol),
1,1,1-trifluoro-N-(4-methoxyphenyl)-N-((trifluoromethyl)sulfonyl)methane
sulfonamide (22 g, 60 mmol) and TEA (10.0 g, 100 mmol) in DCM (200
mL) was stirred at RT for 16 h. Then the mixture solution was
concentrated and the residue was purified by column chromatography
on silica gel (eluent: EA:PE=1: 10-1:5) to give the product as a
white solid (18.7 g, 95.5%). MS (ESI, m/e) [M+1].sup.+ 392.9 and
394.9.
Step 2: Methyl 3-methoxy-4,5-dimethylbenzoate
##STR00110##
[0297] Under a nitrogen atmosphere, a mixture solution of methyl
3-bromo-5-methoxy-4-(((trifluoromethyl)sulfonyl)oxy)benzoate (3.93
g, 10 mmol), methylboronic acid (3.0 g, 50 mmol), Pd(dppf)Cl.sub.2
(731 mg, 1 mmol) and Cs.sub.2CO.sub.3 (6.5 g, 20 mmol) in
1,4-dioxane (100 mL) was stirred at 90.degree. C. for 16 h, then
the reaction mixture was concentrated and the residue was purified
by column chromatography on silica gel (eluent: EA:PE=0-30%) to
give the product as a white solid (1.5 g, 77.3%). MS (ESI, m/e)
[M+1].sup.+ 195.1.
Step 3: 3-methoxy-4,5-dimethylbenzoic acid
##STR00111##
[0299] A mixture solution of methyl 3-methoxy-4,5-dimethylbenzoate
(2.7 g, 13.9 mmol), and NaOH (3 N, 10 mL) in ethanol (30 mL) was
stirred at 80.degree. C. for 2 h. Then the ethanol was removed in
vacuum, the residue was acidified by 2 N HCl, extracted with EA (20
mL.times.3), the combined organic layers were dried over
Na.sub.2SO.sub.4, filtered and concentrated to give the product as
a white solid (1.78 g, 70.4%). MS (ESI, m/e) [M+1].sup.+ 181.1.
Step 4:
7-(1-acryloylpiperidin-4-yl)-2-(3-methoxy-4,5-dimethylphenyl)-4,5,-
6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00112##
[0301] Example A34 was then synthesized from
3-methoxy-4,5-dimethylbenzoic acid following the procedures similar
to those in Example A1. Pd/C was used in the reduction step.
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 6.91 (s, 1H), 6.81 (s,
1H), 6.56 (dd, J=16.0, 10.0 Hz, 1H), 6.26 (d, J=16.0 Hz, 1H), 5.71
(d, J=10.0 Hz, 1H), 4.86-4.68 (m, 1H), 4.22-3.99 (m, 2H), 3.84 (s,
3H), 3.54-3.41 (m, 2H), 3.16-2.98 (m, 1H), 2.74-2.58 (m, 1H),
2.56-2.34 (m, 1H), 2.30 (s, 3H), 2.17 (s, 3H), 2.16-2.02 (m, 2H),
1.90-1.75 (m, 1H), 1.74-1.59 (m, 1H), 1.57-1.43 (m, 1H), 1.42-1.28
(m, 1H). MS (ESI, m/e) [M+1].sup.+ 438.2.
Example A35:
7-(1-acryloylpiperidin-4-yl)-2-(3-chloro-5-methoxy-4-methylphenyl)-4,5,6,-
7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00113##
[0302] Step 1: tert-butyl
4-(3-carbamoyl-2-(3-chloro-5-methoxy-4-methylphenyl)-4,5,6,7-tetrahydropy-
razolo[1,5-a]pyrimidin-7-yl)piperidine-1-carboxylate
##STR00114##
[0304] A mixture solution of tert-butyl
4-(2-(4-bromo-3-chloro-5-methoxyphenyl)-3-carbamoyl-4,5,6,7-tetrahydropyr-
azolo[1,5-a]pyrimidin-7-yl)piperidine-1-carboxylate (568 mg, 1.0
mmol, intermediate from Example A23), methylboronic acid (90 mg,
1.5 mmol), Pd(dppf)Cl.sub.2 (73 mg, 0.1 mmol) and K.sub.2CO.sub.3
(276 mg, 2.0 mmol) in dioxane (10 mL) was stirred at 90.degree. C.
for 16 h under N.sub.2 atmosphere. The reaction was cooled to
ambient temperature and concentrated under reduced pressure to
remove dioxane, the residue was dissolved in DCM (50 mL). The
organic phase was washed with water (10 mL.times.2), dried over
anhydrous Na.sub.2SO.sub.4, filtered and concentrated to give the
product as a yellow solid (470 mg, 93.4%). MS (ESI, m/e)
[M+1].sup.+ 504.4.
Step 2:
7-(1-acryloylpiperidin-4-yl)-2-(3-chloro-5-methoxy-4-methylphenyl)-
-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00115##
[0306] Example A35 was then synthesized from tert-butyl
4-(3-carbamoyl-2-(3-chloro-5-methoxy-4-methylphenyl)-4,5,6,7-tetrahydropy-
razolo[1,5-a]pyrimidin-7-yl)piperidine-1-carboxylate following the
procedures similar to those in Example A2. .sup.1H NMR (400 MHz,
CDCl.sub.3)) .delta. 7.15 (s, 1H), 6.88 (s, 1H), 6.57 (dd, J=16.6,
10.2 Hz, 1H), 6.26 (d, J=16.6 Hz, 1H), 5.70 (d, J=10.2 Hz, 1H),
4.85-4.69 (m, 1H), 4.18-4.00 (m, 2H), 3.86 (s, 3H), 3.51-3.41 (m,
2H), 3.15-2.98 (m, 1H), 2.75-2.57 (m, 1H), 2.57-2.32 (m, 1H), 2.31
(s, 3H), 2.24-2.00 (m, 2H), 1.88-1.75 (m, 1H), 1.74-1.58 (m, 1H),
1.56-1.42 (m, 1H), 1.42-1.28 (m, 1H). MS (ESI, m/e)[M+1].sup.+
458.1.
Example A36:
7-(1-acryloylpiperidin-4-yl)-2-(3-chloro-4-cyclopropyl-5-methoxyphenyl)-4-
,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00116##
[0308] Example A36 was synthesized from tert-butyl
4-(2-(4-bromo-3-chloro-5-methoxyphenyl)-3-carbamoyl-4,5,6,7-tetrahydropyr-
azolo[1,5-a]pyrimidin-7-yl)piperidine-1-carboxylate (intermediate
from Example A23) and cyclopropylboronic acid following the
procedures similar to those in Example A35. .sup.1H NMR (400 MHz,
DMSO-d.sub.6)) .delta. 7.11 (d, J=0.8 Hz, 1H), 7.04 (d, J=0.8 Hz,
1H), 6.86-6.71 (m, 1H), 6.07 (dd, J=17.0, 2.2 Hz, 1H), 5.64 (dd,
J=12.0, 2.2 Hz, 1H), 4.52-4.42 (m, 1H), 4.15-3.85 (m, 2H), 3.78 (s,
3H), 3.33-3.24 (m, 2H), 3.06-2.91 (m, 1H), 2.68-2.50 (m, 1H),
2.32-2.15 (m, 1H), 2.08-1.95 (m, 1H), 1.95-1.84 (m, 1H), 1.82-1.65
(m, 2H), 1.60-1.50 (m, 1H), 1.35-1.12 (m, 2H), 1.00-0.91 (m, 2H),
0.86-0.79 (m, 2H). MS (ESI, m/e) [M+1].sup.+ 484.1.
Example A37:
7-(1-acryloylpiperidin-4-yl)-2-(4-cyclopentyl-3,5-dimethoxyphenyl)-4,5,6,-
7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00117##
[0309] Step 1: methyl
4-(cyclopent-1-en-1-yl)-3,5-dimethoxybenzoate
##STR00118##
[0311] A mixture of methyl 4-bromo-3,5-dimethoxybenzoate (1.0 g,
3.64 mmol), cyclopent-1-en-1-ylboronic acid (1.24 g, 10.92 mmol),
Pd(OAc).sub.2 (408 mg, 1.82 mmol), t-Bu.sub.3P (10% in Hexane, 10
mL) and K.sub.3PO.sub.4 (2.32 g, 10.92 mmol) in dioxane (50 mL) was
heated to 100.degree. C. for two days under N.sub.2. The mixture
was filtered and the filtrate was concentrated to get crude product
which was purified by column chromatograph on silica (eluent:
EA:PE=1:10) to afford the product as a yellow solid (240 mg,
25.2%). MS (ESI) m/e [M+1].sup.+ 263.2.
Step 2: methyl 4-cyclopentyl-3,5-dimethoxybenzoate
##STR00119##
[0313] A mixture of methyl
4-(cyclopent-1-en-1-yl)-3,5-dimethoxybenzoate (240 mg, 0.92 mmol)
and Pd(OH).sub.2/C (50 mg, 10% w/w) in MeOH (20 mL) and DCM (20 mL)
was stirred at RT for 16 h under H.sub.2 atmosphere. The mixture
was filtered and the filtrate was concentrated to give the product
as a yellow solid (180 mg, 75%). MS (ESI) m/e [M+1].sup.+
265.2.
Step 3: 4-cyclopentyl-3,5-dimethoxybenzoic acid
##STR00120##
[0315] A mixture of methyl 4-cyclopentyl-3,5-dimethoxybenzoate (500
mg, 1.89 mmol) and LiOH.H.sub.2O (398 mg, 9.47 mmol) in THF (10 mL)
and H.sub.2O (10 mL) was heated to reflux for 4 h. The mixture was
cooled to ambient temperature and concentrated under reduced
pressure, the residue was adjusted to the pH value of 1-2 with con.
