U.S. patent application number 16/902490 was filed with the patent office on 2020-12-24 for tetrazole-substituted pyrazolopyrimidine inhibitors of jak kinases and uses thereof.
This patent application is currently assigned to Genentech, Inc.. The applicant listed for this patent is Genentech, Inc.. Invention is credited to Marian C. Bryan, Yun-Xing Cheng, Jessica Grandner, Naomi S. Rajapaksa, F. Anthony Romero, Daniel G.M. Shore, Mark Edward Zak.
Application Number | 20200399274 16/902490 |
Document ID | / |
Family ID | 1000004925632 |
Filed Date | 2020-12-24 |
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United States Patent
Application |
20200399274 |
Kind Code |
A1 |
Zak; Mark Edward ; et
al. |
December 24, 2020 |
TETRAZOLE-SUBSTITUTED PYRAZOLOPYRIMIDINE INHIBITORS OF JAK KINASES
AND USES THEREOF
Abstract
Compounds of formula (I) ##STR00001## wherein R.sup.1, R.sup.2,
R.sup.3, R.sup.4, R.sup.5 and R.sup.6 are as defined herein, and
salts thereof, that are useful as JAK kinase inhibitors are
described herein. Also provided are pharmaceutical compositions
that include such a JAK inhibitor and a pharmaceutically acceptable
carrier, adjuvant or vehicle, and methods of treating or lessening
the severity of a disease or condition responsive to the inhibition
of a Janus kinase activity in a patient.
Inventors: |
Zak; Mark Edward; (South San
Francisco, CA) ; Rajapaksa; Naomi S.; (South San
Francisco, CA) ; Cheng; Yun-Xing; (Beijing, CN)
; Grandner; Jessica; (South San Francisco, CA) ;
Shore; Daniel G.M.; (Redwood City, CA) ; Romero; F.
Anthony; (Redwood City, CA) ; Bryan; Marian C.;
(Fort Washington, PA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Genentech, Inc. |
South San Francisco |
CA |
US |
|
|
Assignee: |
Genentech, Inc.
South San Francisco
CA
|
Family ID: |
1000004925632 |
Appl. No.: |
16/902490 |
Filed: |
June 16, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
63036046 |
Jun 8, 2020 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 37/06 20180101;
C07D 487/04 20130101 |
International
Class: |
C07D 487/04 20060101
C07D487/04; A61P 37/06 20060101 A61P037/06 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 18, 2019 |
CN |
PCT/CN2019/091709 |
Claims
1. A compound of Formula (I) ##STR00181## or a stereoismer or
pharmaceutically acceptable salt thereof, wherein: R.sup.1 is:
hydroxyl-C.sub.1-C.sub.6alkyl;
--(CR.sup.a1R.sup.a2).sub.m-het.sup.1;
--(CR.sup.a1R.sup.a2).sub.nNR.sup.bR.sup.c; or
--(CR.sup.a1R.sup.a2).sub.m--C.sub.3-6 cycloalkyl wherein the
C.sub.3-6cycloalkyl moiety is substituted once with R.sup.d;
R.sup.2 is: halo; halo C.sub.1-C.sub.6alkoxy;
C.sub.1-C.sub.6alkylthio; --SF.sub.2; or C.sub.3-C.sub.6cycloalkyl;
R.sup.3 is: hydrogen; or C.sub.1-C.sub.6alkyl; R.sup.4 is:
hydrogen; or C.sub.1-C.sub.6alkyl; R.sup.5 is: hydrogen; or
C.sub.1-C.sub.6alkyl; R.sup.6 is: hydrogen; or
C.sub.1-C.sub.6alkyl; or R.sup.2 and R.sup.6 together with the
atoms to which they are attached may form a six-membered ring
containing two heteroatoms each independently selected from O, N
and S; m is from 0 to 2; n is from 0 to 3; each R.sup.a1 is
independently: hydrogen; or C.sub.1-C.sub.6alkyl; each R.sup.a2 is
independently: hydrogen; halo; or C.sub.1-C.sub.6alkyl; R.sup.b is:
hydrogen; or C.sub.1-C.sub.6alkyl; R.sup.c is: hydrogen;
C.sub.1-C.sub.6alkyl; an amino protecting group; or azetidinyl
which may be unsubstituted or substituted once with
C.sub.1-C.sub.6alkyl; het.sup.1 is a heterocyclyl selected from:
azetidinyl; pyrrolidinyl; piperazinyl; piperidinyl; morpholinyl;
and oxetanyl; each of which may be unsubstituted or substituted
once with R.sup.d and once or twice with R.sup.g; R.sup.d is:
--(CR.sup.a1R.sup.a2).sub.p-het.sup.2;
--(CR.sup.a1R.sup.a2).sub.q--NR.sup.eR.sup.f; or
--(CR.sup.a1R.sup.a2).sub.p--C.sub.3-6cycloalkyl wherein the
C.sub.3-6cycloalkyl moiety is substituted once with
--NR.sup.eR.sup.f; p is from 0 to 2; q is from 0 to 4; R.sup.e is:
hydrogen; or C.sub.1-C.sub.6alkyl; R.sup.f is: hydrogen;
C.sub.1-C.sub.6alkyl; or --CH.sub.2C.sub.2N(CH.sub.3).sub.2; each
R.sup.g is: C.sub.1-C.sub.6alkyl; or halo; and Het.sup.2 is a
heterocycle selected from: tetrahydropyranyl; azetidinyl; and
pyrrolidinyl; each of which may be unsubstituted or substituted
once with C.sub.1-C.sub.6alkyl or --NR.sup.eR.sup.f.
2. The compound of claim 1 or a stereoisomer or pharmaceutically
acceptable salt thereof, wherein R.sup.1 is
--(CHR.sup.a).sub.m-het.sup.1; or
--(CHR.sup.a).sub.n--NR.sup.bR.sup.c, wherein the het.sup.1 may be
unsubstituted or substituted once with R.sup.d.
3. The compound of claim 1 or a stereoisomer or pharmaceutically
acceptable salt thereof, wherein R.sup.2 is: chloro;
difluoromethoxy; methylethio; or cyclopropyl.
4. The compound of claim 1 or a stereoisomer or pharmaceutically
acceptable salt thereof, wherein R.sup.3, R.sup.4, R.sup.5, R.sup.6
and R.sup.a are hydrogen.
5. The compound of claim 1 or a stereoisomer or pharmaceutically
acceptable salt thereof, wherein R.sup.2 and R.sup.6 together with
the atoms to which they are attached form a six-membered ring
containing two heteroatoms each independently selected from O, N
and S.
6. The compound of claim 1 or a stereoisomer or pharmaceutically
acceptable salt thereof, wherein het.sup.1 is azetidinyl, which may
be unsubstituted or substituted once with R.sup.d.
7. The compound of claim 1, wherein R is selected from:
##STR00182## ##STR00183## ##STR00184## or a stereoisomer or
pharmaceutically acceptable salt thereof.
8. The compound of claim 1, wherein the compound is of formula (II)
##STR00185## or a stereoisomer or pharmaceutically acceptable salt
thereof.
9. The compound of claim 1, wherein the compound is of formula (IV)
##STR00186## or a stereoisomer or pharmaceutically acceptable salt
thereof, wherein X is --O-- or --S--, and R.sup.g is hydrogen or
C.sub.1-C.sub.6alkyl.
10. A method of preventing, treating or lessening the severity of a
disease or condition responsive to the inhibition of a Janus kinase
activity in a patient, comprising administering to the patient a
therapeutically effective amount of a compound of claim 1 or a
stereoisomer or pharmaceutically acceptable salt thereof.
11. The method of claim 10, wherein the disease or condition is
cancer, stroke, diabetes, hepatomegaly, cardiovascular disease,
multiple sclerosis, Alzheimer's disease, cystic fibrosis, viral
disease, autoimmune diseases, atherosclerosis, restenosis,
psoriasis, rheumatoid arthritis, inflammatory bowel disease,
asthma, allergic disorders, inflammation, neurological disorders, a
hormone-related disease, conditions associated with organ
transplantation (e.g., transplant rejection), immunodeficiency
disorders, destructive bone disorders, proliferative disorders,
infectious diseases, conditions associated with cell death,
thrombin-induced platelet aggregation, liver disease, pathologic
immune conditions involving T cell activation, CNS disorders or a
myeloproliferative disorder.
12. A pharmaceutical composition comprising a compound of claim 1
or a stereoisomer or pharmaceutically acceptable salt thereof,
wherein the pharmaceutical composition comprises microparticles of
the compound suitable for inhaled delivery.
13. The pharmaceutical composition of claim 12, wherein the
microparticles are prepared by spray-drying, freeze-drying or
micronisation.
14. A kit comprising: (a) a first pharmaceutical composition
comprising a compound of claim 1 or a stereoisomer or
pharmaceutically acceptable salt thereof; and (b) instructions for
use.
15. The kit of claim 14, further comprising a second pharmaceutical
composition comprising an agent for treatment of an inflammatory
disorder, or a chemotherapeutic agent.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to International
Application No. PCT/CN2019/091709 filed on Jun. 18, 2019, and U.S.
Provisional Application No. 63/036,046 filed on Jun. 8, 2020, the
disclosures of which are incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The invention relates to compounds that are inhibitors of a
Janus kinase, such as JAK1 and JAK2, as well as compositions
containing these compounds, and methods of use including, but not
limited to, diagnosis or treatment of patients suffering from a
condition responsive to the inhibition of a JAK kinase.
BACKGROUND OF INVENTION
[0003] Cytokine pathways mediate a broad range of biological
functions, including many aspects of inflammation and immunity.
Janus kinases (JAK), including JAK1, JAK2, JAK3 and TYK2, are
cytoplasmic protein kinases that associate with type I and type II
cytokine receptors and regulate cytokine signal transduction.
Cytokine engagement with cognate receptors triggers activation of
receptor associated JAKs and this leads to JAK-mediated tyrosine
phosphorylation of signal transducer and activator of transcription
(STAT) proteins and ultimately transcriptional activation of
specific gene sets (Schindler et al., 2007, J. Biol. Chem. 282:
20059-63). JAK1, JAK2 and TYK2 exhibit broad patterns of gene
expression, while JAK3 expression is limited to leukocytes.
Cytokine receptors are typically functional as heterodimers, and as
a result, more than one type of JAK kinase is usually associated
with cytokine receptor complexes. The specific JAKs associated with
different cytokine receptor complexes have been determined in many
cases through genetic studies and corroborated by other
experimental evidence. Exemplary therapeutic benefits of the
inhibition of JAK enzymes are discussed, for example, in
International Application No. WO 2013/014567.
[0004] JAK1 was initially identified in a screen for novel kinases
(Wilks A. F., 1989, Proc. Natl. Acad. Sci. U.S.A. 86:1603-1607).
Genetic and biochemical studies have shown that JAK1 is
functionally and physically associated with the type I interferon
(e.g., IFNalpha), type II interferon (e.g., IFNgamma), and IL-2 and
IL-6 cytokine receptor complexes (Kisseleva et al., 2002, Gene
285:1-24; Levy et al., 2005, Nat. Rev. Mol. Cell Biol. 3:651-662;
O'Shea et al., 2002, Cell, 109 (suppl.): S121-S131). JAK1 knockout
mice die perinatally due to defects in LIF receptor signaling
(Kisseleva et al., 2002, Gene 285:1-24; O'Shea et al., 2002, Cell,
109 (suppl.): 5121-S131). Characterization of tissues derived from
JAK1 knockout mice demonstrated critical roles for this kinase in
the IFN, IL-10, IL-2/IL-4 and IL-6 pathways. A humanized monoclonal
antibody targeting the IL-6 pathway (Tocilizumab) was approved by
the European Commission for the treatment of moderate-to-severe
rheumatoid arthritis (Scheinecker et al., 2009, Nat. Rev. Drug
Discov. 8:273-274).
[0005] CD4 T cells play an important role in asthma pathogenesis
through the production of TH2 cytokines within the lung, including
IL-4, IL-9 and IL-13 (Cohn et al., 2004, Annu. Rev. Immunol.
22:789-815). IL-4 and IL-13 induce increased mucus production,
recruitment of eosinophils to the lung, and increased production of
IgE (Kasaian et al., 2008, Biochem. Pharmacol. 76(2): 147-155).
IL-9 leads to mast cell activation, which exacerbates the asthma
symptoms (Kearley et al., 2011, Am. J. Resp. Crit. Care Med.,
183(7): 865-875). The IL-4R.alpha. chain activates JAK1 and binds
to either IL-4 or IL-13 when combined with the common gamma chain
or the IL-13R.alpha.1 chain respectively (Pernis et al., 2002, J.
Clin. Invest. 109(10):1279-1283). The common gamma chain can also
combine with IL-9R.alpha. to bind to IL-9, and IL-9R.alpha.
activates JAK1 as well (Demoulin et al., 1996, Mol. Cell Biol.
16(9):4710-4716). While the common gamma chain activates JAK3, it
has been shown that JAK1 is dominant over JAK3, and inhibition of
JAK1 is sufficient to inactivate signaling through the common gamma
chain despite JAK3 activity (Haan et al., 2011, Chem. Biol.
18(3):314-323). Inhibition of IL-4, IL-13 and IL-9 signaling by
blocking the JAK/STAT signaling pathway can alleviate asthmatic
symptoms in pre-clinical lung inflammation models (Mathew et al.,
2001, J. Exp. Med. 193(9): 1087-1096; Kudlacz et. al., 2008, Eur.
J. Pharmacol. 582(1-3): 154-161).
[0006] Biochemical and genetic studies have shown an association
between JAK2 and single-chain (e.g., EPO), IL-3 and interferon
gamma cytokine receptor families (Kisseleva et al., 2002, Gene
285:1-24; Levy et al., 2005, Nat. Rev. Mol. Cell Biol. 3:651-662;
O'Shea et al., 2002, Cell, 109 (suppl.): S121-S131). Consistent
with this, JAK2 knockout mice die of anemia (O'Shea et al., 2002,
Cell, 109 (suppl.): S121-S131). Kinase activating mutations in JAK2
(e.g., JAK2 V617F) are associated with myeloproliferative disorders
in humans. Additionally, JAK2 associates with the receptors for
cytokines such as IL-5 and Thymic stromal lymphopoietin (TSLP).
IL-5 is the key cytokine responsible for eosinophil
differentiation, growth, activation, survival, and recruitment to
airways (Pelaia et al., 2019, Front. Physiol., 10: 1514; Stirling
et al., 2001, Am. J. Respir. Crit. Care Med., 164: 1403-9;
Fulkerson and Rothenberg, 2013, Nat. Rev. Drug Discov., 12: 117-9;
Varricchi and Canonica, 2016, Expert. Rev. Clin. Immunol., 12:
903-5). Three monoclonal antibody drugs targeting either IL-5
(Mepolizumab, Reslizumab) or the alpha chain of its receptor
(Benralizumab) have been approved as treatments for asthma with an
eosinophilic phenotype. TSLP is an epithelial-cell-derived cytokine
that plays an important role in the regulation of type II immunity
and serves as an alarmin upstream of TH2 cytokine production
(Kitajima et al., 2011, Eur J Immunol., 41: 1862-71). Tezepelumab
is an antagonist antibody to TSLP. Results from a phase 2 trial
indicate it successfully reduced asthma exacerbations in patients
both with and without Type 2-high signatures (Corren et al., 2017,
377: 936-46).
[0007] JAK3 associates exclusively with the gamma common cytokine
receptor chain, which is present in the IL-2, IL-4, IL-7, IL-9,
IL-15 and IL-21 cytokine receptor complexes. JAK3 is critical for
lymphoid cell development and proliferation and mutations in JAK3
result in severe combined immunodeficiency (SCID) (O'Shea et al.,
2002, Cell, 109 (suppl.): S121-S131). Based on its role in
regulating lymphocytes, JAK3 and JAK3-mediated pathways have been
targeted for immunosuppressive indications (e.g., transplantation
rejection and rheumatoid arthritis) (Baslund et al., 2005,
Arthritis & Rheumatism 52:2686-2692; Changelian et al., 2003,
Science 302: 875-878).
[0008] TYK2 associates with the type I interferon (e.g., IFNalpha),
IL-6, IL-10, IL-12 and IL-23 cytokine receptor complexes (Kisseleva
et al., 2002, Gene 285:1-24; Watford, W. T. & O'Shea, J. J.,
2006, Immunity 25:695-697). Consistent with this, primary cells
derived from a TYK2 deficient human are defective in type I
interferon, IL-6, IL-10, IL-12 and IL-23 signaling. A fully human
monoclonal antibody targeting the shared p40 subunit of the IL-12
and IL-23 cytokines (Ustekinumab) was recently approved by the
European Commission for the treatment of moderate-to-severe plaque
psoriasis (Krueger et al., 2007, N. Engl. J. Med. 356:580-92; Reich
et al., 2009, Nat. Rev. Drug Discov. 8:355-356). In addition, an
antibody targeting the IL-12 and IL-23 pathways underwent clinical
trials for treating Crohn's Disease (Mannon et al., 2004, N. Engl.
J. Med. 351:2069-79).
[0009] International Patent Application Publication Numbers WO
2010/051549, WO 2011/003065, WO 2015/177326 and WO 2017/089390
discuss certain pyrazolopyrimidine compounds that are reported to
useful as inhibitors of one or more Janus kinases. Data for certain
specific compounds showing inhibition of JAK1 as well as JAK2,
JAK3, and/or TYK2 kinases is presented therein.
[0010] Currently there remains a need for additional compounds that
are inhibitors of Janus kinases. For example, there is a need for
compounds that possess useful potency as inhibitors of one or more
Janus kinases (e.g., JAK1 and JAK2)--in combination with other
pharmacological properties that are necessary to achieve a useful
therapeutic benefit. For example, there is a need for potent
compounds that demonstrate selectivity for one Janus kinase over
other kinases in general (e.g., selectivity for JAK1 and/or JAK2
over other kinases such as leucine-rich repeat kinase 2 (LRRK2)).
There is also a need for potent compounds that demonstrate
selectivity for one Janus kinase over other Janus kinases (e.g.,
selectivity for JAK1 and/or JAK2 over JAK3 and/or TYK2). Compounds
demonstrating selectivity for both JAK1 and JAK2 over JAK3 and TYK2
could provide a therapeutic benefit, in conditions responsive to
the inhibition of JAK1. Additionally there is currently a need for
potent JAK1 inhibitors that possess other properties (e.g., melting
point, pK, solubility, etc.) necessary for formulation and
administration by inhalation. Such compounds would be particularly
useful for treating conditions such as, for example, asthma.
[0011] There accordingly exists a need in the art for additional or
alternative treatments of conditions mediated by JAK kinases, such
as those described above. There is in particular a need for JAK1
and JAK2 kinase inhibitors usable for inhaled delivery in the
treatment of airway inflammation indications such as asthma.
SUMMARY OF THE INVENTION
[0012] Provided herein are pyrazolopyrimidines that inhibit JAK
kinase, such as selected from a compound of Formula (I) a
stereoisomer or salt thereof, such as a pharmaceutically acceptable
salt thereof. The JAK kinase may be JAK1, JAK2, or both.
[0013] One embodiment provides a compound of Formula (I):
##STR00002##
or a stereoisomer or a pharmaceutically acceptable salt thereof,
wherein:
[0014] R.sup.1 is: hydroxyl-C.sub.1-C.sub.6alkyl;
--(CR.sup.a1R.sup.a2).sub.m-het.sup.1;
--(CR.sup.a1R.sup.a2).sub.n--NR.sup.bR.sup.c; or
--(CR.sup.a1R.sup.a2).sub.m--C.sub.3-6 cycloalkyl wherein the
C.sub.3-6cycloalkyl moiety is substituted once with R.sup.d;
[0015] R.sup.2 is: halo; halo C.sub.1-C.sub.6alkoxy;
C.sub.1-C.sub.6alkylthio; --SF.sub.2; or
C.sub.3-C.sub.6cycloalkyl;
[0016] R.sup.3 is: hydrogen; or C.sub.1-C.sub.6alkyl;
[0017] R.sup.4 is: hydrogen; or C.sub.1-C.sub.6alkyl;
[0018] R.sup.5 is: hydrogen; or C.sub.1-C.sub.6alkyl;
[0019] R.sup.6 is: hydrogen; or C.sub.1-C.sub.6alkyl;
[0020] or R.sup.2 and R.sup.6 together with the atoms to which they
are attached may form a six-membered ring containing two
heteroatoms each independently selected from O, N and S;
[0021] m is from 0 to 2;
[0022] n is from 0 to 3;
[0023] each R.sup.a1 is independently: hydrogen; or
C.sub.1-C.sub.6alkyl;
[0024] each R.sup.a2 is independently: hydrogen; halo; or
C.sub.1-C.sub.6alkyl;
[0025] R.sup.b is: hydrogen; or C.sub.1-C.sub.6alkyl;
[0026] R.sup.c is: hydrogen; C.sub.1-C.sub.6alkyl; an amino
protecting group; or azetidinyl which may be unsubstituted or
substituted once with C.sub.1-C.sub.6alkyl;
[0027] het.sup.1 is a heterocyclyl selected from: azetidinyl;
pyrrolidinyl; piperazinyl; piperidinyl; morpholinyl; and oxetanyl;
each of which may be unsubstituted or substituted once with R.sup.d
and once or twice with R.sup.g;
[0028] R.sup.d is: --(CR.sup.a1R.sup.a2).sub.p-het.sup.2;
--(CR.sup.a1R.sup.a2).sub.q--NR.sup.eR.sup.f; or
--(CR.sup.a1R.sup.a2).sub.p--C.sub.3-6cycloalkyl wherein the
C.sub.3-6cycloalkyl moiety is substituted once with
--NR.sup.eR.sup.f;
[0029] p is from 0 to 2;
[0030] q is from 0 to 4;
[0031] R.sup.e is: hydrogen; or C.sub.1-C.sub.6alkyl;
[0032] R.sup.f is: hydrogen; C.sub.1-C.sub.6alkyl; or
--CH.sub.2C.sub.2N(CH.sub.3).sub.2; [0033] each R.sup.g is:
C.sub.1-C.sub.6alkyl; or halo; and
[0034] Het.sup.2 is a heterocycle selected from: tetrahydropyranyl;
azetidinyl; and pyrrolidinyl; each of which may be unsubstituted or
substituted once with C.sub.1-C.sub.6alkyl or
--NR.sup.eR.sup.f.
[0035] In certain embodiments R.sup.1 is: C.sub.1-C.sub.6alkyl;
hydroxyl-C.sub.1-C.sub.6alkyl;
--(CR.sup.a1R.sup.a2).sub.m-het.sup.1; or
(CR.sup.a1R.sup.a2).sub.n--NR.sup.bR.sup.c, wherein het.sup.1 may
be unsubstituted or substituted once with R.sup.d.
[0036] Also provided is a pharmaceutical composition comprising a
JAK inhibitor as described herein, or a pharmaceutically acceptable
salt thereof, and a pharmaceutically acceptable carrier, dilient or
excipient.
[0037] Also provided is the use of a JAK inhibitor as described
herein, or a pharmaceutically acceptable salt thereof in therapy,
such as in the treatment of an inflammatory disease (e.g., asthma).
Also provided is the use of a JAK inhibitor as described herein or
a pharmaceutically acceptable salt thereof for the preparation of a
medicament for the treatment of an inflammatory disease. Also
provided is a method of preventing, treating or lessening the
severity of a disease or condition responsive to the inhibition of
a Janus kinase activity in a patient, comprising administering to
the patient a therapeutically effective amount of a JAK inhibitor
as described herein or a pharmaceutically acceptable salt
thereof.
[0038] The most validated cytokines in asthma (IL-4, IL-5, IL-9,
IL-13, and TSLP) all signal through JAK1 and/or JAK2. The compounds
of the invention are active for both JAK1 and JAK2. Certain of
these compounds optimally have well-balanced co-activity for both
JAK1 and JAK2, or have slightly higher affinity for JAK1 over JAK2,
rather than having a much greater activity for one of these kinases
over the other. The subject compounds also have good selectivity
against off-target kinases such as LRRK2, which has been associated
with pulmonary toxicity.
[0039] While many compounds may exhibit high affinity for both JAK1
and JAK2 in simple biochemical assays, not all such compounds are
effective at mediating the relevant cytokines associated with JAK1
and JAK2. Certain compounds of the invention, in addition to being
active for both JAK1 and JAK2, are also shown in cell-based assays
to be effective at mediation of asthma-relevant cytokines
associated with JAK1 and JAK2.
[0040] Compounds of the invention also exhibit favorable
pharmacokinetic (PK) properties in lung tissue and are useful for
inhaled therapies. When dosed via the inhaled route using
techniques such as dry powder inhalation (DPI) or intranasal (IN)
delivery, certain compounds unexpectedly show sustained retention
within the lung tissue, with much lower concentrations in systemic
circulation. Such improved PK properties can advantageously result
in smaller dosages and less frequent dosing requirements for
effective therapies. Certain compounds exhibit unexpected improved
solubility, again providing inproved efficacy in lung. Certain
compounds of the invention also exhibit unexpected reduction in
cytotoxicity in comparison to other JAK inhibitors.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0041] "Halogen" or "halo" refers to F, Cl, Br or I. Additionally,
terms such as "haloalkyl," are meant to include monohaloalkyl and
polyhaloalkyl, wherein one or more halogens replace a hydrogen(s)
of an alkyl group.
[0042] The term "alkyl" refers to a saturated linear or
branched-chain monovalent hydrocarbon radical, wherein the alkyl
radical may be optionally substituted. In one example, the alkyl
radical is one to eighteen carbon atoms (C.sub.1-C.sub.18). In
other examples, the alkyl radical is C.sub.0-C.sub.6,
C.sub.0-C.sub.5, C.sub.0-C.sub.3, C.sub.1-C.sub.12,
C.sub.1-C.sub.10, C.sub.1-C.sub.8, C.sub.1-C.sub.6,
C.sub.1-C.sub.5, C.sub.1-C.sub.4, or C.sub.1-C.sub.3. C.sub.0 alkyl
refers to a bond. Examples of alkyl groups include methyl (Me,
--CH.sub.3), ethyl (Et, --CH.sub.2CH.sub.3), 1-propyl (n-Pr,
n-propyl, --CH.sub.2CH.sub.2CH.sub.3), 2-propyl (i-Pr, i-propyl,
--CH(CH.sub.3).sub.2), 1-butyl (n-Bu, n-butyl,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.3), 2-methyl-1-propyl (i-Bu,
i-butyl, --CH.sub.2CH(CH.sub.3).sub.2), 2-butyl (s-Bu, s-butyl,
--CH(CH.sub.3)CH.sub.2CH.sub.3), 2-methyl-2-propyl (t-Bu, t-butyl,
--C(CH.sub.3).sub.3), 1-pentyl (n-pentyl,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.3), 2-pentyl
(--CH(CH.sub.3)CH.sub.2CH.sub.2CH.sub.3), 3-pentyl
(--CH(CH.sub.2CH.sub.3).sub.2), 2-methyl-2-butyl
(--C(CH.sub.3).sub.2CH.sub.2CH.sub.3), 3-methyl-2-butyl
(--CH(CH.sub.3)CH(CH.sub.3).sub.2), 3-methyl-1-butyl
(--CH.sub.2CH.sub.2CH(CH.sub.3).sub.2), 2-methyl-1-butyl
(--CH.sub.2CH(CH.sub.3)CH.sub.2CH.sub.3), 1-hexyl
(--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.3), 2-hexyl
(--CH(CH.sub.3)CH.sub.2CH.sub.2CH.sub.2CH.sub.3), 3-hexyl
(--CH(CH.sub.2CH.sub.3)(CH.sub.2CH.sub.2CH.sub.3)),
2-methyl-2-pentyl (--C(CH.sub.3).sub.2CH.sub.2CH.sub.2CH.sub.3),
3-methyl-2-pentyl (--CH(CH.sub.3)CH(CH.sub.3)CH.sub.2CH.sub.3),
4-methyl-2-pentyl (--CH(CH.sub.3)CH.sub.2CH(CH.sub.3).sub.2),
3-methyl-3-pentyl (--C(CH.sub.3)(CH.sub.2CH.sub.3).sub.2),
2-methyl-3-pentyl (--CH(CH.sub.2CH.sub.3)CH(CH.sub.3).sub.2),
2,3-dimethyl-2-butyl (--C(CH.sub.3).sub.2CH(CH.sub.3).sub.2),
3,3-dimethyl-2-butyl (--CH(CH.sub.3)C(CH.sub.3).sub.3, 1-heptyl and
1-octyl. In some embodiments, substituents for "optionally
substituted alkyls" include one to four instances of F, Cl, Br, I,
OH, SH, CN, NH.sub.2, NHCH.sub.3, N(CH.sub.3).sub.2, NO.sub.2,
N.sub.3, C(O)CH.sub.3, COOH, CO.sub.2CH.sub.3, methyl, ethyl,
propyl, iso-propyl, butyl, isobutyl, cyclopropyl, methoxy, ethoxy,
propoxy, oxo, trifluoromethyl, difluoromethyl, sulfonylamino,
methanesulfonylamino, SO, SO.sub.2, phenyl, piperidinyl,
piperizinyl, and pyrimidinyl, wherein the alkyl, phenyl and
heterocyclic portions thereof may be optionally substituted, such
as by one to four instances of substituents selected from this same
list.
[0043] The term "alkenyl" refers to linear or branched-chain
monovalent hydrocarbon radical with at least one site of
unsaturation, i.e., a carbon-carbon double bond, wherein the
alkenyl radical may be optionally substituted, and includes
radicals having "cis" and "trans" orientations, or alternatively,
"E" and "Z" orientations. In one example, the alkenyl radical is
two to eighteen carbon atoms (C.sub.2-C.sub.18). In other examples,
the alkenyl radical is C.sub.2-C.sub.12, C.sub.2-C.sub.10,
C.sub.2-C.sub.8, C.sub.2-C.sub.6 or C.sub.2-C.sub.3. Examples
include, but are not limited to, ethenyl or vinyl
(--CH.dbd.CH.sub.2), prop-1-enyl (--CH.dbd.CHCH.sub.3), prop-2-enyl
(--CH.sub.2CH.dbd.CH.sub.2), 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.
In some embodiments, substituents for "optionally substituted
alkenyls" include one to four instances of F, Cl, Br, I, OH, SH,
CN, NH.sub.2, NHCH.sub.3, N(CH.sub.3).sub.2, NO.sub.2, N.sub.3,
C(O)CH.sub.3, COOH, CO.sub.2CH.sub.3, methyl, ethyl, propyl,
iso-propyl, butyl, isobutyl, cyclopropyl, methoxy, ethoxy, propoxy,
oxo, trifluoromethyl, difluoromethyl, sulfonylamino,
methanesulfonylamino, SO, SO.sub.2, phenyl, piperidinyl,
piperizinyl, and pyrimidinyl, wherein the alkyl, phenyl and
heterocyclic portions thereof may be optionally substituted, such
as by one to four instances of substituents selected from this same
list.
[0044] The term "alkynyl" refers to a linear or branched monovalent
hydrocarbon radical with at least one site of unsaturation, i.e., a
carbon-carbon, triple bond, wherein the alkynyl radical may be
optionally substituted. In one example, the alkynyl radical is two
to eighteen carbon atoms (C.sub.2-C.sub.1). In other examples, the
alkynyl radical is C.sub.2-C.sub.12, C.sub.2-C.sub.10,
C.sub.2-C.sub.8, C.sub.2-C.sub.6 or C.sub.2-C.sub.3. Examples
include, but are not limited to, ethynyl (--C.dbd.CH), prop-1-ynyl
(--C.dbd.CCH.sub.3), prop-2-ynyl (propargyl, --CH.sub.2C.dbd.CH),
but-1-ynyl, but-2-ynyl and but-3-ynyl. In some embodiments,
substituents for "optionally substituted alkynyls" include one to
four instances of F, Cl, Br, I, OH, SH, CN, NH.sub.2, NHCH.sub.3,
N(CH.sub.3).sub.2, NO.sub.2, N.sub.3, C(O)CH.sub.3, COOH,
CO.sub.2CH.sub.3, methyl, ethyl, propyl, iso-propyl, butyl,
isobutyl, cyclopropyl, methoxy, ethoxy, propoxy, oxo,
trifluoromethyl, difluoromethyl, sulfonylamino,
methanesulfonylamino, SO, SO.sub.2, phenyl, piperidinyl,
piperizinyl, and pyrimidinyl, wherein the alkyl, phenyl and
heterocyclic portions thereof may be optionally substituted, such
as by one to four instances of substituents selected from this same
list.
[0045] "Alkylene" refers to a saturated, branched or straight chain
hydrocarbon group having two monovalent radical centers derived by
the removal of two hydrogen atoms from the same or two different
carbon atoms of a parent alkane. In one example, the divalent
alkylene group is one to eighteen carbon atoms (C.sub.1-C.sub.1).
In other examples, the divalent alkylene group is C.sub.0-C.sub.6,
C.sub.0-C.sub.5, C.sub.0-C.sub.3, C.sub.1-C.sub.12,
C.sub.1-C.sub.1, C.sub.1-C.sub.8, C.sub.1-C.sub.6, C.sub.1-C.sub.5,
C.sub.1-C.sub.4, or C.sub.1-C.sub.3. The group C.sub.0 alkylene
refers to a bond. Example alkylene groups include methylene
(--CH.sub.2--), 1,1-ethyl (--CH(CH.sub.3)--), (1,2-ethyl
(--CH.sub.2CH.sub.2--), 1,1-propyl (--CH(CH.sub.2CH.sub.3)--),
2,2-propyl (--C(CH.sub.3).sub.2--), 1,2-propyl
(--CH(CH.sub.3)CH.sub.2--), 1,3-propyl
(--CH.sub.2CH.sub.2CH.sub.2--), 1,1-dimethyleth-1,2-yl
(--C(CH.sub.3).sub.2CH.sub.2--), 1,4-butyl
(--CH.sub.2CH.sub.2CH.sub.2CH.sub.2--), and the like.
[0046] The term "heteroalkyl" refers to a straight or branched
chain monovalent hydrocarbon radical, consisting of the stated
number of carbon atoms, or, if none are stated, up to 18 carbon
atoms, and from one to five heteroatoms selected from the group
consisting of O, N, Si and S, and wherein the nitrogen and sulfur
atoms can optionally be oxidized and the nitrogen heteroatom can
optionally be quaternized. In some embodiments, the heteroatom is
selected from O, N and S, wherein the nitrogen and sulfur atoms can
optionally be oxidized and the nitrogen heteroatom can optionally
be quaternized. The heteroatom(s) can be placed at any interior
position of the heteroalkyl group, including the position at which
the alkyl group is attached to the remainder of the molecule (e.g.,
--O--CH.sub.2--CH.sub.3). Examples include
--CH.sub.2--CH.sub.2--O--CH.sub.3,
--CH.sub.2--CH.sub.2--NH--CH.sub.3,
--CH.sub.2--CH.sub.2--N(CH.sub.3)--CH.sub.3,
--CH.sub.2--S--CH.sub.2--CH.sub.3, --S(O)--CH.sub.3,
--CH.sub.2--CH.sub.2--S(O).sub.2--CH.sub.3, --Si(CH.sub.3).sub.3
and --CH.sub.2--CH.dbd.N--OCH.sub.3. Up to two heteroatoms can be
consecutive, such as, for example, --CH.sub.2--NH--OCH.sub.3 and
--CH.sub.2--O--Si(CH.sub.3).sub.3. Heteroalkyl groups can be
optionally substituted. In some embodiments, substituents for
"optionally substituted heteroalkyls" include one to four instances
of F, Cl, Br, I, OH, SH, CN, NH.sub.2, NHCH.sub.3,
N(CH.sub.3).sub.2, NO.sub.2, N.sub.3, C(O)CH.sub.3, COOH,
CO.sub.2CH.sub.3, methyl, ethyl, propyl, iso-propyl, butyl,
isobutyl, cyclopropyl, methoxy, ethoxy, propoxy, oxo,
trifluoromethyl, difluoromethyl, sulfonylamino,
methanesulfonylamino, SO, SO.sub.2, phenyl, piperidinyl,
piperizinyl, and pyrimidinyl, wherein the alkyl, phenyl and
heterocyclic portions thereof may be optionally substituted, such
as by one to four instances of substituents selected from this same
list.
[0047] "Amino" means primary (i.e., --NH.sub.2), secondary (i.e.,
--NRH), tertiary (i.e., --NRR) and quaternary (i.e., --N(+)RRR)
amines, that are optionally substituted, in which each R is the
same or different and selected from alkyl, cycloalkyl, aryl, and
heterocyclyl, wherein the alkyl, cycloalkyl, aryl and heterocyclyl
groups are as defined herein. Particular secondary and tertiary
amines are alkylamine, dialkylamine, arylamine, diarylamine,
aralkylamine and diaralkylamine, wherein the alkyl and aryl
portions can be optionally substituted. Particular secondary and
tertiary amines are methylamine, ethylamine, propylamine,
isopropylamine, phenylamine, benzylamine, dimethylamine,
diethylamine, dipropylamine and diisopropylamine. In some
embodiments, R groups of a quarternary amine are each independently
optionally substituted alkyl groups.
[0048] "Aryl" refers to a carbocyclic aromatic group, whether or
not fused to one or more groups, having the number of carbon atoms
designated, or if no number is designated, up to 14 carbon atoms.
One example includes aryl groups having 6-14 carbon atoms. Another
example includes aryl groups having 6-10 carbon atoms. Examples of
aryl groups include phenyl, naphthyl, biphenyl, phenanthrenyl,
naphthacenyl, 1,2,3,4-tetrahydronaphthalenyl, 1H-indenyl,
2,3-dihydro-1H-indenyl, and the like (see, e.g., Lang's Handbook of
Chemistry (Dean, J. A., ed.) 13.sup.th ed. Table 7-2 [1985]). A
particular aryl is phenyl. Substituted phenyl or substituted aryl
means a phenyl group or aryl group substituted with one, two,
three, four or five substituents, for example, 1-2, 1-3 or 1-4
substituents, such as chosen from groups specified herein (see
"optionally substituted" definition), such as F, Cl, Br, I, OH, SH,
CN, NH.sub.2, NHCH.sub.3, N(CH.sub.3).sub.2, NO.sub.2, N.sub.3,
C(O)CH.sub.3, COOH, CO.sub.2CH.sub.3, methyl, ethyl, propyl,
iso-propyl, butyl, isobutyl, cyclopropyl, methoxy, ethoxy, propoxy,
oxo, trifluoromethyl, difluoromethyl, sulfonylamino,
methanesulfonylamino, SO, SO.sub.2, phenyl, piperidinyl,
piperizinyl, and pyrimidinyl, wherein the alkyl, phenyl and
heterocyclic portions thereof may be optionally substituted, such
as by one to four instances of substituents selected from this same
list. Examples of the term "substituted phenyl" include a mono- or
di(halo)phenyl group such as 2-chlorophenyl, 2-bromophenyl,
4-chlorophenyl, 2,6-dichlorophenyl, 2,5-dichlorophenyl,
3,4-dichlorophenyl, 3-chlorophenyl, 3-bromophenyl, 4-bromophenyl,
3,4-dibromophenyl, 3-chloro-4-fluorophenyl, 2-fluorophenyl,
2,4-difluorophenyl and the like; a mono- or di(hydroxy)phenyl group
such as 4-hydroxyphenyl, 3-hydroxyphenyl, 2,4-dihydroxyphenyl, the
protected-hydroxy derivatives thereof and the like; a nitrophenyl
group such as 3- or 4-nitrophenyl; a cyanophenyl group, for
example, 4-cyanophenyl; a mono- or di(alkyl)phenyl group such as
4-methylphenyl, 2,4-dimethylphenyl, 2-methylphenyl,
4-(isopropyl)phenyl, 4-ethylphenyl, 3-(n-propyl)phenyl and the
like; a mono or di(alkoxy)phenyl group, for example,
3,4-dimethoxyphenyl, 3-methoxy-4-benzyloxyphenyl, 3-ethoxyphenyl,
4-(isopropoxy)phenyl, 4-(t-butoxy)phenyl, 3-ethoxy-4-methoxyphenyl
and the like; 3- or 4-trifluoromethylphenyl; a mono- or
dicarboxyphenyl or (protected carboxy)phenyl group such
4-carboxyphenyl, a mono- or di(hydroxymethyl)phenyl or (protected
hydroxymethyl)phenyl such as 3-(protected hydroxymethyl)phenyl or
3,4-di(hydroxymethyl)phenyl; a mono- or di(aminomethyl)phenyl or
(protected aminomethyl)phenyl such as 2-(aminomethyl)phenyl or
2,4-(protected aminomethyl)phenyl; or a mono- or
di(N-(methylsulfonylamino))phenyl such as
3-(N-methylsulfonylamino))phenyl. Also, the term "substituted
phenyl" represents disubstituted phenyl groups where the
substituents are different, for example, 3-methyl-4-hydroxyphenyl,
3-chloro-4-hydroxyphenyl, 2-methoxy-4-bromophenyl,
4-ethyl-2-hydroxyphenyl, 3-hydroxy-4-nitrophenyl,
2-hydroxy-4-chlorophenyl, 2-chloro-5-difluoromethoxy and the like,
as well as trisubstituted phenyl groups where the substituents are
different, for example 3-methoxy-4-benzyloxy-6-methyl
sulfonylamino, 3-methoxy-4-benzyloxy-6-phenyl sulfonylamino, and
tetrasubstituted phenyl groups where the substituents are different
such as 3-methoxy-4-benzyloxy-5-methyl-6-phenyl sulfonylamino. In
some embodiments, a substituent of an aryl, such as phenyl,
comprises an amide. For example, an aryl (e.g., phenyl) substituent
may be --(CH.sub.2).sub.0-4CONR'R'', wherein R' and R'' each
independently refer to groups including, for example, hydrogen;
unsubstituted C.sub.1-C.sub.6alkyl; C.sub.1-C.sub.6alkyl
substituted by halogen, OH, CN, unsubstituted C.sub.1-C.sub.6alkyl,
unsubstituted C.sub.1-C.sub.6 alkoxy, oxo or NR'R''; unsubstituted
C.sub.1-C.sub.6 heteroalkyl; C.sub.1-C.sub.6 heteroalkyl
substituted by halogen, OH, CN, unsubstituted C.sub.1-C.sub.6alkyl,
unsubstituted C.sub.1-C.sub.6 alkoxy, oxo or NR'R''; unsubstituted
C.sub.6-C.sub.10 aryl; C.sub.6-C.sub.10 aryl substituted by
halogen, OH, CN, unsubstituted C.sub.1-C.sub.6akkyl, unsubstituted
C.sub.1-C.sub.6 alkoxy, or NR'R''; unsubstituted 3-11 membered
heterocyclyl (e.g., 5-6 membered heteroaryl containing 1 to 4
heteroatoms selected from O, N and S or 4-11 membered
heterocycloalkyl containing 1 to 4 heteroatoms selected from O, N
and S); and 3-11 membered heterocyclyl (e.g., 5-6 membered
heteroaryl containing 1 to 4 heteroatoms selected from O, N and S
or 4-11 membered heterocycloalkyl containing 1 to 4 heteroatoms
selected from O, N and S) substituted by halogen, OH, CN,
unsubstituted C.sub.1-C.sub.6alkyl, unsubstituted C.sub.1-C.sub.6
alkoxy, oxo or NR'R''; or R' and R'' can be combined with the
nitrogen atom to form a 3-, 4-, 5-, 6-, or 7-membered ring wherein
a ring atom is optionally substituted with N, O or S and wherein
the ring is optionally substituted with halogen, OH, CN,
unsubstituted C.sub.1-C.sub.6alkyl, unsubstituted C.sub.1-C.sub.6
alkoxy, oxo or NR'R''.
[0049] "Cycloalkyl" refers to a non-aromatic, saturated or
partially unsaturated hydrocarbon ring group wherein the cycloalkyl
group may be optionally substituted independently with one or more
substituents described herein. In one example, the cycloalkyl group
is 3 to 12 carbon atoms (C.sub.3-C.sub.12). In other examples,
cycloalkyl is C.sub.3-C.sub.8, C.sub.3-C.sub.10 or
C.sub.5-C.sub.10. In other examples, the cycloalkyl group, as a
monocycle, is C.sub.3-C.sub.8, C.sub.3-C.sub.6 or C.sub.5-C.sub.6.
In another example, the cycloalkyl group, as a bicycle, is
C.sub.7-C.sub.12. In another example, the cycloalkyl group, as a
spiro system, is C.sub.5-C.sub.12. Examples of monocyclic
cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl,
1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl,
cyclohexyl, perdeuteriocyclohexyl, 1-cyclohex-1-enyl,
1-cyclohex-2-enyl, 1-cyclohex-3-enyl, cyclohexadienyl, cycloheptyl,
cyclooctyl, cyclononyl, cyclodecyl, cycloundecyl and cyclododecyl.
