U.S. patent application number 15/207677 was filed with the patent office on 2017-05-11 for pyrazolo[1,5-a]pyrimidine-based compounds, compositions comprising them, and methods of their use.
The applicant listed for this patent is Lexion Pharmaceuticals, Inc.. Invention is credited to Yingzhi BI, Kenneth Gordon CARSON, Giovanni CIANCHETTA, Michael Alan GREEN, Godwin KUMI, Alan MAIN, Yulian ZHANG, Glenn Gregory ZIPP.
Application Number | 20170129896 15/207677 |
Document ID | / |
Family ID | 47892052 |
Filed Date | 2017-05-11 |
United States Patent
Application |
20170129896 |
Kind Code |
A1 |
BI; Yingzhi ; et
al. |
May 11, 2017 |
PYRAZOLO[1,5-a]PYRIMIDINE-BASED COMPOUNDS, COMPOSITIONS COMPRISING
THEM, AND METHODS OF THEIR USE
Abstract
Pyrazolo[1,5-a]pyrimidine-based compounds of the formula:
##STR00001## are disclosed, wherein R.sub.1, R.sub.2 and R.sub.3
are defined herein. Compositions comprising the compounds and
methods of their use to treat, manage and/or prevent diseases and
disorders mediated by mediated by adaptor associated kinase 1
activity are also disclosed.
Inventors: |
BI; Yingzhi; (Plainsboro,
NJ) ; CARSON; Kenneth Gordon; (Princeton, NJ)
; CIANCHETTA; Giovanni; (Waltham, MA) ; GREEN;
Michael Alan; (Easton, PA) ; KUMI; Godwin;
(Rocky Hill, CT) ; MAIN; Alan; (Far Hills, NJ)
; ZHANG; Yulian; (Acton, MA) ; ZIPP; Glenn
Gregory; (Robbinsville, NJ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Lexion Pharmaceuticals, Inc. |
The Woodlands |
TX |
US |
|
|
Family ID: |
47892052 |
Appl. No.: |
15/207677 |
Filed: |
July 12, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14483898 |
Sep 11, 2014 |
9403832 |
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15207677 |
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13785355 |
Mar 5, 2013 |
8946415 |
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14483898 |
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61608765 |
Mar 9, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/519 20130101;
A61P 29/02 20180101; A61P 25/04 20180101; C07B 59/002 20130101;
A61P 25/00 20180101; A61P 25/02 20180101; C07B 2200/05 20130101;
C07D 487/04 20130101; A61P 43/00 20180101; A61P 25/18 20180101;
A61P 25/16 20180101; A61P 25/28 20180101 |
International
Class: |
C07D 487/04 20060101
C07D487/04 |
Claims
1-63. (canceled)
64. A compound of the formula: ##STR00103## or a pharmaceutically
acceptable salt thereof, wherein: R.sub.2C is --OR.sub.2D,
--N(R.sub.2D).sub.2, --C(O)R.sub.2D, --C(O)OR.sub.2D,
--C(O)N(R.sub.2D).sub.2, --N(R.sub.2D)C(O)OR.sub.2D, cyano, halo,
or optionally substituted C.sub.1-12 hydrocarbyl or 2-12-membered
heterocarbyl, which optional substitution is with one or more with
one or more amino, cyano, halo, hydroxyl, or R.sub.2D; and each
R.sub.2D is independently hydrogen or optionally substituted
C.sub.1-12 hydrocarbyl or 2-12-membered heterocarbyl, which
optional substitution is with one or more of amino, cyano, halo, or
hydroxyl.
65. The compound of claim 64, wherein R.sub.2C is
--C(O)OR.sub.2D.
66. The compound of claim 65, wherein R.sub.2D is C.sub.1-12
hydrocarbyl.
67. The compound of claim 65, wherein R.sub.2D is 2-12-membered
heterocarbyl.
68. A compound, which compound is isopropyl
4-(3-(2-methoxypyridin-3-yl)pyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-ca-
rboxylate: ##STR00104## or a pharmaceutically acceptable salt
thereof.
69. A compound, which compound is 2-methoxyethyl
4-(3-(2-methoxypyridin-3-yl)pyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-ca-
rboxylate: ##STR00105## or a pharmaceutically acceptable salt
thereof.
70. A compound, which compound is 3-methyloxetan-3-yl
4-(3-(2-methoxypyridin-3-yl)pyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-ca-
rboxylate: ##STR00106## or a pharmaceutically acceptable salt
thereof.
71. A pharmaceutical composition comprising a compound of claim 64
and a pharmaceutically acceptable excipient or diluent.
72. A method of inhibiting AAK1 activity, which comprises
contacting AAK1 with a compound of claim 64.
73. A method of treating or managing a disease or disorder mediated
by AAK1 activity, which comprises administering to a patient in
need thereof a therapeutically effective amount of a compound of
claim 64.
74. The method of claim 73, wherein the disease or disorder is
Alzheimer's disease, bipolar disorder, pain, Parkinson's disease,
or schizophrenia.
75. The method of claim 74, wherein the pain is neuropathic
pain.
76. The method of claim 75, wherein the neuropathic pain is
fibromyalgia or peripheral neuropathy.
77. The method of claim 76, wherein the peripheral neuropathy is
diabetic neuropathy.
Description
[0001] This application is a continuation of U.S. patent
application Ser. No. 14/483,898, filed Sep. 11, 2014, which is a
continuation of U.S. patent application Ser. No. 13/785,355, filed
Mar. 5, 2013, and now U.S. Pat. No. 8,946,415, which claims
priority to provisional patent application No. 61/608,765, filed
Mar. 9, 2012, the entireties of which are incorporated herein by
reference.
1. FIELD OF THE INVENTION
[0002] This invention is directed to
pyrazolo[1,5-a]pyrimidine-based compounds useful as inhibitors of
adaptor associated kinase 1 (AAK1), compositions comprising them,
and methods of their use.
2. BACKGROUND OF THE INVENTION
[0003] Adaptor associated kinase 1 (AAK1) is a member of the
Ark1/Prk1 family of serine/threonine kinases. AAK1 mRNA exists in
two splice forms termed short and long. The long form predominates
and is highly expressed in brain and heart (Henderson and Conner,
Mol. Biol. Cell. 2007, 18, 2698-2706). AAK1 is enriched in
synaptosomal preparations and is co-localized with endocytic
structures in cultured cells. AAK1 modulates clatherin coated
endocytosis, a process that is important in synaptic vesicle
recycling and receptor-mediated endocytosis. AAK1 associates with
the AP2 complex, a hetero-tetramer which links receptor cargo to
the clatherin coat. The binding of clatherin to AAK1 stimulates
AAK1 kinase activity (Conner et. al., Traffic 2003, 4, 885-890;
Jackson et. al., J. Cell. Biol. 2003, 163, 231-236). AAK1
phosphorylates the mu-2 subunit of AP-2, which promotes the binding
of mu-2 to tyrosine containing sorting motifs on cargo receptors
(Ricotta et. al., J. Cell Bio. 2002, 156, 791-795; Conner and
Schmid, J. Cell Bio. 2002, 156, 921-929). Mu2 phosphorylation is
not required for receptor uptake, but phosphorylation enhances the
efficiency of internalization (Motely et. al., Mol. Biol. Cell.
2006, 17, 5298-5308).
[0004] AAK1 has been identified as an inhibitor of
Neuregulin-1/ErbB4 signaling in PC12 cells. Loss of AAK1 expression
through RNA interference mediated gene silencing or treatment with
the kinase inhibitor K252a (which inhibits AAK1 kinase activity)
results in the potentiation of Neuregulin-1 induced neurite
outgrowth. These treatments result in increased expression of ErbB4
and accumulation of ErbB4 in or near the plasma membrane (Kuai et.
al., Chemistry and Biology 2011, 18, 891-906). NRG1 and ErbB4 are
putative schizophrenia susceptibility genes (Buonanno, Brain Res.
Bull. 2010, 83, 122-131). SNPs in both genes have been associated
with multiple schizophrenia endophenotypes (Greenwood et. al., Am.
J. Psychiatry 2011, 168, 930-946). Neuregulin 1 and ErbB4 KO mouse
models have shown schizophrenia relevant morphological changes and
behavioral phenotypes (Jaaro-Peled et. al., Schizophrenia Bulletin
2010, 36, 301-313; Wen et. al., Proc. Natl. Acad. Sci. USA. 2010,
107, 1211-1216). In addition, a single nucleotide polymorphism in
an intron of the AAK1 gene has been associated with the age of
onset of Parkinson's disease (Latourelle et. al., BMC Med. Genet.
2009, 10, 98). These results suggest that inhibition of AAK1
activity may have utility in the treatment of schizophrenia,
cognitive deficits in schizophrenia, Parkinson's disease, bipolar
disorder, and Alzheimer's disease.
3. SUMMARY OF THE INVENTION
[0005] This invention is directed, in part, to AAK1 inhibitors of
the formula:
##STR00002##
and pharmaceutically acceptable salt thereof, wherein: R.sub.1 is
R.sub.1A or optionally substituted C.sub.1-12 hydrocarbyl or
2-12-membered heterocarbyl, which optional substitution is with one
or more R.sub.1A; each R.sub.1A is independently --OR.sub.1C,
--N(R.sub.1C).sub.2, --C(O)R.sub.1C, --C(O)OR.sub.1C,
--C(O)N(R.sub.1C).sub.2, --N(R.sub.1C)C(O)OR.sub.1C, cyano, halo,
or optionally substituted C.sub.1-12 hydrocarbyl or 2-12-membered
heterocarbyl, which optional substitution is with one or more
R.sub.1B; each R.sub.1B is independently --OR.sub.1C,
--N(R.sub.1C).sub.2, --C(O)R.sub.1C, --C(O)OR.sub.1C,
--C(O)N(R.sub.1C).sub.2, --N(R.sub.1C)C(O)OR.sub.1C, cyano or halo;
each R.sub.1C is independently hydrogen or optionally substituted
C.sub.1-12 hydrocarbyl or 2-12-membered heterocarbyl, which
optional substitution is with one or more of cyano, halo or
hydroxyl; R.sub.2 is --NR.sub.2AR.sub.2B, wherein R.sub.2A is
hydrogen and R.sub.2B is optionally substituted C.sub.1-12
hydrocarbyl or 2-12-membered heterocarbyl, which optional
substitution is with one or more R.sub.2C; or R.sub.2A and R.sub.2B
are taken together to form a 4-7-membered heterocycle optionally
substituted with one or more R.sub.2C; each R.sub.2C is
independently --OR.sub.2D, --N(R.sub.2D).sub.2, --C(O)R.sub.2D,
--C(O)OR.sub.2D, --C(O)N(R.sub.2D).sub.2,
--N(R.sub.2D)C(O)OR.sub.2D, cyano, halo, or optionally substituted
C.sub.1-12 hydrocarbyl or 2-12-membered heterocarbyl, which
optional substitution is with one or more with one or more
R.sub.2D; each R.sub.2D is independently hydrogen or optionally
substituted C.sub.1-12 hydrocarbyl or 2-12-membered heterocarbyl,
which optional substitution is with one or more of cyano, halo or
hydroxyl; and R.sub.3 is hydrogen or C.sub.1-6 alkyl optionally
substituted with one or more of cyano, halo or hydroxyl.
[0006] One embodiment of the invention encompasses pharmaceutical
compositions and dosage forms comprising a compound disclosed
herein (i.e., a compound of the invention).
[0007] Another embodiment of this invention encompasses methods of
inhibiting adaptor associated kinase 1 (AAK1), both in vitro and in
vivo, which comprise contacting AAK1 with a compound fo the
invention.
[0008] Another embodiment encompasses methods of treating and
managing diseases and disorders mediated by AAK1 activity. Examples
of such diseases and disorders are believed to include Alzheimer's
disease, bipolar disorder, pain, Parkinson's disease, and
schizophrenia (including cognitive deficits in schizophrenia).
4. BRIEF DESCRIPTION OF THE FIGURES
[0009] Aspects of the invention are illustrated in FIG. 1, which
shows results obtained from a formalin pain model using AAK1
homozygous (-/-) knockout mice and their wild-type (+/+)
littermates. The AAK1 homozygous (-/-) knockout mice show a clear
reduction in both acute and tonic pain response as compared to
their wild-type (+/+) littermates.
5. DETAILED DESCRIPTION OF THE INVENTION
[0010] This invention is based, in part, on the discovery that AAK1
knockout mice exhibit a high resistance to pain. That discovery
prompted research that ultimately led to the discovery of AAK1
inhibitors, compositions comprising them, and methods of their
use.
5.1. DEFINITIONS
[0011] Unless otherwise indicated, the phrases "compounds of the
invention," "compounds of the present disclosure," and the like
refer to the compounds disclosed herein.
[0012] Unless otherwise indicated, the term "hydrocarbyl" means an
aliphatic or alicyclic moiety having an all-carbon backbone and
consisting of carbon and hydrogen atoms. Examples of hydrocarbyl
groups include those having 1-20, 1-12, 1-6, and 1-4 carbon atoms
(referred to as C.sub.1-20 hydrocarbyl, C.sub.1-12 hydrocarbyl,
C.sub.1-6 hydrocarbyl, and C.sub.1-4 hydrocarbyl, respectively).
Particular examples include alkyl, alkenyl, alkynyl, aryl, benzyl,
cycloalkyl, cycloalkenyl, cycloalkylalkyl, cycloalkenylalkyl,
napthyl, phenyl, and phenylethyl.
[0013] Examples of alkyl moeites include straight-chain and
branched moieties having 1-20, 1-12, 1-6, 1-4 and 1-3 carbon atoms
(referred to as C.sub.1-20 alkyl, C.sub.1-12 alkyl, C.sub.1-6
alkyl, C.sub.1-4 alkyl and C.sub.1-3 alkyl, respectively).
Particular examples include methyl, ethyl, propyl, isopropyl,
n-butyl, t-butyl, isobutyl, pentyl, hexyl, isohexyl, heptyl,
4,4-dimethylpentyl, octyl, 2,2,4-trimethylpentyl, nonyl, decyl,
undecyl and dodecyl.
[0014] Examples of alkenyl moieties include straight-chain and
branched C.sub.2-20, C.sub.2-12 and C.sub.2-6 alkenyl. Particular
examples include vinyl, allyl, 1-butenyl, 2-butenyl, isobutylenyl,
1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl,
2,3-dimethyl-2-butenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl,
1-heptenyl, 2-heptenyl, 3-heptenyl, 1-octenyl, 2-octenyl,
3-octenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, 1-decenyl, 2-decenyl
and 3-decenyl.
[0015] Examples of alkynyl moeites include straight-chain and
branched C.sub.2-20, C.sub.2-12 and C.sub.2-6 alkynyl. Particular
examples include ethynyl and 2-propynyl (propargyl).
[0016] Examples of aryl moeites include anthracenyl, azulenyl,
fluorenyl, indan, indenyl, naphthyl, phenyl and phenanthrenyl.
[0017] Examples of cycloalkyl moeites include C.sub.3-12,
C.sub.3-7, C.sub.4-6 and C.sub.6 cycloalkyl. Particular examples
include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and
adamantyl.
[0018] Unless otherwise indicated, the term "halo" encompass
fluoro, chloro, bromo, and iodo.
[0019] Unless otherwise indicated, the term "heterocarbyl" refers
to a moiety having a backbone made up of one or more carbon atoms
and one or more heteroatoms. Particular heteroatoms are nitrogen,
oxygen and sulfur. A heterocarbyl moieties can be thought of as a
hydrocarbyl moiety wherein at least one carbon atom, CH, CH.sub.2,
or CH.sub.3 group is replaced with one or more heteroatoms and the
requisite number of hydrogen atoms to satisy valencies. Examples of
heterocarbyl include 2-20, 2-12, 2-8, 2-6 and 2-4 membered
heterocarbyl moieties, wherein the number range refers to the sum
total of carbon, nitrogen, oxygen, and/or sulfur atoms in the
moiety. The term "2-12 membered heterocarbyl" thus refers to a
heterocarbyl moiety having a total of 2-12 carbon, nitrogen,
oxygen, and/or sulfur atoms. Particular heterocarbyl moeites
include straight chain and branched heteroalkyl, heteroalkenyl, and
heteroalkynyl, as well as heterocycle and heteroaryl.
[0020] Examples of heteroalkyl moieties include 2-8-membered,
2-6-membered and 2-4-membered heteroalkyl moieties. Particular
examples include alkoxyl, acyl (e.g., formyl, acetyl, benzoyl),
alkylamino (e.g., di-(C.sub.1-3-alkyl)amino), arylamino, aryloxime,
carbamates, carbamides, alkylcarbonyl, arylcarbonyl, aminocarbonyl,
alkylaminocarbonyl, alkylsulfanyl, arylsulfanyl, alkylsulfinyl,
arylsulfinyl, alkylsulfonyl, arylsulfonyl, alkylsulfonylamino, and
arylsulfonylamino.
[0021] Unless otherwise indicated, the term "heterocycle" refers to
a cyclic (monocyclic or polycyclic) heterocarbyl moiety which may
be aromatic, partially aromatic or non-aromatic. Heterocycles
include heteroaryls. Examples include 4-10-membered, 4-7-membered,
6-membered, and 5-membered heterocycles. Particular examples
include benzo[1,3]dioxolyl, 2,3-dihydro-benzo[1,4]dioxinyl,
cinnolinyl, furanyl, hydantoinyl, morpholinyl, oxetanyl, oxiranyl,
piperazinyl, piperidinyl, pyrrolidinonyl, pyrrolidinyl,
tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl,
tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl
and valerolactamyl. Because the term "heterocycle" refers to a
ring, standing alone it does not encompass moieties such as
oxazolidinone and imidazolidinone: such moieties are considered
substituted heterocycles, viz. heterocycles substituted with
oxo.
[0022] Examples of heteroaryl moieties include acridinyl,
benzimidazolyl, benzofuranyl, benzoisothiazolyl, benzoisoxazolyl,
benzoquinazolinyl, benzothiazolyl, benzoxazolyl, furyl, imidazolyl,
indolyl, isothiazolyl, isoxazolyl, oxadiazolyl, oxazolyl,
phthalazinyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridyl,
pyrimidinyl, pyrimidyl, pyrrolyl, quinazolinyl, quinolinyl,
tetrazolyl, thiazolyl, and triazinyl.
[0023] Unless otherwise indicated, the term "include" has the same
meaning as "include, but are not limited to," and the term
"includes" has the same meaning as "includes, but is not limited
to." Similarly, the term "such as" has the same meaning as the term
"such as, but not limited to."
[0024] Unless otherwise indicated, the terms "manage," "managing"
and "management" encompass preventing the recurrence of the
specified disease or disorder in a patient who has already suffered
from the disease or disorder, and/or lengthening the time that a
patient who has suffered from the disease or disorder remains in
remission. The terms encompass modulating the threshold,
development and/or duration of the disease or disorder, or changing
the way that a patient responds to the disease or disorder.
[0025] Unless otherwise indicated, a "therapeutically effective
amount" of a compound is an amount sufficient to provide a
therapeutic benefit in the treatment or management of a disease or
condition, or to delay or minimize one or more symptoms associated
with the disease or condition. A "therapeutically effective amount"
of a compound means an amount of therapeutic agent, alone or in
combination with other therapies, that provides a therapeutic
benefit in the treatment or management of the disease or condition.
The term "therapeutically effective amount" can encompass an amount
that improves overall therapy, reduces or avoids symptoms or causes
of a disease or condition, or enhances the therapeutic efficacy of
another therapeutic agent.
[0026] Unless otherwise indicated, the terms "treat," "treating"
and "treatment" contemplate an action that occurs while a patient
is suffering from the specified disease or disorder, which reduces
the severity of the disease or disorder, or retards or slows the
progression of the disease or disorder.
[0027] Unless otherwise indicated, one or more adjectives
immediately preceding a series of nouns is to be construed as
applying to each of the nouns. For example, the phrase "optionally
substituted alky, aryl, or heteroaryl" has the same meaning as
"optionally substituted alky, optionally substituted aryl, or
optionally substituted heteroaryl."
5.2. COMPOUNDS
[0028] This invention encompasses compounds of the formula:
##STR00003##
and pharmaceutically acceptable salt thereof, wherein: R.sub.1 is
R.sub.1A or optionally substituted C.sub.1-12 hydrocarbyl or
2-12-membered heterocarbyl, which optional substitution is with one
or more R.sub.1A; each R.sub.1A is independently --OR.sub.1C,
--N(R.sub.1C).sub.2, --C(O)R.sub.1C, --C(O)OR.sub.1C,
--C(O)N(R.sub.1C).sub.2, --N(R.sub.1C)C(O)OR.sub.1C, cyano, halo,
or optionally substituted C.sub.1-12 hydrocarbyl or 2-12-membered
heterocarbyl, which optional substitution is with one or more
R.sub.1B; each R.sub.1B is independently --OR.sub.1C,
--N(R.sub.1C).sub.2, --C(O)R.sub.1C, --C(O)OR.sub.1C,
--C(O)N(R.sub.1C).sub.2, --N(R.sub.1C)C(O)OR.sub.1C, cyano or halo;
each R.sub.1C is independently hydrogen or optionally substituted
C.sub.1-12 hydrocarbyl or 2-12-membered heterocarbyl, which
optional substitution is with one or more of cyano, halo or
hydroxyl; R.sub.2 is --NR.sub.2AR.sub.2B, wherein R.sub.2A is
hydrogen and R.sub.2B is optionally substituted C.sub.1-12
hydrocarbyl or 2-12-membered heterocarbyl, which optional
substitution is with one or more R.sub.2C; or R.sub.2A and R.sub.2B
are taken together to form a 4-7-membered heterocycle optionally
substituted with one or more R.sub.2C; each R.sub.2C is
independently --OR.sub.2D, --N(R.sub.2D).sub.2, --C(O)R.sub.2D,
--C(O)OR.sub.2D, --C(O)N(R.sub.2D).sub.2,
--N(R.sub.2D)C(O)OR.sub.2D, cyano, halo, or optionally substituted
C.sub.1-12 hydrocarbyl or 2-12-membered heterocarbyl, which
optional substitution is with one or more with one or more
R.sub.2D; each R.sub.2D is independently hydrogen or optionally
substituted C.sub.1-12 hydrocarbyl or 2-12-membered heterocarbyl,
which optional substitution is with one or more of cyano, halo or
hydroxyl; and R.sub.3 is hydrogen or C.sub.1-6 alkyl optionally
substituted with one or more of cyano, halo or hydroxyl.
[0029] In particular compounds, R.sub.1 is R.sub.1A. In some,
R.sub.1 is optionally substituted C.sub.1-12 hydrocarbyl. In some,
R.sub.1 is optionally substituted phenyl. In some, R.sub.1 is
optionally substituted 2-12-membered heterocarbyl (e.g., 2-8
membered heterocarbyl, 2-6 membered heterocarbyl, 2-6 membered
heterocarbyl). In some, R.sub.1 is optionally substituted
pyridinyl, thiophen, or imidazol.
[0030] In particular compounds, R.sub.1A is halo. In some, R.sub.1A
is --OR.sub.1C, --N(R.sub.1C).sub.2, --C(O)R.sub.1C,
--C(O)OR.sub.1C, or --C(O)N(R.sub.1C).sub.2. In some, R.sub.1A is
--OR.sub.1C.
[0031] In particular compounds, R.sub.1B is --N(R.sub.1C).sub.2,
--OR.sub.1C, halo.
[0032] In particular compounds, R.sub.2A and R.sub.2B are taken
together to form a 4-7-membered heterocycle optionally substituted
with one or more R.sub.2C.
[0033] In particular compounds, R.sub.1C is hydrogen. In some,
R.sub.1C is C.sub.1-12 hydrocarbyl (e.g., C.sub.1-6 hydrocarbyl,
C.sub.1-4 hydrocarbyl such as methyl, ethyl, propyl). In some,
R.sub.2C is --C(O)OR.sub.2D, --C(O)N(R.sub.2D).sub.2, or
--N(R.sub.2D)C(O)OR.sub.2D.
[0034] In particular compounds, R.sub.2D is hydrogen.
[0035] In particular compounds, R.sub.2D is C.sub.1-12 hydrocarbyl
(e.g., C.sub.1-6 hydrocarbyl, C.sub.1-4 hydrocarbyl such as methyl,
ethyl, propyl).
[0036] In particular compounds, R.sub.3 is hydrogen.
[0037] One embodiment of the invention encompasses compounds of the
formula:
##STR00004##
and pharmaceutically acceptable salt thereof, wherein: A is cyclic
C.sub.1-12 hydrocarbyl or 4-7-membered heterocycle; D is
4-7-membered heterocycle; each R.sub.1A is independently
--OR.sub.1C, --N(R.sub.1C).sub.2, --C(O)R.sub.1C, --C(O)OR.sub.1C,
--C(O)N(R.sub.1C).sub.2, --N(R.sub.1C)C(O)OR.sub.1C, cyano, halo,
or optionally substituted C.sub.1-12 hydrocarbyl or 2-12-membered
heterocarbyl, which optional substitution is with one or more
R.sub.1B; each R.sub.1B is independently --OR.sub.1C,
--N(R.sub.1C).sub.2, --C(O)R.sub.1C, --C(O)OR.sub.1C,
--C(O)N(R.sub.1C).sub.2, --N(R.sub.1C)C(O)OR.sub.1C, cyano or halo;
each R.sub.1C is independently hydrogen or optionally substituted
C.sub.1-12 hydrocarbyl or 2-12-membered heterocarbyl, which
optional substitution is with one or more of cyano, halo or
hydroxyl; each R.sub.2C is independently --OR.sub.2D,
--N(R.sub.2D).sub.2, --C(O)R.sub.2D, --C(O)OR.sub.2D,
--C(O)N(R.sub.2D).sub.2, --N(R.sub.2D)C(O)OR.sub.2D, cyano, halo,
or optionally substituted C.sub.1-12 hydrocarbyl or 2-12-membered
heterocarbyl, which optional substitution is with one or more with
one or more R.sub.2D; each R.sub.2D is independently hydrogen or
optionally substituted C.sub.1-12 hydrocarbyl or 2-12-membered
heterocarbyl, which optional substitution is with one or more of
cyano, halo or hydroxyl; n is 1-3; and m is 0-3.
[0038] In particular compounds, D is not piperidinyl.
[0039] In particular compounds, R.sub.2C is not
--N(R.sub.2D).sub.2.
[0040] In particular compounds, A is not phenyl.
[0041] In particular compounds, m is 1.
[0042] In particular compounds, m is 2.
[0043] In particular compounds, R.sub.2D is not ethyl.
[0044] In particular compounds, when D is piperidinyl, A is phenyl,
and R.sub.2C is --N(R.sub.2D).sub.2, R.sub.2D is not ethyl.
[0045] In particular compounds, D is piperazin or pyrrolidin.
[0046] In particular compounds, n is 1.
[0047] In particular compounds, A is pyridinyl, thiophen, or
imidazol.
[0048] Particular compounds are of the formula:
##STR00005##
wherein X is CH or N.
[0049] Another embodiment of the invention encompasses compounds of
the formula:
##STR00006##
and pharmaceutically acceptable salt thereof, wherein: X is CH or
N; each R.sub.1A is independently --OR.sub.1C, --N(R.sub.1C).sub.2,
--C(O)R.sub.1C, --C(O)OR.sub.1C, --C(O)N(R.sub.1C).sub.2,
--N(R.sub.1C)C(O)OR.sub.1C, cyano, halo, or optionally substituted
C.sub.1-12 hydrocarbyl or 2-12-membered heterocarbyl, which
optional substitution is with one or more R.sub.1B; each R.sub.1B
is independently --OR.sub.1C, --N(R.sub.1C).sub.2, --C(O)R.sub.1C,
--C(O)OR.sub.1C, --C(O)N(R.sub.1C).sub.2,
--N(R.sub.1C)C(O)OR.sub.1C, cyano or halo; each R.sub.1C is
independently hydrogen or optionally substituted C.sub.1-12
hydrocarbyl or 2-12-membered heterocarbyl, which optional
substitution is with one or more of cyano, halo or hydroxyl; each
R.sub.2C is independently --OR.sub.2D, --N(R.sub.2D).sub.2,
--C(O)R.sub.2D, --C(O)OR.sub.2D, --C(O)N(R.sub.2D).sub.2,
--N(R.sub.2D)C(O)OR.sub.2D, cyano, halo, or optionally substituted
C.sub.1-12 hydrocarbyl or 2-12-membered heterocarbyl, which
optional substitution is with one or more with one or more
R.sub.2D; each R.sub.2D is independently hydrogen or optionally
substituted C.sub.1-12 hydrocarbyl or 2-12-membered heterocarbyl,
which optional substitution is with one or more of cyano, halo or
hydroxyl; and m is 0-3.
[0050] In particular compounds, R.sub.2C is not optionally
substituted phenyl or pyridinyl.
[0051] In particular compounds, X is N and m is 1 or 2.
