SUBSTITUTED PYRAZOLE ANALOGUES AS RAR ANTAGONISTS
Osteoarthritis is a complex degenerative disease characterized by progressive
destruction of articular cartilage and peri-articular structures including bones, synovial,
and associated fibrous joint tissues. Existing drug therapies can reduce pain associated
with osteoarthritis, but over time become only moderately effective. Each of the current
standard of care therapies has variable risk/benefit considerations. Individuals can
become refractory to specific drug treatments and/or are contraindicated for the
treatments due to pre-existing or emergent cardiovascular and/or gastric intestinal
conditions. Consequently, there remains a need for additional treatment options to treat
and alleviate pain from osteoarthritis.
Retinoids (including RAR agonists), are known to cause and/or exacerbate pain in
animal models, demonstrate catabolic activity for cartilage, and induce osteoarthritis-like
processes in animal models. Compounds which exhibit RAR antagonistic activity may
provide an alternative treatment regime for patients suffering from osteoarthritis pain.
United States Patent 5,464,178 discloses compounds including the compound
below:
which is disclosed as being useful to treat pain associated with inflammation and arthritis.
However the compounds are not described as exhibiting RAR gamma antagonism.
The present invention provides an alternative treatment for osteoarthritis, and in
particular, an alternative treatment for the pain associated with osteoarthritis. The present
invention may also address one or more deficiencies, such as, a reduction in the risks of
undesired interactions with other drugs and the risk of pre-existing or emergent
cardiovascular and/or gastric intestinal conditions under the current standard of care for
osteoarthritis treatment regimes. Further, compounds of the present invention selectively
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bind to RARy and may therefore provide advantages over non-selective RAR antagonists,
which can be accompanied by a broad spectrum of toxic side effects.
The present invention provides a compound having a Formula I below:
wherein: A is CH o ;X is CH o ; Rl is selected from: -S0 2CH3, -S0 2N(CH3)2,
-C(0)N(R3) 2, -C(0)R4, and -NHS0 2CH3; R2 is selected from: -C3-4 alkyl,
-OCH(CH3)2, and -SCH(CH3)2; each R3 is independently selected from: H and -CH3;
R4 is selected from: 4-morpholinyl, 1-piperidinyl, 4-thiomorpholinyl, -NH(CH2)3OH,
and 4-methyl-l-piperazinyl; and provided that when one of A or X is N the other one of
A or X is CH; or pharmaceutically acceptable salts thereof.
The present invention also provides of compounds of Formula I above, or
pharmaceutically acceptable salts thereof, wherein both A and X are CH. In another form
A is N and X is CH. In still yet another form A is CH and X is N.
The present invention also provides compounds of Formula I, or pharmaceutically
acceptable salts thereof, wherein Rl is selected from -C(0)N(R3) 2 or -C(0)R4. In other
embodiments when Rl is selected from -C(0)N(R3) 2 or -C(0)R4; R2 is selected from:
-C3_4 alkyl -OCH(CH3)2, and -SCH(CH3)2; more preferably R2 is isopropyl, -butyl
and -SCH(CH3)2. In another form, when Rl is selected from -C(0)N(R3) 2 or -C(0)R4,
R2 is selected from: isopropyl, tert-butyl -OCH(CH3)2, and -SCH(CH3)2; each R3 is
independently H, or -CH3, and R4 is selected from: 4-morpholinyl, 1-piperidinyl,
4-thiomorpholinyl and 4-methyl-l-piperazinyl. In another form Rl is -C(0)N(R3) 2, R2
is selected from: isopropyl, tert-butyl -OCH(CH3)2, and -SCH(CH3)2; and R3 is
independently H, or -CH3. In another form, Rl is -C(0)R4; R2 is selected from: -C3-4
alkyl, -OCH(CH3)2, and -SCH(CH3)2; and R4 is selected from 4-morpholinyl,
1-piperidinyl, 4-thiomorpholinyl, and 4-methyl-l-piperazinyl. More preferably Rl is -
C(0)R4; R2 is selected from: isopropyl, -butyl and -SCH(CH3)2; and R4 is 4-
morpholinyl, 1-piperidinyl, 4-thiomorpholinyl, and 4-methyl-l-piperazinyl, More
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preferably Rl is -C(0)R4; R2 is selected from: isopropyl, -butyl and -SCH(CH )2;
and R4 is 4-morpholinyl or 4-methyl- 1-piperazinyl. Still yet more preferably, Rl is -
C(0)R4; R2 is -butyl; and R4 is 4-methyl- 1-piperazinyl.
The present invention also provides compounds of Formula I, or pharmaceutically
acceptable salts thereof, wherein, R2 preferably is selected from: -C3-4 alkyl,
and -SCH(CH )2. More preferably R2 is selected from: isopropyl, -butyl, and -
SCH(CH )2. Still more preferably the R2 is isopropyl or tert-butyl.
In one embodiment the present invention also provides compounds of Formula I,
or a pharmaceutically acceptable salt thereof, wherein each R3 is -CH 3. In another
embodiment the present invention provides compounds of Formula I, or a
pharmaceutically acceptable salt thereof, wherein each R3 is H.
The present invention provides compounds of Formula I, or pharmaceutically
acceptable salts thereof, wherein R4 is is selected from: 4-morpholinyl, 1-piperidinyl,
4-thiomorpholinyl, and 4-methyl- 1-piperazinyl or pharmaceutically acceptable salts
thereof. More preferably, R4 is is selected from: 1-piperidinyl, 4-morpholinyl, and
4-methyl- 1-piperazinyl. More preferably R4 is 4-morpholinyl or
4-methyl- 1-piperazinyl. Still more preferably R4 is 4-methyl- 1-piperazinyl.
The present invention also provides compounds of Formula I, or
pharmaceutically acceptable salts thereof, wherein A is CH; Rl is selected from:
-SO2CH3, -S0 2N(CH3)2, -C(0)N(R3) 2, -C(0)R4, and -NHS0 2CH3; R2 is selected
from: -C3-4 alkyl, -OCH(CH3)2, and -SCH(CH3)2; each R3 is independently H or -CH3;
and R4 is selected from: 4-morpholinyl, 1-piperidinyl, 4-thiomorpholinyl,
-NH(CH2)3OH and 4-methyl- 1-piperazinyl.
The present invention also provides compounds of Formula I, or pharmaceutically
acceptable salts thereof, wherein A is CH; X is CH; Rl is -C(0)N(R3) 2, or -C(0)R4;
R2 is selected from: -C3_4 alkyl, -OCH(CH3)2, and -SCH(CH3)2; each R3 is
independently H or -CH3; and R4 is selected from: 4-morpholinyl, 1-piperidinyl, 4-
thiomorpholinyl, -NH(CH2)3OH and 4-methyl- 1-piperazinyl; or pharmaceutically
acceptable salts thereof.
The present invention also provides compounds of Formula I, or pharmaceutically
acceptable salts thereof, wherein A is CH; X is CH; Rl is -C(0)N(R3) 2, or -C(0)R4;
R2 is selected from: -C3_4 alkyl; each R3 is independently H or -CH3; and R4 is selected
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from: 4-morpholinyl, 1-piperidinyl, 4-thiomorpholinyl, - H(CH2) 0 H and
4-methyl- 1-piperazinyl.
The present invention also provides compounds of Formula I, or pharmaceutically
acceptable salts thereof, wherein A is CH; X is N; Rl is -C(0)N(R3) 2 and R2 is -C3-4
alkyl; R3 is H or -CH3. Preferably R2 is -butyl.
The present invention also provides compounds of Formula I, or pharmaceutically
acceptable salts thereof, wherein A is N; X is CH; Rl is -S0 2CH3 or -C(0)N(CH 3)2;
and R2 is -C3-4 alkyl. Preferably R2 is tert-butyl.
A particularly preferred compound of the present invention is 4-[5-(3,5-Di-tertbutylphenyl)-
l-[4-(4-methylpiperazine-l-carbonyl)phenyl]pyrazol-3-yl]benzoic acid, or a
pharmaceutically acceptable salt thereof.
The present invention also provides a pharmaceutical composition that comprises
a compound in any of the forms described above for Formula I, or a pharmaceutically
acceptable salt thereof, and at least one pharmaceutically acceptable carrier, excipient, or
diluent.
The present invention also provides a pharmaceutical composition that comprises
a compound in any of the forms described above for Formula I, or a pharmaceutically
acceptable salt thereof, least one pharmaceutically acceptable carrier, excipient, or diluent
and one or more therapeutic agents.
The present invention provides a method of treating osteoarthritis in a patient in
need of treatment. The method comprises administering an effective amount of a
compound, in any of the forms described above for Formula I, or a pharmaceutically
acceptable salt thereof to the patient.
The present invention also provides a method of treating osteoarthritis in a patient
in need of treatment. The method comprises administering an effective amount of a
pharmaceutical composition comprising a compound in any of the forms described above
for Formula I, or a pharmaceutically acceptable salt thereof to the patient.
The present invention also provides a compound in any of the forms described
above for Formula I or a pharmaceutically acceptable salt thereof for use in therapy.
The present invention also provides a compound in any of the forms described
above for Formula I or a pharmaceutically acceptable salt thereof for use in the treatment
of osteoarthritis, more particularly for the treatment of pain associated with osteoarthritis.
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The present invention also provides a compound in any of the forms described
above for Formula I or a pharmaceutically acceptable salt thereof for use in the
manufacture of a medicament. Prefereably the medicament is for treating osteoarthritis.
Still more preferably the medicament is for treating the pain associated with
osteoarthritis.
The present invention also provides an intermediate according to Formula II
wherein: R is selected from C1-4 alkyl, C1-4 haloalkyl, C 3-6cycloalkyl, C1-4 alkyl-C3-6
cycloalkyl, phenyl, and C1-5 alkylphenyl; A is CH or N; X is CH or N Rl is selected
from: -S0 2CH3, -S0 2N(CH3)2, -C(0)N(R3) 2, -C(0)R4, and -NHS0 2CH3; R2 is
selected from: -C3-4 alkyl, -OCH(CH3)2, and -SCH(CH3)2; each R3 is independently
selected from: H and -CH3; R4 is selected from: 4-morpholinyl, 1-piperidinyl,
4-thiomorpholinyl, -NH(CH2)30H, and 4-methyl-l-piperazinyl; and provided that when
one of A or X is N, the other one of A or X is CH.
The present invention also provides a process of preparing a compound of
Formula I,
A is CH o ; X is CH o ; Rl is selected from: -S0 2CH3, -S0 2N(CH3)2,
-C(0)N(R4) 2, -C(0)R4, and -NHS0 2CH3; R2 is selected from: -C3-4 alkyl,
-OCH(CH3)2, and -SCH(CH3)2; each R3 is independently selected from: H, and -CH3
R4 is selected from: 4-morpholinyl, 1-piperidinyl, 4-thiomorpholinyl, -NH(CH2)30H,
and 4-methyl-l-piperazinyl. The process comprising de-esterifying a compound of
Formula II;
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wherein R1-R4 are as described above and R is selected from: C1-4 alkyl, C1-4 haloalkyl,
C3-6 cycloalkyl, C1-4 alkyl-C3-6 cycloalkyl, phenyl, and C1-5 alkylphenyl to provide a
compound of formula I, or a pharmaceutically acceptable salt thereof.
The present invention also provides a compound which is 4-[5-(3,5-Di-tertbutylphenyl)-
l-[4-(4-methylpiperazine-l-carbonyl)phenyl]pyrazol-3-yl]benzoic acid in
crystalline form characterized by an X-ray powder diffraction pattern obtained from a
CuKa source (l=1.54056 A) which comprises peaks at: a) 5.4, 7.5, 14.6, and 19.9 +/-0.2
in 2Q; or b) 5.4, 7.5, 14.6, 16.0, 19.4, and 19.9 +/- 0.2 in 2Q; or c) 5.4, 7.5, 14.6, 15.7,
16.0, 19.4, 19.9 and 22.1 +/-0.2 in 2Q.
Figure 1 is a spectrogram of a representative XRD pattern for crystalline 4-[5-
(3,5-Di-tert-butylphenyl)-l-[4-(4-methylpiperazine-l-carbonyl)phenyl]pyrazol-3-
yl]benzoic acid. The XRD spectrogram was obtained as described below.
The term alkyl as used herein refers to a carbon substituent which can be a straight
chain, e.g., -CH2CH2CH3, -CH2CH2CH2CH3 or a branched chain, i.e., -CH(CH3)2,
-C(CH3)3 or -CH2CH(CH3)2.
Preferably for all the forms of the compounds described above, the alkyl chain for
the R2 substituent group is a branched alkyl chain, preferably an isopropyl alkyl group or
a tert-butyl group.
The term C1-4 haloalkyl as used herein refers to a hydrocarbon substituent of one
to four carbons where one or more of the hydrogens is replaced with a halogen. The
haloalkyl can be a perhalo alkyl where all the hydrogen atoms are replaced with a halogen
atom. Alternatively 1, 2, 3, or more hydrogens, can be replaced by a halogen. Further the
halogens need not be attached to the same carbon atom.
A "patient" refers to a mammal, preferably a human.
The phrase "pharmaceutically-acceptable salt" refers to salts of the compounds of
the invention considered to be acceptable for clinical and/or veterinary use.