HCl and extracted with EA (20 mL.times.3). The combined organic
layers were washed with brine, dried over Na.sub.2SO.sub.4,
filtered and concentrated to give the product as a yellow solid
(350 mg, 74.0%). MS (ESI) m/e [M+1].sup.+ 251.2.
Step 4:
7-(1-acryloylpiperidin-4-yl)-2-(4-cyclopentyl-3,5-dimethoxyphenyl)-
-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00121##
[0317] Example A37 was then synthesized from
4-cyclopentyl-3,5-dimethoxybenzoic acid following the procedures
similar to those in Example A2 without the Suzuki reaction step.
.sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 6.79 (dd, J=16.6, 10.4
Hz, 1H), 6.73 (br s, 1H), 6.72 (s, 2H), 6.06 (dd, J=16.6, 2.0 Hz,
1H), 5.64 (dd, J=10.4 Hz, 1H), 4.52-4.42 (m, 1H), 4.15-3.98 (m,
2H), 3.76 (s, 6H), 3.65-3.55 (m, 1H), 3.33-3.24 (m, 2H), 3.05-2.90
(m, 1H), 2.60-2.50 (m, 1H), 2.30-2.15 (m, 1H), 2.10-1.95 (m, 1H),
1.95-1.64 (m, 8H), 1.62-1.50 (m, 3H), 1.35-1.12 (m, 2H). MS (ESI,
m/e) [M+1].sup.+ 508.3.
Example A38:
7-(1-acryloylpiperidin-4-yl)-2-(3,5-dimethoxy-4-(trifluoromethyl)phenyl)--
4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00122##
[0318] Step 1: methyl 3,5-dimethoxy-4-(trifluoromethyl)benzoate
##STR00123##
[0320] A mixture of methyl 4-bromo-3,5-dimethoxybenzoate (1.0 g,
3.64 mmol), methyl 2,2-difluoro-2-(fluorosulfonyl)acetate (2.3 mL,
18.2 mmol) and CuI (693 mg, 3.64 mmol) in DMF (10 mL) was heated to
100.degree. C. for 16 h. The mixture was filtered and the filtrate
was concentrated to give the crude product which was purified by
column chromatograph on silica (eluent: EA:PE=1:10) to afford the
product as an off-white solid (820 mg, 83.2%). MS (ESI) m/e
[M+1].sup.+ 265.0.
Step 2: 3,5-dimethoxy-4-(trifluoromethyl)benzoic acid
##STR00124##
[0322] A mixture solution of methyl
3,5-dimethoxy-4-(trifluoromethyl)benzoate (1.5 g, 5.68 mmol) and
LiOH.H.sub.2O (1.2 g, 28.4 mmol) in THF (20 mL) and H.sub.2O (20
mL) was heated to reflux for 4 h. The mixture was cooled to ambient
temperature and removed THF under reduced pressure, the residue was
adjusted to the pH value of 1-2 with con. HCl and extracted with EA
(20 mL.times.3). The combined organic layers were washed with
brine, dried over Na.sub.2SO.sub.4, filtered and concentrated to
give the product as a yellow solid (1.2 g, 84.5%).
Step 3:
7-(1-acryloylpiperidin-4-yl)-2-(3,5-dimethoxy-4-(trifluoromethyl)p-
henyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00125##
[0324] Example A38 was then synthesized from
3,5-dimethoxy-4-(trifluoromethyl)benzoic acid following the
procedures similar to those in Example A2 without the Suzuki
reaction step. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 6.93 (s,
2H), 6.79 (dd, J=16.7, 11.2 Hz, 1H), 6.72 (br s, 1H), 6.07 (dd,
J=16.7, 2.0 Hz, 1H), 5.64 (d, J=11.2 Hz, 1H), 4.55-4.45 (m, 1H),
4.15-4.02 (m, 2H), 3.84 (s, 6H), 3.32-3.25 (m, 2H), 3.08-2.95 (m,
1H), 2.64-2.53 (m, 1H), 2.30-2.15 (m, 1H), 2.10-198 (m, 1H),
1.95-1.85 (m, 1H), 1.75-1.66 (m, 1H), 1.62-1.50 (m, 1H), 1.35-1.20
(m, 2H). MS (ESI, m/e) [M+1].sup.+ 508.2.
Example A39:
7-(1-acryloylpiperidin-4-yl)-2-(4-cyclohexyl-3,5-dimethoxyphenyl)-4,5,6,7-
-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00126##
[0326] Example A39 was synthesized from methyl
4-bromo-3,5-dimethoxybenzoate and cyclohex-1-en-1-ylboronic acid
following the procedures similar to those in Example A37. .sup.1H
NMR (400 MHz, DMSO-d.sub.6) .delta. 6.78 (dd, J=15.8, 10.6 Hz, 1H),
6.71 (br s, 1H), 6.70 (s, 2H), 6.06 (dd, J=15.8, 1.8 Hz, 1H), 5.63
(d, J=10.6 Hz, 1H), 4.52-4.40 (m, 1H), 4.15-3.97 (m, 2H), 3.76 (s,
6H), 3.32-3.28 (m, 2H), 3.25-3.12 (m, 2H), 3.05-2.92 (m, 1H),
2.60-2.50 (m, 1H), 2.34-2.15 (m, 1H), 2.11-1.95 (m, 3H), 1.95-1.85
(m, 1H), 1.80-1.62 (m, 3H), 1.60-1.50 (m, 1H), 1.49-1.40 (m, 2H),
1.38-1.13 (m, 5H). MS (ESI, m/e) [M+1].sup.+ 522.2.
Example A40:
7-(1-acryloylpiperidin-4-yl)-2-(2,6-dimethoxy-2',3',4',5'-tetrahydro-[1,1-
'-biphenyl]-4-yl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamid-
e
##STR00127##
[0328] Example A40 was synthesized from
2,6-dimethoxy-2',3',4',5'-tetrahydro-[1,1'-biphenyl]-4-carboxylic
acid (intermediate prepared from methyl
4-bromo-3,5-dimethoxybenzoate and cyclohex-1-en-1-ylboronic acid
without the Pd(OH).sub.2/C reduction step) following the procedures
similar to those in Example A2 without the Suzuki reaction step.
NaBH.sub.4 was used in the reduction step. .sup.1H NMR (400 MHz,
DMSO-d.sub.6) .delta. 6.79 (dd, J=16.6, 10.4 Hz, 1H), 6.72 (s, 2H),
6.06 (d, J=16.6 Hz, 1H), 5.63 (d, J=10.6 Hz, 1H), 5.45-5.40 (m,
1H), 4.52-4.40 (m, 1H), 4.15-3.92 (m, 2H), 3.71 (s, 6H), 3.35-3.25
(m, 2H), 3.05-2.92 (m, 2H), 2.60-2.52 (m, 1H), 2.32-2.16 (m, 1H),
2.15-1.97 (m, 4H), 1.95-1.85 (m, 1H), 1.80-1.50 (m, 5H), 1.35-1.15
(m, 3H). MS (ESI, m/e) [M+1].sup.+ 520.2.
Example A41:
(E)-7-(1-acryloylpiperidin-4-yl)-2-(3,5-dimethoxy-4-styrylphenyl)-4,5,6,7-
-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00128##
[0330] Example A41 was synthesized from tert-butyl
4-(2-(4-bromo-3,5-dimethoxyphenyl)-3-cyanopyrazolo[1,5-a]pyrimidin-7-yl)p-
iperidine-1-carboxylate and
(E)-4,4,5,5-tetramethyl-2-styryl-1,3,2-dioxaborolane following the
procedures similar to those in Example A2. NaBH.sub.4 was used in
the reduction step. .sup.1H NMR (DMSO-d.sub.6) .delta. 7.58-7.48
(m, 3H), 7.42-7.34 (m, 3H), 7.28-7.22 (m, 1H), 6.83 (s, 2H)
6.82-6.72 (m, 2H), 6.07 (dd, J=16.4, 2.0 Hz, 1H), 5.64 (d, J=10.8
Hz, 1H), 4.54-4.44 (m, 1H), 4.17-4.01 (m, 2H), 3.88 (s, 6H),
3.31-3.25 (m, 2H), 3.07-2.93 (m, 1H), 2.69-2.52 (m, 1H), 2.32-2.19
(m, 1H), 2.11-1.96 (m, 1H), 1.96-1.85 (m, 1H), 1.79-1.69 (m, 1H),
1.63-1.54 (m, 1H), 1.37-1.18 (m, 2H). MS (ESI) m/e [M+1].sup.+
542.2.
Example A42:
7-(1-acryloylpiperidin-4-yl)-2-(3,5-dimethoxy-4-phenethylphenyl)-4,5,6,7--
tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00129##
[0331] Step 1: tert-butyl
4-(3-carbamoyl-2-(3,5-dimethoxy-4-phenethylphenyl)-4,5,6,7-tetrahydropyra-
zolo[1,5-a]pyrimidin-7-yl)piperidine-1-carboxylate
##STR00130##
[0333] To a solution of tert-butyl
(E)-4-(3-carbamoyl-2-(3,5-dimethoxy-4-styrylphenyl)-4,5,6,7-tetrahydropyr-
azolo[1,5-a]pyrimidin-7-yl)piperidine-1-carboxylate (intermediate
from Example 41, 130 mg, 0.22 mmol) in ethanol (15 mL) was added
Pd/C (50 mg), the reaction mixture was exchanged with H.sub.2 for
three times, warmed to 65.degree. C., stirred for about 4 h. A
filtration was performed, the filtrate was concentrated in vacuum
to afford the product as a colorless oil (100 mg, 76.7%). MS (ESI,
m/e) [M+1].sup.+ 590.3.