[0052] Particular compounds are of the formula:
##STR00007##
[0053] Particular compounds are of the formula:
##STR00008##
[0054] Particular compounds are of the formula:
##STR00009##
[0055] Others are of the formula:
##STR00010##
[0056] Another embodiment of the invention encompasses compounds of
the formula:
##STR00011##
and pharmaceutically acceptable salt thereof, wherein: X is CH or
N; each R.sub.1A is independently --OR.sub.1C, --N(R.sub.1C).sub.2,
--C(O)R.sub.1C, --C(O)OR.sub.1C, --C(O)N(R.sub.1C).sub.2,
--N(R.sub.1C)C(O)OR.sub.1C, cyano, halo, or optionally substituted
C.sub.1-12 hydrocarbyl or 2-12-membered heterocarbyl, which
optional substitution is with one or more R.sub.1B; each R.sub.1B
is independently --OR.sub.1C, --N(R.sub.1C).sub.2, --C(O)R.sub.1C,
--C(O)OR.sub.1C, --C(O)N(R.sub.1C).sub.2,
--N(R.sub.1C)C(O)OR.sub.1C, cyano or halo; each R.sub.1C is
independently hydrogen or optionally substituted C.sub.1-12
hydrocarbyl or 2-12-membered heterocarbyl, which optional
substitution is with one or more of cyano, halo or hydroxyl; each
R.sub.2C is independently --OR.sub.2D, --N(R.sub.2D).sub.2,
--C(O)R.sub.2D, --C(O)OR.sub.2D, --C(O)N(R.sub.2D).sub.2,
--N(R.sub.2D)C(O)OR.sub.2D, cyano, halo, or optionally substituted
C.sub.1-12 hydrocarbyl or 2-12-membered heterocarbyl, which
optional substitution is with one or more with one or more
R.sub.2D; each R.sub.2D is independently hydrogen or optionally
substituted C.sub.1-12 hydrocarbyl or 2-12-membered heterocarbyl,
which optional substitution is with one or more of cyano, halo or
hydroxyl; and m is 0-3.
[0057] Particular compounds are of the formula:
##STR00012##
[0058] Particular compounds are of the formula:
##STR00013##
[0059] Others are of the formula:
##STR00014##
[0060] In particular compounds, R.sub.1A is halo. In some, R.sub.1A
is --OR.sub.1C.
[0061] In particular compounds, R.sub.1C is optionally substituted
C.sub.1-12 hydrocarbyl (e.g., C.sub.1-6 hydrocarbyl, C.sub.1-4
hydrocarbyl).
[0062] In particular compounds, R.sub.2C is --C(O)OR.sub.2D,
--C(O)N(R.sub.2D).sub.2, or --N(R.sub.2D)C(O)OR.sub.2D. In some,
R.sub.2C is --C(O)R.sub.2D.
[0063] In particular compounds, R.sub.2D is independently hydrogen
or C.sub.1-12 hydrocarbyl (e.g., C.sub.1-6 hydrocarbyl, C.sub.1-4
hydrocarbyl). In some, R.sub.2D is optionally substituted
C.sub.1-12 hydrocarbyl, which optional substitution is with one or
more of amino, cyano, halo, hydroxyl.
[0064] Compounds of the invention can have one or more asymmetric
centers. Unless otherwise indicated, this invention encompasses all
stereoisomers of the compounds, as well as mixtures thereof.
Individual stereoisomers of compounds can be prepared synthetically
from commercially available starting materials which contain chiral
centers or by preparation of mixtures of enantiomeric products
followed by separation such as conversion to a mixture of
diastereomers followed by separation or recrystallization,
chromatographic techniques, or direct separation of enantiomers on
chiral chromatographic columns. Starting compounds of particular
stereochemistry are either commercially available or can be made
and resolved by techniques known in the art.
[0065] Certain compounds of the present disclosure may also exist
in different stable conformational forms which may be separable.
Torsional asymmetry due to restricted rotation about an asymmetric
single bond, for example because of steric hindrance or ring
strain, may permit separation of different conformers. The present
disclosure includes each conformational isomer of these compounds
and mixtures thereof.
[0066] The present disclosure is intended to include all isotopes
of atoms occurring in the present compounds. Isotopes include those
atoms having the same atomic number but different mass numbers. By
way of general example and without limitation, isotopes of hydrogen
include deuterium and tritium. Isotopes of carbon include .sup.13C
and .sup.14C. Isotopically-labeled compounds of the invention can
generally be prepared by conventional techniques known to those
skilled in the art or by processes analogous to those described
herein, using an appropriate isotopically-labeled reagent in place
of the non-labeled reagent otherwise employed. Such compounds may
have a variety of potential uses, for example as standards and
reagents in determining biological activity. In the case of stable
isotopes, such compounds may have the potential to favorably modify
biological, pharmacological, or pharmacokinetic properties.
[0067] The compounds of the present disclosure can exist as
pharmaceutically acceptable salts. The term "pharmaceutically
acceptable salt," as used herein, represents salts or zwitterionic
forms of the compounds of the present disclosure which are water or
oil-soluble or dispersible, which are, within the scope of sound
medical judgment, suitable for use in contact with the tissues of
patients without excessive toxicity, irritation, allergic response,
or other problem or complication commensurate with a reasonable
benefit/risk ratio, and are effective for their intended use. The
salts can be prepared during the final isolation and purification
of the compounds or separately by reacting a suitable nitrogen atom
with a suitable acid. Representative acid addition salts include
acetate, adipate, alginate, citrate, aspartate, benzoate,
benzenesulfonate, bisulfate, butyrate, camphorate,
camphorsulfonate; digluconate, dihydrobromide, diydrochloride,
dihydroiodide, glycerophosphate, hemisulfate, heptanoate,
hexanoate, formate, fumarate, hydrochloride, hydrobromide,
hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate,
mesitylenesulfonate, methanesulfonate, naphthylenesulfonate,
nicotinate, 2-naphthalenesulfonate, oxalate, palmoate, pectinate,
persulfate, 3-phenylproprionate, picrate, pivalate, propionate,
succinate, tartrate, trichloroacetate, trifluoroacetate, phosphate,
glutamate, bicarbonate, para-toluenesulfonate, and undecanoate.
Examples of acids which can be employed to form pharmaceutically
acceptable addition salts include inorganic acids such as
hydrochloric, hydrobromic, sulfuric, and phosphoric, and organic
acids such as oxalic, maleic, succinic, and citric.
[0068] Basic addition salts can be prepared during the final
isolation and purification of the compounds by reacting a carboxy
group with a suitable base such as the hydroxide, carbonate, or
bicarbonate of a metal cation or with ammonia or an organic
primary, secondary, or tertiary amine. The cations of
pharmaceutically acceptable salts include lithium, sodium,
potassium, calcium, magnesium, and aluminum, as well as nontoxic
quaternary amine cations such as ammonium, tetramethylammonium,
tetraethylammonium, methylamine, dimethylamine, trimethylamine,
triethylamine, diethylamine, ethylamine, tributylamine, pyridine,
N,N-dimethylaniline, N-methylpiperidine, N-methylmorpholine,
dicyclohexylamine, procaine, dibenzylamine,
N,N-dibenzylphenethylamine, and N,N'-dibenzylethylenediamine. Other
representative organic amines useful for the formation of base
addition salts include ethylenediamine, ethanolamine,
diethanolamine, piperidine, and piperazine.
[0069] Particular compounds of the invention inhibit AAK1 with an
IC.sub.50 of less than 0.1, 0.01 or 0.001 .mu.M as measured in the
P81 filter plate assay described below in the Examples. Particular
compounds of the invention inhibit AAK1 with an IC.sub.50 of less
than 0.1, 0.01 or 0.001 .mu.M as measured in the HEK281 cell-based
assay described below in the Examples.
5.3. METHODS OF SYNTHESIS
[0070] Compounds of the present invention (i.e., compounds
disclosed herein) can be prepared using the methods described below
and using methods known to those skilled in the art of organic
chemistry. Particular compounds are of the general formula:
##STR00015##
wherein R.sub.1, R.sub.2 and R.sub.3 are defined herein, and
include salts thereof. These compounds can prepared by the methods
outlined below.
[0071] Compounds of formula shown above can be prepared by the
methods outlined below. In Scheme 1, the chlorine of a compound of
formula 1 is displaced with an amine to produce 2. Bromination of 2
provides 3. Alternatively 1 can be bromanated first to afford 4
then amine displacement to give 3. Suzuki coupling of 3 with an
appropriate boronic acid [R.sub.1B(OH).sub.2] affords compounds of
formula 5.
##STR00016##
[0072] In Scheme 2, the substituted acetonitrile 6 is reacted with
ethyl formate to provide 7. Condensation of 7 with hydrazine
hydrate affords 8. Reaction of 8 with
1,3-dimethylpyrimidine-2,4(1H,3H)-dione provides 9. Further
reaction of 9 with amine gives compounds with formula 5.
##STR00017##
[0073] In Scheme 3, compound with formula 10 can be prepared by
reacting 2 with NIS. Reaction of 10 with methyl
2,2-difluoro-2-(fluorosulfonyl)acetate provides compounds with
formula 11.
##STR00018##
5.4. METHODS OF USE
[0074] One embodiment of this invention encompasses methods of
inhibiting adaptor associated kinase 1 (AAK1), both in vitro and in
vivo, which comprise contacting AAK1 with a compound of the
invention.
[0075] Another embodiment encompasses methods of treating and
managing diseases and disorders mediated by AAK1 activity. Diseases
and disorders mediated by AAK1 activity are diseases and disorders
that have at least one symptom, the severity or manifestation of
which is affected by AAK1 activity. Examples of such diseases and
disorders are believed to include Alzheimer's disease, bipolar
disorder, pain, Parkinson's disease, and schizophrenia (including
cognitive deficits in schizophrenia). Particular methods comprise
administering to a patient (a human or other mammal) in need
thereof a therapeutically or prophylactically effective amount of
an AAK1 inhibitor (e.g., a compound disclosed herein).
[0076] Another embodiment of this invention encompasses a method of
treating or managing a disease or disorder, which comprises
administering to a patient in need thereof a therapeutically or
prophylactically effective amount of an AAK1 inhibitor, wherein the
disease or disorder is Alzheimer's disease, bipolar disorder, pain,
Parkinson's disease, or schizophrenia (including cognitive deficits
in schizophrenia). Particular types of pain include chronic pain,
acute pain, and neuropathic pain. Particular types of neuropathic
pain include fibromyalgia and peripheral neuropathy (e.g., diabetic
neuropathy).
[0077] When used to treat or manage a disease or disorder,
compounds of the invention are preferably administered as part of a
pharmaceutical composition comprising one or more pharmaceutically
acceptable carriers, diluents or excipients.
[0078] Pharmaceutical compositions, or formulations, may be
presented in unit dose forms containing a predetermined amount of
active ingredient per unit dose. Dosage levels of between about
0.01 and about 250 milligram per kilogram ("mg/kg") body weight per
day, preferably between about 0.05 and about 100 mg/kg body weight
per day of the compounds of the present disclosure are typical in a
monotherapy for the prevention and treatment of disease. Typically,
the pharmaceutical compositions of this disclosure will be
administered from about 1 to about 5 times per day or
alternatively, as a continuous infusion. Such administration can be
used as a chronic or acute therapy. The amount of active ingredient
that may be combined with the carrier materials to produce a single
dosage form will vary depending on the condition being treated, the
severity of the condition, the time of administration, the route of
administration, the rate of excretion of the compound employed, the
duration of treatment, and the age, gender, weight, and condition
of the patient. Preferred unit dosage formulations are those
containing a daily dose or sub-dose, as herein above recited, or an
appropriate fraction thereof, of an active ingredient. Treatment
may be initiated with small dosages substantially less than the
optimum dose of the compound. Thereafter, the dosage is increased
by small increments until the optimum effect under the
circumstances is reached. In general, the compound is most
desirably administered at a concentration level that will generally
afford effective results without causing any harmful or deleterious
side effects.
[0079] Compounds of the invention may be administered in
combination with one or more additional therapeutic or prophylactic
agents. For example, when used for the treatment of pain, possible
additional agents include immunosuppressive and anti-inflammatory
agents.
[0080] Immunosuppressants suitable for use in the methods and
compositions of this invention include those known in the art.
Examples include aminopterin, azathioprine, cyclosporin A,
D-penicillamine, gold salts, hydroxychloroquine, leflunomide,
methotrexate, minocycline, rapamycin, sulfasalazine, tacrolimus
(FK506), and pharmaceutically acceptable salts thereof. A
particular immunosuppressant is methotrexate.
[0081] Additional examples include anti-TNF antibodies, such as
adalimumab, certolizumab pegol, etanercept, and infliximab. Others
include interleukin-1 blockers, such as anakinra. Others include
anti-B cell (CD20) antibodies, such as rituximab. Others include T
cell activation blockers, such as abatacept.
[0082] Additional examples include inosine monophosphate
dehydrogenase inhibitors, such as mycophenolate mofetil
(CellCept.RTM.) and mycophenolic acid (Myfortic.RTM.).
[0083] Anti-inflammatory drugs suitable for use in the methods and
compositions of this invention include those known in the art.
Examples include glucocorticoids and NSAIDs.
[0084] Examples of glucocorticoids include aldosterone,
beclometasone, betamethasone, cortisone, deoxycorticosterone,
dexamethasone, fludrocortisones, hydrocortisone,
methylprednisolone, prednisolone, prednisone, triamcinolone, and
pharmaceutically acceptable salts thereof.
[0085] Examples of NSAID include salicylates (e.g., aspirin,
amoxiprin, benorilate, choline magnesium salicylate, diflunisal,
faislamine, methyl salicylate, magnesium salicylate, salicyl
salicylate, and pharmaceutically acceptable salts thereof),
arylalkanoic acids (e.g., diclofenac, aceclofenac, acemetacin,
bromfenac, etodolac, indometacin, nabumetone, sulindac, tolmetin,
and pharmaceutically acceptable salts thereof), arylpropionic acids
(e.g., ibuprofen, carprofen, fenbufen, fenoprofen, flurbiprofen,
ketoprofen, ketorolac, loxoprofen, naproxen, oxaprozin, tiaprofenic
acid, suprofen, and pharmaceutically acceptable salts thereof),
arylanthranilic acids (e.g., meclofenamic acid, mefenamic acid, and
pharmaceutically acceptable salts thereof), pyrazolidine
derivatives (e.g., azapropazone, metamizole, oxyphenbutazone,
phenylbutazone, sulfinprazone, and pharmaceutically acceptable
salts thereof), oxicams (e.g., lornoxicam, meloxicam, piroxicam,
tenoxicam, and pharmaceutically acceptable salts thereof), COX-2
inhibitors (e.g., celecoxib, etoricoxib, lumiracoxib, parecoxib,
rofecoxib, valdecoxib, and pharmaceutically acceptable salts
thereof), and sulphonanilides (e.g., nimesulide and
pharmaceutically acceptable salts thereof).
[0086] Other agents used in the treatment of pain (including but
not limited to neuropathic and inflammatory pain) include agents
such as pregabalin, lidocaine, duloxetine, gabapentin,
carbamazepine, capsaicin, and other
serotonin/norepinephrine/dopamine reuptake inhibitors, and opiates
(such as oxycontin, morphine, and codeine).
[0087] In the treatment of pain caused by a known disease or
condition, such as diabetes, infection (e.g., herpes zoster or HIV
infection), or cancer, compounds of the invention may be
administered in combination with one or more additional therapeutic
or prophylactic agents directed at the underlying disease or
condition. For example, when used to treat diabetic neuropathy,
compounds of the invention may be administered in combination with
one or more anti-diabetic agents, anti-hyperglycemic agents,
hypolipidemic/lipid lowering agents, anti-obesity agents,
anti-hypertensive agents and appetite suppressants. Examples of
anti-diabetic agents include biguanides (e.g., metformin,
phenformin), glucosidase inhibitors (e.g., acarbose, miglitol),
insulins (including insulin secretagogues and insulin sensitizers),
meglitinides (e.g., repaglinide), sulfonylureas (e.g., glimepiride,
glyburide, gliclazide, chlorpropamide, and glipizide),
biguanide/glyburide combinations (e.g., Glucovance),
thiazolidinediones (e.g., troglitazone, rosiglitazone, and
pioglitazone), PPAR-alpha agonists, PPAR-gamma agonists, PPAR
alpha/gamma dual agonists, glycogen phosphorylase inhibitors,
inhibitors of fatty acid binding protein (aP2), glucagon-like
peptide-1 (GLP-1) or other agonists of the GLP-1 receptor,
dipeptidyl peptidase IV (DPP4) inhibitors, and sodium-glucose
co-transporter 2 (SGLT2) inhibitors (e.g., dapagliflozin,
canagliflozin, and LX-4211).
5.5. PHARMACEUTICAL COMPOSITIONS
[0088] Pharmaceutical formulations may be adapted for
administration by any appropriate route, for example by the oral
(including buccal or sublingual), rectal, nasal, topical (including
buccal, sublingual, or transdermal), vaginal, or parenteral
(including subcutaneous, intracutaneous, intramuscular,
intra-articular, intrasynovial, intrasternal, intrathecal,
intralesional, intravenous, or intradermal injections or infusions)
route. Such formulations may be prepared by any method known in the
art of pharmacy, for example by bringing into association the
active ingredient with the carrier(s) or excipient(s). Oral
administration or administration by injection are preferred.
[0089] Pharmaceutical formulations adapted for oral administration
may be presented as discrete units such as capsules or tablets;
powders or granules; solutions or suspensions in aqueous or
non-aqueous liquids; edible foams or whips; or oil-in-water liquid
emulsions or water-in-oil emulsions.
[0090] For instance, for oral administration in the form of a
tablet or capsule, the active drug component can be combined with
an oral, non-toxic pharmaceutically acceptable inert carrier such
as ethanol, glycerol, water, and the like. Powders are prepared by
comminuting the compound to a suitable fine size and mixing with a
similarly comminuted pharmaceutical carrier such as an edible
carbohydrate, as, for example, starch or mannitol. Flavoring,
preservative, dispersing, and coloring agent can also be
present.
[0091] Capsules are made by preparing a powder mixture, as
described above, and filling formed gelatin sheaths. Glidants and
lubricants such as colloidal silica, talc, magnesium stearate,
calcium stearate, or solid polyethylene glycol can be added to the
powder mixture before the filling operation. A disintegrating or
solubilizing agent such as agar-agar, calcium carbonate, or sodium
carbonate can also be added to improve the availability of the
medicament when the capsule is ingested.
[0092] Moreover, when desired or necessary, suitable binders,
lubricants, disintegrating agents, and coloring agents can also be
incorporated into the mixture. Suitable binders include starch,
gelatin, natural sugars such as glucose or beta-lactose, corn
sweeteners, natural and synthetic gums such as acacia, tragacanth
or sodium alginate, carboxymethylcellulose, polyethylene glycol,
and the like. Lubricants used in these dosage forms include sodium
oleate, sodium chloride, and the like. Disintegrators include,
without limitation, starch, methyl cellulose, agar, betonite,
xanthan gum, and the like. Tablets are formulated, for example, by
preparing a powder mixture, granulating or slugging, adding a
lubricant and disintegrant, and pressing into tablets. A powder
mixture is prepared by mixing the compound, suitable comminuted,
with a diluent or base as described above, and optionally, with a
binder such as carboxymethylcellulose, an aliginate, gelating, or
polyvinyl pyrrolidone, a solution retardant such as paraffin, a
resorption accelerator such as a quaternary salt and/or and
absorption agent such as betonite, kaolin, or dicalcium phosphate.
The powder mixture can be granulated by wetting with a binder such
as syrup, starch paste, acadia mucilage, or solutions of cellulosic
or polymeric materials and forcing through a screen. As an
alternative to granulating, the powder mixture can be run through
the tablet machine and the result is imperfectly formed slugs
broken into granules. The granules can be lubricated to prevent
sticking to the tablet forming dies by means of the addition of
stearic acid, a stearate salt, talc, or mineral oil. The lubricated
mixture is then compressed into tablets. The compounds of the
present disclosure can also be combined with a free flowing inert
carrier and compressed into tablets directly without going through
the granulating or slugging steps. A clear or opaque protective
coating consisting of a sealing coat of shellac, a coating of sugar
or polymeric material, and a polish coating of wax can be provided.
Dyestuffs can be added to these coatings to distinguish different
unit dosages.
[0093] Oral fluids such as solution, syrups, and elixirs can be
prepared in dosage unit form so that a given quantity contains a
predetermined amount of the compound. Syrups can be prepared by
dissolving the compound in a suitably flavored aqueous solution,
while elixirs are prepared through the use of a non-toxic vehicle.
Solubilizers and emulsifiers such as ethoxylated isostearyl
alcohols and polyoxyethylene sorbitol ethers, preservatives, flavor
additive such as peppermint oil or natural sweeteners, or saccharin
or other artificial sweeteners, and the like can also be added.
[0094] Where appropriate, dosage unit formulations for oral
administration can be microencapsulated. The formulation can also
be prepared to prolong or sustain the release as for example by
coating or embedding particulate material in polymers, wax, or the
like.
[0095] The compounds of Formula (I), and pharmaceutically
acceptable salts thereof, can also be administered in the form of
liposome delivery systems, such as small unilamellar vesicles,
large unilamellar vesicles, and multilamellar vesicles. Liposomes
can be formed from a variety of phopholipids, such as cholesterol,
stearylamine, or phophatidylcholines.
[0096] The compounds of Formula (I) and pharmaceutically acceptable
salts thereof may also be delivered by the use of monoclonal
antibodies as individual carriers to which the compound molecules
are coupled. The compounds may also be coupled with soluble
polymers as targetable drug carriers. Such polymers can include
polyvinylpyrrolidone, pyran copolymer,
polyhydroxypropylmethacrylamidephenol,
polyhydroxyethylaspartamidephenol, or polyethyleneoxidepolylysine
substituted with palitoyl residues. Furthermore, the compounds may
be coupled to a class of biodegradable polymers useful in achieving
controlled release of a drug, for example, polylactic acid,
polepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters,
polyacetals, polydihydropyrans, polycyanoacrylates, and
cross-linked or amphipathic block copolymers of hydrogels.
[0097] Pharmaceutical formulations adapted for transdermal
administration may be presented as discrete patches intended to
remain in intimate contact with the epidermis of the recipient for
a prolonged period of time. For example, the active ingredient may
be delivered from the patch by iontophoresis as generally described
in Pharmaceutical Research 1986, 3(6), 318.
[0098] Pharmaceutical formulations adapted for topical
administration may be formulated as ointments, creams, suspensions,
lotions, powders, solutions, pastes, gels, sprays, aerosols, or
oils.
[0099] Pharmaceutical formulations adapted for rectal
administration may be presented as suppositories or as enemas.
[0100] Pharmaceutical formulations adapted for nasal administration
wherein the carrier is a solid include a course powder having a
particle size for example in the range 20 to 500 microns which is
administered in the manner in which snuff is taken, i.e., by rapid
inhalation through the nasal passage from a container of the powder
held close up to the nose. Suitable formulations wherein the
carrier is a liquid, for administration as a nasal spray or nasal
drops, include aqueous or oil solutions of the active
ingredient.
[0101] Pharmaceutical formulations adapted for administration by
inhalation include fine particle dusts or mists, which may be
generated by means of various types of metered, dose pressurized
aerosols, nebulizers, or insufflators.
[0102] Pharmaceutical formulations adapted for vaginal
administration may be presented as pessaries, tampons, creams,
gels, pastes, foams, or spray formulations.
[0103] Pharmaceutical formulations adapted for parenteral
administration include aqueous and non-aqueous sterile injection
solutions which may contain anti-oxidants, buffers, bacteriostats,
and soutes which render the formulation isotonic with the blood of
the intended recipient; and aqueous and non-aqueous sterile
suspensions which may include suspending agents and thickening
agents. The formulations may be presented in unit-dose or
multi-dose containers, for example sealed ampoules and vials, and
may be stored in a freeze-dried (lyophilized) condition requiring
only the addition of the sterile liquid carrier, for example water
for injections, immediately prior to use. Extemporaneous injection
solutions and suspensions may be prepared from sterile powders,
granules, and tablets.
[0104] It should be understood that in addition to the ingredients
particularly mentioned above, the formulations may include other
agents conventional in the art having regard to the type of
formulation in question, for example those suitable for oral
administration may include flavoring agents.
5.6. EXAMPLES
[0105] Certain aspects of the invention can be understood from the
following examples.
5.6.1. AAK1 Knockout Mice
[0106] Mice homozygous (-/-) for the disruption of the AAK1 gene
were prepared by two methods; gene trapping and homologous
recombination.
[0107] Gene trapping is a method of random insertional mutagenesis
that uses a fragment of DNA coding for a reporter or selectable
marker gene as a mutagen. Gene trap vectors have been designed to
integrate into introns or genes in a manner that allows the
cellular splicing machinery to splice vector encoded exons to
cellular mRNAs. Commonly, gene trap vectors contain selectable
marker sequences that are preceded by strong splice acceptor
sequences and are not preceded by a promoter. Thus, when such
vectors integrate into a gene, the cellular splicing machinery
splices exons from the trapped gene onto the 5' end of the
selectable marker sequence. Typically, such selectable marker genes
can only be expressed if the vector encoding the gene has
integrated into an intron. The resulting gene trap events are
subsequently identified by selecting for cells that can survive
selective culture.
[0108] Embryonic stem cells (Lex-1 cells from derived murine strain
A129), were mutated by a process involving the insertion of at
least a portion of a genetically engineered vector sequence into
the gene of interest, the mutated embryonic stem cells were
microinjected into blastocysts which were subsequently introduced
into pseudopregnant female hosts and carried to term using
established methods. See, e.g., "Mouse Mutagenesis", 1998,
Zambrowicz et al., eds., Lexicon Press, The Woodlands, Tex. The
resulting chimeric animals were subsequently bred to produce
offspring capable of germline transmission of an allele containing
the engineered mutation in the gene of interest.
[0109] AAK1-gene disrupted mice were also made by homologous
recombination. In this case, the second coding exon of the murine
AAK1 gene (see GenBank Accession Number NM_177762) was removed by
methods known in the art. See, e.g., U.S. Pat. Nos. 5,487,992,
5,627,059, and 5,789,215.
[0110] Mice homozygous (-/-) for the disruption of the AAK1 gene
were studied in conjunction with mice heterozygous (+/-) for the
disruption of the AAK1 gene, and wild-type (+/+) litter mates.
During this analysis, the mice were subject to a medical work-up
using an integrated suite of medical diagnostic procedures designed
to assess the function of the major organ systems in a mammalian
subject. Homozygous (-/-) "knockout" mice were studied in
conjunction with their heterozygous (+/-) and wild-type (+/+)
litter mates. Disruption of the AAK1 gene was confirmed by Southern
analysis. Expression of the murine homolog of AAK1 was detected by
RT-PCR in murine brain; spinal cord; eye; thymus; spleen; lung;
kidney; liver; skeletal muscle; bone; stomach, small intestine and
colon; heart; adipose; asthmatic lung; LPS liver; blood; banded
heart; aortic tree; prostate; and mammary gland (5 week virgin,
mature virgin, 12 DPC, 3 day post-partum (lactating), 3 day
post-weaning (early involution), and 7 day post-weaning (late
involution)).
[0111] AAK1 homozygous (-/-) and their wild-type (+/+) littermates
were tested using the formalin paw test in order to assess their
acute and tonic nociceptive responses. For these tests, Automatic
Nociception Analyzers (purchased from the Ozaki lab at University
of California, San Diego) were used. A metal band was placed around
the left hind paw of each mouse 30 minutes prior to testing. After
the 30-minute acclimation period, 20 .mu.l of 5% formalin is
subcutaneously injected in the dorsal surface of the left hind paw.
Mice were individually housed in cylindrical chambers for 45
minutes. A computer recorded flinches per minute, total flinches
for phase I (acute phase=first 8 minutes), and total flinches for
phase II (tonic phase=time between minutes 20-40) through an
electromagnetic field. See Yaksh T L, Ozaki G, McCumber D, Rathbun
M, Svensson C, Malkmus S, Yaksh M C. An automated flinch detecting
system for use in the formalin nociceptive bioassay. J Appl
Physiol., 2001; 90:2386-402.
[0112] As shown in FIG. 1, phase 1 and phase 2 data were obtained
using homozygous (-/-) mice females (n=16), wild-type females
(n=15), homozygous (-/-) mice males (n=9), and wild-type males
(n=18). In all groups and in both phases, the AAK1 homozygous (-/-)
mice exhibited significantly less recorded paw flinching than their
wild-type (+/+) littermates.