Exemplary arrangements of bicyclic cycloalkyls having 7 to 12 ring
atoms include, but are not limited to, [4,4], [4,5], [5,5], [5,6]
or [6,6] ring systems. Exemplary bridged bicyclic cycloalkyls
include, but are not limited to, bicyclo[2.2.1]heptane,
bicyclo[2.2.2]octane and bicyclo[3.2.2]nonane. Examples of spiro
cycloalkyl include, spiro[2.2]pentane, spiro[2.3]hexane,
spiro[2.4]heptane, spiro[2.5]octane and spiro[4.5]decane. In some
embodiments, substituents for "optionally substituted cycloalkyls"
include one to four instances of F, Cl, Br, I, OH, SH, CN,
NH.sub.2, NHCH.sub.3, N(CH.sub.3).sub.2, NO.sub.2, N.sub.3,
C(O)CH.sub.3, COOH, CO.sub.2CH.sub.3, methyl, ethyl, propyl,
iso-propyl, butyl, isobutyl, cyclopropyl, methoxy, ethoxy, propoxy,
oxo, trifluoromethyl, difluoromethyl, sulfonylamino,
methanesulfonylamino, SO, SO.sub.2, phenyl, piperidinyl,
piperizinyl, and pyrimidinyl, wherein the alkyl, aryl and
heterocyclic portions thereof may be optionally substituted, such
as by one to four instances of substituents selected from this same
list. In some embodiments, a substituent of a cycloalkyl comprises
an amide. For example, a cycloalkyl substituent may be
--(CH.sub.2).sub.0-4CONR'R'', wherein R' and R'' each independently
refer to groups including, for example, hydrogen; unsubstituted
C.sub.1-C.sub.6alkyl; C.sub.1-C.sub.6alkyl substituted by halogen,
OH, CN, unsubstituted C.sub.1-C.sub.6alkyl, unsubstituted
C.sub.1-C.sub.6 alkoxy, oxo or NR'R''; unsubstituted
C.sub.1-C.sub.6 heteroalkyl; C.sub.1-C.sub.6 heteroalkyl
substituted by halogen, OH, CN, unsubstituted C.sub.1-C.sub.6alkyl,
unsubstituted C.sub.1-C.sub.6 alkoxy, oxo or NR'R''; unsubstituted
C.sub.6-C.sub.10 aryl; C.sub.6-C.sub.10 aryl substituted by
halogen, OH, CN, unsubstituted C.sub.1-C.sub.6alkyl, unsubstituted
C.sub.1-C.sub.6 alkoxy, or NR'R''; unsubstituted 3-11 membered
heterocyclyl (e.g., 5-6 membered heteroaryl containing 1 to 4
heteroatoms selected from O, N and S or 4-11 membered
heterocycloalkyl containing 1 to 4 heteroatoms selected from O, N
and S); and 3-11 membered heterocyclyl (e.g., 5-6 membered
heteroaryl containing 1 to 4 heteroatoms selected from O, N and S
or 4-11 membered heterocycloalkyl containing 1 to 4 heteroatoms
selected from O, N and S) substituted by halogen, OH, CN,
unsubstituted C.sub.1-C.sub.6alkyl, unsubstituted C.sub.1-C.sub.6
alkoxy, oxo or NR'R''; or R' and R'' can be combined with the
nitrogen atom to form a 3-, 4-, 5-, 6-, or 7-membered ring wherein
a ring atom is optionally substituted with N, O or S and wherein
the ring is optionally substituted with halogen, OH, CN,
unsubstituted C.sub.1-C.sub.6alkyl, unsubstituted C.sub.1-C.sub.6
alkoxy, oxo or NR'R''.
[0050] "Heterocyclic group", "heterocyclic", "heterocycle",
"heterocyclyl", or "heterocyclo" are used interchangeably and refer
to any mono-, bi-, tricyclic or spiro, saturated or unsaturated,
aromatic (heteroaryl) or non-aromatic (e.g., heterocycloalkyl),
ring system, having 3 to 20 ring atoms (e.g., 3-10 ring atoms),
where the ring atoms are carbon, and at least one atom in the ring
or ring system is a heteroatom selected from nitrogen, sulfur or
oxygen. If any ring atom of a cyclic system is a heteroatom, that
system is a heterocycle, regardless of the point of attachment of
the cyclic system to the rest of the molecule. In one example,
heterocyclyl includes 3-11 ring atoms ("members") and includes
monocycles, bicycles, tricycles and spiro ring systems, wherein the
ring atoms are carbon, where at least one atom in the ring or ring
system is a heteroatom selected from nitrogen, sulfur or oxygen. In
one example, heterocyclyl includes 1 to 4 heteroatoms. In one
example, heterocyclyl includes 1 to 3 heteroatoms. In another
example, heterocyclyl includes 3- to 7-membered monocycles having
1-2, 1-3 or 1-4 heteroatoms selected from nitrogen, sulfur or
oxygen. In another example, heterocyclyl includes 4- to 6-membered
monocycles having 1-2, 1-3 or 1-4 heteroatoms selected from
nitrogen, sulfur or oxygen. In another example, heterocyclyl
includes 3-membered monocycles. In another example, heterocyclyl
includes 4-membered monocycles. In another example, heterocyclyl
includes 5-6 membered monocycles, e.g., 5-6 membered heteroaryl. In
another example, heterocyclyl includes 3-11 membered
heterocycloyalkyls, such as 4-11 membered heterocycloalkyls. In
some embodiments, a heterocycloalkyl includes at least one
nitrogen. In one example, the heterocyclyl group includes 0 to 3
double bonds. Any nitrogen or sulfur heteroatom may optionally be
oxidized (e.g., NO, SO, SO.sub.2), and any nitrogen heteroatom may
optionally be quaternized (e.g., [NR.sub.4].sup.+Cl.sup.-,
[NR.sub.4].sup.+OH.sup.-). Example heterocycles are oxiranyl,
aziridinyl, thiiranyl, azetidinyl, oxetanyl, thietanyl,
1,2-dithietanyl, 1,3-dithietanyl, pyrrolidinyl,
dihydro-1H-pyrrolyl, dihydrofuranyl, tetrahydrofuranyl,
dihydrothienyl, tetrahydrothienyl, imidazolidinyl, piperidinyl,
piperazinyl, isoquinolinyl, tetrahydroisoquinolinyl, morpholinyl,
thiomorpholinyl, 1,1-dioxo-thiomorpholinyl, dihydropyranyl,
tetrahydropyranyl, hexahydrothiopyranyl, hexahydropyrimidinyl,
oxazinanyl, thiazinanyl, thioxanyl, homopiperazinyl,
homopiperidinyl, azepanyl, oxepanyl, thiepanyl, oxazepinyl,
oxazepanyl, diazepanyl, 1,4-diazepanyl, diazepinyl, thiazepinyl,
thiazepanyl, tetrahydrothiopyranyl, oxazolidinyl, thiazolidinyl,
isothiazolidinyl, 1,1-dioxoisothiazolidinonyl, oxazolidinonyl,
imidazolidinonyl, 4,5,6,7-tetrahydro[2H]indazolyl,
tetrahydrobenzoimidazolyl, 4,5,6,7-tetrahydrobenzo[d]imidazolyl,
1,6-dihydroimidazol[4,5-d]pyrrolo[2,3-b]pyridinyl, thiazinyl,
oxazinyl, thiadiazinyl, oxadiazinyl, dithiazinyl, dioxazinyl,
oxathiazinyl, thiatriazinyl, oxatriazinyl, dithiadiazinyl,
imidazolinyl, dihydropyrimidyl, tetrahydropyrimidyl, 1-pyrrolinyl,
2-pyrrolinyl, 3-pyrrolinyl, indolinyl, thiapyranyl, 2H-pyranyl,
4H-pyranyl, dioxanyl, 1,3-dioxolanyl, pyrazolinyl, pyrazolidinyl,
dithianyl, dithiolanyl, pyrimidinonyl, pyrimidindionyl,
pyrimidin-2,4-dionyl, piperazinonyl, piperazindionyl,
pyrazolidinylimidazolinyl, 3-azabicyclo[3.1.0]hexanyl,
3,6-diazabicyclo[3.1.1]heptanyl, 6-azabicyclo[3.1.1]heptanyl,
3-azabicyclo[3.1.1]heptanyl, 3-azabicyclo[4.1.0]heptanyl,
azabicyclo[2.2.2]hexanyl, 2-azabicyclo[3.2.1]octanyl,
8-azabicyclo[3.2.1]octanyl, 2-azabicyclo[2.2.2]octanyl,
8-azabicyclo[2.2.2]octanyl, 7-oxabicyclo[2.2.1]heptane,
azaspiro[3.5]nonanyl, azaspiro[2.5]octanyl, azaspiro[4.5]decanyl,
1-azaspiro[4.5]decan-2-only, azaspiro[5.5]undecanyl,
tetrahydroindolyl, octahydroindolyl, tetrahydroisoindolyl,
tetrahydroindazolyl, 1,1-dioxohexahydrothiopyranyl. Examples of
5-membered heterocycles containing a sulfur or oxygen atom and one
to three nitrogen atoms are thiazolyl, including thiazol-2-yl and
thiazol-2-yl N-oxide, thiadiazolyl, including 1,3,4-thiadiazol-5-yl
and 1,2,4-thiadiazol-5-yl, oxazolyl, for example oxazol-2-yl, and
oxadiazolyl, such as 1,3,4-oxadiazol-5-yl, and
1,2,4-oxadiazol-5-yl. Example 5-membered ring heterocycles
containing 2 to 4 nitrogen atoms include imidazolyl, such as
imidazol-2-yl; triazolyl, such as 1,3,4-triazol-5-yl;
1,2,3-triazol-5-yl, 1,2,4-triazol-5-yl, and tetrazolyl, such as
1H-tetrazol-5-yl. Example benzo-fused 5-membered heterocycles are
benzoxazol-2-yl, benzthiazol-2-yl and benzimidazol-2-yl. Example
6-membered heterocycles contain one to three nitrogen atoms and
optionally a sulfur or oxygen atom, for example pyridyl, such as
pyrid-2-yl, pyrid-3-yl, and pyrid-4-yl; pyrimidyl, such as
pyrimid-2-yl and pyrimid-4-yl; triazinyl, such as
1,3,4-triazin-2-yl and 1,3,5-triazin-4-yl; pyridazinyl, in
particular pyridazin-3-yl, and pyrazinyl. The pyridine N-oxides and
pyridazine N-oxides and the pyridyl, pyrimid-2-yl, pyrimid-4-yl,
pyridazinyl and the 1,3,4-triazin-2-yl groups, are other example
heterocycle groups. Heterocycles may be optionally substituted. For
example, substituents for "optionally substituted heterocycles"
include one to four instances of F, Cl, Br, I, OH, SH, CN,
NH.sub.2, NHCH.sub.3, N(CH.sub.3).sub.2, NO.sub.2, N.sub.3,
C(O)CH.sub.3, COOH, CO.sub.2CH.sub.3, methyl, ethyl, propyl,
iso-propyl, butyl, isobutyl, cyclopropyl, methoxy, ethoxy, propoxy,
oxo, trifluoromethyl, difluoromethyl, sulfonylamino,
methanesulfonylamino, SO, SO.sub.2, phenyl, piperidinyl,
piperizinyl, and pyrimidinyl, wherein the alkyl, aryl and
heterocyclic portions thereof may be optionally substituted, such
as by one to four instances of substituents selected from this same
list. In some embodiments, a substituent of a heterocyclic group,
such as a heteroaryl or heterocycloalkyl, comprises an amide. For
example, a heterocyclic (e.g., heteroaryl or heterocycloalkyl)
substituent may be --(CH.sub.2).sub.0-4CONR'R'', wherein R' and R''
each independently refer to groups including, for example,
hydrogen; unsubstituted C.sub.1-C.sub.6alkyl; C.sub.1-C.sub.6alkyl
substituted by halogen, OH, CN, unsubstituted C.sub.1-C.sub.6alkyl,
unsubstituted C.sub.1-C.sub.6 alkoxy, oxo or NR'R''; unsubstituted
C.sub.1-C.sub.6 heteroalkyl; C.sub.1-C.sub.6 heteroalkyl
substituted by halogen, OH, CN, unsubstituted C.sub.1-C.sub.6alkyl,
unsubstituted C.sub.1-C.sub.6 alkoxy, oxo or NR'R''; unsubstituted
C.sub.6-C.sub.10 aryl; C.sub.6-C.sub.10 aryl substituted by
halogen, OH, CN, unsubstituted C.sub.1-C.sub.6alkyl, unsubstituted
C.sub.1-C.sub.6 alkoxy, or NR'R''; unsubstituted 3-11 membered
heterocyclyl (e.g., 5-6 membered heteroaryl containing 1 to 4
heteroatoms selected from O, N and S or 4-11 membered
heterocycloalkyl containing 1 to 4 heteroatoms selected from O, N
and S); and 3-11 membered heterocyclyl (e.g., 5-6 membered
heteroaryl containing 1 to 4 heteroatoms selected from O, N and S
or 4-11 membered heterocycloalkyl containing 1 to 4 heteroatoms
selected from O, N and S) substituted by halogen, OH, CN,
unsubstituted C.sub.1-C.sub.6alkyl, unsubstituted C.sub.1-C.sub.6
alkoxy, oxo or NR'R''; or R' and R'' can be combined with the
nitrogen atom to form a 3-, 4-, 5-, 6-, or 7-membered ring wherein
a ring atom is optionally substituted with N, O or S and wherein
the ring is optionally substituted with halogen, OH, CN,
unsubstituted C.sub.1-C.sub.6alkyl, unsubstituted C.sub.1-C.sub.6
alkoxy, oxo or NR'R''.
[0051] "Heteroaryl" refers to any mono-, bi-, or tricyclic ring
system where at least one ring is a 5- or 6-membered aromatic ring
containing from 1 to 4 heteroatoms selected from nitrogen, oxygen,
and sulfur, and in an example embodiment, at least one heteroatom
is nitrogen. See, for example, Lang's Handbook of Chemistry (Dean,
J. A., ed.) 13.sup.th ed. Table 7-2 [1985]. Included in the
definition are any bicyclic groups where any of the above
heteroaryl rings are fused to an aryl ring, wherein the aryl ring
or the heteroaryl ring is joined to the remainder of the molecule.
In one embodiment, heteroaryl includes 5-6 membered monocyclic
aromatic groups where one or more ring atoms is nitrogen, sulfur or
oxygen. Example heteroaryl groups include thienyl, furyl,
imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl,
isoxazolyl, triazolyl, thiadiazolyl, oxadiazolyl, tetrazolyl,
thiatriazolyl, oxatriazolyl, pyridyl, pyrimidyl, pyrazinyl,
pyridazinyl, triazinyl, tetrazinyl, tetrazolo[1,5-b]pyridazinyl,
imidazol[1,2-a]pyrimidinyl and purinyl, as well as benzo-fused
derivatives, for example benzoxazolyl, benzofuryl, benzothiazolyl,
benzothiadiazolyl, benzotriazolyl, benzoimidazolyl and indolyl.
Heteroaryl groups can be optionally substituted. In some
embodiments, substituents for "optionally substituted heteroaryls"
include one to four instances of F, Cl, Br, I, OH, SH, CN,
NH.sub.2, NHCH.sub.3, N(CH.sub.3).sub.2, NO.sub.2, N.sub.3,
C(O)CH.sub.3, COOH, CO.sub.2CH.sub.3, methyl, ethyl, propyl,
iso-propyl, butyl, isobutyl, cyclopropyl, methoxy, ethoxy, propoxy,
trifluoromethyl, difluoromethyl, sulfonylamino,
methanesulfonylamino, SO, SO.sub.2, phenyl, piperidinyl,
piperizinyl, and pyrimidinyl, wherein the alkyl, phenyl and
heterocyclic portions thereof may be optionally substituted, such
as by one to four instances of substituents selected from this same
list. In some embodiments, a substituent of a heteroaryl comprises
an amide. For example, a heteroaryl substituent may be
--(CH.sub.2).sub.0-4CONR'R'', wherein R' and R'' each independently
refer to groups including, for example, hydrogen; unsubstituted
C.sub.1-C.sub.6alkyl; C.sub.1-C.sub.6alkyl substituted by halogen,
OH, CN, unsubstituted C.sub.1-C.sub.6alkyl, unsubstituted
C.sub.1-C.sub.6 alkoxy, oxo or NR'R''; unsubstituted
C.sub.1-C.sub.6 heteroalkyl; C.sub.1-C.sub.6 heteroalkyl
substituted by halogen, OH, CN, unsubstituted C.sub.1-C.sub.6alkyl,
unsubstituted C.sub.1-C.sub.6 alkoxy, oxo or NR'R''; unsubstituted
C.sub.6-C.sub.10 aryl; C.sub.6-C.sub.10 aryl substituted by
halogen, OH, CN, unsubstituted C.sub.1-C.sub.6alkyl, unsubstituted
C.sub.1-C.sub.6 alkoxy, or NR'R''; unsubstituted 3-11 membered
heterocyclyl (e.g., 5-6 membered heteroaryl containing 1 to 4
heteroatoms selected from O, N and S or 4-11 membered
heterocycloalkyl containing 1 to 4 heteroatoms selected from O, N
and S); and 3-11 membered heterocyclyl (e.g., 5-6 membered
heteroaryl containing 1 to 4 heteroatoms selected from O, N and S
or 4-11 membered heterocycloalkyl containing 1 to 4 heteroatoms
selected from O, N and S) substituted by halogen, OH, CN,
unsubstituted C.sub.1-C.sub.6alkyl, unsubstituted C.sub.1-C.sub.6
alkoxy, oxo or NR'R''; or R' and R'' can be combined with the
nitrogen atom to form a 3-, 4-, 5-, 6-, or 7-membered ring wherein
a ring atom is optionally substituted with N, O or S and wherein
the ring is optionally substituted with halogen, OH, CN,
unsubstituted C.sub.1-C.sub.6alkyl, unsubstituted C.sub.1-C.sub.6
alkoxy, oxo or NR'R''.
[0052] In particular embodiments, a heterocyclyl group is attached
at a carbon atom of the heterocyclyl group. By way of example,
carbon bonded heterocyclyl groups include bonding arrangements at
position 2, 3, 4, 5, or 6 of a pyridine ring, position 3, 4, 5, or
6 of a pyridazine ring, position 2, 4, 5, or 6 of a pyrimidine
ring, position 2, 3, 5, or 6 of a pyrazine ring, position 2, 3, 4,
or 5 of a furan, tetrahydrofuran, thiofuran, thiophene, pyrrole or
tetrahydropyrrole ring, position 2, 4, or 5 of an oxazole,
imidazole or thiazole ring, position 3, 4, or 5 of an isoxazole,
pyrazole, or isothiazole ring, position 2 or 3 of an aziridine
ring, position 2, 3, or 4 of an azetidine ring, position 2, 3, 4,
5, 6, 7, or 8 of a quinoline ring or position 1, 3, 4, 5, 6, 7, or
8 of an isoquinoline ring.
[0053] In certain embodiments, the heterocyclyl group is
N-attached. By way of example, nitrogen bonded heterocyclyl or
heteroaryl groups include bonding arrangements at position 1 of an
aziridine, azetidine, pyrrole, pyrrolidine, 2-pyrroline,
3-pyrroline, imidazole, imidazolidine, 2-imidazoline,
3-imidazoline, pyrazole, pyrazoline, 2-pyrazoline, 3-pyrazoline,
piperidine, piperazine, indole, indoline, 1H-indazole, position 2
of a isoindole, or isoindoline, position 4 of a morpholine, and
position 9 of a carbazole, or .beta.-carboline.
[0054] The term "alkoxy" refers to a linear or branched monovalent
radical represented by the formula --OR in which R is alkyl, as
defined herein. Alkoxy groups include methoxy, ethoxy, propoxy,
isopropoxy, mono-, di- and tri-fluoromethoxy and cyclopropoxy.
[0055] "Acyl" means a carbonyl containing substituent represented
by the formula --C(O)--R in which R is hydrogen, alkyl, cycloalkyl,
aryl or heterocyclyl, wherein the alkyl, cycloalkyl, aryl and
heterocyclyl are as defined herein. Acyl groups include alkanoyl
(e.g., acetyl), aroyl (e.g., benzoyl), and heteroaroyl (e.g.,
pyridinoyl).
[0056] "Optionally substituted" unless otherwise specified means
that a group may be unsubstituted or substituted by one or more
(e.g., 0, 1, 2, 3, 4, or 5 or more, or any range derivable therein)
of the substituents listed for that group in which said
substituents may be the same or different. In an embodiment, an
optionally substituted group has 1 substituent. In another
embodiment an optionally substituted group has 2 substituents. In
another embodiment an optionally substituted group has 3
substituents. In another embodiment an optionally substituted group
has 4 substituents. In another embodiment an optionally substituted
group has 5 substituents.
[0057] Optional substituents for alkyl radicals, alone or as part
of another substituent (e.g., alkoxy), as well as alkylenyl,
alkenyl, alkynyl, heteroalkyl, heterocycloalkyl, and cycloalkyl,
also each alone or as part of another substituent, can be a variety
of groups, such as those described herein, as well as selected from
the group consisting of halogen; oxo; CN; NO; N.sub.3; --OR';
perfluoro-C.sub.1-C.sub.4 alkoxy; unsubstituted C.sub.3-C.sub.7
cycloalkyl; C.sub.3-C.sub.7 cycloalkyl substituted by halogen, OH,
CN, unsubstituted C.sub.1-C.sub.6alkyl, unsubstituted
C.sub.1-C.sub.6 alkoxy, oxo or NR'R''; unsubstituted
C.sub.6-C.sub.10 aryl (e.g., phenyl); C.sub.6-C.sub.10 aryl
substituted by halogen, OH, CN, unsubstituted C.sub.1-C.sub.6alkyl,
unsubstituted C.sub.1-C.sub.6 alkoxy, or NR'R''; unsubstituted 3-11
membered heterocyclyl (e.g., 5-6 membered heteroaryl containing 1
to 4 heteroatoms selected from O, N and S or 4-11 membered
heterocycloalkyl containing 1 to 4 heteroatoms selected from O, N
and S); 3-11 membered heterocyclyl (e.g., 5-6 membered heteroaryl
containing 1 to 4 heteroatoms selected from O, N and S or 4-11
membered heterocycloalkyl containing 1 to 4 heteroatoms selected
from O, N and S) substituted by halogen, OH, CN, unsubstituted
C.sub.1-C.sub.6alkyl, unsubstituted C.sub.1-C.sub.6 alkoxy, oxo or
NR'R''; --NR'R''; --SR'; --SiR'R''R'''; --OC(O)R'; --C(O)R';
--CO.sub.2R'; --CONR'R''; --OC(O)NR'R''; --NR''C(O)R';
--NR'''C(O)NR'R''; --NR''C(O).sub.2R'; --S(O).sub.2R';
--S(O).sub.2NR'R''; --NR'S(O).sub.2R''; --NR'' 'S(O).sub.2NR'R'';
amidinyl; guanidinyl; --(CH.sub.2).sub.1-4--OR';
--(CH.sub.2).sub.1-4--NR'R''; --(CH.sub.2).sub.1-4--SR';
--(CH.sub.2).sub.1-4--SiR'R''R'''; --(CH.sub.2).sub.1-4--OC(O)R';
--(CH.sub.2).sub.1-4--C(O)R'; --(CH.sub.2).sub.1-4--CO.sub.2R'; and
--(CH.sub.2).sub.1-4CONR'R'', or combinations thereof, in a number
ranging from zero to (2m'+1), where m' is the total number of
carbon atoms in such radical. R', R'' and R''' each independently
refer to groups including, for example, hydrogen; unsubstituted
C.sub.1-C.sub.6alkyl; C.sub.1-C.sub.6alkyl substituted by halogen,
OH, CN, unsubstituted C.sub.1-C.sub.6alkyl, unsubstituted
C.sub.1-C.sub.6 alkoxy, oxo or NR'R''; unsubstituted C.sub.1C
heteroalkyl; C.sub.1C heteroalkyl substituted by halogen, OH, CN,
unsubstituted C.sub.1-C.sub.6alkyl, unsubstituted C.sub.1-C.sub.6
alkoxy, oxo or NR'R''; unsubstituted C.sub.6-C.sub.10 aryl;
C.sub.6-C.sub.10 aryl substituted by halogen, OH, CN, unsubstituted
C.sub.1-C.sub.6alkyl, unsubstituted C.sub.1-C.sub.6 alkoxy, or
NR'R''; unsubstituted 3-11 membered heterocyclyl (e.g., 5-6
membered heteroaryl containing 1 to 4 heteroatoms selected from O,
N and S or 4-11 membered heterocycloalkyl containing 1 to 4
heteroatoms selected from O, N and S); and 3-11 membered
heterocyclyl (e.g., 5-6 membered heteroaryl containing 1 to 4
heteroatoms selected from O, N and S or 4-11 membered
heterocycloalkyl containing 1 to 4 heteroatoms selected from O, N
and S) substituted by halogen, OH, CN, unsubstituted
C.sub.1-C.sub.6alkyl, unsubstituted C.sub.1-C.sub.6 alkoxy, oxo or
NR'R''. When R' and R'' are attached to the same nitrogen atom,
they can be combined with the nitrogen atom to form a 3-, 4-, 5-,
6-, or 7-membered ring wherein a ring atom is optionally
substituted with N, O or S and wherein the ring is optionally
substituted with halogen, OH, CN, unsubstituted
C.sub.1-C.sub.6alkyl, unsubstituted C.sub.1-C.sub.6 alkoxy, oxo or
NR'R''. For example, --NR'R'' is meant to include 1-pyrrolidinyl
and 4-morpholinyl.
[0058] Similarly, optional substituents for the aryl and heteroaryl
groups are varied. In some embodiments, substituents for aryl and
heteroaryl groups are selected from the group consisting of
halogen; CN; NO; N.sub.3; --OR'; perfluoro-C.sub.1-C.sub.4 alkoxy;
unsubstituted C.sub.3-C.sub.7 cycloalkyl; C.sub.3-C.sub.7
cycloalkyl substituted by halogen, OH, CN, unsubstituted
C.sub.1-C.sub.6alkyl, unsubstituted C.sub.1-C.sub.6 alkoxy, oxo or
NR'R''; unsubstituted C.sub.6-C.sub.10 aryl (e.g., phenyl);
C.sub.6-C.sub.10 aryl substituted by halogen, OH, CN, unsubstituted
C.sub.1-C.sub.6alkyl, unsubstituted C.sub.1-C.sub.6 alkoxy, or
NR'R''; unsubstituted 3-11 membered heterocyclyl (e.g., 5-6
membered heteroaryl containing 1 to 4 heteroatoms selected from O,
N and S or 4-11 membered heterocycloalkyl containing 1 to 4
heteroatoms selected from O, N and S); 3-11 membered heterocyclyl
(e.g., 5-6 membered heteroaryl containing 1 to 4 heteroatoms
selected from O, N and S or 4-11 membered heterocycloalkyl
containing 1 to 4 heteroatoms selected from O, N and S) substituted
by halogen, OH, CN, unsubstituted C.sub.1-C.sub.6alkyl,
unsubstituted C.sub.1-C.sub.6 alkoxy, oxo or NR'R''; --NR'R'';
--SR'; --SiR'R''R'''; --OC(O)R'; --C(O)R'; --CO.sub.2R';
--CONR'R''; --OC(O)NR'R''; --NR''C(O)R'; --NR'''C(O)NR'R'';
--NR''C(O).sub.2R'; --S(O).sub.2R'; --S(O).sub.2NR'R'';
--NR'S(O).sub.2R''; --NR'''S(O).sub.2NR'R''; amidinyl; guanidinyl;
--(CH.sub.2).sub.1-4--OR'; --(CH.sub.2).sub.1-4--NR'R'';
--(CH.sub.2).sub.1-4--SR'; --(CH.sub.2).sub.1-4--SiR'R''R''';
--(CH.sub.2).sub.1-4--OC(O)R'; --(CH.sub.2).sub.1-4--C(O)R';
--(CH.sub.2).sub.1-4--CO.sub.2R'; and --(CH.sub.2).sub.1-4CONR'R'',
or combinations thereof, in a number ranging from zero to (2m'+1),
where m' is the total number of carbon atoms in such radical. R',
R'' and R''' each independently refer to groups including, for
example, hydrogen; unsubstituted C.sub.1-C.sub.6alkyl;
C.sub.1-C.sub.6alkyl substituted by halogen, OH, CN, unsubstituted
C.sub.1-C.sub.6alkyl, unsubstituted C.sub.1-C.sub.6 alkoxy, oxo or
NR'R''; unsubstituted C.sub.1-C.sub.6 heteroalkyl; C.sub.1-C.sub.6
heteroalkyl substituted by halogen, OH, CN, unsubstituted
C.sub.1-C.sub.6alkyl, unsubstituted C.sub.1-C.sub.6 alkoxy, oxo or
NR'R''; unsubstituted C.sub.6-C.sub.10 aryl; C.sub.6-C.sub.10 aryl
substituted by halogen, OH, CN, unsubstituted C.sub.1-C.sub.6alkyl,
unsubstituted C.sub.1-C.sub.6 alkoxy, or NR'R''; unsubstituted 3-11
membered heterocyclyl (e.g., 5-6 membered heteroaryl containing 1
to 4 heteroatoms selected from O, N and S or 4-11 membered
heterocycloalkyl containing 1 to 4 heteroatoms selected from O, N
and S); and 3-11 membered heterocyclyl (e.g., 5-6 membered
heteroaryl containing 1 to 4 heteroatoms selected from O, N and S
or 4-11 membered heterocycloalkyl containing 1 to 4 heteroatoms
selected from O, N and S) substituted by halogen, OH, CN,
unsubstituted C.sub.1-C.sub.6alkyl, unsubstituted C.sub.1-C.sub.6
alkoxy, oxo or NR'R''. When R' and R'' are attached to the same
nitrogen atom, they can be combined with the nitrogen atom to form
a 3-, 4-, 5-, 6-, or 7-membered ring wherein a ring atom is
optionally substituted with N, O or S and wherein the ring is
optionally substituted with halogen, OH, CN, unsubstituted
C.sub.1-C.sub.6alkyl, unsubstituted C.sub.1-C.sub.6 alkoxy, oxo or
NR'R''. For example, --NR'R'' is meant to include 1-pyrrolidinyl
and 4-morpholinyl.
[0059] The term "oxo" refers to .dbd.O or (.dbd.O).sub.2.
[0060] As used herein a wavy line "" that intersects a bond in a
chemical structure indicate the point of attachment of the atom to
which the wavy bond is connected in the chemical structure to the
remainder of a molecule, or to the remainder of a fragment of a
molecule. In some embodiments, an arrow together with an asterisk
is used in the manner of a wavy line to indicate a point of
attachment.
[0061] In certain embodiments, divalent groups are described
generically without specific bonding configurations. It is
understood that the generic description is meant to include both
bonding configurations, unless specified otherwise. For example, in
the group R.sup.1-R.sup.2-R.sup.3, if the group R.sup.2 is
described as --CH.sub.2C(O)--, then it is understood that this
group can be bonded both as R.sup.1--CH.sub.2C(O)--R.sup.3, and as
R.sup.1--C(O)CH.sub.2--R.sup.3, unless specified otherwise.
[0062] The terms "compound(s) of the invention," and "compound(s)
of the present invention" and the like, unless otherwise indicated,
include compounds of Formula (I) herein, such as compounds 1-18,
sometimes referred to as JAK inhibitors, including stereoisomers
(including atropisomers), geometric isomers, tautomers, solvates,
metabolites, isotopes, salts (e.g., pharmaceutically acceptable
salts), and prodrugs thereof. In some embodiments, solvates,
metabolites, isotopes or prodrugs are excluded, or any combination
thereof.
[0063] The phrase "pharmaceutically acceptable" refers to molecular
entities and compositions that do not produce an adverse, allergic
or other untoward reaction when administered to an animal, such as,
for example, a human, as appropriate.
[0064] Compounds of the present invention may be in the form of a
salt, such as a pharmaceutically acceptable salt. "Pharmaceutically
acceptable salts" include both acid and base addition salts.
"Pharmaceutically acceptable acid addition salt" refers to those
salts which retain the biological effectiveness and properties of
the free bases and which are not biologically or otherwise
undesirable, formed with inorganic acids such as hydrochloric acid,
hydrobromic acid, sulfuric acid, nitric acid, carbonic acid,
phosphoric acid and the like, and organic acids may be selected
from aliphatic, cycloaliphatic, aromatic, araliphatic,
heterocyclic, carboxylic, and sulfonic classes of organic acids
such as formic acid, acetic acid, propionic acid, glycolic acid,
gluconic acid, lactic acid, pyruvic acid, oxalic acid, malic acid,
maleic acid, maloneic acid, succinic acid, fumaric acid, tartaric
acid, citric acid, aspartic acid, ascorbic acid, glutamic acid,
anthranilic acid, benzoic acid, cinnamic acid, mandelic acid,
embonic acid, phenylacetic acid, methanesulfonic acid,
ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid,
salicyclic acid and the like.
[0065] "Pharmaceutically acceptable base addition salts" include
those derived from inorganic bases such as sodium, potassium,
lithium, ammonium, calcium, magnesium, iron, zinc, copper,
manganese, aluminum salts and the like. Particular base addition
salts are the ammonium, potassium, sodium, calcium and magnesium
salts. Salts derived from pharmaceutically acceptable organic
nontoxic bases include salts of primary, secondary, and tertiary
amines, substituted amines including naturally occurring
substituted amines, cyclic amines and basic ion exchange resins,
such as isopropylamine, trimethylamine, diethylamine,
triethylamine, tripropylamine, ethanolamine, 2-diethylaminoethanol,
tromethamine, dicyclohexylamine, lysine, arginine, histidine,
caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine,
glucosamine, methylglucamine, theobromine, purines, piperizine,
piperidine, N-ethylpiperidine, polyamine resins and the like.
Particular organic non-toxic bases include isopropylamine,
diethylamine, ethanolamine, tromethamine, dicyclohexylamine,
choline, and caffeine.
[0066] In some embodiments, a salt is selected from a
hydrochloride, hydrobromide, trifluoroacetate, sulphate, phosphate,
acetate, fumarate, maleate, tartrate, lactate, citrate, pyruvate,
succinate, oxalate, methanesulphonate, p-toluenesulphonate,
bisulphate, benzenesulphonate, ethanesulphonate, malonate,
xinafoate, ascorbate, oleate, nicotinate, saccharinate, adipate,
formate, glycolate, palmitate, L-lactate, D-lactate, aspartate,
malate, L-tartrate, D-tartrate, stearate, furoate (e.g., 2-furoate
or 3-furoate), napadisylate (naphthalene-1,5-disulfonate or
naphthalene-1-(sulfonic acid)-5-sulfonate), edisylate
(ethane-1,2-disulfonate or ethane-1-(sulfonic acid)-2-sulfonate),
isethionate (2-hydroxyethylsulfonate), 2-mesitylenesulphonate,
2-naphthalenesulphonate, 2,5-dichlorobenzenesulphonate,
D-mandelate, L-mandelate, cinnamate, benzoate, adipate, esylate,
malonate, mesitylate (2-mesitylenesulphonate), napsylate
(2-naphthalenesulfonate), camsylate (camphor-10-sulphonate, for
example (1S)-(+)-10-camphorsulfonic acid salt), glutamate,
glutarate, hippurate (2-(benzoylamino)acetate), orotate, xylate
(p-xylene-2-sulphonate), and pamoic
(2,2'-dihydroxy-1,1'-dinaphthylmethane-3,3'-dicarboxylate).
[0067] A "sterile" formulation is aseptic or free from all living
microorganisms and their spores.
[0068] "Stereoisomers" refer to compounds that have identical
chemical constitution, but differ with regard to the arrangement of
the atoms or groups in space. Stereoisomers include diastereomers,
enantiomers, conformers and the like.
[0069] "Chiral" refers to molecules which have the property of
non-superimposability of the mirror image partner, while the term
"achiral" refers to molecules which are superimposable on their
mirror image partner.
[0070] "Diastereomer" refers to a stereoisomer with two or more
centers of chirality and whose molecules are not mirror images of
one another. Diastereomers have different physical properties,
e.g., melting points, boiling points, spectral properties or
biological activities.
[0071] Mixtures of diastereomers may separate under high resolution
analytical procedures such as electrophoresis and chromatography
such as HPLC.
[0072] "Enantiomers" refer to two stereoisomers of a compound which
are non-superimposable mirror images of one another.
[0073] Stereochemical definitions and conventions used herein
generally follow S. P. Parker, Ed., McGraw-Hill Dictionary of
Chemical Terms (1984) McGraw-Hill Book Company, New York; and
Eliel, E. and Wilen, S., "Stereochemistry of Organic Compounds",
John Wiley & Sons, Inc., New York, 1994. Many organic compounds
exist in optically active forms, i.e., they have the ability to
rotate the plane of plane-polarized light. In describing an
optically active compound, the prefixes D and L, or R and S, are
used to denote the absolute configuration of the molecule about its
chiral center(s). The prefixes d and l or (+) and (-) are employed
to designate the sign of rotation of plane-polarized light by the
compound, with (-) or l meaning that the compound is levorotatory.
A compound prefixed with (+) or d is dextrorotatory. For a given
chemical structure, these stereoisomers are identical except that
they are mirror images of one another. A specific stereoisomer may
also be referred to as an enantiomer, and a mixture of such isomers
is often called an enantiomeric mixture. A 50:50 mixture of
enantiomers is referred to as a racemic mixture or a racemate,
which may occur where there has been no stereoselection or
stereospecificity in a chemical reaction or process. The terms
"racemic mixture" and "racemate" refer to an equimolar mixture of
two enantiomeric species, devoid of optical activity.
[0074] The term "tautomer" or "tautomeric form" refers to
structural isomers of different energies which are interconvertible
via a low energy barrier. For example, proton tautomers (also known
as prototropic tautomers) include interconversions via migration of
a proton, such as keto-enol and imine-enamine isomerizations.
Valence tautomers include interconversions by reorganization of
some of the bonding electrons.
[0075] Certain compounds of the present invention can exist in
unsolvated forms as well as solvated forms, including hydrated
forms. A "solvate" refers to an association or complex of one or
more solvent molecules and a compound of the present invention.
Examples of solvents that form solvates include water, isopropanol,
ethanol, methanol, DMSO, ethyl acetate, acetic acid, and
ethanolamine. Certain compounds of the present invention can exist
in multiple crystalline or amorphous forms. In general, all
physical forms are intended to be within the scope of the present
invention. The term "hydrate" refers to the complex where the
solvent molecule is water.
[0076] A "metabolite" refers to a product produced through
metabolism in the body of a specified compound or salt thereof.
Such products can result, for example, from the oxidation,
reduction, hydrolysis, amidation, deamidation, esterification,
deesterification, enzymatic cleavage, and the like, of the
administered compound.
[0077] Metabolite products typically are identified by preparing a
radiolabelled (e.g., .sup.14C or .sup.3H) isotope of a compound of
the invention, administering it in a detectable dose (e.g., greater
than about 0.5 mg/kg) to an animal such as rat, mouse, guinea pig,
monkey, or to a human, allowing sufficient time for metabolism to
occur (typically about 30 seconds to 30 hours) and isolating its
conversion products from the urine, blood or other biological
samples. These products are easily isolated since they are labeled
(others are isolated by the use of antibodies capable of binding
epitopes surviving in the metabolite). The metabolite structures
are determined in conventional fashion, e.g., by MS, LC/MS or NMR
analysis. In general, analysis of metabolites is done in the same
way as conventional drug metabolism studies well known to those
skilled in the art. The metabolite products, so long as they are
not otherwise found in vivo, are useful in diagnostic assays for
therapeutic dosing of the compounds of the invention.
[0078] A "subject," "individual," or "patient" is a vertebrate. In
certain embodiments, the vertebrate is a mammal. Mammals include,
but are not limited to, farm animals (such as cows), sport animals,
pets (such as guinea pigs, cats, dogs, rabbits and horses),
primates, mice and rats. In certain embodiments, a mammal is a
human. In embodiments comprising administration of a JAK inhibitor
as described herein or a pharmaceutically acceptable salt thereof
to a patient, the patient may be in need thereof.
[0079] The term "Janus kinase" refers to JAK1, JAK2, JAK3 and TYK2
protein kinases. In some embodiments, a Janus kinase may be further
defined as one of JAK1, JAK2, JAK3 or TYK2. In any embodiment, any
one of JAK1, JAK2, JAK3 and TYK2 may be specifically excluded as a
Janus kinase. In some embodiments, a Janus kinase is JAK1. In some
embodiments, a Janus kinase is a combination of JAK1 and JAK2.
[0080] The terms "inhibiting" and "reducing," or any variation of
these terms, includes any measurable decrease or complete
inhibition to achieve a desired result. For example, there may be a
decrease of about, at most about, or at least about 5%, 10%, 15%,
20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,
85%, 90%, 95%, 99%, or more, or any range derivable therein,
reduction of activity (e.g., JAK1 activity) compared to normal.
[0081] "Therapeutically effective amount" means an amount of a
compound or a salt thereof (e.g., a pharmaceutically acceptable
salt thereof) of the present invention that (i) treats or prevents
the particular disease, condition or disorder, or (ii) attenuates,
ameliorates or eliminates one or more symptoms of the particular
disease, condition, or disorder, and optionally (iii) prevents or
delays the onset of one or more symptoms of the particular disease,
condition or disorder described herein. In some embodiments, the
therapeutically effective amount is an amount sufficient to
decrease or alleviate the symptoms of an autoimmune or inflammatory
disease (e.g., asthma). In some embodiments, a therapeutically
effective amount is an amount of a chemical entity described herein
sufficient to significantly decrease the activity or number of
B-cells. In the case of cancer, the therapeutically effective
amount of the drug may reduce the number of cancer cells; reduce
the tumor size; inhibit (i.e., slow to some extent and preferably
stop) cancer cell infiltration into peripheral organs; inhibit
(i.e., slow to some extent and preferably stop) tumor metastasis;
inhibit, to some extent, tumor growth; or relieve to some extent
one or more of the symptoms associated with the cancer.
[0082] To the extent the drug may prevent growth or kill existing
cancer cells, it may be cytostatic or cytotoxic. For cancer
therapy, efficacy can, for example, be measured by assessing the
time to disease progression (TTP) or determining the response rate
(RR).
[0083] "Treatment" (and variations such as "treat" or "treating")
refers to clinical intervention in an attempt to alter the natural
course of the individual or cell being treated, and can be
performed either for prophylaxis or during the course of clinical
pathology. Desirable effects of treatment include preventing
occurrence or recurrence of disease, alleviation of symptoms,
diminishment of any direct or indirect pathological consequences of
the disease, stabilized (i.e., not worsening) state of disease,
decreasing the rate of disease progression, amelioration or
palliation of the disease state, prolonging survival as compared to
expected survival if not receiving treatment and remission or
improved prognosis. In some embodiments, a compound of the
invention or a salt thereof (e.g., a pharmaceutically acceptable
salt thereof), is used to delay development of a disease or
disorder or to slow the progression of a disease or disorder. Those
in need of treatment include those already with the condition or
disorder as well as those prone to have the condition or disorder,
(for example, through a genetic mutation) or those in which the
condition or disorder is to be prevented.
[0084] "Inflammatory disorder" refers to any disease, disorder or
syndrome in which an excessive or unregulated inflammatory response
leads to excessive inflammatory symptoms, host tissue damage, or
loss of tissue function. "Inflammatory disorder" also refers to a
pathological state mediated by influx of leukocytes or neutrophil
chemotaxis.
[0085] "Inflammation" refers to a localized, protective response
elicited by injury or destruction of tissues, which serves to
destroy, dilute, or wall off (sequester) both the injurious agent
and the injured tissue. Inflammation is notably associated with
influx of leukocytes or neutrophil chemotaxis. Inflammation can
result from infection with pathogenic organisms and viruses and
from noninfectious means such as trauma or reperfusion following
myocardial infarction or stroke, immune responses to foreign
antigens, and autoimmune responses. Accordingly, inflammatory
disorders amenable to treatment with a compound or a salt thereof
(e.g., a pharmaceutically acceptable salt thereof) of the present
invention encompass disorders associated with reactions of the
specific defense system as well as with reactions of the
nonspecific defense system.
[0086] "Specific defense system" refers to the component of the
immune system that reacts to the presence of specific antigens.
Examples of inflammation resulting from a response of the specific
defense system include the classical response to foreign antigens,
autoimmune diseases, and delayed type hypersensitivity responses
mediated by T-cells. Chronic inflammatory diseases, the rejection
of solid transplanted tissue and organs, e.g., kidney and bone
marrow transplants, and graft versus host disease (GVHD), are
further examples of inflammatory reactions of the specific defense
system.
[0087] The term "nonspecific defense system" refers to inflammatory
disorders that are mediated by leukocytes that are incapable of
immunological memory (e.g., granulocytes, and macrophages).
Examples of inflammation that result, at least in part, from a
reaction of the nonspecific defense system include inflammation
associated with conditions such as adult (acute) respiratory
distress syndrome (ARDS) or multiple organ injury syndromes;
reperfusion injury; acute glomerulonephritis; reactive arthritis;
dermatoses with acute inflammatory components; acute purulent
meningitis or other central nervous system inflammatory disorders
such as stroke; thermal injury; inflammatory bowel disease;
granulocyte transfusion associated syndromes; and cytokine-induced
toxicity.
[0088] "Autoimmune disease" refers to any group of disorders in
which tissue injury is associated with humoral or cell-mediated
responses to the body's own constituents. Non-limiting examples of
autoimmune diseases include rheumatoid arthritis, lupus and
multiple sclerosis.
[0089] "Allergic disease" as used herein refers to any symptoms,
tissue damage, or loss of tissue function resulting from allergy.
"Arthritic disease" as used herein refers to any disease that is
characterized by inflammatory lesions of the joints attributable to
a variety of etiologies. "Dermatitis" as used herein refers to any
of a large family of diseases of the skin that are characterized by
inflammation of the skin attributable to a variety of etiologies.
"Transplant rejection" as used herein refers to any immune reaction
directed against grafted tissue, such as organs or cells (e.g.,
bone marrow), characterized by a loss of function of the grafted
and surrounding tissues, pain, swelling, leukocytosis, and
thrombocytopenia. The therapeutic methods of the present invention
include methods for the treatment of disorders associated with
inflammatory cell activation.
[0090] "Inflammatory cell activation" refers to the induction by a
stimulus (including, but not limited to, cytokines, antigens or
auto-antibodies) of a proliferative cellular response, the
production of soluble mediators (including but not limited to
cytokines, oxygen radicals, enzymes, prostanoids, or vasoactive
amines), or cell surface expression of new or increased numbers of
mediators (including, but not limited to, major histocompatability
antigens or cell adhesion molecules) in inflammatory cells
(including but not limited to monocytes, macrophages, T
lymphocytes, B lymphocytes, granulocytes (i.e., polymorphonuclear
leukocytes such as neutrophils, basophils, and eosinophils), mast
cells, dendritic cells, Langerhans cells, and endothelial cells).
It will be appreciated by persons skilled in the art that the
activation of one or a combination of these phenotypes in these
cells can contribute to the initiation, perpetuation, or
exacerbation of an inflammatory disorder.