Step 2:
7-(1-acryloylpiperidin-4-yl)-2-(3,5-dimethoxy-4-phenethylphenyl)-4-
,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00131##
[0335] Example A42 was then synthesized from tert-butyl
4-(3-carbamoyl-2-(3,5-dimethoxy-4-phenethylphenyl)-4,5,6,7-tetrahydropyra-
zolo[1,5-a]pyrimidin-7-yl)piperidine-1-carboxylate following the
procedures similar to those in Example A2. .sup.1H NMR
(DMSO-d.sub.6) .delta. 7.30-7.24 (m, 2H), 7.20-7.13 (m, 3H),
6.84-6.72 (m, 2H), 6.70 (m, 2H), 6.07 (dd, J=16.8, 2.4 Hz, 1H),
5.64 (d, J=10.8 Hz, 1H), 4.54-4.43 (m, 1H), 4.15-3.99 (m, 2H), 3.75
(s, 6H), 3.31-3.26 (m, 2H), 3.07-2.93 (m, 1H), 2.90-2.82 (m, 2H),
2.74-2.66 (m, 2H), 2.64-2.53 (m, 1H), 2.32-2.17 (m, 1H), 2.12-1.97
(m, 1H), 1.97-1.84 (m, 1H), 1.79-1.67 (m, 1H), 1.63-1.52 (m, 1H),
1.36-1.13 (m, 2H). MS (ESI) m/e [M+1].sup.+ 544.2.
Example A43:
7-(1-acryloylpiperidin-4-yl)-2-(3,5-dimethoxy-4-(1-phenylvinyl)phenyl)-4,-
5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00132##
[0337] Example A43 was synthesized from tert-butyl
4-(2-(4-bromo-3,5-dimethoxyphenyl)-3-cyanopyrazolo[1,5-a]pyrimidin-7-yl)p-
iperidine-1-carboxylate and
4,4,5,5-tetramethyl-2-(1-phenylvinyl)-1,3,2-dioxaborolane following
the procedures similar to those in Example A2. NaBH.sub.4 was used
in the reduction step. .sup.1H NMR (DMSO-d.sub.6) .delta. 7.31-7.19
(m, 5H), 6.83 (s, 2H), 6.82-6.77 (m, 1H), 6.76 (br s, 1H), 6.07 (d,
J=16.8 Hz, 1H), 5.95 (s, 1H), 5.64 (d, J=10.8 Hz, 1H), 5.11 (s,
1H), 4.56-4.43 (m, 1H), 4.18-4.01 (m, 2H), 3.65 (s, 6H), 3.33-3.25
(m, 2H), 3.08-2.92 (m, 1H), 2.65-2.52 (m, 1H), 2.36-2.19 (m, 1H),
2.12-1.86 (m, 2H), 1.81-1.69 (m, 1H), 1.66-1.52 (m, 1H), 1.38-1.16
(m, 2H). MS (ESI) m/e [M+1].sup.+ 542.2.
Example A44:
7-(1-acryloylpiperidin-4-yl)-2-(3,5-dimethoxy-4-(1-phenylethyl)phenyl)-4,-
5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00133##
[0339] Example A44 was synthesized from tert-butyl
4-(3-carbamoyl-2-(3,5-dimethoxy-4-(1-phenylvinyl)phenyl)-4,5,6,7-tetrahyd-
ropyrazolo[1,5-a]pyrimidin-7-yl)piperidine-1-carboxylate following
the procedures similar to those in Example A42. .sup.1H NMR
(DMSO-d.sub.6) .delta. 7.26-7.18 (m, 4H), 7.14-7.07 (m, 1H),
6.83-6.69 (m, 4H), 6.06 (d, J=16.4 Hz, 1H), 5.63 (d, J=10.0 Hz,
1H), 4.76 (q, J=7.2 Hz, 1H), 4.54-4.41 (m, 1H), 4.15-3.97 (m, 2H),
3.70 (s, 6H), 3.32-3.26 (m, 2H), 3.05-2.91 (m, 1H), 2.63-2.52 (m,
1H), 2.33-2.16 (m, 1H), 2.10-1.84 (m, 2H), 1.62 (d, J=7.2 Hz, 1H),
1.60-1.50 (m, 5H), 1.35-1.13 (m, 2H). MS (ESI) m/e [M+1].sup.+
544.2.
Example A45:
7-(1-acryloylpiperidin-4-yl)-2-(4-cyclopropyl-3-isopropoxy-5-methoxypheny-
l)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00134##
[0340] Step 1: methyl 4-bromo-3-hydroxy-5-methoxybenzoate
##STR00135##
[0342] To a solution of 4-bromo-3,5-dihydroxybenzoic acid (11.6 g,
50.0 mmol) in DMF (150 mL) was added CH.sub.3I (14.2 g, 100.0 mmol)
and K.sub.2CO.sub.3 (20.7 g, 150.0 mmol), the reaction was stirred
at ambient temperature for about 60 h. The reaction was quenched by
water (500 mL), the mixture was extracted with EA (200 mL). The
organic layer was washed with water (100 mL.times.2), concentrated
and purified by column chromatography on silica gel (100-200 mesh,
eluent: PE:EA=5:1) to give the product as a white solid (1.7 g,
13.1%). MS (ESI) m/e [M+1].sup.+ 260.9 and 262.9.
Step 2: methyl 4-bromo-3-isopropoxy-5-methoxybenzoate
##STR00136##
[0344] To a solution of methyl 4-bromo-3-hydroxy-5-methoxybenzoate
(800 mg, 3.07 mmol) in DMF (20 mL) was added 2-chloropropane (585
mg, 6.15 mmol), K.sub.2CO.sub.3 (2.13 g, 15.38 mmol) and KI (100
mg, 0.60 mmol), the mixture was heated to 70.degree. C., stirred
for about 16 h. The reaction was cooled to ambient temperature and
portioned with EA (80 mL) and H.sub.2O (50 mL). The organic layer
was washed with water (30 mL.times.2) and brine (20 mL),
concentrated to give the crude product as a light yellow oil (800
mg, 86.1%).
Step 3:
7-(1-acryloylpiperidin-4-yl)-2-(4-cyclopropyl-3-isopropoxy-5-metho-
xyphenyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00137##
[0346] Example 45 was then synthesized from methyl
4-bromo-3-isopropoxy-5-methoxybenzoate following the procedures
similar to those in Example A33. .sup.1H NMR (DMSO-d.sub.6) .delta.
6.79 (dd, J=16.4, 10.6 Hz, 1H), 6.71 (br s, 1H), 6.65 (s, 1H), 6.64
(s, 1H), 6.07 (dd, J=16.4, 2.2 Hz, 1H), 5.64 (d, J=10.6 Hz, 1H),
4.59-4.43 (m, 2H), 4.15-3.98 (m, 2H), 3.74 (s, 3H), 3.31-3.25 (m,
2H), 3.05-2.91 (m, 1H), 2.63-2.53 (m, 1H), 2.30-2.16 (m, 1H),
2.08-1.96 (m, 1H), 1.96-1.84 (m, 2H), 1.77-1.67 (m, 1H), 1.61-1.51
(m, 1H), 1.34-1.15 (m, 8H), 1.06-1.00 (m, 2H), 0.78-0.70 (m, 2H).
MS (ESI) m/e [M+1].sup.+ 508.3.
Example A46:
7-(1-acryloylpiperidin-4-yl)-2-(4-(1-hydroxyethyl)-3,5-dimethoxyphenyl)-4-
,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00138##
[0348] Example A46 was synthesized as a byproduct (hydroxyl group
was introduced from hydrolysis step of nitrile group) from
tert-butyl
4-(2-(4-bromo-3,5-dimethoxyphenyl)-3-cyanopyrazolo[1,5-a]pyrimidin-7-yl)p-
iperidine-1-carboxylate and
4,4,5,5-tetramethyl-2-vinyl-1,3,2-dioxaborolane following the
procedures similar to those in Example A2. NaBH.sub.4 was used in
the reduction step. .sup.1H NMR (DMSO-d.sub.6) .delta. 6.79 (dd,
J=16.4, 10.6, Hz, 1H)), 6.74 (br s, 1H), 6.73 (s, 2H), 6.06 (dd,
J=16.4, 2.0 Hz, 1H), 5.64 (d, J=10.6 Hz, 1H), 5.25-5.15 (m, 1H),
4.53-4.42 (m, 1H), 4.32 (dd, J=8.8, 1.6 Hz, 1H), 4.15-3.98 (m, 2H),
3.78 (s, 6H), 3.31-3.25 (m, 2H), 3.06-2.91 (m, 1H), 2.64-2.53 (m,
1H), 2.31-2.15 (m, 1H), 2.10-1.84 (m, 2H), 1.78-1.67 (m, 1H),
1.62-1.51 (m, 1H), 1.41 (d, J=6.4 Hz, 3H), 1.35-1.14 (m, 2H). MS
(ESI) m/e [M-17].sup.+ 466.2.
Example A47:
7-(1-acryloylpiperidin-4-yl)-2-(4-bromo-3-chloro-5-ethoxyphenyl)-4,5,6,7--
tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00139##
[0349] Step 1: methyl 3-ethoxy-4-nitrobenzoate
##STR00140##
[0351] To a solution of methyl 3-hydroxy-4-nitrobenzoate (19.7 g,
100 mmol) and potassium carbonate (41.4 g, 300 mmol) in DMF (500
mL) was added iodoethane (15.5 g, 100 mmol). The mixture was
stirred at RT for 16 h. The solvent was removed, H.sub.2O (100 mL)
was added to the residue. The solid was collected by filtration and
evaporated to give a gray solid (19.0 g, 84.4%). MS (ESI, m/e)
[M+1]+226.1.