5.6.2. Synthesis of
[3-(4-Aminomethyl-phenyl)-pyrazolo[1,5-a]pyrimidin-5-yl]-butyl-amine
##STR00019##
[0113] Part A. Butyl-pyrazolo[1,5-a]pyrimidin-5-yl-amine
##STR00020##
[0115] A solution of 5-chloro-pyrazolo[1,5-a]pyrimidine [29274-24-]
(955.4 mg, 6.2 mmol) in butylamine [109-73-9] (6.2 mL, 62.7 mmol),
under N.sub.2 blanket, was magnetically stirred at 65.degree. C.
for 17 h then partitioned between brine (pH adjusted to 10 with
saturated aqueous sodium bicarbonate) and ethyl acetate. The phase
separated extract was dried (MgSO.sub.4) and diluted with heptane
to precipitate butyl-pyrazolo[1,5-a]pyrimidin-5-yl-amine as 1.2 g
of yellow powder, mp. 75-76.degree. C. .sup.1H NMR (400 MHz,
DMSO-d.sub.6) .delta. ppm 0.91 (t, J=7.33 Hz, 3H) 1.37 (dq,
J=15.00, 7.29 Hz, 2H) 1.47-1.59 (m, 2H) 3.24-3.34 (m, 2H) 5.94 (d,
J=1.52 Hz, 1H) 6.22 (d, J=7.58 Hz, 1H) 7.35 (br. s., 1H) 7.74 (d,
J=2.27 Hz, 1H) 8.40 (d, J=7.58 Hz, 1H). .sup.13C NMR (100 MHz,
DMSO-d.sub.6) .delta. ppm 13.69, 19.71, 30.65, 91.00, 99.95,
134.70, 143.45, 148.37, 155.43. LRMS (ESI) m/z 191.1 [(M+H)].sup.+,
calc'd for C.sub.10H.sub.14N.sub.4: 190.25
Part B. (3-Bromo-pyrazolo[1,5-a]pyrimidin-5-yl)-butyl-amine
##STR00021##
[0117] Sodium acetate [127-09-3] (0.7 g, 8.9 mmol) was added to a
solution of butyl-pyrazolo[1,5-a]pyrimidin-5-yl-amine (1.1 g, 5.9
mmol) in glacial acetic acid (100 mL) and allowed to stir at
ambient temperature until all of the solid had dissolved. Bromine
[7726-95-6] (0.3 mL, 6.4 mmol) was added drop by drop into the
ambient temperature, buffered, acetic acid reaction solution over 5
minutes. Upon completion of the bromine addition, the suspension
was allowed to stir for a further 15 minutes then was slowly poured
into 500 mL of stirred water. Saturated aqueous sodium bicarbonate
was added to the stirred suspension until pH of the supernatant was
determined to be approximately 8 by pH paper, then the suspension
was extracted with ethyl acetate. The extract was dried
(MgSO.sub.4) and flash chromatographed (silica gel eluted with 60%
(v/v) ethyl acetate/heptane) to provide 1.2 g of off white solid,
mp. 94-95.degree. C. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta.
ppm 0.93 (t, J=7.33 Hz, 3H) 1.31-1.44 (m, 2H) 1.56 (quin, J=7.20
Hz, 2H) 3.31-3.38 (m, 2H) 6.28 (d, J=7.58 Hz, 1H) 7.61 (t, J=5.05
Hz, 1H) 7.85 (s, 1H) 8.42 (d, J=7.58 Hz, 1H). .sup.13C NMR (100
MHz, DMSO-d.sub.6) .delta. ppm 13.67, 19.64, 30.53, 76.99, 100.87,
135.14, 143.03, 145.13, 156.09. LRMS (ESI) m/z 269.0/271.0
[(M+H)].sup.+, calc'd for C.sub.10H.sub.13BrN.sub.4: 269.15.
Part C.
[3-(4-Aminomethyl-phenyl)-pyrazolo[1,5-a]pyrimidin-5-yl]-butyl-ami-
ne
##STR00022##
[0119] To a mixture of
(3-bromo-pyrazolo[1,5-a]pyrimidin-5-yl)-butyl-amine (350.3 mg, 1.3
mmol), 4-aminomethylphenylboronic acid, hydrochloride [75705-21-4]
(337.1 mg, 1.8 mmol), potassium phosphate tribasic monohydrate
[27176-10-9] (601.6 mg, 2.6 mmol), and [1,1'-bis(diphenylphosphino)
ferrocene]dichloropalladium(II), complex with dichloromethane
[95464-05-4] (117.2 mg, 0.1 mmol) contained in a 50 mL round
bottomed flask was added a solution of 30% (v/v) water in
1,2-dimethoxyethane (25 mL) and a magnetic stir bar. The reaction
pot was fitted to a reflux condenser, lowered into an ambient
temperature oil bath, and the system taken through 10
evacuation/N.sub.2 blanket cycles while being rapidly stirred. The
rapidly stirred, N.sub.2 blanketed, reaction was heated to an oil
bath temperature of 85.degree. C. for 17 h then cooled and
partitioned between brine and ethyl acetate. The phase separated
extract was dried (MgSO.sub.4) and evaporated to afford a brown oil
which was purified by preparative RP-HPLC to yield 71.9 mg of
[3-(4-Aminomethyl-phenyl)-pyrazolo[1,5-a]pyrimidin-5-yl]-butyl-amine
monoacetate salt as a white solid, mp. 171-172.degree. C. .sup.1H
NMR (400 MHz, DMSO-d.sub.6) .delta. ppm 0.97 (t, J=7.39 Hz, 3H)
1.43 (sxt, J=7.36 Hz, 2H) 1.64 (quin, J=7.17 Hz, 2H) 3.43 (q,
J=6.47 Hz, 2H) 3.71-3.84 (m, 2H) 6.29 (d, J=7.50 Hz, 1H) 7.34 (m,
J=8.16 Hz, 2H) 7.62-7.71 (m, 1H) 8.05 (m, J=7.94 Hz, 2H) 8.32 (s,
1H) 8.46 (d, J=7.50 Hz, 1H). .sup.13C NMR (100 MHz, DMSO-d.sub.6)
.delta. ppm 13.03, 14.11, 19.63, 29.11, 30.33, 44.34, 45.62,
103.75, 124.02, 124.14, 126.95, 127.86, 131.85, 135.07, 137.50,
141.15, 141.36, 144.46, 155.50, 172.52. LRMS (ESI) m/z 296.1
[(M+H)].sup.+, calc'd for C.sub.17H.sub.21N.sub.5: 295.39.
5.6.3. Synthesis of
(2-Methoxy-ethyl)-{3-[4-(1H-tetrazol-5-yl)-phenyl]-pyrazolo[1,5-a]pyrimid-
in-5-yl}-amine
##STR00023##
[0120] Part A.
N-(2-methoxyethyl)pyrazolo[1,5-a]pyrimidin-5-amine
##STR00024##
[0122] A stirred mixture of 5-chloro-pyrazolo[1,5-a]pyrimidine
[29274-24-6], (525.5 mg, 3.4 mmol) and 2-methoxy-ethylamine (3.0
mL, 34.5 mmol) was heated to 65.degree. C. for 17 h, cooled and
partitioned between brine (pH adjusted to 8 with saturated aqueous
sodium bicarbonate) and ethyl acetate. Extract was dried
(MgSO.sub.4) and evaporated to yield 706.4 mg of light yellow
solid. LRMS (ESI) m/z 193.1 [(M+H)].sup.+, calc'd for
C.sub.9H.sub.12N.sub.4O: 192.22.
Part B.
(3-Bromo-pyrazolo[1,5-a]pyrimidin-5-yl)-(2-methoxy-ethyl)-amine
##STR00025##
[0124] Sodium acetate [127-09-3] (457.6 mg, 5.6 mmol) was added to
a solution of N-(2-methoxyethyl)pyrazolo[1,5-a]pyrimidin-5-amine
(706.4 mg, 3.7 mmol) in glacial acetic acid (40 mL) and allowed to
stir at ambient temperature until all of the solid had dissolved.
Bromine [7726-95-6] (0.2 mL, 3.7 mmol) was added into the ambient
temperature, buffered acetic acid reaction solution over 2 minutes,
then the reaction solution was slowly poured into stirring water (1
L). The pH was increased by the addition of solid sodium
bicarbonate until the pH of the supernatant was determined to be
approximately 8 by pH paper. Precipitated product was isolated by
filtration, the filter cake washed with water, and allowed to dry
to afford 819.6 mg of white powder. LRMS (ESI) m/z 271.0/273.0
[(M+H)].sup.+, calc'd for C.sub.9H.sub.11BrN.sub.4O: 271.12.
Part C.
(2-Methoxy-ethyl)-{3-[4-(1H-tetrazol-5-yl)-phenyl]-pyrazolo[1,5-a]-
pyrimidin-5-yl}-amine
##STR00026##
[0126] To a mixture of
(3-bromo-pyrazolo[1,5-a]pyrimidin-5-yl)-(2-methoxy-ethyl)-amine
(307.7 mg, 1.1 mmol), (4-(1H-tetrazol-5-yl)phenyl)boronic acid
[179942-55-3] (259.6 mg, 1.4 mmol), potassium phosphate tribasic
monohydrate [27176-10-9] (522.4 mg, 2.3 mmol), and
[1,1'-bis(diphenylphosphino)ferrocene]dichloro-palladium(II),
complex with dichloromethane [95464-05-4] (106.3 mg, 0.1 mmol)
contained in a 50 mL round bottomed flask was added a solution of
30% (v/v) water in 1,2-dimethoxyethane (12 mL) and a magnetic stir
bar. The reaction pot was fitted to a reflux condenser, lowered
into an ambient temperature oil bath, and the system taken through
10 evacuation/N.sub.2 blanket cycles while being rapidly stirred.
The rapidly stirred, N.sub.2 blanketed, reaction was heated to an
oil bath temperature of 85.degree. C. for 4 h then cooled, diluted
with methanol, and gravity filtered. The filtrate was preabsorbed
on silica gel and flash chromatographed (silica gel eluted with 1%
NH.sub.4OH in 10% (v/v) methanol/ethyl acetate). Desired
chromatography fractions were combined, evaporated, redissolved in
2-propanol and gravity filtered into stirring heptane to
precipitate purified product.
(2-Methoxy-ethyl)-{3-[4-(1H-tetrazol-5-yl)-phenyl]-pyrazolo[1,5-a]pyrimid-
in-5-yl}-amine was isolated by filtration as 96.3 mg of solid
material, mp. 264-265.degree. C. (dec.). .sup.1H NMR (400 MHz,
DMSO-d.sub.6) .delta. ppm 3.63 (t, J=4.29 Hz, 5H) 6.41 (d, J=7.58
Hz, 1H) 7.88 (br. s., 1H) 8.04 (m, J=8.59 Hz, 2H) 8.31 (m, J=8.34
Hz, 2H) 8.47 (s, 1H) 8.53 (d, J=7.58 Hz, 1H). .sup.13C NMR (100
MHz, DMSO-d.sub.6) .delta. ppm 40.69, 58.48, 70.48, 100.86, 103.75,
119.86, 125.23, 127.61, 135.97, 137.08, 142.42, 145.63, 156.60,
LRMS (ESI) m/z 337.2 [(M+H)].sup.+, calc'd for
C.sub.16H.sub.16N.sub.8O: 336.36.
5.6.4. Synthesis of
Methyl-[2-(3-thiophen-2-yl-pyrazolo[1,5-a]pyrimidin-5-ylamino)-ethyl]-car-
bamic acid tert-butyl ester
##STR00027##
[0127] Part A. 3-Oxo-2-thiophen-2-yl-propionitrile
##STR00028##
[0129] Thiophen-2-yl-acetonitrile [20893-30-5] (6.7 mL, 62.6 mmol)
was added to a N.sub.2 blanketed, 0.degree. C., stirred solution of
21 wt % sodium ethoxide (25.7 mL, 68.9 mmol) in ethanol (100 mL).
After 5 minutes, ethyl formate [109-94-4] (12.6 mL, 156.7 mmol) was
added and the resultant solution was stirred for 20 minutes. The
ice bath was then removed and replaced with a heating mantle. The
reaction was heated to reflux for 2 h, cooled, and evaporated to
provide a yellow solid. Crude product was dissolved in water, the
solution chilled to 0.degree. C. and acidified with aqueous
hydrochloric acid to precipitate an off white powder. Product was
isolated by filtration, washed with water, and dried to afford 7.4
g. LRMS (ESI) m/z 152.0 [(M+H)].sup.+, calc'd for
C.sub.7H.sub.6NOS: 151.19
Part B. 4-Thiophen-2-yl-2H-pyrazol-3-ylamine
##STR00029##
[0131] Hydrazine hydrate [7803-57-8] (4.8 mL, 99.0 mmol) then
acetic acid [64-19-7] (5.6 mL, 97.8 mmol) were added to a solution
of 3-oxo-2-thiophen-2-yl-propionitrile (7.4 g, 49.0 mmol) in
ethanol (100 mL). The solution was then heated to reflux for 2 h.
The cooled reaction was evaporated to dryness and the resultant
solid triturated in water. Product was isolated by filtration,
washed with water, and dried to provide
4-thiophen-2-yl-2H-pyrazol-3-ylamine as 7.1 g of fine off white
powder, mp. 183-184.degree. C. .sup.1H NMR (400 MHz, DMSO-d.sub.6)
.delta. ppm 2.51 (s, 1H) 7.01-7.10 (m, 2H) 7.27 (dd, J=5.05, 1.01
Hz, 1H). .sup.13C NMR (100 MHz, DMSO-d.sub.6) .delta. ppm 121.07,
121.47, 127.58, 135.85. LRMS (ESI) m/z 166.0 [(M+H)].sup.+, calc'd
for C.sub.7H.sub.7N.sub.3S: 165.22.
Part C. 3-Thiophen-2-yl-4H-pyrazolo[1,5-a]pyrimidin-5-one
##STR00030##
[0133] A 21 wt % solution of sodium ethoxide in ethanol (32.7 mL,
87.6 mmol) was slowly added to an ambient temperature, stirred
suspension of 4-thiophen-2-yl-2H-pyrazol-3-ylamine (4.1 g, 25.0
mmol) and 1,3-dimethylpyrimidine-2,4(1H,3H)-dione [874-14-6] (3.9
g, 27.5 mmol) in ethanol (217 mL). As addition progressed the solid
dissolved to give a clear brown solution. The solution was heated
to reflux for 2 d, cooled, then poured into 5% (w/v) aqueous
ammonium chloride. Ethanol was evaporated out and the reduced
volume product solution chilled, with stirring, in an ice bath to
precipitate 3-thiophen-2-yl-4H-pyrazolo[1,5-a]pyrimidin-5-one as
3.2 g of red-brown fine crystalline powder, mp. 231-232.degree. C.
.sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. ppm 6.13 (br. s., 1H)
7.10 (dd, J=5.05, 3.54 Hz, 1H) 7.38-7.46 (m, 2H) 8.07 (br. s., 1H)
8.51-8.73 (m, 1H) 12.06 (br. s., 1H). .sup.13C NMR (100 MHz,
DMSO-d.sub.6) .delta. ppm 123.66, 127.71, 138.70, 142.47. LRMS
(ESI) m/z 218.0 [(M+H)].sup.+, calc'd for C.sub.10H.sub.7N.sub.3OS:
217.25.
Part D.
Methyl-[2-(3-thiophen-2-yl-pyrazolo[1,5-a]pyrimidin-5-ylamino)-eth-
yl]-carbamic acid tert-butyl ester
##STR00031##
[0135] 1,8-Diazabicycloundec-7-ene (DBU) [6674-22-2] (0.4 mL, 2.5
mmol) was added to an ambient temperature, stirred, turbid solution
of 3-thiophen-2-yl-4H-pyrazolo[1,5-a]pyrimidin-5-one (354.3 mg, 1.6
mmol), (2-amino-ethyl)-methyl-carbamic acid tert-butyl ester
[121492-06-6] (852.2 mg, 4.9 mmol), and
(benzotriazol-1-yloxy)tris(dimethylamino)phosphonium
hexafluorophosphate (BOP) [56602-33-6] (937.7 mg, 2.1 mmol) in
acetonitrile (16 mL). Upon addition of the DBU the turbidity clears
and the solution was permitted to stir for 2 d then partitioned
between brine and ethyl acetate. The extract was dried (MgSO.sub.4)
and flash chromatographed (silica gel, eluted with 10% (v/v)
methanol/ethyl acetate). Evaporation of product fractions yielded
an oil which was crystallized from heptane to provide
methyl-[2-(3-thiophen-2-yl-pyrazolo[1,5-a]pyrimidin-5-ylamino)-ethyl]-car-
bamic acid tert-butyl ester as 333.6 mg of yellow, fine crystalline
powder, mp. 140-141.degree. C. .sup.1H NMR (400 MHz, DMSO-d.sub.6)
.delta. ppm (rotomers present) 1.16-1.28 (m, 5H) 1.37 (br. s., 3H)
2.51 (s, 1H) 2.85 (br. s., 3H) 3.47 (br. s., 1H) 3.55 (br. s., 3H)
6.28 (d, J=6.82 Hz, 1H) 7.04 (dd, J=5.05, 3.54 Hz, 1H) 7.30 (dd,
J=5.05, 1.01 Hz, 1H) 7.41 (br. s., 1H) 7.80 (br. s., 1H) 8.19 (s,
1H) 8.48 (d, J=7.07 Hz, 1H). .sup.13C NMR (100 MHz, DMSO-d.sub.6)
.delta. ppm 27.81, 27.96, 33.75, 38.41, 46.32, 78.13, 100.28,
100.76, 120.16, 121.75, 126.90, 135.01, 135.20, 140.78, 143.74,
154.91, 155.81. LRMS (ESI) m/z 374.1 [(M+H)].sup.+, calc'd for
C.sub.18H.sub.23N.sub.5O.sub.2S: 373.48.
5.6.5. Synthesis of Isopropyl
(2-((3-(3-methoxypyridin-4-yl)pyrazolo[1,5-a]pyrimidin-5-yl)amino)ethyl)(-
methyl)-carbamate
##STR00032##
[0136] Part A. 3-Bromo-5-chloro-pyrazolo[1,5-a]pyrimidine
##STR00033##
[0138] Sodium acetate [127-09-3] (24.1 g, 293.9 mmol) was added to
a solution of 5-chloro-pyrazolo[1,5-a]pyrimidine [29274-24-6],
(30.1 g, 195.7 mmol) in glacial acetic acid (395 mL) and allowed to
stir at ambient temperature until all of the solid had dissolved.
An ice bath was then put in place, and the solution chilled to
0.degree. C. Bromine [7726-95-6] (11.0 mL, 214.7 mmol) was added
drop by drop into the 0.degree. C. buffered acetic acid reaction
solution over 35 minutes which gradually became a thick stirred
suspension. Upon completion of the bromine addition, the suspension
was allowed to stir for a further 15 minutes then was slowly poured
into an aqueous (5% w/v) solution (1 L) of sodium metabisulfite
[7681-57-4] stirred in an ice bath. Solid sodium hydroxide
[1310-73-2] pellets were added to the stirred suspension, at a rate
to maintain an internal temperature of less than 40.degree. C.,
until pH of the supernatant was determined to be approximately 8 by
pH paper. Precipitated product was isolated by filtration, the
filter cake washed with water, and allowed to dry to afford 39.8 g
of light yellow powder, mp. 173-174.degree. C. .sup.1H NMR (400
MHz, DMSO-d.sub.6) .delta. ppm 7.22 (d, J=7.33 Hz, 1H) 8.44 (s, 1H)
9.21 (d, J=7.07 Hz, 1H). .sup.13C NMR (100 MHz, DMSO-d.sub.6)
.delta. ppm 82.84, 109.99, 138.94, 143.76, 145.61, 151.18. LRMS
(ESI) m/z 231.8/233.8/235.8 [(M+H)].sup.+, calc'd for
C.sub.6H.sub.3BrClN.sub.3: 232.47.
Part B. tert-butyl (2-((3-bromopyrazolo
[1,5-a]pyrimidin-5-yl)amino)ethyl)(methyl)carbamate
##STR00034##
[0140] To 465 mg (2.00 mmol) of
3-bromo-5-chloropyrazolo[1,5-a]pyrimidine was added tert-butyl
(2-aminoethyl)(methyl)carbamate (522 mg, 3.00 mmol), 4 mL of
isopropanol, and triethylamine (0.56 mL, 4.00 mmol). The resulting
mixture is heated at 140.degree. C. for 0.5 hr in the microwave. It
was diluted with EtOAc, and washed with water and brine. The
organic layer was dried over MgSO.sub.4, concentrated and purified
on the silica gel column eluting with 20-100% EtOAc/Hex to obtain
607 mg (82%) of the desired product.
Part C.
N1-(3-bromopyrazolo[1,5-a]pyrimidin-5-yl)-N2-methylethane-1,2-diam-
ine
##STR00035##
[0142] The tert-butoxycarbonyl was removed by dissolving 600 mg
(1.62 mmol) of tert-butyl
(2-((3-bromopyrazolo[1,5-a]pyrimidin-5-yl)amino)ethyl)(methyl)carbamate
in 4 mL of DCM, and then adding 4 mL TFA, stirring for 0.5 hr at
rt, and concentrating the mixture to afford the TFA-salt of the
desired product in 100% yield (622 mg).
Part D. Isopropyl
(2-((3-bromopyrazolo[1,5-a]pyrimidin-5-yl)amino)ethyl)(methyl)carbamate
##STR00036##
[0144] To 1.0 equivalent (384 mg, 1.000 mmol) of the amine (TFA
salt) dissolved 20 mL of DCM was added 3.0 equivalents (368 mg,
3.000 mmol) of isopropylchloroformate, 4.0 equivalents
triethylamine (404 mg, 4.000 mmol). The resulting mixture was
stirred at rt for 0.5 hr, and then diluted with DCM (30 mL) washed
with water and brine, dried over MgSO.sub.4, and concentrated. The
crude residue was purified in the silica gel (ISCO) eluting with
1-10% MeOH/DCM to obtain the desired product (87% yield).
Part E. Isopropyl
(2-((3-(3-methoxypyridin-4-yl)pyrazolo[1,5-a]pyrimidin-5-yl)amino)ethyl)(-
methyl)-carbamate
##STR00037##
[0146] To 80 mg (0.22 mmol) of isopropyl
(2-((3-bromopyrazolo[1,5-a]pyrimidin-5-yl)amino)ethyl)(methyl)-carbamate
was added (3-methoxypyridin-4-yl)boronic acid dihydrate (56 mg,
0.27 mmol), K.sub.3PO.sub.4 (143 mg, 0.67 mmol),
PdCl.sub.2(PPh.sub.3).sub.2 (16.8 mg, 0.022 mmol), 3 mL of DME and
1 mL of water. This mixture was microwaved at 140.degree. C. for
0.5 hr. After cooling, it was diluted with EtOAc, and washed with
water and brine. The organic layer was dried over MgSO.sub.4,
concentrated, and purified on the PREP HPLC to afford the desired
product in 77% yield (66.4 mg). .sup.1H NMR (400 MHz, Acetone) 8
ppm 2.87 (br. s., 3H) 2.99 (br. s., 3H) 3.58-3.71 (m, 2H) 3.73-3.82
(m, 2H) 4.09 (s, 3H) 4.82 (td, J=6.25, 2.91 Hz, 1H) 6.45 (br. s.,
1H) 8.21 (br. s., 1H) 8.35 (s, 1H) 8.43 (d, J=6.06 Hz, 1H) 8.58 (s,
1H) 8.77 (br. s., 1H); LRMS (ESI) m/e 385.0 [(M+H).sup.+, calcd for
C.sub.19H.sub.24N.sub.6O.sub.3 384.0].
5.6.6. Synthesis of (S)-tert-butyl
2-(((3-(4-(aminomethyl)phenyl)pyrazolo[1,5-a]pyrimidin-5-yl)amino)methyl)-
pyrrolidine-1-carboxylate
Part A. (S)-tert-butyl
2-(((3-bromopyrazolo[1,5-a]pyrimidin-5-yl)amino)methyl)pyrrolidine-1-carb-
oxylate
##STR00038##
[0148] The reaction of 3-bromo-5-chloropyrazolo[1,5-a]pyrimidine
and (S)-tert-butyl 2-(aminomethyl)pyrrol-idine-1-carboxylate under
the amine displacement conditions described in example 5.6.5
afforded 74% product.
Part B. (S)-tert-butyl
2-(((3-(4-(aminomethyl)phenyl)pyrazolo[1,5-a]pyrimidin-5-yl)amino)methyl)-
-pyrrolidine-1-carboxylate
##STR00039##
[0150] The Suzuki coupling of (5)-tert-butyl
2-(((3-bromopyrazolo[1,5-a]pyrimidin-5-yl)amino)methyl)pyrrolidine-1-carb-
oxylate with (4-(aminomethyl)phenyl)boronic acid under the same
conditions as described in example 5.6.5 to give 73% of the titled
compound. .sup.1H NMR (400 MHz, METHANOL-d.sub.4) .delta. ppm 1.44
(br. s., 9H) 1.90 (dd, J=9.92, 5.51 Hz, 1H) 1.96-2.11 (m, 3H)
3.35-3.45 (m, 4H) 4.07-4.21 (m, 3H) 6.33 (br. s., 1H) 7.46 (d,
J=8.38 Hz, 2H) 8.19 (br. s., 2H) 8.25-8.36 (m, 2H); LRMS (ESI) m/e
423.0 [(M+H).sup.+, calcd for C.sub.23H.sub.30N.sub.6O.sub.2
422.0].
5.6.7. Synthesis of (S)-tert-butyl
2-(((3-(2-methoxyphenyl)pyrazolo[1,5-a]pyrimidin-5-yl)amino)methyl)pyrrol-
idine-1-carboxylate
##STR00040##
[0152] The Suzuki coupling of (5)-tert-butyl
2-(((3-bromopyrazolo[1,5-a]pyrimidin-5-yl)amino)methyl)pyrrolidine-1-carb-
oxylate with (2-methoxyphenyl)boronic acid under the same
conditions as described in example 5.6.5 to give 68% of the titled
compound. .sup.1H NMR (400 MHz, CHLOROFORM-d) .delta. ppm 1.50 (s,
9H) 1.85-2.12 (m, 4H) 3.40 (dd, J=7.58, 4.04 Hz, 2H) 3.46-3.59 (m,
1H) 3.59-3.67 (m, 1H) 3.94 (s, 3H) 4.19 (br. s., 1H) 6.07 (d,
J=7.07 Hz, 1H) 6.99 (d, J=7.58 Hz, 1H) 7.06 (td, J=7.45, 1.01 Hz,
1H) 7.15-7.22 (m, 1H) 8.14-8.28 (m, 1H) 8.45 (d, J=6.82 Hz, 1H)
8.51 (s, 1H); LRMS (ESI) m/e 424.0 [(M+H).sup.+, calcd for
C.sub.23H.sub.29N.sub.5O.sub.3 423.0].
5.6.8. Synthesis of (5)-tert-butyl
2-(((3-(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-5-yl)amino)methyl)pyrrol-
idine-1-carboxylate
##STR00041##
[0153] Part A. (S)-tert-butyl
2-((pyrazolo[1,5-a]pyrimidin-5-ylamino)methyl)pyrrolidine-1-carboxylate
##STR00042##
[0155] The commercially available 5-chloropyrazolo[1,5-a]pyrimidine
is reacted with (S)-tert-butyl
2-(aminomethyl)pyrrolidine-1-carboxylate under the amine
displacement conditions described in example 5.6.5 to give 96%
product.
Part B. (S)-tert-butyl
2-(((3-iodopyrazolo[1,5-a]pyrimidin-5-yl)amino)methyl)pyrrolidine-1-carbo-
xylate
##STR00043##
[0157] The (S)-tert-butyl
2-((pyrazolo[1,5-a]pyrimidin-5-ylamino)methyl)pyrrolidine-1-carboxylate
(790 mg, 2.490 mmol) was dissolved in 10 mL of DMF, was added NIS
(617 mg, 2.740 mmol). This mixture is stirred for 4 hr at rt, and
then quenched with 10 mL of water. It was then diluted with 50 mL
EtOAc and the organic layer was separated, washed twice with brine,
and dried over MgSO.sub.4. It was concentrated and purified on the
silica gel eluting with 20-100% EtOAc/hex to obtain 1.03 g (93%) an
off-white solid.
Part C. (S)-isopropyl
2-(((3-(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-5-yl)amino)methyl)pyrrol-
idine-1-carboxylate
##STR00044##
[0159] To 221.5 mg (0.50 mmol) of the aryl iodide dissolved in 2 mL
DMF was added methyl 2,2-difluoro-2-(fluorosulfonyl)acetate (0.77
g, 4.00 mmol), CuI (762 mg, 2.00 mmol), HMPA (0.41 mL, 3.50 mmol).