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Pharmaceutically acceptable salts and common methodology for preparing them are well
known in the art. See, e.g., P. Stahl, et ah, Handbook of Pharmaceutical Salts: Properties,
Selection and Use, (VCHA/Wiley-VCH, 2002); S.M. Berge, etal, "Pharmaceutical
Salts," Journal of Pharmaceutical Sciences, Vol. 66, No. 1, January 1977.
The terms and abbreviations used in the instant Schemes, Preparations, Examples
and Procedures have their normal meanings unless otherwise designated.
As used herein, the following terms have the meanings indicated: "AcOH" refers
to acetic acid, "ATRA" refers to all-trans retinoic acid; "BOP" refers to benzotriazol-1-
yloxy)tris(dimethylamino)phosphoniumhexafluorophosphate; "CDI" refers to 1,1'-
carbonyldiimidazole; "CHAPS" refers to 3-[(3-cholamidopropyl)dimethylammonio]-lpropanesulfonate
hydrate; "DCM" refers to dichloromethane; "DDQ" refers to 2,3-
dichloro-5,6-dicyano- 1,4-benzoquinone or 4,5-dichloro-3 ,6-dioxo-cyclohexa- 1,4-diene-
1,2-dicarbonitrile; "DMF" refers to dimethylformamide; "DMSO" refers to methyl
sulfoxide; "DTT" refers to dithiothreitol; "ED " refers to l-(3-dimethylaminopropyl)-3-
ethylcarbodiimide hydrochloride; "EtOAc" refers to ethyl acetate; "EtOH" refers to
ethanol; "FBS" refers to fetal bovine serum; "HEPES" refers to 4-(2-hydroxyethyl)-lpiperazineethanesulfonic
acid; "HOBT" refers to 1-hydroxybenzotriazole hydrate; LCES/
MS refers to liquid chromatography electrospray mass spectroscopy; "MeOH" refers
to methanol; "MTBE" refers to methyl t-butyl ether; "PCPNiCl" refers to the reagent
wherein the phosphorous-carbon-phosphorous atoms are bound to the nickel in a pincer
complex; "SPA" refers to scintillation proximity assay; "TFA" refers to trifluoroacetic
acid; "THF" refers to tetrahydrofuran; and "TTNPB" refers to tetrahydro-5, 5,8,8-
tetramethyl-2-naphthalenyl)- 1-propenyljbenzoic acid.
The compounds of the present invention may be prepared by a variety of
procedures known in the art as well as the general procedures illustrated in Schemes 1 - 5
below. However, the following discussion is not intended to be limiting to the scope of
the present invention in any way. For example, the specific synthetic steps for each of the
routes described may be combined in different ways, or in conjunction with steps from
different schemes, to prepare additional compounds of the present invention.
The reagents and starting materials are readily available to one of ordinary skill in
the art or may be made by procedures which are selected from standard techniques of
organic and heterocyclic chemistry, and the procedures described in the Examples below.
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The substituents Rl, R2, R3, A, and X are defined as previously indicated. Other
variables are defined in the text accompanying the Schemes. Unless specified to the
contrary, the naming of the following Preparations and Examples is done using the IUPAC
naming feature in Symyx Draw® version 3.2 (Symyx Solutions, Inc.).
Scheme 1
Scheme 1 illustrates the synthesis of compounds of the invention as shown by
formula (8).
In Step A, an aldehyde of formula (1) (A = CH or N) is condensed with 4-
acetylbenzoic acid (2) to provide a propenyl benzoic acid of formula (3). The reaction
proceeds in a mixture of EtOH and water at a temperature of 10 to 80 °C for 12 h to 2
days.
In Step B, propenyl benzoic acid (3) is esterified to a benzoate (4) using acid
catalysis; preferably the benzoate is methyl benzoate prepared using methanesulfonic acid
in MeOH at -10 to 50 °C for 4 to 24 h.
In Step C, benzoate (4) is reacted with a phenyl or pyridyl hydrazine of formula
(5) (X = CH or N) to provide a dihydropyrazole of formula (6). Preferred conditions use
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a mixture of 1-butanol and acetic acid at a temperature of 70 °C to the reflux temperature
of the solvent for 8 to 24 h.
In Step D, dihydropyrazole (6) is oxidized to a pyrazole benzoate of formula (7).
The literature provides a variety of options to the skilled artisan for such an oxidation.
Preferred conditions make use of manganese (IV) oxide in a mixture of 1,2-
dichloroethane and acetic acid at 50 °C to the reflux temperature of the solvent for 4 to 24
h. Other preferred conditions use DDQ in refluxing toluene.
In Step E, pyrazole benzoate (7) is hydrolyzed to a pyrazole benzoic acid of
formula (8) using an inorganic base, preferably lithium hydroxide in a mixture of
THF/MeOH or THF/MeOH/water for 4 to 24 h at 0 to 60 °C.
The benzaldehydes or pyridine-4-carboxaldehydes of formula (1) are
commercially available or can be readily prepared by literature procedures. Likewise the
phenyl and pyridyl hydrazines of formula (5) are commercially available or can be readily
prepared.
Scheme 2
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Scheme 2 illustrates an alternate means for making compounds of the invention
(12) whereR2a = I or R2, and R3a is N(R3)2 or R4.
In Step A, a benzoic acid or a picolinic acid (9) is amidated to form a benzamide
or pyridinecarboxamide of formula (10). There are a variety of coupling reagents and
reaction condtions available to the skilled artisan for making an amide from a carboxylic
acid. Preferred conditions use BOP as a coupling reagent, in an inert solvent, such as
DMF, with an organic base, such as diisopropylethylamine in the presence of the
appropriate amine. Other preferred conditions use EDCI and HOBT in dichloromethane.
Alternately, the carbonylimididazole is made in situ using CDI and then reacted with the
amine.
In Step B, when R2a = I and A = CH, the iodo ?-butylphenyl (10) is transformed
to the isopropylthiophenyl of formula (11). The reaction is performed in an inert solvent,
such as DMF, using 1,2-diisopropylsulfane, in the presence of zinc and a nickel PCP
pincer complex, such as [NiCl{C6H3-2,6-(OPPh2)2}] (Tetrahedron Lett. 2006, 49, 5059).
The reaction proceeds at a temperature of 80 - 120 °C for 4 - 24 h.
In Step C, the benzoate of formula ( 11) or (10) is hydrolyzed as previously
described for Scheme 1, Step E.
The benzoic acid or picolinic acid (9) can be made by cyclizing the corresponding
hydrazine with a methyl propenoyl benzoate (3) as described for Scheme 1, Step C.
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Scheme 3
Scheme 3 illustrates further chemical modifications leading to compounds of the
invention (15).
The nitrophenyl or 2-nitropyridyl (13) can be made by cyclizing the
corresponding hydrazine with a propenoyl benzoate (4) as described for Scheme 1, Step
C. 4-(Nitrophenyl)hydrazine and 5-hydrazinyl-2-nitro-pyridine are commercially
available or can be made using chemistry known in the art. Alternatively, the aniline or
2-aminopyridine (14) can be obtained using other phenylhydrazine or hydrazinopyridine
intermediates which are then transformed to the free amine by the skilled artisan. If
necessary, appropriate protecting groups can be used.
In Step A, a nitrophenyl or 2-nitropyridyl of formula (13) is reduced to the aniline
or 2-aminopyridyl of formula (14). The reduction is performed in a solvent mixture of
MeOH and water in the presence of iron and ammonium chloride. The reaction is heated
at reflux temperature for 1 - 8 h.
Following the reduction, the resulting amine is sulfonylated in Step B using
methanesulfonyl chloride in the presence of pyridine. Hydrolysis, Step C, is as
previously described in Scheme 1, Step E.
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Scheme 4
Scheme 4 illustrates an alternate route to constructing the pyrazole core, leading to
compounds of the invention (8).
In Step A the phenyl or pyridyl hydrazine (5) is cyclized with 4-(2-
methoxycarbonyl-acetyl)-benzoate (16a, Ra = CC^Me) or with a 4-(2-
cyanoacetyl)benzoate (16b, R = CN) to provide the hydroxypyrazole (17a, Y = OH)
(Synlett 2004, 795) or aminopyrazole (17b, Y = NH2) respectively. The reaction proceeds
in a protic solvent, such as MeOH (for the methyl benzoate), at the refluxing temperature
of the solvent.
In Step B, the hydroxypyrazole (17a) and the aminopyrazole (17b) are
transformed to the bromopyrazole (18a) and the iodopyrazole (18b), respectively. The
bromopyrazole (18a) is formed using phosphorous tribromide in an inert solvent such as
acetonitrile, at the refluxing temperature of the solvent. The iodopyrazole (18b) is formed
by oxidative deamination of the aminopyrazole (17b) using an alkyl nitrite, such as
isoamyl nitrite or ?-butyl nitrite, in the presence of a suitable iodide source such as
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copper(I) iodide with or without the addition of diiodomethane. The reaction takes place
in an inert solvent, such as acetonitrile, at 60 - 85 °C over 1 to 12 h.
In Step C, a tert-butylphenyl or tert-pyridyl pyrazole of formula (7) is obtained
using a cross-coupling reaction between the bromo or iodopyrazole (18a or 18b) and a
phenyl or 4-pyridyl boronate ester (19). Although the boronate ester is shown, it will be
known to one skilled in the art that the boronic acids can work equally as well in Suzuki
reactions such as these. Furthermore, it is known to the skilled artisan that there are
various reaction conditions and Pd catalysts that can be used in such a reaction. The
preferred conditions when Z = Br (18a) use a palladium catalyst, such as
tetrakis(triphenylphosphine)palladium(0), in an inert solvent such as THF, in the presence
of an inorganic base such as aqueous sodium carbonate. The reaction proceeds over 2 -
24 h at about 50 to 65 °C. Preferred conditions when Z = I (18b) make use of
bis(triphenylphosphine)palladium(II) chloride in a solvent mixture of THF/water in the
presence of a inorganic base, such as potassium carbonate. The reaction proceeds over 2
- 24 h at about 60 °C to the reflux temperature of the solvent.
Hydrolysis, Step D, is as previously described in Scheme 1, Step E.
The boronate esters (19) or analogous boronic acids can be readily made using
literature procedures or by adapting literature procedures (see for example Org Syn 2005,
82, 126).
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Scheme 5
Scheme 5 illustrates another synthetic route to making compounds of the
invention (8) where R3a is N(R3)2 or R4.
In Step A, the aminopyrazole benzoic acid or picolinic acid (20) is acylated to
form a benzamide or pyridinecarboxamide of formula (21). There are a variety of
coupling reagents and reaction conditions available to the skilled artisan for making an
amide from a carboxylic acid. Preferred conditions use CDI, in an inert solvent such as
THF, to make the carbonylimidazole in situ. This is followed by reaction with a cyclic
amine, such as, morpholine, thiomorpholine, piperidine, or 1-methylpiperazine at 45 to 70
°C.
In Step B, the aminopyrazole (21) is converted to the iodopyrazole (22) using a
Sandmeyer reaction, as previously described for Scheme 4, Step B.
In Step C, the cross-coupling reaction between the iodopyrazole (22) and the
phenyl or 4-pyridyl boronate ester (or boronic acid) proceeds essentially as previously
described in Scheme 4, Step C, which is followed by hydrolysis in Step D.
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Preparation 1
3,5-Di-/er/-butylbenzaldehyde
Dissolve l-bromo-3,5-di-/er/-butylbenzene (5.00 g, 18.57 mmol) in THF (50 mL)
under a nitrogen atmosphere. Cool to -78 °C. Slowly add w-butyllithium (2.5 M in
hexanes) (22.29 mL, 55.72 mmol) at -78 °C. Stir at -78 °C for about 30 min. Add DMF
(4.3 1 mL, 55.72 mmol) dropwise. Warm the mixture to 0 °C and stir for 2.5 h. Pour
aqueous NH4CI (30 mL) into the mixture. Extract with EtOAc (3 x 20 mL). Dry the
combined organic portions over a2S0 4; filter; collect the filtrate; and concentrate under
reduced pressure. Purify the residue using flash chromatography eluting with a gradient
of 0-10% EtOAc/petroleum ether to afford the title compound (2.96 g, 73%) as a white
solid. LC-ES/MS m/z 219 [M+H]+.
Preparation 2
1-(Bromomethyl)-3 -tert-butyl-5-iodo-benzene
Dissolve l-tert-butyl-3-iodo-5-methylbenzene (1.14 g, 4.14 mmol) in carbon
tetrachloride (20 mL). Add benzoyl peroxide (0.04 g, 0.166 mmol). Heat the mixture to
reflux, and add N-bromosuccinimide (1.47 g, 8.28 mmol). Stir the mixture overnight at
reflux temperature. Pour the reaction into water (100 mL) and extract with
dichloromethane (2 x 50 mL). Wash the combined organics with saturated aHC0 (50
mL), dry over Na2S0 4; filter; collect the filtrate; and concentrate under reduced pressure.