Step 2: methyl 4-amino-3-ethoxybenzoate
##STR00141##
[0353] To a solution of methyl 3-ethoxy-4-nitrobenzoate (10.0 g,
44.4 mmol) in MeOH (100 mL) was added Pd/C (3.0 g). The mixture was
stirred at RT for 16 h under the protection of Hydrogen. The solid
was removed by filtration and the filtrate was evaporated to give
the product as a colorless oil. (8.0 g, 93.2%). MS (ESI, m/e)
[M+1]+196.1.
Step 3:
7-(1-acryloylpiperidin-4-yl)-2-(4-bromo-3-chloro-5-ethoxyphenyl)-4-
,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00142##
[0355] Example A47 was then synthesized from methyl
4-amino-3-ethoxybenzoate following the procedures similar to those
in Example A23. .sup.1H NMR (DMSO-d.sub.6) .delta. 7.30 (s, 1H),
7.17 (s, 1H), 6.85-6.73 (m, 1H), 6.07 (d, J=16.7 Hz, 1H), 5.64 (d,
J=10.5 Hz, 1H), 4.57-4.37 (m, 1H), 4.14 (q, J=7.0 Hz, 2H),
4.05-3.98 (m, 2H), 3.33-3.22 (m, 2H), 3.06-2.90 (m, 1H), 2.70-2.50
(m, 1H), 2.30-2.17 (m, 1H), 2.07-1.96 (m, 1H), 1.95-1.83 (m, 1H),
1.76-1.64 (m, 1H), 1.63-1.50 (m, 1H), 1.38 (t, J=7.0 Hz, 3H),
1.32-1.14 (m, 2H). MS (ESI) m/e [M+1].sup.+ 536.1.
Example A48:
7-(1-acryloylpiperidin-4-yl)-2-(3-chloro-4-cyclopropyl-5-ethoxyphenyl)-4,-
5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00143##
[0356] Step 1: tert-butyl
4-(2-(3-chloro-4-cyclopropyl-5-ethoxyphenyl)-3-cyano-4,5,6,7-tetrahydropy-
razolo[1,5-a]pyrimidin-7-yl)piperidine-1-carboxylate
##STR00144##
[0358] To a solution of tert-butyl
4-(2-(4-bromo-3-chloro-5-ethoxyphenyl)-3-cyano-4,5,6,7-tetrahydropyrazolo-
[1,5-a]pyrimidin-7-yl)piperidine-1-carboxylate (300 mg, 0.53 mmol,
intermediate from Example A47), cyclopropylboronic acid (55 mg,
63.6 mmol) and K.sub.3PO.sub.4 (374 mg, 1.76 mmol) in 1,4-dioxane
(100 mL) was added Pd(dppf)Cl.sub.2 (30 mg, 0.04 mmol). The mixture
was stirred at 90.degree. C. for 16 h. The reaction was quenched
with EA (100 ml) and H.sub.2O (100 ml), the organic phase was
washed with sat. NH.sub.4Cl (50 mL) and sat. NaCl (100 ml), dried
over anhydrous Na.sub.2SO.sub.4 and evaporated to give the crude
product as a black solid (200 mg, 72.2%). MS (ESI, m/e)
[M+1]+526.2.
Step 2:
7-(1-acryloylpiperidin-4-yl)-2-(3-chloro-4-cyclopropyl-5-ethoxyphe-
nyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00145##
[0360] Example A48 was then synthesized from tert-butyl
4-(2-(3-chloro-4-cyclopropyl-5-ethoxyphenyl)-3-cyano-4,5,6,7-tetrahydropy-
razolo[1,5-a]pyrimidin-7-yl)piperidine-1-carboxylate following the
procedures similar to those in Example A2, NaBH.sub.4 was used in
the reduction step. .sup.1H NMR (CDCl.sub.3) .delta. 7.14 (s, 1H),
6.86 (s, 1H), 6.63-6.52 (m, 1H), 6.26 (d, J=16.6 Hz, 1H), 5.68 (d,
J=10.3 Hz, 1H), 5.63-5.43 (br s, 1H), 4.86-4.66 (m, 1H), 4.20-3.99
(m, 3H), 3.61-3.36 (m, 2H), 3.13-2.95 (m, 1H), 2.72-2.44 (m, 2H),
2.31-2.25 (m, 1H), 2.17-1.96 (m, 4H), 1.51-1.38 (m, 3H), 1.38-1.16
(m, 4H), 1.04-0.90 (m, 4H). MS (ESI) m/e [M+1].sup.+ 498.2.
Example A49:
7-(1-acryloylpiperidin-4-yl)-2-(3-chloro-5-ethoxy-4-methylphenyl)-4,5,6,7-
-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00146##
[0362] Example A49 was synthesized from tert-butyl
4-(2-(4-bromo-3-chloro-5-ethoxyphenyl)-3-cyano-4,5,6,7-tetrahydropyrazolo-
[1,5-a]pyrimidin-7-yl)piperidine-1-carboxylate and methylboronic
acid following the procedures similar to those in Example A48.
.sup.1H NMR (CDCl.sub.3) .delta. 7.14 (s, 1H), 6.87 (s, 1H),
6.66-6.53 (m, 2H), 6.27 (d, J=16.7 Hz, 1H), 5.68 (d, J=10.7 Hz,
1H), 4.86-4.66 (m, 1H), 4.20-3.95 (m, 3H), 3.64-3.36 (m, 2H),
3.10-2.97 (m, 1H), 2.72-2.44 (m, 2H), 2.31 (s, 3H), 2.14-2.02 (m,
2H), 1.87-1.73 (m, 1H) 1.7-1.58 (m, 1H), 1.44 (d, J=6.8 Hz, 3H)
1.38-1.16 (m, 4H). MS (ESI) m/e [M+1].sup.+ 472.2.
Example A50:
2-(3,5-dimethoxy-4-methylphenyl)-7-(1-propioloylpiperidin-4-yl)-4,5,6,7-t-
etrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00147##
[0364] To a solution of propiolic acid (175 mg, 2.5 mmol) in DCM
(30 mL) was dropwisely added oxalyl dichloride (1.0 mL) at
0.degree. C., the solution was stirred at 0.degree. C. for 2 h. The
solvent was removed and the residue was dissolved in DCM (2.0 mL),
the solution was added to the mixture of
2-(3,5-dimethoxy-4-methylphenyl)-7-(piperidin-4-yl)-4,5,6,7-tetrahydropyr-
azolo[1,5-a]pyrimidine-3-carboxamide (200 mg, 0.5 mmol,
Intermediate from Example A1) and TEA (150 mg, 1.5 mmol) in DCM (30
mL), the reaction was stirred at RT for 3 h. The organic phase was
washed with sat. NaHCO.sub.3 (100 mL) and sat. NaCl (100 mL), dried
over anhydrous Na.sub.2SO.sub.4, filtered and concentrated, the
residue was purified by pre-TLC (DCM:MeOH=50: 1) to afford the
product (5 mg, 2.3%). .sup.1H NMR (DMSO-d.sub.6) .delta. 6.71 (s,
2H), 4.52-4.46 (m, 1H), 4.40-4.31 (m, 1H), 4.31-4.22 (m, 1H),
4.17-4.01 (m, 1H), 3.78 (s, 6H), 3.33-3.25 (m, 2H), 3.16-3.09 (m,
1H), 2.71-2.57 (m, 1H), 2.31-2.16 (m, 1H), 2.03 (s, 3H), 2.03-1.96
(m, 1H), 1.95-1.86 (m, 1H), 1.84-1.70 (m, 1H), 1.67-1.53 (m, 1H),
1.40-1.18 (m, 2H),. MS (ESI) m/e [M+1].sup.+ 452.2.
Example A51:
7-(1-acryloylpiperidin-4-yl)-2-(3,5-dimethoxy-4-vinylphenyl)-4,5,6,7-tetr-
ahydropyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00148##
[0366] To a solution of
7-(1-acryloylpiperidin-4-yl)-2-(4-bromo-3,5-dimethoxyphenyl)-4,5,6,7-tetr-
ahydropyrazolo[1,5-a]pyrimidine-3-carboxamide (250 mg, 0.48 mmol,
Intermediate from Example 6) in dioxane (5 mL) and 1 M
K.sub.2CO.sub.3 (2.5 mL) was added
4,4,5,5-tetramethyl-2-vinyl-1,3,2-dioxaborolane (149 mg, 0.96 mmol)
and Pd(dppf)Cl.sub.2 (37 mg, 0.05 mmol), the reaction atmosphere
was exchanged with N.sub.2 for three times, warmed to 90.degree. C.
and stirred for about 17 h. Cooled to ambient temperature, the
mixture was concentrated and purified by column chromatograph on
silica gel (100-200 mesh, eluent: DCM:MeOH=30:1) to give the crude
product (100 mg) which was further purified by pre-HPLC to afford
the product (50 mg, 22.3%). .sup.1H NMR (DMSO-d.sub.6) .delta. 6.89
(dd, J=18.0, 12.0 Hz, 1H), 6.84-6.66 (m, 4H), 6.12-6.07 (m, 2H),
5.64 (d, J=11.2 Hz, 1H), 5.39 (dd, J=12.0, 2.8 Hz, 1H), 4.55-4.40
(m, 1H), 4.15-3.97 (m, 2H), 3.82 (s, 6H), 3.32-3.25 (m, 2H),
3.08-2.90 (m, 1H), 2.65-2.52 (m, 1H), 2.33-2.16 (m, 1H), 2.10-1.85
(m, 2H), 1.80-1.67 (m, 1H), 1.64-1.51 (m, 1H), 1.37-1.14 (m, 2H).
MS (ESI) m/e [M+1]-h 466.2.