This mixture was stirred on an oil bath preheated to 90.degree. C.
for 0.75 hr. The reaction was cooled to rt, and quenched with
NH.sub.4Cl saturated aqueous solution. Extracted with 30 mL of
EtOAc, washed with brine and dry over MgSO.sub.4. It was
concentrated and purified on the PREP HPLC to obtain 81 mg (42%) of
the desired product. .sup.1H NMR (400 MHz, METHANOL-d.sub.4)
.delta. ppm 1.44 (s, 9H) 1.86-1.99 (m, 3H) 1.99-2.07 (m, 1H) 3.40
(br. s., 3H) 3.61 (br. s., 1H) 4.11 (br. s., 1H) 6.37 (br. s., 1H)
7.98 (s, 1H) 8.33 (br. s., 1H); LRMS (ESI) m/e 386.0 [(M+H).sup.+,
calcd for C.sub.17H.sub.22F.sub.3N.sub.6O.sub.2 385.0].
5.6.9. Synthesis of
4-[3-(3-Methoxy-pyridin-4-yl)-pyrazolo[1,5-a]pyrimidin-5-yl]-piperazine-1-
-carboxylic acid isopropyl ester
##STR00045##
[0160] Part A.
3-Bromo-5-piperazin-1-yl-pyrazolo[1,5-a]pyrimidine
##STR00046##
[0162] 3-Bromo-5-chloro-pyrazolo[1,5-a]pyrimidine (15.4 g, 66.2
mmol) and piperazine [110-85-0] (56.8 g, 659.5 mmol) were ground
together in a mortar to an intimate mixture and transferred to a
250 mL round bottomed flask containing a magnetic stir bar. The
flask was fitted to a reflux condenser, N.sub.2 blanketed, and the
reaction pot immersed in an ambient temperature oil bath. While the
neat solid mixture stirred, the bath was heated to 120.degree. C.
over 0.5 h and held at nominal temperature for a total of 2 h. The
bath was removed and the molten reaction allowed to cool to
approximately 75.degree. C. Ethyl acetate was cautiously added down
the condenser to dissolve the reaction product to prevent its
setting up into a solid mass. The reaction solution was transferred
to a separatory funnel, further diluted with ethyl acetate, and
washed with water. The ethyl acetate extract was dried (MgSO.sub.4)
and evaporated to obtain 16.1 g of clear yellow oil. The oil was
crystallized from methanol to provide 15.8 g of light yellow
crystalline powder in two crops, mp. 108-109.degree. C. .sup.1H NMR
(400 MHz, DMSO-d.sub.6) .delta. ppm 2.51 (s, 1H) 3.16-3.24 (m, 3H)
3.38 (s, 1H) 3.93-4.00 (m, 3H) 6.85 (d, J=7.83 Hz, 1H) 8.03 (s, 1H)
8.78 (d, J=8.08 Hz, 1H) 9.61 (br. s., 1H). .sup.13C NMR (100 MHz,
DMSO-d.sub.6) .delta. ppm 41.25, 42.16, 77.73, 97.78, 136.77,
144.01, 144.18, 155.53. LRMS (ESI) m/z 282.0/284.0 [(M+H)].sup.+,
calc'd for C.sub.10H.sub.12BrN.sub.5: 282.14.
Part B.
4-(3-Bromo-pyrazolo[1,5-a]pyrimidin-5-yl)-piperazine-1-carboxylic
acid isopropyl ester
##STR00047##
[0164] To a rapidly stirred, 0.degree. C., N.sub.2 blanketed,
solution of 3-bromo-5-piperazin-1-yl-pyrazolo[1,5-a]pyrimidine
(15.8 g, 56.0 mmol) and Hunig's base [7087-68-5] (20.0 mL, 114.8
mmol) in ethyl acetate (280 mL) was steadily added a 1.0M solution
of isopropyl chloroformate in toluene (67.0 mL) over the course of
10 minutes. The reaction was allowed to stir and warm to ambient
temperature over night at which time it was washed with brine,
dried (MgSO.sub.4), preloaded onto silica gel, chromatographed
(Silica gel, eluted with 100% ethyl acetate) and crystallized from
ethyl acetate/heptane to afford 13.2 g of white powder, mp.
78-79.degree. C. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. ppm
1.22 (d, J=6.32 Hz, 6H) 3.44-3.54 (m, 4H) 3.67-3.79 (m, 4H) 4.82
(spt, J=6.23 Hz, 1H) 6.77 (d, J=7.83 Hz, 1H) 7.97 (s, 1H) 8.69 (d,
J=7.83 Hz, 1H). .sup.13C NMR (100 MHz, DMSO-d.sub.6) .delta. ppm
21.94, 42.80, 43.87, 68.17, 77.32, 97.66, 136.47, 143.87, 144.40,
154.29, 155.64. LRMS (ESI) m/z 368.0/370.0 [(M+H)].sup.+, calc'd
for C.sub.14H.sub.18BrN.sub.5O.sub.2: 368.24.
Part C.
4-[3-(3-Methoxy-pyridin-4-yl)-pyrazolo[1,5-a]pyrimidin-5-yl]-piper-
azine-1-carboxylic acid isopropyl ester
##STR00048##
[0166] To a mixture of
4-(3-bromo-pyrazolo[1,5-a]pyrimidin-5-yl)-piperazine-1-carboxylic
acid isopropyl ester (385.0 mg, 1.1 mmol),
3-methoxypyridine-4-boronic acid hydrate [1072952-50-1] (229.7 mg,
1.34 mmol), potassium phosphate tribasic monohydrate [27176-10-9]
(516.3 mg, 2.2 mmol), and
[1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II),
complex with dichloromethane [95464-05-4] (102.4 mg, 0.1 mmol)
contained in a 50 mL round bottomed flask was added a solution of
30% (v/v) water in 1,2-dimethoxyethane (25 mL) and a magnetic stir
bar. The reaction pot was fitted to a reflux condenser, lowered
into an ambient temperature oil bath, and the system taken through
10 evacuation/N.sub.2 blanket cycles while being rapidly stirred.
The rapidly stirred, N.sub.2 blanketed, reaction was heated to an
oil bath temperature of 85.degree. C. for 17 h then cooled and
partitioned between brine (pH adjusted to 10 with 3N aqueous sodium
hydroxide) and ethyl acetate. The phase separated extract was dried
(MgSO.sub.4) and evaporated to afford an orange oil which was
purified by preparative RP-HPLC. Isolated product was dissolved in
methanol, diluted with concentrated hydrochloric acid, evaporated
to dryness, redissolved in methanol and precipitated by the
addition of diethyl ether. Filtration of the precipitate yielded
100.5 mg of the product monohydrochloride salt as a yellow powder.
mp. 206-207.degree. C. (dec.).sup.1H NMR (400 MHz, DMSO-d.sub.6)
.delta. ppm 1.23 (d, J=6.39 Hz, 6H) 2.51 (s, 2H) 3.53-3.59 (m, 4H)
3.85 (br. s., 3H) 4.13 (s, 3H) 4.84 (spt, J=6.25 Hz, 1H) 6.96 (d,
J=7.94 Hz, 1H) 8.42 (d, J=6.39 Hz, 1H) 8.48 (s, 1H) 8.76 (s, 1H)
8.86 (d, J=7.94 Hz, 1H) 9.06 (d, J=6.17 Hz, 1H). .sup.13C NMR (100
MHz, DMSO-d.sub.6) .delta. ppm 21.97, 42.79, 44.23, 57.17, 68.25,
97.56, 98.39, 120.03, 123.90, 137.27, 137.05, 137.95, 146.48,
147.00, 151.80, 154.26, 156.66. LRMS (ESI) m/z 397.1 [(M+H)].sup.+,
calc'd for C.sub.20H.sub.24N.sub.6O.sub.3: 396.45.
5.6.10. Synthesis of
4-[3-(2-Methoxy-phenyl)-pyrazolo[1,5-a]pyrimidin-5-yl]-piperazine-1-carbo-
xylic acid isopropyl ester
##STR00049##
[0168] Prepared similarly to the preparation of example 5.6.9 from
4-(3-bromo-pyrazolo[1,5-a]pyrimidin-5-yl)-piperazine-1-carboxylic
acid isopropyl ester (385.0 mg, 1.1 mmol) and
2-methoxyphenylboronic acid [5720-06-9] (191.8 mg, 1.3 mmol) to
afford 76.4 mg of yellow powder as a free base, mp. 57-59.degree.
C. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. ppm 1.22 (d, J=6.32
Hz, 6H) 3.48-3.54 (m, 4H) 3.68-3.77 (m, 4H) 3.87 (s, 3H) 4.77-4.85
(m, 1H) 6.76 (d, J=7.83 Hz, 1H) 6.94-7.07 (m, 2H) 7.10-7.18 (m, 1H)
8.38 (dd, J=7.71, 1.64 Hz, 1H) 8.43 (s, 1H) 8.71 (d, J=8.08 Hz,
1H). .sup.13C NMR (100 MHz, DMSO-d.sub.6) .delta. ppm 21.95, 44.07,
55.32, 68.14, 96.93, 101.10, 111.29, 120.48, 121.64, 125.83,
127.67, 136.28, 145.00, 154.31, 155.25, 155.51. LRMS (ESI) m/z
396.1 [(M+H)].sup.+, calc'd for C.sub.21H.sub.25N.sub.5O.sub.3:
395.47.
5.6.11. Synthesis of (S)-tert-butyl
2-(((3-(2-ethylphenyl)pyrazolo[1,5-a]pyrimidin-5-yl)amino)methyl)pyrrolid-
ine-1-carboxylate
##STR00050##
[0170] The Suzuki coupling of (5)-tert-butyl
2-(((3-bromopyrazolo[1,5-a]pyrimidin-5-yl)amino)methyl)pyrrolidine-1-carb-
oxylate with (2-ethylphenyl)boronic acid under the same conditions
as described in example 5.6.5 to give 77% of the titled compound.
.sup.1H NMR (400 MHz, CHLOROFORM-d) .delta. ppm 1.20 (t, J=7.58 Hz,
3H) 1.49 (s, 9H) 1.79-1.95 (m, 3H) 1.95-2.05 (m, 1H) 2.82 (q,
J=7.58 Hz, 2H) 3.34-3.44 (m, 3H) 3.44-3.53 (m, 1H) 4.15 (br. s.,
1H) 6.08 (d, J=6.82 Hz, 1H) 7.23-7.31 (m, 3H) 7.32-7.38 (m, 1H)
7.51 (dd, J=7.20, 1.39 Hz, 1H) 7.91 (s, 1H) 8.20 (d, J=6.32 Hz,
1H); LRMS (ESI) m/e 422.0 [(M+H).sup.+, calcd for
C.sub.24H.sub.31N.sub.5O.sub.2 421.0].
5.6.12. Synthesis of (5)-tert-butyl 2-(((3-(2-[D3]methoxy
phenyl)pyrazolo[1,5-a]pyrimidin-5-yl)amino)methyl)pyrrolidine-1-carboxyla-
te
##STR00051##
[0171] Part A.
2-(2-[D3]methoxyphenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane
##STR00052##
[0173] To the 2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol
(100 mg, 0.455 mmol) dissolved in 6 mL of DMSO was added deuterated
methyl iodine (132 mg, 0.909 mmol) followed by 60% NaH (22 mg,
0.909 mmol). The resulting mixture was stirred overnight at rt.
Dilute with 25 mL EtOAc, and wash with water and brine. Dry over
MgSO.sub.4 and concentrate to give 108 mg (100%) of the ether
product.
Part B. (S)-tert-butyl 2-(((3-(2-[D3]methoxy
phenyl)pyrazolo[1,5-a]pyrimidin-5-yl)amino)methyl)-pyrrolidine-1-carboxyl-
ate
##STR00053##
[0175] The reaction of the 2-(2-[D3]methoxy
phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane with (S)-tert-butyl
2-(((3-bromopyrazolo[1,5-a]pyrimidin-5-yl)amino)methyl)pyrrolidine-1-carb-
oxylate under Suzuki conditions described in example 5.6.5 afforded
the titled compound in 70% yield. .sup.1H NMR (400 MHz,
CHLOROFORM-d) .delta. ppm 1.51 (s, 9H) 1.83-2.11 (m, 4H) 3.40 (dd,
J=7.45, 3.92 Hz, 2H) 3.47-3.59 (m, 1H) 3.59-3.66 (m, 1H) 4.20 (br.
s., 1H) 6.07 (d, J=7.07 Hz, 1H) 6.96-7.02 (m, 1H) 7.06 (td, J=7.52,
1.14 Hz, 1H) 7.15-7.24 (m, 1H) 8.19 (d, J=6.32 Hz, 1H) 8.46 (d,
J=6.82 Hz, 1H) 8.51 (s, 1H); LRMS (ESI) m/e 427.0 [(M+H).sup.+,
calcd for C.sub.23H.sub.26D.sub.3N.sub.5O.sub.3 426.0].
5.6.13. Synthesis of
4-[3-(2-Methoxy-pyridin-3-yl)-pyrazolo[1,5-a]pyrimidin-5-yl]-piperazine-1-
-carboxylic acid isopropyl ester
##STR00054##
[0177] Prepared similarly to the preparation of example 5.6.9 from
4-(3-bromo-pyrazolo[1,5-a]pyrimidin-5-yl)-piperazine-1-carboxylic
acid isopropyl ester (338.7 mg, 0.9 mmol) and
2-methoxy-3-pyridineboronic acid [163105-90-6] (169.4 mg, 1.1 mmol)
to afford 76.0 mg of yellow powder as a free base, mp.
131-132.degree. C. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. ppm
1.22 (d, J=6.06 Hz, 6H) 3.47-3.56 (m, 4H) 3.71-3.80 (m, 4H) 4.00
(s, 3H) 4.82 (spt, J=6.23 Hz, 1H) 6.81 (d, J=8.08 Hz, 1H) 7.08 (dd,
J=7.45, 4.93 Hz, 1H) 7.98 (dd, J=4.93, 1.89 Hz, 1H) 8.51 (s, 1H)
8.76 (d, J=7.83 Hz, 1H) 8.82 (dd, J=7.58, 2.02 Hz, 1H). .sup.13C
NMR (100 MHz, DMSO-d.sub.6) .delta. ppm 21.95, 42.85, 44.06, 53.10,
68.16, 97.21, 98.90, 116.40, 117.17, 134.55, 136.43, 141.99,
144.65, 144.71, 154.26, 155.46, 158.89. LRMS (ESI) m/z 397.1
[(M+H)].sup.+, calc'd for C.sub.20H.sub.24N.sub.6O.sub.3:
396.45.
5.6.14. Synthesis of
4-[3-(1-Methyl-2-oxo-1,2-dihydro-pyridin-3-yl)-pyrazolo[1,5-a]pyrimidin-5-
-yl]-piperazine-1-carboxylic acid isopropyl ester
##STR00055##
[0179] Prepared similarly to the preparation of example 5.6.9 from
4-(3-bromo-pyrazolo[1,5-a]pyrimidin-5-yl)-piperazine-1-carboxylic
acid isopropyl ester and
(1-methyl-2-oxo-1,2-dihydropyridin-3-yl)boronic acid to provide a
yellow powder as a free base, mp. 206-207.degree. C. .sup.1H NMR
(400 MHz, DMSO-d.sub.6) .delta. ppm 1.23 (d, J=6.32 Hz, 6H) 3.53
(s, 7H) 3.70-3.82 (m, 4H) 4.83 (spt, J=6.23 Hz, 1H) 6.37 (t, J=6.82
Hz, 1H) 6.78 (d, J=7.83 Hz, 1H) 7.55 (dd, J=6.57, 1.26 Hz, 1H) 8.64
(dd, J=7.20, 1.14 Hz, 1H) 8.72 (d, J=7.83 Hz, 1H) 8.96 (s, 1H).
.sup.13C NMR (100 MHz, DMSO-d.sub.6) .delta. ppm 21.97, 37.39,
42.84, 44.11, 68.16, 96.94, 100.56, 105.49, 122.80, 132.49, 135.00,
136.42, 144.40, 144.56, 154.29, 155.46, 160.29. LRMS (ESI) m/z
397.1 [(M+H)].sup.+, calc'd for C.sub.20H.sub.24N.sub.6O.sub.3:
396.45.
5.6.15. Synthesis of
4-[3-(3-Fluoro-2-methoxy-phenyl)-pyrazolo[1,5-a]pyrimidin-5-yl]-piperazin-
e-1-carboxylic acid isopropyl ester
##STR00056##
[0181] Prepared similarly to the preparation of example 5.6.9 from
4-(3-bromo-pyrazolo[1,5-a]pyrimidin-5-yl)-piperazine-1-carboxylic
acid isopropyl ester (389.9 mg, 1.1 mmol) and
3-fluoro-2-methoxyphenylboronic acid [762287-59-2] (216.2 mg, 1.3
mmol) to afford 153.8 mg of light yellow powder as a free base, mp.
52-53.degree. C. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. ppm
1.22 (d, J=6.32 Hz, 6H) 3.49-3.56 (m, 4H) 3.75 (dd, J=6.44, 4.17
Hz, 4H) 3.83 (d, J=1.01 Hz, 3H) 4.82 (spt, J=6.23 Hz, 1H) 6.81 (d,
J=8.08 Hz, 1H) 7.06 (ddd, J=11.37, 8.08, 1.52 Hz, 1H) 7.16 (td,
J=8.08, 5.81 Hz, 1H) 8.23 (dt, J=7.89, 1.36 Hz, 1H) 8.42 (s, 1H).
.sup.19F NMR (377 MHz, DMSO-d.sub.6) .delta. ppm -131.42. .sup.13C
NMR (100 MHz, DMSO-d.sub.6) .delta. ppm 21.95, 42.83, 44.05, 60.38,
68.16, 97.24, 99.94, 99.98, 112.72, 112.90, 123.22, 124.06, 127.98,
136.39, 143.40, 143.51, 144.25, 144.49, 154.28, 154.63, 155.53,
157.04. LRMS (ESI) m/z 414.1 [(M+H)].sup.+, calc'd for
C.sub.21H.sub.24FN.sub.5O.sub.3: 413.46.
5.6.16. Synthesis of Isopropyl
4-(3-(2-fluoropyridin-3-yl)pyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-car-
boxylate
##STR00057##
[0183] Isopropyl
4-(3-bromopyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-carboxylate (4
g, 10.86 mmol), 2-fluoropyridine-3-boronic acid hydrate (3.44 g,
21.7 mmol), potassium carbonate (4.5 g, 32.6 mmol), Pd(OAc).sub.2
(24 mg, 0.1086 mmol), and x-Phos (146 mg, 0.217 mmol) were taken up
in dioxane and water under nitrogen and stirred at 80.degree. C.
for 2 hours. Reaction then cooled to room temperature, filtered
through celited plug with ethyl acetate. Reduced in vacuo and run
through an isco silica column with hexane:ethyl acetate 75-100%.
Product plus desbromo co elute, reduced in vacuo. Product
recrystallized 3 times from IPAC, washed with heptanes dried to get
889 mg product. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 8.95
(t, J=2.15 Hz, 1H), 8.78 (d, J=7.83 Hz, 1H), 8.29 (d, J=4.29 Hz,
1H), 8.00 (d, J=4.80 Hz, 1H), 7.43 (dt, J=2.40, 4.99 Hz, 1H), 6.85
(d, J=7.83 Hz, 1H), 4.78-4.87 (m, 1H), 3.73-3.83 (m, 4H), 3.48-3.58
(m, 4H), 3.31 (s, 1H), 1.22 (d, J=6.06 Hz, 6H) LRMS (ESI) m/z 385
[(M+H)].sup.+, calc'd for C.sub.19H.sub.21FN.sub.6O.sub.2:
384.42.
5.6.17. Synthesis of Isopropyl
4-(3-(5-fluoro-2-methoxypyridin-3-yl)pyrazolo[1,5-a]pyrimidin-5-yl)pipera-
zine-1-carboxylate
##STR00058##
[0185] To a 200 mL round bottom flask was added 4.00 g (10.87 mmol)
of isopropyl
4-(3-bromopyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-carboxylat- e,
2.89 g (11.41 mmol) of (5-fluoro-2-methoxypyridin-3-yl)boronic
acid, 0.38 g (0.33 mmol) of Pd(PPh.sub.3).sub.4, and a stirring
rod. After adding all the solids, 60 ml of 2% TPGS-750-M solution
was added, followed by the addition of 7.56 mL (54.35 mmol)
triethylamine.
[0186] The reaction flask was plunged into an oil bath pre-heated
to 90.degree. C., and stirred vigorously. It took about 0.25 hr of
stirring for all the suspended solids to dissolve completely.
Several color changes were observed, the last color that remained
through the rest of the reaction was a very dark brown color.
[0187] After 1.5 hr, LCMS showed that reaction was complete. It was
allowed to cool to RT, and diluted with 100 mL EtOAc. The organic
layer was separated, and the aqueous layer was extracted twice with
40 mL EtOAc portions. The combined organic layers was washed with
brine, dried over MgSO.sub.4, and concentrated. A small volume of
DCM was used to dissolve the crude mixture, and then loaded
directly onto a 120 g silica gel column for separation on the ISCO.
A gradient of 40% EtOAc/hex to 100% EtOAc was run for the first 25
minutes. The desired product did not elute, only some impurities
eluted. However, about 10 minutes into running with 100% EtOAc, the
desbromo side product started eluting together with the desired
product. Even though the desbromo compound is spread through all
the fractions containing the desired product, the percentage of the
desbromo compound seem to drastically decrease with time.
Therefore, the initial fractions containing the highest percentages
of the desbromo were discarded, and the remaining fractions were
concentrated to obtain 3.4 g of about 87% purity at 254 nm. (with
about 13% desbromo compound). [Base on .sup.1H NMR, the desbromo is
over-estimated at the 254 nm on the UV, and under estimated on the
UV at 220 nm]. The material was recrystallized twice using IPAC and
heptanes to obtain 3.16 g (70% yield) of the desired product with
purity over 99% on the UV at 254 nm. .sup.1H NMR (400 MHz,
CHLOROFORM-d) .delta. ppm 1.31 (d, J=6.32 Hz, 6H) 3.61-3.70 (m, 4H)
3.77 (br. s., 4H) 4.08 (s, 3H) 5.00 (quin, J=6.19 Hz, 1H) 6.41 (d,
J=7.83 Hz, 1H) 7.83 (d, J=3.03 Hz, 1H) 8.37 (d, J=7.83 Hz, 1H)
8.63-8.71 (m, 2H). LRMS (ESI) m/e 415 [(M+H).sup.+, calcd for
C.sub.20H.sub.23FN.sub.6O.sub.3 414].
5.6.18. Synthesis of Isopropyl
4-(3-(2-ethoxypyridin-3-yl)pyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-car-
boxylate
##STR00059##
[0189] To a 1 liter round bottom flask was added 15.00 g (40.76
mmol) of isopropyl
4-(3-bromopyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-carboxylat- e,
8.85 g (52.99 mmol) of (2-ethoxypyridin-3-yl)boronic acid, 14.06 g
(101.90 mmol) of K.sub.2CO.sub.3, 1.41 g (1.22 mmol) of
P(PPh.sub.3).sub.4, and a stirring rod. After adding all the
solids, 200 ml of MeCN was added, followed by the addition of 100
mL of water. The reaction flask was plunged into an oil bath
pre-heated to 85.degree. C., and stirred vigorously. This reaction
was setup to reflux. After stirring at 85.degree. C. for about 16
hr, it was allowed to cool to RT and diluted with 200 mL of EtOAc.
The organic layer was separated, and the aqueous layer was
extracted twice with 80 mL portions of EtOAc. The combined organic
layers was washed with brine, dried over MgSO.sub.4, and
concentrated. A small volume of DCM was used to dissolve the crude
mixture, and then loaded directly onto a 750 g silica gel column
for separation on the ISCO-XL. A gradient of 50% EtOAc/hex to 100%
EtOAc was run for the first 35 minutes. The desired product did not
elute, only some impurities eluted. However, about 10 minutes into
running with 100% EtOAc, the desbromo side product starts eluting
together with the desired product. Even though the desbromo
compound is spread through all the fractions containing the desired
product, the percentage of the desbromo compound seem to decrease
with time. Therefore, the initial fractions containing the highest
percentages of the desbromo were discarded, and the remaining
fractions were concentrated to obtain 13.46 g of about 86% purity
at 254 nm. (with about 14% desbromo compound). This material was
recrystallized twice, using IPAC and heptanes, to obtain 11.4 g
(68%) of the desired product. .sup.1H NMR (400 MHz, CHLOROFORM-d)
.delta. ppm 1.30 (d, J=6.32 Hz, 6H) 1.53 (t, J=7.07 Hz, 3H)
3.59-3.70 (m, 4H) 3.70-3.81 (m, 4H) 4.53 (q, J=7.07 Hz, 2H) 5.00
(dt, J=12.44, 6.28 Hz, 1H) 6.38 (d, J=7.83 Hz, 1H) 6.98 (dd,
J=7.45, 4.93 Hz, 1H) 8.01 (dd, J=4.93, 1.89 Hz, 1H) 8.37 (d, J=7.83
Hz, 1H) 8.70 (s, 1H) 8.76 (dd, J=7.45, 1.89 Hz, 1H). LRMS (ESI) m/e
411 [(M+H).sup.+, calcd for C.sub.21H.sub.26N.sub.6O.sub.3
410].
5.6.19. Synthesis of 2-Methoxyethyl
4-(3-(2-methoxypyridin-3-yl)pyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-ca-
rboxylate
##STR00060##
[0190] Part A. 2-methoxyethyl
4-(3-bromopyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-carboxylate
##STR00061##
[0192] To 10.00 g (35.44 mmol) of
3-bromo-5-(piperazin-1-yl)pyrazolo[1,5-a]pyrimidine dissolved in
120 mL of EtOAc, at 0.degree. C. was added 5.40 g (38.99 mmol) of
2-methoxyethyl carbonochloridate, followed by 9.86 mL (70.88 mmol)
of TEA. After 5 minutes, the ice bath was removed, and the reaction
mixture stirred at RT. After 5 hr, LCMS showed that the reaction
was complete. It was diluted with EtOAc, quenched with brine, and
the organic layer dried over MgSO.sub.4. It was concentrated and
purified on the ISCO with a 330 g column eluting with 0-9% MeOH/DCM
to obtain the desired product, 12.63 g (93%). .sup.1H NMR (400 MHz,
CHLOROFORM-d) .delta. ppm 3.42 (s, 3H) 3.62-3.69 (m, 6H) 3.73-3.82
(m, 4H) 4.26-4.35 (m, 2H) 6.36 (d, J=7.83 Hz, 1H) 7.86 (s, 1H) 8.27
(d, J=7.83 Hz, 1H). LRMS (ESI) m/e 384 [(M+H).sup.+, (doublet),
calcd for C.sub.14H.sub.18BrN.sub.5O.sub.3 383].
Part B. 2-methoxyethyl
4-(3-(2-methoxypyridin-3-yl)pyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-ca-
rboxylate
##STR00062##
[0194] To a 100 mL round bottom flask was added 15.00 g (39.06
mmol) of 2-methoxyethyl
4-(3-bromopyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-carboxylate,
7.17 g (46.88 mmol) of (2-methoxypyridin-3-yl)boronic acid, 38 mL
(273.44 mmol) of TEA, 200 ml of MeCN was added, 100 mL of water,
followed by the addition of 0.21 g (0.29 mmol) of Pd-132. The
reaction flask was plunged into an oil bath pre-heated to
85.degree. C., and stirred. After 1 hr, the reaction was complete,
and it was allowed to cool to RT, and then diluted with 200 mL of
EtOAc. The organic layer was separated, and the aqueous layer was
extracted twice with 150 mL portions of EtOAc. The combined organic
layers was washed with brine, dried over MgSO.sub.4, and filtered
through a thin pad of silica gel, and the filtrate was concentrated
to obtain 14 g solid product, [90% desired product and 10% desbromo
side product, based on uv absoption at 254 nm].
[0195] The resulting solid was subjected to charcoal treatment, by
dissolving all 14 g in 100 mL DCE and adding 500 mg of Darco. It
was heated and stirred for about 1.5 hr, cooled and then filtered
through celite, and concentrated. The product obtained appeared to
be clean enough to be recrystallized without subjecting it to
silica gel chromatography. The recrystallization was carried out by
dissolving it in a small volume of IPAC at about 75.degree. C. (a
few milliliters MeOH was used to aid the dissolution of the
solids). After complete dissolution, heptane was added and
precipitates begun forming, and allowed to cool at 0.degree. C. for
1 hr. It was then filtered and dried 13.1 g product with purity of
about 97%. This material was recrystallized again, to obtain a pale
yellow solid 12.45 g (77% yield, purity to over 99%) of the desired
product. .sup.1H NMR (400 MHz, CHLOROFORM-d) .delta. ppm 3.42 (s,
3H) 3.61-3.71 (m, 6H) 3.72-3.80 (m, 4H) 4.10 (s, 3H) 4.29-4.34 (m,
2H) 6.38 (d, J=7.83 Hz, 1H) 7.00 (dd, J=7.45, 4.93 Hz, 1H) 8.04
(dd, J=4.93, 1.89 Hz, 1H) 8.37 (d, J=7.83 Hz, 1H) 8.64 (s, 1H) 8.74
(dd, J=7.45, 1.89 Hz, 1H). .sup.13C NMR (101 MHz, CHLOROFORM-d)
.delta. ppm 43.49, 44.87, 53.51, 59.15, 64.96, 71.07, 76.92, 77.24,
77.56, 96.37, 100.99, 116.93, 117.05, 135.42, 136.16, 142.79,
145.37, 146.26, 155.46, 155.52, 160.08. LRMS (ESI) m/e 413
[(M+H).sup.+, calcd for C.sub.20H.sub.24N.sub.6O.sub.4 412].