Purify the crude product by flash chromatography to afford l-(bromomethyl)-3-tertbutyl-
5-iodo-benzene as a mixture with l-ter?-butyl-3-(dibromomethyl)-5-iodobenzene
(1.24 g) (about 1.5/1 ratio of mono-bromo/dibromo. ¾ NMR (300 MHz, CDC13) d 1.29-
1.30 (m, 9H), 1.31-1.32 (m, 6H), 4.39 (s, 2H), 6.53 (s, 0.43H), 7.34 (s, 1H), 7.48 (s,
0.43H), 7.56 (s, 1H), 7.62-7.65 (m, 1.49H), 7.74 (s, 0.56H).
Preparation 3
3-/er/-Butyl-5-iodo-benzaldehyde
Dissolve l-(bromomethyl)-3-/er/-butyl-5-iodo-benzene (3.80 g, 10.76 mmol) in
dimethyl sulfoxide (20 mL). Heat to 100 °C and stir 4 h. Cool the reaction to room
temperature. Partition the mixture between water (40 mL) and EtOAc (40 mL). Dry the
organic portion over Na2S0 4; filter; collect the filtrate; and concentrate under reduced
pressure. Purify by flash chromatography (Biotage® system, 80 g cartridge) with a
gradient of 0-5% EtOAc/petroleum ether to afford the title compound (1.90 g, 61%). H
-16-
NMR (300 MHz, CDC13) d 1.36 (s, 9H), 7.87 (s, 1H), 7.97 (s, 1H), 8.03 (s, 1H), 9.92 (s,
1H).
Preparation 4
3-Bromo-5 -tert-butylphenol
Under a nitrogen atmosphere dissolve l,3-dibromo-5-/er/-butylbenzene (10.00 g,
34.25 mmol) in THF (30 mL). Cool to -78 °C. Slowly add w-butyllithium (2.5 M in
hexanes) (14.38 mL, 35.96 mmol) at -78 °C. Stir the resulting mixture for 30 min at -78
°C. Add trimethoxyborane (4.88 mL, 42.81 mmol) over 10 min. Warm to room
temperature and stir for 1 h. Cool the mixture to 0 °C. Add AcOH (13. 74 mL, 239.72
mmol) and stir for 10 min. Slowly add hydrogen peroxide (4. 11 mL, 134.93 mmol) and
water (0.718 mL) and stir 3 h. Add water (5 mL) and extract with EtOAc. Wash the
combined organic portions with brine. Purify the crude material by flash
chromatography, eluting with petroleum ether/EtOAc (10:1) to afford the title compound
(6.46 g, 82%). LC-ES/MS m/z ( Br/ 1Br) 227/229 [M-H]Preparation
l-Bromo-3-ter/-butyl-5-isopropoxy -benzene
Dissolve 3-bromo-5-/er/-butylphenol (2.00 g, 8.73 mmol) and 2-bromopropane
(1.27 mL, 13.09 mmol) in DMF (10 mL). Add potassium carbonate (3.62 g, 26.19
mmol). Heat to 50 °C and stir 2 h. Dilute with EtOAc (100 mL) and wash the reaction
mixture with water (3 x 20 mL). Dry and concentrate the organic portion under reduced
pressure. Purify the crude mixture by flash chromatography, eluting with petroleum ether
to afford the title compound (2.00 g, 85%) as a clear liquid. H NMR (CDC13 300 MHz)
d 1.28 (s, 9H), 1.32-1.36 (d, 6H), 4.47-4.52 (m, 1H), 6.81-6.86 (m, 2H), 7.06-7.08 (t, 1H).
Preparation 6
3-ter/-Butyl-5-isopropoxy-benzaldehyde
Dissolve l-bromo-3-/er/-butyl-5-isopropoxybenzene (2.00 g, 7.38 mmol) in THF
(50 mL) under an atmosphere of nitrogen. Cool the solution to -78 °C. Add nbutyllithium
(2.5 M in hexanes) (8.85 mL, 22.12 mmol) at -78 °C slowly to keep the
temperature below -70 °C. Stir the mixture for 30 min at -78 °C. Add DMF (1.71 mL,
22. 12 mmol) dropwise into the mixture at -78 °C. Warm the mixture to 0 °C and stir 2.5
h. Quench the reaction with aqueous NH4CI. Extract with EtOAc and dry the combined
organics over Na2S0 4; filter; and concentrate under reduced pressure. Purify the resulting
-17-
residue by flash chromatography on silica, eluting with a gradient of about 0-10%
EtOAc/petroleum ether to afford the title compound (1.35 g, 83%). H NMR (CDC13,
300 MHz) d 1.34 (s, 9H), 1.35-1.37 (t, 6H), 4.59-4.67 (m, 1H), 7.18-7.21 (m, 2H), 7.45-
7.46 (t,lH), 9.95 (s,lH).
Preparation 7
4-[(E)-3-(3,5-di-tert-Butylphenyl)prop-2-enoyl]benzoic acid
Dissolve 4-acetylbenzoic acid (15.00 g, 91.37 mmol) in ethanol (80 mL), and
water (40 mL). Add sodium hydroxide (3.65 g, 91.26 mmol). Stir the mixture at room
temperature for 30 min. Add 3,5-di-tert-butylbenzaldehyde (20.00 g, 91.60 mmol). Stir
the mixture at room temperature for 2 days. Quench the reaction with 2 N HCl (10 mL).
Adjust to about pH = 2 with 2 N HCl (20 mL). Filter the resulting white solid, washing
with ethanol (100 mL). Dry the solid under reduced pressure to afford the title compound
(18.30 g, 55%) as a white solid. LC-ES/MS m/z 365 [M+H]+.
Prepare the intermediates in Table 1 below, by essentially following the
procedure as described in Preparation 7, using the appropriate benzaldehyde with 4-
acetylbenzoic acid and 1.05 - 1.1 eq of solid NaOH or 5 N NaOH. Filter the solids upon
acidification, washing with petroleum ether.
Table 1
5/1 ratio of EtOH/water.
Preparation 10
Methyl-4-[(E)-3-(3,5-di-tert-butylphenyl)prop-2-enoyl]benzoate
Dissolve 4-[(E)-3-(3,5-di-tert-butylphenyl)prop-2-enoyl]benzoic acid (2.30 g ,
6.3 1 mmol) in methanol (250 mL) and cool to 0 °C. Add methanesulfonic acid (4. 14 mL,
63. 10 mmol) at 0 °C. Stir the mixture overnight, allowing to warm to room temperature.
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Concentrate the mixture under reduced pressure. Add EtOAc (100 mL) to the mixture.
Wash the organics with aqueous NaHCC (50 mL). Dry the organic layer over Na2S0 4;
filter; collect the filtrate; and concentrate under reduced pressure. Purify the residue by
flash chromatography eluting with a gradient of 0-10% EtOAc/petroleum ether to afford
the title compound ( 1.90 g, 80%) as a white solid. LC-ES/MS m/z 379 [M+H]+.
Prepare the intermediates in Table 2 below, by essentially following the procedure
as described in Preparation 10, using the appropriate propenoylbenzoic acid.
Table 2
Preparation 13
2-Methylsulfanyl-5-nitro-pyridine
Dissolve 2-chloro-5-nitropyridine (2.20 g, 13.88 mmol) and triethylamine (3.00
mL, 21.52 mmol) in methanol (20 mL). Add sodium methyl mercaptide (1.00 g, 14.27
mmol) in methanol (10 mL) at room temperature and stir for 2 h. Concentrate the
reaction solution under reduced pressure. Add 10% aqueous K2CO 3 to the resulting
residue. Extract the mixture with dichloromethane 3 times. Dry the combined organic
portions over Na2S0 4, filter, and concentrate under reduced pressure to afford the title
compound (2.3 g, 13.51 mmol, 97%) as a yellow solid. LC-ES/MS m/z 171 [M+H]+.
Preparation 14
2-Methylsulfonyl-5-nitro-pyridine
Dissolve 2-(methylthio)-5-nitropyridine (2.30 g, 13.51 mmol) in acetone (20 mL).
Add 2 N sulfuric acid (25 mL, 50.00 mmol) dropwise. Add KMn0 4 (3.00 g, 18.98 mmol)
in water (50 mL) dropwise to the resulting slurry. Stir the mixture at room temperature
overnight. Filter the solid. Stir the solid with a warm mixture of EtOH/MeOH (10: 1).
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Filter the resulting heterogeneous mixture through 2 cm of silica to remove the insoluble
salt. Concentrate the filtrate to afford the title compound (1.80 g, 66%) as a pale yellow
solid. LC-ES/MS m/z 203 [M+H]+.
Preparation 15
6-Methylsulfonylpyridin-3 -amine
Dissolve 2-(methylsulfonyl)-5-nitropyridine (1.80 g, 8.90 mmol) in water (25 mL)
and methanol (25 mL). Add iron (1.49 g, 26.68 mmol), and ammonium chloride (2.86 g,
53.47 mmol). Stir for 1 h at reflux temperature. Filter the mixture, washing with EtOAc.
Extract the filtrate with EtOAc. Dry the organic portion over MgS0 4; filter; collect the
filtrate; and concentrate the filtrate under reduced pressure to afford the title compound
(1.40 g, 91%). LC-ES/MS m/z 173 [M+H]+.
Preparation 16
(6-Methylsulfonyl-3 -pyridyl)hydrazine hydrochloride
Dissolve 6-(methylsulfonyl)pyridin-3-amine (0.50 g, 2.90 mmol) in concentrated
hydrochloric acid (6 mL). Add sodium nitrite (0.24 g, 3.48 mmol) in water (10 mL)
dropwise slowly at -10 to -15 °C. Stir the mixture for 2 h at -10 to -15 °C. Add tin
dichloride (2.20 g, 11.60 mmol) in concentrated hydrochloric acid (15 mL) dropwise at -5
°C. Stir the mixture 1 h at -5 °C. Filter the resulting yellow solid washing with diethyl
ether to afford the title compound (0.270 g, 42%) as a yellow solid. LC-ES/MS m/z 188
[M+H]+.
Preparation 17
4-Amino-N,N-dimethyl-benzenesulfonamide
Dissolve 4-acetamidobenzene-l-sulfonyl chloride (1.13 g, 4.84 mmol) in THF (20
mL). Add dimethylamine (2 M in THF, 10 mL, 20.00 mmol) slowly with stirring. Stir
the mixture overnight. Concentrate the mixture under reduced pressure. Dissolve the
residue in EtOAc (50 mL). Wash the organic portion with 2 N NaOH and brine. Dry
over a2S0 4 filter; collect the filtrate; and concentrate to dryness. Dissolve the resulting
oil in ethanol. Add concentrated hydrochloric acid (10 mL, 116.43 mmol). Heat the
mixture to reflux and stir 4 h. Concentrate the material under reduced pressure. Dissolve
the residue in EtOAc (50 mL) and water (50 mL). Adjust to about pH = 10 with 2 N
NaOH. Wash the organic layer with brine; dry over a2S0 4; filter; collect the filtrate;
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and concentrate the filtrate to dryness to afford the title compound (0.85 g, 88%). LCES/
MS m/z 201 [M+H]+.
Preparation 18
4-Hydrazino-N,N-dimethyl-benzenesulfonamide hydrochloride
Dissolve 4-amino-N,N-dimethylbenzenesulfonamide (200 mg, 0.999 mmol) in
concentrated hydrochloric acid (4 mL). Cool to 0 °C. Add sodium nitrite (80 mg, 1.16
mmol) in water (0.4 mL) dropwise at 0 °C. Stir the mixture at 0 °C for 1 h. Add a
solution of tin dichloride (760 mg, 4.01 mmol) in concentrated HCl (0.8 mL) dropwise to
the mixture at 0 °C. Stir the mixture at 0 °C for 1 h. Adjust the solution to about pH = 10
with 2 N NaOH. Extract the mixture with EtOAc. Concentrate the organic portion under
reduced pressure. Add 2 N HCl (5 mL, 10.00 mmol) and stir the mixture for 1 h.
Concentrate the solution under reduced pressure to afford the title compound (180 mg).
Use the crude product directly in the next step without further purification. LC-ES/MS
m/z 216 [M+H]+.
Preparation 19
4-[3-(3,5-Di-ter?-butylphenyl)-5-(4-methoxycarbonylphenyl)-3,4-dihydropyrazol-2-
yl]benzoic acid
Dissolve (E)-methyl 4-(3-(3,5-di-tert-butylphenyl)acryloyl)benzoate (1.00 g, 2.64
mmol), and 4-hydrazinylbenzoic acid (0.64 g, 4.23 mmol) in 1-butanol (100 mL). Add
acetic acid (58 mL) and heat to 120 °C for 20 h. Concentrate the mixture under reduced
pressure. Wash the solid with MeOH (3 10 mL) to afford the title compound (1.02 g,
75%) as a white solid.
Prepare the intermediates in Table 3 below, by essentially following the procedure
as described in Preparation 19, using the appropriate hydrazine (1.6 - 2 eq) and the
appropriate methyl benzoate in a solvent system of 1-butanol/AcOH varying from a ratio
of 5/4 to 10/3 except where noted.
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Table 3
Use 1-butanol/AcOH ratio of 1/3. Purify by preparatory TLC eluting with 2:1 petroleum
ether/EtOAc.