Example A52:
7-(1-acryloylpiperidin-4-yl)-2-(4-cyclopropyl-3,5-bis(methoxy-d3)phenyl)--
4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00149##
[0368] Example A52 was synthesized from methyl
4-bromo-3,5-dihydroxybenzoate and CD.sub.3I following the
procedures similar to those in Example A2. .sup.1H NMR (400 MHz,
DMSO-d.sub.6) .delta. 6.78 (dd, J=16.8, 10.8 Hz, 1H), 6.73 (br s,
1H), 6.67 (s, 2H), 6.06 (dd, J=16.8, 2.2 Hz, 1H), 5.64 (d, J=10.8
Hz, 1H), 4.54-4.40 (m, 1H), 4.15-3.97 (m, 2H), 3.50-3.20 (m, 3H),
3.05-2.90 (m, 1H), 2.63-2.52 (m, 1H), 2.30-2.15 (m, 1H), 2.10-1.81
(m, 3H), 1.77-1.10 (m, 3H), 1.02-0.90 (m, 2H), 0.82-0.68 (m, 2H).
MS (EST, m/e) [M+1].sup.+ 486.2.
Example A52a and A52b: (S or
R)-7-(1-acryloylpiperidin-4-yl)-2-(4-cyclopropyl-3,5-bis(methoxy-d3)pheny-
l)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide and (R
or
S)-7-(1-acryloylpiperidin-4-yl)-2-(4-cyclopropyl-3,5-bis(methoxy-d3)pheny-
l)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00150##
[0370] Two enantiomers A52a (faster isomer) and A52b (slower
isomer) were separated by chiral preparative HPLC. The chiral
separation conditions are shown below. The faster enantiomer was
eluted at retention time of around 4.2 min. The slower enantiomer
was eluted at retention time of around 5.5 min.
TABLE-US-00006 Column CHIRAL ART Cellulose-SB Column size 2 cm
.times. 25 cm, 5 um Injection 0.5 mL Mobile phase Hex:EtOH = 50:50
Flow rate 20 ml/min Wave length UV 220 nm Temperature 25.degree. C.
Sample solution 11.7 mg/mL in EtOH:DCM = 3:1 Prep-HPLC equipment
Prep-Gilson-HPLC
[0371] A52a was assigned to (S)-configuraton and A52b was assigned
to (R)-configuration:
##STR00151##
Example B1:
7-(1-acryloylazetidin-3-yl)-2-(3,5-dichloro-4-methylphenyl)-4,5,6,7-tetra-
hydropyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00152##
[0373] Example B1 was synthesized from
5-amino-3-(3,5-dichloro-4-methylphenyl)-1H-pyrazole-4-carbonitrile
(intermediate from Example A7) and tert-butyl
3-(3-(dimethylamino)acryloyl)azetidine-1-carboxylate (ref:
WO2014173289A1) following the procedures similar to those in
Example A2 without the Suzuki reaction step. NaBH.sub.4 was used in
the reduction step. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta.
7.56 (s, 2H), 6.62 (s, 1H), 6.39-6.24 (m, 1H), 6.14-6.03 (m, 1H),
5.68-5.58 (m, 1H), 4.45-4.35 (m, 2H), 4.34-3.80 (m, 3H), 3.33-3.20
(m, 1H), 3.08-2.88 (m, 2H), 2.44 (s, 3H), 2.14-2.00 (m, 1H),
1.81-1.66 (m, 1H). MS (ESI, m/e) [M+1].sup.+ 434.1.
Example B2:
7-(1-acryloylazetidin-3-yl)-2-(4-bromo-3,5-dimethoxyphenyl)-4,5,6,7-tetra-
hydropyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00153##
[0375] Example B2 was synthesized from
5-amino-3-(4-bromo-3,5-dimethoxyphenyl)-1H-pyrazole-4-carbonitrile
(intermediate from Example A2) following the procedures similar to
those in Example B1. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta.
6.84 (s, 1H), 6.83 (s, 1H), 6.75 (br s, 1H), 6.36-6.27 (m, 1H),
6.10-6.05 (m, 1H), 5.67-5.60 (m, 1H), 4.45-3.95 (m, 5H), 3.85 (s,
3H), 3.84 (s, 3H), 3.32-3.25 (m, 2H), 3.05-2.95 (m, 1H), 2.15-2.07
(m, 1H), 1.84-1.67 (m, 1H). MS (ESI, m/e) [M+1].sup.+ 490.1 and
492.1.
Example B3:
7-(1-acryloylazetidin-3-yl)-2-(3,5-dimethoxy-4-methylphenyl)-4,5,6,7-tetr-
ahydropyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00154##
[0377] Example B3 was synthesized from
5-amino-3-(3,5-dimethoxy-4-methylphenyl)-1H-pyrazole-4-carbonitrile
(intermediate from Example A1) following the procedures similar to
those in Example B1. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta.
6.76 (br s, 1H), 6.70 (s, 2H), 6.37-6.26 (m, 1H), 6.11-6.03 (m,
1H), 5.68-5.59 (m, 1H), 4.42-4.33 (m, 1H), 4.32-4.25 (m, 0.5H),
4.20-4.14 (m, 0.5H), 4.12-4.06 (m, 0.5H), 4.04-3.95 (m, 1H),
3.90-3.84 (m, 0.5H), 3.78 (s, 6H), 3.33-3.25 (m, 2H), 3.05-2.90 (m,
1H), 2.36-2.23 (m, 1H), 2.15-2.05 (m, 1H), 2.03 (s, 3H), 1.83-1.69
(m, 1H). MS (EST) m/e [M+1].sup.+ 426.2.
Example C1:
7-acryloyl-2-(3,5-dimethoxy-4-methylphenyl)-5,5a,6,7,8,8a-hexahydro-4H-py-
razolo[1,5-a]pyrrolo[3,4-e]pyrimidine-3-carboxamide
##STR00155##
[0379] Example C1 was synthesized from
5-amino-3-(3,5-dimethoxy-4-methylphenyl)-1H-pyrazole-4-carbonitrile
(intermediate from Example A1) and
tert-butyl-3-((dimethylamino)methylene)-4-oxopyrrolidine-1-carboxylate
(ref: WO2014173289) following the procedures similar to those in
Example A1. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 6.87-6.79
(m, 1H), 6.73 (s, 2H), 6.63-6.52 (m, 1H), 6.20-6.05 (m, 1H),
5.72-5.60 (m, 1H), 4.75-4.60 (m, 1H), 4.25-4.05 (m, 1H), 4.02-3.85
(m, 1H), 3.78 (s, 6H), 3.75-3.65 (m, 1H), 3.55-3.45 (m, 2H),
3.17-3.06 (m, 1H), 3.05-2.92 (m, 1H), 2.03 (s, 3H). MS (ESI) m/e
[M+1].sup.+ 411.9.
Example C2:
7-acryloyl-2-(3,4,5-trimethoxyphenyl)-5,5a,6,7,8,8a-hexahydro-4H-pyrazolo-
[1,5-a]pyrrolo[3,4-e]pyrimidine-3-carboxamide
##STR00156##
[0381] Example C2 was synthesized from 3,4,5-trimethoxybenzoic acid
following the procedures similar to those in Example A2 and C1.
.sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 6.78 (s, 2H), 6.56 (dd,
J=16.8, 10.4 Hz, 1H), 6.15-6.06 (m, 1H), 5.70-5.61 (m, 1H),
4.71-4.60 (m, 1H), 4.22-4.06 (m, 1H), 4.00-3.75 (m, 7H), 3.69 (s,
3H), 3.52-3.43 (m, 1H), 3.42-3.34 (m, 1H), 3.33-3.24 (m, 1H),
3.15-3.04 (m, 1H), 3.03-2.90 (m, 1H). MS (ESI) m/e [M+1].sup.+
427.8.
Example D1:
7-acryloyl-2-(3,5-dimethoxy-4-methylphenyl)-4,5,5a,6,7,8,9,9a-octahydropy-
razolo[1,5-a]pyrido[3,4-e]pyrimidine-3-carboxamide
##STR00157##
[0383] Example D1 was synthesized from
5-amino-3-(3,5-dimethoxy-4-methylphenyl)-1H-pyrazole-4-carbonitrile
(intermediate from Example A1) and
tert-butyl-3-((dimethylamino)methylene)-4-oxopiperidine-1-carboxylate
(ref: WO2014173289) following the procedures similar to those in
example A2 without the Suzuki reaction step. NaBH.sub.4 was used in
the reduction step. .sup.1H NMR (DMSO-d.sub.6) .delta. 6.94-6.77
(m, 1H), 6.72 (s, 2H), 6.20-6.06 (m, 1H), 5.75-5.64 (m, 1H),
4.46-4.37 (m, 1H), 3.79 (s, 6H), 3.76-3.63 (m, 3H), 3.35-3.26 (m,
2H), 3.24-3.15 (m, 1H), 2.40-2.39 (m, 1H), 2.16-2.06 (m, 1H), 2.03
(s, 3H), 2.00-1.94 (m, 1H). MS (ESI) m/e [M+1].sup.+ 426.2.
Example D2:
7-acryloyl-2-(3,5-dimethoxy-4-methylphenyl)-4,4a,5,6,7,8,8a,9-octahydropy-
razolo[1,5-a]pyrido[4,3-d]pyrimidine-3-carboxamide
##STR00158##
[0385] Example D2 is a byproduct when preparing Example D1. .sup.1H
NMR (DMSO-d.sub.6) .delta. 6.88 (dd, J=16.5, 10.6 Hz, 1H), 6.73 (s,
2H), 6.55 (br s, 1H), 6.13 (dd, J=16.5, 2.0 Hz, 1H), 5.71 (d,
J=10.6 Hz, 1H), 4.73-4.53 (m, 1H), 4.33-4.11 (m, 2H), 3.78 (s, 6H),
3.70-3.59 (m, 1.5H), 3.31-3.18 (m, 1H), 3.18-3.07 (m, 0.5H),
2.99-2.84 (m, 0.5H), 2.77-2.63 (m, 0.5H), 2.30-2.16 (m, 1H), 2.03
(s, 3H), 1.92-1.73 (m, 1H), 1.49-1.27 (m, 1H). MS (ESI) m/e
[M+1].sup.+ 426.2.