5.6.20. Synthesis of tert-butyl
4-(3-(2-methoxypyridin-3-yl)pyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-ca-
rboxylate
##STR00063##
[0196] Part A. tert-butyl
4-(3-bromopyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-carboxylate
##STR00064##
[0198] 3-bromo-5-chloropyrazolo[1,5-a]pyrimidine (10 g, 43 mmol)
taken up in isopropanol. T-butylpiperazine carboxylate (9.61 g,
51.6 mmol), diisopropylethylamine (22.4 mL, 129 mmol) added and
stirred at 65.degree. C. overnight. Reaction was cooled to room
temperature, diluted with water and solid product filtered off. The
solid was washed with water, dried to obtain 15.84 g crude product
for further reaction, as is. LRMS (ESI) m/z 382/384 [(M+H)].sup.+,
calc'd for C.sub.15H.sub.20BrN.sub.5O.sub.2: 382.2.
Part B. tert-butyl
4-(3-(2-methoxypyridin-3-yl)pyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-ca-
rboxylate
##STR00065##
[0200] Tert-butyl
4-(3-bromopyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-carboxylate
(15.84 g, 41.5 mmol), 2-methoxypyridine-3-boronic acid (12.68 g,
82.9 mmol), potassium carbonate (17.16 g, 124.5 mmol),
Pd(OAc).sub.2 (92 mg, 4.15 mmol), and x-Phos (394 mg, 8.3 mmol)
were taken up in 60 mL dioxane and 30 mL water under nitrogen and
stirred at 75.degree. C. for 2 hours. Reaction then cooled to room
temperature, diluted with toluene washed with brine. The organic
layer filtered through magnesium sulfate and celite plug. Reduced
in vacuo and run through an ISCO silica column with hexane:ethyl
acetate 0-100%. The product plus some desbromo co elute,
Concentrated in vacuo. Product recrystallized 2 times from
IPAC:Heptane 30:70, washed with heptanes dried to get 12 g product.
.sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 8.78-8.85 (m, 1H), 8.74
(d, J=8.08 Hz, 1H), 8.50 (s, 1H), 7.92-8.02 (m, 1H), 7.04-7.13 (m,
1H), 6.80 (d, J=8.08 Hz, 1H), 4.00 (s, 3H), 3.70-3.78 (m, 4H), 3.49
(d, J=5.31 Hz, 4H), 1.44 (s, 9H). LRMS (ESI) m/z 411 [(M+H)].sup.+,
calc'd for C.sub.21H.sub.26N.sub.6O.sub.3: 410.48.
5.6.21. Synthesis of Isopropyl
4-(3-(5-fluoro-2-methoxyphenyl)pyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-
-carboxylate
##STR00066##
[0201] Part A. isopropyl
4-(3-bromopyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-carboxylate
##STR00067##
[0203] 3-bromo-5-(piperazin-1-yl)pyrazolo[1,5-a]pyrimidine (2.5 g,
8.87 mmol) taken up in DCM. Isopropylchloroformate 1M in toluene
(13.3 mL, 13.3 mmol), and triethylamine (2.47 mL, 17.7 mmol) added
and stirred at room temperature 30 minutes. Reaction washed with
water, DCM layer dried over magnesium sulfate filtered reduced in
vacuo. Run on an Isco silica column with DCM:MeOH 0-5%. Product
fractions were combined and reduced in vacuo to obtain 2.86 g crude
product for further reaction as is. LRMS (ESI) m/z 368/370
[(M+H)].sup.+, calc'd for C.sub.14H.sub.18BrN.sub.5O: 368.24.
Part B. isopropyl
4-(3-(5-fluoro-2-methoxyphenyl)pyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-
-carboxylate
##STR00068##
[0205] Isopropyl
4-(3-bromopyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-carboxylate
(2.86 g, 7.8 mmol), 5-fluoro-2-methoxyphenyl boronic acid (2.64 g,
15.5 mmol), potassium carbonate (3.22 g, 23.3 mmol), Pd(OAc).sub.2
(35 mg, 0.155 mmol), and x-Phos (146 mg, 0.31 mmol) were taken up
in 15 mL dioxane and 7 mL water under nitrogen and stirred at
80.degree. C. for 2 hours. Reaction then cooled to room
temperature, filtered through celited plug with ethyl acetate.
Reduced in vacuo and run through an isco silica column with
hexane:ethyl acetate 75-100%. Product plus desbromo co-elute,
reduced in Vacuo. Product recrystallized 2 times from ethyl
acetate:Hexane, washed with heptanes dried to get 12 g product.
.sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 8.73 (d, J=7.78 Hz,
1H), 8.50 (s, 1H), 8.29 (dd, J=3.14, 11.17 Hz, 1H), 7.04 (dd,
J=5.02, 9.03 Hz, 1H), 6.95 (dd, J=3.26, 7.78 Hz, 1H), 6.79 (d,
J=8.03 Hz, 1H), 4.77-4.85 (m, 1H), 3.87 (s, 3H), 3.70-3.78 (m, 4H),
3.48-3.56 (m, 4H), 1.21 (d, J=6.27 Hz, 6H). LRMS (ESI) m/z 414
[(M+H)].sup.+, calc'd for C.sub.21H.sub.24N.sub.5O.sub.3:
413.4.
5.6.22. Synthesis of Isopropyl
4-(3-(2-methoxy-6-methylpyridin-3-yl)pyrazolo[1,5-a]pyrimidin-5-yl)pipera-
zine-1-carboxylate
##STR00069##
[0207] To a 100 mL liter round bottom flask was added 2.00 g (5.43
mmol) of isopropyl
4-(3-bromopyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-carboxylate,
1.00 g (5.98 mmol) of (2-methoxy-6-methylpyridin-3-yl)boronic acid,
0.19 g (0.16 mmol) of Pd(PPh.sub.3).sub.4, and a stirring rod.
[0208] After adding all the solids, 30 ml of 2% TPGS-750-M solution
was added, followed by the addition of 3.80 mL (27.15 mmol)
triethylamine. The reaction flask was plunged into an oil bath
pre-heated to 85.degree. C., and stirred vigorously. It took about
0.25 hr of stirring for all the suspended solids to dissolve
completely. After stirring at 85.degree. C. for about 2 hr, LCMS
showed that the reaction was complete. It was allowed to cool to
RT, dilute with 40 mL EtOAc. The organic layer was separated, and
the aqueous layer was extracted twice with 30 mL portions EtOAc.
The combined organic layers was washed with brine, dried over
MgSO.sub.4, and concentrated. A small volume of DCM was used to
dissolve the crude mixture, and then loaded directly onto a 80 g
silica gel column for separation on the ISCO. A gradient of 50%
EtOAc/hex to 100% EtOAc was run for the first 15 minutes, and then
the column was run with only EtOAc. The desired product begun
eluting about 8 minutes into running with only EtOAc. The desbromo
side product elutes together with the desired product. Even though
the desbromo compound is spread through all the fractions
containing the desired product, the percentage of the desbromo
compound seem to decrease with time. Therefore, the first few tubes
containing the highest percentages of the desbromo were discarded,
and the remaining fractions were concentrated to obtain 1.73 g of
about 84% purity at 254 nm (with about 16% desbromo compound).
[Base on .sup.1H nmr, the desbromo is over-estimated at the 254 nm
on the UV, and under estimated on the UV at 220 nm].
[0209] The material was recrystallized twice using IPAC and
heptanes, to afford 1.45 g (65% yield) of the desired product.
.sup.1H NMR (400 MHz, CHLOROFORM-d) .delta. ppm 1.30 (d, J=6.32 Hz,
6H) 2.49 (s, 3H) 3.59-3.68 (m, 4H) 3.73 (dd, J=6.19, 3.66 Hz, 4H)
4.07 (s, 3H) 4.99 (dt, J=12.44, 6.28 Hz, 1H) 6.36 (d, J=7.83 Hz,
1H) 6.85 (d, J=7.58 Hz, 1H) 8.35 (d, J=7.83 Hz, 1H) 8.55-8.63 (m,
2H); .sup.13C NMR (101 MHz, CHLOROFORM-d) .delta. ppm 22.48, 24.10,
43.39, 44.94, 53.38, 69.37, 96.32, 101.34, 113.40, 116.03, 135.94,
136.12, 145.14, 146.15, 152.13, 155.35, 155.45, 159.49. LRMS (ESI)
m/e 411 [(M+H).sup.+, calcd for C.sub.21H.sub.26N.sub.6O.sub.3
410].
5.6.23. Synthesis of Isopropyl
4-(3-(6-methoxypyridin-2-yl)pyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-ca-
rboxylate
##STR00070##
[0210] Part A. isopropyl
4-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrazolo[1,5-a]pyrimidi-
n-5-yl)piperazine-1-carboxylate
##STR00071##
[0212] To 184 mg (0.50 mmol) of isopropyl
4-(3-bromopyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-carboxylate in
a 50 mL round bottom flask was added 191 mg (0.75 mmol) of
4,4,4',4',5,5,5',5'-octamethyl-2,2'-bi(1,3,2-dioxaborolane), 147 mg
(1.50 mmol) of KOAc, 8 mL of DMSO followed by 41 mg (0.05 mmol) of
PdCl.sub.2(dppf).sub.2.DCM. The air was replaced with N.sub.2, and
the mixture heated to 80.degree. C. with stirring. Next morning
LCMS shows reaction had gone to completion. It was cooled to RT,
diluted with EtOAc, washed with brine, and the organic layer dried
over MgSO.sub.4. It was concentrated and purified in the ISCO using
a 12 g column, and eluting with 25-100% EtOAc/hex to obtain 107 mg
of the desired product [Purity was only about 85%, about 15% was
hydrolyzed to the corresponding boronic acid].
Part B. Isopropyl
4-(3-(6-methoxypyridin-2-yl)pyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-ca-
rboxylate
[0213] To 65 mg (0.157 mmol) of isopropyl
4-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrazolo[1,5-a]pyrimidi-
n-5-yl)piperazine-1-carboxylate in a microwave vial was added, 29
mg (0.157 mmol) of 2-bromo-6-methoxypyridine, 43 mg (0.313 mmol) of
K.sub.2CO.sub.3, 18 mg (0.016 mmol) Pd(PPh.sub.3).sub.4, 3 mL of
DME and then 1 mL water. The resulting mixture was microwaved at
125.degree. C. for 0.33 hr. It was diluted with EtOAc, washed with
brine, and the organic layer dried over MgSO.sub.4. It was
concentrated and purified on the neutral PREP HPLC to obtain 42 mg
of the desired product. .sup.1H NMR (400 MHz, CHLOROFORM-d) .delta.
ppm 1.30 (d, J=6.32 Hz, 6H) 3.60-3.74 (m, 4H) 3.81 (br. s., 4H)
4.04 (s, 3H) 5.00 (quin, J=6.25 Hz, 1H) 6.39 (d, J=7.83 Hz, 1H)
6.55 (dd, J=8.08, 0.76 Hz, 1H) 7.62 (dd, J=8.08, 7.58 Hz, 1H)
7.83-7.93 (m, 1H) 8.37 (d, J=7.83 Hz, 1H) 8.66 (s, 1H). LRMS (ESI)
m/e 397 [(M+H).sup.+, calcd for C.sub.20H.sub.24N.sub.6O.sub.3
396].
5.6.24. Synthesis of
5-isopropyl-3-(4-(3-(2-methoxypyridin-3-yl)pyrazolo[1,5-a]pyrimidin-5-yl)-
piperazin-1-yl)-1,2,4-oxadiazole
##STR00072##
[0214] Part A.
4-(3-bromopyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-carbonitrile
##STR00073##
[0216] 3-bromo-5-(piperazin-1-yl)pyrazolo[1,5-a]pyrimidine (500 mg,
1.77 mmol) taken up in 20 mL DCM and stirred at 0.degree. C. in an
ice bath. Sodium bicarbonate (744 mg, 8.86 mmol) in 6 mL water
added and stirred. Cyanogen bromide (225 mg, 2.12 mmol) added and
stirred at 0.degree. C. for 30 minutes then allowed to reach room
temperature and stirred overnight. Reaction diluted with DCM washed
with water, DCM layer dried over magnesium sulfate reduced in vacuo
for 500 mg crude product for further reaction as is. LRMS (ESI) m/z
307/309 [(M+H)].sup.+, calc'd for C.sub.11H.sub.11BrN.sub.6:
307.15.
Part B.
3-(4-(3-bromopyrazolo[1,5-a]pyrimidin-5-yl)piperazin-1-yl)-5-isopr-
opyl-1,2,4-oxadiazole
##STR00074##
[0218]
4-(3-bromopyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-carbonitrile
(450 mg, 1.46 mmol) in 9 mL DMF and sodium carbonate (155 mg, 1.46
mmol) stirred room temperature 15 minutes, hydroxylamine HCl (203
mg, 2.9 mmol) added and reaction stirred 30 minutes at 80.degree.
C. Then 12 mL toluene added and pyridine (474 uL, 5.86 mmol) and
isobutyricanhydride (973 uL, 5.86 mmol) added and reaction stirred
at 80.degree. C. for 1.5 hours. Reaction cooled to room temperature
reduced in vacuo then taken up in ethyl acetate and washed with
water, brine, water. Organic layer dried over magnesium sulfate and
reduced in vacuo. For 250 mg crude product to use as is in further
reactions. LRMS (ESI) m/z 392/394 [(M+H)].sup.+, calc'd for
C.sub.15H.sub.18BrN.sub.7O: 392.26.
Part C.
5-isopropyl-3-(4-(3-(2-methoxypyridin-3-yl)pyrazolo[1,5-a]pyrimidi-
n-5-yl)piperazin-1-yl)-1,2,4-oxadiazole
[0219]
3-(4-(3-bromopyrazolo[1,5-a]pyrimidin-5-yl)piperazin-1-yl)-5-isopro-
pyl-1,2,4-oxadiazole (200 mg, 0.51 mmol),
2-methoxypyridine-3-boronic acid (156 mg, 1.0 mmol), potassium
carbonate (211 mg, 1.53 mmol), Pd(OAc).sub.2 (3 mg, 0.01 mmol), and
x-Phos (10 mg, 0.02 mmol) were taken up in 2 mL dioxane and 1 mL
water in a sealed tube and heated at 85.degree. C. for 2 hours.
Reaction then cooled to room temperature filtered through a celite
plug with acetonitrile and DCM, reduced in vacuo. Purified on
Shimadzu neutral phase prep lyophilized to get 12 mg product.
.sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 8.83 (dd, J=1.77, 7.58
Hz, 1H), 8.77 (d, J=7.83 Hz, 1H), 8.51 (s, 1H), 7.99 (dd, J=1.89,
4.93 Hz, 1H), 7.10 (dd, J=4.80, 7.58 Hz, 1H), 6.86 (d, J=8.08 Hz,
1H), 4.00 (s, 3H), 3.84-3.90 (m, 4H), 3.48-3.53 (m, 4H), 3.15 (t,
J=7.07 Hz, 1H), 1.29 (d, J=6.82 Hz, 6H). LRMS (ESI) m/z 421
[(M+H)].sup.+, calc'd for C.sub.21H.sub.24N.sub.8O.sub.2:
420.48.
5.6.25. Synthesis of
(S)-2-amino-1-(4-(3-(2-methoxypyridin-3-yl)pyrazolo[1,5-a]pyrimidin-5-yl)-
piperazin-1-yl)-4-methylpentan-1-one
##STR00075##
[0220] Part A.
(S)-2-amino-1-(4-(3-bromopyrazolo[1,5-a]pyrimidin-5-yl)piperazin-1-yl)-4--
methylpentan-1-one
##STR00076##
[0222] 3-bromo-5-(piperazin-1-yl)pyrazolo[1,5-a]pyrimidine (200 mg,
0.71 mmol), Boc-L-Leu (196 mg, 0.85 mmol), HATU (245 mg, 1.06
mmol), and triethylamine (394 uL, 2.83 mmol) taken up in DMF and
stirred at room temperature overnight. Reaction was diluted with
DCM and washed with water. The organic layer was dried over
magnesium sulfate, filtered and reduced in vacuo. The residue was
taken up in 20 mL of a 40% TFA in DCM solution and stirred at
35.degree. C. overnight. The mixture was concentrated in vacuo and
passed through a silica plug with 10% MeOH in DCM. Solvent was
removed and the residue was dried to give 600 mg crude product as
TFA salt for further reactions. LRMS (ESI) m/z 395/397
[(M+H)].sup.+, calc'd for C.sub.16H.sub.23BrN.sub.6O: 395.31.
Part B.
(S)-2-amino-1-(4-(3-(2-methoxypyridin-3-yl)pyrazolo[1,5-a]pyrimidi-
n-5-yl)piperazin-1-yl)-4-methylpentan-1-one
[0223]
(S)-2-amino-1-(4-(3-bromopyrazolo[1,5-a]pyrimidin-5-yl)piperazin-1--
yl)-4-methylpentan-1-one (300 mg, 0.59 mmol),
2-methoxypyridine-3-boronic acid (135 mg, 0.88 mmol), potassium
carbonate (326 mg, 2.36 mmol), Pd(OAc).sub.2 (3 mg, 0.011 mmol),
and x-Phos (11 mg, 0.022 mmol) were taken up in 2 mL dioxane and 1
mL water in a sealed tube and heated at 85.degree. C. for 2 hours.
Reaction then cooled to room temperature filtered through a celite
plug with acetonitrile and DCM, reduced in vacuo. Purified on
shimadzu neutral phase prep, lyophilized to get 28.1 mg of formic
acid salt product. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 8.82
(dd, J=2.02, 7.58 Hz, 1H), 8.77 (d, J=7.83 Hz, 1H), 8.51 (s, 1H),
8.27 (s, 1H), 7.99 (dd, J=1.77, 4.80 Hz, 1H), 7.09 (dd, J=4.80,
7.58 Hz, 1H), 6.83 (d, J=8.08 Hz, 1H), 4.00 (s, 3H), 3.62-3.93 (m,
9H), 1.72-1.88 (m, 1H), 1.23-1.42 (m, 2H), 0.91 (dd, J=6.57, 10.11
Hz, 6H). LRMS (ESI) m/z 424 [(M+H)].sup.+, calc'd for
C.sub.22H.sub.29N.sub.7O.sub.2: 423.52.
5.6.26. Synthesis of
(S)-2-amino-N--((S)-1-(3-(2-methoxypyridin-3-yl)pyrazolo[1,5-a]pyrimidin--
5-yl)pyrrolidin-3-yl)-N,4-dimethylpentanamide
##STR00077##
[0224] Part A. (S)-tert-butyl
(1-(3-bromopyrazolo[1,5-a]pyrimidin-5-yl)pyrrolidin-3-yl)carbamate
##STR00078##
[0226] 3-bromo-5-chloropyrazolo[1,5-a]pyrimidine (2.2 g, 9.5 mmol)
(S)-3-Boc-aminopyrrolidine (2.6 g, 14.2 mmol), and triethylamine
(5.27 mL, 37.9 mmol) were taken up in isopropanol in a sealed tube
and microwaved at 140.degree. C. for 30 minutes. Reaction stripped
down in vacuo taken up in ethyl acetate washed with water then
passed through a silica plug with ethyl acetate. Solvent was
removed in vacuo to give 3.6 g crude product that was carried on as
is to part B. LRMS (ESI) m/z 382/384 [(M+H)].sup.+, calc'd for
C.sub.15H.sub.19BrN.sub.5O.sub.2: 382.2.
Part B.
(S)-1-(3-bromopyrazolo[1,5-a]pyrimidin-5-yl)-N-methylpyrrolidin-3--
amine
##STR00079##
[0228] (S)-tert-butyl
(1-(3-bromopyrazolo[1,5-a]pyrimidin-5-yl)pyrrolidin-3-yl)carbamate
(3.6 g, 9.4 mmol) taken up in 15 mL DMF and cooled to 0.degree. C.
in an ice bath. Iodomethane (1.026 mL, 16.5 mmol) added and stirred
5 minutes. Then sodium hydride 60% in oil (754 mg, 18.8 mmol) was
slowly added. Reaction stirred 5 minutes at 0.degree. C. then
removed from ice bath and stirred 30 minutes at room temperature.
Reaction was quenched with ice then extracted with ethyl acetate
2.times.. Ethyl acetate fractions combined dried over magnesium
sulfate filtered reduce in vacuo. This was then taken up in 26 mL
DCM and 4 mL TFA added. Stirred 1 hour until complete by LC/MS.
Reaction washed with IN NaOH, DCM layer removed dried over
magnesium sulfate filtered, reduced in vacuo to get 2.72 g crude
product carried on as is to part C. LRMS (ESI) m/z 296/298
[(M+H)].sup.+, calc'd for C.sub.11H.sub.14BrN.sub.5: 296.17.
Part C.
(S)-2-amino-N--((S)-1-(3-bromopyrazolo[1,5-a]pyrimidin-5-yl)pyrrol-
idin-3-yl)-N,4-dimethylpentanamide
##STR00080##
[0230]
(S)-1-(3-bromopyrazolo[1,5-a]pyrimidin-5-yl)-N-methylpyrrolidin-3-a-
mine (200 mg, 0.68 mmol) Boc-L-Leu (187 mg, 0.81 mmol), HATU (385
mg, 1.01 mmol), and triethylamine (375 uL, 2.7 mmol) Taken up in
DMF and Stirred at room temp overnight. Reaction diluted with DCM,
washed with water, DCM layer then dried over magnesium sulfate
filtered and reduced in vacuo. This was then taken up in 26 mL DCM
and 4 mL TFA added. Stirred 1 hour until complete by LC/MS.
Reaction washed with IN NaOH, DCM layer removed dried over
magnesium sulfate filtered, reduced in vacuo for 226 mg crude
product used as is in part D. LRMS (ESI) m/z 409/411 [(M+H)].sup.+,
calc'd for C.sub.17H.sub.24BrN.sub.6O: 409.
Part D.
(S)-2-amino-N--((S)-1-(3-(2-methoxypyridin-3-yl)pyrazolo[1,5-a]pyr-
imidin-5-yl)pyrrolidin-3-yl)-N,4-dimethylpentanamide
[0231]
(S)-2-amino-N--((S)-1-(3-bromopyrazolo[1,5-a]pyrimidin-5-yl)pyrroli-
din-3-yl)-N,4-dimethylpentanamide (220 mg, 0.54 mmol),
2-methoxypyridine-3-boronic acid (164 mg, 1.1 mmol), potassium
carbonate (291 mg, 2.15 mmol), Pd(OAc).sub.2 (2 mg, 0.011 mmol) and
x-phos (10 mg, 0.022 mmol) were taken up in 2 mL acetonitrile and 1
mL water and heated in a sealed tube at 85.degree. C. for 2 hours.
Reaction was then filtered through celite with acetonitrile and DCM
reduced in vacuo, then purified on Shimadzu neutral phase prep and
product fractions lyophilized to get
(S)-2-amino-N--((S)-1-(3-(2-methoxypyridin-3-yl)pyrazolo[1,5-a]pyrimidin--
5-yl)pyrrolidin-3-yl)-N,4-dimethylpentanamide 41.3 mg as 0.5 eq
formic salt. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 8.87-8.97
(m, 1H), 8.72 (s, 1H), 8.50 (s, 1H), 8.30 (s, 1H), 7.96 (dd,
J=2.02, 4.80 Hz, 1H), 7.00-7.11 (m, 1H), 6.51 (d, J=7.83 Hz, 1H),
4.00 (s, 3H), 3.48-3.64 (m, 1H), 3.43 (br. s., 3H), 2.96 (s, 2H),
2.81 (s, 1H), 2.11-2.28 (m, 2H), 1.72-1.89 (m, 1H), 1.19-1.39 (m,
2H), 0.90 (dd, J=1.89, 6.69 Hz, 6H). (ESI) m/z 438 [(M+H)].sup.+,
calc'd for C.sub.23H.sub.31N.sub.7O.sub.2: 437.5.
5.6.27. Synthesis of
(S)-2-amino-1-(4-(3-(2-methoxypyridin-3-yl)pyrazolo[1,5-a]pyrimidin-5-yl)-
piperazin-1-yl)-3-methylbutan-1-one
##STR00081##
[0232] Part A.
(S)-2-amino-1-(4-(3-bromopyrazolo[1,5-a]pyrimidin-5-yl)piperazin-1-yl)-3--
methylbutan-1-one
##STR00082##
[0234] 3-bromo-5-(piperazin-1-yl)pyrazolo[1,5-a]pyrimidine (400 mg,
1.42 mmol) Boc-L-Leucine (392 mg, 1.7 mmol), HATU (490 mg, 2.13
mmol), and triethylamine (788 uL, 5.7 mmol) taken up in DMF and
stirred at room temperature overnight. Reaction diluted with DCM
washed with water, DCM layer then dried over magnesium sulfate
filtered and reduced in Vacuo. This was then taken up in 26 mL DCM
and 4 mL TFA added. Stirred 1 hour until complete by LC/MS.
Reaction was washed with IN NaOH, DCM layer removed dried over
magnesium sulfate filtered, reduced in vacuo to obtain 349 mg crude
product for further reaction as is. LRMS (ESI) m/z 381/383
[(M+H)].sup.+, calc'd for C.sub.15H.sub.21BrN.sub.6O: 381.28.
Part B.
(S)-2-amino-1-(4-(3-(2-methoxypyridin-3-yl)pyrazolo[1,5-a]pyrimidi-
n-5-yl)piperazin-1-yl)-3-methylbutan-1-one
[0235]
(S)-2-amino-1-(4-(3-bromopyrazolo[1,5-a]pyrimidin-5-yl)piperazin-1--
yl)-3-methylbutan-1-one (110 mg, 0.29 mmol),
(2-methoxypyridin-3-yl)boronic acid (88 mg, 0.58 mmol), potassium
carbonate (120 mg, 0.87 mmol), Pd(OAc).sub.2 (1 mg, 0.0058 mmol),
and x-Phos (5 mg, 0.012 mmol) were taken up in 3 mL dioxane and 1
mL water under nitrogen and stirred at 85.degree. C. for 2 hours.
Reaction then cooled to room temperature filtered through celited
plug with acetonitrile and DCM. Reduced in vacuo and purified on
shimadzu neutral phase prep, lyopholized to get 18 mg 1.5 eq formic
salt product. .sup.1H NMR (400 MHz, MSO-d6) .delta. 8.83 (dd,
J=1.77, 7.58 Hz, 1H), 8.78 (d, J=7.83 Hz, 1H), 8.52 (s, 1H), 8.35
(s, 2H), 7.99 (dd, J=1.89, 4.93 Hz, 1H), 7.09 (dd, J=4.93, 7.45 Hz,
1H), 6.85 (d, J=7.83 Hz, 1H), 4.01 (s, 3H), 3.81-3.89 (m, 1H), 3.74
(d, J=5.31 Hz, 3H), 3.68 (d, J=7.58 Hz, 3H), 1.76-1.89 (m, 1H),
0.94 (d, J=6.82 Hz, 3H), 0.86 (d, J=6.82 Hz, 3H). LRMS (ESI) m/z
410 [(M+H)].sup.+, calc'd for C.sub.21H.sub.27N.sub.7O.sub.2:
409.5.
5.6.28. Synthesis of
(S)-2-amino-3-methoxy-1-(4-(3-(2-methoxypyridin-3-yl)pyrazolo[1,5-a]pyrim-
idin-5-yl)piperazin-1-yl)propan-1-one
##STR00083##
[0236] Part A.
(S)-2-amino-1-(4-(3-bromopyrazolo[1,5-a]pyrimidin-5-yl)piperazin-1-yl)-3--
methoxypropan-1-one
##STR00084##
[0238] 3-Bromo-5-(piperazin-1-yl)pyrazolo[1,5-a]pyrimidine (300 mg,
1.1 mmol) taken up in 10 mL DMF. Boc-Ser(Me)-OH (280 mg, 1.3 mmol),
HATU (368 mg, 1.6 mmol), and triethylamine (590 uL, 4.25 mmol) was
added and stirred at room temp overnight. Reaction was diluted with
DCM and washed with water. DCM layer was dried over magnesium
sulfate reduced in vacuo. This was then taken up in 30% TFA in DCM
solution and stirred at room temperature for 2 hours. It was then
cooled to 0.degree. C. and conc Aq NaOH added slowly until aqueous
layer remains basic. DCM layer extracted dried over magnesium
sulfate, reduced in vacuo for 405 mg crude product for further
reaction as is. LRMS (ESI) m/z 383/385 [(M+H)].sup.+, calc'd for
C.sub.14H.sub.19BrN.sub.6O.sub.2: 383.25.