**Use 4 eq of 5-hydrazinyl-2-(methylsulfonyl)pyridine.
Preparation 24
5-Bromopyridine-2-carboxylic acid
Add 5-bromopicolinonitrile ( 1 g, 5.46 mmol) to concentrated HC1 (13.4 mL,
139.66 mmol) in a round bottomed flask. Heat the mixture to reflux with stirring
overnight. Cool the mixture to room temperature. Filter the resulting white solid,
washing with water. Dry the solid under reduced pressure to give 5-bromopyridine-2-
carboxylic acid (0.707 g, 64%) as a white solid. LC-ES/MS m/z 202 [M+H]+.
Preparation 25
5-Bromo-N,N-dimethyl-pyridine-2-carboxamide
Add 5-bromopyridine-2-carboxylic acid (0.71 g, 3.50 mmol) to a solution of
dimethylamine hydrochloride (0.32 g, 3.92 mmol), EDCI (0.77 g, 4.02 mmol), HOBT
(0.35 g, 2.29 mmol), and triethylamine (1.47 mL, 10.55 mmol) in DMF (10 mL). Stir the
mixture for 40 h at room temperature. Concentrate the mixture under reduced
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pressure. Dissolve the residue in dichloromethane (20 mL) and water (5 mL). Wash the
mixture with aqueous NaHCC (2 10 mL). Dry the combined organics over Na2S0 4;
filter; collect the filtrate; and concentrate under reduced pressure. Purify the resulting
residue by flash chromatography, eluting with a gradient of 0-60% EtOAc/petroleum
ether over 20 min, to afford the title compound (0.67 g, 84%). LC-ES/MS m/z ( Br/ 1Br)
229/231 [M+H]+.
Preparation 26
-Butyl N-[[6-(dimethylcarbamoyl)-3-pyridyl]amino]carbamate
Dissolve tert-butyl carbazate (2.18 g, 16.49 mmol), 5-bromo-N,N-dimethylpyridine-
2-carboxamide (3.44 g, 15.02 mmol), Pd(OAc)2 (340 mg, 1.50 mmol), sodium tbutoxide
(2.05 g, 21.01 mmol]), and 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene
(0.89 g, 1.51 mmol), in toluene (50 mL). Purge the reaction vessel 3 times with
nitrogen. Heat the mixture to 85 °C and stir for 6 h. Filter the material through
diatomaceous earth, washing with EtOAc (60 mL). Concentrate the mixture under
reduced pressure. Purify the resulting residue by flash chromatography, eluting with a
gradient of 0-15% MeOH/DCM over 30 min to afford the title compound (0.42 g, 10%).
LC-ES/MS m/z 281[M+H] +.
Preparation 27
Methyl 4-[3-(3,5-di-tert-butylphenyl)-2-[6-(dimethylcarbamoyl)-3-pyridyl]-3,4-
dihydropyrazol-5-yl]benzoate
Dissolve di- -butyl N-[[6-(dimethylcarbamoyl)-3-pyridyl]amino]carbamate
(420 mg, 1.50 mmol) in DCM (20 mL). Add TFA (5 mL) in a single portion with
stirring. Stir at room temperature for 2 h. Concentrate the mixture under reduced
pressure to afford an oil. Dissolve the oil in 1-butanol (20 mL) and AcOH (5 mL). Add
methyl 4-[(E)-3-(3,5-ditert-butylphenyl)prop-2-enoyl]benzoate (600 mg, 1.59 mmol) to
the reaction mixture. Purge the reaction vessel 3 times with nitrogen. Heat the mixture to
120 °C and stir for 10 h. Concentrate the mixture under reduced pressure. Purify the
resulting residue by preparatory TLC, eluting with 1:1 DCM/EtOAc to afford the title
compound (85 mg, 11%). LC-ES/MS m/z 541 [M+H]+.
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Preparation 28
Methyl 4-[3-(3,5-di-tert-butylphenyl)-2-(4-nitrophenyl)-3,4-
dihydropyrazol-5-yl]benzoate
Dissolve methyl 4-[(E)-3-(3,5-di-tert-butylphenyl)prop-2-enoyl]benzoate (200
mg, 0.528 mmol) and (4-nitrophenyl)hydrazine (90 mg, 0.588 mmol) in MeOH (4 mL).
Add methanesulfonic acid (0.14 mL, 2.08 mmoles). Heat the solution to 120 °C with
microwave irradiation for 30 min. Quench the reaction with aqueous a2C0 (0.2 mL).
Filter the resulting solid, washing the solid with MeOH to afford the title compound (270
mg, quantitative) as a yellow solid. LC-ES/MS m/z 514 [M+H]+.
Preparation 29
4-[5-(3,5-Di-tert-butylphenyl)-3-(4-methoxycarbonylphenyl)pyrazol-l-yl]benzoic acid
issolve 4-[3-(3,5-di-tert-butylphenyl)-5-(4-methoxycarbonylphenyl)-3,4-
dihydropyrazol-2-yl]benzoic acid (1.02 g, 1.99 mmol) in 1,2-dichloroethane (20 mL).
Add acetic acid (77 mL) and manganese (IV) oxide (4.84 g, 55.71 mmol). Heat the
mixture to 70 °C and stir overnight. Filter the mixture, washing with
dichloromethane. Concentrate the mixture under reduced pressure. Purify the crude
product using flash chromatography, eluting with 1:1 dichloromethane:petroleum ether to
afford the title compound (1.01 g, 99%) as a white solid. LC-ES/MS m/z 5 11 [M+H]+.
Preparation 30
Methyl 4-[5-(3,5-di-tert-butylphenyl)-l-[4-(piperidine-l-carbonyl)phenyl]
pyrazol-3-yl]benzoate
Dissolve piperidine (0.029 g, 0.353 mmoles) in DMF (6 mL). Add 4-[5-(3,5-ditert-
butylphenyl)-3-(4-methoxycarbonylphenyl)pyrazol-l-yl]benzoic acid (0.120 g, 0.235
mmol) and diisopropylethylamine (0.05 mL, 0.282 mmol). Stir the mixture for about 10
min. Add BOP (0. 124 g, 0.282 mmol) and stir the mixture for about 3 h at room
temperature. Add water (3 mL) and extract with EtOAc (10 mL). Dry the organic layer
over a2S0 4; filter; collect the filtrate; and concentrate the filtrate to dryness under
reduced pressure. Purify the crude product by preparatory TLC, eluting with 4 :1
petroleum ether/EtOAc to afford the title compound (0.108 g, 80%). LC-ES/MS m/z 578
[M+H]+.
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Prepare the intermediates in Table 4 below, by essentially following the procedure
as described in Preparation 30, using the appropriate amine. For example, in Preparation
3 1 use ammonia (2.0 M solution in methanol).
Table 4
Preparation 37
4-[5-(3-tert-Butyl-5-iodo-phenyl)-3-(4-methoxycarbonylphenyl)pyrazol-l-yl]benzoic
acid
Dissolve 4-[5-(3-tert-butyl-5-iodo-phenyl)-3-(4-methoxycarbonylphenyl)pyrazoll-
yl]benzoic acid (1.34 g, 2.30 mmol) in 1,2-dichloroethane (50 mL). Add AcOH (10
mL) and manganese (IV) oxide (5.60 g, 64.42 mmol). Stir the mixture overnight at room
temperature. Filter the mixture, washing with DCM. Concentrate the filtrate under
reduced pressure. Purify the crude material by flash chromatography, eluting with a
gradient of 3-25% EtOAc/petroleum ether to afford the title compound (0.98 g, 73%).
LC-ES/MS m/z 581 [M+H]+.
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Preparation 38
Methyl 4-[5-(3-tert-butyl-5-iodo-phenyl)-l-[4-(dimethylcarbamoyl)phenyl]pyrazol-3-
yljbenzoate
Dissolve 4-[5-(3-tert-butyl-5-iodo-phenyl)-3-(4-methoxycarbonylphenyl)pyrazoll-
yl]benzoic acid (1.40 g, 2.41 mmol), dimethylamine hydrochloride (0.43 g, 5.31
mmol), EDCI (1.16 g, 6.03 mmol), and HOBT (0.92 g, 6.03 mmol) in DCM (20 mL).
Stir at room temperature overnight. Quench the reaction with aqueous aHC0 (10 mL).
Extract with DCM (20 mL). Wash the combined organic portion with aqueous aHC0
(2 10 mL), dry over Na2S0 4, filter, and concentrate under reduced pressure. Purify the
resulting residue by flash chromatography on silica (Biotage® system, 40 g cartridge @
25 mL/min) with a gradient of 0-60% EtOAc/petroleum ether over 40 min to afford the
title compound (1.20 g, 82%). LC-ES/MS m/z 608 [M+H]+.
Preparation 39
Methyl-4-[5-(3-/er/-butyl-5-iodo-phenyl)-l-[4-(4-methylpiperazine-lcarbonyl)
phenyl]pyrazol-3 -yljbenzoate
Prepare the title compound, by essentially following the procedure as described in
Preparation 38, using 1-methylpiperazine with 4-[5-(3-tert-butyl-5-iodo-phenyl)-3-(4-
methoxycarbonylphenyl)pyrazol-l-yl]benzoic acid. LC-ES/MS m/z 663 [M+H]+.
Preparation 40
Methyl 4-[5-(3-tert-butyl-5-isopropylsulfanyl-phenyl)-l-[4-(4-methylpiperazine-lcarbonyl)
phenyl]pyrazol-3-yl]benzoate
Dissolve methyl 4-(5-(3-tert-butyl-5-iodophenyl)- 1-(4-(dimethylcarbamoyl)
phenyl)- lH-pyrazol-3-yl)benzoate (0.28 g, 0.461 mmol) and 1,2-diisopropylsulfane
(0.037 mL, 0.232 mmol) in dry DMF (2 mL). Add zinc (0.03 g, 0.454 mmol), and (SP-4-
30-[2,6-bis[(dimethylphosphino -KP)oxy]phenyl-KC]chloro-nickel ((PCP)NiCl) (0.01 g,
0.017 mmol) (reagent prepared according to Tetrahedron Lett. 2006, 49, 5059). Purge the
reaction vessel 3 times with nitrogen. Heat the mixture at 110 °C with stirring for 4 h.
Quench the mixture with water (20 mL) and extract with EtOAc (3 x 20 mL). Wash the
combined extracts with brine (2 x 10 mL), dry over a2S0 4, filter, and concentrate.
Purify the crude mixture by flash chromatography on silica (Biotage® system, 20 g
cartridge @ 25 mL/min) eluting with a gradient of 0-20% EtOAc/petroleum ether over 30
min to give a mixture of methyl 4-[5-(3-tert-butyl-5-isopropylsulfanyl-phenyl)-l-[4-
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(dimethylcarbamoyl)phenyl]pyrazol-3-yl]benzoate and starting material (methyl 4-(5-(3-
tert-butyl-5-iodophenyl)-l-(4-(dimethylcarbamoyl)phenyl)-lH-pyrazol-3-yl)benzoate) as
a white solid (210 mg). Dissolve the mixture (210 mg) in dry DMF (2 mL). Add 1,2-
diisopropylsulfane (0.037 mL, 0.232 mmol), zinc (0.03 g, 0.454 mmol), and (PCP)NiCl
(0.01 g, 0.017 mmol). Purge the reaction vessel 3 times with nitrogen. Heat the mixture
at 110 °C and stir overnight. Quench the mixture with water (20 mL) and extract with
EtOAc (3 20 mL). Wash the combined extracts with brine (2 10 mL), dry over
Na2S0 4, filter, and concentrate under reduced pressure. Purify the crude mixture by flash
chromatography on silica (Biotage® system, 20 g cartridge @ 25 mL/min) eluting with a
gradient of 0-20% EtOAc/petroleum ether over 30 min to afford 190 mg of crude
material. Purify the crude product by preparatory HPLC (Spring Column™ CI 8, 250 x
250 mm, 10 mih particle, eluting with a gradient of 75-100% acetonitrile with 0.05% TFA
in water) to afford the title compound (0. 10 g, 39%) as an oil. LC-MS m/z 556 [M+H]+.
Preparation 41
Methyl 4-[5-(3-/er/-butyl-5-isopropylsulfanyl-phenyl)-l-[4-(4-methylpiperazine-lcarbonyl)
phenyl]pyrazol-3-yl]benzoate
Prepare the title compound, by essentially following the procedure as described in
Preparation 40, using diisopropylsulfane and methyl 4-[5-(3-tert-butyl-5-iodo-phenyl)-l-
[4-(4-methylpiperazine-l-carbonyl)phenyl]pyrazol-3-yl]benzoate. LC-ES/MS m/z 6 11
[M+H]+.
Preparation 42
4-[5-(3-tert-Butyl-5-isopropoxy-phenyl)-3-(4-methoxycarbonylphenyl)pyrazol-lyl]
benzoic acid
Dissolve 4-[3-(3-tert-butyl-5-isopropoxy-phenyl)-5-(4-methoxycarbonylphenyl)-
3,4-dihydropyrazol-2-yl]benzoic acid (0.50 g, 0.971 mmol) in toluene (10 mL). Add
DDQ (0.44 g, 1.94 mmol) and heat the mixture to reflux with stirring for 2 h.
Concentrate the reaction under reduced pressure. Purify the residue by preparatory TLC,
eluting with 30:1 dichloromethane/MeOH to afford the title compound (0.47 g, 94%).
LC-ES/MS m/z 513 [M+H]+.