Example E1:
7-(acrylamidomethyl)-2-(3,5-dimethoxy-4-methylphenyl)-4,5,6,7-tetrahydrop-
yrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00159##
[0386] Step 1: 2-(2-oxopropyl)isoindoline-1,3-dione
##STR00160##
[0388] To a solution of potassium 1,3-dioxoisoindolin-2-ide (125.0
g, 678.1 mmol) in DMF (500 mL) was added 1-chloropropan-2-one (57
g, 594.0 mmol), the reaction mixture was stirred at ambient
temperature for about 60 h. The reaction mixture was poured into
ice water (1.0 L), the resulting mixture was stirred for about 1 h.
The precipitate was collected by filtration and dried in vacuum to
afford the product as a white solid (101.2 g, 81.3%). MS (ESI, m/e)
[M+1].sup.+ 203.9.
Step 2:
2-(4-(dimethylamino)-2-oxobut-3-en-1-yl)isoindoline-1,3-dione
##STR00161##
[0390] A solution of 2-(2-oxopropyl)isoindoline-1,3-dione (101.0 g,
500.0 mmol) in 1,1-dimethoxy-N,N-dimethylmethanamine (400 mL) was
heated at 70.degree. C. for about 16 h. Cooled to ambient
temperature, PE (200 mL) was added, the mixture was stirred at
ambient temperature for 1 h. The precipitate was collected by
filtration. The solids were placed into a 500 ml round-bottom
flask, HOAc (120 mL) was added, the mixture was stirred at ambient
temperature for 40 min and filtrated, the filtrate was concentrated
to remove HOAc. The residue was recrystallized from EA (150 mL) to
give the product as a yellow solid (18 g, 14.0%). MS (ESI, m/e)
[M+1].sup.+ 258.9.
Step 3:
2-(3,5-dimethoxy-4-methylphenyl)-7-((1,3-dioxoisoindolin-2-yl)meth-
yl) pyrazolo[1,5-a]pyrimidine-3-carbonitrile
##STR00162##
[0392] To a solution of
2-(4-(dimethylamino)-2-oxobut-3-en-1-yl)isoindoline-1,3-dione (1.0
g, 4.0 mmol) in toluene (30 mL) was added
5-amino-3-(3,5-dimethoxy-4-methylphenyl)-1H-pyrazole-4-carbonitrile
(1.0 g, 4.0 mmol, intermediate from Example A1) and HOAc (2.0 mL),
the reaction was stirred at 95.degree. C. for about 16 h. The
reaction mixture was cooled to ambient temperature and concentrated
under reduced pressure. The residue was recrystallized from EA (25
mL) to afford the product as a brown solid (1.15 g, 65.5%). MS
(ESI, m/e) [M+1].sup.+ 454.1.
Step 4:
7-(aminomethyl)-2-(3,5-dimethoxy-4-methylphenyl)-4,5,6,7-tetrahydr-
opyrazolo[1,5-a]pyrimidine-3-carbonitrile
##STR00163##
[0394] To a solution of
2-(3,5-dimethoxy-4-methylphenyl)-7-((1,3-dioxoisoindolin-2-yl)methyl)pyra-
zolo[1,5-a]pyrimidine-3-carbonitrile (1.1 g, 2.43 mmol) in DCM (20
mL) and methanol (10 mL) was added NaBH.sub.4 (460 mg, 12.13 mmol)
in portions, the reaction was stirred at ambient temperature for 1
h. EtOH (30 mL) and Hydrazine hydrate (20 mL) was added into above
solution, then heated at 90.degree. C. for about 16 h. Cooled to
ambient temperature, the mixture was concentrated to remove organic
solvent, then EtOH (20 mL) and Hydrazine hydrate (10 mL) was added,
the resulting mixture was heated at 105.degree. C. for about 6 h.
Cooled to ambient temperature, concentrated to remove EtOH, the
resulting mixture was extracted with EA (50 mL.times.2). The
combined organic phases were concentrated and purified by column
chromatograph on silica gel (100-200 mesh, eluent: DCM:MeOH=20:1)
to afford the product as a white solid (300 g, 37.8%). MS (ESI,
m/e) [M+1].+-.327.3.
Step 5:
N-((3-cyano-2-(3,5-dimethoxy-4-methylphenyl)-4,5,6,7-tetrahydropyr-
azolo[1,5-a]pyrimidin-7-yl)methyl)acrylamide
##STR00164##
[0396] To a solution of
7-(aminomethyl)-2-(3,5-dimethoxy-4-methylphenyl)-4,5,6,7-tetrahydropyrazo-
lo[1,5-a]pyrimidine-3-carbonitrile (150 mg, 0.46 mmol) in DCM (10
mL) was added TEA (230 mg, 2.30 mmol) and acryloyl chloride (83 mg,
0.92 mmol), the mixture was stirred at ambient temperature for
about 20 min. The mixture was diluted with DCM (20 mL), washed with
H.sub.2O (10 mL.times.2) and sat. NaCl (10 mL), dried over
anhydrous Na.sub.2SO.sub.4, filtered and concentrated to give the
crude product as a yellow solid (150 mg, 85.8%). 103471 Step 5:
7-(acrylamidomethyl)-2-(3,5-dimethoxy-4-methylphenyl)-4,5,6,7-tetrahydrop-
yrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00165##
[0397]
N-((3-cyano-2-(3,5-dimethoxy-4-methylphenyl)-4,5,6,7-tetrahydropyra-
zolo[1,5-a]pyrimidin-7-yl)methyl)acrylamide (150 mg, 0.39 mmol) was
dissolved in MsOH (5.0 mL), the solution was stirred at 80.degree.
C. for about 2.5 h. Cooled to about 0.degree. C., the pH of the
mixture was adjusted to around 14 with 3 N NaOH, the resulting
mixture was extracted with DCM (20 mL.times.3). The combined
organic phases were washed with sat. NaCl (10 mL), dried over
anhydrous Na.sub.2SO.sub.4, filtered and concentrated to give the
crude product (180 mg) which was purified by pre-TLC
(DCM:MeOH=25:1) to give the product (70 mg, 44.5%). .sup.1H NMR
(400 MHz, DMSO-d.sub.6) .delta. 8.35 (t, J=5.8 Hz, 1H), 6.76 (br s,
1H), 6.74 (s, 2H), 6.28 (dd, J=17.0, 10.2 Hz, 1H), 6.12 (d, J=17.0
Hz, 1H), 5.63 (d, J=10.2 Hz, 1H), 4.25-4.15 (m, 1H), 3.83-3.74 (m,
1H), 3.79 (s, 6H), 3.45-3.37 (m 1H), 3.36-3.26 (m, 2H), 2.08-1.90
(m, 2H), 2.03 (s, 3H). MS (ESI) m/e [M+1].sup.+ 400.2.
Example F1:
7-(7-acryloyl-7-azaspiro[3.5]nonan-2-yl)-2-(3,5-dimethoxy-4-methylphenyl)-
-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00166##
[0399] Example F1 was synthesized from
5-amino-3-(3,5-dimethoxy-4-methylphenyl)-1H-pyrazole-4-carbonitrile
(intermediate from Example A1) and tert-butyl
2-(3-(dimethylamino)acryloyl)-7-azaspiro[3.5]nonane-7-carboxylate
(ref: WO2014173289) following the procedures similar to those in
example A2 without the Suzuki reaction step. .sup.1H NMR (400 MHz,
DMSO-d.sub.6) .delta. 6.83-6.72 (m, 1H), 6.70 (br s, 1H), 6.69 (s,
2H), 6.04 (dd, J=16.8, 2.3 Hz, 1H), 5.62 (dd, J=10.6, 2.3 Hz, 1H),
4.14-4.04 (m, 1H), 3.78 (s, 6H), 3.50-3.42 (m, 2H), 3.40-3.35 (m,
2H), 3.27-3.20 (m, 2H), 2.63-2.53 (m, 1H), 2.03 (s, 3H), 2.00-1.76
(m, 5H), 1.75-1.65 (m, 1H), 1.60-1.50 (m, 2H), 1.45-1.35 (m, 2H).
MS (ESI, m/e) [M+1].sup.+ 494.3.
Example G1:
7-(3-acryloyl-3-azabicyclo[3.2.1]octan-8-yl)-2-(3,5-dimethoxy-4-methylphe-
nyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00167##
[0401] Example G1 was synthesized from
5-amino-3-(3,5-dimethoxy-4-methylphenyl)-1H-pyrazole-4-carbonitrile
(intermediate from Example A1) and tert-butyl
8-(3-(dimethylamino)acryloyl)-3-azabicyclo[3.2.1]octane-3-carboxylate
(ref: WO2014173289) following the procedures similar to those in
example A2 without the Suzuki reaction step. NaBH.sub.4 was used in
the reduction step. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta.
6.84-6.73 (m, 1H), 6.71 (s, 2H), 6.06 (d, J=16.4 Hz, 1H), 5.63 (d,
J=10.8 Hz, 1H), 4.32-4.16 (m, 1H), 3.95-3.80 (m, 3H), 3.78 (s, 6H),
3.45-3.30 (m, 2H), 3.20-3.00 (m, 1H), 2.77-2.56 (m, 1H), 2.30-2.20
(m, 1H), 2.15-2.05 (m, 1H), 2.03 (s, 3H), 2.02-1.95 (m 2H),
1.93-1.69 (m, 2H), 1.40-1.30 (m, 2H). MS (ESI, m/e) [M+1].sup.+
508.2.