[0091] In some embodiments, inflammatory disorders which can be
treated according to the methods of this invention include, but are
not limited to, asthma, rhinitis (e.g., allergic rhinitis),
allergic airway syndrome, atopic dermatitis, bronchitis, rheumatoid
arthritis, psoriasis, contact dermatitis, chronic obstructive
pulmonary disease (COPD) and delayed hypersensitivity
reactions.
[0092] The terms "cancer" and "cancerous", "neoplasm", and "tumor"
and related terms refer to or describe the physiological condition
in mammals that is typically characterized by unregulated cell
growth. A "tumor" comprises one or more cancerous cells. Examples
of cancer include carcinoma, blastoma, sarcoma, seminoma,
glioblastoma, melanoma, leukemia, and myeloid or lymphoid
malignancies. More particular examples of such cancers include
squamous cell cancer (e.g., epithelial squamous cell cancer) and
lung cancer including small-cell lung cancer, non-small cell lung
cancer ("NSCLC"), adenocarcinoma of the lung and squamous carcinoma
of the lung. Other cancers include skin, keratoacanthoma,
follicular carcinoma, hairy cell leukemia, buccal cavity, pharynx
(oral), lip, tongue, mouth, salivary gland, esophageal, larynx,
hepatocellular, gastric, stomach, gastrointestinal, small
intestine, large intestine, pancreatic, cervical, ovarian, liver,
bladder, hepatoma, breast, colon, rectal, colorectal,
genitourinary, biliary passage, thyroid, papillary, hepatic,
endometrial, uterine, salivary gland, kidney or renal, prostate,
testis, vulval, peritoneum, anal, penile, bone, multiple myeloma,
B-cell lymphoma, central nervous system, brain, head and neck,
Hodgkin's, and associated metastases. Examples of neoplastic
disorders include myeloproliferative disorders, such as
polycythemia vera, essential thrombocytosis, myelofibrosis, such as
primary myelofibrosis, and chronic myelogenous leukemia (CML).
[0093] A "chemotherapeutic agent" is an agent useful in the
treatment of a given disorder, for example, cancer or inflammatory
disorders. Examples of chemotherapeutic agents are well-known in
the art and include examples such as those disclosed in U.S. Publ.
Appl. No. 2010/0048557, incorporated herein by reference.
Additionally, chemotherapeutic agents include pharmaceutically
acceptable salts, acids or derivatives of any of chemotherapeutic
agents, as well as combinations of two or more of them.
[0094] "Package insert" is used to refer to instructions
customarily included in commercial packages of therapeutic products
that contain information about the indications, usage, dosage,
administration, contraindications or warnings concerning the use of
such therapeutic products.
[0095] Unless otherwise stated, structures depicted herein include
compounds that differ only in the presence of one or more
isotopically enriched atoms. Exemplary isotopes that can be
incorporated into compounds of the present invention include
isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur,
fluorine, chlorine, and iodine, such as .sup.2H, .sup.3H, .sup.11C,
.sup.13C, .sup.14C, .sup.13N, .sup.15N, .sup.15O, .sup.17O,
.sup.18O, .sup.32P .sup.33P, .sup.35S, .sup.18F, .sup.36Cl,
.sup.123I, and .sup.125, respectively. Isotopically-labeled
compounds (e.g., those labeled with 3H and .sup.14C) can be useful
in compound or substrate tissue distribution assays. Tritiated
(i.e., .sup.3H) and carbon-14 (i.e., .sup.14C) isotopes can be
useful for their ease of preparation and detectability. Further,
substitution with heavier isotopes such as deuterium (i.e.,
.sup.2H) may afford certain therapeutic advantages resulting from
greater metabolic stability (e.g., increased in vivo half-life or
reduced dosage requirements). In some embodiments, one or more
hydrogen atoms are replaced by .sup.2H or 3H, or one or more carbon
atoms are replaced by .sup.13C- or .sup.14C-enriched carbon.
Positron emitting isotopes such as .sup.15O, .sup.13N, .sup.11C,
and .sup.18F are useful for positron emission tomography (PET)
studies to examine substrate receptor occupancy. Isotopically
labeled compounds can generally be prepared by procedures analogous
to those disclosed in the Schemes or in the Examples herein, by
substituting an isotopically labeled reagent for a non-isotopically
labeled reagent.
[0096] It is specifically contemplated that any limitation
discussed with respect to one embodiment of the invention may apply
to any other embodiment of the invention. Furthermore, any compound
or a salt thereof (e.g., a pharmaceutically acceptable salt
thereof) or composition of the invention may be used in any method
of the invention, and any method of the invention may be used to
produce or to utilize any compound or a salt thereof (e.g., a
pharmaceutically acceptable salt thereof) or composition of the
invention.
[0097] The use of the term "or" is used to mean "and/or" unless
explicitly indicated to refer to alternatives only or the
alternatives are mutually exclusive, although the disclosure
supports a definition that refers to only alternatives and
"and/or."
[0098] Throughout this application, the term "about" is used to
indicate that a value includes the standard deviation of error for
the device or method being employed to determine the value.
[0099] As used herein, "a" or "an" means one or more, unless
clearly indicated otherwise. As used herein, "another" means at
least a second or more.
[0100] Headings used herein are intended only for organizational
purposes.
Inhibitors of Janus Kinases
[0101] One embodiment provides a compound of Formula (I):
##STR00003##
or a stereoisomer or pharmaceutically acceptable salt thereof,
wherein:
[0102] R.sup.1 is: hydroxyl-C.sub.1-C.sub.6alkyl;
--(CR.sup.a1R.sup.a2).sub.m-het.sup.1;
--(CR.sup.a1R.sup.a2).sub.n--NR.sup.bR.sup.c; or
--(CR.sup.a1R.sup.a2).sub.m--C.sub.3-6cycloalkyl wherein the
C.sub.3-6cycloalkyl moiety is substituted once with R.sup.4;
[0103] R.sup.2 is: halo; halo C.sub.1-C.sub.6alkoxy;
C.sub.1-C.sub.6alkylthio; --SF.sub.2; or
C.sub.3-C.sub.6cycloalkyl;
[0104] R.sup.3 is: hydrogen; or C.sub.1-C.sub.6alkyl;
[0105] R.sup.4 is: hydrogen; or C.sub.1-C.sub.6alkyl;
[0106] R.sup.5 is: hydrogen; or C.sub.1-C.sub.6alkyl;
[0107] R.sup.6 is: hydrogen; or C.sub.1-C.sub.6alkyl;
[0108] or R.sup.2 and R.sup.6 together with the atoms to which they
are attached may form a six-membered ring containing two
heteroatoms each independently selected from O, N and S;
[0109] m is from 0 to 2;
[0110] n is from 0 to 3;
[0111] each R.sup.a1 is independently: hydrogen; or
C.sub.1-C.sub.6alkyl;
[0112] each R.sup.a2 is independently: hydrogen; halo; or
C.sub.1-C.sub.6alkyl;
[0113] R.sup.b is: hydrogen; or C.sub.1-C.sub.6alkyl;
[0114] R.sup.c is: hydrogen; C.sub.1-C.sub.6alkyl; an amino
protecting group; or azetidinyl which may be unsubstituted or
substituted once with C.sub.1-C.sub.6alkyl;
[0115] het.sup.1 is a heterocyclyl selected from: azetidinyl;
pyrrolidinyl; piperazinyl; piperidinyl; morpholinyl; and oxetanyl;
each of which may be unsubstituted or substituted once with R.sup.d
and once or twice with R.sup.g;
[0116] R.sup.d is: --(CR.sup.a1R.sup.a2).sub.p-het.sup.2;
--(CR.sup.a1R.sup.a2).sub.q--NR.sup.eR.sup.f; or
--(CR.sup.a1R.sup.a2).sub.p--C.sub.3-6cycloalkyl wherein the
C.sub.3-6cycloalkyl moiety is substituted once with
--NR.sup.eR.sup.f;
[0117] p is from 0 to 2;
[0118] q is from 0 to 4;
[0119] R.sup.e is: hydrogen; or C.sub.1-C.sub.6alkyl;
[0120] R.sup.f is: hydrogen; C.sub.1-C.sub.6alkyl; or
--CH.sub.2C.sub.2N(CH.sub.3).sub.2;
[0121] each R.sup.g is: C.sub.1-C.sub.6alkyl; or halo; and
[0122] Het.sup.2 is a heterocycle selected from: tetrahydropyranyl;
azetidinyl; and pyrrolidinyl; each of which may be unsubstituted or
substituted once with C.sub.1-C.sub.6alkyl or
--NR.sup.eR.sup.f.
[0123] In certain embodiments R is: C.sub.1-C.sub.6alkyl;
hydroxyl-C.sub.1-C.sub.6alkyl;
--(CR.sup.a1R.sup.a2).sub.m-het.sup.1; or
(CR.sup.a1R.sup.a2).sub.n--NR.sup.bR.sup.c, wherein het.sup.1 may
be unsubstituted or substituted once with R.sup.d.
[0124] In certain embodiments R.sup.1 is: C.sub.1-C.sub.6alkyl;
--(CR.sup.a1R.sup.a2).sub.m-het; or
--(CHR.sup.a).sub.n--NR.sup.bR.sup.c, wherein het.sup.1
[0125] In certain embodiments R.sup.1 is:
--(CR.sup.a1R.sup.a2).sub.m-het.sup.1; or
--(CR.sup.a1R.sup.a2).sub.n--NR.sup.bR.sup.c, wherein het.sup.1 may
be unsubstituted or substituted once with R.sup.d.
[0126] In certain embodiments R.sup.1 is --(CHR.sup.a).sub.m-het,
wherein het.sup.1 may be unsubstituted or substituted once with
R.sup.d.
[0127] In certain embodiments R.sup.1 is
--(CR.sup.a1R.sup.a2)--NR.sup.bR.sup.c.
[0128] In certain embodiments R.sup.2 is halo; halo
C.sub.1-C.sub.6alkoxy; or C.sub.1-C.sub.6alkylthio.
[0129] In certain embodiments R.sup.2 is halo.
[0130] In certain embodiments R.sup.2 is halo
C.sub.1-C.sub.6alkoxy.
[0131] In certain embodiments R.sup.2 is
C.sub.1-C.sub.6alkylthio.
[0132] In certain embodiments R.sup.2 is: chloro; difluoromethoxy;
methylethio; or cyclopropyl.
[0133] In certain embodiments R.sup.2 is chloro.
[0134] In certain embodiments R.sup.2 is difluoromethoxy.
[0135] In certain embodiments R.sup.2 is methylethio.
[0136] In certain embodiments R.sup.3 is hydrogen.
[0137] In certain embodiments R.sup.4 is hydrogen.
[0138] In certain embodiments R.sup.5 is hydrogen.
[0139] In certain embodiments R.sup.6 is hydrogen.
[0140] In certain embodiments R.sup.2 and R.sup.6 together with the
atoms to which they are attached form a six-membered ring
containing two heteroatoms each independently selected from O, N
and S.
[0141] In certain embodiments m is 0. In embodiments wherein m is 0
and R is het, it should be understood that the bond connecting het
to the tetrazole ring is made with a carbon atom of het.sup.1 and
not a heteroatom.
[0142] In certain embodiments m is 0.
[0143] In certain embodiments m is 1.
[0144] In certain embodiments m is 2.
[0145] In certain embodiments n is 0.
[0146] In certain embodiments n is 1.
[0147] In certain embodiments n is 2.
[0148] In certain embodiments R.sup.a1 is hydrogen.
[0149] In certain embodiments R.sup.a2 is hydrogen.
[0150] In certain embodiments R.sup.b is hydrogen.
[0151] In certain embodiments R.sup.b is C.sub.1-C.sub.6alkyl.
[0152] In certain embodiments R.sup.c is hydrogen.
[0153] In certain embodiments R.sup.c is C.sub.1-C.sub.6alkyl.
[0154] In certain embodiments R.sup.c is
1-methyl-azetidin-3-yl.
[0155] In certain embodiments het.sup.1 is azetidinyl, which may be
unsubstituted or substituted once with R.sup.d.
[0156] In certain embodiments het.sup.1 is pyrrolidinyl, which may
be unsubstituted or substituted once with R.sup.d.
[0157] In certain embodiments het.sup.1 is piperazinyl, which may
be unsubstituted or substituted once with R.sup.d.
[0158] In certain embodiments het.sup.1 is piperidinyl, which may
be unsubstituted or substituted once with R.sup.d.
[0159] In certain embodiments het.sup.1 is morpholinyl.
[0160] In certain embodiments het.sup.1 is oxetanyl.
[0161] In certain embodiments p is 0.
[0162] In certain embodiments p is 1.
[0163] In certain embodiments p is 2.
[0164] In certain embodiments q is 0.
[0165] In certain embodiments q is 2.
[0166] In certain embodiments q is 3.
[0167] In certain embodiments q is 4.
[0168] In certain embodiments R.sup.e is hydrogen.
[0169] In certain embodiments R.sup.e is C.sub.1-C.sub.6alkyl.
[0170] In certain embodiments R.sup.f is hydrogen.
[0171] In certain embodiments R.sup.f is C.sub.1-C.sub.6alkyl.
[0172] In certain embodiments Het.sup.2 is tetrahydropyranyl.
[0173] In certain embodiments Het.sup.2 is azetidinyl which may be
unsubstituted or substituted once with C.sub.1-C.sub.6alkyl.
[0174] In certain embodiments Het.sup.2 is pyrrolidinyl which may
be unsubstituted or substituted once with C.sub.1-C.sub.6alkyl.
[0175] In certain embodiments R is selected from:
##STR00004## ##STR00005## ##STR00006##
[0176] In certain embodiments R.sup.1 is selected from:
##STR00007## ##STR00008##
[0177] In certain embodiments, the subject compounds are of formula
(II)
##STR00009##
or a stereoisomer or pharmaceutically acceptable salt thereof,
wherein R.sup.1, R.sup.2 and R.sup.6 are as defined herein.
[0178] In certain embodiments, the subject compounds are of formula
(III)
##STR00010##
or a stereoisomer or pharmaceutically acceptable salt thereof,
wherein R.sup.1, R.sup.2 and R.sup.6 are as defined herein.
[0179] In certain embodiments, the subject compounds are of formula
(IV)
##STR00011##
or a stereoisomer or pharmaceutically acceptable salt thereof,
wherein X is --O-- or --S--, R.sup.9 is hydrogen or
C.sub.1-C.sub.6alkyl, and R is as defined herein.
[0180] In certain embodiments, the subject compounds are of formula
(V)
##STR00012##
or a stereoisomer or pharmaceutically acceptable salt thereof,
wherein R.sup.2 and R.sup.d are as defined herein.
[0181] In certain embodiments, the subject compounds are of formula
(VI)
##STR00013##
or a stereoisomer or pharmaceutically acceptable salt thereof,
wherein R.sup.2 and R.sup.d are as defined herein.
[0182] Also provided is a pharmaceutical composition comprising a
JAK inhibitor as described herein, or a pharmaceutically acceptable
salt thereof, and a pharmaceutically acceptable carrier, dilient or
excipient.
[0183] Also provided is the use of a JAK inhibitor as described
herein, or a pharmaceutically acceptable salt thereof in therapy,
such as in the treatment of an inflammatory disease (e.g., asthma).
Also provided is the use of a JAK inhibitor as described herein or
a pharmaceutically acceptable salt thereof for the preparation of a
medicament for the treatment of an inflammatory disease. Also
provided is a method of preventing, treating or lessening the
severity of a disease or condition responsive to the inhibition of
a Janus kinase activity in a patient, comprising administering to
the patient a therapeutically effective amount of a JAK inhibitor
as described herein or a pharmaceutically acceptable salt
thereof.
[0184] In one embodiment the disease or condition for therapy is
cancer, polycythemia vera, essential thrombocytosis, myelofibrosis,
chronic myelogenous leukemia (CML), rheumatoid arthritis,
inflammatory bowel syndrome, Crohn's disease, psoriasis, contact
dermatitis or delayed hypersensitivity reactions.
[0185] In one embodiment the use of a JAK inhibitor as described
herein or a pharmaceutically acceptable salt thereof, for the
treatment of cancer, polycythemia vera, essential thrombocytosis,
myelofibrosis, chronic myelogenous leukemia (CML), rheumatoid
arthritis, inflammatory bowel syndrome, Crohn's disease, psoriasis,
contact dermatitis or delayed hypersensitivity reactions is
provided.
[0186] In one embodiment a composition that is formulated for
administration by inhalation is provided.
[0187] In one embodiment a metered dose inhaler that comprises a
compound of the present invention or a pharmaceutically acceptable
salt thereof is provided.
[0188] In one embodiment a JAK inhibitor as described herein or a
pharmaceutically acceptable salt thereof is at least ten-times more
potent as an inhibitor of JAK1 than as an inhibitor of LRRK2.
[0189] In one embodiment a method for treating hair loss in a
mammal comprising administering a JAK inhibitor as described herein
or a pharmaceutically acceptable salt thereof to the mammal is
provided.
[0190] In one embodiment the use of a JAK inhibitor as described
herein or a pharmaceutically acceptable salt thereof for the
treatment of hair loss is provided.
[0191] In one embodiment the use of a JAK inhibitor as described
herein or a pharmaceutically acceptable salt thereof to prepare a
medicament for treating hair loss in a mammal is provided.
[0192] Compounds of the invention may contain one or more
asymmetric carbon atoms. Accordingly, the compounds may exist as
diastereomers, enantiomers or mixtures thereof. The syntheses of
the compounds may employ racemates, diastereomers or enantiomers as
starting materials or as intermediates. Mixtures of particular
diastereomeric compounds may be separated, or enriched in one or
more particular diastereomers, by chromatographic or
crystallization methods. Similarly, enantiomeric mixtures may be
separated, or enantiomerically enriched, using the same techniques
or others known in the art. Each of the asymmetric carbon or
nitrogen atoms may be in the R or S configuration and both of these
configurations are within the scope of the invention.
[0193] In the structures shown herein, where the stereochemistry of
any particular chiral atom is not specified, then all stereoisomers
are contemplated and included as the compounds of the invention.
Where stereochemistry is specified by a solid wedge or dashed line
representing a particular configuration, then that stereoisomer is
so specified and defined. Unless otherwise specified, if solid
wedges or dashed lines are used, relative stereochemistry is
intended.
[0194] Another aspect includes prodrugs of the compounds described
herein, including known amino-protecting and carboxy-protecting
groups which are released, for example hydrolyzed, to yield the
compound of the present invention under physiologic conditions.
[0195] The term "prodrug" refers to a precursor or derivative form
of a pharmaceutically active substance that is less efficacious to
the patient compared to the parent drug and is capable of being
enzymatically or hydrolytically activated or converted into the
more active parent form. See, e.g., Wilman, "Prodrugs in Cancer
Chemotherapy" Biochemical Society Transactions, 14, pp. 375-382,
615th Meeting Belfast (1986) and Stella et al., "Prodrugs: A
Chemical Approach to Targeted Drug Delivery," Directed Drug
Delivery, Borchardt et al., (ed.), pp. 247-267, Humana Press
(1985). Prodrugs include, but are not limited to,
phosphate-containing prodrugs, thiophosphate-containing prodrugs,
sulfate-containing prodrugs, peptide-containing prodrugs, D-amino
acid-modified prodrugs, glycosylated prodrugs,
.beta.-lactam-containing prodrugs, optionally substituted
phenoxyacetamide-containing prodrugs or optionally substituted
phenylacetamide-containing prodrugs, and 5-fluorocytosine and
5-fluorouridine prodrugs.
[0196] A particular class of prodrugs are compounds in which a
nitrogen atom in an amino, amidino, aminoalkyleneamino,
iminoalkyleneamino or guanidino group is substituted with a hydroxy
group, an alkylcarbonyl (--CO--R) group, an alkoxycarbonyl
(--CO--OR), or an acyloxyalkyl-alkoxycarbonyl
(--CO--O--R--O--CO--R) group where R is a monovalent or divalent
group, for example alkyl, alkylene or aryl, or a group having the
Formula --C(O)--O--CP1P2-haloalkyl, where P1 and P2 are the same or
different and are hydrogen, alkyl, alkoxy, cyano, halogen, alkyl or
aryl. In a particular embodiment, the nitrogen atom is one of the
nitrogen atoms of the amidino group. Prodrugs may be prepared by
reacting a compound with an activated group, such as acyl groups,
to bond, for example, a nitrogen atom in the compound to the
exemplary carbonyl of the activated acyl group. Examples of
activated carbonyl compounds are those containing a leaving group
bonded to the carbonyl group, and include, for example, acyl
halides, acyl amines, acyl pyridinium salts, acyl alkoxides, acyl
phenoxides such as p-nitrophenoxy acyl, dinitrophenoxy acyl,
fluorophenoxy acyl, and difluorophenoxy acyl. The reactions are
generally carried out in inert solvents at reduced temperatures
such as -78.degree. C. to about 50.degree. C. The reactions may
also be carried out in the presence of an inorganic base, for
example potassium carbonate or sodium bicarbonate, or an organic
base such as an amine, including pyridine, trimethylamine,
triethylamine, triethanolamine, or the like.
[0197] Additional types of prodrugs are also encompassed. For
instance, a free carboxyl group of a JAK inhibitor as described
herein can be derivatized as an amide or alkyl ester. As another
example, compounds of the present invention comprising free hydroxy
groups can be derivatized as prodrugs by converting the hydroxy
group into a group such as, but not limited to, a phosphate ester,
hemisuccinate, dimethylaminoacetate, or
phosphoryloxymethyloxycarbonyl group, as outlined in Fleisher, D.
et al., (1996) Improved oral drug delivery: solubility limitations
overcome by the use of prodrugs Advanced Drug Delivery Reviews,
19:115. Carbamate prodrugs of hydroxy and amino groups are also
included, as are carbonate prodrugs, sulfonate esters and sulfate
esters of hydroxy groups. Derivatization of hydroxy groups as
(acyloxy)methyl and (acyloxy)ethyl ethers, wherein the acyl group
can be an alkyl ester optionally substituted with groups including,
but not limited to, ether, amine and carboxylic acid
functionalities, or where the acyl group is an amino acid ester as
described above, are also encompassed. Prodrugs of this type are
described in J. Med. Chem., (1996), 39:10. More specific examples
include replacement of the hydrogen atom of the alcohol group with
a group such as (C.sub.1-C.sub.6)alkanoyloxymethyl,
1-((C.sub.1-C.sub.6)alkanoyloxy)ethyl,
1-methyl-1-((C.sub.1-C.sub.6)alkanoyloxy)ethyl,
(C.sub.1-C.sub.6)alkoxycarbonyloxymethyl,
N--(C.sub.1-C.sub.6)alkoxycarbonylaminomethyl, succinoyl, (C.sub.1.
C.sub.6)alkanoyl, alpha-amino(C.sub.1-C.sub.4)alkanoyl, arylacyl
and alpha-aminoacyl, or alpha-aminoacyl-alpha-aminoacyl, where each
alpha-aminoacyl group is independently selected from the naturally
occurring L-amino acids, P(O)(OH).sub.2,
--P(O)(O(C.sub.1-C.sub.6)alkyl).sub.2 or glycosyl (the radical
resulting from the removal of a hydroxyl group of the hemiacetal
form of a carbohydrate).
[0198] "Leaving group" refers to a portion of a first reactant in a
chemical reaction that is displaced from the first reactant in the
chemical reaction. Examples of leaving groups include, but are not
limited to, halogen atoms, alkoxy and sulfonyloxy groups. Example
sulfonyloxy groups include, but are not limited to,
alkylsulfonyloxy groups (for example methyl sulfonyloxy (mesylate
group) and trifluoromethylsulfonyloxy (triflate group)) and
arylsulfonyloxy groups (for example p-toluenesulfonyloxy (tosylate
group) and p-nitrosulfonyloxy (nosylate group)).
Synthesis of Janus Kinase Inhibitor Compounds
[0199] Compounds may be synthesized by synthetic routes described
herein. In certain embodiments, processes well-known in the
chemical arts can be used, in addition to, or in light of, the
description contained herein. The starting materials are generally
available from commercial sources such as Aldrich Chemicals
(Milwaukee, Wis.) or are readily prepared using methods well known
to those skilled in the art (e.g., prepared by methods generally
described in Louis F. Fieser and Mary Fieser, Reagents for Organic
Synthesis, v. 1-19, Wiley, N.Y. (1967-1999 ed.), Beilsteins
Handbuch der organischen Chemie, 4, Aufl. ed. Springer-Verlag,
Berlin, including supplements (also available via the Beilstein
online database)), or Comprehensive Heterocyclic Chemistry, Editors
Katrizky and Rees, Pergamon Press, 1984.
[0200] Compounds may be prepared singly or as compound libraries
comprising at least 2, for example 5 to 1,000 compounds, or 10 to
100 compounds. Libraries of compounds may be prepared by a
combinatorial `split and mix` approach or by multiple parallel
syntheses using either solution phase or solid phase chemistry, by
procedures known to those skilled in the art. Thus according to a
further aspect of the invention there is provided a compound
library comprising at least 2 compounds of the present
invention.
[0201] For illustrative purposes, reaction Schemes depicted below
provide routes for synthesizing the compounds of the present
invention as well as key intermediates. For a more detailed
description of the individual reaction steps, see the Examples
section below. Those skilled in the art will appreciate that other
synthetic routes may be used. Although some specific starting
materials and reagents are depicted in the Schemes and discussed
below, other starting materials and reagents can be substituted to
provide a variety of derivatives or reaction conditions. In
addition, many of the compounds prepared by the methods described
below can be further modified in light of this disclosure using
conventional chemistry well known to those skilled in the art.
[0202] In the preparation of compounds of the present invention,
protection of remote functionality (e.g., primary or secondary
amine) of intermediates may be necessary. The need for such
protection will vary depending on the nature of the remote
functionality and the conditions of the preparation methods.
Suitable amino-protecting groups include acetyl, trifluoroacetyl,
benzyl, phenylsulfonyl, t-butoxycarbonyl (BOC), benzyloxycarbonyl
(CBz) and 9-fluorenylmethyleneoxycarbonyl (Fmoc). The need for such
protection is readily determined by one skilled in the art. For a
general description of protecting groups and their use, see T. W.
Greene, Protective Groups in Organic Synthesis, John Wiley &
Sons, New York, 1991.
[0203] Other conversions commonly used in the synthesis of
compounds of the present invention, and which can be carried out
using a variety of reagents and conditions, include the following:
[0204] (1) Reaction of a carboxylic acid with an amine to form an
amide. Such a transformation can be achieved using various reagents
known to those skilled in the art but a comprehensive review can be
found in Tetrahedron, 2005, 61, 10827-10852. [0205] (2) Reaction of
a primary or secondary amine with an aryl halide or pseudo halide,
e.g., a triflate, commonly known as a "Buchwald-Hartwig
cross-coupling," can be achieved using a variety of catalysts,
ligands and bases. A review of these methods is provided in
Comprehensive Organic Name Reactions and Reagents, 2010, 575-581.
[0206] (3) A palladium cross-coupling reaction between an aryl
halide and a vinyl boronic acid or boronate ester. This
transformation is a type of "Suzuki-Miyaura cross-coupling," a
class of reaction that has been thoroughly reviewed in Chemical
Reviews, 1995, 95(7), 2457-2483. [0207] (4) The hydrolysis of an
ester to give the corresponding carboxylic acid is well known to
those skilled in the art and conditions include: for methyl and
ethyl esters, the use of a strong aqueous base such as lithium,
sodium or potassium hydroxide or a strong aqueous mineral acid such
as HCl; for a tert-butyl ester, hydrolysis would be carried out
using acid, for example, HCl in dioxane or trifluoroacetic acid
(TFA) in dichloromethane (DCM).
##STR00014## ##STR00015##
[0208] Reaction Scheme 1 illustrates a synthesis for compounds of
the invention. Compound 1 can be arylated under palladium catalyzed
conditions to generate compound 2. The nitro group of compound 2
can be reduced with conditions such as iron and ammonium chloride
to generate amino aniline 3. Amide bond coupling with commercially
available pyrazolo[1,5-a]pyrimidine-3-carboxylic acid in the
presence of a coupling reagent such as, but not limited to, PyAOP,
with an organic base such as, but not limited to DIPEA, and DMAP in
an organic solvent such as, but not limited to, DMF provides
compound 4. Removal of the SEM protecting group of compound 4,
using an acid such as, but not limited to HCl in a solvent such as,
but not limited to, 1,4-dioxane, results in compound 5. Compound 5
can then undergo N-alkylation with a protected tetrazole compound.
In certain embodiments the protecting group PG may be
tetrahydropyranyl, so that the tetrazole reagent is
5-(chloromethyl)-2-(tetrahydro-2H-pyran-2-yl)-2H-tetrazole to
afford compound 6. Deprotection using HCl or other acid yields
tetrazole compound 7. Compound 7 in turn undergoes an N-alkylation
by reaction with R'--X wherein X is halo such as iodo, to provide
compound 8 and compound 9, which are compounds of formula (I) in
accordance with the invention
##STR00016## ##STR00017##
[0209] Reaction Scheme 2 illustrates a synthesis for compounds of
Formula VII therein. Commercially available
4-(difluoromethoxy)phenol can be treated with a brominating agent
such as, but not limited to, NBS in a solvent such as, but not
limited, acetic acid to yield 12. Difluoromethylation of 12 to form
compound 13 can be accomplished by treatment of compound 12 with
diethyl (bromodifluoromethyl)phosphonate with a base such as, but
not limited to, aqueous potassium hydroxide in a solvent such as,
but not limited to, acetonitrile. Compound 13 can be treated with
4-nitro-1-[[2-(trimethylsilyl)ethoxy]methyl]-1H-pyrazole
4-bromo-1-(difluoromethoxy)-2-iodobenzene under palladium catalyzed
conditions with a base such as, but not limited to, potassium
carbonate in a solvent such as, but not limited to, DMA to generate
compound 14. The nitro group of compound 14 can be reduced with
conditions such as iron and ammonium chloride to generate amino
pyrazole 15. Amide bond coupling of compound 15 with commercially
available pyrazolo[1,5-a]pyrimidine-3-carboxylic acid in the
presence of a coupling reagent such as, but not limited to, PyAOP,
with an organic base such as, but not limited to, DIPEA and DMAP in
a solvent such as, but not limited to, DMF provides compound 16.
Removal of the SEM protecting group of compound 16 can be
accomplished with an acid such as, but not limited to, HCl in an
organic solvent such as, but not limited to, 1,4-dioxane to
generate intermediate compound 5 which may be used to make
compounds of the invention as shown in Reaction Scheme 1.
[0210] It will be appreciated that where appropriate functional
groups exist, compounds of various formulae or any intermediates
used in their preparation may be further derivatised by one or more
standard synthetic methods employing condensation, substitution,
oxidation, reduction, or cleavage reactions. Particular
substitution approaches include conventional alkylation, arylation,
heteroarylation, acylation, sulfonylation, halogenation, nitration,
formylation and coupling procedures.
[0211] In a further example, primary amine or secondary amine
groups may be converted into amide groups (--NHCOR' or --NRCOR') by
acylation. Acylation may be achieved by reaction with an
appropriate acid chloride in the presence of a base, such as
triethylamine, in a suitable solvent, such as dichloromethane, or
by reaction with an appropriate carboxylic acid in the presence of
a suitable coupling agent such HATU
(0-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate) in a suitable solvent such as dichloromethane.
Similarly, amine groups may be converted into sulphonamide groups
(--NHSO.sub.2R' or --NR''SO.sub.2R') groups by reaction with an
appropriate sulphonyl chloride in the presence of a suitable base,
such as triethylamine, in a suitable solvent such as
dichloromethane. Primary or secondary amine groups can be converted
into urea groups (--NHCONR'R'' or --NRCONR'R'') by reaction with an
appropriate isocyanate in the presence of a suitable base such as
triethylamine, in a suitable solvent, such as dichloromethane.
[0212] An amine (--NH.sub.2) may be obtained by reduction of a
nitro (--NO.sub.2) group, for example by catalytic hydrogenation,
using for example hydrogen in the presence of a metal catalyst, for
example palladium on a support such as carbon in a solvent such as
ethyl acetate or an alcohol e.g., methanol. Alternatively, the
transformation may be carried out by chemical reduction using for
example a metal, e.g., tin or iron, in the presence of an acid such
as hydrochloric acid.
[0213] In a further example, amine (--CH.sub.2NH.sub.2) groups may
be obtained by reduction of nitriles (--CN), for example by
catalytic hydrogenation using for example hydrogen in the presence
of a metal catalyst, for example palladium on a support such as
carbon, or Raney nickel, in a solvent such as an ether e.g., a
cyclic ether such as tetrahydrofuran, at an appropriate
temperature, for example from about -78.degree. C. to the reflux
temperature of the solvent.
[0214] In a further example, amine (--NH.sub.2) groups may be
obtained from carboxylic acid groups (--CO.sub.2H) by conversion to
the corresponding acyl azide (--CON.sub.3), Curtius rearrangement
and hydrolysis of the resultant isocyanate (--N.dbd.C.dbd.O).
[0215] Aldehyde groups (--CHO) may be converted to amine groups
(--CH.sub.2NR'R'')) by reductive amination employing an amine and a
borohydride, for example sodium triacetoxyborohydride or sodium
cyanoborohydride, in a solvent such as a halogenated hydrocarbon,
for example dichloromethane, or an alcohol such as ethanol, where
necessary in the presence of an acid such as acetic acid at around
ambient temperature.
[0216] In a further example, aldehyde groups may be converted into
alkenyl groups (--CH.dbd.CHR') by the use of a Wittig or
Wadsworth-Emmons reaction using an appropriate phosphorane or
phosphonate under standard conditions known to those skilled in the
art.
[0217] Aldehyde groups may be obtained by reduction of ester groups
(such as --CO.sub.2Et) or nitriles (--CN) using diisobutylaluminium
hydride in a suitable solvent such as toluene. Alternatively,
aldehyde groups may be obtained by the oxidation of alcohol groups
using any suitable oxidising agent known to those skilled in the
art.
[0218] Ester groups (--CO.sub.2R') may be converted into the
corresponding acid group (--CO.sub.2H) by acid- or base-catalused
hydrolysis, depending on the nature of R. If R is t-butyl,
acid-catalysed hydrolysis can be achieved for example by treatment
with an organic acid such as trifluoroacetic acid in an aqueous
solvent, or by treatment with an inorganic acid such as
hydrochloric acid in an aqueous solvent.
[0219] Carboxylic acid groups (--CO.sub.2H) may be converted into
amides (CONHR' or --CONR' R'') by reaction with an appropriate
amine in the presence of a suitable coupling agent, such as HATU,
in a suitable solvent such as dichloromethane.
[0220] In a further example, carboxylic acids may be homologated by
one carbon (i.e --CO.sub.2H to --CH.sub.2CO.sub.2H) by conversion
to the corresponding acid chloride (--COCl) followed by
Arndt-Eistert synthesis.
[0221] In a further example, --OH groups may be generated from the
corresponding ester (e.g., --CO.sub.2R'), or aldehyde (--CHO) by
reduction, using for example a complex metal hydride such as
lithium aluminium hydride in diethyl ether or tetrahydrofuran, or
sodium borohydride in a solvent such as methanol. Alternatively, an
alcohol may be prepared by reduction of the corresponding acid
(--CO.sub.2H), using for example lithium aluminium hydride in a
solvent such as tetrahydrofuran, or by using borane in a solvent
such as tetrahydrofuran.
[0222] Alcohol groups may be converted into leaving groups, such as
halogen atoms or sulfonyloxy groups such as an alkylsulfonyloxy,
e.g., trifluoromethylsulfonyloxy or arylsulfonyloxy, e.g.,
p-toluenesulfonyloxy group using conditions known to those skilled
in the art. For example, an alcohol may be reacted with thioyl
chloride in a halogenated hydrocarbon (e.g., dichloromethane) to
yield the corresponding chloride. A base (e.g., triethylamine) may
also be used in the reaction.
[0223] In another example, alcohol, phenol or amide groups may be
alkylated by coupling a phenol or amide with an alcohol in a
solvent such as tetrahydrofuran in the presence of a phosphine,
e.g., triphenylphosphine and an activator such as diethyl-,
diisopropyl, or dimethylazodicarboxylate. Alternatively alkylation
may be achieved by deprotonation using a suitable base e.g., sodium
hydride followed by subsequent addition of an alkylating agent,
such as an alkyl halide.
[0224] Aromatic halogen substituents in the compounds may be
subjected to halogen-metal exchange by treatment with a base, for
example a lithium base such as n-butyl or t-butyl lithium,
optionally at a low temperature, e.g., around -78.degree. C., in a
solvent such as tetrahydrofuran, and then quenched with an
electrophile to introduce a desired substituent. Thus, for example,
a formyl group may be introduced by using N,N-dimethylformamide as
the electrophile. Aromatic halogen substituents may alternatively
be subjected to metal (e.g., palladium or copper) catalysed
reactions, to introduce, for example, acid, ester, cyano, amide,
aryl, heteraryl, alkenyl, alkynyl, thio- or amino substituents.
Suitable procedures which may be employed include those described
by Heck, Suzuki, Stille, Buchwald or Hartwig.
[0225] Aromatic halogen substituents may also undergo nucleophilic
displacement following reaction with an appropriate nucleophile
such as an amine or an alcohol. Advantageously, such a reaction may
be carried out at elevated temperature in the presence of microwave
irradiation.
Methods of Separation
[0226] In each of the exemplary Schemes it may be advantageous to
separate reaction products from one another or from starting
materials. The desired products of each step or series of steps is
separated or purified (hereinafter separated) to the desired degree
of homogeneity by the techniques common in the art. Typically such
separations involve multiphase extraction, crystallization or
trituration from a solvent or solvent mixture, distillation,
sublimation, or chromatography. Chromatography can involve any
number of methods including, for example: reverse-phase and normal
phase; size exclusion; ion exchange; supercritical fluid; high,
medium, and low pressure liquid chromatography methods and
apparatus; small scale analytical; simulated moving bed (SMB) and
preparative thin or thick layer chromatography, as well as
techniques of small scale thin layer and flash chromatography.
[0227] Another class of separation methods involves treatment of a
mixture with a reagent selected to bind to or render otherwise
separable a desired product, unreacted starting material, reaction
by product, or the like. Such reagents include adsorbents or
absorbents such as activated carbon, molecular sieves, ion exchange
media, or the like. Alternatively, the reagents can be acids in the
case of a basic material, bases in the case of an acidic material,
binding reagents such as antibodies, binding proteins, selective
chelators such as crown ethers, liquid/liquid ion extraction
reagents (LIX), or the like.
[0228] Selection of appropriate methods of separation depends on
the nature of the materials involved. Example separation methods
include boiling point, and molecular weight in distillation and
sublimation, presence or absence of polar functional groups in
chromatography, stability of materials in acidic and basic media in
multiphase extraction, and the like. One skilled in the art will
apply techniques most likely to achieve the desired separation.
[0229] Diastereomeric mixtures can be separated into their
individual diastereoisomers on the basis of their physical chemical
differences by methods well known to those skilled in the art, such
as by chromatography or fractional crystallization. Enantiomers can
be separated by converting the enantiomeric mixture into a
diastereomeric mixture by reaction with an appropriate optically
active compound (e.g., chiral auxiliary such as a chiral alcohol or
Mosher's acid chloride), separating the diastereoisomers and
converting (e.g., hydrolyzing) the individual diastereoisomers to
the corresponding pure enantiomers. Also, some of the compounds of
the present invention may be atropisomers (e.g., substituted
biaryls) and are considered as part of this invention. Enantiomers
can also be separated by use of a chiral HPLC column or
supercritical fluid chromatography.
[0230] A single stereoisomer, e.g., an enantiomer, substantially
free of its stereoisomer may be obtained by resolution of the
racemic mixture using a method such as formation of diastereomers
using optically active resolving agents (Eliel, E. and Wilen, S.,
Stereochemistry of Organic Compounds, John Wiley & Sons, Inc.,
New York, 1994; Lochmuller, C. H., J. Chromatogr., 113(3):283-302
(1975)). Racemic mixtures of chiral compounds of the invention can
be separated and isolated by any suitable method, including: (1)
formation of ionic, diastereomeric salts with chiral compounds and
separation by fractional crystallization or other methods, (2)
formation of diastereomeric compounds with chiral derivatizing
reagents, separation of the diastereomers, and conversion to the
pure stereoisomers, and (3) separation of the substantially pure or
enriched stereoisomers directly under chiral conditions. See: Drug
Stereochemistry, Analytical Methods and Pharmacology, Irving W.
Wainer, Ed., Marcel Dekker, Inc., New York (1993).
[0231] Diastereomeric salts can be formed by reaction of
enantiomerically pure chiral bases such as brucine, quinine,
ephedrine, strychnine, .alpha.-methyl-.beta.-phenylethylamine
(amphetamine), and the like with asymmetric compounds bearing
acidic functionality, such as carboxylic acid and sulfonic acid.
The diastereomeric salts may be induced to separate by fractional
crystallization or ionic chromatography. For separation of the
optical isomers of amino compounds, addition of chiral carboxylic
or sulfonic acids, such as camphorsulfonic acid, tartaric acid,
mandelic acid, or lactic acid can result in formation of the
diastereomeric salts.
[0232] Alternatively, the substrate to be resolved is reacted with
one enantiomer of a chiral compound to form a diastereomeric pair
(Eliel, E. and Wilen, S., Stereochemistry of Organic Compounds,
John Wiley & Sons, Inc., New York, 1994, p. 322).
Diastereomeric compounds can be formed by reacting asymmetric
compounds with enantiomerically pure chiral derivatizing reagents,
such as menthyl derivatives, followed by separation of the
diastereomers and hydrolysis to yield the pure or enriched
enantiomer. A method of determining optical purity involves making
chiral esters, such as a menthyl ester, e.g., (-) menthyl
chloroformate in the presence of base, or Mosher ester,
.alpha.-methoxy-.alpha.-(trifluoromethyl)phenyl acetate (Jacob, J.
Org. Chem. 47:4165 (1982)), of the racemic mixture, and analyzing
the NMR spectrum for the presence of the two atropisomeric
enantiomers or diastereomers. Stable diastereomers of atropisomeric
compounds can be separated and isolated by normal- and
reverse-phase chromatography following methods for separation of
atropisomeric naphthyl-isoquinolines (WO 96/15111, incorporated
herein by reference). By method (3), a racemic mixture of two
enantiomers can be separated by chromatography using a chiral
stationary phase (Chiral Liquid Chromatography W. J. Lough, Ed.,
Chapman and Hall, New York, (1989); Okamoto, J. of Chromatogr.
513:375-378 (1990)). Enriched or purified enantiomers can be
distinguished by methods used to distinguish other chiral molecules
with asymmetric carbon atoms, such as optical rotation and circular
dichroism. The absolute stereochemistry of chiral centers and
enatiomers can be determined by x-ray crystallography.
[0233] Positional isomers and intermediates for their synthesis may
be observed by characterization methods such as NMR and analytical
HPLC. For certain compounds where the energy barrier for
interconversion is sufficiently high, the E and Z isomers may be
separated, for example by preparatory HPLC.
Pharmaceutical Compositions and Administration
[0234] The compounds with which the invention is concerned are JAK
kinase inhibitors, such as JAK1 inhibitors, and are useful in the
treatment of several diseases, for example, inflammatory diseases,
such as asthma.
[0235] Accordingly, another embodiment provides pharmaceutical
compositions or medicaments containing a compound of the invention
or a pharmaceutically acceptable salt thereof, and a
pharmaceutically acceptable carrier, diluent or excipient, as well
as methods of using the compounds of the invention to prepare such
compositions and medicaments.
[0236] In one example, a compound of the invention or a
pharmaceutically acceptable salt thereof may be formulated by
mixing at ambient temperature at the appropriate pH, and at the
desired degree of purity, with physiologically acceptable carriers,
i.e., carriers that are non-toxic to recipients at the dosages and
concentrations employed into a galenical administration form. The
pH of the formulation depends mainly on the particular use and the
concentration of compound, but typically ranges anywhere from about
3 to about 8. In one example, a compound of the invention or a
pharmaceutically acceptable salt thereof is formulated in an
acetate buffer, at pH 5. In another embodiment, the compounds of
the present invention are sterile. The compound may be stored, for
example, as a solid or amorphous composition, as a lyophilized
formulation or as an aqueous solution.
[0237] Compositions are formulated, dosed, and administered in a
fashion consistent with good medical practice. Factors for
consideration in this context include the particular disorder being
treated, the particular mammal being treated, the clinical
condition of the individual patient, the cause of the disorder, the
site of delivery of the agent, the method of administration, the
scheduling of administration, and other factors known to medical
practitioners.
[0238] It will be understood that the specific dose level for any
particular patient will depend upon a variety of factors including
the activity of the specific compound employed, the age, body
weight, general health, sex, diet, time of administration, route of
administration, rate of excretion, drug combination and the
severity of the particular disease undergoing treatment. Optimum
dose levels and frequency of dosing will be determined by clinical
trial, as is required in the pharmaceutical art. In general, the
daily dose range for oral administration will lie within the range
of from about 0.001 mg to about 100 mg per kg body weight of a
human, often 0.01 mg to about 50 mg per kg, for example 0.1 to 10
mg per kg, in single or divided doses. In general, the daily dose
range for inhaled administration will lie within the range of from
about 0.1 .mu.g to about 1 mg per kg body weight of a human,
preferably 0.1 .mu.g to 50 .mu.g per kg, in single or divided
doses. On the other hand, it may be necessary to use dosages
outside these limits in some cases.
[0239] The compounds of the invention or a pharmaceutically
acceptable salt thereof, may be administered by any suitable means,
including oral, topical (including buccal and sublingual), rectal,
vaginal, transdermal, parenteral, subcutaneous, intraperitoneal,
intrapulmonary, intradermal, intrathecal, inhaled and epidural and
intranasal, and, if desired for local treatment, intralesional
administration. Parenteral infusions include intramuscular,
intravenous, intraarterial, intraperitoneal, or subcutaneous
administration. In some embodiments, inhaled administration is
employed.