Prepare the intermediates in Table 5 below, by essentially following the procedure
as described in Preparation 42, using the appropriate dihydropyrazole. Purify the crude
products using preparatory TLC, eluting with petroleum ether/EtOAc.
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Table 5
Preparation 47
Methyl 4-[5-(3 -tert-butyl-5-isopropoxy -phenyl)- 1-[4-(4-methylpiperazine- 1-
carbonyl)phenyl]pyrazol-3-yl]benzoate
Dissolve 1-methylpiperazine (70 mg, 0.702 mmol) and 4-[5-(3-tert-butyl-5-
isopropoxy-phenyl)-3-(4-methoxycarbonylphenyl)pyrazol-l-yl]benzoic acid (180 mg,
0.351 mmol) in dichloromethane (10 mL). Add 1-hydroxybenzotriazole ( 118 mg, 0.878
mmol) and EDCI (168 mg, 0.878 mmol). Stir the mixture 3 h at room temperature. Add
water (3 mL) and aqueous aHC0 (10 mL) and extract with EtOAc (3 10 mL). Dry
the combined organics over Na2S0 4, filter, and concentrate to dryness. Purify the
resulting residue by preparatory TLC, eluting with 10: 1DCM/MeOH to afford the title
compound (140 mg, 67%). LC-ES/MS m/z 595 [M+H]+.
Preparation 48
Methyl 4-[l-(4-aminophenyl)-5-(3,5-di-tert-butylphenyl)pyrazol-3-yl]benzoate
Dissolve methyl 4-(5-(3,5-di-tert-butylphenyl)-l-(4-nitrophenyl)-lH-pyrazol-3-
yl)benzoate (230 mg, 0.450 mmol) in MeOH (8 mL) and water (8 mL). Add iron (80 mg,
1.430 mmol) and ammonium chloride (120 mg, 2.240 mmol) in a single portion. Heat the
mixture to reflux and stir 2 h. Filter the mixture, washing with EtOAc. Extract the
mixture 3 times with EtOAc. Dry the combined organic portions over Na2S0 4, filter, and
concentrate under reduced pressure to afford the title compound (210 mg, 97%). LCES/
MS m/z 482 [M+H]+.
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Preparation 49
Methyl 4-[5-(3,5-di-tert-butylphenyl)-l-[4-(methanesulfonamido)phenyl]pyrazol-3-
yljbenzoate
Dissolve methyl 4-[l-(4-aminophenyl)-5-(3,5-di-tert-butylphenyl)pyrazol-3-
yl]benzoate (210 mg, 0.436 mmol) in DCM (10 mL). Add pyridine (0.04 mL, 0.495
mmol) and stir for 5 min. Add methanesulfonyl chloride (0.04 mL, 0.517 mmol) in a
single portion and stir overnight. Quench the reaction with aqueous a2C0 . Extract the
mixture 3 times with EtOAc. Dry the combined organic portion over a2S0 4, filter, and
concentrate to dryness under reduced pressure. Purify the resulting residue by flash
chromatography (Biotage® system, 20 g cartridge @ 25 mL/min) eluting with a gradient
of 8-60% EtOAc/petroleum ether to afford the title compound (230 mg, 94%). LCES/
MS m/z 560 [M+H]+.
Preparation 50
2-(3,5-Di-tert-butylphenyl)-4,4,5,5-tetramethyl-l,3,2-dioxaborolane
Bubble nitrogen through DMF (150 mL) for about 15 min prior to adding the
reagents. Then dissolve l-bromo-3,5-di-tert-butylbenzene (20.93 g, 77.74 mmol),
bis(pinacolato)diboron (22.70 g, 89.40 mmol), and ( I , -
bis(diphenylphosphino)ferrocene)palladium(II) chloride (3.17 g, 3.89 mmol) in the DMF.
Stir for 10 min. Add potassium acetate (22.89 g, 233.23 mmol) and bubble argon through
the solution for 7 min. Heat the reaction to 85 °C with stirring for 24 h. Dilute the
reaction with water (1.5 L). Collect the resulting brown precipitate by vacuum filtration
and washing with water. Dissolve the residue in dichloromethane. Dry this solution over
sodium sulfate, filter; collect the filtrate; and concentrate under reduced
pressure. Triturate the resulting solids with hot hexanes (400 mL) and filter, washing
with hexanes. Concentrate the filtrate to a volume of approximately 300 mL. Place the
solution in a freezer overnight. Collect the solid by vacuum filtration and rinsing with
cold hexanes. Concentrate the filtrate under reduced pressure to a volume of 150 mL.
Cool this mixture in the freezer for about 1.5 h. Collect the resulting solids by vacuum
filtration and rinse with cold hexanes. Combine the 2 crops and dry under high vacuum
to afford the title compound (21.85 g, 89%) as a light tan crystalline solid. H NMR (400
MHz, CDC13) d 1.33 (s, 12H), 1.33 (s, 18H), 7.53 (t, J = 2.0 Hz, 1H), 7.65 (d, J = 2.0 Hz,
2H).
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Preparation 50A: Alternate Procedure
2-(3,5-Di-tert-butylphenyl)-4,4,5,5-tetramethyl-l,3,2-dioxaborolane
Charge a reactor with l-bromo-3,5-di-tert-butylbenzene (182.0 g, 1.1 15 mol), bis
(pinacolato)diboron (197.4 g, 1.281 mol), [l,l'-bis(diphenylphosphino)ferrocene]
palladium(II) chloride (27.6 g, 0.056 mol), potassium acetate (199.0 g, 3.347 mol) and
DMF (1.2 L). Heat the resulting solution to 85 °C for 5 h. Cool the reaction mixture to
25 °C and add water (6 L) to form a brown precipitate. Filter and wash the solid with
water. Collect the solid. Dissolve the solid in DCM (1.82 L), dry over Na2S0 4, filter;
collect the filtrate; and evaporate the solvent. Triturate the residue with hot hexane (3.2
L) and filter to remove the catalyst. Concentrate the filtrate to approximately 1.8 L. Cool
this solution to 15 °C and stir for 48 h. Filter to collect the solid and dry in the open air to
provide the title compound (150.0 g, 70%). LC-ES/MS m/z 317 [M+H]+.
Preparation 51
3-tert-Butyl-5-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)phenol
Dissolve 3-bromo-5-tert-butylphenol (2.29 g, 10.00 mmol), 4„4,4',4',5,5,5',5'-
octamethyl-2,2'-bi-l,3,2-dioxaborolane (3.046 g, 12.0 mmol), (1,1'-
bis(diphenylphosphino)ferrocene)palladium(II) chloride (0.820 g, 1.00 mmol), 1,1'-
bis(diphenylphosphino)ferrocene (0.560 g, 1.00 mmol), and potassium acetate (2.94 g,
30.00 mmol) in 1,4-dioxane (80 mL). Purge the reaction vessel 3 times with
nitrogen. Heat the mixture to 80 °C and stir overnight. Filter the mixture through
diatomaceous earth, rinsing the solid cake with EtOAc. Concentrate the filtrate under
reduced pressure. Purify the crude mixture by flash chromatography on silica (ISCO®
system, 20 g cartridge @ 25 mL/min) eluting with a gradient of 0-20% EtOAc/petroleum
ether over 30 min to afford the title compound (2.26 g; 82%). LC-ES/MS m/z 275 [MH]-.
Preparation 52
2-(3-tert-Butyl-5-isopropoxy-phenyl)-4,4,5,5-tetramethyl-l,3,2-dioxaborolane
Dissolve 3-tert-butyl-5-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)phenol (200
mg, 0.724 mmol), and 2-bromopropane (178 mg, 1.448 mmol) in DMF (3 mL). Add
potassium carbonate (300 mg, 2.149 mmol) in a single portion with stirring. Heat the
mixture to 90 °C while stirring overnight. Cool the mixture to room temperature and
dilute with EtOAc (50 mL). Wash the combined organics with water and brine; dry over
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Na2S0 4; filter; collect the filtrate; and concentrate under reduced pressure. Purify the
crude mixture by flash chromatography on silica (ISCO® system, 20 g cartridge @ 30
mL/min) eluting with a gradient of 0-50% EtOAc/petroleum ether over 20 min to afford
the title compound (152 mg, 66%). LC-ES/MS m/z 319 [M+H]+.
Preparation 53
2,6-Di-/er/-butyl-4-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)pyridine
Add l,5-cyclooctadiene)(methoxy)iridium (I) dimer (Ir(OMe)(COD)) 2) (0.05 g,
0.075 mmol), 4-/er/-butyl-2-(4-/er/-butyl-2-pyridyl)pyridine (0.04 g, 0.15 mmol), and
bis(pinacolato)diboron (2.67 g, 10.50 mmol) to hexane (30 mL) which has been purged
with nitrogen for 20 min. Place in a preheated oil bath at 55 °C. Stir for 10 min. Add
2,6-di-tert-butylpyridine (3.81 g, 19.90 mmol) and heat at 55 °C for 72 h. Cool the
mixture to room temperature and concentrate under reduced pressure to give the title
compound (5.20 g, 82%). LC-ES/MS (m/z) 318 [M+H]+.
Preparation 54
3-Bromo-5-formyl-tert-butylbenzene
Dissolve 1,3-dibromo-tert-butylbenzene (6.093 g, 20.87 mmol) in 50 mL THF (50
mL) and cool to -78 °C. Add «-butyl lithium (2.5 M in hexanes) (9.18 mL, 22.95 mmol)
dropwise over 10 min and stir for 15 min. Add DMF (3.23 mL, 41.73 mmol) in one
portion and stir for 30 min. Dilute the reaction mixture with EtOAc (40 mL) and 1N HC1
(40 mL). Extract the aqueous layer with EtOAc (2 x 40 mL). Wash the combined
organic portions with brine (100 mL). Dry over sodium sulfate; filter; collect filtrate; and
concentrate under reduced pressure. Purify the resulting residue by flash
chromatography, eluting with a gradient of hexanes to 10% EtOAc/hexanes over 30 min
to give the title compound (4.02 g) as a light yellow oil. H NMR (400 MHz, DMSO-d 6) :
d 1.27-1.27 (m, 9H), 7.83 (s, 2H), 7.89-7.88 (m, 1H), 9.93 (s, 1H).
Preparation 55
l-(3-Bromo-5-tert-butylphenyl)ethanol
Dissolve 3-bromo-5-formyl-tert-butylbenzene (3.624 g, 15.03 mmol) in diethyl
ether (50 mL). Add methylmagnesium bromide (5.51 mL, 16.53 mmol) slowly over 10
min. Stir the reaction mixture for 18 h. Pour the reaction mixture into saturated
ammonium chloride (50 mL) and extract with EtOAc (2 x 50 mL). Dry the organic
portions over sodium sulfate, filter, and concentrate under reduced pressure. Purify the
-31-
resulting residue by flash chromatography, eluting with a gradient of hexanes to 10%
EtOAc/hexanes over 30 min to give the title compound (3.49 g) as a clear oil. H MR
(400 MHz, DMSO-d 6) d 1.23-1.22 (s, 9H), 1.25 (d, J = 6.5 Hz, 3H), 4.68-4.62 (m, 1H),
5. 19 (d, J = 4.4 Hz, 1H), 7.32-7.28 (m, 3H).
Preparation 56
l-(3-Bromo-5-tert-butylphenyl)ethanone
Dissolve l-(3-bromo-5-tert-butylphenyl)ethanol (3.49 g, 13.57 mmol) in
chloroform (100 mL) and then add pyridinium chlorochromate (4.48 g, 20.36 mmol). Stir
the mixture for 72 h at room temperature. Add 5 N NaOH (150 mL) and stir until the
precipitate dissolves. Extract the aqueous layer with dichloromethane (2 x 100
mL). Wash the combined organic portions with 1N HC1 (150 mL). Dry the organics
over sodium sulfate; filter; collect the filtrate; and concentrate under reduced pressure.
Purify the residue by flash chromatography eluting with a gradient of hexanes to 20%
EtOAc/hexanes over 30 min to give the title compound (2.62 g) as a clear oil. H NMR
(400 MHz, DMSO- 6) d 1.26 (s, 9H), 2.55 (s, 3H), 7.77 (t, J = 1.8 Hz, 1H), 7.85 (t, J= 1.6
Hz, 1H), 7.87 (t, J= 1.6 Hz, 1H).
Preparation 57
2-(3-Bromo-5-tert-butyl-phenyl)propan-2-ol
Dissolve l-(3-bromo-5-tert-butylphenyl)ethanone (0.80 g, 3.15 mmol) in diethyl
ether (40 mL). Add methylmagnesium bromide (1.58 mL, 4.73 mmol) and then stir for
72 h. Pour the reaction into 1N hydrochloric acid (50 mL). Extract with diethyl ether (2
x 50 mL). Dry the combined organic portions over sodium sulfate; filter; concentrate the
filtrate; and concentrate under reduced pressure to afford the title compound (0.82 g,
96%) as a white solid. H NMR (400 MHz, DMSO-i¾) d 1.24 (s, 9H), 1.38 (s, 6H), 5.09
(s, 1H), 7.31 (t, J = 1.8 Hz, 1H), 7.40 (t, J = 1.7 Hz, 1H), 7.44 (t, J = 1.6 Hz, 1H).