Example H1:
7-(2-acryloyl-2-azaspiro[3.3]heptan-6-yl)-2-(3,5-dimethoxy-4-methylphenyl-
)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00168##
[0403] Example H1 was synthesized from
5-amino-3-(3,5-dimethoxy-4-methylphenyl)-1H-pyrazole-4-carbonitrile
(intermediate from Example A1) and tert-butyl
6-(3-(dimethylamino)acryloyl)-2-azaspiro[3.3]heptane-2-carboxylate
(ref: WO2014173289) following the procedures similar to those in
example A2 without the Suzuki reaction step. NaBH.sub.4 was used in
the reduction step. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta.
6.71 (s 1H), 6.70 (s 1H), 6.55 (br s, 1H), 6.32-6.18 (m, 1H),
6.10-6.00 (m, 1H), 5.68-5.58 (m, 1H), 4.28-4.23 (m, 1H), 4.15-4.10
(m, 0.5H), 4.08-4.00 (m, 1H), 4.00-3.92 (m, 2H), 3.88-3.82 (m,
0.5H), 3.78 (s, 6H), 3.30-3.17 (m, 2H), 2.32-2.20 (m, 3H),
2.15-2.05 (m, 1H), 2.00-1.90 (m, 2H), 1.80-1.70 (m, 1H). MS (EST,
m/e) [M+1].sup.+ 466.2.
[0404] Some compounds in WO2014173289A1 are listed in the Table
I.
TABLE-US-00007 Compounds in WO2014173289A1 Structure Description
167.sup.# ##STR00169## No 167* (faster isomer) 167.sup.$ (slower
isomer) ##STR00170## Separated from compound 167 in WO2014173289A1
using chiral HPLC 174 ##STR00171## No Compound in WO2014173289A1
##STR00172## No 144 ##STR00173## No 158 ##STR00174## No 169.sup.#
##STR00175## No 169* (faster isomer) 169.sup.$ (slower isomer)
##STR00176## Separated from compound 169 in WO2014173289A1 using
chiral HPLC 176 ##STR00177## No
[0405] Btk Kinase Enzymatic Assays
[0406] Compounds disclosed herein were tested for inhibition of Btk
kinase (aa2-659, Carna Biosciences) in assays based on the
time-resolved fluorescence-resonance energy transfer (TR-FRET)
methodology. The assays were carried out in 384-well low volume
black plates in a reaction mixture containing Btk kinase, 30 .mu.M
ATP, 0.4 .mu.M peptide substrate and serially diluted compound in
buffer containing 50 mM Tris pH7.4, 10 mM MgCl.sub.2, 2 mM
MnCl.sub.2, 0.1 mM EDTA, 1 mM DTT, 0.005% Tween-20, 20 nM SEB and
0.01% BSA. The kinase was incubated with compound for 60 minutes at
room temperature and the reaction was initiated by the addition of
ATP and peptide substrate. After reaction at room temperature for
60 minutes, an equal volume of stop/detection solution was added
according to the manufacture's instruction (CisBio Bioassays). The
stop/detection solution contained Eu.sup.3+ cryptate-conjugated
mouse monoclonal antibody (PT66) anti-phosphotyrosine and
XL665-conjugated streptavidin in buffer containing 50 mM HEPES
pHn7.0, 800 mM KF, 20 mM EDTA, and 0.1% BSA. Plates were sealed and
incubated at room temperature for 1 hour, and the TR-FRET signals
(ratio of fluorescence emission at 665 nm over emission at 620 nm
with excitation at 337 nm wavelength) were recorded on a PHERAstar
FS plate reader (BMG Labtech). Phosphorylation of peptide substrate
led to the binding of anti-phosphotyrosine antibody to the
biotinylated peptide substrate, which places fluorescent donor
(Eu.sup.3+ crypate) in close proximity to the accepter
(Streptavidin-XL665), thus resulting in a high degree of
fluorescence resonance energy transfer from the donor fluorophore
(at 620 nm) to the acceptor fluorophore (at 665 nm). Inhibition of
Btk kinase activity resulted in decrease of the TR-FRET signal. The
IC.sub.50 for each compound was derived from fitting the data to
the four-parameter logistic equation by Graphpad Prism
software.
[0407] Biochemical Kinase Selectivity
[0408] IC.sub.50 determination of Tec: The protocol of Tec assay is
similar to Btk assay except for the following modifications: 1) 280
.mu.M ATP and 0.083 ug/ml Poly-GT substrate were used in the kinase
reaction; 2) the reaction buffer doesn't contain SEB.
[0409] IC.sub.50 determination of EGFR: The protocol of EGFR assay
is similar to Btk assay except for the following modifications: 1)
20 .mu.M ATP, 0.72 .mu.M TK substrate-biotin (one universal
substrate for tyrosine kinases) and serially diluted compound (the
final concentration of 4.76% DMSO) were used in the kinase
reaction; 2) the reaction buffer contains 50 mM HEPES pH7.5, 10 mM
MgCl.sub.2, 1 mM EGTA, 0.01% Brij-35, 2.5 mM DTT and 0.1% BSA; 3)
the stop/detection solution buffer contains 25 mM HEPES pH7.38, 400
mM KF, 50 mM EDTA, 0.01% Triton-X100 and 0.1% BSA.
[0410] Btk pY223 Cellular Assay
[0411] Btk pY223 cellular assay is an HTRF based assay intended to
determine the endogenous levels of phosphorylated Btk at Tyr223.
Auto-phosphorylation of Tyr223 is necessary for full activation of
Btk. The assay is performed using Ramos cells (CRL-1596, ATCC) with
a Btk pY223 assay kit (63ADKPEG, Cisbio).
[0412] Briefly, Ramos cells were serum starved in 0.5%
FBS-containing RPMI1640 overnight. The cells were then incubated
with interested compounds at various concentrations in a CO.sub.2
incubator for 3 hours. After the incubation, cells were stimulated
with 1 mM pervanadate (PV) or Na.sub.3VO.sub.4 (OV) for 20 min.
Then, the cells were collected and lysed with 1.times. lysis buffer
at RT for 10 min (4.times. lysis buffer supplied in the kit).
1.times. antibody mix was prepared by diluting anti-Btk-d.sub.2 and
anti-pBtk-K in detection buffer (supplied in the kit). 18 .mu.l of
cell lysates was transferred to the OptiPlate-384 assay plate
(6005620, Perkin Elmer) pre-loaded with 2 .mu.l/well of 1.times.
antibody mixture. After mixing gently and spinning briefly, the
plate was sealed up and kept in dark at RT for 18 hours. The
fluorescence emission was measured at two different wavelengths
(665 nm and 620 nm) on a compatible HTRF reader (PHERAstar FS,
BMG). The potency of compounds was calculated based on the
inhibition of ratio between signal intensities at 665 nm and 620
nm. IC.sub.50 values were calculated with GraphPad Prism software
using the sigmoidal dose-response function.
[0413] Effects of Compounds on Tumor Cell Proliferation in Rec-1
Haematological Cancer Line
[0414] MCL cell line (Rec-1) was used in this study. Cells were
reinstated from frozen stocks that were laid down within 30
passages from the original purchase. Cells were maintained in
RPMI-1640 supplemented with 10% fetal bovine serum/FB S (Thermo
Scientific); 100 units/ml penicillin (Gibco) and 0.1 mg/ml
streptomycin (Gibco) at 37.degree. C. in a humidified atmosphere of
5% CO.sub.2.
[0415] The growth-inhibitory activity of compounds in Rec-1 cells
was determined using CellTiter-Glo luminescent cell viability assay
(Promega). The number of cells seeded per well of a 96-well plate
was optimized to ensure logarithmic growth over 6 days treatment
period. Cells were treated in triplicate with a 10-point dilution
series. Following a 6-day exposure to the compound, 100 ul of cell
media were removed and 30 ul of CellTiter-Glo reagent was added
into the cell culture. Mixture was agitated on an orbital shaker
for 2 minutes to ensure cell lysis, followed by 10 minutes
incubation at RT to allow development and stabilization of
luminescent signals, which corresponded to quantity of ATP and thus
the quantity of metabolically active cells. Luminescent signals
were measured using PHERAstar FS reader (BMG). Mean IC.sub.50
values for cell viability were determined with GraphPad Prism
software.
[0416] Cellular Btk Occupation Assay
[0417] Cellular Btk occupation assay is an ELISA based assay
intended to determine the occupation of inhibitor to endogenous
Btk. Occupied Btk protein loses its kinase activity and the
occupation prevents other inhibitors from binding to the kinase
site. The assay was performed using Z-138 cells (CRL-3001, ATCC).
The detection probe is developed in-house.
[0418] Briefly, Z-138 cells (1.times.10.sup.6 cells/well) were
treated with increasing concentrations of each compound for 3
hours, washed, lysed, and loaded to ELISA plate pre-immobilized
with the detection probe. After overnight incubation, the plate was
washed with PBST for 3 times. Probe conjugated Btk protein was
detected by a Btk antibody (611116, BD) followed by a secondary
antibody (w4021, Promega). The potency of compounds was calculated
based on the inhibition of ratio between signal intensities at
OD450 nm and OD550 nm. IC50 values were calculated with GraphPad
Prism software using the sigmoidal dose-response function.
[0419] Representative compounds as disclosed herein were tested and
found to inhibit kinase Btk, autophosphorylation of Btk at Tyr-223,
proliferation in Rec-1 cell line and cellular Btk occupation assay
with IC.sub.50 values ranging from subnanomolar to 10
micromolar.