Part B.
(S)-2-amino-3-methoxy-1-(4-(3-(2-methoxypyridin-3-yl)pyrazolo[1,5--
a]pyrimidin-5-yl)piperazin-1-yl)propan-1-one
[0239]
3(S)-2-amino-1-(4-(3-bromopyrazolo[1,5-a]pyrimidin-5-yl)piperazin-1-
-yl)-3-methoxypropan-1-one (400 mg, 1.04 mmol),
2-methoxypyridine-3-boronic acid (240 mg, 1.56 mmol), potassium
carbonate (435 mg, 3.12 mmol), Pd(OAc).sub.2 (5 mg, 0.021 mmol),
and x-Phos (14 mg, 0.042 mmol) were taken up in 2 mL dioxane and 1
mL water in a sealed tube and heated at 85.degree. C. for 2 hours.
Reaction then cooled to room temperature filtered through a celite
plug with acetonitrile and DCM, reduced in vacuo. Purified on
Shimadzu neutral phase prep lyophilized to get 39 mg as the formic
acid salt product. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 8.82
(dd, J=1.89, 7.45 Hz, 1H), 8.76 (d, J=7.83 Hz, 1H), 8.51 (s, 1H),
8.29 (s, 1H), 7.99 (dd, J=1.89, 4.93 Hz, 1H), 7.09 (dd, J=4.93,
7.45 Hz, 1H), 6.84 (d, J=7.83 Hz, 1H), 3.98-4.02 (m, 4H), 3.61-3.85
(m, 8H), 3.31-3.45 (m, 2H) LRMS (ESI) m/z 412 [(M+H)].sup.+, calc'd
for C.sub.20H.sub.25N.sub.7O.sub.3: 411.47.
5.6.29. Synthesis of
(S)-2-amino-1-(4-(3-(2-methoxypyridin-3-yl)pyrazolo[1,5-a]pyrimidin-5-yl)-
piperazin-1-yl)-3,3-dimethylbutan-1-one
##STR00085##
[0240] Part A.
(S)-2-amino-1-(4-(3-bromopyrazolo[1,5-a]pyrimidin-5-yl)piperazin-1-yl)-3,-
3-dimethylbutan-1-one
##STR00086##
[0242] 3-bromo-5-(piperazin-1-yl)pyrazolo[1,5-a]pyrimidine (300 mg,
1.1 mmol) N-Boc-L-tert-Leucine (295 mg, 1.28 mmol), HATU (368 mg,
1.6 mmol), and triethylamine (590 uL, 4.26 mmol) taken up in DMF
and stirred at room temperature overnight. Reaction diluted with
DCM washed with water, DCM layer then dried over magnesium sulfate
filtered and reduced in vacuo. This was then taken up in 26 mL DCM
and 4 mL TFA added. Stirred 1 hour until complete by LC/MS.
Reaction washed with IN NaOH, DCM layer removed dried over
magnesium sulfate filtered, reduced in vacuo to obtain 420 mg crude
product for further reaction as is. LRMS (ESI) m/z 395/397
[(M+H)].sup.+, calc'd for C.sub.16H.sub.23BrN.sub.6O: 395.31.
Part B.
(S)-2-amino-1-(4-(3-(2-methoxypyridin-3-yl)pyrazolo[1,5-a]pyrimidi-
n-5-yl)piperazin-1-yl)-3,3-dimethylbutan-1-one
[0243]
(S)-2-amino-1-(4-(3-bromopyrazolo[1,5-a]pyrimidin-5-yl)piperazin-1--
yl)-3,3-dimethylbutan-1-one (420 mg, 1.1 mmol),
(2-methoxypyridin-3-yl)boronic acid (240 mg, 1.6 mmol), potassium
carbonate (435 mg, 3.2 mmol), Pd(OAc).sub.2 (5 mg, 0.021 mmol), and
x-Phos (14 mg, 0.043 mmol) were taken up in 4 mL dioxane and 1 mL
water under nitrogen and stirred at 85.degree. C. for 2 hours.
Reaction then cooled to room temperature, filtered through celite
plug with acetonitrile and DCM. Reduced in vacuo and purified on
shimadzu neutral phase prep, lyopholized to get 18 mg 1 eq formic
salt product. .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 8.81 (dd,
J=1.77, 7.58 Hz, 1H), 8.75 (d, J=7.83 Hz, 1H), 8.51 (s, 1H), 8.29
(s, 1H), 7.97-8.03 (m, 1H), 7.06-7.09 (m, 1H), 6.83 (d, J=7.83 Hz,
1H), 4.00 (s, 3H), 3.69-3.78 (m, 3H), 3.80 (s, 4H), 3.84 (s, 2H),
0.94 (s, 9H) LRMS (ESI) m/z 424 [(M+H)].sup.+, calc'd for
C.sub.22H.sub.29N.sub.7O.sub.2: 423.52.
5.6.30. Synthesis of (R)-Tetrahydrofuran-3-yl
4-(3-(2-methoxypyridin-3-yl)pyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-ca-
rboxylate
##STR00087##
[0244] Part A.
4-(3-Bromo-pyrazolo[1,5-a]pyrimidin-5-yl)-piperazine-1-carboxylic
acid 4-nitro-phenyl ester
##STR00088##
[0246] To 2.00 g (7.09 mmol) of
3-bromo-5-piperazin-1-yl-pyrazolo[1,5-a]pyrimidine dissolved in 40
mL of EtOAc at 0.degree. C., was added 1.71 g (8.51 mmol) of
4-nitrophenyl carbonochloridate, followed by 1.97 mL (14.18 mmol)
of TEA. After 5 minutes stirring at 0.degree. C., the ice bath was
removed, and stirring continued at RT for about 2 hr. It was
diluted with 50 mL of 50% EtOAc/hex. The desired product was
filtered out, and dried to obtain 3.17 g (100% yield) of an
off-white solid.
Part B.
4-(3-Bromo-pyrazolo[1,5-a]pyrimidin-5-yl)-piperazine-1-carboxylic
acid (R)-(tetrahydro-furan-3-yl) ester
##STR00089##
[0248] To 112 mg (0.25 mmol) of 4-nitrophenyl
4-(3-bromopyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-carboxylate
dissolved in 10 mL of THF, was added (R)-tetrahydrofuran-3-ol (44
mg, 0.5 mmol), followed by 60% NaH (40 mg, 1.0 mmol) and stirred at
RT for 2 hr. After 2 hr it was diluted with 20 mL of EtOAc and
slowly quench with brine. The organic layer was dried over MgSO4
and concentrated, it was purified over 12 gram silica gel column
eluting with 0-10% MeOH/DCM to obtain 71 mg (72% yield) of the
desired product.
Part C. (R)-Tetrahydrofuran-3-yl
4-(3-(2-methoxypyridin-3-yl)pyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-ca-
rboxylate
[0249] Suzuki coupling reaction was carried out under the same
conditions as describe about for the synthesis of
6-[1-(2,2-Dimethyl-propyl)-1H-pyrazol-4-yl]-3-(2-methoxy-pyridin-3-yl)-im-
idazo-[1,2-]pyridazine yielding 72% of the desired product. .sup.1H
NMR (400 MHz, CHLOROFORM-d) .delta. ppm 1.98-2.14 (m, 1H) 2.23
(dtd, J=14.02, 8.17, 8.17, 6.27 Hz, 1H) 3.66 (d, J=5.02 Hz, 4H)
3.71-3.82 (m, 4H) 3.83-4.02 (m, 4H) 4.10 (s, 3H) 5.27-5.39 (m, 1H)
6.38 (d, J=7.78 Hz, 1H) 7.00 (dd, J=7.53, 5.02 Hz, 1H) 8.04 (dd,
J=4.77, 1.76 Hz, 1H) 8.37 (d, J=7.78 Hz, 1H) 8.63 (s, 1H) 8.73 (dd,
J=7.53, 1.76 Hz, 1H).
5.6.31. Synthesis of 1-Methoxypropan-2-yl
4-(3-(2-methoxypyridin-3-yl)pyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-ca-
rboxylate
##STR00090##
[0250] Part A. 1-methoxypropan-2-yl
4-(3-bromopyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-carboxylate
##STR00091##
[0252] This was made by using 1-methoxypropan-2-ol and following
the Part B in the procedure described above for making
(R)-tetrahydrofuran-3-yl
4-(3-bromopyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-carboxylate.
Part B. 1-Methoxypropan-2-yl
4-(3-(2-methoxypyridin-3-yl)pyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-ca-
rboxylate
[0253] The Suzuki coupling reaction was carried out by following
the procedure that was used for other examples. .sup.1H NMR (400
MHz, CHLOROFORM-d) .delta. ppm 1.29 (d, J=6.57 Hz, 3H) 3.40 (s, 3H)
3.42-3.56 (m, 2H) 3.61-3.70 (m, 4H) 3.72-3.81 (m, 4H) 4.10 (s, 3H)
5.06 (td, J=6.25, 4.17 Hz, 1H) 6.37 (d, J=7.83 Hz, 1H) 7.00 (dd,
J=7.58, 5.05 Hz, 1H) 8.03 (dd, J=4.80, 1.77 Hz, 1H) 8.36 (d, J=7.83
Hz, 1H) 8.63 (s, 1H) 8.73 (dd, J=7.45, 1.89 Hz, 1H).
5.6.32. Synthesis of Tetrahydro-2H-pyran-4-yl
4-(3-(2-methoxypyridin-3-yl)pyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-ca-
rboxylate
##STR00092##
[0254] Part A. Tetrahydro-2H-pyran-4-yl
4-(3-bromopyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-carboxylate
##STR00093##
[0256] This was made by using tetrahydro-2H-pyran-4-ol and
following the Part B in the procedure described above for making
(R)-tetrahydrofuran-3-yl
4-(3-bromopyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-carboxylate.
Part B. Tetra hydro-2H-pyran-4-yl
4-(3-(2-methoxypyridin-3-yl)pyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-ca-
rboxylate
[0257] This was synthesized by following the Suzuki procedure
described above for making (R)-tetrahydrofuran-3-yl
4-(3-(2-methoxypyridin-3-yl)pyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-ca-
rboxylate. .sup.1H NMR (400 MHz, CHLOROFORM-d) .delta. ppm
1.66-1.80 (m, 2H) 1.95-2.02 (m, 2H) 3.51-3.62 (m, 2H) 3.67 (d,
J=4.55 Hz, 4H) 3.71-3.82 (m, 4H) 3.88-4.00 (m, 2H) 4.09 (s, 3H)
4.94 (tt, J=8.43, 3.95 Hz, 1H) 6.36 (d, J=7.58 Hz, 1H) 6.99 (dd,
J=7.07, 5.31 Hz, 1H) 8.03 (d, J=4.80 Hz, 1H) 8.35 (d, J=7.58 Hz,
1H) 8.62 (s, 1H) 8.72 (d, J=7.33 Hz, 1H). LRMS (ESI) m/e 439
[(M+H).sup.+, calcd for C.sub.22H.sub.26FN.sub.6O.sub.4 438].
5.6.33. Synthesis of (R)-tetrahydrofuran-3-yl
4-(3-(5-fluoro-2-methoxypyridin-3-yl)pyrazolo[1,5-a]pyrimidin-5-yl)pipera-
zine-1-carboxylate
##STR00094##
[0259] (R)-tetrahydrofuran-3-yl
4-(3-bromopyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-carboxylate
(100 mg, 0.25 mmol),
5-fluoro-2-methoxy-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-
e (127 mg, 0.5 mmol), triethylamine (211 uL, 1.5 mmol),
Pd(OAc).sub.2 (1 mg, 0.0025 mmol), and x-Phos (5 mg, 0.005 mmol)
were taken up in 4 mL dioxane and 1 mL water under nitrogen and
stirred at 85.degree. C. for 2 hours. Reaction then cooled to room
temperature, filtered through celite plug with acetonitrile and
DCM. Reduced in vacuo and purified on Shimadzu neutral phase prep,
lyopholized to get 30 mg product. .sup.1H NMR (400 MHz,
DMSO-d.sub.6) .delta. 8.80 (d, J=7.83 Hz, 1H), 8.72 (dd, J=3.03,
10.36 Hz, 1H), 8.56 (s, 1H), 7.94 (d, J=3.03 Hz, 1H), 6.86 (d,
J=7.83 Hz, 1H), 5.18 (d, J=1.77 Hz, 1H), 4.00 (s, 3H), 3.71-3.83
(m, 8H), 3.32 (s, 2H), 2.08 (s, 1H), 2.13 (s, 1H), 1.89-2.00 (m,
1H) LRMS (ESI) m/z 443 [(M+H)].sup.+, calc'd for
C.sub.21H.sub.23FN.sub.6O.sub.4: 442.45.
5.6.34. Synthesis of (S)-tetrahydrofuran-3-yl
4-(3-(2-methoxypyridin-3-yl)pyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-ca-
rboxylate
##STR00095##
[0260] Part A. (S)-tetrahydrofuran-3-yl
4-(3-bromopyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-carboxylate
##STR00096##
[0262] To 224 mg (0.50 mmol) of 4-nitrophenyl
4-(3-bromopyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-carboxylate
dissolved in 18 mL of THF, was added (S)-tetrahydrofuran-3-ol (88
mg, 1.0 mmol), followed by 60% NaH (80 mg, 2.0 mmol) and stirred at
RT for 2 hr. After 2 hr it was diluted with 30 mL of EtOAc and
slowly quench with brine. The organic layer was dried over MgSO4
and concentrated, it was purified using a 40 g silica gel column
eluting with 0-10% MeOH/DCM to obtain 301 mg (76% yield) of the
desired product. 1H NMR (400 MHz, CHLOROFORM-d) .delta. ppm
2.01-2.13 (m, 1H) 2.17-2.29 (m, 1H) 3.57-3.70 (m, 4H) 3.74-3.84 (m,
4H) 3.86-4.01 (m, 4H) 5.27-5.38 (m, 1H) 6.37 (d, J=7.83 Hz, 1H)
7.87 (s, 1H) 8.28 (d, J=7.83 Hz, 1H)
Part B. (S)-tetrahydrofuran-3-yl
4-(3-(2-methoxypyridin-3-yl)pyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-ca-
rboxylate
[0263] The Suzuki coupling reaction was carried out under the same
conditions as described for the synthesis of LP-943795, yielding
61% of the desired product. .sup.1H NMR (400 MHz, CHLOROFORM-d)
.delta. ppm 2.02-2.14 (m, 1H) 2.16-2.30 (m, 1H) 3.66 (d, J=4.80 Hz,
4H) 3.72-3.82 (m, 4H) 3.84-4.01 (m, 4H) 4.10 (s, 3H) 5.33 (td,
J=3.85, 1.89 Hz, 1H) 6.38 (d, J=7.83 Hz, 1H) 7.01 (dd, J=7.45, 4.93
Hz, 1H) 8.04 (dd, J=5.05, 1.77 Hz, 1H) 8.38 (d, J=7.83 Hz, 1H) 8.64
(s, 1H) 8.73 (dd, J=7.33, 1.77 Hz, 1H). LRMS (ESI) m/e 425
[(M+H).sup.+, calcd for C.sub.21H.sub.24N.sub.6O.sub.4 424].
5.6.35. Synthesis of (S)-1-methoxypropan-2-yl
4-(3-(2-methoxypyridin-3-yl)pyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-ca-
rboxylate
##STR00097##
[0264] Part A. (S)-1-methoxypropan-2-yl
4-(3-bromopyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-carboxylate
##STR00098##
[0266] This was carried out under same conditions and scale as used
above [(S)-tetrahydrofuran-3-yl
4-(3-bromopyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-carboxylate]
to obtain 71% of the desired product. 1H NMR (400 MHz,
CHLOROFORM-d) .delta. ppm 1.29 (d, J=6.27 Hz, 3H) 3.30-3.41 (m, 3H)
3.41-3.54 (m, 2H) 3.58-3.69 (m, 4H) 3.71-3.83 (m, 4H) 4.98-5.12 (m,
1H) 6.35 (d, J=8.03 Hz, 1H) 7.86 (s, 1H) 8.26 (d, J=7.78 Hz,
1H).
Part B. (S)-1-methoxypropan-2-yl
4-(3-(2-methoxypyridin-3-yl)pyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-ca-
rboxylate
[0267] The Suzuki coupling reaction was carried out under the
typical conditions used for the synthesis of other examples,
yielding 64% of the desired product. .sup.1H NMR (400 MHz,
CHLOROFORM-d) .delta. ppm 1.29 (d, J=6.32 Hz, 3H) 3.40 (s, 3H)
3.42-3.54 (m, 2H) 3.61-3.70 (m, 4H) 3.71-3.81 (m, 4H) 4.09 (s, 3H)
5.06 (quind, J=6.32, 6.32, 6.32, 6.32, 4.04 Hz, 1H) 6.38 (d, J=7.83
Hz, 1H) 7.00 (dd, J=7.45, 4.93 Hz, 1H) 8.03 (dd, J=4.93, 1.89 Hz,
1H) 8.36 (d, J=7.83 Hz, 1H) 8.63 (s, 1H) 8.74 (dd, J=7.58, 2.02 Hz,
1H). LRMS (ESI) m/e 427 [(M+H)+, calcd for
C.sub.21H.sub.26N.sub.6O.sub.4 426].
5.6.36. Synthesis of 2-Methoxyethyl
4-(3-(2-methoxypyridin-3-yl)pyrazolo[1,5-a]pyrimidin-5-yl)piperazine(d.su-
b.8)-1-carboxylate
##STR00099##
[0268] Part A.
3-Bromo-5-(piperazin-1-yl)pyrazolo[1,5-a]pyrimidine(d.sub.8)
##STR00100##
[0270] A mixture of 3-bromo-5-chloropyrazolo[1,5-a]pyrimidine (930
mg, 4 mmol), piperazine-d8 HCl (1 g, 6 mmol) and TEA (2.23 mL, 16
mmol) in isopropanol (8 mL) was heated in a microwave oven at
140.degree. C. for 30 min. The mixture was concentrated to give the
titled compound that was used for next step without further
purification.
Part B. 2-Methoxyethyl
4-(3-bromopyrazolo[1,5-a]pyrimidin-5-yl)piperazine(d8)-1-carboxylate
##STR00101##
[0272] To a mixture of
3-bromo-5-(piperazin-1-yl)pyrazolo[1,5-a]pyrimidine-d8 (.about.2
mmol) and DIEA (1.74 mL, 10 mmol) in THF (20 mL) was added
methoxyethyl chloroformate (279 uL, 2.4 mmol). The resulting
mixture was stirred at rt for overnight. The mixture was
concentrated and the residue was subjected to ISCO then prep HPLC
to give the titled compound (382 mg). .sup.1H NMR (400 MHz,
CHLOROFORM-d) .delta. ppm 3.39 (s, 3H) 3.57-3.70 (m, 2H) 4.24-4.31
(m, 2H) 6.32 (d, J=7.83 Hz, 1H) 7.84 (s, 1H) 8.24 (d, J=8.08 Hz,
1H).
Part C. 2-Methoxyethyl
4-(3-(2-methoxypyridin-3-yl)pyrazolo[1,5-a]pyrimidin-5-yl)piperazine(d8)--
1-carboxylate
[0273] Under typical Suzuki coupling conditions with 2-methoxyethyl
4-(3-bromopyrazolo[1,5-a]pyrimidin-5-yl)piperazine(d.sub.8)-1-carboxylate
and appropriate boronic acid to provide the titled compound.
.sup.1H NMR (400 MHz, CHLOROFORM-d) .delta. ppm 3.41 (s, 3H)
3.58-3.70 (t, J=3.40 Hz, 2H) 4.09 (s, 3H) 4.27-4.32 (t, J=3.40 Hz,
2H) 6.35 (d, J=7.83 Hz, 1H) 6.99 (dd, J=7.58, 4.80 Hz, 1H) 8.02
(dd, J=4.80, 1.77 Hz, 1H) 8.35 (d, J=7.83 Hz, 1H) 8.62 (s, 1H) 8.72
(dd, J=7.45, 1.89 Hz, 1H). LRMS (ESI) m/e 421.2 [(M+H).sup.+, calcd
for C.sub.21H.sub.26N.sub.6O.sub.4 420.5].
5.6.37. Synthesis of Part A. Isopropyl
4-(3-bromopyrazolo[1,5-a]pyrimidin-5-yl)piperazine(d.sub.8)-1-carboxylate
##STR00102##
[0275] To a mixture of
3-bromo-5-(piperazin-1-yl)pyrazolo[1,5-a]pyrimidine-d.sub.8
(.about.2 mmol) and DIEA (1.74 mL, 10 mmol) in THF (20 mL) was
added isopropyl chloroformate (2 M solution, 1.2 mL, 2.4 mmol)
dropwise. The resulting mixture was stirred at rt for overnight.
The mixture was concentrated and the residue was subjected to ISCO
to give the titled compound (449 mg). .sup.1H NMR (400 MHz,
CHLOROFORM-d) .delta. ppm 1.26 (d J=6.32 Hz, 6H) 4.90-5.00 (m, 1H)
6.31 (d, J=7.83 Hz, 1H) 7.83 (s, 1H) 8.23 (d, J=7.83 Hz, 1H).
Part B. Isopropyl
4-(3-(2-methoxy-6-methylpyridin-3-yl)pyrazolo[1,5-a]pyrimidin-5-yl)pipera-
zine(d8)-1-carboxylate
[0276] Under typical Suzuki coupling conditions with 2-isopropyl
4-(3-bromopyrazolo[1,5-a]pyrimidin-5-yl)piperazine(d8)-1-carboxylate
and appropriate boronic acid to provide the titled compound. 1H NMR
(400 MHz, CHLOROFORM-d) .delta. ppm 1.29 (d, J=6.32 Hz, 6H) 2.48
(s, 3H) 4.07 (s, 3H) 4.98 (quin, J=6.25 Hz, 1H) 6.32 (d, J=7.83 Hz,
1H) 6.84 (d, J=7.58 Hz, 1H) 8.33 (d, J=7.83 Hz, 1H) 8.56-8.60 (m,
2H). LRMS (ESI) m/e 419.2 [(M+H)+, calcd for
C.sub.21H.sub.26N.sub.6O.sub.4 418.5].
5.6.38. P81 Filter Plate Assay
[0277] Compounds were serially diluted into a Labcyte LDV plate
(Labcyte, cat# LP-0200) using a Mutiprobe (PerkinElmer) and Biomek
FX (Beckman Coulter) so that the highest compound concentration was
at 96 .mu.M. Compounds were then pinged (75 nL per well) into a
Greiner 384-well reaction plate (Greiner, #781076) using an ECHO
550 Liquid Handler (Labcyte). A total of 12 .mu.l reaction buffer
(IMAP buffer containing Tween and DTT, from Molecular Devices) was
then added to each well of columns 1 and 13 for the negative
controls and 12 .mu.l of 2.times.AAK1 (0.2 nM full-length human
protein, NCBI accession no. NP_055726.2) was added to the remaining
wells. Enzyme was then pre-incubated with compound for 10 minutes
at RT. Reactions were initiated upon Minitrak (PerkinElmer)
addition of 12 .mu.l substrate mix containing 2.times. Mu2 (0.2
.mu.M, full length human protein), 2.times. cold ATP (2 .mu.M), and
1.3 .mu.Ci of hot .sup.33P-ATP. Reactions proceeded for one hour at
RT. Meanwhile, Millipore 384-well P81 filter plates (Millipore,
catalog # MZPHN0W10) were placed on a plate washer (Zoom ZW, from
Titertek) and pre-wet with 50 .mu.l 1% phosphoric acid. Kinase
reactions were then stopped upon addition of 24 .mu.l of 2%
phosphoric acid to each well and the Minitrak was then used to
transfer 40 .mu.l from each well into the pre-wet Millipore
384-well P81 filter plates. Reaction mixtures were incubated for 10
minutes at RT in the P81 plates, followed by washing five times
with 100 .mu.l/well of 1% phosphoric acid using the Zoom filter
washer. The bottom of each filter plate was sealed followed by
addition of 20 .mu.l Microscint 40 to each well, sealing the top of
the plates with Flashplate cover, and then waiting one hour until
reading on the TopCount (PerkinElmer).
5.6.39. HEK281 Cell-Based Assay
[0278] HEK293F cells were cultured in media containing DMEM (Gibco,
cat. #11965), 10% FBS (SAFC Biosciences, cat. #12103C), 1.times.GPS
(glutamine, penicillin and streptomycin). On day one, cells were
plated on a 10 cm dish so that they are .about.80% confluent at
time of transfection. Roughly 12 million cells were in a 10 cm dish
at time of transfection. On day two, each dish was transfected with
48 ug DNA and 144 ul Lipofectamine 2000 (Invitrogen, cat.
#11668-019). The DNA was comprised of a mixture (per 10 cm dish)
containing 3 ug AAK1/HA/pIRES (full length human, NCBI accession
no. NP_055726.2), 45 .mu.g Flag/AP2MI/pcDNA (full length human),
and 1.5 ml OPTI-MEM. The Lipofectamine 2000 is made up of a mixture
(per 10 cm dish) containing 144 .mu.l Lipofectamine 2000 and 1.5 ml
OPTI-MEM. Each mixture was transferred to individual 15 ml tubes
and incubated at RT for 5 minutes, and then the two mixes were
combined and incubated at RT for 20 minutes. Growth media was then
aspirated from each 10 cm plate and replaced with 10 ml of DMEM+10%
FBS (no GPS). Finally, 3 ml DNA/Lipofectamine mix was added to each
10 cm dish and mix gently followed by incubate of plate overnight
at 37.degree. C. and 5% CO.sub.2.
[0279] On day three, compounds were diluted in 100% DMSO at
1000.times. final concentration, followed by 3-fold serial
dilutions for a total of 5 concentrations tested. Four compounds
were tested per 10 cm dish. One ul of each compound dilution was
then pipetted into a deep-well, 96-well plate, followed by addition
of 500 .mu.l DMEM+0.5% FBS into each well for a 2.times. final
concentration of each compound. Cells were resuspended in a 10 cm
dish by simple pipetting (HEK293 cells come off the plate that easy
at this point) and then transferred to a 50 ml conical tube and
pelleted by centrifugation at 1000 rpm for 5 min. Cell pellets were
then resuspended in 2.75 ml DMEM+0.5% FBS per 10 cm dish and 100
.mu.l of cell suspension transferred into each well of 96-well TC
plate. Finally, 100 .mu.l of 2.times. compound diluted in DMEM+0.5%
FBS was then added into wells containing cell suspension for a
1.times. final concentration. Plates were then incubated at
37.degree. C. and 5% CO.sub.2 for 3 hours followed by transferring
of cell suspensions from each well into 12-tube PCR strips. The PCR
strips were spun in a tip rack at 1000 rpm for 5 minutes to pellet
cells and media was then removed by pipetting without disturbing
the cell pellet.
[0280] To prepare for Western Blot analysis, cell pellets were
resuspend in 40 ul 1.times.LDS-PAGE sample buffer (Invitrogen, cat.
#NP0008)+2.times. Halt phophatase and protease inhibitor cocktail
(Thermo Scientific, cat. #1861284), followed by sonicating each
with microtip sonicator set at 5 for 8-10 seconds. Five ul of
10.times. NuPage Sample Reducing Agent (with 50 mM DTT) was to each
sample followed by heat denaturing at 70 C for 10 min on PCR
machine. A total of 10 .mu.l per sample was loaded into each lane
of a 4-20% Tris-Glycine Criterion 26-well gel (Biorad, cat.
#345-0034) for the phospho-mu2 blot and 10 .mu.l per lane in a
4-12% Bis-Tris (+MES buffer) NuPAGE 26-well gel (Invitrogen, cat. #
WG1403BX10) for the mu2 blot. For controls, 2 ng of phospho-mu2 or
20 ng mu2/Flag proteins were loaded in the last well of each gel.
After SDS-PAGE, samples on each gel were transferred to PVDF
membrane using an iBlot and membranes were blocked for one hour in
TBST+5% milk, followed by wash 3.times. for 5-10 min with TBST.
Criterion gels were probed with rabbit anti-phospho-mu2 (1:5000; a
rabbit polyclonal antibody produced by New England Peptide and
affinity purified at Lexicon) in TBST+5% BSA, whereas, NuPAGE gels
were probed with mouse anti-Flag (1:500; Sigma, cat. # F1804) in
TBST+5% milk, and these primary antibodies were incubated overnight
at 4.degree. C. on a rocker.
[0281] On day four, Western blots were washed 3.times. for 5-10
minutes with TBST, probe with anti-rabbit-HRP (1:2000; BioRad, cat.