[0240] The compounds of the present invention or a pharmaceutically
acceptable salt thereof, may be administered in any convenient
administrative form, e.g., tablets, powders, capsules, lozenges,
granules, solutions, dispersions, suspensions, syrups, sprays,
vapors, suppositories, gels, emulsions, patches, etc. Such
compositions may contain components conventional in pharmaceutical
preparations, e.g., diluents (e.g., glucose, lactose or mannitol),
carriers, pH modifiers, buffers, sweeteners, bulking agents,
stabilizing agents, surfactants, wetting agents, lubricating
agents, emulsifiers, suspending agents, preservatives,
antioxidants, opaquing agents, glidants, processing aids,
colorants, perfuming agents, flavoring agents, other known
additives as well as further active agents.
[0241] Suitable carriers and excipients are well known to those
skilled in the art and are described in detail in, e.g., Ansel,
Howard C., et al., Ansel's Pharmaceutical Dosage Forms and Drug
Delivery Systems. Philadelphia: Lippincott, Williams & Wilkins,
2004; Gennaro, Alfonso R., et al. Remington: The Science and
Practice of Pharmacy. Philadelphia: Lippincott, Williams &
Wilkins, 2000; and Rowe, Raymond C. Handbook of Pharmaceutical
Excipients. Chicago, Pharmaceutical Press, 2005. For example,
carriers include solvents, dispersion media, coatings, surfactants,
antioxidants, preservatives (e.g., antibacterial agents, antifungal
agents), isotonic agents, absorption delaying agents, salts,
preservatives, drugs, drug stabilizers, gels, binders, excipients,
disintegration agents, lubricants, sweetening agents, flavoring
agents, dyes, such like materials and combinations thereof, as
would be known to one of ordinary skill in the art (see, for
example, Remington's Pharmaceutical Sciences, pp 1289-1329, 1990).
Except insofar as any conventional carrier is incompatible with the
active ingredient, its use in the therapeutic or pharmaceutical
compositions is contemplated. Exemplary excipients include
dicalcium phosphate, mannitol, lactose, starch, magnesium stearate,
sodium saccharine, cellulose, magnesium carbonate or combinations
thereof. A pharmaceutical composition may comprise different types
of carriers or excipients depending on whether it is to be
administered in solid, liquid or aerosol form, and whether it need
to be sterile for such routes of administration.
[0242] For example, tablets and capsules for oral administration
may be in unit dose presentation form, and may contain conventional
excipients such as binding agents, for example syrup, acacia,
gelatin, sorbitol, tragacanth, or polyvinyl-pyrrolidone; fillers,
for example, lactose, sugar, maize-starch, calcium phosphate,
sorbitol or glycine; tabletting lubricant, for example, magnesium
stearate, talc, polyethylene glycol or silica; disintegrants, for
example, potato starch, or acceptable wetting agents such as sodium
lauryl sulfate. The tablets may be coated according to methods well
known in normal pharmaceutical practice. Oral liquid preparations
may be in the form of, for example, aqueous or oily suspensions,
solutions, emulsions, syrups or elixirs, or may be presented as a
dry product for reconstitution with water or other suitable vehicle
before use. Such liquid preparations may contain conventional
additives such as suspending agents, for example, sorbitol, syrup,
methyl cellulose, glucose syrup, gelatin hydrogenated edible fats;
emulsifying agents, for example, lecithin, sorbitan monooleate, or
acacia; non-aqueous vehicles (which may include edible oils), for
example, almond oil, fractionated coconut oil, oily esters such as
glycerine, propylene glycol, or ethyl alcohol; preservatives, for
example, methyl or propyl p-hydroxybenzoate or sorbic acid, and if
desired conventional flavoring or coloring agents.
[0243] For topical application to the skin, a compound may be made
up into a cream, lotion or ointment. Cream or ointment formulations
which may be used for the drug are conventional formulations well
known in the art, for example as described in standard textbooks of
pharmaceutics such as the British Pharmacopoeia.
[0244] Compounds of the invention or a pharmaceutically acceptable
salt thereof may also be formulated for inhalation, for example, as
a nasal spray, or dry powder or aerosol inhalers. For delivery by
inhalation, the compound is typically in the form of
microparticles, which can be prepared by a variety of techniques,
including spray-drying, freeze-drying and micronisation. Aerosol
generation can be carried out using, for example, pressure-driven
jet atomizers or ultrasonic atomizers, such as by using
propellant-driven metered aerosols or propellant-free
administration of micronized compounds from, for example,
inhalation capsules or other "dry powder" delivery systems.
[0245] By way of example, a composition of the invention may be
prepared as a suspension for delivery from a nebulizer or as an
aerosol in a liquid propellant, for example, for use in a
pressurized metered dose inhaler (PMDI). Propellants suitable for
use in a PMDI are known to the skilled person, and include CFC-12,
HFA-134a, HFA-227, HCFC-22 (CCl.sub.2F2) and HFA-152 (CH.sub.4F2
and isobutane).
[0246] In some embodiments, a composition of the invention is in
dry powder form, for delivery using a dry powder inhaler (DPI).
Many types of DPI are known.
[0247] Microparticles for delivery by administration may be
formulated with excipients that aid delivery and release. For
example, in a dry powder formulation, microparticles may be
formulated with large carrier particles that aid flow from the DPI
into the lung. Suitable carrier particles are known, and include
lactose particles; they may have a mass median aerodynamic diameter
of, for example, greater than 90 .mu.m.
[0248] In the case of an aerosol-based formulation, an example
is:
[0249] Compound of the invention* 24 mg/canister
[0250] Lecithin, NF Liq. Conc. 1.2 mg/canister
[0251] Trichlorofluoromethane, NF 4.025 g/canister
[0252] Dichlorodifluoromethane, NF 12.15 g/canister.
[0253] * or a pharmaceutically acceptable salt thereof
[0254] A compound of the invention or a pharmaceutically acceptable
salt thereof may be dosed as described depending on the inhaler
system used. In addition to the compound, the administration forms
may additionally contain excipients as described above, or, for
example, propellants (e.g., Frigen in the case of metered
aerosols), surface-active substances, emulsifiers, stabilizers,
preservatives, flavorings, fillers (e.g., lactose in the case of
powder inhalers) or, if appropriate, further active compounds.
[0255] For the purposes of inhalation, a large number of systems
are available with which aerosols of optimum particle size can be
generated and administered, using an inhalation technique which is
appropriate for the patient. In addition to the use of adaptors
(spacers, expanders) and pear-shaped containers (e.g.,
Nebulator.RTM., Volumatic.RTM.), and automatic devices emitting a
puffer spray (Autohaler@), for metered aerosols, in the case of
powder inhalers in particular, a number of technical solutions are
available (e.g., Diskhaler.RTM., Rotadisk.RTM., Turbohaler.RTM. or
the inhalers, for example, as described in U.S. Pat. No. 5,263,475,
incorporated herein by reference). Additionally, compounds of the
invention or a pharmaceutically acceptable salt thereof, may be
delivered in multi-chamber devices thus allowing for delivery of
combination agents.
[0256] The compound or a pharmaceutically acceptable salt thereof,
may also be administered parenterally in a sterile medium.
Depending on the vehicle and concentration used, the compound can
either be suspended or dissolved in the vehicle. Advantageously,
adjuvants such as a local anaesthetic, preservative or buffering
agent can be dissolved in the vehicle.
Targeted Inhaled Drug Delivery
[0257] Compounds of the present invention may be used for targeted
inhaled delivery. Optimisation of drugs for delivery to the lung by
topical (inhaled) administration has been recently reviewed
(Cooper, A. E. et al. Curr. Drug Metab. 2012, 13, 457-473).
[0258] Due to limitations in delivery devices, the dose of an
inhaled drug may be limited humans, which necessitates highly
potent molecules with good lung pharmacokinetic properties. High
potency against the target of interest is especially important for
an inhaled drug due to factors such as the limited amount of drug
that can be delivered in a single puff from an inhaler, and the
safety concerns related to a high aerosol burden in the lung (for
example, cough or irritancy). For example, in some embodiments, a
Ki of about 0.5 nM or less in a JAK1 biochemical assay such as
described herein, and an IC50 of about 20 nM or less in a JAK1
dependent cell based assay such as described herein, may be
desirable for an inhaled JAK1 inhibitor. In other embodiments, the
projected human dose of a compound of the present invention, or a
pharmaceutically acceptable salt thereof, is at least two times
less than the projected human dose of a compound known in the art.
Accordingly, in some embodiments, compounds (or a pharmaceutically
acceptable salt thereof) described herein demonstrate such potency
values. The procedures below were used to evaluate the subject
compounds for potential use as inhaled drugs.
[0259] IL13 signaling. IL13 signaling is strongly implicated in
asthma pathogenesis. IL13 is a cytokine that requires active JAK1
in order to signal. Thus, inhibition of JAK1 also inhibits IL13
signaling, which may provide benefit to asthma patients. Inhibition
of IL13 signaling in an animal model (e.g., a mouse model) may
predict future benefit to human asthmatic patients. Thus, it may be
beneficial for an inhaled JAK1 inhibitor to show suppression of
IL13 signaling in an animal model. Methods of measuring such
suppression are known in the art. For example, as discussed herein
and is known in the art, JAK1-dependent STAT6 phosphorylation is
known to be a downstream consequence of IL13 stimulation.
Accordingly, in some embodiments, compounds (or a pharmaceutically
acceptable salt thereof) described herein demonstrate inhibition of
lung pSTAT6 induction. To examine pharmacodynamic effects on pSTAT6
levels, compounds of the invention were co-dosed intra-nasally with
1 .mu.g IL13 to female Balb/c mice. Compounds were formulated in
0.2% (v:v) Tween 80 in saline and mixed 1:1 (v:v) with IL13
immediately prior to administration. The intranasal doses were
administered to lightly anaesthetised (isoflurane) mice by
dispensing a fixed volume (50 .mu.L) directly into the nostrils by
pipette to achieve the target dose level (3 mg/kg, 1 mg/kg, 0.3
mg/kg, 0.1 mg/kg). At 0.25 hr post dose, blood samples (ca 0.5 mL)
were collected by cardiac puncture and plasma generated by
centrifugation (1500 g, 10 min, +4.degree. C.). The lungs were
perfused with chilled phosphate buffer saline (PBS), weighed and
snap frozen in liquid nitrogen. All samples were stored at ca.
-80.degree. C. until analysis. Defrosted lung samples were weighed
and homogenised following the addition of 2 mL HPLC grade water for
each gram of tissue, using an Omni-Prep Bead Ruptor at 4.degree. C.
Plasma and lung samples were extracted by protein precipitation
with three volumes of acetonitrile containing Tolbutamide (50
ng/mL) and Labetalol (25 ng/mL) as analytical internal standards.
Following vortex mixing and centrifugation for 30 minutes at 3200 g
and 4.degree. C., the supernatants were diluted appropriately
(e.g., 1:1 v:v) with HPLC grade water in a 96-well plate.
Representative aliquots of plasma and lung samples were assayed for
the parent compound by LC-MS/MS, against a series of matrix matched
calibration and quality control standards. The standards were
prepared by spiking aliquots of control Balb/c mouse plasma or lung
homogenate (2:1 in HPLC grade water) with test compound and
extracting as described for the experimental samples. A lung:plasma
ratio was determined as the ratio of the mean lung concentration
(.mu.M) to the mean plasma concentration (.mu.M) at the sampling
time (0.25 h
[0260] To measure pSTAT6 levels, mouse lungs were stored frozen at
-80.degree. C. until assay and homogenised in 0.6 ml ice-cold cell
lysis buffer (Cell Signalling Technologies, catalogue #9803S)
supplemented with 1 mM PMSF and a cocktail of protease (Sigma
Aldrich, catalogue #P8340) and phosphatase (Sigma Aldrich,
catalogue #P5726 and P0044) inhibitors. Samples were centrifuged at
16060.times.g for 4 minutes at 4.degree. C. to remove tissue debris
and protein concentration of homogenates determined using the
Pierce BCA protein assay kit (catalogue #23225). Samples were
diluted to a protein concentration of 5 mg/ml in ice-cold distilled
water and assayed for pSTAT6 levels by Meso Scale Discovery
electro-chemiluminescent immuno-assay. Briefly, 5 .mu.l/well 150
.mu.g/ml STAT6 capture antibody (R&D Systems, catalogue #MAB
2169) was coated onto 96 well Meso Scale Discovery High Binding
Plates (catalogue #L15XB-3) and air-dried for 5 hours at room
temperature. Plates were blocked by addition of 150 .mu.l/well 30
mg/ml Meso Scale Discovery Blocker A (catalogue #R93BA-4) and
incubation for 2 hours at room temperature on a microplate shaker.
Blocked plates were washed 4 times with Meso Scale Discovery TRIS
wash buffer (catalogue #R61TX-1), followed by transfer of 50
.mu.l/well lung homogenate to achieve a protein loading of 250
.mu.g/well. Assay plates were incubated overnight at 4.degree. C.
and washed 4 times with TRIS wash buffer before addition of 25
.mu.l/well 2.5 .mu.g/ml sulfotag-labelled pSTAT6 detection antibody
(BD Pharmingen, catalogue #558241) for 2 hours at room temperature
on a microplate shaker. Plates were washed 4 times with TRIS wash
buffer and 150 .mu.l/well 1.times. Meso Scale Discovery Read Buffer
T (catalogue #R92TC-1) added. Lung homogenate pSTAT6 levels were
quantified by detection of electro-chemiluminescence on a Meso
Scale Discovery SECTOR S 600 instrument.
[0261] JAK and JAK2 inhibition Compounds inhibiting both JAK1 and
JAK2 are potentially useful for treatment of different types of
asthma. Selectivity between JAK1 and JAK2 may also be important for
an inhaled JAK1 inhibitor. For example, GMCSF
(granulocyte-macrophage colony-stimulating factor) is a cytokine
that signals through JAK2 exclusively. Neutralization of GMCSF
activity is associated with pulmonary alveolar proteinosis (PAP) in
the lung. However, submaximal JAK2 suppression does not appear to
be associated with PAP. Thus, even modest JAK1 vs JAK2 selectivity,
or approximately equivalent inhibition of JAK1 and JAK2, may be of
benefit in avoiding full suppression of the GMCSF pathway and
avoiding PAP. For example, in certain embodiments compounds that
are equipotent for JAK1 and JAK2 are desirable. In other
embodiments compounds with about 2.times.-5.times. selectivity for
JAK1 over JAK2 may be of benefit for an inhaled JAK1 inhibitor.
Accordingly, in some embodiments, compounds (or a pharmaceutically
acceptable salt thereof) described herein demonstrate such
selectivity. Methods of measuring JAK1 and JAK2 selectivity are
known in the art, and information can also be found in the Examples
herein.
[0262] Kinase profiling. Additionally, it may be desirable for an
inhaled JAK1 or JAK1/JAK2 inhibitor to be selective over one or
more other kinases to reduce the likelihood of potential toxicity
due to off-target kinase pathway suppression. Thus, it may also be
of benefit for an inhaled JAK1 inhibitor to be selective against a
broad panel of non-JAK kinases, such as in protocols available from
ThermoFisher Scientific's SelectScreen.TM. Biochemical Kinase
Profiling Service using Adapta.TM. Screening Protocol Assay
Conditions (Revised Jul. 29, 2016), LanthaScreen.TM. Eu Kinase
Binding Assay Screening Protocol and Assay Conditions (Revised Jun.
7, 2016), and/or Z'LYTE.TM. Screening Protocol and Assay Conditions
(Revised Sep. 16, 2016). For example, a compound of the present
invention, or a pharmaceutically acceptable salt thereof, exhibits
at least 50-fold selectivity for JAK1 versus a panel of non-JAK
kinases. Accordingly, in some embodiments, compounds (or a
pharmaceutically acceptable salt thereof) described herein
demonstrate such selectivity.
[0263] Cytotoxicity assays. Hepatocyte toxicity, general
cytotoxicity or cytotoxicity of unknown mechanism is an undesirable
feature for a potential drug, including inhaled drugs. It may be of
benefit for an inhaled JAK or JAK1/JAK2 inhibitor to have low
intrinsic cytotoxicity against various cell types. Typical cell
types used to assess cytotoxicity include both primary cells such
as human hepatocytes, and proliferating established cell lines such
as Jurkat and HEK-293. Accordingly, in some embodiments, compounds
(or a pharmaceutically acceptable salt thereof) described herein
demonstrate such values. Methods of measuring cytotoxicity are
known in the art. In some embodiments, compounds described herein
were tested as follows:
[0264] (a) Jurkat and HEK293T cells were maintained at a sub
confluent density in T175 flasks. Cells were plated at 450 cells/45
.mu.l medium in Greiner 384 well black/clear tissue culture treated
plates. (Greiner Catalog #781091). After dispensing cells, plates
were equilibrated at room temperature for 30 minutes. After 30
minutes at room temperature, cells were incubated overnight at
37.degree. C. in a CO.sub.2 and humidity controlled incubator. The
following day, cells were treated with compounds diluted in 100%
DMSO (final DMSO concentration on cells=0.5%) with a 10 point
dose-response curve with a top concentration of 50 .mu.M. Cells and
compounds were then incubated for 72 hours overnight at 37.degree.
C. in a CO.sub.2 and humidity controlled incubator. After 72 hours
of incubation, viability was measured using CellTiterGlo.RTM.
(Promega Catalog #G7572) to all wells. After incubation at room
temperature for 20 minutes, plates were read on EnVision.TM.
(Perkin Elmer Life Sciences) using luminescence mode;
[0265] (b) using human primary hepatocytes: the test compound was
prepared as a 10 mM solution in DMSO. Additionally, a positive
control such as Chlorpromazine was prepared as a 10 mM solution in
DMSO. Test compounds were typically assessed using a 7-point dose
response curve with 2-fold dilutions. Typically, the maximum
concentration tested was 50-100 .mu.M. The top concentration was
typically dictated by solubility of the test compound.
Cryopreserved primary human hepatocytes (BioreclamationIVT) (lot
IZT) were thawed in InVitroGro.TM. HT thawing media
(BioreclamationIVT) at 37.degree. C., pelleted and resuspended.
Hepatocyte viability was assessed by Trypan blue exclusion and
cells were plated in black-walled, BioCoat.TM. collagen 384-well
plates (Corning BD) at a density of 13,000 cells/well in
InVitroGro.TM. CP plating media supplemented with 1% Torpedo.TM.
Antibiotic Mix (BioreclamationIVT) and 5% fetal bovine serum. Cells
were incubated overnight for 18 hours (37.degree. C., 5% CO.sub.2)
prior to treatment. Following 18 hours incubation, plating media
was removed and hepatocytes were treated with compounds diluted in
InVitroGro.TM. HI incubation media containing 1% Torpedo.TM.
Antibiotic Mix and 1% DMSO (serum-free conditions). Hepatocytes
were treated with test compounds at concentrations such as 0.78,
1.56, 3.12, 6.25, 12.5, 25, and 50 .mu.M at a final volume of 50
.mu.L. A positive control (e.g., Chlorpromazine) was included in
the assay, typically at the same concentrations as the test
compound. Additional cells were treated with 1% DMSO as a vehicle
control. All treatments were for a 48 hour time period (at
37.degree. C., 5% CO.sub.2) and each treatment condition was
performed in triplicate. Following 48 hours of compound treatment,
CellTiter-Glo@ cell viability assay (Promega) was used as the
endpoint assay to measure ATP content as a determination of cell
viability. The assay was performed according to manufacture
instructions. Luminescence was determined on an EnVision.TM.
Muliplate Reader (PerkinElmer, Waltham, Mass., USA). Luminescence
data was normalized to vehicle (1% DMSO) control wells. Inhibition
curves and IC.sub.50 estimates were generated by non-linear
regression of log-transformed inhibitor concentrations (7-point
serial dilutions including vehicle) vs. normalized response with
variable Hill slopes, with top and bottom constrained to constant
values of 100 and 0, respectively (GraphPad Prism.TM., GraphPad
Software, La Jolla, Calif., USA).
[0266] hERG Inhibition. Inhibition of the hERG (human
ether-a-go-go-related gene) potassium channel may lead to long QT
syndrome and cardiac arrhythmias. Although plasma levels of an
inhaled JAK1 or JAK1/JAK2 inhibitor are expected to be low,
lung-deposited compound exiting the lung via pulmonary absorption
into the bloodstream will circulate directly to the heart. Thus,
local heart concentrations of an inhaled JAK1 inhibitor may be
transiently higher than total plasma levels, particularly
immediately after dosing. Thus, it may be of benefit to minimize
hERG inhibition of an inhaled JAK1 inhibitor. For example, in some
embodiments, a hERG IC50 greater than 30.times. over the free-drug
plasma Cmax is preferred. Accordingly, in some embodiments,
compounds (or a pharmaceutically acceptable salt thereof) of the
invention demonstrate minimized hERG inhibition under conditions
such as:
[0267] (a) using hERG 2pt automatic patch clamp conditions to
examine in vitro effects of a compound on hERG expressed in
mammalizan cells, evaluated at room temperature using the QPatch
HT.RTM. (Sophion Bioscience A/S, Denmark), an automatic parallel
patch clamp system. In some cases, compounds were tested at only
one or two concentrations such as 1 or 10 uM. In other cases a more
extensive concentration response relationship was established to
allow estimation of IC50. For example, test compound concentrations
were selected to span the range of approximately 10-90% inhibition
in half-log increments. Each test article concentration was tested
in two or more cells (n.gtoreq.2). The duration of exposure to each
test article concentration was a minimum of 3 minutes; and/or
[0268] (b) those described in WO 2014/074775, in the Examples,
under "Effect on Cloned hERG Potassium Channels Expressed in
Mammalian Cells," a ChanTest.TM., a Charles River Company, protocol
with the following changes: cells stably expressing hERG were held
at -80 mV. Onset and steady state inhibition of hERG potassium
current due to compound were measured using a pulse pattern with
fixed amplitudes (conditioning prepulse: +20 mV for 1 s;
repolarizing test ramepto -90 mV (-0.5 V/s) repeated at 5 s
intervals). Each recording ended with a final application of a
supramaximal concentration of a reference substance, E-4021 (500
nM) (Charles River Company). The remaining uninhibited current was
subtracted off-line digitially from the data to determine the
potency of the test substance for hERG inhibition.
[0269] CYP (cytochrome P450) inhibition assay. CYP inhibition may
not be a desirable feature for an inhaled JAK1 or JAK1/JAK2
inhibitor. For example, a reversible or time dependent CYP
inhibitor may cause an undesired increase in its own plasma levels,
or in the plasma levels of other co-administered drugs (drug-drug
interactions). Additionally, time dependent CYP inhibition is
sometimes caused by biotransformation of parent drug to a reactive
metabolite. Such reactive metabolites may covalently modify
proteins, potentially leading to toxicity. Thus, minimizing
reversible and time dependent CYP inhibition may be of benefit to
an inhaled JAK1 inhibitor. Accordingly, in some embodiments,
compounds (or a pharmaceutically acceptable salt thereof) of the
present invention demonstrate minimal or no reversible and/or time
dependent CYP inhibition. Methods of measuring CYP inhibition are
known in the art. CYP inhibition of compounds described herein were
assessed over a concentration range of 0.16-10 uM of compound using
pooled (n=150) human liver microsomes (Corning, Tewksbury, Mass.)
using methods previously reported (Halladay et al., Drug Metab.
Lett. 2011, 5, 220-230). Incubation duration and protein
concentration was dependent on the CYP isoform and the probe
substrate/metabolites assessed. The following
substrate/metabolites, and incubation times and protein
concentrations for each CYP were used: CYP1A2,
phenacetin/acetaminophen, 30 min, 0.03 mg/ml protein; CYP2C9,
warfarin/7-hydroxywarfarin, 30 min, 0.2 mg/ml protein; CYP2C19,
mephenytoin/4-hydroxymephenytoin, 40 min, 0.2 mg/ml protein;
CYP2D6, dextromethorphan/dextrorphan, 10 min, 0.03 mg/ml protein;
CYP3A4, midazolam/1-hydroxymidazolam, 10 min, 0.03 mg/ml protein
and CYP3A4 testosterone/6-hydroxytestosterone, 10 min, 0.06 mg/ml
protein. These conditions were previously determined to be in the
linear rate of formation for the CYP-specific metabolites. All
reaction were initiated with 1 mM NADPH and terminated by the
addition of 0.1% formic acid in acetonitrile containing appropriate
stable labeled internal standard. Samples were analyzed by
LC-MS/MS.
[0270] Mouse lung tissue binding. A high bound fraction or
percentage of JAK1/JAK2 inhibitors to lung tissue may be
undesirable since it can reduce the amount of free drug available
to inhibit JAK1 or JAK2.
[0271] (a) Tissue binding experiments were performed in triplicate
(n=3) using a Single-Use RED Plate by following the standard
protocol. Initially, individual drugs were spiked to tissue
homogenates (pH 7.4) to achieve a final concentration of 1 .mu.M,
and then 300 .mu.L of drug-tissue homogenate mixtures were
transferred to the donor wells of the RED plate which was
pre-loaded with 500 .mu.L phosphate buffer saline (133 mM) on the
receiver wells. The RED plate was sealed with a gas permeable
membrane and placed in a shaking incubator (450 rpm, VWR
Symphony.TM.) for 6 hr at 37.degree. C. with 5% CO.sub.2. At the
end of incubation, aliquots of 30 .mu.L samples were taken out of
the RED device and matrix equalized with an equal volume of tissue
homogenates or buffer, and resulting samples were then immediately
quenched with ice cold acetonitrile (sample:acetonitrile 1:4)
containing either propranolol or labetalol as an internal standard.
After shaking for 15 min at 500 rpm on a Thermo Scientific Compact
Digital MicroPlate Shaker, all samples were then subjected to
centrifugation at 3700 rpm for 15 min (Beckman Coulter Allegra X
12R) to remove plasma protein. Subsequently, supernatants were
collected and then diluted with an equal volume of water prior to
LC-MS/MS analysis.
[0272] (b) In an alternate procedure, the extent of lung tissue
binding of test compound to mouse lung homogenate may also be
determined by equilibrium dialysis using Pierce RED (rapid
equilibrium dialysis) devices (Fisher Scientific 89811 &
89809). A 10 mM solution of compound in DMSO was prepared and
diluted to 1 mM with DMSO. An aliquot of this 1 mM (4 .mu.L) was
added into lung homogenate (dilution factor of 1:9, lung
tissue:potassium phosphate buffer (0.05 M, pH 7.4)) to give a final
compound incubation concentration of 5 .mu.M with solvent
accounting for 0.5% (v/v) of the final incubation volume.
[0273] For each assay, the percentage lung tissue bound was
determined in triplicate. Lung homogenate (200 .mu.L) was loaded
into one side of a RED device insert, in triplicate, and 350 .mu.L
of potassium phosphate buffer was loaded into the other side. The
RED devices were sealed and incubated for 4 hours at ca. 37.degree.
C. on an orbital shaker (150 rpm).
[0274] Following incubation, an aliquot of lung homogenate (8
.mu.L) and an aliquot of dialysate (72 .mu.L) were matrix matched
(lung homogenate with 72 .mu.L phosphate buffer, dialysate with 8
.mu.L lung homogenate) ahead of analysis. Protein was precipitated
from the samples with the addition of 160 .mu.L of acetonitrile
containing internal standard. The same matrix matching and protein
precipitation procedure was performed on lung homogenate aliquots
sampled at the start of the experiment (t=0 min samples), for the
assessment of the mass balance. The quenched samples were
centrifuged (4000 rpm, 30 min, 4.degree. C.) and the resultant
supernatant diluted with water (3:1 (v/v), supernatant: water) and
the samples analysed for parent compound by liquid chromatography
mass spectrometry assay.
[0275] The unbound fraction (fu) in lung homogenate was determined
from the ratio of the dialysate to homogenate peak area, corrected
to take into account the lung homogenate dilution (D) to enable an
estimate of whole lung tissue binding using the following
equations:
Undiluted fu=(1/D)/[((1/Apparent fu)-1)+(1/D)]
Corrected fraction bound (%)=(1-undiluted fu)*100
[0276] Kinetic solubility. Good aqueous solubility for JAK1/JAK2
inhibitors for inhaled delivery may be desirable. In one procedure
to measure kinetic solubility, 4 .mu.L of a 10 mM DMSO stock
solution of test compound is added to 196 .mu.L of pH 7.4 phosphate
buffered saline solution in a Millipore Multiscreen.RTM. 96-well
filter plate to give a test concentration of 200 .mu.M with 2%
residual DMSO. The filter plate is sealed with aluminum sealing
film and shaken at room temperature for 24 hours, then the mixtures
were vacuum filtered into a clean 96-well plate. The filtrate
samples are diluted by a factor of two using pH 7.4 phosphate
buffered saline solution, then 5 .mu.L of the resulting solutions
are analyzed by ultra-high performance liquid chromatography
(UHPLC) with chemiluminescence nitrogen detection (CLND) and
ultraviolet (UV) detection at a wavelength of 254 nm. Sample
concentration is typically quantified by the CLND intensity, which
is related to the number of nitrogens in the compound. The UV
detection is used primarily to confirm sample purity except for
rare cases when test compounds contained no nitrogens. In those
cases, a compound-specific calibration curve is collected based on
UV absorbance. This curve is then used to determine sample
concentration.
[0277] Lipophilicity. Lipophilicity is relevant to the solubility,
absorption, tissue penetration, protein binding, distribution, and
ADME and PK properties generally of potential drugs. Calculated log
P (c Log P), the logarithm of partition coefficient of a compound
between n-octanol and water (i.e. log(concentration of the compound
in n-octanol/concentration of compound in water), thus can be an
important consideration for JAK1/JAK inhibitors for inhaled
delivery.
[0278] Liver Microsomal Stability. In order to minimize systemic
exposure of an inhaled JAK1/JAK2 inhibitor it may be beneficial to
optimize for rapid metabolism in the liver. Liver microsomal
stability assay was performed on a BioCel 1200 liquid handling
workstation (Agilent Technologies, Santa Clara, Calif.). Compounds
(1.0 .mu.M) were incubated for 5 min at 37.degree. C. in 100 .mu.L
of a reaction mixture containing 100 mM phosphate buffer (pH 7.4)
and 0.5 mg/mL liver microsomes and 1 mM NADPH. At different time
intervals (0, 20, 40 and 60 min), aliquots of 20 .mu.L of reaction
mixtures were taken out and mixed with 4-volumes of acetonitrile
(ACN) containing 0.1 M propranolol as the internal standard to stop
metabolic reaction. The samples were then centrifuged at
3250.times.g for 40 min to remove precipitated protein. The
supernatants were subsequently transferred to a new 96-well plate
and diluted 2-fold using deionized water, and were then subjected
to LC-MS/MS analysis using an ABI Sciex 5500 QTRAP.RTM. mass
spectrometer (Applied Biosystems, Foster City, Calif.) coupled with
a Agilent 1260 HPLC (Agilent Technologies, Santa Clara, Calif.).
Percent of remaining was calculated using peak area ratio of test
compound to the internal standard at different time points relative
to the control (T=0 min). See B. Williamson, C. Wilson, G. Dagnell,
R J Riley. Harmonised high throughput microsomal stability assay.
J. Pharmacol. Toxicol. Methods. 2017; 84:31-36.
[0279] Solid state properties. For compounds destined to be
delivered via dry powder inhalation there is also a requirement to
be able to generate crystalline forms of the compound that can be
micronized to 1-5 .mu.m in size. Particle size is an important
determinant of lung deposition of an inhaled compound. Particles
with a diameter of less than 5 microns (.mu.m) are typically
defined as respirable. Particles with a diameter larger than 5
.mu.m are more likely to deposit in the oropharynx and are
correspondingly less likely to be deposited in the lung.
Additionally, fine particles with a diameter of less than 1 .mu.m
are more likely than larger particles to remain suspended in air,
and are correspondingly more likely to be exhaled from the lung.
Thus, a particle diameter of 1-5 .mu.m may be of benefit for an
inhaled medication whose site of action is in the lung. Typical
methods used to measure particle size include laser diffraction and
cascade impaction. Typical values used to define particle size
include: [0280] D10, D50, and D90. These are measurements of
particle diameter that indicate, respectively, 10%, 50%, or 90% of
the sample is below that value. For example a D50 of 3 .mu.m
indicates that 50% of the sample is below 3 .mu.m in size. [0281]
Mass mean aerodynamic diameter (MMAD). MMAD is the diameter at
which 50% of the particles by mass are larger and 50% are smaller.
MMAD is a measure of central tendency. [0282] Geometric Standard
Deviation (GSD). GSD is a measure of the magnitude in dispersity
from the MMAD, or the spread in aerodynamic particle size
distribution. A common formulation for inhaled medications is a dry
powder preparation including the active pharmaceutical ingredient
(API) blended with a carrier such as lactose with or without
additional additives such as magnesium stearate. For this
formulation and others, it may be beneficial for the API itself to
possess properties that allow it to be milled to a respirable
particle size of 1-5 .mu.m. Agglomeration of particles is to be
avoided, which can be measured by methods known in the art, such as
examining D90 values under different pressure conditions.
Accordingly, in some embodiments, compounds (or a pharmaceutically
acceptable salt thereof) of the present invention can be prepared
with such a respirable particle size with little or no
agglomeration.
[0283] As for crystallinity, for some formulations of inhaled
drugs, including lactose blends, it is important that API of a
specific crystalline form is used. Crystallinity and crystalline
form may impact many parameters relevant to an inhaled drug
including but not limited to: chemical and aerodynamic stability
over time, compatibility with inhaled formulation components such
as lactose, hygroscopicity, lung retention, and lung irritancy.
Thus, a stable, reproducible crystalline form may be of benefit for
an inhaled drug. Additionally, the techniques used to mill
compounds to the desired particle size are often energetic and may
cause low melting crystalline forms to convert to other crystalline
forms, or to become fully or partially amorphous. A crystalline
form with a melting point of less than 150.degree. C. may be
incompatible with milling, while a crystalline form with a melting
point of less than 100.degree. C. is likely to be non-compatible
with milling. Thus, it may be beneficial for an inhaled medication
to have a melting point of at least greater than 100.degree. C.,
and ideally greater than 150.degree. C. Accordingly, in some
embodiments, compounds (or a pharmaceutically acceptable salt
thereof) described herein demonstrate such properties.
[0284] Additionally, minimizing molecular weight may help to lower
the efficacious dose of an inhaled JAK1 inhibitor. Lower molecular
weight results in a corresponding higher number of molecules per
unit mass of the active pharmaceutical ingredient (API). Thus, it
may be of benefit to find the smallest molecular weight inhaled
JAK1 inhibitor that retains all the other desired properties of an
inhaled drug.
[0285] Finally, the compound needs to maintain a sufficient
concentration in the lung over a given time period so as to be able
to exert a pharmacological effect of the desired duration, and for
pharmacological targets where systemic inhibition of said target is
undesired, to have a low systemic exposure. The lung has an
inherently high permeability to both large molecules (proteins,
peptides) as well as small molecules with concomitant short lung
half-lives, thus it may be necessary to attenuate the lung
absorption rate through modification of one or more features of the
compounds: minimizing membrane permeability, optimized pKa, cLogP,
solubility, dissolution rate, or introducing a degree of basicity
(e.g., introducing an amine) into the compound to enhance binding
to the phospholipid-rich lung tissue or through trapping in acidic
sub-cellular compartments such as lysosomes (pH 5). Methods of
measuring such properties are known in the art.
[0286] Accordingly, in some embodiments, a compound of the present
invention (or a pharmaceutically acceptable salt thereof) favorably
exhibits one or more of the above features. Further, in some
embodiments, a compound of the present invention favorably exhibits
one or more of these features relative to a compound known in the
art--this may be particularly true for compounds of the art
intended as oral drugs versus inhaled. For example, compounds with
rapid oral absorption are typically poorly retained in the lung on
inhalation.
Methods of Treatment with and Uses of Janus Kinase Inhibitors
[0287] The compounds of the present invention or a pharmaceutically
acceptable salt thereof, inhibit the activity of a Janus kinase,
such as JAK1 kinase. For example, a compound or a pharmaceutically
acceptable salt thereof inhibits the phosphorylation of signal
transducers and activators of transcription (STATs) by JAK1 kinase
as well as STAT mediated cytokine production. Compounds of the
present invention are useful for inhibiting JAK1 kinase activity in
cells through cytokine pathways, such as IL-6, IL-15, IL-7, IL-2,
IL-4, IL-9, IL-10, IL-13, IL-21, G-CSF, IFNalpha, IFNbeta or
IFNgamma pathways. Accordingly, in one embodiment is provided a
method of contacting a cell with a compound of the present
invention or a pharmaceutically acceptable salt thereof, to inhibit
a Janus kinase activity in the cell (e.g., JAK1 activity).
[0288] The compounds can be used for the treatment of immunological
disorders driven by aberrant IL-6, IL-15, IL-7, IL-2, IL-4, IL9,
IL-10, IL-13, IL-21, G-CSF, IFNalpha, IFNbeta or IFNgamma cytokine
signaling.
[0289] Accordingly, one embodiment includes a compound of the
present invention or a pharmaceutically acceptable salt thereof,
for use in therapy.
[0290] In some embodiments, there is provided use of a compound of
the present invention or a pharmaceutically acceptable salt
thereof, in the treatment of an inflammatory disease.
[0291] Further provided is use of a compound of the present
invention or a pharmaceutically acceptable salt thereof for the
preparation of a medicament for the treatment of an inflammatory
disease, such as asthma. Also provided is a compound of the present
invention or a pharmaceutically acceptable salt thereof for use in
the treatment of an inflammatory disease, such as asthma.
[0292] Another embodiment includes a method of preventing, treating
or lessening the severity of a disease or condition, such as
asthma, responsive to the inhibition of a Janus kinase activity,
such as JAK1 kinase activity, in a patient. The method can include
the step of administering to a patient a therapeutically effective
amount of a compound of the present invention or a pharmaceutically
acceptable salt thereof. In one embodiment, the disease or
condition responsive to the inhibition of a Janus kinase, such as
JAK1 kinase, is asthma.
[0293] In one embodiment, the disease or condition is cancer,
stroke, diabetes, hepatomegaly, cardiovascular disease, multiple
sclerosis, Alzheimer's disease, cystic fibrosis, viral disease,
autoimmune diseases, atherosclerosis, restenosis, psoriasis,
rheumatoid arthritis, inflammatory bowel disease, asthma, allergic
disorders, inflammation, neurological disorders, a hormone-related
disease, conditions associated with organ transplantation (e.g.,
transplant rejection), immunodeficiency disorders, destructive bone
disorders, proliferative disorders, infectious diseases, conditions
associated with cell death, thrombin-induced platelet aggregation,
liver disease, pathologic immune conditions involving T cell
activation, CNS disorders or a myeloproliferative disorder.
[0294] In one embodiment, the inflammatory disease is rheumatoid
arthritis, psoriasis, asthma, inflammatory bowel disease, contact
dermatitis or delayed hypersensitivity reactions. In one
embodiment, the autoimmune disease is rheumatoid arthritis, lupus
or multiple sclerosis.
[0295] In another embodiment, a compound of the present invention
or a pharmaceutically acceptable salt thereof may be used to treat
lung diseases such as a fibrotic lung disease or an interstitial
lung disease (e.g., an interstitial pneumonia). In some
embodiments, a compound of the present invention or a
pharmaceutically acceptable salt thereof may be used to treat
idiopathic pulmonary fibrosis (IPF), systemic sclerosis
interstitial lung disease (SSc-ILD)), nonspecific interstitial
pneumonia (NSIP), rheumatoid arthritis-associated interstitial lung
disease (RA-ILD), sarcoidosis, hypersensitivity pneumonitis, or ILD
secondary to connective tissue disease beyond scleroderma (e.g.,
polymyositis, dermatomyositis, rheumatoid arthritis, systemic lupus
erythematosus (SLE), or mixed connective tissue disease).
[0296] In one embodiment, the cancer is breast, ovary, cervix,
prostate, testis, penile, genitourinary tract, seminoma, esophagus,
larynx, gastric, stomach, gastrointestinal, skin, keratoacanthoma,
follicular carcinoma, melanoma, lung, small cell lung carcinoma,
non-small cell lung carcinoma (NSCLC), lung adenocarcinoma,
squamous carcinoma of the lung, colon, pancreas, thyroid,
papillary, bladder, liver, biliary passage, kidney, bone, myeloid
disorders, lymphoid disorders, hairy cells, buccal cavity and
pharynx (oral), lip, tongue, mouth, salivary gland, pharynx, small
intestine, colon, rectum, anal, renal, prostate, vulval, thyroid,
large intestine, endometrial, uterine, brain, central nervous
system, cancer of the peritoneum, hepatocellular cancer, head
cancer, neck cancer, Hodgkin's or leukemia.
[0297] In one embodiment, the disease is a myeloproliferative
disorder. In one embodiment, the myeloproliferative disorder is
polycythemia vera, essential thrombocytosis, myelofibrosis or
chronic myelogenous leukemia (CML).
[0298] Another embodiment includes the use of a compound of the
present invention or a pharmaceutically acceptable salt thereof,
for the manufacture of a medicament for the treatment of a disease
described herein (e.g., an inflammatory disorder, an immunological
disorder or cancer). In one embodiment, the invention provides a
method of treating a disease or condition as described herein e.g.,
an inflammatory disorder, an immunological disorder or cancer) by
targeting inhibition of a JAK kinase, such as JAK1.
Combination Therapy
[0299] The compounds may be employed alone or in combination with
other agents for treatment. The second or further (e.g., third)
compound of a pharmaceutical composition or dosing regimen
typically has complementary activities to the compound of this
invention such that they do not adversely affect each other. Such
agents are suitably present in combination in amounts that are
effective for the purpose intended. The compounds may be
administered together in a unitary pharmaceutical composition or
separately and, when administered separately this may occur
simultaneously or sequentially. Such sequential administration may
be close or remote in time.
[0300] For example, other compounds may be combined with a compound
of the present invention or a pharmaceutically acceptable salt
thereof for the prevention or treatment of inflammatory diseases,
such as asthma. Suitable therapeutic agents for a combination
therapy include, but are not limited to: an adenosine A2A receptor
antagonist; an anti-infective; a non-steroidal Glucocorticoid
Receptor (GR Receptor) agonist; an antioxidant; a .quadrature.2
adrenoceptor agonist; a CCR1 antagonist; a chemokine antagonist
(not CCR1); a corticosteroid; a CRTh2 antagonist; a DP1 antagonist;
a formyl peptide receptor antagonist; a histone deacetylase
activator; a chloride channel hCLCA1 blocker; an epithelial sodium
channel blocker (ENAC blocker; an inter-cellular adhesion molecule
1 blocker (ICAM blocker); an IKK2 inhibitor; a JNK inhibitor; a
transient receptor potential ankyrin 1 (TRPA1) inhibitor; a
Bruton's tyrosine kinase (BTK) inhibitor (e.g., fenebrutinib); a
spleen tyrosine kinase (SYK) inhibitor; a tryptase-beta antibody;
an ST2 receptor antibody (e.g., AMG 282); a cyclooxygenase
inhibitor (COX inhibitor); a lipoxygenase inhibitor; a leukotriene
receptor antagonist; a dual .quadrature.2 adrenoceptor agonist/M3
receptor antagonist (MABA compound); a MEK-1 inhibitor; a
myeloperoxidase inhibitor (MPO inhibitor); a muscarinic antagonist;
a p38 MAPK inhibitor; a phosphodiesterase PDE4 inhibitor; a
phosphatidylinositol 3-kinase .delta. inhibitor (PI3-kinase .delta.
inhibitor); a phosphatidylinositol 3-kinase .quadrature. inhibitor
(P3-kinase .quadrature. inhibitor); a peroxisome proliferator
activated receptor agonist (PPAR .quadrature. agonist); a protease
inhibitor; a retinoic acid receptor modulator (RAR .quadrature.
modulator); a statin; a thromboxane antagonist; a TLR7 receptor
agonist; or a vasodilator.