TABLE-US-00008 TABLE II Assay data for representative compounds
IC50 (nM) IC50 (nM) in cell Fold of Fold of Btk Compound No. Btk
EGFR EGFR/Btk Tec Tec/Btk pY223 A1 1.2 3326 2772 253 210 60 A1a
(Faster isomer) 1.0 687 687 172 172 17.2 A1b >100 ND ND ND ND ND
A2 1.3 >1000 ND 180 138 28 A2a (Faster isomer) 0.7 450 643 83
119 7.0 A2b (Slower isomer) 94.3 ND ND ND ND ND A3 1.5 >1000 ND
134 89 29.1 A4 0.97 >1000 ND 50 52 20 A5 1.2 >1000 ND 145 121
21.3 A6 1.1 1999 1817 359 326 33.5 A6a (Faster isomer) 0.57 1092
1915 98 172 10.8 A6b 41 ND ND ND ND ND A7 4.1 1186 289 254 62 148.2
A8 0.93 1382 1486 130 140 14.8 A9 41 ND ND ND ND ND A10 51 ND ND ND
ND ND A11 1.9 1846 972 271 143 39 A11a 108 ND ND ND ND ND A11b 1.1
ND ND ND ND ND A12 4.7 >10000 ND 289 61 37.3 A13 >100 ND ND
ND ND ND A14 15 ND ND >1000 ND ND A15 20 ND ND 1002 50 ND A16
7.1 2367 333 556 78 ND A17 2.0 713 356 189 94.5 136.2 A18 2.0 603
302 297 149 74.8 A19 7.2 >10000 ND 455 63.2 ND A20 3.3 496 150
352 107 142.6 A21 21 ND ND ND ND ND A22 2.3 1760 765 551 240 54.2
A23 1.2 656 547 107 89 28.2 A24 28 ND ND ND ND ND A25 23 ND ND
>1000 ND ND A26 41 ND ND ND ND ND A27 18 ND ND >1000 ND ND
A28 16 ND ND 369 23 ND A29 16 ND ND >1000 ND ND A30 53 ND ND
>1000 ND ND A31 >100 ND ND ND ND ND A32 52 ND ND ND ND ND A33
2.7 9394 3479 499 185 42.9 A34 1.8 405 225 207 115 41.8 A35 2.3
1149 500 174 75.6 65 A36 1.1 2573 2339 137 125 50.5 A37 1.5 4496
2997 50 33 49.4 A38 2.8 2249 803 264 94 36.7 A39 6.8 6082 894 102
15 ND A40 11 ND ND 253 23 ND A41 3.3 1513 458 101 31 66.8 A42 3.0
2005 668 53 18 ND A43 6.8 6181 909 39 5.7 ND A44 4.9 3171 647 35
7.1 ND A45 4.4 >10000 ND 362 82 ND A46 5.9 3813 646 974 165
257.5 A47 17 ND ND ND ND ND A48 16 ND ND ND ND ND A49 18 ND ND ND
ND ND A50 0.34 599 1762 3.2 9.4 12.3 A51 ND ND ND 136 ND ND A52 1.3
ND ND ND ND ND A52a 0.7 ND ND ND ND ND A52b 39.9 ND ND ND ND ND B1
13 ND ND ND ND ND B2 5.0 1752 350 172 34 45.7 B3 5.9 1913 324 157
27 93.1 C1 3.4 536 158 380 112 49.6 C2 34 ND ND ND ND ND D1 10 ND
ND ND ND ND D2 63 ND ND >1000 ND ND E1 33 ND ND ND ND ND F1
>100 ND ND ND ND ND G1 12 ND ND ND ND ND H1 35 ND ND ND ND ND
167.sup.# 2.4 9.5 4 ND ND ND 167* (faster isomer) 1.0 14 14 157 157
83.5 167.sup.$ (slower isomer) >100 ND ND ND ND ND 174 4.8 281
58.5 64 13 ND ##STR00178## 22 ND ND ND ND ND 144 140 ND ND 491 3.5
ND 158 2.9 0.49 0.2 56 19 ND 169.sup.# 0.15 0.54 3.6 ND ND ND 169*
(faster isomer) 0.46 1.0 2.2 37 80 28.2 169.sup.$(slower isomer)
>100 ND ND ND ND ND ND-no data
TABLE-US-00009 TABLE III ##STR00179## ##STR00180## ##STR00181##
##STR00182## ##STR00183## ##STR00184## ##STR00185## ##STR00186##
##STR00187## ##STR00188## ##STR00189## ##STR00190## ##STR00191##
##STR00192## ##STR00193##
[0420] The examples of the present invention and compounds
disclosed in WO2014173289A1 were tested in in biochemical assay,
cellular assay, pharmacodynamic evaluation in mice and
pharmacokinetic evaluation in rat. The results are shown in Tables
II, IV and FIG. 1A to 2B. Table II surprisingly demonstrates these
compounds having high affinity for Btk and comparatively less
affinity for both Tec and EGFR (>100 fold selectivity), for
example compounds A1a, A2a, A5, A6a, A8, A11, A18, A20, A22, A33,
A34, A36, A46 and C1, most of which have been found to have a
specific combination of a tri-substituted phenyl ring and a
piperidine ring (particularly N-substituted acryloyl piperidine
ring) in the molecule.
[0421] To the contrast, Compounds 167.sup.# or 167* and 169.sup.#
or 169*, disclosed in WO2014173289A1, also bears a tri-substituted
phenyl ring (with substituent R.sup.1 is methoxy) in combination
with another phenyl ring on the right side. These compounds do
exhibit high or comparable inhibitory activity against Btk, and
exhibit good selectivity of Btk over Tec. However, they showed poor
selectivity of Btk against EGFR, lower cellular potency,
pharmacodynamic property and oral bioavailability. Compound 174 in
WO2014173289A1 having a combination of di-substituents on the top
phenyl ring and a piperidine ring on the right side showed low
affinity for Btk and almost no selectivity against Tec.
[0422] Starting from Compounds 167 and 169 in WO2014173289A1, the
inventors also prepared two analogs A14 and A15 which have a
different combination of a tri-substituted phenyl ring and a
piperidine ring in the molecule. However, both compounds showed
poor affinity for Btk as shown in Table II of the present
specification.
[0423] In Vivo Mice PD Assay
[0424] ICR mice were randomly assigned into different groups with 4
mice per group. Mice were treated with different dose levels of
compounds as indicated and euthanized using carbon dioxide at 4 hrs
after dosing. Treatments were administered by oral gavage (p.o.).
For peripheral blood mononuclear cell (PBMC) and spleen
preparation, mouse spleens were collected from euthanized mice and
grinded in protein lysis buffer. Mouse blood was lysed with protein
lysis buffer. Samples were centrifuged at 4.degree. C. for 15
minutes. Supernatant was transferred to a fresh tube and protein
concentration was determined using BCA protein analysis. Ninety-six
well plates was coated with neutravidin and incubated with a
biotinylated probe. Cell lysates or recombinant human Btk protein
samples were added and the plates were incubated at 4.degree. C.
overnight. Plates were washed with PBST and blocked with blocking
buffer for 1 hr. Primary anti-Btk rabbit polyclonal antibody was
added and incubated for 2 hrs and secondary anti-rabbit-HRP
antibody was added and incubated for 1 hr. Following standard ELISA
protocols using TMB as substrate, OD450 nm was read using
microplate reader. The amount of unoccupied Btk proteins in PBMC or
spleen lysates was calculated based on the standard curve and
presented as percentage of that of vehicle-treated animals.
[0425] Result
[0426] The PD activity of compounds A1a, A2a, A6a, 167* and 169*
was assessed by Btk occupancy assay compared to compound 176, which
is designed based on direct quantification of the covalent bonding
to Btk protein after drug exposure. As shown in FIGS. 1A and 1B,
A1a and A2a were more potent than 176 in both of PBMC and spleen.
A6a showed similar activity with that of 176. However, 167* and
169* were 2.about.3 fold less potent when compared with 176 in both
of PBMC and spleen (FIGS. 2A and 2B).
[0427] Rat Pharmacokinetic Study
[0428] 8 weeks old Male Sprague Dawley rats with jugular vein
catheter were purchased from SKILLS Model Animal Research
Technology Co., Ltd. (Beijing, China). All procedures involving
animals were conducted in accordance with the Institutional Animal
Care and Use Committee (IACUC) of BeiGene. Compound was dissolved
in 32% DMA, 36% ethanol, and 32% propylene glycol for intravenous
(IV) injection, and was suspended in 0.5% (w/v) methylcellulose
(MC) solution for oral gavage (PO). The rats were fasted overnight
prior to the treatment until 4 hours post-dose, and divided into
two groups for 3 animals in each group. The first group was given a
single IV injection of compound at 1 mgkg.sup.-1 via tail vein.
Blood samples (.about.0.15 mL) were collected at Pre-dose, 5, 15,
30 min, 1, 2, 4, 8, and 24 h post-dose via jugular vein cannula
into EDTA-coagulated tubes. The second group was given a single PO
administration at 5 mgkg.sup.-1, respectively. Blood samples were
collected at Pre-dose, 15, 30 min, 1, 2, 4, 8, and 24 h post-dose.
All whole blood was processed for plasma by centrifugation at 3000
g for 10 min. Plasma samples were collected and kept below
-30.degree. C. The concentration of compound in plasma was
determined by LC-MS/MS method.
[0429] Result
[0430] In the rat PK study as shown in Table IV, A1a, A2a and A11b
exhibited lower plasma clearance values, higher Cmax, AUC and oral
bioavailability compared to compound 167*.
TABLE-US-00010 TABLE IV Rat PK results Cl Cmax AUC.sub.0-inf
Compound (mL/min/kg) (ng/mL) (h ng mL.sup.-1) F (%) A1a (Faster
24.4 667 964 26.4 isomer) A11b 13.7 2250 4799 69.5 A2a (Faster 42.8
859 1710 73.7 isomer) 167* 151.6 60.4 65.1 11.8
* * * * *