Preparation 58
1-Bromo-3 -tert-butyl-5 -isopropyl-benzene
Dissolve 2-(3-bromo-5-tert-butyl-phenyl)propan-2-ol (0.82 g, 3.02 mmol) in
dichloromethane (20 mL). Add TFA (2.29 mL, 30.24 mmol) and then triethylsilane (2.42
mL, 15.12 mmol). Stir the reaction for 18 h. Pour the reaction into saturated sodium
bicarbonate (50 mL) and extract two times with DCM (2 x 40 mL). Dry the combined
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organic portions over sodium sulfate; filter; collect the filtrate; and concentrate under
reduced pressure. Purify the resulting residue by flash chromatography, eluting with a
gradient of hexanes to 10% EtOAc/hexanes over 30 min to give the title compound (0.60
g, 78%) as a clear liquid. H NMR (400 MHz, DMSO-i¾) d 1.16 (d, J = 6.9 Hz, 6H), 1.23
(s, 9H), 2.89-2.82 (m, 1H), 7.22-7.20 (m, 2H), 7.30 (t, J = 1.8 Hz, 1H).
Preparation 59
(3-tert-Butyl-5-isopropyl-phenyl)boronic acid
Dissolve l-bromo-3-tert-butyl-5-isopropylbenzene (0.35 g, 1.37 mmol) in THF
(10 mL) under a nitrogen atmosphere. Cool the solution to -78 °C. Add w-butyllithium
(2.5 M in hexanes) (0.66 mL, 1.65 mmol) and stir the mixture at -78 °C for 20 min.
Add trimethylborane (0.2 mL, 1.76 mmol) at -78 °C and stir at -78 °C for 2 h. Quench
the reaction with water (20 mL). Extract the mixture with EtOAc (2 40 mL). Collect
the EtOAc extracts and purify the crude mixture using preparatory TLC, eluting with 1:5
EtOAc/petroleum ether to afford the title compound (0. 19 g, 63%). LC-ES/MS m/z 219
[M-H] .
Preparation 60
Methyl 4-[5-hydroxy-l-(4-methylsulfonylphenyl)pyrazol-3-yl]benzoate
Dissolve 4-methylsulfonylphenylhydrazine hydrochloride (13.40 g, 57.15 mmol),
and 4-(2-methoxycarbonyl-acetyl)-benzoic acid methyl ester (10.00 g, 42.3 mmol) in
MeOH (150 mL). Heat the mixture to reflux and stir overnight. Cool the reaction to
room temperature. Add MeOH (50 mL) and cool to 0 °C. Filter the solids using vacuum
filtration; then wash the solids with cold MeOH. Dry the solids under reduced pressure to
give the title compound (14.3 g, 91%) as a light tan solid. LC-ES/MS m/z 373 [M+H]+.
Preparation 61
Methyl 4-[5-bromo-l-(4-methylsulfonylphenyl)pyrazol-3-yl]benzoate
Dissolve methyl 4-[5-hydroxy- 1-(4-methylsulfonylphenyl)pyrazol-3-yl]benzoate
(2.00 g, 5.371 mmol) in acetonitrile (8 mL). Add phosphorus tribromide (2.55 mL, 26.85
mmol). Heat the reaction to reflux and stir overnight. Add phosphorus tribromide (1.273
mL, 13.43 mmol) and stir for 72 h. Add phosphorus tribromide (1.27 mL, 13.43 mmol)
and stir for 24 h. Cool the reaction to room temperature. Slowly pour the mixture over
saturated aqueous sodium bicarbonate. Extract the resulting mixture with DCM.
Concentrate the combined extracts under reduced pressure. Purify the resulting residue
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by flash chromatography over silica gel (40 g) with a gradient of 0-5% EtOAc/DCM to
afford the title compound (0.95 g, 41%) as a white crystalline solid. LC-ES/MS m/z
( Br/ 1Br) 435/437 [M+H]+.
Preparation 62
Methyl 4-[5-(3,5-di-/er/-butylphenyl)-l-(4-methylsulfonylphenyl)pyrazol-3-yl]benzoate
Add methyl 4-[5-bromo-l-(4-methylsulfonylphenyl)pyrazol-3-yl]benzoate (75
mg, 0.172 mmol), 2-(3,5-di-ter/-butylphenyl)-4,4,5,5-tetramethyl-l,3,2-dioxaborolane (82
mg, 0.258 mmol), (tetrakis(triphenylphosphine)palladium (40 mg, 0.034 mmol), THF (1.7
mL), and 2 N aqueous sodium carbonate (0.284 mL, 0.569 mmol) to an 8 mL screw cap
vial (containing septa) equipped with a stir bar. Purge with argon for 1-2 min. Place the
reaction flask in a 65 °C oil bath and stir the reaction overnight. Cool the reaction to
room temperature and dilute with water. Extract the aqueous with EtOAc. Concentrate
the combined extracts under reduced pressure. Purify by radial chromatography (silica
gel, 2 mm plate) eluting with 20-50% EtOAc gradient in hexane to afford the title
compound (60 mg, 64%) as a white solid foam. LC-ES/MS m/z 545 [M+H]+.
Preparation 63
Methyl 4-[5-amino-l-(4-cyanophenyl)pyrazol-3-yl]benzoate
Suspend of hydrazinylbenzonitrile hydrochloride (1.86 g, 10.97 mmol) and methyl
4-(2-cyanoacetyl)benzoate (2.03 g, 9.99 mmol) in MeOH (40 mL). Heat the reaction to
reflux while stirring overnight. Cool the reaction mixture to room temperature. Filter the
mixture, washing the solid with petroleum ether (40 mL). Dry the resulting solid in
vacuo to afford product (2.02 g). Concentrate the filtrate under reduced pressure. Purify
the resulting residue by flash chromatography on silica gel (Biotage® system, 40 g
cartridge @ 40mL/min) with a gradient of 0-70% EtOAc/DCM (40 min) to afford
additional product (0.42 g). Combine the two lots of purified product (2.44 g, 77%). LCES/
MS m/z 319 [M+H]+.
Preparation 64
Methyl 4-[ 1-(4-cyanophenyl)-5-iodo-pyrazol-3-yl]benzoate
Suspend methyl 4-[5-amino-l-(4-cyanophenyl)pyrazol-3-yl]benzoate (2.02 g, 6.4
mmol) and copper(I) iodide (1.21 g, 6.4 mmol) in acetonitrile (80 mL). Add t-butyl
nitrite (1.3 1 g, 12.7 mmol) while stirring. Heat the mixture to 75 °C while stirring for 3 h.
Dilute the mixture with EtOAc (50 mL). Wash the organics with dilute a2S20 (3x) and
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brine. Dry the organics over a2S0 4; filter, collect the filtrate; and concentrate to dryness
under reduced pressure. Purify the crude mixture by flash chromatography on silica
(ISCO® system, 20 g cartridge @ 25 mL/min) with a gradient of 0-50%
EtOAc/petroleum ether over 30 min to afford the title compound (1.54 g, 57%). LCES/
MS m/z 430 [M+H]+.
Preparation 65
Methyl 4-[l-(4-carbamoylphenyl)-5-iodo-pyrazol-3-yl]benzoate
Dissolve methyl 4-[l-(4-cyanophenyl)-5-iodo-pyrazol-3-yl]benzoate (0.206 g,
0.480 mmol) in TFA (3 mL). Add sulfuric acid (0.75 mL) slowly while stirring the
mixture. Heat the mixture to 45 °C with stirring overnight. Pour the mixture into ice
water and extract with isopropanol/DCM (1:2 ratio, 3 x 50 mL). Wash the combined
organics with brine; dry over Na2S0 4; filter; collect the filtrate; and concentrate to
dryness under reduced pressure to afford the title compound (0.205 g, 96%). LC-ES/MS
m/z 448 [M+H]+.
Preparation 66
4-[5-Amino-3-(4-methoxycarbonylphenyl)pyrazol-l-yl]benzoic acid
Suspend 4-hydrazinylbenzoic acid (1.67 g, 10.98 mmol) and methyl 4-(2-
cyanoacetyl)benzoate (2.03 g, 9.99 mmol) in methanol (40 mL). Heat to reflux and stir
overnight. Cool the reaction mixture to room temperature. Filter the resulting solid,
washing with petroleum ether to afford the title compound (2.92 g, 87%). LC-ES/MS
m/z 338 [M+H]+.
Preparation 66A: Alternate Procedure
4-[5-Amino-3-(4-methoxycarbonylphenyl)pyrazol-l-yl]benzoic acid
Charge a reactor with acetic acid (25 L), 4-hydrazinobenzoic acid hydrochloride
( 1115.0 g, 5.91 1 mol) and methyl 4-cyanoacetylbenzoate (1200.0 g, 5.91 1 mol) at 13 °C.
Heat the mixture to 80 °C and stir for 20 h. Cool the reaction mixture to 20 °C and filter
to give a yellow filter cake. Triturate the filter cake with hexane (5 L) and filter to give
the title compound (1621 g, 81%) as a yellow solid. LC-ES/MS m/z 338 [M+H]+.
Preparation 67
Methyl 4-[5-amino-l-[4-(4-methylpiperazine-l-carbonyl)phenyl]pyrazol-3-yl]benzoate
Charge a reactor with THF (18.6 L) and 4-[5-amino-3-(4-
methoxycarbonylphenyl)pyrazol- 1-yl]benzoic acid (620 g, 1.840 moles) at 15 °C. Add
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CDI (387 g, 2.392 mol) in batches, heat the mixture to reflux, and stir for 2.5 h. Add Nmethylpiperizine
(360 mL, 2.760 mol) dropwise at reflux temperature over 25 min. Then
stir for 17 h at reflux. Cool to 20 °C and add water (7 L) and EtOAc (18 L). Separate the
two phases. Extract the aqueous with EtOAc (2 10 L). Combine the organic layers;
wash with 1M NaOH (2.5 L); dry with Na2S0 4; filter; collect the filtrate; and concentrate
under reduced pressure to give a yellow solid. Add a solvent mixture of
heptane/MTBE/MeOH (4/4/1, 10 L) and stir for 0.5 h. Collect the solid by filtration. Dry
the resulting material to give the title compound (610 g, 77%) as a yellow solid. LCES/
MS m/z 420 [M+H]+.
Preparation 68
Methyl 4-[5-iodo-l-[4-(4-methylpiperazine-l-carbonyl)phenyl]pyrazol-3-yl]benzoate
Charge a reactor with acetonitrile (21 L), methyl 4-[5-amino-l-[4-(4-
methylpiperazine-l-carbonyl)phenyl]pyrazol-3-yl]benzoate (1070 g, 2.554 mol),
diiodomethane (1362 g, 5.101 mol) and copper (I) iodide (970 g, 5.105 mol). Heat the
mixture to 80 °C with stirring. Add isoamyl nitrite (896 g, 7.658 mol) dropwise at 80 °C,
and then stir the resultant suspension at 80 °C for 1 h. Cool the mixture to 17 °C and
allow to stand for 48 h. Add saturated aqueous solution of NH4C 1 (10 L) and EtOAc (50
L). Separate the two phases. Wash the organic layer with a saturated aqueous solution of
a2S203 (10 L), and dry over Na2S0 4. Filter; collect the filtrate; and evaporate the
filtrate to give an orange oil. Purify the residue by column chromatography, eluting with
DCM/MeOH (from 50/1 to 5/1) to provide the title compound (381 g, 28%) as a brown
solid. LC-ES/MS m/z 531 [M+H]+.
Preparation 69
Methyl 4-[5-iodo-l-[4-(morpholine-4-carbonyl)phenyl]pyrazol-3-yl]benzoate
Suspend 4-(5-amino-3-(4-(methoxycarbonyl)phenyl)-lH-pyrazol-l-yl)benzoic
acid (2.56 g, 7.60 mmol) and copper(I) iodide (1.44 g, 7.60 mmol) in acetonitrile (80
mL). Add ?-butyl nitrite (1.8 mL, 15.1 mmol) in a single portion. Heat the mixture to 75
°C and stir 3 h. Filter the mixture, washing the solid with acetonitrile to afford a crude
mixture of 4-(5-iodo-3-(4-(methoxycarbonyl)phenyl)-lH-pyrazol-l-yl)benzoic acid (3.7
g). Dissolve 4-(5-iodo-3-(4-(methoxycarbonyl)phenyl)-lH-pyrazol-l-yl)benzoic acid
(3.7 g) in dry DMF (50 mL). Add diisopropylethylamine (1.8 mL, 10.3 mmol) and
morpholine (1.2 mL, 13.8 mmol) and stir 10 min. Add BOP (4.52 g, 10.2 mmol) and stir
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the mixture 24 h. Quench the mixture with water (200 mL). Extract the mixture with
EtOAc (4 60 mL). Wash the combined organic portions with water (4 x 50 mL) and
brine (2 x 50 mL); dry over a2S0 4; filter; collect the filtrate; and concentrate under
reduced pressure. Purify the crude product by preparatory HPLC (Waters Sunfire™
column C18, 4.6 mm x 150 mm, 5 mih particle size) eluting with a gradient of 45-100%
acetonitrile with 0.05% TFA in water to afford the title compound (0.88 g, 23%). LCES/
MS m/z 518 [M+H]+.