[0301] In addition, a compound of the present invention or a
pharmaceutically acceptable salt thereof, may be combined with: (1)
corticosteroids, such as alclometasone dipropionate, amelometasone,
beclomethasone dipropionate, budesonide, butixocort propionate,
biclesonide, clobetasol propionate, desisobutyrylciclesonide,
dexamethasone, etiprednol dicloacetate, fluocinolone acetonide,
fluticasone furoate, fluticasone propionate, loteprednol etabonate
(topical) or mometasone furoate; (2) .beta.2-adrenoreceptor
agonists such as salbutamol, albuterol, terbutaline, fenoterol,
bitolterol, carbuterol, clenbuterol, pirbuterol, rimoterol,
terbutaline, tretoquinol, tulobuterol and long acting
.beta.2-adrenoreceptor agonists such as metaproterenol,
isoproterenol, isoprenaline, salmeterol, indacaterol, formoterol
(including formoterol fumarate), arformoterol, carmoterol,
abediterol, vilanterol trifenate, or olodaterol; (3)
corticosteroid/long acting .beta.2 agonist combination products
such as salmeterol/fluticasone propionate (Advair, also sold as
Seretide.RTM.), formoterol/budesonide (Symbicort.RTM.),
formoterol/fluticasone propionate (Flutiform.RTM.),
formoterol/ciclesonide, formoterol/mometasone furoate,
indacaterol/mometasone furoate, vilanterol trifenate/fluticasone
furoate (BREO ELLIPTA), or arformoterol/ciclesonide; (4)
anticholinergic agents, for example, muscarinic-3 (M3) receptor
antagonists such as ipratropium bromide, tiotropium bromide,
aclidinium bromide (LAS-34273), glycopyrronium bromide, or
umeclidinium bromide; (5) M3-anticholinergic/.beta.2-adrenoreceptor
agonist combination products such as vilanterol/umeclidinium
(Anoro.RTM. Ellipta.RTM.), olodaterol/tiotropium bromide,
glycopyrronium bromide/indacaterol (Ultibro.RTM., also sold as
Xoterna.RTM.), fenoterol hydrobromide/ipratropium bromide
(Berodual.RTM.), albuterol sulfate/ipratropium bromide
(Combivent.RTM.), formoterol fumarate/glycopyrrolate, or aclidinium
bromide/formoterol; (6) dual pharmacology
M3-anticholinergic/.beta.2-adrenoreceptor agonists such as
batefenterol succinate, AZD-2115 or LAS-190792; (7) leukotriene
modulators, for example, leukotriene antagonists such as
montelukast, zafirulast or pranlukast or leukotriene biosynthesis
inhibitors such as zileuton, or LTB4 antagonists such as amelubant,
or FLAP inhibitors such as fiboflapon, GSK-2190915; (8)
phosphodiesterase-IV (PDE-IV) inhibitors (oral or inhaled), such as
roflumilast, cilomilast, oglemilast, rolipram, tetomilast,
AVE-8112, revamilast, CHF 6001; (9) antihistamines, for example,
selective histamine-1 (H1) receptor antagonists such as
fexofenadine, citirizine, loratidine or astemizole or dual H1/H3
receptor antagonists such as GSK 835726, or GSK 1004723; (10)
antitussive agents, such as codeine or dextramorphan; (11) a
mucolytic, for example, N-acetyl cysteine or fudostein; (12) a
expectorant/mucokinetic modulator, for example, ambroxol,
hypertonic solutions (e.g., saline or mannitol) or surfactant; (13)
a peptide mucolytic, for example, recombinant human
deoxyribonoclease I (dornase-alpha and rhDNase) or helicidin; (14)
antibiotics, for example azithromycin, tobramycin or aztreonam;
(15) non-selective COX-1/COX-2 inhibitors, such as ibuprofen or
ketoprofen; (16) COX-2 inhibitors, such as celecoxib and rofecoxib;
(17) VLA-4 antagonists, such as those described in WO 97/03094 and
WO 97/02289, each incorporated herein by reference; (18) TACE
inhibitors and TNF-.alpha. inhibitors, for example anti-TNF
monoclonal antibodies, such as Remicade.RTM. and CDP-870 and TNF
receptor immunoglobulin molecules, such as Enbrel.RTM.; (19)
inhibitors of matrix metalloprotease, for example MMP-12; (20)
human neutrophil elastase inhibitors, such as BAY-85-8501 or those
described in WO 2005/026124, WO 2003/053930 and WO 2006/082412,
each incorporated herein by reference; (21) A2b antagonists such as
those described in WO 2002/42298, incorporated herein by reference;
(22) modulators of chemokine receptor function, for example
antagonists of CCR3 and CCR8; (23) compounds which modulate the
action of other prostanoid receptors, for example, a thromboxane
A.sub.2 antagonist; DP1 antagonists such as laropiprant or
asapiprant CRTH2 antagonists such as OC000459, fevipiprant, ADC
3680 or ARRY 502; (24) PPAR agonists including PPAR alpha agonists
(such as fenofibrate), PPAR delta agonists, PPAR gamma agonists
such as pioglitazone, rosiglitazone and balaglitazone; (25)
methylxanthines such as theophylline or aminophylline and
methylxanthine/corticosteroid combinations such as
theophylline/budesonide, theophylline/fluticasone propionate,
theophylline/ciclesonide, theophylline/mometasone furoate and
theophylline/beclometasone dipropionate; (26) A2a agonists such as
those described in EP1052264 and EP1241176; (27) CXCR2 or IL-8
antagonists such as AZD-5069, AZD-4721, or danirixin; (28) IL-R
signalling modulators such as kineret and ACZ 885; (29) MCP-1
antagonists such as ABN-912; (30) a p38 MAPK inhibitor such as
BCT197, JNJ49095397, losmapimod or PH-797804; (31) TLR7 receptor
agonists such as AZD 8848; (32) PI3-kinase inhibitors such as
RV1729 or GSK2269557 (nemiralisib); (33) triple combination
products such as TRELEGY ELLIPTA (fluticasone furoate, umeclidinium
bromide, and vilanterol); or (34) small molecule inhibitors of
TRPA1, BTK, or SYK.
[0302] In some embodiments a compound of the present invention or a
pharmaceutically acceptable salt thereof, can be used in
combination with one or more additional drugs, for example
anti-hyperproliferative, anti-cancer, cytostatic, cytotoxic,
anti-inflammatory or chemotherapeutic agents, such as those agents
disclosed in U.S. Publ. Appl. No. 2010/0048557, incorporated herein
by reference. A compound of the present invention or a
pharmaceutically acceptable salt thereof, can be also used in
combination with radiation therapy or surgery, as is known in the
art.
[0303] Combinations of any of the foregoing with a compound of the
present invention or a pharmaceutically acceptable salt thereof are
specifically contemplated.
Articles of Manufacture
[0304] Another embodiment includes an article of manufacture (e.g.,
a kit) for treating a disease or disorder responsive to the
inhibition of a Janus kinase, such as a JAK1 kinase. The kit can
comprise:
[0305] (a) a first pharmaceutical composition comprising a compound
of the present invention or a pharmaceutically acceptable salt
thereof; and
[0306] (b) instructions for use.
[0307] In another embodiment, the kit further comprises:
[0308] (c) a second pharmaceutical composition, such as a
pharmaceutical composition comprising an agent for treatment as
described above, such as an agent for treatment of an inflammatory
disorder, or a chemotherapeutic agent.
[0309] In one embodiment, the instructions describe the
simultaneous, sequential or separate administration of said first
and second pharmaceutical compositions to a patient in need
thereof.
[0310] In one embodiment, the first and second compositions are
contained in separate containers. In another embodiment, the first
and second compositions are contained in the same container.
[0311] Containers for use include, for example, bottles, vials,
syringes, blister pack, etc. The containers may be formed from a
variety of materials such as glass or plastic. The container
includes a compound of the present invention or a pharmaceutically
acceptable salt thereof, which is effective for treating the
condition and may have a sterile access port (for example the
container may be an intravenous solution bag or a vial having a
stopper pierceable by a hypodermic injection needle). The label or
package insert indicates that the compound is used for treating the
condition of choice, such as asthma or cancer. In one embodiment,
the label or package inserts indicates that the compound can be
used to treat a disorder. In addition, the label or package insert
may indicate that the patient to be treated is one having a
disorder characterized by overactive or irregular Janus kinase
activity, such as overactive or irregular JAK1 activity. The label
or package insert may also indicate that the compound can be used
to treat other disorders.
[0312] Alternatively, or additionally, the kit may further comprise
a second (or third) container comprising a pharmaceutically
acceptable buffer, such as bacteriostatic water for injection
(BWFI), phosphate-buffered saline, Ringer's solution or dextrose
solution. It may further include other materials desirable from a
commercial and user standpoint, including other buffers, diluents,
filters, needles, and syringes.
[0313] In order to illustrate the invention, the following examples
are included. However, it is to be understood that these examples
do not limit the invention and are only meant to suggest a method
of practicing the invention. Persons skilled in the art will
recognize that the chemical reactions described may be readily
adapted to prepare other compounds of the present invention, and
alternative methods for preparing the compounds are within the
scope of this invention. For example, the synthesis of
non-exemplified compounds according to the invention may be
successfully performed by modifications apparent to those skilled
in the art, e.g., by appropriately protecting interfering groups,
by utilizing other suitable reagents known in the art other than
those described, or by making routine modifications of reaction
conditions. Alternatively, other reactions disclosed herein or
known in the art will be recognized as having applicability for
preparing other compounds of the invention.
EXAMPLES
[0314] The following representative compounds of Table 1 were
prepared using procedures similar to those described in the Schemes
and Examples herein. Absolute stereochemistry of each compound
below may not be depicted: therefore, structures may appear more
than once, each representing asingle stereoisomer. LC-MS method,
retention time and m/z are also shown in Table 1
TABLE-US-00001 TABLE 1 Exemplary JAK Inhibitors LC/ Ret. Structure
Name MS time m/z 1 ##STR00018## N-[3-[5-chloro-2-
(difluoromethoxy)phenyl]- 1-[[2-(2- hydroxyethyl)tetrazol-5-
yl]methyl]pyrazol-4- yl]pyrazolo[1,5- a]pyrimidine-3- carboxamide A
1.33 531.2 2 ##STR00019## N-[3-[5-chloro-2-
(difluoromethoxy)phenyl]- 1-[[2-(2- morpholinoethyl)tetrazol-
5-yl]methyl]pyrazol-4- yl]pyrazolo[1,5- a]pyrimidine-3- carboxamide
S 2.73 600.2 3 ##STR00020## N-[3-[5-chloro-2-
(difluoromethoxy)phenyl]- 1-[[2-(1-methyl-4- piperidyl)tetrazol-5-
yl]methyl]pyrazol-4- yl]pyrazolo[1,5- a]pyrimidine-3- carboxamide N
1.42 584.3 4 ##STR00021## N-[3-[5-chloro-2-
(difluoromethoxy)phenyl]- 1-[[2-(oxetan-3- yl)tetrazol-5-
yl]methyl]pyrazol-4- yl]pyrazolo[1,5- a]pyrimidine-3- carboxamide T
1.22 543.2 5 ##STR00022## N-[3-[5-chloro-2-
(difluoromethoxy)phenyl]- 1-[[1-(2- morpholinoethyl)tetrazol-
5-yl]methyl]pyrazol-4- yl]pyrazolo[1,5- a]pyrimidine-3- carboxamide
S 2.37 600.3 6 ##STR00023## N-[3-[5-chloro-2-
(difluoromethoxy)phenyl]- 1-[[2-(4-piperidyl)tetrazol-
5-yl]methyl]pyrazol-4- yl]pyrazolo[1,5- a]pyrimidine-3- carboxamide
V 3.33 570.2 7 ##STR00024## N-[3-[5-chloro-2-
(difluoromethoxy)phenyl]- 1-[[2-(1-methylazetidin-3- yl)tetrazol-5-
yl]methyl]pyrazol-4- yl]pyrazolo[1,5- a]pyrimidine-3- carboxamide S
2.71 556.2 8 ##STR00025## N-[1-[[2-(azetidin-3-
yl)tetrazol-5-yl]methyl]-3- [5-chloro-2- (difluoromethoxy)phenyl]
pyrazol-4-yl]pyrazolo[1,5- a]pyrimidine-3- carboxamide A 1.32 542.2
9 ##STR00026## N-[3-[5-chloro-2- (difluoromethoxy)phenyl]-
1-[[2-(1-tetrahydropyran- 4-yl-4-piperidyl)tetrazol-5-
yl]methyl]pyrazol-4- yl]pyrazolo[1,5- a]pyrimidine-3- carboxamide A
1.38 654.3 10 ##STR00027## N-[3-[2,5- bis(difluoromethoxy)
phenyl]-1-[[2-(1- methylazetidin-3- yl)tetrazol-5-
yl]methyl]pyrazol-4- yl]pyrazolo[1,5- a]pyrimidine-3- carboxamide S
2.21 588.3 11 ##STR00028## N-[1-[[2-(azetidin-3-
yl)tetrazol-5-yl]methyl]-3- [2,5- bis(difluoromethoxy)
phenyl]pyrazol-4- yl]pyrazolo[1,5- a]pyrimidine-3- carboxamide S
2.34 574.3 12 ##STR00029## N-[1-[[1-(azetidin-3-
yl)tetrazol-5-yl]methyl]-3- [2,5- bis(difluoromethoxy)
phenyl]pyrazol-4- yl]pyrazolo[1,5- a]pyrimidine-3- carboxamide E
2.17 574.1 13 ##STR00030## N-[1-[[2-(2- aminoethyl)tetrazol-5-
yl]methyl]-3-[2,5- bis(difluoromethoxy) phenyl]pyrazol-4-
yl]pyrazolo[1,5- a]pyrimidine-3- carboxamide E 2.24 562.2 14
##STR00031## N-[1-[[1-(2- aminoethyl)tetrazol-5- yl]methyl]-3-[2,5-
bis(difluoromethoxy) phenyl]pyrazol-4- yl]pyrazolo[1,5-
a]pyrimidine-3- carboxamide E 2.1 562.2 15 ##STR00032##
N-[3-[5-chloro-2- (difluoromethoxy)phenyl]-
1-[[1-(1-methylazetidin-3- yl)tetrazol-5- yl]methyl]pyrazol-4-
yl]pyrazolo[1,5- a]pyrimidine-3- carboxamide E 2.31 556.1 16
##STR00033## N-[3-[2,5- bis(difluoromethoxy) phenyl]-1-[[2-[2-
(dimethylamino)ethyl] tetrazol-5-yl]methyl] pyrazol-4-
yl]pyrazolo[1,5- a]pyrimidine-3- carboxamide E 2.18 590.2 17
##STR00034## N-[3-[2,5- bis(difluoromethoxy) phenyl]-1-[[1-[2-
(dimethylamino)ethyl] tetrazol-5-yl]methyl] pyrazol-4-
yl]pyrazolo[1,5- a]pyrimidine-3- carboxamide E 2.25 590.2 18
##STR00035## N-[3-[2,5- bis(difluoromethoxy) phenyl]-1-[[2-(4-
piperidyl)tetrazol-5- yl]methyl]pyrazol-4- yl]pyrazolo[1,5-
a]pyrimidine-3- carboxamide D 2.11 602.2 19 ##STR00036## N-[3-[2,5-
bis(difluoromethoxy) phenyl]-1-[[1-[2- (methylamino)ethyl]
tetrazol-5-yl]methyl] pyrazol-4- yl]pyrazolo[1,5- a]pyrimidine-3-
carboxamide E 2.2 576.2 20 ##STR00037## N-[3-[2,5-
bis(difluoromethoxy) phenyl]-1-[[2-[2- (methylamino)ethyl]
tetrazol-5-yl]methyl] pyrazol-4- yl]pyrazolo[1,5- a]pyrimidine-3-
carboxamide E 2.47 576.2 21 ##STR00038## N-[3-[2,5-
bis(difluoromethoxy) phenyl]-1-[[2-[1-[2- (dimethylamino)ethyl]
azetidin-3-yl]tetrazol-5- yl]methyl]pyrazol-4- yl]pyrazolo[1,5-
a]pyrimidine-3- carboxamide E 2.22 645.2 22 ##STR00039## N-[3-[2,5-
bis(difluoromethoxy) phenyl]-1-[2-(1-methyl-4-
piperidyl)tetrazol-5- yl]methyl]pyrazol-4- yl]pyrazolo[1,5-
a]pyrimidine-3- carboxamide S 2.5 616.3 23 ##STR00040##
N-[3-[5-cyclopropyl-2- (difluoromethoxy)phenyl]-
1-[[2-(1-methylazetidin-3- yl)tetrazol-5- yl]methyl]pyrazol-4-
yl]pyrazolo[1,5- a]pyrimidine-3- carboxamide E 2.48 562.2 24
##STR00041## N-[3-[2,5- bis(difluoromethoxy) phenyl]-1-[[1-(4-
piperidyl)tetrazol-5- yl]methyl]pyrazol-4- yl]pyrazolo[1,5-
a]pyrimidine-3- carboxamide A 1.29 602.1 25 ##STR00042##
N-[1-[[2-[1-(2- aminoethyl)-4- piperidyl]tetrazol-5-
yl]methyl]-3-[2,5- bis(difluoromethoxy) phenyl]pyrazol-4-
yl]pyrazolo[1,5- a]pyrimidine-3- carboxamide E 2.22 645.2 26
##STR00043## N-[3-[2,5- bis(difluoromethoxy) phenyl]-1-[[1-(1-
methylpyrrolidin-3- yl)tetrazol-5- yl]methyl]pyrazol-4-
yl]pyrazolo[1,5- a]pyrimidine-3- carboxamide E 2.33 602.1 27
##STR00044## N-[3-[2,5- bis(difluoromethoxy)
phenyl]-1-[(2-pyrrolidin-3- yltetrazol-5- yl)methyl]pyrazol-4-
yl]pyrazolo[1,5- a]pyrimidine-3- carboxamide E 1.67 588.1 28
##STR00045## N-[3-[2,5- bis(difluoromethoxy)
phenyl]-1-[(2-pyrrolidin-3- yltetrazol-5- yl)methyl]pyrazol-4-
yl]pyrazolo[1,5- a]pyrimidine-3- carboxamide E 2.36 588.1 29
##STR00046## N-[3-[2,5- bis(difluoromethoxy) phenyl]-1-[[2-(1-
methylpyrrolidin-3- yl)tetrazol-5- yl]methyl]pyrazol-4-
yl]pyrazolo[1,5- a]pyrimidine-3- carboxamide T 1.08 602.2 30
##STR00047## N-[3-[2,5- bis(difluoromethoxy) phenyl]-1-[[2-(1-
methylpyrrolidin-3- yl)tetrazol-5- yl]methyl]pyrazol-4-
yl]pyrazolo[1,5- a]pyrimidine-3- carboxamide E 2.49 624.1 31
##STR00048## tert-butyl N-[2-[3-[5-[[3- [2,5- bis(difluoromethoxy)
phenyl]-4-(pyrazolo[1,5- a]pyrimidine-3- carbonylamino)pyrazol-1-
yl]methyl]tetrazol-2- yl]azetidin-1-yl]ethyl]-N- methyl-carbamate S
1.85 731.3 32 ##STR00049## N-[3-[2,5- bis(difluoromethoxy)
phenyl]-1-[[2-[(1- methylazetidin-3- yl)methyl]tetrazol-5-
yl]methyl]pyrazol-4- yl]pyrazolo[1,5- a]pyrimidine-3- carboxamide N
1.3 602.2 33 ##STR00050## N-[3-[2,5- bis(difluoromethoxy)
phenyl]-1-[[1-[(1- methylazetidin-3- yl)methyl]tetrazol-5-
yl]methyl]pyrazol-4- yl]pyrazolo[1,5- a]pyrimidine-3- carboxamide S
2.14 602.2 34 ##STR00051## N-[3-[2,5- bis(difluoromethoxy)
phenyl]-1-[[2-[1-[2- (methylamino)ethyl] azetidin-3-yl]tetrazol-5-
yl]methyl]pyrazol-4- yl]pyrazolo[1,5- a]pyrimidine-3- carboxamide A
1.2 631.3 35 ##STR00052## N-[1-[[2-(2- aminoethyl)tetrazol-5-
yl]methyl]-3-[2- (difluoromethoxy)-5- methylsulfanyl-
phenyl]pyrazol-4- yl]pyrazolo[1,5- a]pyrimidine-3- carboxamide A
1.33 542.1 36 ##STR00053## N-[3-[2- (difluoromethoxy)-5-
methylsulfanyl-phenyl]-1- [[2-[2- (dimethylamino)ethyl]
tetrazol-5-yl]methyl] pyrazol-4- yl]pyrazolo[1,5- a]pyrimidine-3-
carboxamide E 2.23 570.2 37 ##STR00054## N-[3-[5-chloro-2-
(difluoromethoxy)phenyl]- 1-[[2-[1-[2- (dimethylamino)ethyl]
azetidin-3-yl]tetrazol-5- yl]methyl]pyrazol-4- yl]pyrazolo[1,5-
a]pyrimidine-3- carboxamide A 1.23 613.2 38 ##STR00055## N-[3-[2,5-
bis(difluoromethoxy) phenyl]-1-[[2-[1- (1-methyl-4-
piperidyl)azetidin-3- yl]tetrazol-5- yl]methyl]pyrazol-4-
yl]pyrazolo[1,5- a]pyrimidine-3- carboxamide A 1.19 671.3 39
##STR00056## N-[3-[5-chloro-2- (difluoromethoxy)phenyl]-
1-[[2-[2-(4- methylpiperazin-1- yl)ethyl]tetrazol-5-
yl]methyl]pyrazol-4- yl]pyrazolo[1,5- a]pyrimidine-3- carboxamide T
1.08 613.2 40 ##STR00057## N-[3-[5-chloro-2-
(difluoromethoxy)phenyl]- 1-[[2-(2-piperazin-1- ylethyl)tetrazol-5-
yl]methyl]pyrazol-4- yl]pyrazolo[1,5- a]pyrimidine-3- carboxamide E
2.64 599.2 41 ##STR00058## N-[3-[2- (difluoromethoxy)-5-
methylsulfanyl-phenyl]-1- [[1-[2- (dimethylamino)ethyl]
tetrazol-5-yl]methyl] pyrazol-4- yl]pyrazolo[1,5- a]pyrimidine-3-
carboxamide E 2.16 570.2 42 ##STR00059## N-[3-[5-chloro-2-
(difluoromethoxy)phenyl]- 1-[[1-[2-(4- methylpiperazin-1-
yl)ethyl]tetrazol-5- yl]methyl]pyrazol-4- yl]pyrazolo[1,5-
a]pyrimidine-3- carboxamide E 3.21 613.2 43 ##STR00060## N-[3-[6-
(difluoromethoxy)-3,4- dihydro-2H-1,4- benzoxazin-7-yl]-1[[2-[2-
(dimethylamino)ethyl] tetrazol-5-yl]methyl] pyrazol-4-
yl]pyrazolo[1,5- a]pyrimidine-3- carboxamide A 1.15 581.3 44
##STR00061## N-[3-[6- (difluoromethoxy)-3,4- dihydro-2H-1,4-
benzoxazin-7-yl]-1-[[1-[2- (dimethylamino)ethyl]
tetrazol-5-yl]methyl] pyrazol-4- yl]pyrazolo[1,5- a]pyrimidine-3-
carboxamide A 1.13 581.3 45 ##STR00062## N-[3-[2,5-
bis(difluoromethoxy) phenyl]-1-[[1-[2- [methyl-(1-
methylazetidin-3- yl)amino]ethyl]tetrazol-5- yl]methyl]pyrazol-4-
yl]pyrazolo[1,5- a]pyrimidine-3- carboxamide 46 ##STR00063##
N-[3-[2,5- bis(difluoromethoxy) phenyl]-1-[[2- [1-[[rac-(2S)-1-
methylpyrrolidin-2- yl]methyl]azetidin-3- yl]tetrazol-5-
yl]methyl]pyrazol-4- yl]pyrazolo[1,5- a]pyrimidine-3- carboxamide;
formic acid 47 ##STR00064## N-[3-[2- (difluoromethoxy)-5-
methylsulfanyl-phenyl]-1- [[2-[1-[2- (dimethylamino)ethyl]
azetidin-3-yl]tetrazol-5- yl]methyl]pyrazol-4- yl]pyrazolo[1,5-
a]pyrimidine-3- carboxamide A 1.24 625.3 48 ##STR00065## N-[3-[2,5-
bis(difluoromethoxy) phenyl]-1-[[2-[2- [methyl-(1-
methylazetidin-3- yl)amino]ethyl]tetrazol-5- yl]methyl]pyrazol-4-
yl]pyrazolo[1,5- a]pyrimidine-3- carboxamide A 1.34 645.3 49
##STR00066## N-[3-[2,5- bis(difluoromethoxy)
phenyl]-1-[[2-[[rac-(2R)-1- methylpyrrolidin-2-
yl]methyl]tetrazol-5- yl]methyl]pyrazol-4- yl]pyrazolo[1,5-
a]pyrimidine-3- carboxamide F 1.78 616.3 50 ##STR00067##
N-[3-[5-cyclopropyl-2- (difluoromethoxy)phenyl]- 1-[[2-[1-[2-
(dimethylamino)ethyl] azetidin-3-yl]tetrazol-5-
yl]methyl]pyrazol-4- yl]pyrazolo[1,5- a]pyrimidine-3- carboxamide A
1.27 619.3 51 ##STR00068## N-[3-[2,5- bis(difluoromethoxy)
phenyl]-1-[[2-[1-[3- (dimethylamino)propyl]
azetidin-3-yl]tetrazol-5- yl]methyl]pyrazol-4- yl]pyrazolo[1,5-
a]pyrimidine-3- carboxamide E 2.09 659.3 52 ##STR00069## N-[3-[2,5-
bis(difluoromethoxy) phenyl]-1-[[2- [1-[[rac-(2R)-1-
methylpyrrolidin-2- yl]methyl]azetidin-3- yl]tetrazol-5-
yl]methyl]pyrazol-4- yl]pyrazolo[1,5- a]pyrimidine-3- carboxamide N
1.48 671.4 53 ##STR00070## N-[3-[2,5- bis(difluoromethoxy)
phenyl]-1-[[2- [[rac-(2S)-1- methylpyrrolidin-2-
yl]methyl]tetrazol-5- yl]methyl]pyrazol-4- yl]pyrazolo[1,5-
a]pyrimidine-3- carboxamide A 1.34 616.3 54 ##STR00071## N-[3-[2,5-
bis(difluoromethoxy) phenyl]-1-[[1- [[rac-(2S)-1-
methylpyrrolidin-2- yl]methyl]tetrazol-5- yl]methyl]pyrazol-4-
yl]pyrazolo[1,5- a]pyrimidine-3- carboxamide F 1.86 616.2 55
##STR00072## N-[3-[2,5- bis(difluoromethoxy) phenyl]-1-[[2-[1-[4-
(dimethylamino)butyl] azetidin-3-yl]tetrazol-5-
yl]methyl]pyrazol-4- yl]pyrazolo[1,5- a]pyrimidine-3- carboxamide E
2.25 673.3 56 ##STR00073## N-[3-[2,5- bis(difluoromethoxy)
phenyl]-1-[[2-[1-[(1- methylazetidin-3- yl)methyl]azetidin-3-
yl]tetrazol-5- yl]methyl]pyrazol-4- yl]pyrazolo[1,5-
a]pyrimidine-3- carboxamide E 2.23 657.2 57 ##STR00074##
N-[3-[5-cyclopropyl-2- (difluoromethoxy)phenyl]- 1-[[2-[1-[3-
(dimethylamino)propyl] azetidin-3-yl]tetrazol-5-
yl]methyl]pyrazol-4- yl]pyrazolo[1,5- a]pyrimidine-3- carboxamide A
1.21 633.4 58 ##STR00075## N-[3-[2- (difluoromethoxy)-5-
methylsulfanyl-phenyl]-1- [[2-[1-[3- (dimethylamino)propyl]
azetidin-3-yl]tetrazol-5- yl]methyl]pyrazol-4- yl]pyrazolo[1,5-
a]pyrimidine-3- carboxamide A 1.17 639.3
59 ##STR00076## N-[3-[2,5- bis(difluoromethoxy)
phenyl]-1-[[2-[2-[4- (dimethylamino)-1- piperidyl]ethyl]tetrazol-5-
yl]methyl]pyrazol-4- yl]pyrazolo[1,5- a]pyrimidine-3- carboxamide A
1.17 673.3 60 ##STR00077## N-[3-[2,5- bis(difluoromethoxy)
phenyl]-1-[[1-[2-[4- (dimethylamino)-1- piperidyl]ethyl]tetrazol-5-
yl]methyl]pyrazol-4- yl]pyrazolo[1,5- a]pyrimidine-3- carboxamide N
1.44 673.4 61 ##STR00078## N-[3-[2,5- bis(difluoromethoxy)
phenyl]-1-[[2-[1-[2- (dimethylamino)ethyl]-4- piperidyl]tetrazol-5-
yl]methyl]pyrazol-4- yl]pyrazolo[1,5- a]pyrimidine-3- carboxamide N
1.47 673.4 62 ##STR00079## N-[3-[5-chloro-2-
(difluoromethoxy)phenyl] 1-[[2-[1-[3- (dimethylamino)propyl]
azetidin-3-yl]tetrazol-5- yl]methyl]pyrazol-4- yl]pyrazolo[1,5-
a]pyrimidine-3- carboxamide A 1.17 627.3 63 ##STR00080## N-[3-[2,5-
bis(difluoromethoxy) phenyl]-1-[[2-[3- (dimethylamino) cyclobutyl]
tetrazol-5- yl]methyl]pyrazol-4- yl]pyrazolo[1,5- a]pyrimidine-3-
carboxamide E 1.77 616.3 64 ##STR00081## N-[3-[2,5-
bis(difluoromethoxy) phenyl]-1-[[2-[3- (dimethylamino) cyclobutyl]
tetrazol-5- yl]methyl]pyrazol-4- yl]pyrazolo[1,5- a]pyrimidine-3-
carboxamide; formic acid A 1.33 616.3 65 ##STR00082## N-[3-[2,5-
bis(difluoromethoxy) phenyl]-1-[[1-[1-[3- (dimethylamino)propyl]
azetidin-3-yl]tetrazol-5- yl]methyl]pyrazol-4- yl]pyrazolo[1,5-
a]pyrimidine-3- carboxamide E 2.16 659.3 66 ##STR00083## N-[3-[2,5-
bis(difluoromethoxy) phenyl]-1-[[2-[4- (dimethylamino) cyclohexyl]
tetrazol-5- yl]methyl]pyrazol-4- yl]pyrazolo[1,5- a]pyrimidine-3-
carboxamide A 1.42 644.4 67 ##STR00084## N-[3-[2,5-
bis(difluoromethoxy) phenyl]-1-[[2-[1-[2- (dimethylamino)ethyl]
pyrrolidin-3-yl]tetrazol-5- yl]methyl]pyrazol-4- yl]pyrazolo[1,5-
a]pyrimidine-3- carboxamide A 1.3 659.2 68 ##STR00085## N-[3-[2,5-
bis(difluoromethoxy) phenyl]-1-[[2-[1-[2- (dimethylamino)ethyl]
pyrrolidin-3-yl]tetrazol-5- yl]methyl]pyrazol-4- yl]pyrazolo[1,5-
a]pyrimidine-3- carboxamide A 1.3 659.2 69 ##STR00086##
N-[1-[[1-(4- aminocyclohexyl)tetrazol- 5-yl]methyl]-3-[2,5-
bis(difluoromethoxy) phenyl]pyrazol-4- yl]pyrazolo[1,5-
a]pyrimidine-3- carboxamide A 1.31 616.3 70 ##STR00087## N-[3-[2,5-
bis(difluoromethoxy) phenyl]-1-[[2-[1-[3- (dimethylamino)
cyclobutyl] azetidin-3-yl]tetrazol-5- yl]methyl]pyrazol-4-
yl]pyrazolo[1,5- a]pyrimidine-3- carboxamide A 1.19 671.3 71
##STR00088## N-[3-[2,5- bis(difluoromethoxy) phenyl]-1-[[2-[1-[3-
(dimethylamino) cyclobutyl] azetidin-3-yl]tetrazol-5-
yl]methyl]pyrazol-4- yl]pyrazolo[1,5- a]pyrimidine-3- carboxamide F
1.42 671.3 72 ##STR00089## N-[3-[2,5- bis(difluoromethoxy)
phenyl]-1-[[2-[1-(1- methylpyrrolidin-3-
yl)azetidin-3-yl]tetrazol-5- yl]methyl]pyrazol-4- yl]pyrazolo[1,5-
a]pyrimidine-3- carboxamide A 1.29 657.3 73 ##STR00090## N-[3-[2,5-
bis(difluoromethoxy) phenyl]-1-[[2-[1-(1- methylpyrrolidin-3-
yl)azetidin-3-yl]tetrazol-5- yl]methyl]pyrazol-4- yl]pyrazolo[1,5-
a]pyrimidine-3- carboxamide A 1.32 657.3 74 ##STR00091## N-[3-[2,5-
bis(difluoromethoxy) phenyl]-1-[[2-[1-[3- (dimethylamino)
cyclopentyl] azetidin-3-yl]tetrazol-5- yl]methyl]pyrazol-4-
yl]pyrazolo[1,5- a]pyrimidine-3- carboxamide A 1.24 685.2 75
##STR00092## N-[3-[2,5- bis(difluoromethoxy) phenyl]-1-[[2-[1-[3-
(dimethylamino) cyclopentyl] azetidin-3-yl]tetrazol-5-
yl]methyl]pyrazol-4- yl]pyrazolo[1,5- a]pyrimidine-3- carboxamide U
2.23 685.3 76 ##STR00093## N-[3-[2,5- bis(difluoromethoxy)
phenyl]-1-[[2-[1-[3- (dimethylamino) cyclopentyl]
azetidin-3-yl]tetrazol-5- yl]methyl]pyrazol-4- yl]pyrazolo[1,5-
a]pyrimidine-3- carboxamide U 2.26 685.3 77 ##STR00094## N-[3-[2,5-
bis(difluoromethoxy) phenyl]-1-[[2-[1-[3- (dimethylamino)-3-
methyl-butyl]azetidin-3- yl]tetrazol-5- yl]methyl]pyrazol-4-
yl]pyrazolo[1,5- a]pyrimidine-3- carboxamide A 1.24 687.3 78
##STR00095## N-[3-[2,5- bis(difluoromethoxy) phenyl]-1-[[2-[1-(1-
methylazetidin-3- yl)azetidin-3-yl]tetrazol-5- yl]methyl]pyrazol-4-
yl]pyrazolo[1,5- a]pyrimidine-3- carboxamide; formic acid B 2.33
643.2 79 ##STR00096## N-[3-[2- (difluoromethoxy)-5-
isopropylsulfanyl- phenyl]- 1-[[2-[1-(1-methyl-4-
piperidyl)azetidin-3- yl]tetrazol-5- yl]methyl]pyrazol-4-
yl]pyrazolo[1,5- a]pyrimidine-3- carboxamide U 2.59 679.3 80
##STR00097## N-[3-[2,5- bis(difluoromethoxy)
phenyl]-1-[[2-[1-[2-[1- (dimethylamino) cyclopropyl]
ethyl]azetidin-3- yl]tetrazol-5- yl]methyl]pyrazol-4-
yl]pyrazolo[1,5- a]pyrimidine-3- carboxamide; formic acid H 0.94
685.3 81 ##STR00098## N-[3-[2,5- bis(difluoromethoxy)
phenyl]-1-[[2-[3-[2- (dimethylamino)ethyl- methyl-
amino]cyclobutyl]tetrazol- 5-yl]methyl]pyrazol-4- yl]pyrazolo[1,5-
a]pyrimidine-3- carboxamide; formic acid A 1.22 673.3 82
##STR00099## N-[3-[2,5- bis(difluoromethoxy) phenyl]-1-[[2-[3-[2-
(dimethylamino)ethyl- methyl- amino]cyclobutyl]tetrazol-
5-yl]methyl]pyrazol-4- yl]pyrazolo[1,5- a]pyrimidine-3-
carboxamide; formic acid A 1.22 673.3 83 ##STR00100## N-[3-[2,5-
bis(difluoromethoxy) phenyl]-1-[[2-[1-[3- (dimethylamino)-2-fluoro-
propyl]azetidin-3- yl]tetrazol-5- yl]methyl]pyrazol-4-
yl]pyrazolo[1,5- a]pyrimidine-3- carboxamide A 1.24 677.2 84
##STR00101## N-[3-[2,5- bis(difluoromethoxy) phenyl]-1-[[2-[1-[3-
(dimethylamino)-2-fluoro- propyl]azetidin-3- yl]tetrazol-5-
yl]methyl]pyrazol-4- yl]pyrazolo[1,5- a]pyrimidine-3- carboxamide A
1.24 677.2
General Experimental Details
[0315] All solvents and commercial reagents were used as received
unless otherwise stated.
[0316] Where products were purified by chromatography on silica
this was carried out using either a glass column manually packed
with silica gel (Kieselgel 60, 220-440 mesh, 35-75 .mu.m) or an
Isolute.RTM. SPE Si II cartridge. `Isolute SPE Si cartridge` refers
to a pre-packed polypropylene column containing unbonded activated
silica with irregular particles with average size of 50 .mu.m and
nominal 60A porosity. Where an Isolute.RTM. SCX-2 cartridge was
used, `Isolute.RTM. SCX-2 cartridge` refers to a pre-packed
polypropylene column containing a non-end-capped propylsulphonic
acid functionalised silica strong cation exchange sorbent.
LCMS Conditions
[0317] Method A
[0318] Experiments were performed on a SHIMADZU LCMS-2020 with a
C18-reverse-phase column (50.times.3 mm Shim-Pack XR-ODS, 2.2 .mu.m
particle size), elution with solvent A: water+0.05% trifluoroacetic
acid; solvent B: acetonitrile+0.05% trifluoroacetic acid.
Gradient:
TABLE-US-00002 Gradient - Time flow ml/min % A % B 0.00 1.2 95 5
2.00 1.2 5 95 2.70 1.2 5 95 2.75 1.2 95 5 Detection - UV (220 and
254 nm) and ELSD
[0319] Method B
[0320] Experiments were performed on a SHIMADZU LCMS-2020 with a
C18-reverse-phase column (50.times.3 mm Shim-Pack XR-ODS, 2.2 .mu.m
particle size), elution with solvent A: water+0.05% trifluoroacetic
acid; solvent B: acetonitrile+0.05% trifluoroacetic acid.
Gradient:
TABLE-US-00003 Gradient - Time flow ml/min % A % B 0.00 1.2 80 20
3.60 1.2 40 60 4.00 1.2 0 100 4.70 1.2 0 100 4.75 1.2 95 5
Detection - UV (220 and 254 nm) and ELSD
[0321] Method C
[0322] Experiments were performed on a SHIMADZU LCMS-2020 with a
C18-reverse-phase column (50.times.3 mm Shim-Pack XR-ODS, 2.2 .mu.m
particle size), elution with solvent A: water+0.05% trifluoroacetic
acid; solvent B: acetonitrile+0.05% trifluoroacetic acid.
Gradient:
TABLE-US-00004 Gradient - Time flow ml/min % A % B 0.00 1.2 95 5
3.00 1.2 5 95 3.70 1.2 5 95 3.75 1.2 95 5 Detection - UV (220 and
254 nm) and ELSD
[0323] Method D
[0324] Experiments were performed on a SHIMADZU LCMS-2020 with a
C18-reverse-phase column (50.times.3 mm Shim-Pack XR-ODS, 2.2 .mu.m
particle size), elution with solvent A: water+0.05% trifluoroacetic
acid; solvent B: acetonitrile+0.05% trifluoroacetic acid.
Gradient:
TABLE-US-00005 Gradient - Time flow ml/min % A % B 0.00 1.2 95 5
3.50 1.2 30 70 3.70 1.2 0 100 4.50 1.2 0 100 4.75 1.2 95 5
Detection - UV (220 and 254 nm) and ELSD
[0325] Method E
[0326] Experiments were performed on a SHIMADZU LCMS-2020 with a
C18-reverse-phase column (50.times.3 mm Shim-Pack XR-ODS, 2.2 .mu.m
particle size), elution with solvent A: water+0.05% trifluoroacetic
acid; solvent B: acetonitrile+0.05% trifluoroacetic acid.
Gradient:
TABLE-US-00006 Gradient - Time flow ml/min % A % B 0.00 1.2 95 5
3.50 1.2 40 60 3.70 1.2 0 100 4.70 1.2 0 100 4.75 1.2 95 5
Detection - UV (220 and 254 nm) and ELSD
[0327] Method F
[0328] Experiments were performed on a SHIMADZU LCMS-2020 with a
C18-reverse-phase column (50.times.3 mm Shim-Pack XR-ODS, 2.2 .mu.m
particle size), elution with solvent A: water+0.05% trifluoroacetic
acid; solvent B: acetonitrile+0.05% trifluoroacetic acid.
Gradient:
TABLE-US-00007 Gradient - Time flow ml/min % A % B 0.00 1.2 70 30
3.50 1.2 30 70 3.70 1.2 0 100 4.50 1.2 0 100 4.75 1.2 95 5
Detection - UV (220 and 254 nm) and ELSD
[0329] Method G
[0330] Experiments were performed on a SHIMADZU 20A HPLC with a
C18-reverse-phase column (50.times.2.1 mm Ascentis Express C18, 2.7
.mu.m particle size), elution with solvent A: water+0.05%
trifluoroacetic acid; solvent B: acetonitrile+0.05% trifluoroacetic
acid. Gradient:
TABLE-US-00008 Gradient - Time flow ml/min % A % B 0.00 1.0 95 5
1.10 1.0 0 100 1.60 1.0 0 100 1.70 1.0 95 5 Detection - UV (220 and
254 nm) and ELSD
[0331] Method H
[0332] Experiments were performed on a SHIMADZU LCMS-2020 with a
C18-reverse-phase column (50.times.3 mm Shim-Pack XR-ODS, 2.2 .mu.m
particle size), elution with solvent A: water+0.05% trifluoroacetic
acid; solvent B: acetonitrile+0.05% trifluoroacetic acid.
Gradient:
TABLE-US-00009 Gradient - Time flow ml/min % A % B 0.00 1.2 95 5
1.10 1.2 0 100 1.70 1.2 0 100 1.75 1.2 95 5 Detection - UV (220 and
254 nm) and ELSD
[0333] Method I
[0334] Experiments were performed on a SHIMADZU 20A HPLC with
Poroshell HPH-Cis, column (50.times.3 mm, 2.7 .mu.m particle size),
elution with solvent A: water/5 mM NH.sub.4HCO.sub.3; solvent B:
acetonitrile. Gradient:
TABLE-US-00010 Gradient - Time flow ml/min % A % B 0.00 1.2 90 10
1.10 1.2 5 95 1.60 1.2 5 95 1.70 1.2 90 10 Detection - UV (220 and
254 nm) and ELSD
[0335] Method J
[0336] Experiments were performed on a SHIMADZU LCMS-2020 with a
C18-reverse-phase column (50.times.3 mm Kinetex XB-Cis, 2.6 .mu.m
particle size), elution with solvent A: water+0.05% trifluoroacetic
acid; solvent B: acetonitrile+0.05% trifluoroacetic acid.
Gradient:
TABLE-US-00011 Gradient - Time flow ml/min % A % B 0.00 1.5 95 5
1.20 1.5 0 100 1.70 1.5 0 100 1.80 1.5 95 5 Detection - UV (220 and
254 nm) and ELSD
[0337] Method K
[0338] Experiments were performed on a SHIMADZU LCMS-2020 with a
C18-reverse-phase column (50.times.3 mm Shim-Pack XR-ODS, 2.2 .mu.m
particle size), elution with solvent A: water+0.05% trifluoroacetic
acid; solvent B: acetonitrile+0.05% trifluoroacetic acid.
Gradient:
TABLE-US-00012 Gradient - Time flow ml/min % A % B 0.00 1.0 95 5
2.20 1.0 0 100 3.20 1.0 0 100 3.30 1.0 95 5 Detection - UV (220 and
254 nm) and ELSD
[0339] Method L
[0340] Experiments were performed on a SHIMADZU LCMS-2020 with a
C18-reverse-phase column (50.times.2.1 mm Kinetex XB-Cis 100A, 2.6
.mu.m particle size), elution with solvent A: water+0.05%
trifluoroacetic acid; solvent B: acetonitrile+0.05% trifluoroacetic
acid. Gradient:
TABLE-US-00013 Gradient - Time flow ml/min % A % B 0.00 1.0 95 5
1.10 1.0 0 100 1.60 1.0 0 100 1.70 1.0 95 5 Detection - UV (220 and
254 nm) and ELSD
[0341] Method M
[0342] Experiments were performed on a SHIMADZU LCMS-2020 with a
C18-reverse-phase column (30.times.2.1 mm Kinetex C.sub.18-100A,
1.7 .mu.m particle size), elution with solvent A: water+0.05%
trifluoroacetic acid; solvent B: acetonitrile+0.05% trifluoroacetic
acid. Gradient:
TABLE-US-00014 Gradient - Time flow ml/min % A % B 0.01 1.0 95 5
0.60 1.0 0 100 1.00 1.0 0 100 1.05 1.0 95 5 Detection - UV (220 and
254 nm) and ELSD
[0343] Method N
[0344] Experiments were performed on a SHIMADZU LCMS-2020 with a
C18-reverse-phase column (50.times.3.0 mm Poroshell HPH-C18, 2.7
.mu.m particle size), elution with solvent A: water+5 mM ammonium
bicarbonate; solvent B: acetonitrile. Gradient:
TABLE-US-00015 Gradient - Time flow ml/min % A % B 0.01 1.0 90 10
2.00 1.0 5 95 2.70 1.0 5 95 2.80 1.0 90 10 Detection - UV (220 and
254 nm) and ELSD
[0345] Method O
[0346] Experiments were performed on a SHIMADZU LCMS-2020 with a
C18-reverse-phase column (50.times.3.0 mm Titank C18, 3.0 .mu.m
particle size), elution with solvent A: water+5 mM ammonium
bicarbonate; solvent B: acetonitrile. Gradient:
TABLE-US-00016 Gradient - Time flow ml/min % A % B 0.01 1.0 90 10
2.00 1.0 5 95 2.70 1.0 5 95 2.80 1.0 90 10 Detection - UV (220 and
254 nm) and ELSD
[0347] Method P
[0348] Experiments were performed on a SHIMADZU LCMS-2020 with a
C18-reverse-phase column (30.times.2.1 mm Halo C18, 2.0 .mu.m
particle size), elution with solvent A: water+0.05% trifluoroacetic
acid; solvent B: acetonitrile+0.05% trifluoroacetic acid.
Gradient:
TABLE-US-00017 Gradient - Time flow ml/min % A % B 0.00 1.0 95 5
1.30 1.0 0 100 1.80 1.0 0 100 1.90 1.0 95 5 Detection - UV (220 and
254 nm) and ELSD
[0349] Method Q
[0350] Experiments were performed on a SHIMADZU LCMS-2020 with a
C18-reverse-phase column (50.times.3.0 mm YMC-Triart C18, 2.5 .mu.m
particle size), elution with solvent A: water+0.1% formic acid;
solvent B: acetonitrile+0.1% formic acid. Gradient:
TABLE-US-00018 Gradient - Time flow ml/min % A % B 0.01 1.0 95 5
3.00 1.0 5 95 3.70 1.0 5 95 3.75 1.0 95 5 Detection - UV (220 and
254 nm) and ELSD
[0351] Method R
[0352] Experiments were performed on a SHIMADZU LCMS-2020 with a
C18-reverse-phase column (50.times.3 mm Shim-Pack XR-ODS, 2.2 .mu.m
particle size), elution with solvent A: water+0.05% trifluoroacetic
acid; solvent B: acetonitrile+0.05% trifluoroacetic acid.