#170-6515) or anti-mouse-HRP (1:2000; Biorad, cat. #170-6516) in
TBST+5% milk for 1 hour at RT, washed 3.times. for 10 minutes with
TBST, and developed with ECL reagent (GE Healthcare, cat. #RPN2132)
on a Versadoc. Finally, the camera was set up to take a picture
every 30 seconds for 10 minutes and the best image saved for each
blot with no saturated signal (when the signal is saturated, the
bands will be highlighted red). A volume analysis on each band was
performed to obtain density values. Percent inhibition was
calculated for each sample by first normalizing to total Mu2
expression levels and then comparing to 0% and 100% controls.
IC.sub.50 values were then calculated using Excel fitting
software.
5.6.40. In Vitro Data
[0282] In vitro data obtained for various compounds of the
invention are provided below in Table 1, wherein "MW" means
molecular weight, "P81 Assay" refers to the P81 filter plate assay
described above, "CBA" refers to the HEK281 cell-based assay
described above, "-" means that results for the given assay were
not obtained, "*" means less than or equal to 1.0 .mu.M, "**" means
a value of less than or equal to 0.1 .mu.M, and "***" means less
than or equal to 0.01 .mu.M.
TABLE-US-00001 TABLE 1 Compound MW CBA IC.sub.50 .mu.M P81
IC.sub.50 .mu.M
(4-(3-(2-methoxypyridin-3-yl)pyrazolo[1,5-a]pyrimidin-5- 407.5 **
*** yl)piperazin-1-yl)(pyrrolidin-1-yl)methanone
(S)-1-(2-(((3-(2-methoxyphenyl)pyrazolo[1,5-a]pyrimidin- 393.5 **
*** 5-yl)amino)methyl)pyrrolidin-1-yl)butan-1-one
(S)-1-(2-(((3-(2-methoxyphenyl)pyrazolo[1,5-a]pyrimidin- 421.5 ***
*** 5-yl)amino)methyl)pyrrolidin-1-yl)-3,3-dimethylbutan-1- one
(S)-1-(3,3-dimethylbutyl)-5-(((3-(2- 435.6 ** ***
ethoxyphenyl)pyrazolo[1,5-a]pyrimidin-5-
yl)amino)methyl)pyrrolidin-2-one
(S)-1-(3,3-dimethylbutyl)-5-(((3-(2- 421.5 *** ***
methoxyphenyl)pyrazolo[1,5-a]pyrimidin-5-
yl)amino)methyl)pyrrolidin-2-one
(S)-1-(3,3-dimethylbutyl)-5-(((3-(2-methoxypyridin-3- 422.5 ** ***
yl)pyrazolo[1,5-a]pyrimidin-5-yl)amino)methyl)pyrrolidin- 2-one
(S)-1-(3,3-dimethylbutyl)-5-(((3-(3-methoxypyridin-4- 422.5 ** ***
yl)pyrazolo[1,5-a]pyrimidin-5-yl)amino)methyl)pyrrolidin- 2-one
(S)-1-(3,3-dimethylbutyl)-5-(((3-phenylpyrazolo[1,5- 391.5 -- ***
a]pyrimidin-5-yl)amino)methyl)pyrrolidin-2-one
(S)-2-(((3-bromopyrazolo[1,5-a]pyrimidin-5- 395.3 *** ***
yl)amino)methyl)-N-(tert-butyl)pyrrolidine-1-carboxamide
(S)-2-cyclopropyl-N-methyl-N-(1-(3-(pyridin-2- 376.5 -- **
yl)pyrazolo[1,5-a]pyrimidin-5-yl)pyrrolidin-3-yl)acetamide
(S)-3,3,3-trifluoro-1-(2-(((3-(2- 433.4 ** ***
methoxyphenyl)pyrazolo[1,5-a]pyrimidin-5-
yl)amino)methyl)pyrrolidin-1-yl)propan-1-one
(S)-3,3,3-trifluoro-1-(2-(((3-(pyridin-4-yl)pyrazolo[1,5- 404.4 **
*** a]pyrimidin-5-yl)amino)methyl)pyrrolidin-1-yl)propan-1- one
(S)-3,3,3-trifluoro-N-(1-(3-(2-methoxyphenyl)pyrazolo[1,5- 433.4 --
** a]pyrimidin-5-yl)pyrrolidin-3-yl)-N-methylpropanamide
(S)-3,3,3-trifluoro-N-(1-(3-(2-methoxypyridin-3- 434.4 *** ***
yl)pyrazolo[1,5-a]pyrimidin-5-yl)pyrrolidin-3-yl)-N-
methylpropanamide (S)-3,3,3-trifluoro-N-methyl-N-(1-(3-(pyridin-2-
404.4 *** *** yl)pyrazolo[1,5-a]pyrimidin-5-yl)pyrrolidin-3-
yl)propanamide (S)-5-(((3-bromopyrazolo[1,5-a]pyrimidin-5- 394.3 **
*** yl)amino)methyl)-1-(3,3-dimethylbutyl)pyrrolidin-2-one
(S)-ethyl 2-(((3-(2-methoxyphenyl)pyrazolo[1,5- 395.5 *** ***
a]pyrimidin-5-yl)amino)methyl)pyrrolidine-1-carboxylate
(S)-isopropyl (1-(3-(2-methoxyphenyl)pyrazolo[1,5- 409.5 -- **
a]pyrimidin-5-yl)pyrrolidin-3-yl)(methyl)carbamate (S)-isopropyl
(1-(3-(2-methoxypyridin-3-yl)pyrazolo[1,5- 410.5 -- **
a]pyrimidin-5-yl)pyrrolidin-3-yl)(methyl)carbamate (S)-isopropyl
2-(((3-(2-methoxyphenyl)pyrazolo[1,5- 409.5 *** ***
a]pyrimidin-5-yl)amino)methyl)pyrrolidine-1-carboxylate
(S)-isopropyl methyl(1-(3-(pyridin-2-yl)pyrazolo[1,5- 380.4 -- *
a]pyrimidin-5-yl)pyrrolidin-3-yl)carbamate (S)-isopropyl
methyl(1-(3-(pyridin-4-yl)pyrazolo[1,5- 380.4 -- **
a]pyrimidin-5-yl)pyrrolidin-3-yl)carbamate (S)-methyl
2-(((3-(2-methoxyphenyl)pyrazolo[1,5- 381.4 ** ***
a]pyrimidin-5-yl)amino)methyl)pyrrolidine-1-carboxylate
(S)--N-(1-(3-(2-methoxyphenyl)pyrazolo[1,5-a]pyrimidin-5- 393.5 *
*** yl)pyrrolidin-3-yl)-N-methylbutyramide
(S)--N-(1-(3-(2-methoxypyridin-3-yl)pyrazolo[1,5- 394.5 * ***
a]pyrimidin-5-yl)pyrrolidin-3-yl)-N-methylbutyramide
(S)--N-(1-(3-bromopyrazolo[1,5-a]pyrimidin-5-yl)pyrrolidin- 406.2
-- ** 3-yl)-3,3,3-trifluoro-N-methylpropanamide
(S)--N-(1-(3-bromopyrazolo[1,5-a]pyrimidin-5-yl)pyrrolidin- 393.3
-- ** 3-yl)-N-methylpyrrolidine-1-carboxamide
(S)--N-(tert-butyl)-2-(((3-(2-methoxyphenyl)pyrazolo[1,5- 422.5 **
*** a]pyrimidin-5-yl)amino)methyl)pyrrolidine-1-carboxamide
(S)--N-methyl-N-(1-(3-(pyridin-2-yl)pyrazolo[1,5- 364.4 -- *
a]pyrimidin-5-yl)pyrrolidin-3-yl)butyramide (S)-tert-butyl
(1-(3-(2-methoxyphenyl)pyrazolo[1,5- 423.5 ** ***
a]pyrimidin-5-yl)pyrrolidin-3-yl)(methyl)carbamate (S)-tert-butyl
2-(((3-(2- 437.5 ** ***
(methoxymethyl)phenyl)pyrazolo[1,5-a]pyrimidin-5-
yl)amino)methyl)pyrrolidine-1-carboxylate (S)-tert-butyl
2-(((3-(2-ethylphenyl)pyrazolo[1,5- 421.5 ** ***
a]pyrimidin-5-yl)amino)methyl)pyrrolidine-1-carboxylate
(S)-tert-butyl 2-(((3-(2-hydroxyphenyl)pyrazolo[1,5- 409.5 ** ***
a]pyrimidin-5-yl)amino)methyl)pyrrolidine-1-carboxylate
(S)-tert-butyl 2-(((3-(2-isopropoxyphenyl)pyrazolo[1,5- 451.6 ***
*** a]pyrimidin-5-yl)amino)methyl)pyrrolidine-1-carboxylate
(S)-tert-butyl 2-(((3-(2-methoxyphenyl)pyrazolo[1,5- 423.5 *** --
a]pyrimidin-5-yl)amino)methyl)pyrrolidine-1-carboxylate
(S)-tert-butyl 2-(((3-(3-methoxyphenyl)pyrazolo[1,5- 423.5 *** ***
a]pyrimidin-5-yl)amino)methyl)pyrrolidine-1-carboxylate
(S)-tert-butyl 2-(((3-(4-(aminomethyl)phenyl)pyrazolo[1,5- 422.5
*** *** a]pyrimidin-5-yl)amino)methyl)pyrrolidine-1-carboxylate
(S)-tert-butyl 2-(((3-(4-carbamoylphenyl)pyrazolo[1,5- 436.5 * ***
a]pyrimidin-5-yl)amino)methyl)pyrrolidine-1-carboxylate
(S)-tert-butyl 2-(((3-(4-methoxyphenyl)pyrazolo[1,5- 423.5 *** ***
a]pyrimidin-5-yl)amino)methyl)pyrrolidine-1-carboxylate
(S)-tert-butyl 2-(((3-(4-methoxypyridin-3-yl)pyrazolo[1,5- 424.5
*** *** a]pyrimidin-5-yl)amino)methyl)pyrrolidine-1-carboxylate
(S)-tert-butyl 2-(((3-(pyridin-4-yl)pyrazolo[1,5-a]pyrimidin- 394.5
*** -- 5-yl)amino)methyl)pyrrolidine-1-carboxylate (S)-tert-butyl
2-(((3-(pyridin-4-yl)pyrazolo[1,5-a]pyrimidin- 394.5 *** --
5-yl)amino)methyl)pyrrolidine-1-carboxylate (S)-tert-butyl
2-(((3-(trifluoromethyl)pyrazolo[1,5- 385.4 ** ***
a]pyrimidin-5-yl)amino)methyl)pyrrolidine-1-carboxylate
(S)-tert-butyl 2-(((3-iodopyrazolo[1,5-a]pyrimidin-5- 443.3 ** --
yl)amino)methyl)pyrrolidine-1-carboxylate (S)-tert-butyl
2-(((3-phenylpyrazolo[1,5-a]pyrimidin-5- 393.5 ** ***
yl)amino)methyl)pyrrolidine-1-carboxylate (S)-tert-butyl
methyl(1-(3-(2-methylpyridin-4- 408.5 ** ***
yl)pyrazolo[1,5-a]pyrimidin-5-yl)pyrrolidin-3-yl)carbamate
(S)-tert-butyl methyl(1-(3-(pyridin-2-yl)pyrazolo[1,5- 394.5 -- *
a]pyrimidin-5-yl)pyrrolidin-3-yl)carbamate
1-(4-(3-(2-methoxypyridin-3-yl)pyrazolo[1,5-a]pyrimidin-5- 394.5 **
*** yl)piperazin-1-yl)-3-methylbutan-1-one 2,2,2-trifluoroethyl
4-(3-(2-methoxypyridin-3- 436.4 *** ***
yl)pyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-carboxylate
2-fluoroethyl 4-(3-(2-methoxypyridin-3-yl)pyrazolo[1,5- 400.4 ***
*** a]pyrimidin-5-yl)piperazine-1-carboxylate 2-methoxyethyl
4-(3-(2-methoxyphenyl)pyrazolo[1,5- 411.5 -- ***
a]pyrimidin-5-yl)piperazine-1-carboxylate 2-methoxyethyl
4-(3-(2-methoxypyridin-3-yl)pyrazolo[1,5- 412.4 ** ***
a]pyrimidin-5-yl)piperazine-1-carboxylate
3-(2-methoxypyridin-3-yl)-N-(4,4,4- 351.3 ** ***
trifluorobutyl)pyrazolo[1,5-a]pyrimidin-5-amine
3-(3-methoxypyridin-4-yl)-N-(2- 429.4 *** --
(trifluoromethoxy)phenethyl)pyrazolo[1,5-a]pyrimidin-5- amine
3-(4-(aminomethyl)phenyl)-N-(2- 351.4 ** --
(cyclopentyloxy)ethyl)pyrazolo[1,5-a]pyrimidin-5-amine
3-(4-(aminomethyl)phenyl)-N-(2- 353.5 ** --
(neopentyloxy)ethyl)pyrazolo[1,5-a]pyrimidin-5-amine
3-(4-(aminomethyl)phenyl)-N-(2-(tert- 339.4 ** --
butoxy)ethyl)pyrazolo[1,5-a]pyrimidin-5-amine
3-(4-(aminomethyl)phenyl)-N-(2- 427.4 *** --
(trifluoromethoxy)phenethyl)pyrazolo[1,5-a]pyrimidin-5- amine
3-(4-(aminomethyl)phenyl)-N-(2- 297.4 ** --
methoxyethyl)pyrazolo[1,5-a]pyrimidin-5-amine
3-(4-(aminomethyl)phenyl)-N-(3- 365.5 ** --
(cyclopentyloxy)propyl)pyrazolo[1,5-a]pyrimidin-5-amine
3-(4-(aminomethyl)phenyl)-N-butylpyrazolo[1,5- 295.4 ** --
a]pyrimidin-5-amine 3-(4-methoxypyridin-3-yl)-N-(2- 429.4 ** --
(trifluoromethoxy)phenethyl)pyrazolo[1,5-a]pyrimidin-5- amine
3-bromo-N-((1-(2,2,2-trifluoroethyl)pyrrolidin-2- 378.2 ** ***
yl)methyl)pyrazolo[1,5-a]pyrimidin-5-amine
3-bromo-N-(3-(cyclopentyloxy)propyl)pyrazolo[1,5- 339.2 * --
a]pyrimidin-5-amine
4-(5-(butylamino)pyrazolo[1,5-a]pyrimidin-3-yl)-N-(2- 366.5 ** --
(methylamino)ethyl)benzamide
5-(4-(isobutylsulfonyl)piperazin-1-yl)-3-(2-methoxypyridin- 430.5
** 3-yl)pyrazolo[1,5-a]pyrimidine cyclopentyl
4-(3-(2-methoxypyridin-3-yl)pyrazolo[1,5- 422.5 *** ***
a]pyrimidin-5-yl)piperazine-1-carboxylate ethyl
4-(3-(2-ethoxypyridin-3-yl)pyrazolo[1,5-a]pyrimidin- 396.4 *** ***
5-yl)piperazine-1-carboxylate ethyl
4-(3-(2-methoxypyridin-3-yl)pyrazolo[1,5- 382.4 ** ***
a]pyrimidin-5-yl)piperazine-1-carboxylate ethyl
5-(4-(isopropoxycarbonyl)piperazin-1- 361.4 **
yl)pyrazolo[1,5-a]pyrimidine-3-carboxylate ethyl methyl(2-((3-(4-
396.4 ** -- (methylcarbamoyl)phenyl)pyrazolo[1,5-a]pyrimidin-5-
yl)amino)ethyl)carbamate isobutyl
(2-((3-(4-carbamoylphenyl)pyrazolo[1,5- 410.5 *** --
a]pyrimidin-5-yl)amino)ethyl)(methyl)carbamate isobutyl
methyl(2-((3-(4- 424.5 * --
(methylcarbamoyl)phenyl)pyrazolo[1,5-a]pyrimidin-5-
yl)amino)ethyl)carbamate isopropyl
(1-(3-(2-methoxypyridin-3-yl)pyrazolo[1,5- 396.4 -- **
a]pyrimidin-5-yl)azetidin-3-yl)(methyl)carbamate isopropyl
(2-((3-(3-methoxypyridin-4-yl)pyrazolo[1,5- 384.4 *** --
a]pyrimidin-5-yl)amino)ethyl)(methyl)carbamate isopropyl
(2-((3-(4-(aminomethyl)phenyl)pyrazolo[1,5- 382.5 ** --
a]pyrimidin-5-yl)amino)ethyl)(methyl)carbamate isopropyl
(2-((3-(4-carbamoylphenyl)pyrazolo[1,5- 396.4 *** --
a]pyrimidin-5-yl)amino)ethyl)(methyl)carbamate isopropyl
(2-((3-(4-methoxypyridin-3-yl)pyrazolo[1,5- 384.4 ** --
a]pyrimidin-5-yl)amino)ethyl)(methyl)carbamate isopropyl
4-(3-(1,3,5-trimethyl-1H-pyrazol-4- 397.5 -- **
yl)pyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-carboxylate isopropyl
4-(3-(1-isobutyl-1H-pyrazol-4-yl)pyrazolo[1,5- 411.5 -- **
a]pyrimidin-5-yl)piperazine-1-carboxylate isopropyl
4-(3-(1-methyl-2-oxo-1,2-dihydropyridin-3- 396.4 ** ***
yl)pyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-carboxylate isopropyl
4-(3-(2-(methoxymethyl)phenyl)pyrazolo[1,5- 409.5 -- *
a]pyrimidin-5-yl)piperazine-1-carboxylate isopropyl
4-(3-(2-(methylthio)pyridin-3-yl)pyrazolo[1,5- 412.5 -- **
a]pyrimidin-5-yl)piperazine-1-carboxylate isopropyl
4-(3-(2,6-dimethoxypyridin-3-yl)pyrazolo[1,5- 426.5 *** ***
a]pyrimidin-5-yl)piperazine-1-carboxylate isopropyl
4-(3-(2-aminopyridin-3-yl)pyrazolo[1,5- 381.4 -- **
a]pyrimidin-5-yl)piperazine-1-carboxylate isopropyl
4-(3-(2-chloropyridin-3-yl)pyrazolo[1,5- 400.9 * **
a]pyrimidin-5-yl)piperazine-1-carboxylate isopropyl
4-(3-(2-ethoxypyridin-3-yl)pyrazolo[1,5- 410.5 ** ***
a]pyrimidin-5-yl)piperazine-1-carboxylate isopropyl
4-(3-(2-fluoropyridin-3-yl)pyrazolo[1,5- 384.4 ** ***
a]pyrimidin-5-yl)piperazine-1-carboxylate isopropyl
4-(3-(2-hydroxypyridin-3-yl)pyrazolo[1,5- 382.4 ** ***
a]pyrimidin-5-yl)piperazine-1-carboxylate isopropyl
4-(3-(2-isopropoxypyridin-3-yl)pyrazolo[1,5- 424.5 ** ***
a]pyrimidin-5-yl)piperazine-1-carboxylate isopropyl
4-(3-(2-methoxy-5-methylpyridin-3- 410.5 *** ***
yl)pyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-carboxylate isopropyl
4-(3-(2-methoxy-6-methylpyridin-3- 410.5 ** ***
yl)pyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-carboxylate isopropyl
4-(3-(2-methoxyphenyl)pyrazolo[1,5- 395.5 -- ***
a]pyrimidin-5-yl)piperazine-1-carboxylate isopropyl
4-(3-(2-methoxypyridin-3-yl)pyrazolo[1,5- 396.4 -- ***
a]pyrimidin-5-yl)piperazine-1-carboxylate isopropyl
4-(3-(2-methoxypyridin-3-yl)pyrazolo[1,5- 410.4 *** ***
a]pyrimidin-5-yl)-3-oxopiperazine-1-carboxylate isopropyl
4-(3-(2-methoxypyridin-3-yl)pyrazolo[1,5- 424.5 -- **
a]pyrimidin-5-yl)-2,2-dimethylpiperazine-1-carboxylate isopropyl
4-(3-(2-methylpyridin-3-yl)pyrazolo[1,5- 380.4 -- **
a]pyrimidin-5-yl)piperazine-1-carboxylate isopropyl
4-(3-(3,6-dimethoxypyridazin-4-yl)pyrazolo[1,5- 427.5 *** ***
a]pyrimidin-5-yl)piperazine-1-carboxylate isopropyl
4-(3-(3-ethoxyphenyl)pyrazolo[1,5-a]pyrimidin- 409.5 -- ***
5-yl)piperazine-1-carboxylate isopropyl
4-(3-(3-fluoro-2-methoxyphenyl)pyrazolo[1,5- 413.4 -- **
a]pyrimidin-5-yl)piperazine-1-carboxylate isopropyl
4-(3-(3-methoxypyridin-2-yl)pyrazolo[1,5- 396.4 -- *
a]pyrimidin-5-yl)piperazine-1-carboxylate isopropyl
4-(3-(3-methoxypyridin-4-yl)pyrazolo[1,5- 396.4 ** ***
a]pyrimidin-5-yl)piperazine-1-carboxylate isopropyl
4-(3-(4-fluoropyridin-3-yl)pyrazolo[1,5- 384.4 -- **
a]pyrimidin-5-yl)piperazine-1-carboxylate isopropyl
4-(3-(4-methoxypyridin-2-yl)pyrazolo[1,5- 396.4 -- ***
a]pyrimidin-5-yl)piperazine-1-carboxylate isopropyl
4-(3-(4-methoxypyridin-3-yl)pyrazolo[1,5- 396.4 ** ***
a]pyrimidin-5-yl)piperazine-1-carboxylate isopropyl
4-(3-(5-fluoro-2-methoxyphenyl)pyrazolo[1,5- 413.4 *** ***
a]pyrimidin-5-yl)piperazine-1-carboxylate isopropyl
4-(3-(5-fluoro-2-methoxypyridin-3- 414.4 ** ***
yl)pyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-carboxylate isopropyl
4-(3-(5-fluoropyridin-3-yl)pyrazolo[1,5- 384.4 -- ***
a]pyrimidin-5-yl)piperazine-1-carboxylate isopropyl
4-(3-(5-methoxypyridin-2-yl)pyrazolo[1,5- 396.4 *** ***
a]pyrimidin-5-yl)piperazine-1-carboxylate isopropyl
4-(3-(5-methoxypyridin-3-yl)pyrazolo[1,5- 396.4 ** ***
a]pyrimidin-5-yl)piperazine-1-carboxylate isopropyl
4-(3-(6-fluoropyridin-3-yl)pyrazolo[1,5- 384.4 -- **
a]pyrimidin-5-yl)piperazine-1-carboxylate isopropyl
4-(3-(6-methoxypyridin-3-yl)pyrazolo[1,5- 396.4 * ***
a]pyrimidin-5-yl)piperazine-1-carboxylate isopropyl
4-(3-(ethylcarbamoyl)pyrazolo[1,5-a]pyrimidin- 360.4 -- **
5-yl)piperazine-1-carboxylate isopropyl
4-(3-(isopropylcarbamoyl)pyrazolo[1,5- 374.4 -- *
a]pyrimidin-5-yl)piperazine-1-carboxylate
isopropyl 4-(3-(methylcarbamoyl)pyrazolo[1,5- 346.4 -- **
a]pyrimidin-5-yl)piperazine-1-carboxylate isopropyl
4-(3-(pyrazin-2-yl)pyrazolo[1,5-a]pyrimidin-5- 367.4 -- **
yl)piperazine-1-carboxylate isopropyl
4-(3-(pyridin-2-yl)pyrazolo[1,5-a]pyrimidin-5- 366.4 ** ***
yl)piperazine-1-carboxylate isopropyl
4-(3-(pyridin-3-yl)pyrazolo[1,5-a]pyrimidin-5- 366.4 -- ***
yl)piperazine-1-carboxylate isopropyl
4-(3-(pyridin-4-yl)pyrazolo[1,5-a]pyrimidin-5- 366.4 ** ***
yl)piperazine-1-carboxylate isopropyl
4-(3-(pyrimidin-5-yl)pyrazolo[1,5-a]pyrimidin-5- 367.4 -- **
yl)piperazine-1-carboxylate isopropyl
4-(3-isopropylpyrazolo[1,5-a]pyrimidin-5- 331.4 -- **
yl)piperazine-1-carboxylate isopropyl
4-(pyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1- 289.3 -- ***
carboxylate isopropyl methyl(2-((3-(4-((2- 453.5 ** --
(methylamino)ethyl)carbamoyl)phenyl)pyrazolo[1,5-
a]pyrimidin-5-yl)amino)ethyl)carbamate isopropyl methyl(2-((3-(4-
410.5 *** -- (methylcarbamoyl)phenyl)pyrazolo[1,5-a]pyrimidin-5-
yl)amino)ethyl)carbamate isopropyl methyl(2-((3-(4-(pyrrolidin-2-
422.5 *** -- yl)phenyl)pyrazolo[1,5-a]pyrimidin-5-
yl)amino)ethyl)carbamate methyl
4-(3-(2-methoxypyridin-3-yl)pyrazolo[1,5- 368.4 ** **
a]pyrimidin-5-yl)piperazine-1-carboxylate
N-(2-(cyclopentyloxy)ethyl)-3-(3-methoxypyridin-4- 353.4 ** --
yl)pyrazolo[1,5-a]pyrimidin-5-amine
N-(2-(tert-butoxy)ethyl)-3-(3-methoxypyridin-4- 341.4 ** --
yl)pyrazolo[1,5-a]pyrimidin-5-amine N-(2-aminoethyl)-4-(5-((2-
354.4 * -- methoxyethyl)amino)pyrazolo[1,5-a]pyrimidin-3-
yl)benzamide N-(2-aminoethyl)-4-(5-((3,3- 380.5 * --
dimethylbutyl)amino)pyrazolo[1,5-a]pyrimidin-3- yl)benzamide
N-(2-aminoethyl)-4-(5-(butylamino)pyrazolo[1,5- 352.4 ** --
a]pyrimidin-3-yl)benzamide
N-(2-methoxyethyl)-3-phenylpyrazolo[1,5-a]pyrimidin-5- 268.3 * --
amine N-(3-(2-methoxypyridin-3-yl)pyrazolo[1,5-a]pyrimidin-5-yl)-
393.4 -- * N-(4,4,4-trifluorobutyl)acetamide
N-(3-(cyclopentyloxy)propyl)-3-(3-methoxypyridin-4- 367.4 ** --
yl)pyrazolo[1,5-a]pyrimidin-5-amine
N-(tert-butyl)-4-(3-(2-methoxypyridin-3-yl)pyrazolo[1,5- 409.5 **
*** a]pyrimidin-5-yl)piperazine-1-carboxamide
N-(tert-butyl)-4-(3-(2-methoxypyridin-3-yl)pyrazolo[1,5- 423.5 **
*** a]pyrimidin-5-yl)-N-methylpiperazine-1-carboxamide
N-isopropyl-4-(3-(2-methoxypyridin-3-yl)pyrazolo[1,5- 395.5 *** ***
a]pyrimidin-5-yl)piperazine-1-carboxamide
N-isopropyl-4-(3-(2-methoxypyridin-3-yl)pyrazolo[1,5- 409.5 ** ***
a]pyrimidin-5-yl)-N-methylpiperazine-1-carboxamide tert-butyl
(1-(3-(2-methoxypyridin-3-yl)pyrazolo[1,5- 410.5 -- *
a]pyrimidin-5-yl)azetidin-3-yl)(methyl)carbamate tert-butyl
(2-((3-(3-methoxypyridin-4-yl)pyrazolo[1,5- 398.5 *** --
a]pyrimidin-5-yl)amino)ethyl)(methyl)carbamate tert-butyl
(2-((3-(4-carbamoylphenyl)pyrazolo[1,5- 410.5 *** --
a]pyrimidin-5-yl)amino)ethyl)(methyl)carbamate tert-butyl
(2-(4-(5-((2-methoxyethyl)amino)pyrazolo[1,5- 454.5 * --
a]pyrimidin-3-yl)benzamido)ethyl)carbamate tert-butyl
4-(3-(2-methoxypyridin-3-yl)pyrazolo[1,5- 410.5 *** ***
a]pyrimidin-5-yl)piperazine-1-carboxylate tert-butyl
methyl(2-((3-(4- 424.5 *** --
(methylcarbamoyl)phenyl)pyrazolo[1,5-a]pyrimidin-5-
yl)amino)ethyl)carbamate
(4-(3-(2-methoxypyridin-3-yl)pyrazolo[1,5-a]pyrimidin-5- 421.5 **
*** yl)piperazin-1-yl)(piperidin-1-yl)methanone isopropyl
4-(3-(6-methoxypyridin-2-yl)pyrazolo[1,5- 396.4 ** ***
a]pyrimidin-5-yl)piperazine-1-carboxylate isopropyl
4-(3-(5-fluoro-2-methoxy-4- 427.5 *** ***
methylphenyl)pyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-
carboxylate isopropyl
4-(3-(2,4-dimethoxypyrimidin-5-yl)pyrazolo[1,5- 427.5 ** ***
a]pyrimidin-5-yl)piperazine-1-carboxylate isopropyl
4-(3-(3-fluoro-4-methylphenyl)pyrazolo[1,5- 397.4 -- **
a]pyrimidin-5-yl)piperazine-1-carboxylate isopropyl
4-(3-iodopyrazolo[1,5-a]pyrimidin-5- 415.2 -- **
yl)piperazine-1-carboxylate (S)-isopropyl
2-((methyl(pyrazolo[1,5-a]pyrimidin-5- 317.4 -- *
yl)amino)methyl)pyrrolidine-1-carboxylate (S)-isopropyl
2-(((3-(2-methoxypyridin-3-yl)pyrazolo[1,5- 424.5 *** ***
a]pyrimidin-5-yl)(methyl)amino)methyl)pyrrolidine-1- carboxylate
isopropyl 4-(3-(2-methoxypyridin-4-yl)pyrazolo[1,5- 396.4 -- **
a]pyrimidin-5-yl)piperazine-1-carboxylate (E)-isopropyl
4-(3-(3-methoxyprop-1-en-1-yl)pyrazolo[1,5- 359.4 -- *