Preparation 70
Methyl 4-[l-[4-(dimethylcarbamoyl)phenyl]-5-iodo-pyrazol-3-yl]benzoate
Dissolve methyl 4-[l-(4-carbamoylphenyl)-5-iodo-pyrazol-3-yl]benzoate (223
mg, 0.499 mmol) in DMF (5 mL). Cool to 0 °C. Add sodium hydride (60% in oil) (60
mg, 1.5 mmol) in small portions over 10 min with stirring at 0 °C. Stir the mixture 1 h
and add methyl iodide (93 , 1.5 mmol) in a single portion. Allow the mixture to warm
to room temperature and stir 18 h. Quench the reaction with aqueous N 4CI (20 mL).
Extract the mixture with EtOAc (3 x 20 mL). Wash the combined organic portions with
brine (2 x 20 mL), dry over Na2S0 4, filter, and concentrate to dryness. Purify the crude
mixture by flash chromatography on silica (Biotage® system, 20 g cartridge @ 25
mL/min) eluting with a gradient of 0-40% EtOAc/dichloromethane to afford the title
compound (175 mg, 74%). LC-ES/MS m/z 476 [M+H]+.
Preparation 71: Alternate route to intermediate of Preparation 36
Methyl 4-[5-(3,5-ditert-butylphenyl)-l-[4-(4-methylpiperazine-lcarbonyl)
phenyl]pyrazol-3-yl]benzoate
Charge a reactor with methyl 4-[5-iodo-l-[4-(4-methylpiperazine-lcarbonyl)
phenyl]pyrazol-3-yl]benzoate (380 g, 0.720 mol), 2-(3,5-di-tert-butylphenyl)-
4,4,5,5-tetramethyl-l,3,2-dioxaborolane (296 g, 0.936 mol), bis(triphenylphosphine)
palladium(II) chloride (50 g, 0.072 mol), potassium carbonate (297 g, 2.150 mol), THF
( 11.4 L) and water (0.76 L). Heat the reaction to 85 °C for 1 h. Cool to 25 °C and filter
to give the two-layer filtrate. Separate the phases, and extract the aqueous layer with
EtOAc (2 x 5 L). Combine the organic layers; dry with a2S0 4; filter; collect the filtrate;
and evaporate the solvent to give a thick, dark brown oil. Purify the oil by column
chromatography, eluting with C C MeOH (from 50/1 to 20/1) to give the product as a
thick brown oil. Triturate the oil in a solvent mixture of acetonitrile/hexane/MTBE
X-1 93 17
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(2/1/1 , 2L) for 1 h. Filter to collect the solid and dry in the open air to give the product of
the title compound (360 g, 84%) as a gray solid. LC-ES/MS m/z 593 [M+H]+.
Preparation 72
Methyl 4-[5-(3-tert-butyl-5-isopropoxy-phenyl)-l -(4-carbamoylphenyl)pyrazol-3-
yljbenzoate
Dissolve methyl 4-[ l-(4-carbamoylphenyl)-5-iodo-pyrazol-3-yl]benzoate ( 116
mg, 0.259 mmol), 2-(3-tert-butyl-5-isopropoxy-phenyl)-4,4,5,5-tetramethyl- l,3,2-
dioxaborolane (100 mg, 0.3 14 mmol), and potassium carbonate (108 mg, 0.78 1 mmol) in
THF (15 mL), and water (3 mL). Add bis(triphenylphosphine)palladium(II) chloride (30
mg, 0.043 mmol). Purge the reaction vessel 3 times with nitrogen. Heat the mixture to
85 °C with stirring for 4 h. Dilute the mixture with EtOAc (50 mL). Wash the combined
organics with brine; dry over Na2S0 4; filter; collect the filtrate; and concentrate to
dryness under reduced pressure. Purify the crude mixture by flash chromatography on
silica (Biotage® system, 12 g cartridge @ 25 mL/min) eluting with a gradient of 0-50%
EtOAc/dichloromethane over 25 min to afford the title compound (105 mg, 79%). LCES/
MS m/z 512 [M+H]+.
Prepare the intermediates in Table 6 below, by essentially following the procedure
as described in Preparation 72, using the appropriate iodopyrazole and boronic acid or
ester ( 1.2 - 1.5 eq).
Table 6
Boronic Acid or LC-ES/MS
Prep Structure and Chemical Name
Ester (m/z)
2-(3-tert-Butyl-5-
Methyl 4-[5-(3-tert-butyl-5-isopropoxy- isopropoxyphenyl) 582
73 phenyl)- 1-[4-(morpholine-4- -4,4,5,5- [M+H]+
carbonyl)phenyl]pyrazol-3-yl]benzoate tetramethyl- 1,3,2-
dioxaborolane
2,6-Di-tert-butyl-
Methyl 4-[ l-(4-cyanophenyl)-5-(2,6-di-tert- 493
74 4-(4,4,5,5-
butyl-4-pyridyl)pyrazol-3-yl]benzoate [M+H]+
tetramethyl- 1,3,2-
X-1 93 17
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Preparation 79
Methyl 4-[ l-(4-carbamoylphenyl)-5-(2,6-di-ter?-butyl-4-pyridyl)pyrazol-3-yl]benzoate
Dissolve methyl 4-(5-(3-tert-butyl-5-isopropoxyphenyl)- l-(4-cyanophenyl)- lHpyrazol-
3-yl)benzoate (220 mg, 0.447 mmol) in TFA (4 mL). Add sulfuric acid ( 1 mL)
slowly. Heat the mixture to 45 °C and stir overnight. Pour the mixture into ice water and
adjust to about pH = 8 with 2 N NaOH ( 15 mL). Extract the mixture with EtOAc (3 50
mL). Wash the combined organic portions with brine (3 x 10 mL); dry over Na S0 4;
filter; collect the filtrate; and concentrate to dryness to afford the title compound (2 10 mg,
92%) as a white solid. LC-ES/MS m/z 511 [M+H]+.
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Example 1
4-[5-(3,5-Di-tert-butylphenyl)-l-[4-(4-methylpiperazine-l-carbonyl)phenyl]pyrazol-3-
yl]benzoic acid
Charge a reactor with THF (1790 L), methyl 4-[5-(3,5-di-tert-butylphenyl)-l-[4-
(4-methylpiperazine-l-carbonyl)phenyl]pyrazol-3-yl]benzoate (358 g, 0.605 mol) and
water ( 1140 mL) at 8 - 12 °C. Add lithium hydroxide monohydrate (38 g, 0.905 mol) in
one portion. Stir the mixture for 16 h at 8 - 12 °C. Add EtOAc (30 L) and adjust the
mixture to pH = 5 with 1N HC1. Separate the two phases and extract the aqueous layer
with EtOAc (2 10 L). Combine the organic layers, dry with sodium sulfate, filter, and
remove the solvent to give a solid. Purify the material by column chromatography,
eluting with DCM/MeOH (20/1) to give the product as a pale yellow solid. Triturate the
solid in a solvent mixture of acetonitrile/MTBE (1/3, 4 L) for 1 h. Filter to collect the
solid and dry under vacuum at 50 °C for 72 h to give the title compound (264.2 g, 75%)
as a white solid. LC-ES/MS m/z 579 [M+H]+.
Crystallization Procedure:
The crystalline free base of 4-[5-(3,5-Di-tert-butylphenyl)-l-[4-(4-
methylpiperazine-l-carbonyl)phenyl]pyrazol-3-yl]benzoic acid is prepared by placing
63.6 mg of f4-[5-(3,5-Di-tert-butylphenyl)-l -[4-(4-methylpiperazine- 1-
carbonyl)phenyl]pyrazol-3-yl]benzoic acid in a 20 mL vial. Add 4 mL of MeOH to
prepare a slurry including a white solid. Place the vial with the slurry stirplate heated to
60 °C and stir at 1000 rpm for 2 hours. Thereafter, allow the sample to cool to room
temperature. Isolate the resulting white solid by vacuum filtration dry overnight in a
vaccum oven set to 70 °C overnight.
Alternative Crystallization Procedure:
Crystalline 4-[5-(3 ,5-Di-tert-butylphenyl)- 1-[4-(4-methylpiperazine- 1-
carbonyl)phenyl]pyrazol-3-yl]benzoic acid can also be prepared placing 69 mg of of 4-[5-
(3,5-Di-tert-butylphenyl)-l-[4-(4-methylpiperazine-l-carbonyl)phenyl]pyrazol-3-
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yljbenzoic acid and 3 mg of seed crystals of the same form in a 20 mL vial and add 2 mL
of MeOH to prepare a slurry containing white solid. The slurry is heated to 60 °C and
stirred at 1000 rpm for four hours. Thereafter stop the stirring and allow the sample to
cool to room temperature and stand until morning to yield a thick layer of white solid
under a clear but slightly yellow supernatant. Isolate the white solid by vacuum filtration
and dry under nitrogen stream for 10 minutes before being placed in a new tared vial.
This resulting material can be examined by X-Ray Powder Diffraction as described
below. Additional this material can be placed in a vaccum oven set to 70 °C to dry
completely.
Example 2
4-[5-(3,5-Di-tert-butylphenyl)-l-[4-(piperidine-l-carbonyl)phenyl]pyrazol-3-yl]benzoic
acid
Dissolve methyl 4-[5-(3,5-di-tert-butylphenyl)-l-[4-(piperidine-lcarbonyl)
phenyl]pyrazol-3-yl]benzoate (121 mg, 0.209 mmol) in water ( 1 mL), THF (3
mL), and methanol ( 1 mL). Add LiOH ( 11 mg, 251 mmol). Stir the mixture for about 3
h. Adjust the pH of the mixture with 2 N HC1 to pH = 7. Dilute the mixture with EtOAc
(20 mL). Wash the organics with 2 N HC1 (4 mL), and saturated aqueous NaCl (10 mL).
Dry the organics over Na2S0 4, filter, and concentrate under reduced pressure. Purify the
crude product by preparatory TLC eluting with 10:1 dichloromethane/methanol to afford
the title compound (75 mg, 64%). LC-ES/MS m/z 564 [M+H]+.
Prepare the examples in Table 7 below, by essentially following the procedure as
described in Example 2, using the appropriate methyl benzoate precursor.
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Table 7
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Example 12
-Butyl-5-isopropylsulfanyl-phenyl)-l-[4-(dimethylcarbamoyl)phenyl]pyrazol-
3-yl]benzoic acid
Dissolve methyl 4-[5-(3-tert-butyl-5-isopropylsulfanyl-phenyl)-l-[4-
(dimethylcarbamoyl)phenyl]pyrazol-3-yl]benzoate (100 mg, 0.180 mmol) in methanol (2
mL) and THF (6 mL). Add 1M LiOH (2 mL) in a single portion with stirring. Stir the
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mixture for 3 h at room temperature. Acidify the mixture with 2 N HC1 to about pH = 2
and extract with EtOAc (3 x 20 mL). Wash the combined organic portions with brine (2
10 mL), dry over Na2S0 4, filter and concentrate under reduced pressure to afford the
title compound (82 mg, 84%) as a white solid. LC-ES/MS m/z 542 [M+H]+.
Example 13
4-[5-(3-tert-Butyl-5-isopropylsulfanyl-phenyl)-l-[4-(4-methylpiperazine-lcarbonyl)
phenyl]pyrazol-3 -yl]benzoic acid
Dissolve methyl 4-[5-(3-tert-butyl-5-isopropylsulfanyl-phenyl)-l-[4-(4-
methylpiperazine-l-carbonyl)phenyl]pyrazol-3-yl]benzoate (30 mg, 0.049 mmoles) in
THF (3 mL) and methanol ( 1 mL). Add 1N lithium hydroxide (1.0 mL, 1.0 mmol). Stir
the reaction for 6 h. Adjust to about pH = 6 with 1N HC1. Extract with EtOAc (40
mL). Wash the organic portion with water (2 x 20 mL), dry over a2S0 4, filter, and
concentrate to afford the title compound (25 mg, 85%). LC-ES/MS m/z 597 [M+H]+.
Example 14
4-[5-(3-tert-Butyl-5-isopropoxy-phenyl)-l-[4-(4-methylpiperazine-lcarbonyl)
phenyl]pyrazol-3 -yl]benzoic acid
Dissolve methyl 4-[5-(3-tert-butyl-5-isopropoxy -phenyl)- 1-[4-(4-
methylpiperazine-l-carbonyl)phenyl]pyrazol-3-yl]benzoate (0.14 g, 0.235 mmol) in THF
(3 mL), methanol ( 1 mL), and water ( 1 mL). Add LiOH (0.015 g, 0.353 mmol) and
stir for 4 h. Adjust to about pH = 7 with 2 N HC1. Separate the layers and wash the
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organic layer with aqueous NaCl (10 mL). Dry the organics over a2S0 4, filter, and
concentrate to afford the title compound (135 mg, 99%). LC-ES/MS m/z 581 [M+H]+.