Gradient:
TABLE-US-00019 Gradient - Time flow ml/min % A % B 0.00 1.2 70 30
3.10 1.2 0 100 3.70 1.2 0 100 3.75 1.2 95 5 Detection - UV (220 and
254 nm) and ELSD
[0353] Method S
[0354] Experiments were performed on a SHIMADZU LCMS-2020 with a
C18-reverse-phase column (50.times.3.0 mm Poroshell HPH-C18, 2.7
.mu.m particle size), elution with solvent A: water+5 mM ammonium
bicarbonate; solvent B: acetonitrile. Gradient:
TABLE-US-00020 Gradient - Time flow ml/min % A % B 0.01 1.0 90 10
3.50 1.0 40 60 4.00 1.0 5 95 4.70 1.0 5 95 4.80 1.0 90 10 Detection
- UV (220 and 254 nm) and ELSD
[0355] Method T
[0356] Experiments were performed on a SHIMADZU 20A HPLC with a
C18-reverse-phase column (50.times.2.1 mm Ascentis Express C18, 2.7
.mu.m particle size), elution with solvent A: water+0.05%
trifluoroacetic acid; solvent B: acetonitrile+0.05% trifluoroacetic
acid. Gradient:
TABLE-US-00021 Gradient - Time flow ml/min % A % B 0.00 1.0 95 5
2.00 1.0 0 100 2.70 1.0 0 100 2.80 1.0 95 5 Detection - UV (220 and
254 nm) and ELSD
[0357] Method U
[0358] Experiments were performed on a SHIMADZU LCMS-2020 with a
C18-reverse-phase column (50.times.3 mm Shim-Pack XR-ODS, 2.2 .mu.m
particle size), elution with solvent A: water+0.05% trifluoroacetic
acid; solvent B: acetonitrile+0.05% trifluoroacetic acid.
Gradient:
TABLE-US-00022 Gradient - Time flow ml/min % A % B 0.01 1.2 95 5
3.50 1.2 50 50 3.70 1.2 0 100 4.70 1.2 0 100 4.75 1.2 95 5
Detection - UV (220 and 254 nm) and ELSD
[0359] Method V
[0360] Experiments were performed on a SHIMADZU LCMS-2020 with a
C18-reverse-phase column (50.times.3.0 mm Poroshell HPH-C18, 2.7
.mu.m particle size), elution with solvent A: water+5 mM ammonium
bicarbonate; solvent B: acetonitrile. Gradient:
TABLE-US-00023 Gradient - Time flow ml/min % A % B 0.01 1.0 90 10
3.50 1.0 60 40 4.00 1.0 5 95 4.70 1.0 5 95 4.80 1.0 90 10 Detection
- UV (220 and 254 nm) and ELSD
[0361] Method W
[0362] Experiments were performed on a SHIMADZU LCMS-2020 with a
C18-reverse-phase column (50.times.2.1 mm Waters Acquity BEH, 1.7
.mu.m particle size), elution with solvent A: water+0.1% formic
acid; solvent B: acetonitrile+0.1% formic acid. Gradient:
TABLE-US-00024 Gradient - Time flow ml/min % A % B 0.00 0.8 95 5
1.60 0.8 0 100 1.80 0.8 0 100 2.00 0.8 95 5 Detection - UV (220 and
254 nm) and ELSD
[0363] Method X
[0364] Experiments were performed on an Agilent 1290 UHPLC coupled
with Agilent MSD (6140) mass spectrometer using ESI as ionization
source. The LC separation was using a Phenomenex XB-C18, 1.7 um,
50.times.2.1 mm column at a flow rate of 0.4 ml/minute. Mobile
phase A was water with 0.1% formic acid and mobile phase B was
acetonitrile with 0.1% formic acid. The gradient started at 2% B
and ended at 98% B over 7 min and was held at 98% B for 1.5 min
following equilibration for 1.5 min. LC column temperature was
40.degree. C. UV absorbance were collected at 220 nm and 254 nm and
mass spec full scan was applied to all experiments.
List of Common Abbreviations
[0365] ACN Acetonitrile [0366] Brine Saturated aqueous sodium
chloride solution [0367] CH.sub.3OD Deuterated Methanol [0368]
CDCl.sub.3 Deuterated Chloroform [0369] DCM Dichloromethane [0370]
DIEA or DIPEA Diisopropylethylamine [0371] DMA Dimethylacetamide
[0372] DMAP 4-Dimethylaminopyridine [0373] DMF Dimethylformamide
[0374] DMSO Dimethylsulfoxide [0375] DMSO-d6 Deuterated
dimethylsulfoxide [0376] DTAD Di-tert-butyl azodicarboxylate [0377]
EDC or EDCI 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide [0378]
ESI Electrospray ionization [0379] EtOAc Ethyl acetate [0380] EtOH
Ethanol [0381] FA Formic Acid [0382] HOAc Acetic acid [0383] g Gram
[0384] h hour [0385] HATU
(O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate) [0386] HCl Hydrochloric acid [0387] HOBt
Hydroxybenzotriazole [0388] HPLC High performance liquid
chromatography [0389] IMS Industrial methylated spirits [0390] L
Liter [0391] LCMS Liquid chromatography-mass spectrometry [0392]
LiHMDS or LHMDS Lithium hexamethydisylazide [0393] MDAP Mass
directed automated purification [0394] MeCN Acetonitrile [0395]
MeOH Methanol [0396] .mu.m Micrometer [0397] min minute [0398] mg
Milligram [0399] mL Milliliter [0400] mm Millimeter [0401] M Molar
[0402] nm Nanometer [0403] NMR Nuclear magnetic resonance
spectroscopy [0404] Pd.sub.2(dba).sub.3.CHCl.sub.3
Tris(dibenzylideneacetone)dipalladium(0)-chloroform adduct [0405]
PE Petroleum ether [0406] Prep-HPLC Preparative high performance
liquid chromatography [0407] PyAOP
(7-Azabenzotriazol-1-yloxy)tripyrrolidinophosphonium
hexafluorophosphate [0408] SCX-2 Strong cation exchange [0409] TBAF
Tetra-n-butylammonium fluoride [0410] THF Tetrahydrofuran [0411]
TFA Trifluoroacetic acid [0412] Xantphos
4,5-Bis(diphenylphosphino)-9,9-dimethylxanthine [0413] ZnCl.sub.2
Zinc chloride
##STR00102##
[0413]
N-[3-[2,5-bis(difluoromethoxy)phenyl]-1H-pyrazol-4-yl]pyrazolo[1,5-
-a]pyrimidine-3-carboxamide
Step 1: Synthesis of 1-(benzyloxy)-4-(difluoromethoxy)benzene
##STR00103##
[0415] Into a 3000-mL round-bottom flask purged and maintained with
an inert atmosphere of nitrogen, was placed N,N-dimethylformamide
(1500 mL), 4-(benzyloxy)phenol (200 g, 999 mmol) and cesium
carbonate (651 g, 1.99 mol). The reaction vessel was equipped with
an outlet for CO.sub.2 release. This was followed by the addition
of sodium 2-chloro-2,2-difluoroacetate (228 g, 1.50 mol, 1.50
equiv) in several batches at 120.degree. C. The reaction was
stirred at 120.degree. C. in an oil bath until gas evolution ceased
(1 h), and then allowed to cool to room temperature. The reaction
mixture was slowly added to 3000 mL of water/ice with stirring. The
resulting mixture was extracted with ethyl acetate (3.times.4000
mL). The organic layers were combined and dried over anhydrous
sodium sulfate and concentrated under reduced pressure. The residue
was purified by flash chromatography on silica gel eluting with
ethyl acetate/petroleum ether (1/19). The appropriate fractions
were combined and concentrated under reduced pressure. This
reaction was repeated four times. This resulted in 450 g (36%) of
1-(benzyloxy)-4-(difluoromethoxy)benzene as a white solid in
total.
Step 2: Synthesis of 4-(difluoromethoxy)phenol
##STR00104##
[0417] Into a 3000-mL round-bottom flask was placed methanol (1500
mL), 1-(benzyloxy)-4-(difluoromethoxy)benzene (140 g, 559 mmol) and
10% Palladium carbon (15 g, 141 mmol). The resulting mixture was
stirred under hydrogen (45 psi) overnight at room temperature. The
catalysts were filtered out. The filtrate was concentrated under
reduced pressure. This reaction was repeated three times. This
resulted in 300 g (78%) of 4-(difluoromethoxy)phenol as a yellow
oil.
Step 3: Synthesis of 2-bromo-4-(difluoromethoxy)phenol
##STR00105##
[0419] Into a 1000-mL round-bottom flask was placed acetic acid
(500 mL), 4-(difluoromethoxy)phenol (50 g, 312 mmol) and NBS (55.6
g, 312 mmol). The reaction mixture was stirred for 1 h at
15.degree. C. The resulting mixture was then added slowly to 1000
mL of water/ice with stirring. The resulting solution was extracted
with ethyl acetate (3.times.1000 mL). The organic layers were
combined, dried over anhydrous sodium sulfate and concentrated
under reduced pressure. The residue was purified by flash
chromatography on silica gel eluting with dichloromethane/petroleum
ether (30/70). The appropriate fractions were collected and
concentrated under reduced pressure. This resulted in 50 g (67%) of
2-bromo-4-(difluoromethoxy)phenol as a light yellow oil.
Step 4: Synthesis of 2-bromo-1,4-bis(difluoromethoxy)benzene
##STR00106##
[0421] Into a 2000-mL round-bottom flask, was placed CH.sub.3CN
(500 mL), water (500 mL), 2-bromo-4-(difluoromethoxy)phenol (54 g,
226 mmol) and potassium hydroxide (94 g, 1.68 mol). The flask was
placed in an ice batch and the reaction mixture was stirred for 30
min in an ice batch. Diethyl (bromodifluoromethyl)phosphonate (120
g, 449 mmol) was then added dropwise to the reaction mixture at
0.degree. C. Upon completion of diethyl
(bromodifluoromethyl)phosphonate addition, the reaction mixture was
stirred for 1 h in a water/ice bath. The resulting solution was
extracted with ethyl acetate (3.times.300 mL). The organic layers
were combined and dried over anhydrous sodium sulfate and
concentrated under reduced pressure. The residue was purified by
flash chromatography on silica gel eluting with ethyl
acetate/petroleum ether (1/19), and the appropriate fractions were
collected and concentrated under reduced pressure. This resulted in
54 g (83%) of 2-bromo-1,4-bis(difluoromethoxy)benzene as light
yellow oil.
Step 5: Synthesis of
5-[2,5-bis(difluoromethoxy)phenyl]-4-nitro-1-[[2-(trimethylsilyl)ethoxy]m-
ethyl]-1H-pyrazole
##STR00107##
[0423] Into a 1000-mL round-bottom flask purged and maintained with
an inert atmosphere of nitrogen, was placed DMA (500 mL), potassium
carbonate (112 g, 810 mmol),
4-nitro-1-[[2-(trimethylsilyl)ethoxy]methyl]-1H-pyrazole (66 g, 271
mmol), 2-bromo-1,4-bis(difluoromethoxy)benzene (79 g, 273 mmol),
2,2-dimethylpropanoic acid (8.3 g, 81.3 mmol), Pd(OAc).sub.2 (6.0
g, 26.7 mmol) and bis(adamantan-1-yl)(butyl)phosphane (19 g, 52.9
mmol, 0.195 equiv). The reaction mixture was stirred at 120.degree.
C. overnight in an oil bath, and allowed to cool to room
temperature. The reaction mixture was then added to 1000 mL of
water/ice with stirring. The resulting solution was extracted with
ethyl acetate (3.times.1000 mL). The organic layers were combined
and dried over anhydrous sodium sulfate and concentrated under
reduced pressure. The residue was purified by flash chromatography
on silica gel eluting with ethyl acetate/petroleum ether (1/1). The
appropriate fractions were collected and concentrated under reduced
pressure. This resulted in 100 g (82%) of
5-[2,5-bis(difluoromethoxy)phenyl]-4-nitro-1-[[2-(trimethylsilyl)ethoxy]m-
ethyl]-1H-pyrazole as a solid. LC/MS (Method H, ESI): [M+H]*=452.1,
R.sub.T=1.49 min
Step 6: Synthesis of
5-[2,5-bis(difluoromethoxy)phenyl]-1-[[2-(trimethylsilyl)ethoxy]methyl]-1-
H-pyrazol-4-amine
##STR00108##
[0425] Into a 3000-mL 3-necked round-bottom flask was placed
ethanol (1500 mL), water (150 mL),
5-[2,5-bis(difluoromethoxy)phenyl]-4-nitro-1-[[2-(trimethylsilyl)ethoxy]m-
ethyl]-1H-pyrazole (100 g, 221 mmol), iron powder (124 g, 2.22 mol)
and NH.sub.4Cl (59.2 g, 1.11 mol). The resulting mixture was
stirred at reflux temperature in an oil bath for 2 h before being
cooled to room temperature. The solids were filtered off and washed
with ethanol. The filtrate was concentrated under reduced pressure.
The residue was dissolved in 3000 mL of ethyl acetate. The ethyl
acetate solution was washed with 1.times.1000 mL of brine, dried
over anhydrous sodium sulfate and concentrated under reduced
pressure. This resulted in 100 g of crude
5-[2,5-bis(difluoromethoxy)phenyl]-1-[[2-(trimethylsilyl)ethoxy]methyl]-1-
H-pyrazol-4-amine as light yellow oil, which was used directly
without purification. LC/MS (Method H, ESI): [M+H]*=422.1, RT=1.25
min.
Step 7: Synthesis of
N-[5-[2,5-bis(difluoromethoxy)phenyl]-1-[[2-(trimethylsilyl)ethoxy]methyl-
]-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00109##
[0427] Into a 2000-mL round-bottom flask was placed DMA (1000 mL),
5-[2,5-bis(difluoromethoxy)phenyl]-1-[[2-(trimethylsilyl)ethoxy]methyl]-1-
H-pyrazol-4-amine, pyrazolo[1,5-a]pyrimidine-3-carboxylic acid
(58.1 g, 356 mmol),
7-Azabenzotriazol-1-yloxy)tripyrrolidinophosphonium
hexafluorophosphate (PyAOP) (186 g, 356 mmol),
4-dimethylaminopyridine (2.90 g, 23.7 mmol) and DIPEA (92.0 g, 712
mmol). The resulting solution was stirred overnight at 65.degree.
C. in an oil bath. The reaction mixture was then added slowly to
2000 mL of water with stirring. The resulting solution was
extracted with ethyl acetate (3.times.2000 mL). The combined
organic phases were washed with 1000 mL of brine, dried over
anhydrous sodium sulfate and concentrated in under pressure. The
residue was purified by flash chromatography on silica gel eluting
with ethyl acetate/petroleum ether (40/60). The appropriate
fractions were combined and concentrated under reduced pressure to
afford 120 g of
N-[5-[2,5-bis(difluoromethoxy)phenyl]-1-[[2-(trimethylsilyl)ethoxy]methyl-
]-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide as a
white solid. LC/MS (Method G, ESI): [M+H]*=567.2, RT=1.05 min.
Step 8: Synthesis of
N-[3-[2,5-bis(difluoromethoxy)phenyl]-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyri-
midine-3-carboxamide
##STR00110##
[0429] Into a 2000-mL round-bottom flask was placed methanol (800
mL), concentrated hydrochloric acid (400 mL, 12N) and
N-[5-[2,5-bis(difluoromethoxy)phenyl]-1-[[2-(trimethylsilyl)ethoxy]methyl-
]-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide (80 g,
141 mmol). The resulting solution was stirred for 4 h at 25.degree.
C. The solids were collected by filtration. The solids were added
to a 1 L flask and H.sub.2O (200 mL) was added. A saturated
NaHCO.sub.3 aqueous solution was added dropwise with stirring until
the solution reached pH.about.8. The solids were collected by
filtration, washed with water and dried to afford 55 g (89%) of
N-[3-[2,5-bis(difluoromethoxy)phenyl]-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyri-
midine-3-carboxamide as a light yellow solid. .sup.1H NMR (300 MHz,
CD.sub.3OD) .delta. 9.08 (dd, J=7.2, 1.5 Hz, 1H), 8.65-8.61 (m,
2H), 8.28 (s, 1H), 7.46 (d, J=9.0 Hz, 1H), 7.40 (d, J=3.0 Hz, 1H),
7.34 (dd, J=8.9, 2.9 Hz, 1H), 7.19 (dd, J=6.7, 4.4 Hz, 1H), 6.87
(t, J=73.7 Hz, 1H), 6.73 (t, J=73.7 Hz, 1H). LC/MS (Method H, ESI):
[M+H].sup.+=437.1, R.sub.T=1.12 min.
##STR00111##
N-(5-(5-bromo-2-(difluoromethoxy)phenyl)-1-((2-(trimethylsilyl)ethoxy)met-
hyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide
Step 1: Synthesis of 4-bromo-1-(difluoromethoxy)-2-iodobenzene
##STR00112##
[0431] To a solution of 4-bromo-2-iodophenol (282 g, 943 mmol) in
N,N-dimethylformamide (2000 mL) and water (500 mL) was added sodium
2-chloro-2,2-difluoroacetate (216 g, 1.42 mol) and cesium carbonate
(617 g, 1.89 mol). The reaction vessel was equipped with a gas
outlet for CO.sub.2 release. The resulting mixture was stirred
overnight at 120.degree. C., allowed to cool to room temperature
and poured into ice water (3000 mL). The resulting solution was
extracted with ethyl acetate (3.times.1500 mL) and the organic
layers were combined. The ethyl acetate extracts were washed with
brine (1000 mL), dried over anhydrous sodium sulfate and
concentrated under reduced pressure. The residue was purified by
flash chromatography on silica gel eluting with ethyl
acetate/petroleum ether (1/10) to afford 300 g (91%) of
4-bromo-1-(difluoromethoxy)-2-iodobenzene as a yellow oil. .sup.1H
NMR (300 MHz, CDCl.sub.3) .delta. 7.96 (dd, J=5.7 Hz, 2.4 Hz, 1H),
7.45 (dd, J=8.7 Hz, 2.4 Hz, 1H), 7.03 (d, J=8.7 Hz, 1H), 6.39 (t,
J=72.9 Hz, 1H).
Step 2: Synthesis of
5-[5-bromo-2-(difluoromethoxy)phenyl]-4-nitro-1-[[2-(trimethylsilyl)ethox-
y]methyl]-1H-pyrazole
##STR00113##
[0433] To a solution of
4-nitro-1-[[2-(trimethylsilyl)ethoxy]methyl]-1H-pyrazole (100 g,
411 mmol) in anhydrous THF (1000 mL) was added dropwise to a
solution of LiHMDS (490 mL, 1.0 mol/L in THF) with stirring at
-70.degree. C. under nitrogen. The resulting solution was stirred
for 1 h at -50.degree. C. and then cooled to -70.degree. C.
ZnCl.sub.2 (500 mL, 0.7 mol/L in THF) was added dropwise at
-70.degree. C. The resulting solution was allowed to warm to room
temperature and stirred at room temperature for 1 h. To the mixture
was added 4-bromo-1-(difluoromethoxy)-2-iodobenzene (150 g, 860
mmol), Pd(PPh.sub.3).sub.4 (24.0 g, 20.8 mmol). The resulting
solution was heated at reflux temperature overnight, allowed to
cool to room temperature, and concentrated under reduced pressure.
This reaction at this scale was repeated one more time, and the
crude products from the two runs were combined for purification.
The residue was purified by flash chromatography on silica gel
eluting with ethyl acetate/petroleum ether (1/20). The appropriate
fractions were combined and concentrated under reduced pressure.
This resulted in 300 g (79%) of
5-[5-bromo-2-(difluoromethoxy)phenyl]-4-nitro-1-[[2-(trimethylsilyl)ethox-
y]methyl]-1H-pyrazole as a light yellow solid in all. .sup.1H NMR
(300 MHz, CDCl.sub.3) .delta. 8.27 (s, 1H), 7.68 (dd, J=8.7, 2.4
Hz, 1H), 7.62 (d, J=2.4 Hz, 1H), 7.19 (d, J=8.4 Hz, 1H), 6.39 (t,
J=72.5 Hz, 1H), 5.44-5.19 (m, 2H), 3.72-3.54 (m, 2H), 0.94-0.89 (m,
2H), 0.02 (s, 9H).
Step 3: Synthesis of
5-(5-bromo-2-(difluoromethoxy)phenyl)-1-((2-(trimethylsilyl)ethoxy)methyl-
)-1H-pyrazol-4-amine
##STR00114##
[0435] To a solution of
5-(5-bromo-2-(difluoromethoxy)phenyl)-4-nitro-1-((2-(trimethylsilyl)ethox-
y)methyl)-1H-pyrazole (50.1 g, 108 mmol) in ethanol (2000 mL) and
water (200 mL) was added iron powder (60.1 g, 1.07 mol) and
NH.sub.4Cl (28.0 g, 0.523 mol). The reaction mixture was stirred at
reflux temperature for 3 h under nitrogen. The solids were filtered
out, and washed with ethanol (100 mL). The filtrate was
concentrated under reduced pressure. The residue was dissolved in
3000 mL of ethyl acetate. The ethyl acetate solution was washed
with 1.times.500 mL of brine, dried over anhydrous sodium sulfate
and concentrated under reduced pressure to give 50.1 g of crude
5-(5-bromo-2-(difluoromethoxy)phenyl)-1-((2-(trimethylsilyl)ethoxy)-
methyl)-1H-pyrazol-4-amine as a yellow oil. The crude product was
used for next step without further purification. LC/MS (Method G,
ESI): [M+H].sup.+=434.2, R.sub.T=0.93 min.
Step 4: Synthesis of
N-(5-(5-bromo-2-(difluoromethoxy)phenyl)-1-((2-(trimethylsilyl)ethoxy)met-
hyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00115##
[0437] To a solution of
5-(5-bromo-2-(difluoromethoxy)phenyl)-1-((2-(trimethylsilyl)ethoxy)methyl-
)-1H-pyrazol-4-amine (50.1 g, 115 mmol) in DMA (1500 mL) was added
pyrazolo[1,5-a]pyrimidine-3-carboxylic acid (32.1 g, 196.0 mmol),
PyAOP (102 g, 196 mmol), DMAP (1.41 g, 11.0 mmol) and DIPEA (44.1
g, 0.341 mol). The resulting solution was stirred for 3 h at
60.degree. C. in an oil bath, and then allowed to cool to room
temperature. The reaction mixture was then partitioned between
water/ice (2000 mL) and ethyl acetate (2000 mL). The aqueous phase
was extracted with ethyl acetate (2.times.). The organic layers
were combined, washed with brine (1000 mL), dried over anhydrous
sodium sulfate and concentrated under reduced pressure. The residue
was purified by flash chromatography on silica gel eluting with
ethyl acetate/petroleum ether (4:1). The appropriate fractions were
combined and concentrated under reduced pressure. Water (150 mL)
was added to the residue and the mixture was stirred in water for 1
h at room temperature. The solid was collected by filtration and
air-dried to afford 60.1 g (91%) of
N-(5-(5-bromo-2-(difluoromethoxy)phenyl)-1-((2-(trimethylsilyl)ethoxy)met-
hyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide as
alight yellow solid. LC/MS (Method G, ESI): [M+H]*=579.1 &
581.1, RT=1.10 min. .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 9.62
(s, 1H), 8.80 (dd, J=6.9, 1.7 Hz, 1H), 8.73 (s, 1H), 8.53 (dd,
J=4.2, 1.7 Hz, 1H), 8.38 (s, 1H), 7.79 (d, J=2.4 Hz, 1H), 7.67 (dd,
J=8.8, 2.5 Hz, 1H), 7.29 (d, J=1.4 Hz, 1H), 7.00 (dd, J=6.9, 4.2
Hz, 1H), 6.43 (t, J=72.6 Hz, 1H), 5.53-5.27 (m, 2H), 3.73-3.50 (m,
2H), 0.88 (ddd, J=9.5, 6.4, 4.4 Hz, 2H), 0.00 (s, 9H).
##STR00116##
N-[3-[5-bromo-2-(difluoromethoxy)phenyl]-1H-pyrazol-4-yl]pyrazolo[1,5-a]p-
yrimidine-3-carboxamide
[0438]
N-[5-[5-bromo-2-(difluoromethoxy)phenyl]-1-[[2-(trimethylsilyl)etho-
xy]methyl]-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide
(Intermediate 2, 5.00 g, 8.63 mmol) was treated with HCl/dioxane
(150 mL, 4 M) overnight at room temperature. The resulting mixture
was concentrated under reduced pressure. This resulted in 3.80 g of
N-[3-[5-bromo-2-(difluoromethoxy)phenyl]-1H-pyrazol-4-yl]pyrazolo[1,5-a]p-
yrimidine-3-carboxamide as a yellow solid. The purity of the
intermediate was sufficient for use in the next step without
further purification. LC/MS (Method I, ESI): [M+H].sup.+=449.0,
RT=1.02 min. .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 9.11 (dd,
J=6.8, 1.6 Hz, 1H), 8.67-8.64 (m, 2H), 8.32 (s, 1H), 7.80 (d, J=2.4
Hz, 1H), 7.72 (dd, J=8.8, 2.4 Hz, 1H), 7.37 (d, J=8.8 Hz, 1H), 7.23
(dd, J=7.0, 4.2 Hz, 1H), 6.81 (t, J=73.2 Hz, 1H).
##STR00117##
N-[3-[2-(difluoromethoxy)-5-iodophenyl]-1H-pyrazol-4-yl]pyrazolo[1,5-a]py-
rimidine-3-carboxamide
Step 1: Synthesis of
N-[5-[2-(difluoromethoxy)-5-iodophenyl]-1-[[2-(trimethylsilyl)ethoxy]meth-
yl]-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00118##
[0440] To a solution of
N-[5-[5-bromo-2-(difluoromethoxy)phenyl]-1-[[2-(trimethylsilyl)ethoxy]met-
hyl]-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide (100
mg, 0.173 mmol) in t-BuOH (2 mL) was added
N,N-dimethylethane-1,2-diamine (2.28 mg, 0.0259 mmol), NaI (155 mg,
1.04 mmol), CuI (4.93 mg, 0.026 mmol) under nitrogen. The resulting
solution was stirred for 14 h at 120.degree. C. in an oil bath
under nitrogen before being cooled to room temperature. The
resulting mixture was concentrated under reduced pressure. The
residue was purified by flash chromatography on silica gel eluting
with ethyl acetate/petroleum ether (1/1) to afford 80 mg (74%) of
N-[5-[2-(difluoromethoxy)-5-iodophenyl]-1-[[2-(trimethylsilyl)ethoxy]meth-
yl]-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide as a
yellow solid. LC/MS (Method J, ESI): [M+H]*=627.1, R.sub.T=1.31
min.
Step 2: Synthesis of
N-[3-[2-(difluoromethoxy)-5-iodophenyl]-1H-pyrazol-4-yl]pyrazolo[1,5-a]py-
rimidine-3-carboxamide
##STR00119##
[0442]
N-[5-[2-(difluoromethoxy)-5-iodophenyl]-1-[[2-(trimethylsilyl)ethox-
y]methyl]-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide
(80.0 mg, 0.128 mmol) was treated with CF.sub.3CO.sub.2H (3.0 mL)
for 30 min at room temperature. The resulting mixture was
concentrated under reduced pressure. The residue was dissolved in
water. Saturated sodium bicarbonate was slowly added until the
solution was adjusted pH 8. The solid was collected by filtration.
The solid was purified by flash chromatography on silica gel
eluting with ethyl acetate/petroleum ether (2/1) to give 23.0 mg
(36%) of
N-[3-[2-(difluoromethoxy)-5-iodophenyl]-1H-pyrazol-4-yl]pyrazolo[1,5-a]py-
rimidine-3-carboxamide as alight yellow solid. LC/MS (Method K,
ESI): [M+H].sup.+=497.1, R.sub.T=1.74 min. .sup.1H NMR (300 MHz,
CD.sub.3OD) .delta. 9.08 (dd, J=6.9, 1.5 Hz, 1H), 8.65-8.61 (m,
2H), 8.27 (s, 1H), 7.94 (s, 1H), 7.87 (d, J=8.7 Hz, 1H), 7.21-7.18
(m, 2H), 6.78 (t, J=73.2 Hz, 1H).
##STR00120##
N-[3-[5-cyclopropyl-2-(difluoromethoxy)phenyl]-1H-pyrazol-4-yl]pyrazolo[1-
,5-a]pyrimidine-3-carboxamide
Step 1: Synthesis of
N-[3-[5-cyclopropyl-2-(difluoromethoxy)phenyl]-1-[[2-(trimethylsilyl)etho-
xy]methyl]-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00121##
[0444] To a solution of
N-(5-(5-bromo-2-(difluoromethoxy)phenyl)-1-((2-(trimethylsilyl)ethoxy)met-
hyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide
(Intermediate 2, 1.40 g, 2.41 mmol) in dioxane (15 mL) and water
(3.0 mL) was added cyclopropylboronic acid (314 mg, 3.66 mmol),
Pd(dppf)Cl.sub.2--CH.sub.2Cl.sub.2 (200 mg, 0.245 mmol) and cesium
carbonate (1.56 g, 4.79 mmol) under nitrogen. The reaction mixture
was stirred overnight at 80.degree. C. under nitrogen. The
resulting mixture was concentrated under reduced pressure. The
residue was passed through a short pad of silica gel eluting with
dichloromethane/methanol (94/6). The appropriate fractions were
combined and concentrated under reduced pressure to give 1.40 g
(purity=85% at 254 nm) of
N-[3-[5-cyclopropyl-2-(difluoromethoxy)phenyl]-1-[[2-(trimethylsilyl)etho-
xy]methyl]-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide
as a dark red solid. LC/MS (Method G, ESI): [M+H].sup.+=541.2,
RT=1.12 min. The intermediate was used without further
purification.
Step 2: Synthesis of
N-[3-[5-cyclopropyl-2-(difluoromethoxy)phenyl]-1H-pyrazol-4-yl]pyrazolo[1-
,5-a]pyrimidine-3-carboxamide
##STR00122##
[0446]
N-[3-[5-cyclopropyl-2-(difluoromethoxy)phenyl]-1-[[2-(trimethylsily-
l)ethoxy]methyl]-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide
(145 mg) from previous step was treated with HCl/dioxane (5.0 mL, 4
M) for 2 h at 25.degree. C. The solution was concentrated under
reduced pressure. The residue was purified by Prep-HPLC with the
following conditions: Column: Xbridge C18, 19*150 mm, 5 um; Mobile
Phase A: Water/0.05% NH.sub.4HCO.sub.3, Mobile Phase B: ACN; Flow
rate: 30 mL/min; Gradient: 20% B to 85% B in 10 min; 254 nm to
obtain 44.9 mg (41%) of
N-[3-[5-cyclopropyl-2-(difluoromethoxy)phenyl]-1H-pyrazol-4-yl]pyrazolo[1-
,5-a]pyrimidine-3-carboxamide as a yellow solid. LC/MS (Method H,
ESI): [M+H].sup.+=411.2, RT=1.14 min. .sup.1H NMR (300 MHz,
CD.sub.3OD) .delta. 9.09 (dd, J=6.9, 1.5 Hz, 1H), 8.63-8.61 (m,
2H), 8.27 (s, 1H), 7.28-7.25 (m, 3H), 7.20 (dd, J=7.2, 4.2 Hz, 1H),
6.68 (t, J=73.8 Hz, 1H), 2.04-1.95 (m, 1H), 1.03-0.97 (m, 2H),
0.79-0.71 (m, 2H).
##STR00123##
Pyrazolo[1,5-a]pyrimidine-3-carboxylic acid
[3-(5-chloro-2-difluoromethoxyphenyl)-1H-pyrazol-4-yl] amide
Step 1: Synthesis of
2-bromo-4-chloro-1-(difluoromethoxy)benzene
##STR00124##
[0448] To a solution of 2-bromo-4-chlorophenol (4.98 g, 24.0 mmol)
in DMF (25 mL) was added sodium chlorodifluoroacetate (8.42 g, 55.2
mmol), cesium carbonate (10.97 g, 33.67 mmol) and water (2.5 mL).
The reaction was stirred at 100.degree. C. for 16 hours. The
reaction mixture was partitioned between ethyl acetate and water,
the organic portion washed with brine, dried (MgSO.sub.4), and
evaporated. The crude product was purified by flash chromatography
on silica eluting with 0-20% EtOAc in heptanes to yield
2-bromo-4-chloro-1-(difluoromethoxy)benzene as a clear, colorless
oil (2.98 g, 48%). 1H NMR (400 MHz, DMSO-d.sub.6) .delta.: (ppm)
7.90 (d, 1H), 7.54 (dd, 1H), 7.38 (d, 1H), 7.28 (t, 1H).
Step 2: Synthesis of
5-(5-chloro-2-difluoromethoxyphenyl)-4-nitro-1-(2-trimethylsilanylethoxym-
ethyl)-1H-pyrazole
##STR00125##
[0450] To a solution of
4-nitro-1-(2-trimethylsilanylethoxymethyl)-1H-pyrazole (preparation
described in WO2011003065) (46.5 g, 191 mmol) in DMA (350 mL) was
added 2-bromo-4-chloro-1-difluoromethoxybenzene (64.0 g, 248 mmol),
palladium (II) acetate (2.15 g, 9.6 mmol),
di-(adamantyl)-n-butylphosphine (5.0 g, 13.4 mmol), potassium
carbonate (79.2 g, 573 mmol) and trimethylacetic acid (5.27 g, 51.6
mmol). The mixture was degassed with nitrogen for 10 min then
heated at 130.degree. C. for 8 h. The reaction mixture was allowed
to cool to room temperature, diluted with ethyl acetate and washed
with water and brine, dried (MgSO.sub.4), filtered and evaporated.
The resultant crude material was purified by flash chromatography
on silica eluting with 0-10% EtOAc in cyclohexane to afford
5-(5-chloro-2-difluoromethoxyphenyl)-4-nitro-1-(2-trimethylsilanylethoxym-
ethyl)-1H-pyrazole (62.4 g, 78%). .sup.1H NMR (400 MHz, CDCl.sub.3)
.delta.: (ppm) 8.24 (s, 1H), 7.52-7.53 (m, 2H), 6.39 (t, 1H),
5.29-5.30 (m, 2H), 3.63-3.64 (m, 2H), 0.90 (s, 9H).
Step 3: Synthesis of
5-(5-chloro-2-(difluoromethoxy)phenyl)-1-((2-(trimethylsilyl)ethoxy)methy-
l)-1H-pyrazol-4-amine
##STR00126##
[0452] To a solution of
5-(5-chloro-2-difluoromethoxyphenyl)-4-nitro-1-(2-trimethylsilanylethoxym-
ethyl)-1H-pyrazole (62 g, 148 mmol) in ethanol (600 mL) was added
water (200 mL), ammonium chloride (32 g, 590 mmol) and iron powder
(41 g, 740 mmol). The mixture was heated at 80.degree. C. for 2
hours then allowed to cool to room temperature. The residual solid
was removed by filtration through Celite@. The filtrate was
evaporated under reduced pressure, diluted with water and extracted
twice with DCM. The combined organic extracts were washed with
water and brine, dried (MgSO.sub.4) and evaporated to afford a dark
oil. The oil was purified by flash chromatography on silica eluting
with 0-25% EtOAc in DCM. Appropriate fractions were collected and
the solvent removed in vacuo to afford
5-(5-chloro-2-(difluoromethoxy)phenyl)-1-((2-(trimethylsilyl)ethoxy)methy-
l)-1H-pyrazol-4-amine as a brown oil (30.8 g, 54%). .sup.1H NMR
(400 MHz, CDCl3) .delta.: (ppm) 7.56 (d, 1H), 7.44 (dd, 1H), 7.34
(s, 1H), 7.30-7.25 (m, 1H), 6.37 (t, 1H), 5.29 (s, 2H), 3.56 (t,
2H), 0.88 (dd, 2H), 0.00 (s, 9H).
Step 4: Synthesis of
N-(5-(5-chloro-2-(difluoromethoxy)phenyl)-1-((2-(trimethylsilyl)ethoxy)me-
thyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00127##
[0454] A solution of
5-(5-chloro-2-(difluoromethoxy)phenyl)-1-((2-(trimethylsilyl)ethoxy)methy-
l)-1H-pyrazol-4-amine (60.0 g, 154 mmol) in THF (100 mL) was added
drop wise over 30 minutes to an ice/water cooled mixture of
pyrazolo[1,5-a]pyrimidine-3-carbonyl chloride (27.8 g, 153 mmol),
and DIPEA (49.5 g, 383 mmol) in THF (300 mL). On complete addition
the mixture was left to stir at room temperature for 1 h. The
solvent was evaporated and the residue diluted with 0.5 N aqueous
HCl and extracted with ethyl acetate. The combined organic extract
was passed through Celite@ to remove the residual solid and the
filtrate washed with 1 M aqueous potassium carbonate, water and
brine, dried (sodium sulfate) and evaporated to give a red solid.
The solid was triturated with 10% diethyl ether in cyclohexane. The
solid was collected by filtration, washed with 1:1 diethyl ether in
cyclohexane and left to air dry to afford
N-(5-(5-chloro-2-(difluoromethoxy)phenyl)-1-((2-(trimethylsilyl)ethoxy)me-
thyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide as an
off-white solid (59.2 g, 73%). .sup.1H NMR (300 MHz, CDCl.sub.3):
.delta. (ppm) 9.61 (s, 1H), 8.77-8.78 (m, 1H), 8.51 (dd, 1H), 8.36
(s, 1H), 7.65 (d, 1H), 7.52 (dd, 1H), 7.36 (d, 1H), 7.29 (s, 1H),
7.01 (dd, 1H), 6.42 (t, 1H), 5.39-5.41 (m, 2H), 3.60-3.64 (m, 2H),
0.87-0.89 (m, 2H), 0.09 (s, 9H).
Step 5: Synthesis of
N-(3-(5-chloro-2-(difluoromethoxy)phenyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]-
pyrimidine-3-carboxamide
##STR00128##
[0456] A suspension of
N-(5-(5-chloro-2-(difluoromethoxy)phenyl)-1-((2-(trimethylsilyl)ethoxy)me-
thyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (59.0
g, 110 mmol) in methanol (420 mL) was treated with 6N HCl (80 mL)
and the mixture heated at 60.degree. C. for 4 h. The solvent was
evaporated and the residue triturated with water. The solid was
collected by filtration, washed with water and left to air dry. The
solid was triturated with a minimum volume of acetonitrile,
collected by filtration, washed with diethyl ether and dried at
60.degree. C. under high vacuum to afford the title compound as a
yellow solid (42.9 g, 96%). .sup.1H NMR (400 MHz, DMSO-d.sub.6)
.delta.: (ppm) 9.71 (s, 1H), 9.34 (dd, 1H), 8.68-8.69 (m, 1H), 8.66
(s, 1H), 8.25 (s, 1H), 7.62 (dd, 2H), 7.43-7.46 (m, 1H), 7.29 (dd,
1H), 7.23 (d, 1H).
##STR00129##
N-(3-(2-(difluoromethoxy)-5-(methylthio)phenyl)-1H-pyrazol-4-yl)pyrazolo[-
1,5-a]pyrimidine-3-carboxamide
Step 1: Synthesis of
5-(2-(difluoromethoxy)-5-(methylthio)phenyl)-4-nitro-1-((2-(trimethylsily-
l)ethoxy)methyl)-1H-pyrazole
##STR00130##
[0458] Into a 1000-mL round-bottom flask purged and maintained with
an inert atmosphere of nitrogen, was placed toluene (500 mL),
5-[5-bromo-2-(difluoromethoxy)phenyl]-4-nitro-1-[[2-(trimethylsilyl)ethox-
y] methyl]-1H-pyrazole (60 g, 129 mmol), NaSMe (26 g, 371 mmol),
Pd.sub.2(dba).sub.3.CHCl.sub.3 (6.7 g, 6.47 mmol), XantPhos (7.5 g,
12.96 mmol). The resulting mixture was stirred overnight at
85.degree. C. The resulting mixture was concentrated under vacuum.
This reaction was repeated three times. The residue was applied
onto a silica gel column eluting with ethyl acetate/petroleum ether
(1:20). The appropriate fractions were combined and concentrated
under vacuum. This resulted in 171 g of
5-(2-(difluoromethoxy)-5-(methylthio)phenyl)-4-nitro-1-((2-(trim-
ethylsilyl)ethoxy)methyl)-1H-pyrazole as a yellow solid in all.
LC/MS (Method F, ESI): [M+H]+=432.1, RT=1.23 min; .sup.1H NMR (300
MHz, CDCl3) .delta.: (ppm) 8.25 (s, 1H), 7.42 (dd, J=8.7, 2.4 Hz,
1H), 7.34 (d, J=2.1 Hz, 1H), 7.23 (d, J=8.7 Hz, 1H), 6.39 (t,
J=72.9 Hz, 1H), 5.36-5.22 (m, 2H), 3.74-3.55 (m, 2H), 2.51 (s, 3H),
0.94-0.90 (m, 2H), 0.02 (s, 9H).
Step 2: Synthesis of
5-(2-(difluoromethoxy)-5-(methylthio)phenyl)-1-((2-(trimethylsilyl)ethoxy-
)methyl)-1H-pyrazol-4-amine
##STR00131##
[0460] To a mixture of
5-[2-(difluoromethoxy)-5-(methylsulfanyl)phenyl]-4-nitro-1-[[2-(trimethyl-
silyl)ethoxy]methyl]-1H-pyrazole (171 g, 408 mmol), ethanol (2000
mL), water (200 mL) was added iron powder (228 g, 4.08 mol),
NH.sub.4Cl (120 g, 2.24 mol). The reaction mixture was stirred at
reflux for 3 h under nitrogen, and cooled to room temperature. The
solids were filtered out. The filtrate was concentrated under
vacuum. The residue was dissolved in 3000 mL of ethyl acetate and
washed with 1.times.500 mL of brine. The organic phase was dried
over anhydrous sodium sulfate and concentrated under vacuum. This
resulted in 148 g of
5-(2-(difluoromethoxy)-5-(methylthio)phenyl)-1-((2-(trimethylsilyl)ethoxy-
)methyl)-1H-pyrazol-4-amine as yellow oil. LC/MS (Method F, ESI):
[M+H]+=402.1, R.sub.T=0.93 min.
Step 3: Synthesis of
N-(5-(2-(difluoromethoxy)-5-(methylthio)phenyl)-1-((2-(trimethylsilyl)eth-
oxy)methyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00132##
[0462] Into a 3000-mL 3-necked round-bottom flask, was placed DMA
(1500 mL),
5-(2-(difluoromethoxy)-5-(methylthio)phenyl)-1-((2-(trimethylsilyl)e-
thoxy)methyl)-1H-pyrazol-4-amine (148 g),
pyrazolo[1,5-a]pyrimidine-3-carboxylic acid (102 g), HATU (325 g),
4-dimethylaminopyridine (4.5 g), DIPEA (142 g). The resulting
solution was stirred for 3 h at 60.degree. C., poured into ice
water (2000 mL), extracted with 3.times.2000 mL of ethyl acetate
and the organic layers combined. The resulting mixture was washed
with 1.times.1000 mL of brine. The mixture was dried over anhydrous
sodium sulfate and concentrated under vacuum. The residue was
applied onto a silica gel column eluting with ethyl
acetate/petroleum ether (4:1) to give 200 g of
N-(5-(2-(difluoromethoxy)-5-(methylthio)phenyl)-1-((2-(trimethylsilyl)eth-
oxy)methyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide
as a light yellow solid. LC/MS (Method A, ESI): [M+H]+=547.2,
RT=1.10 min; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.: (ppm) 9.63
(s, 1H), 8.77 (dd, J=7.0, 1.7 Hz, 1H), 8.73 (s, 1H), 8.51 (dd,
J=4.2, 1.8 Hz, 1H), 8.38 (s, 1H), 7.50 (d, J=2.4 Hz, 1H), 7.39 (dd,
J=8.7, 2.4 Hz, 1H), 7.30 (d, J=8.7 Hz, 1H), 6.98 (dd, J=6.9, 4.2
Hz, 1H), 6.39 (t, J=73.2 Hz, 1H), 5.46-5.38 (m, 2H), 3.70-3.59 (m,
2H), 2.52 (s, 3H), 0.92-0.85 (m, 2H), 0.03 (s, 9H).
Step 4: Synthesis of
N-(3-(2-(difluoromethoxy)-5-(methylthio)phenyl)-1H-pyrazol-4-yl)pyrazolo[-
1,5-a]pyrimidine-3-carboxamide
##STR00133##
[0464] To a solution of
N-(5-(2-(difluoromethoxy)-5-(methylthio)phenyl)-1-((2-(trimethylsilyl)eth-
oxy)methyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide
(60 g) in methanol (600 mL) was added concentrated HCl solution
(300 mL). The resulting solution was stirred overnight at
35.degree. C. The resulting mixture was concentrated under vacuum.
The solids were collected by filtration. The solid was suspended in
200 mL of water. The pH value of the solution was adjusted to 8
with saturated sodium bicarbonate. The product was collected by
filtration, dried to give 30 g (66%) of
N-(3-(2-(difluoromethoxy)-5-(methylthio)phenyl)-1H-pyrazol-4-yl)pyrazolo[-
1,5-a]pyrimidine-3-carboxamide as alight yellow solid. LC/MS
(Method G, ESI): [M+H]+=417.0, R.sub.T=0.80 min; .sup.1H NMR (300
MHz, DMSO-d.sub.6) .delta.: (ppm) 13.02 (s, 1H), 9.71 (s, 1H), 9.33
(dd, J=6.9, 1.5 Hz, 1H), 8.68 (dd, J=4.1, 1.4 Hz, 1H), 8.66 (s,
1H), 8.24 (s, 1H), 7.47-7.36 (m, 3H), 7.27 (dd, J=6.9, 4.2 Hz, 1H),
7.17 (t, J=73.8 Hz, 1H), 2.51 (s, 3H).
##STR00134##
N-(5-(5-bromo-4-chloro-2-(difluoromethoxy)phenyl)-1-((2-(trimethylsilyl)e-
thoxy)methyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide
Step 1: Synthesis of 4-bromo-5-chloro-2-iodophenol
##STR00135##
[0466] To a solution of 5-chloro-2-iodophenol (100 g, 393 mmol) in
acetonitrile (1000 mL) was added CuBr.sub.2 (264 g, 1.18 mol) in
several batches with stirring at 70.degree. C. The resulting
mixture was stirred for 4 h at 70.degree. C., cooled to room
temperature and concentrated under vacuum. The residue was then
quenched by the addition of 3000 mL of water/ice, and extracted
with 3.times.2000 mL of ethyl acetate and the organic layers
combined. The extracts were washed with 1.times.1000 mL of brine,
dried over sodium sulfate, and concentrated under vacuum. The
residue was purified by flash chromatography on silica gel eluting
with ethyl acetate/petroleum ether (1:30). The appropriate
fractions were collected and concentrated under vacuum. The
reaction was repeated one more time. This resulted in 140 g (53%)
of 4-bromo-5-chloro-2-iodophenol as a white solid. H NMR (400 MHz,
CDCl.sub.3): .delta. (ppm) 7.89 (s, 1H), 7.14 (s, 1H), 5.32 (s,
1H).