a]pyrimidin-5-yl)piperazine-1-carboxylate (E)-isopropyl
4-(3-(2-ethoxyvinyl)pyrazolo[1,5-a]pyrimidin- 359.4 -- **
5-yl)piperazine-1-carboxylate isopropyl
4-(3-chloropyrazolo[1,5-a]pyrimidin-5- 323.8 -- **
yl)piperazine-1-carboxylate isopropyl
4-(3-(2-d.sub.3-methoxypyridin-3-yl)pyrazolo[1,5- 399.4 *** ***
a]pyrimidin-5-yl)piperazine-1-carboxylate (S)-isopropyl
2-(((3-(2-methoxypyridin-3-yl)pyrazolo[1,5- 410.5 *** ***
a]pyrimidin-5-yl)amino)methyl)pyrrolidine-1-carboxylate tert-butyl
4-(3-bromopyrazolo[1,5-a]pyrimidin-5-yl)-d.sub.8- 390.2 -- **
piperazine-1-carboxylate isopropyl
4-(3-(2-methoxypyridin-3-yl)pyrazolo[1,5- 404.4 *** ***
a]pyrimidin-5-yl)-d.sub.8-piperazine-1-carboxylate isopropyl
4-(3-(1,6-dimethyl-2-oxo-1,2-dihydropyridin-3- 410.5 -- ***
yl)pyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-carboxylate isopropyl
3-(3-(2-methoxypyridin-3-yl)pyrazolo[1,5- 396.4 -- *
a]pyrimidin-5-yl)-2-oxoimidazolidine-1-carboxylate isopropyl
4-(3-(1H-benzo[d]imidazol-4-yl)pyrazolo[1,5- 405.5 -- **
a]pyrimidin-5-yl)piperazine-1-carboxylate
2-isobutyl-7-(3-(2-methoxypyridin-3-yl)pyrazolo[1,5- 421.5 -- ***
a]pyrimidin-5-yl)hexahydroimidazo[1,5-a]pyrazin-3(2H)- one
(1R,5S)-tert-butyl 3-(3-(2-methoxypyridin-3- 422.5 -- **
yl)pyrazolo[1,5-a]pyrimidin-5-yl)-3,6-
diazabicyclo[3.1.1]heptane-6-carboxylate isopropyl
4-(3-(2-methoxy-4-methylphenyl)pyrazolo[1,5- 409.5 ** ***
a]pyrimidin-5-yl)piperazine-1-carboxylate (1R,5S)-isopropyl
3-(3-(2-methoxypyridin-3- 408.5 -- **
yl)pyrazolo[1,5-a]pyrimidin-5-yl)-3,6-
diazabicyclo[3.1.1]heptane-6-carboxylate isopropyl
(2-((3-(2-methoxypyridin-3-yl)pyrazolo[1,5- 398.5 -- **
a]pyrimidin-5-yl)(methyl)amino)ethyl)(methyl)carbamate isopropyl
4-(3-(2-(2-methoxyethoxy)phenyl)pyrazolo[1,5- 439.5 -- **
a]pyrimidin-5-yl)piperazine-1-carboxylate isopropyl
4-(3-(2-(2-methoxyethoxy)pyridin-3- 440.5 -- **
yl)pyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-carboxylate ethyl
4-(3-(2-methoxy-5-methylpyridin-3-yl)pyrazolo[1,5- 396.4 ** ***
a]pyrimidin-5-yl)piperazine-1-carboxylate isopropyl
(2-((3-(2-methoxypyridin-3-yl)pyrazolo[1,5- 370.4 -- **
a]pyrimidin-5-yl)amino)ethyl)carbamate isopropyl
(2-((3-(2-methoxy-6-methylpyridin-3- 412.5 -- **
yl)pyrazolo[1,5-a]pyrimidin-5-
yl)(methyl)amino)ethyl)(methyl)carbamate isopropyl
(2-((3-(2-methoxy-5-methylpyridin-3- 412.5 -- **
yl)pyrazolo[1,5-a]pyrimidin-5-
yl)(methyl)amino)ethyl)(methyl)carbamate isopropyl
(2-((3-(5-fluoro-2-methoxypyridin-3- 416.4 -- ***
yl)pyrazolo[1,5-a]pyrimidin-5-
yl)(methyl)amino)ethyl)(methyl)carbamate ethyl
4-(3-(5-fluoro-2-methoxypyridin-3-yl)pyrazolo[1,5- 400.4 ** ***
a]pyrimidin-5-yl)piperazine-1-carboxylate ethyl
4-(3-(5-fluoro-2-methoxyphenyl)pyrazolo[1,5- 399.4 -- ***
a]pyrimidin-5-yl)piperazine-1-carboxylate ethyl
4-(3-(2-methoxy-6-methylpyridin-3-yl)pyrazolo[1,5- 396.4 ** ***
a]pyrimidin-5-yl)piperazine-1-carboxylate ethyl
4-(3-(2,6-dimethoxypyridin-3-yl)pyrazolo[1,5- 412.4 -- ***
a]pyrimidin-5-yl)piperazine-1-carboxylate isopropyl
(3-((3-(2-methoxypyridin-3-yl)pyrazolo[1,5- 412.5 ** ***
a]pyrimidin-5-yl)(methyl)amino)propyl)(methyl)carbamate isopropyl
(3-((3-(2-methoxy-6-methylpyridin-3- 426.5 ** ***
yl)pyrazolo[1,5-a]pyrimidin-5-
yl)(methyl)amino)propyl)(methyl)carbamate ethyl
4-(3-(2-d.sub.3-methoxypyridin-3-yl)pyrazolo[1,5- 385.4 ** ***
a]pyrimidin-5-yl)piperazine-1-carboxylate isopropyl
4-(3-(2-(2-(dimethylamino)ethoxy)pyridin-3- 453.5 -- **
yl)pyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-carboxylate isopropyl
(3-((3-(5-fluoro-2-methoxypyridin-3- 430.5 -- ***
yl)pyrazolo[1,5-a]pyrimidin-5-
yl)(methyl)amino)propyl)(methyl)carbamate
N-(2-(tert-butoxy)ethyl)-3-(2-methoxypyridin-3- 341.4 -- ***
yl)pyrazolo[1,5-a]pyrimidin-5-amine
N-(2-(tert-butoxy)ethyl)-3-(2-methoxy-6-methylpyridin-3- 355.4 --
** yl)pyrazolo[1,5-a]pyrimidin-5-amine tert-butyl
(2-((3-(2-methoxypyridin-3-yl)pyrazolo[1,5- 399.4 -- ***
a]pyrimidin-5-yl)oxy)ethyl)(methyl)carbamate isopropyl
(2-((3-(2-methoxypyridin-3-yl)pyrazolo[1,5- 385.4 -- **
a]pyrimidin-5-yl)oxy)ethyl)(methyl)carbamate (S)-isopropyl
(1-(3-(2,6-dimethoxypyridin-3- 440.5 -- **
yl)pyrazolo[1,5-a]pyrimidin-5-yl)pyrrolidin-3- yl)(methyl)carbamate
(S)-isopropyl (1-(3-(5-fluoro-2-methoxypyridin-3- 428.5 -- **
yl)pyrazolo[1,5-a]pyrimidin-5-yl)pyrrolidin-3- yl)(methyl)carbamate
(S)-isopropyl (1-(3-(2-methoxy-5-methylpyridin-3- 424.5 -- ***
yl)pyrazolo[1,5-a]pyrimidin-5-yl)pyrrolidin-3- yl)(methyl)carbamate
(S)-isopropyl (1-(3-(1,6-dimethyl-2-oxo-1,2-dihydropyridin- 424.5
-- * 3-yl)pyrazolo[1,5-a]pyrimidin-5-yl)pyrrolidin-3-
yl)(methyl)carbamate (S)-isopropyl
(1-(3-(2-d.sub.3-methoxy-6-methylpyridin-3- 424.5 ** ***
yl)pyrazolo[1,5-a]pyrimidin-5-yl)pyrrolidin-3- yl)(methyl)carbamate
(S)-isopropyl (1-(3-(2-methoxypyridin-3-yl)pyrazolo[1,5- 413.4 **
*** a]pyrimidin-5-yl)pyrrolidin-3-yl)(methyl)carbamate isopropyl
4-(3-(imidazo[1,2-a]pyridin-5-yl)pyrazolo[1,5- 405.5 -- **
a]pyrimidin-5-yl)piperazine-1-carboxylate (S)-isopropyl
(1-(3-(5-fluoro-2- 427.5 ** ***
methoxyphenyl)pyrazolo[1,5-a]pyrimidin-5-yl)pyrrolidin-3-
yl)(methyl)carbamate isopropyl
4-(3-(imidazo[1,2-a]pyridin-8-yl)pyrazolo[1,5- 405.5 -- **
a]pyrimidin-5-yl)piperazine-1-carboxylate propyl
4-(3-(2-methoxypyridin-3-yl)pyrazolo[1,5- 396.4 ** ***
a]pyrimidin-5-yl)piperazine-1-carboxylate propyl
4-(3-(2-methoxy-6-methylpyridin-3-yl)pyrazolo[1,5- 410.5 *** ***
a]pyrimidin-5-yl)piperazine-1-carboxylate propyl
4-(3-(5-fluoro-2-methoxypyridin-3-yl)pyrazolo[1,5- 414.4 ** ***
a]pyrimidin-5-yl)piperazine-1-carboxylate
3-(2-methoxypyridin-3-yl)-5-(4-(3,3,3- 406.4 -- *
trifluoropropyl)piperazin-1-yl)pyrazolo[1,5-a]pyrimidine
3-(5-fluoro-2-methoxypyridin-3-yl)-5-(4-(3,3,3- 424.4 -- *
trifluoropropyl)piperazin-1-yl)pyrazolo[1,5-a]pyrimidine
3-bromo-5-(4-(2,2,2-trifluoroethyl)piperazin-1- 364.2 -- **
yl)pyrazolo[1,5-a]pyrimidine 3-(2-methoxypyridin-3-yl)-5-(4-(2,2,2-
392.4 -- ** trifluoroethyl)piperazin-1-yl)pyrazolo[1,5-a]pyrimidine
3-(2-methoxy-6-methylpyridin-3-yl)-5-(4-(2,2,2- 406.4 -- **
trifluoroethyl)piperazin-1-yl)pyrazolo[1,5-a]pyrimidine
3-(5-fluoro-2-methoxypyridin-3-yl)-5-(4-(2,2,2- 410.4 -- *
trifluoroethyl)piperazin-1-yl)pyrazolo[1,5-a]pyrimidine tert-butyl
4-(3-(2-d.sub.3-methoxypyridin-3-yl)pyrazolo[1,5- 413.4 *** ***
a]pyrimidin-5-yl)piperazine-1-carboxylate tert-butyl
4-(3-(2-methoxy-6-methylpyridin-3- 424.5 *** ***
yl)pyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-carboxylate
tert-butyl 4-(3-(2-ethoxypyridin-3-yl)pyrazolo[1,5- 424.5 *** ***
a]pyrimidin-5-yl)piperazine-1-carboxylate tert-butyl
4-(3-(5-fluoro-2-methoxypyridin-3- 428.5 ** ***
yl)pyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-carboxylate
tert-butyl 4-(3-(5-fluoro-2-methoxyphenyl)pyrazolo[1,5- 427.5 ***
*** a]pyrimidin-5-yl)piperazine-1-carboxylate
N-(3-(tert-butoxy)propyl)-3-(2-methoxypyridin-3- 355.4 ** ***
yl)pyrazolo[1,5-a]pyrimidin-5-amine
N-(3-(tert-butoxy)propyl)-3-(2-methoxy-6-methylpyridin-3- 369.5 --
*** yl)pyrazolo[1,5-a]pyrimidin-5-amine
3-bromo-N-(3-(tert-butoxy)propyl)pyrazolo[1,5- 327.2 -- *
a]pyrimidin-5-amine isopropyl
4-(3-(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-5- 357.3 -- *
yl)piperazine-1-carboxylate isopropyl
4-(3-(pyridazin-4-yl)pyrazolo[1,5-a]pyrimidin-5- 367.4 -- *
yl)piperazine-1-carboxylate
N-(3-(tert-butoxy)propyl)-3-(2-methoxy-6-methylpyridin-3- 383.5 --
** yl)-N-methylpyrazolo[1,5-a]pyrimidin-5-amine
N-(3-(tert-butoxy)propyl)-3-(2-methoxypyridin-3-yl)-N- 369.5 -- **
methylpyrazolo[1,5-a]pyrimidin-5-amine
5-isopropyl-3-(4-(3-(2-methoxypyridin-3-yl)pyrazolo[1,5- 420.5 --
** a]pyrimidin-5-yl)piperazin-1-yl)-1,2,4-oxadiazole
(S)-2-amino-1-(4-(3-(2-methoxypyridin-3-yl)pyrazolo[1,5- 423.5 ***
*** a]pyrimidin-5-yl)piperazin-1-yl)-4-methylpentan-1-one isopropyl
4-(3-(4-(2-aminoethoxy)phenyl)pyrazolo[1,5- 424.5 ** ***
a]pyrimidin-5-yl)piperazine-1-carboxylate
(S)-2-amino-N-((S)-1-(3-(2-methoxypyridin-3- 437.5 *** ***
yl)pyrazolo[1,5-a]pyrimidin-5-yl)pyrrolidin-3-yl)-N,4-
dimethylpentanamide
(S)-2-amino-N-(2-((3-(2-methoxypyridin-3-yl)pyrazolo[1,5- 425.5 --
*** a]pyrimidin-5-yl)(methyl)amino)ethyl)-N,4- dimethylpentanamide
(S)-2-amino-1-(4-(3-bromopyrazolo[1,5-a]pyrimidin-5- 395.3 -- **
yl)piperazin-1-yl)-4-methylpentan-1-one isopropyl
4-(3-(4-(methoxycarbonyl)phenyl)pyrazolo[1,5- 423.5 -- **
a]pyrimidin-5-yl)piperazine-1-carboxylate
(S)-2-amino-1-(4-(3-(5-fluoro-2- 440.5 *** ***
methoxyphenyl)pyrazolo[1,5-a]pyrimidin-5-yl)piperazin-1-
yl)-4-methylpentan-1-one
(S)-2-amino-4-methyl-1-(4-(3-phenylpyrazolo[1,5- 392.5 *** ***
a]pyrimidin-5-yl)piperazin-1-yl)pentan-1-one isopropyl
4-(3-(4-(aminomethyl)phenyl)pyrazolo[1,5- 394.5 *** ***
a]pyrimidin-5-yl)piperazine-1-carboxylate
(S)-2-amino-1-(4-(3-(2-methoxypyridin-3-yl)pyrazolo[1,5- 409.5 ***
*** a]pyrimidin-5-yl)piperazin-1-yl)-3-methylbutan-1-one
(R)-2-amino-1-(4-(3-(2-methoxypyridin-3-yl)pyrazolo[1,5- 423.5 --
** a]pyrimidin-5-yl)piperazin-1-yl)-4-methylpentan-1-one isopropyl
4-(3-(2-fluoro-6-methoxypyridin-3- 414.4 -- **
yl)pyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-carboxylate
(S)-2-amino-1-(4-(3-(2-fluorophenyl)pyrazolo[1,5- 396.5 *** ***
a]pyrimidin-5-yl)piperazin-1-yl)-3-methylbutan-1-one
(S)-2-amino-1-(4-(3-chloropyrazolo[1,5-a]pyrimidin-5- 350.8 -- **
yl)piperazin-1-yl)-4-methylpentan-1-one
(S)-2-amino-1-(4-(3-fluoropyrazolo[1,5-a]pyrimidin-5- 334.4 -- *
yl)piperazin-1-yl)-4-methylpentan-1-one 2-methoxyethyl
4-(3-(5-fluoro-2- 429.4 *** ***
methoxyphenyl)pyrazolo[1,5-a]pyrimidin-5-yl)piperazine-
1-carboxylate 2-methoxyethyl 4-(3-(5-fluoro-2-methoxypyridin-3-
430.4 ** ***
yl)pyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-carboxylate
2-methoxyethyl 4-(3-(2-methoxy-6-methylpyridin-3- 426.5 ** ***
yl)pyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-carboxylate
(S)-1-(4-(3-(2-methoxypyridin-3-yl)pyrazolo[1,5- 437.5 *** ***
a]pyrimidin-5-yl)piperazin-1-yl)-4-methyl-2-
(methylamino)pentan-1-one
(S)-2-amino-3-methoxy-1-(4-(3-(2-methoxypyridin-3- 411.5 *** ***
yl)pyrazolo[1,5-a]pyrimidin-5-yl)piperazin-1-yl)propan-1- one
(1-aminocyclopentyl)(4-(3-(2-methoxypyridin-3- 421.5 *** ***
yl)pyrazolo[1,5-a]pyrimidin-5-yl)piperazin-1-yl)methanone
(S)-2-amino-1-(4-(3-(2-methoxypyridin-3-yl)pyrazolo[1,5- 423.5 ***
*** a]pyrimidin-5-yl)piperazin-1-yl)-3,3-dimethylbutan-1-one
2-amino-1-(4-(3-(2-methoxypyridin-3-yl)pyrazolo[1,5- 395.5 -- **
a]pyrimidin-5-yl)piperazin-1-yl)-2-methylpropan-1-one
(S)-(4-(3-(2-methoxypyridin-3-yl)pyrazolo[1,5-a]pyrimidin- 407.5 **
*** 5-yl)piperazin-1-yl)(pyrrolidin-2-yl)methanone 2-methoxyethyl
4-(3-(2-ethoxypyridin-3-yl)pyrazolo[1,5- 426.5 -- ***
a]pyrimidin-5-yl)piperazine-1-carboxylate
(S)-2-amino-1-(4-(3-(3-fluorophenyl)pyrazolo[1,5- 396.5 *** ***
a]pyrimidin-5-yl)piperazin-1-yl)-3-methylbutan-1-one
(S)-2-amino-3-methyl-1-(4-(3-phenylpyrazolo[1,5- 378.5 *** ***
a]pyrimidin-5-yl)piperazin-1-yl)butan-1-one
(S)-2-amino-1-(4-(3-(4-fluorophenyl)pyrazolo[1,5- 396.5 *** ***
a]pyrimidin-5-yl)piperazin-1-yl)-3-methylbutan-1-one
(S)-2-amino-1-(4-(3-(2,5-difluorophenyl)pyrazolo[1,5- 414.5 *** ***
a]pyrimidin-5-yl)piperazin-1-yl)-3-methylbutan-1-one
(S)-2-amino-1-(4-(3-(2-fluoro-4- 410.5 ** ***
methylphenyl)pyrazolo[1,5-a]pyrimidin-5-yl)piperazin-1-
yl)-3-methylbutan-1-one
(S)-2-amino-1-(4-(3-(2-methoxyphenyl)pyrazolo[1,5- 408.5 *** ***
a]pyrimidin-5-yl)piperazin-1-yl)-3-methylbutan-1-one
3-methoxypropyl 4-(3-(2-methoxypyridin-3- 426.5 -- ***
yl)pyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-carboxylate
2-ethoxyethyl 4-(3-(2-methoxypyridin-3-yl)pyrazolo[1,5- 426.5 -- **
a]pyrimidin-5-yl)piperazine-1-carboxylate 2-(2-methoxyethoxy)ethyl
4-(3-(2-methoxypyridin-3- 456.5 -- **
yl)pyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-carboxylate isopropyl
4-(3-(4-methoxypyrimidin-5-yl)pyrazolo[1,5- 397.4 -- **
a]pyrimidin-5-yl)piperazine-1-carboxylate
(S)-2-amino-3,3-dimethyl-1-(4-(3-phenylpyrazolo[1,5- 392.5 ** ***
a]pyrimidin-5-yl)piperazin-1-yl)butan-1-one
(S)-2-amino-1-(4-(3-(2-methoxyphenyl)pyrazolo[1,5- 422.5 *** ***
a]pyrimidin-5-yl)piperazin-1-yl)-3,3-dimethylbutan-1-one
(S)-2-amino-1-(4-(3-(5-fluoro-2- 440.5 *** ***
methoxyphenyl)pyrazolo[1,5-a]pyrimidin-5-yl)piperazin-1-
yl)-3,3-dimethylbutan-1-one
(S)-1-(4-(3-(2-methoxypyridin-3-yl)pyrazolo[1,5- 437.5 ** ***
a]pyrimidin-5-yl)piperazin-1-yl)-3,3-dimethyl-2-
(methylamino)butan-1-one
(S)-1-(4-(3-(2-methoxypyridin-3-yl)pyrazolo[1,5- 409.5 -- **
a]pyrimidin-5-yl)piperazin-1-yl)-4-methylpentan-2-amine
(S)-2-amino-1-(4-(3-(5-fluoro-2- 426.5 *** ***
methoxyphenyl)pyrazolo[1,5-a]pyrimidin-5-yl)piperazin-1-
yl)-3-methylbutan-1-one
(1-(3-(2-methoxypyridin-3-yl)pyrazolo[1,5-a]pyrimidin-5- 322.3 --
** yl)-1H-imidazol-4-yl)methanol (R)-tetrahydrofuran-3-yl
4-(3-(2-methoxypyridin-3- 424.5 *** ***
yl)pyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-carboxylate
1-methoxypropan-2-yl 4-(3-(2-methoxypyridin-3- 426.5 *** ***
yl)pyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-carboxylate
tetrahydro-2H-pyran-4-yl 4-(3-(2-methoxypyridin-3- 438.5 ** ***
yl)pyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-carboxylate
1-methoxy-2-methylpropan-2-yl 4-(3-(2-methoxypyridin-3- 440.5 * ***
yl)pyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-carboxylate
5-(4-(methoxymethyl)-1H-imidazol-1-yl)-3-(2- 336.3 -- **
methoxypyridin-3-yl)pyrazolo[1,5-a]pyrimidine
(2S,3R)-2-amino-3-methoxy-1-(4-(3-(2-methoxypyridin-3- 425.5 ** ***
yl)pyrazolo[1,5-a]pyrimidin-5-yl)piperazin-1-yl)butan-1- one
(S)-(4-(3-(2-methoxypyridin-3-yl)pyrazolo[1,5-a]pyrimidin- 423.5 --
*** 5-yl)piperazin-1-yl)(morpholin-3-yl)methanone
(3-aminotetrahydrofuran-3-yl)(4-(3-(2-methoxypyridin-3- 423.5 **
*** yl)pyrazolo[1,5-a]pyrimidin-5-yl)piperazin-1-yl)methanone
2-(dimethylamino)ethyl 4-(3-(2-methoxypyridin-3- 425.5 -- **
yl)pyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-carboxylate
2-(tert-butoxy)ethyl 4-(3-(2-methoxypyridin-3- 454.5 -- ***
yl)pyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-carboxylate
1,3-dimethoxypropan-2-yl 4-(3-(2-methoxypyridin-3- 456.5 -- **
yl)pyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-carboxylate
(R)-tetrahydrofuran-3-yl 4-(3-(5-fluoro-2-methoxypyridin- 442.4 ***
*** 3-yl)pyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1- carboxylate
(R)-tetrahydrofuran-3-yl 4-(3-(5-fluoro-2- 441.5 *** ***
methoxyphenyl)pyrazolo[1,5-a]pyrimidin-5-yl)piperazine-
1-carboxylate (R)-tetrahydrofuran-3-yl
4-(3-(2-methoxy-6-methylpyridin- 438.5 ** ***
3-yl)pyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1- carboxylate
oxetan-3-yl 4-(3-(2-methoxypyridin-3-yl)pyrazolo[1,5- 410.4 *** ***
a]pyrimidin-5-yl)piperazine-1-carboxylate (S)-tetrahydrofuran-3-yl
4-(3-(2-methoxypyridin-3- 424.5 *** ***
yl)pyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-carboxylate
(S)-1-methoxypropan-2-yl 4-(3-(2-methoxypyridin-3- 426.5 *** ***
yl)pyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-carboxylate
(R)-1-methoxypropan-2-yl 4-(3-(2-methoxypyridin-3- 426.5 * ***
yl)pyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-carboxylate
2-methoxy-2-oxoethyl 4-(3-(2-methoxypyridin-3- 426.4 -- **
yl)pyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-carboxylate isopropyl
4-(3-bromopyrazolo[1,5-a]pyrimidin-5-yl)-d.sub.8- 376.2 -- **
piperazine-1-carboxylate 2-methoxyethyl
4-(3-(2-methoxypyridin-3-yl)pyrazolo[1,5- 420.4 ** ***
a]pyrimidin-5-yl)-d.sub.8-piperazine-1-carboxylate isopropyl
4-(3-(2-methoxy-6-methylpyridin-3- 418.4 *** ***
yl)pyrazolo[1,5-a]pyrimidin-5-yl)-d.sub.8-piperazine-1- carboxylate
N-(3-(2-methoxypyridin-3-yl)pyrazolo[1,5-a]pyrimidin-5- 311.3 -- **
yl)butyramide
5.6.41. Pharmacological Effects
[0283] Studies of AAK1 knockout mice showed that disruption of the
AAK1 gene affects pain response as measured using the formalin paw
test. See Example 5.6.1, above. The same test was used to confirm
that the administration of an AAK1 inhibitor can also affect pain
response.
[0284] Mice were tested for nociception with Automatic Nociception
Analyzers (purchased from the Ozaki lab at University of
California, San Diego). A metal band was placed around the left
hind paw of each mouse with superglue 30 minutes prior to testing.
After the 30-minute acclimation period, 20 .mu.l of 5% formalin was
subcutaneously injected in the dorsal surface of the left hind paw.
Mice were individually housed in cylindrical chambers for 45
minutes. Fresh 5% formalin solution was prepared by diluting
formaldehyde (Formalde-fresh 20%, Fisher Scientific, Fair Lawn,
N.J.) with distilled water. Investigatory compounds were
administered 30 minutes prior to formalin injection.
[0285] A computer software recorded flinches per minute, total
flinches for Phase I (acute phase=first 8 minutes), and total
flinches for Phase II (tonic phase between 20-40 minutes) through
an electromagnetic field. See Yaksh T L, Ozaki G, McCumber D,
Rathbun M, Svensson C, Malkmus S, Yaksh M C. An automated flinch
detecting system for use in the formalin nociceptive bioassay. J
Appl Physiol., 2001; 90:2386-402.
[0286] Various compounds of the invention were tested at different
doses. Gabapentin and pregabalin were used as positive controls.
Results are shown below in Table 2, wherein "*" means an effect
equal to or greater than 50 percent of that of gabapentin at 200
mpk, "**" means an effect equal to or greater than 100 percent of
that of gabapentin at 200 mpk, "sc" means subcutaneous
administration, and "po" means oral administration.
TABLE-US-00002 TABLE 2 Compound Dose (mpk) Effect Gabapentin 50 sc
* Gabapentin 200 sc ** Pregabalin 50 sc * (S)-tert-butyl
2-(((3-(2-methoxyphenyl)pyrazolo[1,5-a]pyrimidin-5- 10 sc *
yl)amino)methyl)pyrrolidine-1-carboxylate (S)-tert-butyl
2-(((3-(2-methoxyphenyl)pyrazolo[1,5-a]pyrimidin-5- 60 sc *
yl)amino)methyl)pyrrolidine-1-carboxylate (S)-tert-butyl
2-(((3-(pyridin-4-yl)pyrazolo[1,5-a]pyrimidin-5- 30 sc **
yl)amino)methyl)pyrrolidine-1-carboxylate isopropyl
4-(3-(2-methoxyphenyl)pyrazolo[1,5-a]pyrimidin-5- 30 sc **
yl)piperazine-1-carboxylate isopropyl
4-(3-(2-methoxypyridin-3-yl)pyrazolo[1,5-a]pyrimidin-5- 10 po *
yl)piperazine-1-carboxylate isopropyl
4-(3-(2-methoxypyridin-3-yl)pyrazolo[1,5-a]pyrimidin-5- 30 po **
yl)piperazine-1-carboxylate
These results demonstrate that AAK1 inhibitors can be used to treat
pain.
[0287] All publications (e.g., patents and patent applications)
cited above are incorporated herein by reference in their
entireties.
* * * * *