Step 2: Synthesis of
1-bromo-2-chloro-4-(difluoromethoxy)-5-iodobenzene
##STR00136##
[0468] To a solution of 4-bromo-5-chloro-2-iodophenol (140 g, 420
mmol) in DMF (1200 mL) were added sodium
2-chloro-2,2-difluoroacetate (95.8 g, 628 mmol), cesium carbonate
(274 g, 840 mmol). The reaction mixture was stirred for 2 h at
120.degree. C. in an oil bath, cooled to room temperature, and then
quenched by the addition of 2500 mL of water/ice. The resulting
solution was extracted with 3.times.2000 mL of ethyl acetate and
the organic layers combined. The extracts were washed with
1.times.1000 mL of brine, dried over anhydrous sodium sulfate and
concentrated under vacuum. The residue was purified by flash
chromatography on silica gel eluting with ethyl acetate/petroleum
ether (1/30). The appropriate fractions were combined and
concentrated under vacuum to give 130 g (81%) of
1-bromo-2-chloro-4-(difluoromethoxy)-5-iodobenzene as a light
yellow solid.
Step 3: Synthesis of
5-(5-bromo-4-chloro-2-(difluoromethoxy)phenyl)-4-nitro-1-((2-(trimethylsi-
lyl)ethoxy)methyl)-1H-pyrazole
##STR00137##
[0470] To a solution of
4-nitro-1-[[2-(trimethylsilyl)ethoxy]methyl]-1H-pyrazole (67.0 g,
275 mmol) in tetrahydrofuran (1000 mL) was added LiHMDS (340 mL, 1
M in THF) dropwise with stirring at -70.degree. C. under nitrogen.
The resulting solution was stirred for 1 h at -70.degree. C. To
this solution was added ZnCl.sub.2 (400 mL, 0.7 M in THF) dropwise
with stirring at -70.degree. C. The resulting solution was stirred
for 1 h at -70.degree. C. under nitrogen. To the mixture was added
1-bromo-2-chloro-4-(difluoromethoxy)-5-iodobenzene (105 g, 274
mmol), Pd(PPh.sub.3).sub.4 (16.0 g, 13.9 mmol) under nitrogen. The
resulting solution was stirred overnight at 90.degree. C., allowed
to cool to room temperature and concentrated under vacuum. The
residue was purified by flash chromatography on silica gel eluting
with ethyl acetate/petroleum ether (1:20). The appropriate
fractions were collected and concentrated under vacuum. This
resulted in 115 g (84%) of
5-[5-bromo-4-chloro-2-(difluoromethoxy)phenyl]-4-nitro-1-[[2-(trimethylsi-
lyl)ethoxy]methyl]-1H-pyrazole as a light yellow solid. LC/MS
(Method B, ESI): [M+H]+=498.0 & 500.0, Rt=1.27 min.
Step 4: Synthesis of
5-[5-bromo-4-chloro-2-(difluoromethoxy)phenyl]-1-[[2-(trimethylsilyl)etho-
xy]methyl]-1H-pyrazol-4-amine
##STR00138##
[0472] To a solution of
5-(5-bromo-4-chloro-2-(difluoromethoxy)phenyl)-4-nitro-1-((2-(trimethylsi-
lyl)ethoxy)methyl)-1H-pyrazole (102 g, 205 mmol) in ethanol (1000
mL) and water (100 mL) was added iron powder (102 g, 1.82 mol) and
ammonium chloride (53 g, 1.00 mol). The reaction mixture was
stirred for 3 h at 100.degree. C. in an oil bath. The solids were
filtered out. The filtrate was concentrated under vacuum. The
residue was dissolved in 2000 mL of ethyl acetate. The organic
solution was washed with 1.times.500 mL of brine, dried over
anhydrous sodium sulfate and concentrated under vacuum. This
resulted in 102 g (crude) of
5-[5-bromo-4-chloro-2-(difluoromethoxy)phenyl]-1-[[2-(trimethylsilyl)etho-
xy]methyl]-1H-pyrazol-4-amine as light yellow oil. LC/MS (Method E,
ESI): [M+H]+=467.9 & 469.9, R.sub.T=1.29 min.
Step 5: Synthesis of
N-(5-(5-bromo-4-chloro-2-(difluoromethoxy)phenyl)-1-((2-(trimethylsilyl)e-
thoxy)methyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00139##
[0474] To a solution of
5-[5-bromo-4-chloro-2-(difluoromethoxy)phenyl]-1-[[2-(trimethylsilyl)etho-
xy]methyl]-1H-pyrazol-4-amine (100 g, 213 mmol) in DMA (800 mL) was
added pyrazolo[1,5-a]pyrimidine-3-carboxylic acid (52.0 g, 319
mmol), PyAOP (166 g, 319 mmol), DIPEA (82.3 g, 638 mmol) and
4-dimethylaminopyridine (2.59 g, 21.2 mmol). The resulting solution
was stirred overnight at 60.degree. C. in an oil bath. The reaction
was then quenched by the addition of 2000 mL of water/ice. The
resulting solution was extracted with 3.times.1500 mL of ethyl
acetate and the organic layers combined. The combined organic
layers were washed with 500 mL of brine, dried over anhydrous
sodium sulfate and concentrated under vacuum. The residue was
purified by flash chromatography on silica gel eluting with ethyl
acetate/petroleum ether (2:1). The appropriate fractions were
combined and concentrated under vacuum. The residue was suspended
in water (800 mL) and stirred for 1 h. The solids were collected by
filtration. This resulted in 113 g (86%) of
N-(5-(5-bromo-4-chloro-2-(difluoromethoxy)phenyl)-1-((2-(trimethylsilyl)e-
thoxy)methyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide
as an off-white solid. LC/MS (Method A, ESI): [M+H]+=613.2 &
615.2, RT=2.29 min. .sup.1H NMR (400 MHz, CDCl.sub.3): .delta.
(ppm) 9.56 (s, 1H), 8.81 (dd, J=6.8, 1.5 Hz, 1H), 8.73 (s, 1H),
8.53 (d, J=4.0 Hz, 1H), 8.33 (s, 1H), 7.92 (s, 1H), 7.54 (s, 1H),
7.03 (dd, J=6.8 Hz, 4.0 Hz, 1H), 6.45 (t, J=72.2 Hz, 1H), 5.43 (d,
J=11.2 Hz, 1H), 5.35 (d, J=11.2 Hz, 1H), 3.68-3.56 (m, 2H),
0.94-0.84 (m, 2H), 0.00 (s, 9H).
##STR00140##
N-(3-(6-(difluoromethoxy)-3,4-dihydro-2H-benzo[b][1,4]oxazin-7-yl)-1H-pyr-
azol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide
Step 1: Synthesis of tert-butyl
(2-(2-chloro-4-(difluoromethoxy)-5-(4-(pyrazolo[1,5-a]pyrimidine-3-carbox-
amido)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-5-yl)phenoxy)ethyl)-
carbamate
##STR00141##
[0476] To a solution of Intermediate 8 (200 mg, 0.326 mmol) in
toluene (10 mL) was added tert-butyl N-(2-hydroxyethyl)carbamate
(105 mg, 0.651 mmol), [PdCl(allyl)]2 (6.01 mg, 0.0161 mmol),
t-BuBrettPhos (16.0 mg, 0.0329 mmol) and cesium carbonate (213 mg,
0.654 mmol) under nitrogen. The resulting solution was stirred for
4 h at 60.degree. C. and concentrated under vacuum. The residue was
purified by flash chromatography on silica gel eluting with
dichloromethane/methanol (19/1). The appropriate fractions were
combined and concentrated under vacuum to give 182 mg (80%) of
tert-butyl
(2-(2-chloro-4-(difluoromethoxy)-5-(4-(pyrazolo[1,5-a]pyrimidine-3-carbox-
amido)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-5-yl)phenoxy)ethyl)-
carbamate as yellow oil. LC/MS (Method C, ESI): [M+H]+=694.1,
Rt=1.54 min.
Step 2: Synthesis of tert-butyl
6-(difluoromethoxy)-7-(4-(pyrazolo[1,5-a]pyrimidine-3-carboxamido)-1-((2--
(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-5-yl)-2,3-dihydro-4H-benzo[b][1,-
4]oxazine-4-carboxylate
##STR00142##
[0478] To a solution of tert-butyl
(2-(2-chloro-4-(difluoromethoxy)-5-(4-(pyrazolo[1,5-a]pyrimidine-3-carbox-
amido)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-5-yl)phenoxy)ethyl)-
carbamate (182 mg, 0.270 mmol) in t-BuOH (15 mL) was added
BrettPhos Palladacycle Gen. 3 (CAS 1470372-59-8, vendor J&K
Scientific Ltd) (48.0 mg, 0.0530 mmol), BrettPhos (56.0 mg, 0.104
mmol) and potassium carbonate (73.0 mg, 0.528 mmol) under nitrogen.
The resulting solution was stirred for 20 h at 110.degree. C.,
allowed to cool to room temperature, concentrated under vacuum. The
residue was purified by flash chromatography on silica gel eluting
with ethyl acetate/petroleum ether (1/1). The appropriate fractions
were combined and concentrated under vacuum to give 95.0 mg (53%)
of tert-butyl
6-(difluoromethoxy)-7-(4-(pyrazolo[1,5-a]pyrimidine-3-carboxamido)-1-((2--
(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-5-yl)-2,3-dihydro-4H-benzo[b][1,-
4]oxazine-4-carboxylate as a yellow solid. LC/MS (Method B, ESI):
[M+H]+=658.1, Rt=1.17 min.
Step 3: Synthesis of
N-(3-(6-(difluoromethoxy)-3,4-dihydro-2H-benzo[b][1,4]oxazin-7-yl)-1H-pyr-
azol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00143##
[0480] To a solution of tert-butyl
6-(difluoromethoxy)-7-(4-(pyrazolo[1,5-a]pyrimidine-3-carboxamido)-1-((2--
(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-5-yl)-2,3-dihydro-4H-benzo[b][1,-
4]oxazine-4-carboxylate (80.0 mg, 0.122 mmol) in methanol (8.0 mL)
was added aqueous HCl solution (6 mol/L in water) (4.0 mL). The
resulting solution was stirred for 4 h at 25.degree. C. and
concentrated under vacuum. The crude product (50.0 mg) was purified
by Prep-HPLC with the following conditions: Column, XBridge Shield
RP18 OBD Column, 19* 150 mm, 5 um; mobile phase, 10 mM
NH.sub.4HCO.sub.3 in water and acetonitrile (10.0% acetonitrile up
to 38.0% in 10 min); Detector, UV 254 nm to obtain 16.1 mg (24%) of
N-(3-(6-(difluoromethoxy)-3,4-dihydro-2H-benzo[b][1,4]oxazin-7-yl)-1H-pyr-
azol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide as a yellow
solid. LC/MS (Method D, ESI): [M+H]+=428.0, Rt=2.05 min; .sup.1H
NMR (400 MHz, CD3OD): .delta. (ppm) 8.98 (d, J=6.8 Hz, 1H),
8.57-8.51 (m, 2H), 8.12 (s, 1H), 7.10 (dd, J=7.0, 4.2 Hz, 1H), 6.79
(s, 1H), 6.51 (s, 1H), 6.40 (t, J=75.2 Hz, 1H), 4.13-4.11 (m, 2H),
3.39-3.32 (m, 2H).
##STR00144##
N-(1-((2H-tetrazol-5-yl)methyl)-3-(2,5-bis(difluoromethoxy)phenyl)-1H-pyr-
azol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide
Step 1: Synthesis of
N-(3-(2,5-bis(difluoromethoxy)phenyl)-1-((2-(tetrahydro-2H-pyran-2-yl)-2H-
-tetrazol-5-yl)methyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxa-
mide and
N-(3-(2,5-bis(difluoromethoxy)phenyl)-1-((1-(tetrahydro-2H-pyran--
2-yl)-1H-tetrazol-5-yl)methyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-
-carboxamide as a red oil. (Mixture of Regioisomers)
##STR00145##
[0482] To a suspension of
N-[3-[2,5-bis(difluoromethoxy)phenyl]-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyri-
midine-3-carboxamide (Intermediate 1, 10.0 g, 22.9 mmol), cesium
carbonate (22.3 g, 68.6 mmol) and TBAI (423 mg, 1.15 mmol) in
N,N-dimethylformamide (100 mL) was added
5-(chloromethyl)-2-tetrahydropyran-2-yl-tetrazole (11.6 g, 57.3
mmol) at rt. The reaction mixture was stirred for 1.5 h, after
which it was diluted with brine (300 mL), and extracted with ethyl
acetate (3.times.300 mL). The organic layers were combined, washed
with brine (300 mL), dried over anhydrous sodium sulfate, and
concentrated under vacuum. The residue was purified by flash
chromatography on silica gel eluting with DCM (1% TEA)/EA (1/2) to
afford a mixture of
N-(3-(2,5-bis(difluoromethoxy)phenyl)-1-((2-(tetrahydro-2H-pyran-2-yl)-2H-
-tetrazol-5-yl)methyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxa-
mide and
N-(3-(2,5-bis(difluoromethoxy)phenyl)-1-((1-(tetrahydro-2H-pyran--
2-yl)-1H-tetrazol-5-yl)methyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-
-carboxamide as a red oil. LC/MS (Method I, ESI): [M+H]+=603.25,
Rt=1.02 min
Step 2: Synthesis of
N-(1-((2H-tetrazol-5-yl)methyl)-3-(2,5-bis(difluoromethoxy)phenyl)-1H-pyr-
azol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00146##
[0484] A solution of
N-[3-[2,5-bis(difluoromethoxy)phenyl]-1-[(2-tetrahydropyran-2-yltetrazol--
5-yl)methyl]pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide
(mixture of regioisomers, 4.21 g, 6.98 mmol) in HCl/MeOH (60.0 mL,
240 mmol) was stirred for overnight at rt. The residue was purified
by flash chromatography on a C18 column eluting with 38.7%
ACN/water to afford
N-(1-((2H-tetrazol-5-yl)methyl)-3-(2,5-bis(difluoromethoxy)phenyl)-1H-pyr-
azol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (2.50 g, 4.83
mmol, 69.2% yield) as a white solid. LC/MS (Method I, ESI):
[M+H]+=519.3, Rt=0.71 min.
##STR00147##
N-(1-((2H-tetrazol-5-yl)methyl)-3-(2-(difluoromethoxy)-5-(methylthio)phen-
yl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide
Step 1: Synthesis of
N-(3-(2-(difluoromethoxy)-5-(methylthio)phenyl)-1-((2-(tetrahydro-2H-pyra-
n-2-yl)-2H-tetrazol-5-yl)methyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-
-3-carboxamide (together with tetrazole alkylation isomer)
##STR00148##
[0486] A solution of
N-[3-[2-(difluoromethoxy)-5-methylsulfanyl-phenyl]-1H-pyrazol-4-yl]pyrazo-
lo[1,5-a]pyrimidine-3-carboxamide (Intermediate 7, 2.02 g, 4.85
mmol) in N,N-dimethylformamide (20 mL) was stirred at rt. Then
5-(chloromethyl)-2-tetrahydropyran-2-yl-tetrazole (2.81 g, 13.9
mmol), cesium carbonate (4.71 g, 14.5 mmol), TBAI (88.7 mg, 0.24
mmol) was added and stirred at rt for 1.5 h. After filtration, the
filtrate was diluted with water (50 mL). The resulting solution was
extracted with EA (50*3 mL) and the organic layers were combined.
The organic layer was dried over anhydrous sodium sulfate and
concentrated under vacuum. The residue was purified by flash
chromatography on silica gel eluting with EA/DCM (1% TEA) (46%) to
afford a mixture of
N-[3-[2-(difluoromethoxy)-5-methylsulfanyl-phenyl]-1-[(2-tetrahydropyran--
2-yltetrazol-5-yl)methyl]pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxam-
ide along with the tetrazole alkylation regioisomer (3.51 g, 6.02
mmol) as a white solid. LC/MS (Method M, ESI): [M+H]+=583.1,
Rt=0.66 min.
Step 2: Synthesis of
N-(1-((2H-tetrazol-5-yl)methyl)-3-(2-(difluoromethoxy)-5-(methylthio)phen-
yl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00149##
[0488] A solution of
N-[3-[2-(difluoromethoxy)-5-methylsulfanyl-phenyl]-1-[(2-tetrahydropyran--
2-yltetrazol-5-yl)methyl]pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxam-
ide (mixture of regioisomers, 8.34 g, 14.3 mmol) in methanol (30
mL) was stirred at rt. Then 1,4-dioxane (80 mL) (4 M HCl) was added
and the mixture was stirred at rt for 2 h. The solvent was
concentrated under vacuum, and the crude product was used without
further purification. LC/MS (Method M, ESI): [M+H]+=499.1, RT=0.61
min
##STR00150##
N-(1-((2H-tetrazol-5-yl)methyl)-3-(5-chloro-2-(difluoromethoxy)phenyl)-1H-
-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide
Step 1: Synthesis of
N-[3-[5-chloro-2-(difluoromethoxy)phenyl]-1-[(2-tetrahydropyran-2-yltetra-
zol-5-yl)methyl]pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide
(Together with Tetrazole Alkylation Isomer)
##STR00151##
[0490] A solution of
N-[3-[5-chloro-2-(difluoromethoxy)phenyl]-1H-pyrazol-4-yl]pyrazolo[1,5-a]-
pyrimidine-3-carboxamide (3.2 g, 7.92 mmol) in
N,N-dimethylformamide (30 mL) was added cesium carbonate (5.2 g,
15.99 mmol) and TBAI (178 mg, 0.480 mmol) and
5-(chloromethyl)-2-tetrahydropyran-2-yl-tetrazole (4.05 g, 20.0
mmol) at rt. The resulting solution was stirred for 2 h at rt. The
reaction mixture was diluted with water (150 mL). The resulting
solution was extracted with EA (300*3 mL) and the organic layers
were combined. The organic layer was concentrated under vacuum. The
residue was purified by flash chromatography on silica gel eluting
with EA/DCM (24%) to afford a mixture of
N-[3-[5-chloro-2-(difluoromethoxy)phenyl]-1-[(2-tetrahydropyran-2-yltetra-
zol-5-yl)methyl]pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide
(3.20 g, 5.33 mmol, 67.3% yield) along with the tetrazole
alkylation regioisomer as a yellow oil. LC/MS (Method M, ESI):
[M+H]+=471.1, RT=0.66 min
Step 2: Synthesis of
N-(1-((2H-tetrazol-5-yl)methyl)-3-(5-chloro-2-(difluoromethoxy)phenyl)-1H-
-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00152##
[0492] A solution of
N-[3-[5-chloro-2-(difluoromethoxy)phenyl]-1-[(2-tetrahydropyran-2-yltetra-
zol-5-yl)methyl]pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide
(mixture of regioisomers, 3.21 g, 4.5 mmol) in 4 M HCl/MeOH (40 mL,
4.5 mmol) was stirred for 2 h at rt. The organic layer was
concentrated under vacuum. The residue was purified by flash
chromatography on C.sub.18 gel eluting with ACN/H.sub.2O (TFA)
(35%) to afford
N-[3-[5-chloro-2-(difluoromethoxy)phenyl]-1-(2H-tetrazol-5-ylmethyl)pyraz-
ol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide (1.81 g, 3.35 mmol,
74.4% yield) as a yellow solid. LC/MS (Method H, ESI):
[M+H]+=487.1, RT=1.12 min.
##STR00153##
N-(1-((2H-tetrazol-5-yl)methyl)-3-(5-cyclopropyl-2-(difluoromethoxy)pheny-
l)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide
Step 1: Synthesis of
N-(3-(5-cyclopropyl-2-(difluoromethoxy)phenyl)-1-((2-(tetrahydro-2H-pyran-
-2-yl)-2H-tetrazol-5-yl)methyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine--
3-carboxamide
##STR00154##
[0493] Step 1: Synthesis of
N-[3-[5-cyclopropyl-2-(difluoromethoxy)phenyl]-1-[(2-tetrahydropyran-2-yl-
tetrazol-5-yl)methyl]pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide
[0494] A solution of
N-[3-[5-cyclopropyl-2-(difluoromethoxy)phenyl]-1H-pyrazol-4-yl]pyrazolo[1-
,5-a]pyrimidine-3-carboxamide (Intermediate 5, 3.02 g, 7.35 mmol),
TBAI (271 mg, 0.740 mmol) and cesium carbonate (7.18 g, 22.1 mmol)
in N,N-dimethylformamide (40 mL) was
5-(chloromethyl)-2-tetrahydropyran-2-yl-tetrazole (3.75 g, 18.5
mmol) added at rt. The resulting solution was stirred for 1.5 h at
rt. The residue was filtered through Celite.RTM., and the filtrate
was diluted with water (80 mL). The resulting mixture was extracted
with EA (80*3 mL). The organic layers were washed with brine (100*3
mL), dried over anhydrous sodium sulfate, and concentrated under
vacuum. The residue was purified by flash chromatography on silica
gel eluting with EA/PE (0.1% TEA)=4/1 to afford a mixture of
N-[3-[5-cyclopropyl-2-(difluoromethoxy)phenyl]-1-[(2-tetrahydropyran-2-yl-
tetrazol-5-yl)methyl]pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide
and the tetrazole alkylation regioisomer (3.26 g, 5.65 mmol, 76.8%
yield) as a yellow oil. LC/MS (Method G, ESI): [M+H]+=577.2,
RT=0.98 min.
Step 2: Synthesis of
N-[3-[5-cyclopropyl-2-(difluoromethoxy)phenyl]-1-[(2-tetrahydropyran-2-yl-
tetrazol-5-yl)methyl]pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00155##
[0496] A solution of
N-[3-[5-cyclopropyl-2-(difluoromethoxy)phenyl]-1-[(2-tetrahydropyran-2-yl-
tetrazol-5-yl)methyl]pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide
(mixture of regioisomers, 3.26 g, 5.65 mmol) in 4 M HCl/methanol
(20 mL) was stirred for 2 h at room temperature. The resulting
solution was concentrated under vacuum. The resulting residue was
purified by reverse phase chromatography (acetonitrile 0-45/0.1%
TFA in water) to afford
N-[3-[5-cyclopropyl-2-(difluoromethoxy)phenyl]-1-(2H-tetrazol-5-ylmethyl)-
pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide (1.85 g, 3.8
mmol, 66.5% yield) as light yellow solid. LC/MS (Method H, ESI):
[M+H]+=493.2, RT=1.16 min.
EXAMPLES
Example 1
##STR00156##
[0497]
N-(3-(5-chloro-2-(difluoromethoxy)phenyl)-1-((2-(2-hydroxyethyl)-2H-
-tetrazol-5-yl)methyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxa-
mide
[0498] Into a 100-mL round-bottom flask, was placed
N-[3-[5-chloro-2-(difluoromethoxy)phenyl]-1-(2H-1,2,3,4-tetrazol-5-ylmeth-
yl)-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide
(Intermediate 12, 200 mg, 0.412 mmol), 1,3-dioxolan-2-one (122 mg,
1.38 mmol, 3.35 equiv), sodium hydroxide (44 mg, 1.10 mmol, 2.67
equiv), N,N-dimethylformamide (30 mL). The resulting solution was
stirred for 4 h at 120.degree. C. in an oil bath. The resulting
mixture was concentrated under vacuum. The residue was applied onto
a silica gel column with dichloromethane/petroleum ether (11.5:1).
The crude product was purified by Chiral-Prep-HPLC with the
following conditions (Prep-HPLC-009): Column, Phenomenex Lux 5 u
Cellulose-4 XIA Packed, 2.12*25 cm, 5 um; mobile phase, Hex and
ethanol (hold 60.0% ethanol-in 32 min); Detector, UV 220/254 nm.
This resulted in 19.6 mg (9%) of
N-[3-[5-chloro-2-(difluoromethoxy)phenyl]-1-[[2-(2-hydroxyethyl)-2H-1,2,3-
,4-tetrazol-5-yl]methyl]-1H-pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carbo-
xamide as a white solid. LC/MS (Method N, ESI): [M+H]+=531.2,
RT=1.33 min. .sup.1H NMR (300 MHz, DMSO-d.sub.6): .delta. (ppm)
9.76 (s, 1H), 9.35 (dd, J=6.9, 1.5 Hz, 1H), 8.68-8.66 (m, 2H), 8.52
(s, 1H), 7.64 (dd, J=8.9, 2.9 Hz, 1H), 7.57-6.99 (m, 4H), 5.78 (s,
2H), 5.07 (t, J=5.6 Hz, 1H), 4.72 (t, J=5.1 Hz, 2H), 3.94-3.88 (m,
2H).
Example 21
N-(3-(2,5-bis(difluoromethoxy)phenyl)-1-((2-(1-(2-(dimethylamino)ethyl)aze-
tidin-3-yl)-2H-tetrazol-5-yl)methyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimi-
dine-3-carboxamide
##STR00157##
[0499] Step 1: Synthesis of tert-butyl
3-(5-((3-(2,5-bis(difluoromethoxy)phenyl)-4-(pyrazolo[1,5-a]pyrimidine-3--
carboxamido)-1H-pyrazol-1-yl)methyl)-2H-tetrazol-2-yl)azetidine-1-carboxyl-
ate
##STR00158##
[0501] 1-Boc-3-iodoazetidine (2.42 g, 8.55 mmol) was added into a
mixture of
N-[3-[2,5-bis(difluoromethoxy)phenyl]-1-(2H-tetrazol-5-ylmethyl)pyrazo-
l-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide (Intermediate 1,
1.87 g, 3.61 mmol) and potassium carbonate (2.43 g, 17.6 mmol) in
N,N-dimethylformamide (20 mL). The resulting solution was stirred
for 3 h at 60.degree. C. and overnight at 75.degree. C. The mixture
was brought to rt and filtered over Celite.RTM.. The filtrate was
diluted with brine (120 mL). The resulting mixture was extracted
with EA (3.times.50 mL) and the organic layers were combined. The
organic layer was washed with brine (2*50 mL). The residue was
purified by flash chromatography on silica gel eluting with DCM (1%
of TEA)/EA (1% of TEA)(1/2) to afford tert-butyl
3-[5-[[3-[2,5-bis(difluoromethoxy)phenyl]-4-(pyrazolo[1,5-a]pyrimidine-3--
carbonylamino)pyrazol-1-yl]methyl]tetrazol-2-yl]azetidine-1-carboxylate
(1.4 g, 92%) as yellow oil and 330 mg of tert-butyl
3-[5-[[3-[2,5-bis(difluoromethoxy)phenyl]-4-(pyrazolo[1,5-a]pyrimidine-3--
carbonylamino)pyrazol-1-yl]methyl]tetrazol-2-yl]azetidine-1-carboxylate
(mixture of diastereomers) as yellow oil.
[0502] The 1.4 g of tert-butyl
3-[5-[[3-[2,5-bis(difluoromethoxy)phenyl]-4-(pyrazolo[1,5-a]pyrimidine-3--
carbonylamino)pyrazol-1-yl]methyl]tetrazol-2-yl]azetidine-1-carboxylate
was purified by reverse phase chromatography (acetonitrile
0-60/0.1% NH.sub.4HCO.sub.3 in water) to afford tert-butyl
3-[5-[[3-[2,5-bis(difluoromethoxy)phenyl]-4-(pyrazolo[1,5-a]pyrimidine-3--
carbonylamino)pyrazol-1-yl]methyl]tetrazol-2-yl]azetidine-1-carboxylate
(1.00 g, 1.5 mmol, 41.2% yield) as a white solid. LC/MS (Method C,
ESI): [M+H]+=674.2 RT=2.63 min.
[0503] The 330 mg of mixture of diastereomers was purified by flash
chromatography on silica gel eluting with DCM (1% of TEA)/EA (1% of
TEA)(1/2) to afford tert-butyl
3-[5-[[3-[2,5-bis(difluoromethoxy)phenyl]-4-(pyrazolo[1,5-a]pyrimidine-3--
carbonylamino)pyrazol-1-yl]methyl]tetrazol-1-yl]azetidine-1-carboxylate
(140 mg, 0.208 mmol, 5.8% yield) as yellow solid. LC/MS (Method G,
ESI): [M+H]+=674.2 RT=0.97 min.
Step 2: Synthesis of
N-(1-((2-(azetidin-3-yl)-2H-tetrazol-5-yl)methyl)-3-(2,5-bis(difluorometh-
oxy)phenyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide
##STR00159##
[0505] A solution of tert-butyl
3-(5-((3-(2,5-bis(difluoromethoxy)phenyl)-4-(pyrazolo[1,5-a]pyrimidine-3--
carboxamido)-1H-pyrazol-1-yl)methyl)-2H-tetrazol-2-yl)azetidine-1-carboxyl-
ate (1.05 g, 1.56 mmol) in 2,2,2-trifluoroacetic acid (3
mL)/dichloromethane (12 mL) was stirred at rt for 2 h. The reaction
mixture was concentrated under vacuum, and the resulting product
was used without further purification. LC/MS (Method R, ESI):
[M+H]+=574.2 RT=1.58 min.
Step 3: Synthesis of tert-butyl
(2-(3-(5-((3-(2,5-bis(difluoromethoxy)phenyl)-4-(pyrazolo[1,5-a]pyrimidin-
e-3-carboxamido)-1H-pyrazol-1-yl)methyl)-2H-tetrazol-2-yl)azetidin-1-yl)et-
hyl)carbamate
##STR00160##
[0507] To a solution of
N-(1-((2-(azetidin-3-yl)-2H-tetrazol-5-yl)methyl)-3-(2,5-bis(difluorometh-
oxy)phenyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide
(821 mg, 1.43 mmol) in 1,4-dioxane (30 mL) was added potassium
carbonate (593 mg, 4.29 mmol) at rt. The resulting solution was
stirred for 10 min, after which tert-butyl 2-bromoethylcarbamate
(1.29 g, 5.73 mmol) was added. The resulting reaction mixture was
stirred at 70.degree. C. overnight. The mixture was concentrated
under vacuum, and the residue was purified by flash chromatography
on a C.sub.18 column, eluting with 42% ACN/NH.sub.4HCO.sub.3
(0.05%) to afford tert-butyl
(2-(3-(5-((3-(2,5-bis(difluoromethoxy)phenyl)-4-(pyrazolo[1,5-a]pyrimidin-
e-3-carboxamido)-1H-pyrazol-1-yl)methyl)-2H-tetrazol-2-yl)azetidin-1-yl)et-
hyl)carbamate (715 mg, 0.998 mmol, 69.8% yield) as a yellow oil.
LC/MS (Method M, ESI): [M+H]+=717.4, RT=0.67 min.
Step 4: Synthesis of
N-(1-((2-(1-(2-aminoethyl)azetidin-3-yl)-2H-tetrazol-5-yl)methyl)-3-(2,5--
bis(difluoromethoxy)phenyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-ca-
rboxamide
##STR00161##
[0509] A solution
tert-butyl(2-(3-(5-((3-(2,5-bis(difluoromethoxy)phenyl)-4-(pyrazolo[1,5-a-
]pyrimidine-3-carboxamido)-1H-pyrazol-1-yl)methyl)-2H-tetrazol-2-yl)azetid-
in-1-yl)ethyl)carbamate (720 mg, 1 mmol) in dichloromethane (4 mL)
and trifluoroacetic acid (1 mL) was stirred for 2 h at rt. The
reaction mixture was concentrated under vacuum. The residue was
purified by flash chromatography on a C18 column, eluting with 45%
ACN/water (0.05% HCl) to
N-(1-((2-(1-(2-aminoethyl)azetidin-3-yl)-2H-tetrazol-5-yl)methyl)-3-(2,5--
bis(difluoromethoxy)phenyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-ca-
rboxamide (615 mg, 0.10 mmol, 99.3% yield) as a yellow oil. LC/MS
(Method M, ESI): [M+H]+=617.4, RT=0.54 min.
Step 5: Synthesis of
N-(3-(2,5-bis(difluoromethoxy)phenyl)-1-((2-(1-(2-(dimethylamino)ethyl)az-
etidin-3-yl)-2H-tetrazol-5-yl)methyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrim-
idine-3-carboxamide
##STR00162##
[0511] To a solution of
N-[1-[[2-[1-(2-aminoethyl)azetidin-3-yl]tetrazol-5-yl]methyl]-3-[2,5-bis(-
difluoromethoxy)phenyl]pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamid-
e (615 mg, 1 mmol) in methanol (10 mL) at rt was added
HCHO/H.sub.2O (256 mg, 3.15 mmol). The resulting solution was
stirred for 2 h at 25.degree. C. NaBH(AcO).sub.3 (846 mg, 3.99
mmol) was added, and the reaction mixture was stirred at 25.degree.
C. for 3 h, after which it was concentrated under vacuum. The
residue was purified by flash chromatography on silica gel eluting
with ACN/water (0.05% NH.sub.4HCO.sub.3). A slurry of 560 mg of
product in ethanol (4 mL) was stirred slowly at room temperature
for 2 days to afford
N-[3-[2,5-bis(difluoromethoxy)phenyl]-1-[[2-[1-[2-(dimethylamino)ethyl]az-
etidin-3-yl]tetrazol-5-yl]methyl]pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3--
carboxamide (433 mg, 0.663 mmol, 66.4% yield) as a white solid.
LC/MS (Method X, ESI): [M+H]+=645.3, RT=3.38 min. .sup.1H NMR (400
MHz, DMSO-d.sub.6): .delta. (ppm) 9.76 (s, 1H), 9.34 (dd, J=7.2,
1.6 Hz, 1H), 8.67-8.65 (m, 2H), 8.52 (s, 1H), 7.49-7.46 (m, 1H),
7.40-6.99 (m, 5H), 5.80 (s, 2H), 5.58-5.54 (m, 1H), 3.83-3.79 (m,
2H), 3.57-3.53 (m, 2H), 2.60-2.56 (m, 2H), 2.22-2.20 (m, 2H), 2.12
(s, 6H).
Example 10
##STR00163##
[0512]
N-(3-(2,5-bis(difluoromethoxy)phenyl)-1-((2-(1-methylazetidin-3-yl)-
-2H-tetrazol-5-yl)methyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carb-
oxamide
[0513] A solution of
N-[1-[[2-(azetidin-3-yl)tetrazol-5-yl]methyl]-3-[2,5-bis(difluoromethoxy)-
phenyl]pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide (670.5
mg, 1.17 mmol) and HCHO/H.sub.2O (113 mg, 1.4 mmol) in methanol (10
mL) was stirred at 25.degree. C. for 2 h. Then
NaBH(CH.sub.3COO).sub.3 (301 mg, 1.42 mmol) was added and stirred
at 25.degree. C. overnight. The solvent was concentrated under
vacuum, and the resulting residue was diluted with water (20 mL).
The resulting solution was extracted with EA (50*3 mL) and the
organic layers were combined. The organic layer was dried over
anhydrous sodium sulfate and concentrated under vacuum. The product
was purified by Prep-HPLC with the following conditions Column:
XBridge Prep OBD C.sub.18 Column 30× 150 mm 5 um; Mobile Phase
A:Water (10 mmol/L NH.sub.4HCO.sub.3), Mobile Phase B: ACN; Flow
rate: 60 mL/min; Gradient: 27% B to 37% B in 10 min; 254/220 nm;
Rt: 13 min to afford
N-[3-[2,5-bis(difluoromethoxy)phenyl]-1-[[2-(1-methylazetidin-3-yl)tetraz-
ol-5-yl]methyl]pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide
(507 mg, 0.858 mmol, 73.4% yield) as a white solid. LC/MS (Method
E, ESI): [M+H]+=588.2, RT=2.28 min. .sup.1H NMR (400 MHz,
DMSO-d.sub.6): .delta. (ppm) 9.77 (s, 1H), 9.35 (dd, J=7.2, 1.2 Hz,
1H), 8.68-8.64 (m, 2H), 8.52 (s, 1H), 7.50-7.00 (m, 6H), 5.80 (s,
2H), 5.58-5.52 (m, 1H), 3.84-3.80 (m, 2H), 3.59-3.52 (m, 2H), 2.33
(s, 3H).
Example 12
##STR00164##
[0514]
N-(1-((1-(azetidin-3-yl)-1H-tetrazol-5-yl)methyl)-3-(2,5-bis(difluo-
romethoxy)phenyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide
[0515] A solution of tert-butyl
3-[5-[[3-[2,5-bis(difluoromethoxy)phenyl]-4-(pyrazolo[1,5-a]pyrimidine-3--
carbonylamino)pyrazol-1-yl]methyl]tetrazol-1-yl]azetidine-1-carboxylate
(200 mg, 0.30 mmol) in dichloromethane (5 mL) was stirred at rt.
Then TFA (1 mL, 0.30 mmol) was added and stirred at rt for 2 h. The
solvent was concentrated under vacuum. The residue was purified by
flash chromatography on silica gel eluting with H.sub.2O (0.1%
TFA)/ACN (69%) to afford
N-[1-[[1-(azetidin-3-yl)tetrazol-5-yl]methyl]-3-[2,5-bis(difluo-
romethoxy)phenyl]pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide
(184 mg) as a yellow oil. LC/MS (Method H, ESI): [M+H]+=574.2,
RT=0.97 min. .sup.1H NMR (300 MHz, DMSO-d.sub.6): .delta. (ppm)
9.78 (s, 1H), 9.36 (dd, J=6.9, 1.5 Hz, 1H), 8.70-8.64 (m, 2H), 8.59
(s, 1H), 7.61-6.97 (m, 6H), 6.10 (s, 2H), 5.94-5.88 (m, 1H),
4.47-4.16 (m, 4H).
Example 78
##STR00165##
[0516]
N-(3-(2,5-bis(difluoromethoxy)phenyl)-1-((2-(1'-methyl-[1,3'-biazet-
idin]-3-yl)-2H-tetrazol-5-yl)methyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimi-
dine-3-carboxamide
Step 1: Synthesis of tert-butyl
3-(5-((3-(2,5-bis(difluoromethoxy)phenyl)-4-(pyrazolo[1,5-a]pyrimidine-3--
carboxamido)-1H-pyrazol-1-yl)methyl)-2H-tetrazol-2-yl)-[1,3'-biazetidine]--
1'-carboxylate
##STR00166##
[0518] A solution of
N-[1-[[2-(azetidin-3-yl)tetrazol-5-yl]methyl]-3-[2,5-bis(difluoromethoxy)-
phenyl]pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide (1.0 g,
1.74 mmol) and 1-boc-3-azetidinone (896 mg, 5.23 mmol) in the
mixture of methanol (20 mL) and acetic acid (1 mL) was stirred at
25.degree. C. for 2 h. Then NaBH(OAc).sub.3 (1.1 g, 5.19 mmol) was
added and the mixture was stirred at room temperature for 3 h. Upon
completion of reaction, the solvent was removed under vacuum. The
residue was purified by reverse phase separation column [Mobile
Phase A: Water (0.1% NH.sub.4HCO.sub.3), Mobile Phase B:
acetonitrile; Gradient: 10% B to 70% B in 35 min] to give
tert-butyl
3-(5-((3-(2,5-bis(difluoromethoxy)phenyl)-4-(pyrazolo[1,5-a]pyrimidine-3--
carboxamido)-1H-pyrazol-1-yl)methyl)-2H-tetrazol-2-yl)-[1,3'-biazetidine]--
1'-carboxylate (650 mg, 0.890 mmol, 50.1% yield) as a light yellow
solid. LC/MS (Method P, ESI): [M+H]+=729.15, RT=0.85 min.
Step 2: Synthesis of
N-(1-((2-([1,3'-biazetidin]-3-yl)-2H-tetrazol-5-yl)methyl)-3-(2,5-bis(dif-
luoromethoxy)phenyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxami-
de
##STR00167##
[0520] A solution of tert-butyl
3-[3-[5-[[3-[2,5-bis(difluoromethoxy)phenyl]-4-(pyrazolo[1,5-a]pyrimidine-
-3-carbonylamino)pyrazol-1-yl]methyl]tetrazol-2-yl]azetidin-1-yl]azetidine-
-1-carboxylate (650 mg, 0.890 mmol) in dichloromethane (6 mL) and
2,2,2-trifluoroacetic acid (2 mL) was stirred at room temperature
for 3 h. Upon completion of reaction, the solvent was removed under
vacuum to give 600 mg of the crude product as a yellow solid. LC/MS
(Method G, ESI): [M+H]+=629.2, RT=0.92 min.
Step 3: Synthesis of
N-(3-(2,5-bis(difluoromethoxy)phenyl)-1-((2-(1'-methyl-[1,3'-biazetidin]--
3-yl)-2H-tetrazol-5-yl)methyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-
-carboxamide
##STR00168##
[0522] A solution of
N-(1-((2-([1,3'-biazetidin]-3-yl)-2H-tetrazol-5-yl)methyl)-3-(2,5-bis(dif-
luoromethoxy)phenyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxami-
de (600 mg, 0.950 mmol) and formaldehyde/water (2 mL) in the
mixture of acetic acid (1 mL) and methanol (10 mL) was stirred at
room temperature for 1 h. Then NaBH(OAc).sub.3 (1.0 g, 4.72 mmol)
was added and the mixture was stirred for 3 h. Upon completion of
reaction, the solvent was removed under vacuum. The residue was
purified by reverse phase separation column [Mobile Phase A: Water
(0.1% NH.sub.4HCO.sub.3), Mobile Phase B: acetonitrile; Gradient:
10% B to 70% B in 30 min] to give
N-(3-(2,5-bis(difluoromethoxy)phenyl)-1-((2-(1'-methyl-[1,3'-biazetidin]--
3-yl)-2H-tetrazol-5-yl)methyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-
-carboxamide (400 mg, 0.603 mmol, 63.3% yield) as a light yellow
solid. LC/MS (Method 0, ESI): [M+H]+=643.3, RT=1.42 min. .sup.1H
NMR (300 MHz, DMSO-d.sub.6): .delta. (ppm) 9.78 (s, 1H), 9.35 (dd,
J=7.2, 1.7 Hz, 1H), 8.67-8.64 (m, 2H), 8.53 (s, 1H), 7.53-6.96 (m,
6H), 5.82 (s, 2H), 5.64-5.59 (m, 1H), 3.87-3.82 (m, 2H), 3.71-3.35
(m, 10H).
Example 51
##STR00169##
[0523]
N-[3-[2,5-bis(difluoromethoxy)phenyl]-1-[[2-[1-[3-(dimethylamino)pr-
opyl]azetidin-3-yl]tetrazol-5-yl]methyl]pyrazol-4-yl]pyrazolo[1,5-a]pyrimi-
dine-3-carboxamide
Step 1: Synthesis of
tert-butyl(3-(3-(5-((3-(2,5-bis(difluoromethoxy)phenyl)-4-(pyrazolo[1,5-a-
]pyrimidine-3-carboxamido)-1H-pyrazol-1-yl)methyl)-2H-tetrazol-2-yl)azetid-
in-1-yl)propyl)carbamate
##STR00170##
[0525] A solution of
N-[1-[[2-(azetidin-3-yl)tetrazol-5-yl]methyl]-3-[2,5-bis(difluoromethoxy)-
phenyl]pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide (500
mg, 0.870 mmol), acetic acid (104 mg, 1.74 mmol) and tert-butyl
N-(3-oxopropyl)carbamate (302 mg, 1.74 mmol) in methanol (8 mL) at
rt. The resulting solution was stirred for 2 h at rt. Then
NaBH(AcO).sub.3 (555 mg, 2.62 mmol) was added and stirred at rt for
3 h. The reaction mixture was concentrated under vacuum. The
residue was purified by flash chromatography on silica gel eluting
with ACN/water (0.05% NH.sub.4HCO.sub.3)(35%) to afford tert-butyl
N-[3-[3-[5-[[3-[2,5-bis(difluoromethoxy)phenyl]-4-(pyrazolo[1,5-a]pyrimid-
ine-3-carbonylamino)pyrazol-1-yl]methyl]tetrazol-2-yl]azetidin-1-yl]propyl-
]carbamate (470 mg, 0.643 mmol, 73.8% yield) as a yellow oil. LC/MS
(Method Q, ESI): [M+H]+=731.3, RT=1.35 min.
Step 2: Synthesis of
N-(1-((2-(1-(3-aminopropyl)azetidin-3-yl)-2H-tetrazol-5-yl)methyl)-3-(2,5-
-bis(difluoromethoxy)phenyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-c-
arboxamide
##STR00171##
[0527] A solution of tert-butyl
N-[3-[3-[5-[[3-[2,5-bis(difluoromethoxy)phenyl]-4-(pyrazolo[1,5-a]pyrimid-
ine-3-carbonylamino)pyrazol-1-yl]methyl]tetrazol-2-yl]azetidin-1-yl]propyl-
]carbamate (152 mg, 0.210 mmol) in dichloromethane (4 mL) and
2,2,2-trifluoroacetic acid (1 mL) at rt. The resulting solution was
stirred for 2 h at rt. The reaction mixture was concentrated under
vacuum. The residue was purified by flash chromatography on C18
column to gel eluting with ACN/water (0.05% HCl) (45%) to afford
N-[1-[[2-[1-(3-aminopropyl)azetidin-3-yl]tetrazol-5-yl]methyl]-3-[2,5-bis-
(difluoromethoxy)phenyl]pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxami-
de (80.2 mg, 0.127 mmol, 61.1% yield) as a yellow oil. LC/MS
(Method M, ESI): [M+H]+=631.4, RT=0.55 min.
Step 3: Synthesis of
N-[3-[2,5-bis(difluoromethoxy)phenyl]-1-[[2-[1-[3-(dimethylamino)propyl]a-
zetidin-3-yl]tetrazol-5-yl]methyl]pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-
-carboxamide
##STR00172##
[0529] A solution of HCHO/H.sub.2O (39.4 mg, 0.490 mmol) and
N-[1-[[2-[1-(3-aminopropyl)azetidin-3-yl]tetrazol-5-yl]methyl]-3-[2,5-bis-
(difluoromethoxy)phenyl]pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxami-
de (85.1 mg, 0.130 mmol) in methanol (5 mL) at rt. The resulting
solution was stirred for 2 h at 25.degree. C. Then NaBH(AcO).sub.3
(114 mg, 0.540 mmol) was added and stirred at 25.degree. C. for 3
h. Then, NaBH.sub.3CN (14 mg, 0.23 mmol) was added and the mixture
was stirred for 1 h. The reaction mixture was concentrated under
vacuum. The residue was purified by HPLC on condition: Column:
XBridge Prep OBD C18 Column, 19*250 mm, 5 um; Mobile Phase A:Water
(0 mmol/L NH.sub.4HCO.sub.3), Mobile Phase B: ACN; Flow rate: 25
mL/min; Gradient: 20 B to 50 B in 7 min; 254 nm; RT1: 5.88; to
afford
N-[3-[2,5-bis(difluoromethoxy)phenyl]-1-[[2-[1-[3-(dimethylamino)propyl]a-
zetidin-3-yl]tetrazol-5-yl]methyl]pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-
-carboxamide (25.4 mg, 0.038 mmol, 27.9% yield) as a yellow solid.
LC/MS (Method A, ESI): [M+H]+=659.3, RT=1.17 min. H NMR (300 MHz,
DMSO-d.sub.6): .delta. (ppm) 9.78 (s, 1H), 9.36 (dd, J=9.4, 2.2 Hz,
1H), 8.68-8.64 (m, 2H), 8.53 (s, 1H), 7.54-6.95 (m, 6H), 5.81 (s,
2H), 5.57 (m, 1H), 3.82-3.77 (m, 2H), 3.52-3.48 (m, 2H), 2.21-2.16
(m, 2H), 2.08 (s, 6H), 1.49-1.32 (m, 2H).
Example 80
##STR00173##
[0530]
N-(3-(2,5-bis(difluoromethoxy)phenyl)-1-((2-(1-(2-(1-(dimethylamino-
)cyclopropyl)ethyl)azetidin-3-yl)-2H-tetrazol-5-yl)methyl)-1H-pyrazol-4-yl-
)pyrazolo[1,5-a]pyrimidine-3-carboxamide
Step 1: Synthesis of tert-butyl
(1-(2-(3-(5-((3-(2,5-bis(difluoromethoxy)phenyl)-4-(pyrazolo[1,5-a]pyrimi-
dine-3-carboxamido)-1H-pyrazol-1-yl)methyl)-2H-tetrazol-2-yl)azetidin-1-yl-
)ethyl)cyclopropyl)carbamate
##STR00174##
[0532] A solution of tert-butyl
N-[1-(2-oxoethyl)cyclopropyl]carbamate (69.4 mg, 0.350 mmol),
N-[1-[[2-(azetidin-3-yl)tetrazol-5-yl]methyl]-3-[2,5-bis(difluoromethoxy)-
phenyl]pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide
(Intermediate 1, 200 mg, 0.350 mmol) and acetic acid (62.6 mg, 1.04
mmol) in methanol (10 mL) was stirred at room temperature for 1 h.
Then NaBH(OAc).sub.3 (148 mg, 0.700 mmol) was added and the mixture
was stirred at room temperature for further 1 h. Then NaBH.sub.3CN
(21.9 mg, 0.350 mmol) was added and the mixture was stirred at room
temperature for further 1 h. Upon completion of reaction, the
solvent was removed in vacuo. The residue was purified by reverse
phase separation column [Mobile Phase A: Water (0.1% TFA), Mobile
Phase B: acetonitrile; Gradient: 30% B to 70% B in 30 min] to
afford tert-butyl
(1-(2-(3-(5-((3-(2,5-bis(difluoromethoxy)phenyl)-4-(pyrazolo[1,5-a]pyrimi-
dine-3-carboxamido)-1H-pyrazol-1-yl)methyl)-2H-tetrazol-2-yl)azetidin-1-yl-
)ethyl)cyclopropyl)carbamate (136 mg, 0.18 mmol, 51.5% yield) as
alight yellow solid. LC/MS (Method H, ESI): [M+H]+=757.3, RT=1.10
min.
Step 2: Synthesis of
N-(1-((2-(1-(2-(1-aminocyclopropyl)ethyl)azetidin-3-yl)-2H-tetrazol-5-yl)-
methyl)-3-(2,5-bis(difluoromethoxy)phenyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]-
pyrimidine-3-carboxamide
##STR00175##
[0534] A solution of tert-butyl
N-[1-[2-[3-[5-[[3-[2,5-bis(difluoromethoxy)phenyl]-4-(pyrazolo[1,5-a]pyri-
midine-3-carbonylamino)pyrazol-1-yl]methyl]tetrazol-2-yl]azetidin-1-yl]eth-
yl]cyclopropyl]carbamate (126 mg, 0.170 mmol)] in dichloromethane
(4 mL) and 2,2,2-trifluoroacetic acid (1 mL) was stirred at rt for
2 h. The solvent was concentrated under vacuum. The product was
used without further purification. LC/MS (Method H, ESI):
[M+H]+=657.3, RT=0.94 min.
Step 3: Synthesis of
N-(1-((2-(1-(2-(1-aminocyclopropyl)ethyl)azetidin-3-yl)-2H-tetrazol-5-yl)-
methyl)-3-(2,5-bis(difluoromethoxy)phenyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]-
pyrimidine-3-carboxamide
##STR00176##
[0536] A solution of
N-(1-((2-(1-(2-(1-aminocyclopropyl)ethyl)azetidin-3-yl)-2H-tetrazol-5-yl)-
methyl)-3-(2,5-bis(difluoromethoxy)phenyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]-
pyrimidine-3-carboxamide (120 mg, 0.180 mmol) and formaldehyde
(43.1 mg, 0.570 mmol) in methanol (4 mL) was stirred at rt for 2 h.
Then NaBH(OAc).sub.3 (154 mg, 0.730 mmol) was added and stirred at
35.degree. C. for 3 h. Finally, NaBH.sub.3CN (9.3 mg, 0.150 mmol)
was added and stirred at 35.degree. C. for 1 h. The extract was
washed with water and brine, dried over anhydrous sodium sulfate,
and then concentrated in vacuo. The reaction was repeated on the
same scale, and the combined crude product was purified by HPLC:
YMC-Actus Triart C18, 30*250.5 um; Mobile Phase A: Water (10 mmol/L
NH.sub.4HCO.sub.3), Mobile Phase B: ACN; Flow rate: 60 mL/min;
Gradient: 38 B to 44 B in 7 min; 220 nm to afford
N-(3-(2,5-bis(difluoromethoxy)phenyl)-1-((2-(1-(2-(1-(dimethylamino)cyclo-
propyl)ethyl)azetidin-3-yl)-2H-tetrazol-5-yl)methyl)-1H-pyrazol-4-yl)pyraz-
olo[1,5-a]pyrimidine-3-carboxamide (15 mg, 0.022 mmol, 6% yield).
LC/MS (Method H, ESI): [M+H]+=685.3, RT=0.94 min. .sup.1H NMR (400
MHz, DMSO-d.sub.6): .delta. (ppm) 9.78 (s, 1H), 9.38-9.38 (m, 1H),
8.67-8.64 (m, 2H), 8.52 (s, 1H), 7.47-7.01 (m, 6H), 5.80 (s, 2H),
5.60-5.55 (m, 1H), 3.78-3.74 (m, 2H), 3.49-3.46 (m, 2H), 2.50-2.44
(m, 2H), 2.21 (s, 6H), 1.50-1.23 (m, 3H).
Example 83
##STR00177##
[0537]
N-(3-(2,5-bis(difluoromethoxy)phenyl)-1-((2-(1-(3-(dimethylamino)-2-
-fluoropropyl)azetidin-3-yl)-2H-tetrazol-5-yl)methyl)-1H-pyrazol-4-yl)pyra-
zolo[1,5-a]pyrimidine-3-carboxamide
Step 1: Synthesis of tert-butyl
(3-(3-(5-((3-(2,5-bis(difluoromethoxy)phenyl)-4-(pyrazolo[1,5-a]pyrimidin-
e-3-carboxamido)-1H-pyrazol-1-yl)methyl)-2H-tetrazol-2-yl)azetidin-1-yl)-2-
-fluoropropyl)carbamate
##STR00178##
[0539] A solution of
N-[1-[[2-(azetidin-3-yl)tetrazol-5-yl]methyl]-3-[2,5-bis(difluoromethoxy)-
phenyl]pyrazol-4-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide
(Intermediate 1, 300 mg, 0.520 mmol) and tert-butyl
N-(2-fluoro-3-oxo-propyl)carbamate (220 mg, 1.15 mmol) in the
mixture of acetic acid (0.50 mL) and methanol (5 mL) was stirred at
room temperature for 1 h. Then NaBH(OAc).sub.3 (333 mg, 1.57 mmol)
was added and the mixture was stirred for further 2 h. Upon
completion of reaction, the organic solvent was removed under
vacuum. The residue was purified by reverse phase separation column
[Mobile Phase A: Water (0.1% NH.sub.4HCO.sub.3), Mobile Phase B:
acetonitrile; Gradient: 20% B to 70% B in 30 min] to give
tert-butyl
(3-(3-(5-((3-(2,5-bis(difluoromethoxy)phenyl)-4-(pyrazolo[1,5-a]pyrimidin-
e-3-carboxamido)-1H-pyrazol-1-yl)methyl)-2H-tetrazol-2-yl)azetidin-1-yl)-2-
-fluoropropyl)carbamate (290 mg, 0.387 mmol, 74% yield) as a light
yellow solid. LC/MS (Method Q, ESI): [M+H]+=749.25, RT=1.98
min.
Step 2: Synthesis of
N-(1-((2-(1-(3-amino-2-fluoropropyl)azetidin-3-yl)-2H-tetrazol-5-yl)methy-
l)-3-(2,5-bis(difluoromethoxy)phenyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrim-
idine-3-carboxamide
##STR00179##
[0541] A solution of tert-butyl
(3-(3-(5-((3-(2,5-bis(difluoromethoxy)phenyl)-4-(pyrazolo[1,5-a]pyrimidin-
e-3-carboxamido)-1H-pyrazol-1-yl)methyl)-2H-tetrazol-2-yl)azetidin-1-yl)-2-
-fluoropropyl)carbamate (290 mg, 0.390 mmol) in a mixture of
2,2,2-trifluoroacetic acid (1 mL) and dichloromethane (3 mL) was
stirred at room temperature for 3 h. Upon completion of the
reaction, the solvent was concentrated under vacuum. The residue
was purified by Prep Chiral HPLC [Column: Chiralpak ID-2, 2*25 cm,
5 um; Mobile Phase A: MTBE (0.3% IPA), Mobile Phase B: MeOH; Flow
rate: 20 mL/min; Gradient: 10 B to 10 B in 26 min; 220/254 nm; RT1:
18.047; RT2: 22.229; Injection Volumn: 0.6 mL; Number Of Runs: 12]
to give two stereoisomers (65 mg (Peak 1) & 52 mg (Peak 2)).
LC/MS (Method I, ESI): [M+H]+=649.3, RT=0.96 min.
Step 3: Synthesis of
N-(3-(2,5-bis(difluoromethoxy)phenyl)-1-((2-(1-(3-(dimethylamino)-2-fluor-
opropyl)azetidin-3-yl)-2H-tetrazol-5-yl)methyl)-1H-pyrazol-4-yl)pyrazolo[1-
,5-a]pyrimidine-3-carboxamide
##STR00180##
[0543] A solution of the second-eluting enantiomer (Peak
2)N-(1-((2-(1-(3-amino-2-fluoropropyl)azetidin-3-yl)-2H-tetrazol-5-yl)met-
hyl)-3-(2,5-bis(difluoromethoxy)phenyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyr-
imidine-3-carboxamide (52 mg, 0.080 mmol) and HCHO (60 mg, 0.80
mmol) in a mixture of methanol (2 mL) and acetic acid (0.20 mL) was
stirred at room temperature for 1 h. NaBH(OAc).sub.3 (51 mg, 0.24
mmol) was added and the mixture was stirred at room temperature for
1 h. NaBH.sub.3CN (5.0 mg, 0.08 mmol) was added and the mixture was
further stirred at room temperature for 1 h. The solvent was
removed under vacuum. The residue was purified by Prep-HPLC
[Column: Xselect CSH OBD Column 30*150 mm 5 um; Mobile Phase A:
Water (10 mmol/L NH.sub.4HCO3), Mobile Phase B: ACN:MeOH=4:1; Flow
rate: 60 mL/min; Gradient: 27 B to 49 B in 11 min; 220 nm; RT1:
10.08 to give
N-(3-(2,5-bis(difluoromethoxy)phenyl)-1-((2-(1-(3-(dimethylamino)-2-fluor-
opropyl)azetidin-3-yl)-2H-tetrazol-5-yl)methyl)-1H-pyrazol-4-yl)pyrazolo[1-
,5-a]pyrimidine-3-carboxamide (10.6 mg, 0.016 mmol, 19.3% yield) as
a light yellow solid. LC/MS (Method A, ESI): [M+H]+=677.2, RT=1.24
min. .sup.1H NMR (400 MHz, CDCl.sub.3): .delta. (ppm) 9.87 (s, 1H),
8.80 (dd, J=7.2, 1.6 Hz, 1H), -9.38 (m, 1H), 8.72 (s, 1H),
8.54-8.52 (m, 2H), 7.47 (d, J=2.8 Hz, 1H), 7.36-7.33 (m, 1H),
7.25-7.22 (m, 1H), 7.03-7.00 (m, 1H), 6.73-6.29 (m, 2H), 5.67 (s,
2H), 5.56-5.25 (m, 1H), 4.80-4.60 (m, 1H), 4.04-4.02 (m, 2H),
3.82-3.78 (m, 2H), 2.92-2.82 (m, 2H), 2.62-2.58 (m, 1H), 2.52-2.44
(m, 1H), 2.30 (s, 6H).
Assays
Test Agents
[0544] Test agent samples were provided as solutions at a
concentration of 10 mM in dimethyl sulfoxide (DMSO) and were stored
in the dark at room temperature before use.
JAK1 and JAK2 Biochemical Assays
[0545] The in vitro biochemical assays quantify JAK-catalyzed
phosphorylation of a synthetic peptide, as detected using a
LabChip.RTM. EZ Reader II microfluidic mobility shift instrument
(PerkinElmer; Waltham, Mass.). The substrate peptide Y-1B has the
sequence 5-FAM-VALVDGYFRLTT-NH.sub.2. Y-1B is fluorescently labeled
on the N-terminus with 5-FAM (5-carboxyfluorescein) and contains a
single tyrosine residue (Y) that can be phosphorylated by JAK
activity. The substrate peptide stock is prepared in DMSO at 5 mM.
Purified recombinant human JAK1 kinase domain protein (Residues
854-1154) was expressed in insect cells and procured from Proteros
Biostructures GmbH (Martinsried, Germany). Recombinant human JAK2
kinase domain protein (Residues 812-1132) was expressed in insect
cells and purified at Genentech, Inc. (South San Francisco,
Calif.). The kinase reaction mixtures contained 100 mM
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer
(pH 7.2), 10 mM magnesium chloride, 0.015% Brij.RTM. 35, 4 mM
dithiothreitol, 1.5 M Y-1B peptide substrate, 25 M adenosine
triphosphate (ATP), 1 nM total JAK1 or 0.2 nM total JAK2, and up to
1000 nM test compound in a final concentration of 2% (volume to
volume [v/v]) DMSO. In each titration experiment, test compound was
tested in duplicate at each of the twelve concentrations. Blank
reactions contained ATP, peptide, and DMSO, but no JAK or test
compound, whereas uninhibited control reactions contained ATP,
peptide, JAK, and DMSO, but no test compound.
[0546] Peptide plus ATP mixture (24 .mu.L) was added to 1 .mu.L of
test compound in DMSO (or DMSO alone). The reactions were initiated
by adding 25 .mu.L of JAK enzyme to the inhibitor/peptide/ATP
mixture before thoroughly mixing the resultant solution. Reactions
were incubated at room temperature (22.degree. C.-23.degree. C.) in
a final volume of 50 .mu.L per well in 384-well plates. After a
30-minute incubation, the reactions were stopped by adding 25 .mu.L
of 150 mM ethylenediaminetetraacetic acid in 100 mM HEPES buffer
(pH 7.2) containing 0.015% Brij 35 to each well.
[0547] In each reaction, the residual Y-1B substrate and the
phospho-peptide product generated were separated using the EZ
Reader II instrument. Electrophoretic separation of molecules of
product from molecules of substrate was achieved using downstream
and upstream voltages of -500 and -2600 V, respectively, at an
operating pressure of -1.3 psi. The 5-FAM group present on both the
substrate and product peptides was excited at 488 nm, the
fluorescence was detected at 530 nm, and the peak heights were
reported.
Data Analysis
[0548] The extent (or percent) of conversion of substrate to
product was calculated from the corresponding peak heights in the
electropherogram using HTS Well Analyzer software, Version 5.2
(PerkinElmer), and the following equation (Equation 1):
% conversion=[P/(S+P)].times.100 Equation 1
where S and P represent the peak heights of the substrate and
product, respectively. After any baseline signal from blank wells
containing no JAK was subtracted from the signal of all test wells,
the % conversion data were converted to fractional activity as
shown in Equation 2, where .nu..sub.i and .nu..sub.o are the %
conversion in the presence and absence of test compound,
respectively. The % conversion observed in the uninhibited control
reactions containing JAK and DMSO vehicle, but no test compound,
was defined to have fractional activity=1 (with no inhibitor
present, .nu..sub.i=.nu..sub.o), whereas blank wells with no JAK
were defined as having fractional activity=0. Fractional activity
was plotted against test compound concentration and the data were
fitted using XLfit software (IDBS; Guildford, United Kingdom) to a
tight-binding apparent inhibition constant (K.sub.i.sup.app)
quadratic equation (see Equation 2) (Williams J W, Morrison J F.
The kinetics of reversible tight-binding inhibition. Methods
Enzymol 1979; 63:437-67), which was used to calculate fractional
activity and K.sub.i.sup.app
Fractional activity = v i v o = 1 - ( [ E ] T + [ I ] T + K i app )
- ( [ E ] T + [ I ] T + K i app ) 2 - 4 [ E ] T [ I ] T 2 [ E ] T
Equation 2 ##EQU00001##
where [E].sub.T and [I].sub.T are the total concentrations of
active enzyme (initial estimates of 0.15 nM for JAK1 and 0.048 nM
for JAK2) and inhibitor (the varied parameter), respectively.
Finally, the K.sub.i was calculated from the K.sub.i.sup.app by
applying the competitive inhibition relationship (Equation 3)
K.sub.i=K.sub.i.sup.app/(1+[ATP]/K.sub.m.sup.app) Equation 3
where [ATP] is the concentration of ATP=25 .mu.M, K.sub.m.sup.app
is the apparent ATP Michaelis constant=32.1 .mu.M for JAK1, and
K.sub.m.sup.app=11.7 .mu.M for JAK2. By applying the tight-binding
Equation 2 to account for any depletion of inhibitor, and the
competitive-inhibition relationship Equation 3, the sensitivity of
the assay can extend at least to a calculated K.sub.i of 0.008 nM
for JAK1 and 0.0015 nM for JAK2.
Kinase Selectivity
[0549] The in vitro kinase selectivity of test agents was assessed
at a concentration of 1 .mu.M in a panel of recombinant human
kinase activity and binding assays, including cytoplasmic and
receptor tyrosine kinases, serine/threonine kinases, and lipid
kinases (SelectScreen.RTM. Kinase Profiling Services, ThermoFisher
Scientific, Madison, Wis.). The kinase activity assays measure
peptide phosphorylation (Z'-LYTE.RTM.) or ADP production
(Adapta.RTM.) while the binding assays monitor displacement of ATP
site binding probes (LanthaScreen.RTM.). The ATP concentrations
used in the activity assays were typically within 2-fold of the
experimentally determined apparent Michaelis constant
(K.sub.m.sup.app) value for each kinase while the competitive
binding tracer concentrations used in the binding assays were
generally within 3-fold of the experimentally determined
dissociation constant (K.sub.d) values. Inhibitors were tested in
duplicate against each kinase and the mean % Inhibition values are
reported. For kinases that were inhibited by close to or greater
than 50% at the initial 1-.mu.M test concentration, 10-point
inhibitor titrations using the same assays were carried out in
order to determine the inhibitor concentrations that caused 50%
inhibition (IC.sub.5). The total JAK1 concentration used in this
assay panel was 75 nM. If 100% of the 75 nM JAK1 protein were
catalytically active, the limit of JAK1 inhibitor sensitivity from
the vendor's JAK1 assay would theoretically be an IC.sub.50 value
of 37.5 nM (one-half of the total enzyme concentration). However,
the SelectScreen.RTM. JAK1 assay generated JAK1 IC.sub.50 values
for several inhibitors that are much lower than 37.5 nM and which
are in agreement with our internal determinations. Thus, the active
JAK1 enzyme concentration in the SelectScreen.RTM. assay must be
much lower than the total nominal JAK1 protein concentration of 75
nM used in the assay, and the observed sensitivity of this assay is
much better than the theoretical sensitivity IC.sub.50 limit of
37.5 nM.
Data Analysis
[0550] For fitting the data in concentration-kinase inhibition
plots, the SelectScreen.RTM. Kinase Profiling Services used XLfit
software (IDBS), Model No. 205 (sigmoidal concentration-response
model), which is a four-parameter logistic fit model described by
Equation 4
[001]y=A+{(B-A)/[1+(C/x).sup.D]} Equation 4
where x is the inhibitor concentration, y is the observed %
inhibition, A is the minimum y-value, B is the maximum y-value, C
is the IC.sub.50 value, and D is the Hill slope. In certain cases,
a three-parameter logistic fit was used. For example, if the
plateau of the curve at infinitely low inhibitor concentration did
not fit between -20% and 20% inhibition, that lower plateau was set
to 0% inhibition, whereas if the plateau of the curve at infinite
inhibitor concentration did not fit between 70% and 130%
inhibition, that upper plateau was set to 100% inhibition.
TF-1 Cell Line Phospho-STAT JAK1 and JAK2 Pathway Selectivity
Assays
[0551] TF-1 human erythroleukemia cells (ATCC.RTM.; Manassas, Va.;
Catalog No. CRL-2003.TM.) were grown in Roswell Park Memorial
Institute (RPMI) medium supplemented with 10% heat-inactivated
fetal bovine serum (FBS), 2 ng/mL granulocyte-macrophage
colony-stimulating factor, 1.times. non-essential amino acids
(NEAA), and 1 mM sodium pyruvate. The day before the assay, the
cultures were transferred to Opti-MEM.TM., 1.times.NEAA, 1 mM
sodium pyruvate, and 0.5% charcoal-stripped FBS (starve medium).
Inhibitor stock solutions (5 mM in DMSO) were serially diluted 1:2
in DMSO to generate a 10-point concentration titration (at
500.times. test concentration), which was further diluted by a
50-fold dilution in Assay Medium (RPMI containing 1.times.NEAA and
1 mM sodium pyruvate) to generate a 10.times. concentration
titration (in 2% DMSO). The cells (300,000 cells/well in 35 .mu.L
of Assay Medium) were seeded in 384-well Greiner plates. Diluted
inhibitor at 10.times. concentration (5 .mu.L) was added to the
cells and the plates were incubated for 30 minutes at 37.degree. C.
in a humidified incubator. Cells were stimulated with the human
recombinant cytokine at the respective EC.sub.90 concentrations, as
previously determined for each individual lot. For the
phosphorylated signal transducer and activator of transcription 6
(P-STAT6) TF-1+Interleukin-13 (IL-13) assay, 10 L of 250 ng/mL
IL-13 (R&D Systems; Minneapolis, Minn.) was added to the cells,
which were then incubated for 10 minutes at 37.degree. C. For the
P-STAT5 TF-1+Erythropoietin (EPO) assay, 10 .mu.L of 110 IU/mL EPO
(Gibco Life Technologies, Catalog No. PHC2054) was added to the
cells, which were then incubated for 30 min at 37.degree. C. For
both assays, the incubation was followed by addition to the cells
of 5 .mu.L of ice-cold 10.times. cell lysis buffer (Cell Signaling
Technologies; Danvers, Mass.; Catalog No. 9803S) containing 1 mM
phenylmethylsulfonyl fluoride (PMSF). Assay plates were frozen at
-80.degree. C. for a minimum of 1 hour. In the IL-13 assay, P-STAT6
was measured by coating goat anti-rabbit (GAR) plates (Meso Scale
Discovery [MSD]; Rockville, Md.; Catalog No. MSD L21RA-1) with
rabbit anti-human total STAT6 antibody (Cell Signaling
Technologies; Catalog No. 9362S), incubating the cell lysates in
the coated plates overnight at 4.degree. C., and then detecting
with mouse anti-P-STAT6 (Tyr641) Clone 16E12 antibody
(MilliporeSigma; Burlington, Mass.; Catalog No. 05-590, custom
labeled by MSD with SULFO-tag) using standard MSD plate processing,
washing, and detection protocols. In the EPO assay, P-STAT5 was
detected using the phospho-STAT5a,b Whole Cell Lysate Kit (MSD;
Catalog No. K150IGD-1). The electrochemiluminescence (ECL) signal
of wells was read on the MESO SECTOR S600 (MSD) reader.
Data Analysis
[0552] Data analysis was performed by subtracting the negative
control (cytokine stimulated and 20 .mu.M control inhibitor-treated
cells) mean ECL value from the ECL value of all wells, determining
percent of control for test compound well ECL values relative to
the positive control (cytokine stimulated and DMSO-treated cells)
mean ECL value, and determining the IC.sub.50 for test compounds
with a four-parameter logistic fit model as shown in Equation
4.
P-STAT6 BEAS-2B+IL-13 Cell Assay
[0553] In order to study the effect of JAK1 inhibitors in a cell
line that is relevant to the cell biology of human asthma, an
IL-13-stimulated STAT6 phosphorylation assay in the human lung
bronchial epithelial BEAS-2B cell line was developed.
BEAS-2B cells (ATCC.RTM. CRL-9609.TM.) were grown in Bronchial
Epithelial Growth Medium (BEGM) (Lonza Catalog No. CC-3170;
Walkersville, Md.; or PromoCell Catalog No. C-21060; Heidelberg,
Germany). Test compound stock solutions (0.5 mM in DMSO) were
serially diluted 1:2 in DMSO to generate a 10-point concentration
curve (at 500.times. test concentration), which was further diluted
by a 50-fold dilution step in BEGM to generate a 10.times.
concentration curve (in 2% DMSO). Cells were plated at 100,000
cells/well in 200 .mu.L of BEGM in 96-well plates and incubated for
48 hours at 37.degree. C. in a humidified incubator. Medium was
aspirated from the cells and replaced with 70 .mu.L of fresh BEGM.
Diluted test compound (10 .mu.L; or 2% DMSO in assay medium) was
added to the cells, and the plates were incubated for 1 hour at
37.degree. C. in a humidified incubator. Twenty .mu.L of 250 ng/mL
human recombinant IL-13 (Bio Techne Catalog No. 213-ILB) was then
added to the cells and incubated for 15 minutes at 37.degree. C.
Medium was aspirated from the cells and 60 .mu.L of ice-cold
1.times. cell lysis buffer (Cell Signaling Technologies; Catalog
No. 9803S) containing 1 mM PMSF was added to the cells. Assay
plates were incubated at -80.degree. C. for at least 1 hour.
P-STAT6 was measured by coating GAR plates (MSD; Catalog No.
L45RA-1) with rabbit anti-human total STAT6 antibody (Cell
Signaling Technologies; Catalog No. 9362S), incubating the cell
lysates in the coated plates overnight at 4.degree. C., and then
detecting with mouse anti-phospho-STAT6 (Tyr641) Clone 16E12
antibody (Millipore; Catalog No. 05-590, custom labeled by MSD with
SULFO-tag) using standard MSD plate processing, washing, and
detection protocols. Plates were read on the MESO SECTOR S600.
Data Analysis
[0554] Data analysis was performed by subtracting negative control
values from all wells and determining percent of control using the
positive control values; the IC.sub.50 was determined with a
four-parameter logistic fit model as shown in Equation 4.
P-STAT6 BEAS-2B+IL-13 Cell Assay with Inhibitor Washout (WO)
[0555] In order to assess the ability of JAK1 inhibitors to retain
their ability to inhibit IL-13-stimulated STAT6 phosphorylation
following cells washing to remove free unbound inhibitor, an
inhibitor washout (WO) assay in the human lung bronchial epithelial
BEAS-2B cell line was developed. Retention of inhibitory activity
after inhibitor washout is consistent with durable binding of
inhibitor to the JAK1 protein and/or retention of inhibitor
molecules within the cell after washout.
As with the standard BEAS-2B cell assay (vide supra), BEAS-2B cells
were grown in Bronchial Epithelial Growth Medium (BEGM). Test
compound stock solutions (0.5 mM in DMSO) were serially diluted 1:2
in DMSO to generate a 10-point concentration curve (at 500.times.
test concentration), which was further diluted by a 50-fold
dilution step in BEGM to generate a 10.times. concentration curve
(in 2% DMSO). Cells were plated at 100,000 cells/well in 200 .mu.L
of BEGM in 96-well plates and incubated for 48 hours at 37.degree.
C. in a humidified incubator. Medium was aspirated from the cells
and replaced with 70 .mu.L of fresh BEGM. Diluted test compound (10
.mu.L; or 2% DMSO in assay medium) was added to the cells, and the
plates were incubated for 1 hour at 37.degree. C. in a humidified
incubator. The medium was aspirated from the cells and replaced
with 80 .mu.L of fresh BEGM to wash away the inhibitor from the
cells, and then the cell plate was incubated for 10 minutes at
37.degree. C. in a humidified incubator. This washout procedure was
repeated two more times. After the third wash step, the cell plate
was returned to the 37.degree. C. humidified incubator and
incubated for 1 hour. Twenty .mu.L of 250 ng/mL IL-13 was then
added to the cells and incubated for 15 minutes at 37.degree. C.
Medium was aspirated from the cells and 60 .mu.L of ice-cold
1.times. cell lysis buffer (Cell Signaling Technologies; Catalog
No. 9803S) containing 1 mM PMSF was added to the cells. Assay
plates were incubated at -80.degree. C. for at least 1 hour.
P-STAT6 was measured by coating GAR plates (MSD; Catalog No.
L45RA-1) with rabbit anti-human total STAT6 antibody (Cell
Signaling Technologies; Catalog No. 9362S), incubating the cell
lysates in the coated plates overnight at 4.degree. C., and then
detecting with mouse anti-phospho-STAT6 (Tyr641) Clone 16E12
antibody (Millipore; Catalog No. 05-590, custom labeled by MSD with
SULFO-tag) using standard MSD plate processing, washing, and
detection protocols. Plates were read on the MESO SECTOR S600.
Data Analysis
[0556] Data analysis was performed by subtracting negative control
values from all wells and determining percent of control using the
positive control values; the IC.sub.50 was determined with a
four-parameter logistic fit model as shown in Equation 4.
Cell Cytotoxicity Assays
[0557] A549 (ATCC.RTM. CCL-185.TM.), Jurkat clone E6-1 (ATCC.RTM.
TIB-152.TM.), and HEK-293T (ATCC.RTM. CRL-1573.TM.) cells
maintained at a sub-confluent density in T175 flasks were used.
Cells in exponential growth phase were plated (450 cells in 45
.mu.L of medium) in Greiner 384-well black/clear tissue culture
treated plates (Greiner Catalog No. 781091). After dispensing
cells, plates were allowed to equilibrate at room temperature for
30 minutes, after which time the cell plates were placed overnight
in a 37.degree. C. CO.sub.2 and humidity-controlled incubator. The
following day, cells were treated with test agent diluted in 100%
DMSO (0.5% final DMSO concentration on cells) with a 10-point
titration and atop concentration of 50 .mu.M. Cells and compounds
were then incubated for 72 hours in a 37.degree. C. CO.sub.2 and
humidity-controlled incubator, after which time cell viability was
measured by adding CellTiter-Glo.RTM. (Promega G7572) reagent to
all wells. Plates were incubated at room temperature for 20 minutes
and then the well luminescence was read on anEnVision plate reader
(Perkin Elmer Life Sciences).
[0558] Data from the above JAK1 and JAK2 assays for the compounds
of Table 1 are shown in Table 2 below
TABLE-US-00025 TABLE 2 Cellular JAK1 Cellular Cellular (JAK2
Biochem Biochem PSTAT6 PSTAT6 PSTAT5 JAK1 JAK2 BEAS2B + TF-1 + TF1
+ Ki (nM) Ki (nM) IL13 (nM) IL13 (nM) EPO (nM) 1 0.15 0.089 6.7 2
0.54 0.29 14 3 0.58 0.43 14 4 0.23 0.12 6.8 5 0.4 0.23 15 6 7 0.42
0.17 9.8 5 13 8 0.5 0.28 9.3 8.4 13 9 0.48 0.24 13 10 0.33 0.24 17
4 5.1 11 0.39 0.27 23 9.5 10 12 0.21 0.18 31 22 51 13 0.25 0.16 11
18 10 14 0.26 0.12 39 45 46 15 0.22 0.092 8.6 3.4 7.2 16 0.45 0.27
18 12 12 17 0.43 0.23 19 2.7 9.7 18 0.51 0.43 35 20 19 19 0.22 0.15
21 7 21 20 0.57 0.46 30 45 15 21 0.4 0.39 15 40 32 22 0.44 0.3 19
4.8 18 23 0.42 0.24 17 7.4 8.2 24 0.83 0.91 470 25 0.77 0.8 300 26
0.51 0.39 16 11 18 27 0.35 0.38 12 13 17 28 0.3 0.36 12 7.1 19 29
0.43 0.25 15 30 0.4 0.31 17 31 0.34 0.25 22 13 8.6 32 0.72 0.62 22
33 0.55 0.56 51 34 0.4 0.29 180 35 0.37 0.18 13 36 0.37 0.22 17 37
0.48 0.36 16 6.9 16 38 0.48 0.44 18 21 42 39 1.3 0.69 36 40 0.78
0.68 86 41 0.32 0.2 14 42 0.33 0.22 16 43 0.86 0.34 36 44 0.71 0.47
25 45 0.81 0.65 81 46 0.49 0.4 18 47 0.46 0.32 16 48 0.63 0.54 15
49 0.53 0.41 19 50 0.5 0.44 16 51 0.37 0.37 12 52 0.38 0.39 19 53
0.48 0.32 35 54 0.75 0.45 51 55 0.6 0.58 44 56 0.84 0.73 80 57 0.71
0.55 73 58 0.41 0.38 57 51 24 59 1 0.71 62 60 0.81 0.97 70 61 0.63
0.52 32 33 36 62 0.66 0.38 38 63 0.64 0.46 22 64 0.87 0.55 17 65
0.41 0.38 660 66 0.41 0.3 32 67 0.53 0.42 56 68 0.46 0.43 41 69
0.75 0.97 1000 70 0.64 0.54 27 30 12 71 1.4 1.1 23 72 0.61 0.48 36
39 51 73 0.64 0.55 43 74 0.55 0.47 93 75 1.1 1.1 200 76 2.4 1.9 74
77 0.38 0.39 110 78 0.44 0.41 40 62 47 79 0.5 0.44 40 80 0.46 0.41
40 81 0.52 0.47 29 82 0.54 0.47 32 83 0.43 0.39 17 84 0.55 0.5
20
[0559] As can be seen from Table 2, compounds of the invention have
good, balanced affinity for both JAK1 and JAK2, and many compounds
are active in the cell based assays.
Animal Models
Mouse House Dust Mite (HDM) Model
[0560] Seven to eight week old female C57BL/6J mice purchased from
Jackson West. Mice are immunized on day 0 &14
withmitraperitoneal administration of House Dust Mite (HDM, D.
Pteronyssinus, purchased from Greer Laboratories, normalized to
0.918 ug DerP1 content per mouse) mixed with 2 mg of alum (Thermo
Scientific) diluted in sterile PBS. On days 21 & 24, mice were
challenged with HDM (again normalized for 0.918 ug DerP1 content)
in PBS, dosed by intra-tracheal inhalation. Prior to each inhaled
HDM challenge (and in asubset of groups, also on days 22 &23),
animals receive test compound via nose-only inhalation (using dry
powder inhalation equipment from Electro-Medical Measurement
Systems (EMMS), including a Wright dust feeder and a
4-layer/24-port or 2-layer/12-port, directed flow, nose-only
inhalation tower) ending 1 hour prior to challenge. Control animals
receive air-only nose only inhalation. 24 hours after the final
treatment, mice are bled retro-orbitally for plasma PK, and then
euthanized by CO2 inhalation. Post-euthanasia, BAL fluid is
collected for total (by FACS, using a known quantity of spike-in
reference beads) and differential (by Wright Giemsa-stained
cytospin) cell counts. Lungs and spleens are collected, weighed,
and frozen for PK. There were 5 or 6 animals per group.
[0561] In addition, to validate lung-delivered dose, PK satellite
groups of 3 naive animals each are dosed with test compound via
nose-only inhalation for a single day or for four consecutive days.
Directly after the final inhalation dosing, PK satellite animals
are bled retro-orbitally for plasma PK, and then euthanized by
CO.sub.2 inhalation. Lungs and spleens are collected and weighed
for PK analysis.
Rat OVA Model
[0562] Six week old male Brown Norway rats from Charles
River-Kingston. Rats are immunized on day 0 with intraperitoneal
administration of 150 ug OVA (Sigma) mixed with 40 mg of alum
(Thermo Scientific) diluted in sterile PBS. 28 days after
sensitization, rats are challenged with 2% OVA in PBS aerosolized
via a nebulizer for 30 minutes for three consecutive days. Prior to
each OVA challenge, animals receive JAK1/JAK2 test compound via
nose-only inhalation (using dry powder inhalation equipment from
Electro-Medical Measurement Systems (EMMS), including a Wright dust
feeder and a 4-layer, 24-port, directed flow, nose-only inhalation
tower) ending 1 hour prior to challenge. Control animals receive
either MCT buffer orally, or air-only nose only inhalation. 24
hours after the final treatment, rats are euthanized by CO.sub.2
inhalation. They are bled from the abdominal aorta for plasma PK
and whole blood FACS analysis. Post-euthanasia, BAL fluid is
collected for total (by FACS, using a known quantity of spike-in
reference beads) and differential (by Wright Giemsa-stained
cytospin) cell counts. Lungs are collected, weighed, and frozen for
PK. Spleens are weighed and cut in half for PK and for FACS
analysis. Blood and spleen samples are analyzed by FACS for total
cell counts and % NK cells (CD161a positive). There are 6 animals
per group, except for the naive control group, which contains 5
animals.
[0563] In addition, to validate lung-delivered dose, PK satellite
groups of 3 naive animals each received JAK1/JAK2 test compound via
nose-only inhalation for a single day or for three days. Directly
after the final inhalation dosing, PK satellite animals are
euthanized by CO.sub.2 inhalation. They are bled from the abdominal
aorta for plasma PK. Lungs and spleens was collected and weighed
for PK analysis.
[0564] Plasma and lung levels of test compounds and ratios thereof
are determined in the following manner. BALB/c mice from Charles
River Laboratories are used in the assay. Test compounds are
individually formulated in 0.2% Tween 80 in saline and the dosing
solution is introduced into the trachea of a mouse by oral
aspiration. At various time points (typically 0.167, 2, 6, 24 hr)
post dosing, blood samples are removed via cardiac puncture and
intact lungs are excised from the mice. Blood samples are
centrifuged (Eppendorf centrifuge, 5804R) for 4 minutes at
approximately 12,000 rpm at 4.degree. C. to collect plasma. Lungs
are padded dry, weighed, and homogenized at a dilution of 1:3 in
sterile water. Plasma and lung levels of test compound are
determined by LC-MS analysis against analytical standards
constructed into a standard curve in the test matrix. A lung to
plasma ratio is determined as the ratio of the lung AUC in micro g
hr/g to the plasma AUC in micro g hr/mL, where AUC is
conventionally defined as the area under the curve of test compound
concentration vs. time.
Pharmacokinetics in Plasma and Lung in Mouse
[0565] Plasma and lung levels of test compounds and ratios thereof
are determined in the following manner. BALB/c mice from Charles
River Laboratories are used in the assay. Test compounds are
individually formulated in 20% propylene glycol in pH 4 citrate
buffer at a concentration of 0.2 mg/mL and 50 uL of the dosing
solution is introduced into the trachea of a mouse by oral
aspiration. At various time points (typically 0.167, 2, 6, 24 hr)
post dosing, blood samples are removed via cardiac puncture and
intact lungs are excised from the mice. Blood samples are
centrifuged (Eppendorf centrifuge, 5804R) for 4 minutes at
approximately 12,000 rpm at 4.degree. C. to collect plasma. Lungs
are padded dry, weighed, and homogenized at a dilution of 1:3 in
sterile water. Plasma and lung levels of test compound are
determined by LC-MS analysis against analytical standards
constructed into a standard curve in the test matrix. A lung to
plasma ratio is determined as the ratio of the lung AUC in micro g
hr/g to the plasma AUC in micro g hr/mL, where AUC is
conventionally defined as the area under the curve of test compound
concentration vs. time.
Pharmacokinetics in Plasma and Lung in Mouse
[0566] The pharmacokinetics of a compound is determined in female
Balb/c mice following administration of a target dose of 0.3 mg/kg
formulated in 0.2% Tween 80 in saline by single intra-nasal (IN)
bolus solution/suspension administration. 7-8 Week old female
Balb/c mice a may be purchased from Charles River. Mice are housed
under specific pathogen-free conditions until used in a study.
[0567] Animals are not fasted before dosing. Blood samples are
taken from 3 animals per time-point at 0.083, 2, 7 and 24 hours
post-dose, under anesthesia (intraperitoneal injection of
pentobarbitone), via cardiac puncture into EDTA-coated
microtainers. Blood samples are centrifuged (1500 g, 10 min at
4.degree. C.) to separate plasma. Plasma samples frozen at
approximately -80.degree. C. After intra-nasal dosing, prior to
lung perfusion, the spleens are removed, weighed and snap frozen.
Following confirmation of death, the lungs of the dosed animals are
perfused with chilled PBS to remove residual blood from the
pulmonary vasculature. The lungs are then excised and weighed (all
weights recorded). All tissue samples are frozen by immersion in
liquid nitrogen. Tissue samples are stored frozen (ca. -80.degree.
C.) until analysis.
[0568] Prior to PK analysis defrosted tissue samples (spleen and
lung) are weighed and homogenised following the addition of 4 mL
HPLC grade water for each gram of tissue, using an Omni-Prep Bead
Ruptor (Omni Inc., Kennesaw, Ga.) at 4.degree. C. Plasma and tissue
homogenate samples are extracted using protein precipitation with
four volumes of acetonitrile containing Tolbutamide (200 ng/mL) or
Labetalol (100 ng/mL) as internal standard. Samples are mixed and
centrifuged at 3200 g and 4.degree. C. for 30 minutes to remove
precipitated proteins, and the supernatant diluted appropriately
(e.g. 1:1, v/v) with HPLC grade water in a 96-well plate.
Representative aliquots of plasma, spleen and lung samples are
assayed for compound concentrations by LC-MS/MS in positive ion
mode using a Waters Xevo TQ-S (Waters, Elstree, UK) against matrix
matched calibration curves and quality control standards. The
standards are prepared by spiking aliquots of control plasma,
spleen and lung tissue homogenate with compound and extracted as
described for the experimental samples. The assay limit of
detection 0.168 mg/mL-4000 ng/mL in all matrices. Concentrations
below the lower limit of quantitation (LLOQ) are treated as zero
for the calculation of mean and SD. Mean concentrations measured in
samples are used to construct semi-logarithmic concentration-time
curve profiles. Pharmacokinetic (PK) analysis is performed using
non-compartmental methods in Biobook (E-WorkbookIDBS).
Murine Model of Alternaria alternata-Induced Eosinophilic
Inflammation of the Lung
[0569] Airway eosinophilia is a hallmark of human asthma.
Alternaria alternata is a fungal aeroallergen that can exacerbate
asthma in humans and induces eosinophilic inflammation in the lungs
of mice (Havaux et al. Clin Exp Immunol. 2005, 139(2):179-88). In
mice, it has been demonstrated that alternaria indirectly activates
tissue resident type 2 innate lymphoid cells in the lung, which
respond to (e.g. IL-2 and IL-7) and release JAK-dependent cytokines
(e.g. IL-5 and IL-13) and coordinate eosinophilic inflammation
(Bartemes et al. J Immunol. 2012, 188(3):1503-13).
[0570] Seven- to nine-week old male C57 mice from Taconic are used
in the study. On the day of study, animals are lightly anesthetized
with isoflurane and administered either vehicle or test compound
via oropharyngeal aspiration. Animals are placed in lateral
recumbency post dose and monitored for full recovery from
anesthesia before being returned to their home cage. One hour
later, animals are once again briefly anesthetized and challenged
with either vehicle or alternaria extract via oropharyngeal
aspiration before being monitored for recovery from anesthesia and
returned to their home cage. Forty-eight hours after alternaria
administration, bronchoalveolar lavage fluid (BALF) is collected
and eosinophils are counted in the BALF using the Advia 120
Hematology System (Siemens).
[0571] Compound activity in the model is evidenced by a decrease in
the level of eosinophils present in the BALF of treated animals at
forty-eight hours compared to the vehicle treated, alternaria
challenged control animals. Data are expressed as percent
inhibition of the vehicle treated, alternaria challenged BALF
eosinophils response. To calculate percent inhibition, the number
of BALF eosinophils for each condition is converted to percent of
the average vehicle treated, alternaria challenged BALF eosinophils
and subtracted from one-hundred percent.
* * * * *