Example 15
4-[5-(3,5-Di-tert-butylphenyl)-l-(6-methylsulfonyl-3-pyridyl)pyrazol-3-yl]benzoic acid
Dissolve methyl 4-[5-(3,5-di-tert-butylphenyl)- 1-(6-methylsulfonyl-3-
pyridyl)pyrazol-3-yl]benzoate (102 mg, 0.187 mmol) in water (3 mL), THF (9 mL), and
methanol (3 mL). Add LiOH (16 mg, 0.374 mmol) in a single portion. Stir the mixture
overnight. Adjust the solution to about pH = 2 with 2 N HC1. Extract the mixture 3 times
with EtOAc. Wash the combined organics with brine (3 x 50 mL), dry over Na2S0 4,
filter, and concentrate under reduced pressure. Purify the resulting residue by preparatory
TLC to afford the title compound (85 mg, 86%). LC-ES/MS m/z 532 [M+H]+.
Example 16
4-[5-(3,5-Di-tert-butylphenyl)-l-[6-(dimethylcarbamoyl)-3-pyridyl]pyrazol-3-yl]benzoic
Dissolve methyl 4-[5-(3,5-di-tert-butylphenyl)-l-[6-(dimethylcarbamoyl)-3-
pyridyl]pyrazol-3-yl]benzoate prep 27 is wrong use prep 44 instead (60 mg, 0.111
mmol) in MeOH (4 mL) and THF ( 1 mL). Add 1M LiOH (0.5 mL) in a single portion.
Stir the mixture for 5 h at room temperature. Acidify the mixture to about pH = 6-7 with
2 N HC1. Extract with EtOAc (50 mL). Wash the organic portion with brine (2 x 20
mL), dry over Na2S0 4, filter, and concentrate under reduced pressure. Purify the
resulting residue by preparatory TLC eluting with 10: 1 DMC/MeOH to afford the title
compound (30 mg, 51%). LC-ES/MS m/z 525 [M+H]+.
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Example 17
4-[5-(3,5-Di-tert-butylphenyl)-l-[4-(methanesulfonamido)phenyl]pyrazol-3-yl]benzoic
acid
Dissolve methyl 4-[5-(3,5-di-tert-butylphenyl)-l-[4-
(methanesulfonamido)phenyl]pyrazol-3-yl]benzoate (230 mg, 411 mmol) in methanol (3
mL) and THF (3 mL). Add 1M LiOH ( 1 mL, 1.0 mmol) in a single portion. Stir the
mixture overnight. Quench the reaction with dilute aqueous HC1. Extract the mixture 3
times with EtOAc. Dry the combined organic portions over a2S0 4, filter, and
concentrate to dryness under reduced pressure. Purify the resulting residue
by preparatory TLC, eluting with 2:1 EtOAc/petroleum ether to afford the title
compound (200 mg, 89%). LC-ES/MS m/z 546 [M+H]+.
Example 18
4-[5-(3,5-Di-tert-butylphenyl)-l-[4-(dimethylsulfamoyl)phenyl]pyrazol-3-yl]benzoic acid
Dissolve methyl 4-[5-(3,5-di-tert-butylphenyl)-l-[4-
(dimethylsulfamoyl)phenyl]pyrazol-3-yl]benzoate (0.1 1 g, 0.192 mmol) in methanol ( 1
mL) and THF (6 mL). Add 1N LiOH (40 mg, 0.953 mmol) in a single portion. Stir the
mixture at room temperature overnight. Dilute the reaction mixture with water. Extract
with EtOAc (30 mL). Dry the combined organic portions over a2S0 4, filter, and
concentrate under reduced pressure to afford the title compound ( 110 mg, 100%). LCES/
MS m/z 560 [M+H]+.
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Example 19
4-[5-(3,5-Di-tert-butylphenyl)-l-(4-methylsulfonylphenyl)pyrazol-3-yl]benzoic acid
Dissolve methyl 4-[5-(3,5-di-tert-butylphenyl)-l-(4-
methylsulfonylphenyl)pyrazol-3-yl]benzoate (58 mg, 0.106 mmol) in ethanol (2.5 mL)
and THF (3 mL). Add sodium hydroxide (0.064 mL, 0.3 19 mmol) at room
temperature. Heat the reaction at 50 °C with stirring for 2 h. Cool the reaction to room
temperature. Dilute the reaction with water and adjust to about pH = 1-2 with 1N HC1.
Stir for 10 min and cool to 4 °C. Collect the resulting crystals by vacuum filtration,
rinsing with water to afford the title compound (49 mg, 87%) as a white crystalline solid.
LC-ES/MS m/z 531 [M+H]+.
Example 20
4-[5-(3-tert-Butyl-5-isopropoxy -phenyl)- l-(4-carbamoylphenyl)pyrazol-3-yl]benzoic acid
Dissolve methyl 4-[5-(3-tert-butyl-5-isopropoxy-phenyl)-l-(4-
carbamoylphenyl)pyrazol-3-yl]benzoate (105 mg, 0.205 mmol) in methanol (6 mL), and
THF (2 mL). Add 1M LiOH (2 mL) in a single portion with stirring. Stir the mixture for
18 h. Add 2 N HC1 to pH = 6. Extract with EtOAc (50 mL). Wash the organic portion
with brine (2 x 20 mL), dry over Na2S0 4, filter, and concentrate to dryness under reduced
pressure. Purify the crude mixture using flash chromatography on silica (ISCO® system,
12 g cartridge @ 25 mL/min) with a gradient of 0-20% methanol/dichloromethane over
25 min to afford the title compound (93 mg, 91%). LC-ES/MS m/z 498 [M+H]+.
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Example 21
-Butyl-5-isopropoxy-phenyl)-l-[4-(morpholine-4-carbonyl)phenyl]pyrazol-3-
yl]benzoic acid
Dissolve methyl 4-(5-(3-tert-butyl-5-isopropoxyphenyl)-l-(4-(morpholine-4-
carbonyl)phenyl)-pyrazol-3-yl)benzoate check prep 73 (160 mg, 0.275 mmol) in
methanol (6 mL) and THF (2 mL). Add 1M LiOH (2 mL) in a single portion and stir
overnight. Adjust to about pH = 6 with 2 N HC1. Extract with EtOAc (50 mL). Wash
the combined organics with brine (2 x 20 mL), dry over a2S0 4, filter, and concentrate to
dryness to afford the title compound (136 mg, 87%). LC-ES/MS m/z 568 [M+H]+.
Example 22
4-[l-(4-Carbamoylphenyl)-5-(2,6-di-tert-butyl-4-pyridyl)pyrazol-3-yl]benzoic acid
Dissolve methyl 4-[l-(4-carbamoylphenyl)-5-(2,6-di-tert-butyl-4-pyridyl)pyrazol-
3-yl]benzoate (210 mg, 0.41 1 mmol) in methanol (6 mL) and THF (2 mL). Add 1M
LiOH ( 1 mL) in a single portion. Stir the mixture 18 h. Adjust the mixture about pH = 6
with 2 N HC1 and extract with EtOAc (50 mL). Wash the combined organic portions
with brine (2 x 20 mL), dry over a2S0 4, filter, and concentrate to dryness to afford the
title compound (180 mg, 88%) as a white solid. LC-ES/MS m/z 497 [M+H]+.
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X-Ray Powder Diffraction
The XRD patterns of crystalline 4-[5-(3,5-Di-tert-butylphenyl)-l-[4-(4-
methylpiperazine-l-carbonyl)phenyl]pyrazol-3-yl]benzoic acid which can be prepared as
described above for Example 1 is obtained on a Bruker D4 Endeavor X-ray powder
diffractometer, equipped with a CuKa source l = 1.54060 A) and a Vantec detector,
operating at 35 kV and 50 mA. The sample is scanned between 4 and 40° in 2Q, with a
step size of 0.009° in 2Qand a scan rate of 0.5 seconds/step, and with 0.6 mm divergence,
5.28 fixed anti-scatter, and 9.5 mm detector slits. The dry powder is packed on a quartz
sample holder and a smooth surface is obtained using a glass slide. The crystal form
diffraction patterns are collected at ambient temperature and relative humidity. A peak
position variability of ± 0.2 in 2Qtakes into account potential variations without hindering
the unequivocal identification of the indicated crystal form. Confirmation of a crystal
form may be made based on any unique combination of distinguishing peaks (in units of °
2Q), typically the more prominent peaks. The crystal form diffraction pattern, collected at
ambient temperature and relative humidity, was adjusted based on NIST 675 standard
peaks at 8.853 and 26.774 degrees 2-theta.
Thus, a prepared sample of the free base of 4-[5-(3,5-Di-tert-butylphenyl)-l-[4-(4-
methylpiperazine-l-carbonyl)phenyl]pyrazol-3-yl]benzoic acid prepared as described
above for Example 1 is characterized by an XRD pattern using CuKa radiation as having
diffraction peaks (2-theta values) as described in Table 8 below, and in particular having
peaks at 5.414 in combination with one or more of the peaks selected from the group
consisting of 19.851, 7.498, and 14.588; with a tolerance for the diffraction angles of 0.2
degrees.
Table 8 :
X-ray powder diffraction peaks of the free base of 4-[5-(3,5-Di-tert-butylphenyl)-l-[4-(4-
methylpiperazine- 1-carbonyl)phenyl]pyrazol-3 -yl]benzoic acid.
Crystalline 4-[5-(3,5-Di-tert-butylphenyl)-l-[4-(4-
methylpiperazine- 1-carbonyl)phenyl]pyrazol-3 -yl]benzoic acid
Peak Positions
Relative Intensity
Angle (°2-Theta) +/- (% of most intense d value
Peak 0.2° peak) (angstroms)
1 5.4 100 16.30899
2 19.9 49.5 4.46887
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Assays
The following assay protocols and result(s) thereof demonstrating the utility and
efficacy of the compounds and/or methods of the current invention are given for the
purpose of illustration and are not meant to be limiting in any way.
RARa, b and g Binding Assay
Compounds can be evaluated for binding to RARa, b and g by measuring their
ability to competitively bind to the RAR receptors when dimerized with the binding
partner RXRa. Competitive binding assays may be carried out by Scintillation Proximity
Assay (SPA) technology using the RARa, b or g heterodimer (with RXRa as a partner
with all the RARs) receptors prepared in a baculovirus expression system. Use the
biotinylated oligonucleotide:
5'-ATAATGTAGGTAATAGGTCACCAGGAGGTCAAAGG-3' for binding of receptor
to yttrium silicate streptavidin- coated SPA beads. Per well, preincuabate 0.1 nM with
82.7 mg SPA beads in a binding buffer containing 10 mM HEPES pH 7.8, 80 mM KC1,
0.5 mM MgCl2, 1mM DTT, 0.5% CHAPS and 16.6 mg bovine serum albumin for 30 min
at room temperature. Then spin the mixture at 2,000 rpm for 3 min to pelletize the beadsoligo
mix. Remove the supernatant and resuspend the beads-oligo pellet in the same
binding buffer as above, but which in addition now also contain 14% glycerol, 5 mg of
sheared salmon sperm DNA, 0.5 mg of RARa, 1.0 mg of RAR , or 0.25 mg of RAR
receptor, respectively. Carry out the binding assays in the presence of ~ 11.3 m ϊ of H 4-
[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naph thalenyl)-l-propenyl]benzoic acid
(TTNPB), and multiple concentrations of test compound ranging from (5 nM to 10 mM) .
Non-specific binding may be determined in the presence of 1 mM unlabeled TTNPB. Use
the data to calculate an IC50 for compounds after fitting the dose-response curves to a
4-paratmeter logistic fit. Use the Cheng-Prusoff equation to convert IC50 (nM) values for
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compounds to K , and the ¾ may be determined by saturation binding. All of the
compounds listed as Examples disclosed herein demonstrate activity in the RARy binding
assay substantially as described herein with a measured ¾ of less than 20 nM. All of the
compounds listed as Examples disclosed herein demonstrate low activity in the RAR b
binding assay substantially as described herein with a measured K of greater than 100
nM. All of the compounds listed as Examples disclosed herein exhibit low activity in the
RARa binding assay substantially as described herein with a measured K of greater than
100 nM. The results of four of the Examples are shown in Table 9 below:
RARa, b and g Binding Assay
Table 9
Ki RARy Ki RARa Ki RAR
Compound name
(nM) (nM) (nM)
Example 1
4-[5-(3,5-Di-tert-butylphenyl)-l-[4-(4- 1.69 >970 >1820
methylpiperazine- 1- n = 1 n = 1 n = 1
carbonyl)phenyl]pyrazol-3 -yl]benzoic acid
Example 8
1.58 357 1400
4-[5-(2,6-Di-tert-butyl-4-pyridyl)-l-[4-
n = 1 n = 1 n = 1
(morpholine-4-carbonyl)phenyl]pyrazol-3-
yl]benzoic acid
Example 12
4-[5-(3 -tert-Buty 1-5 -isopropylsulfanyl- 3.06 386 609
phenyl)-l-[4-(dimethylcarbamoyl) n = 1 n = 1 n = 1
phenyl]pyrazol-3-yl]benzoic acid
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The results of this assay support that the of the Examples disclosed herein bind to
the RARy receptor and the selectivity of the Examples for the RARy receptor over the
RARa, and RAR receptors.
Gal4 reporter assay to determine RAR receptor antagonist activity
For cell-based assays, human embryonic kidney HEK 293 cells are transfected
with receptor and reporter gene plasmids using Fugene. The reporter plasmid containing
five Gal4 binding sites and a major late promoter of adenovirus upstream of the luciferase
reporter cDNA is transfected together with a plasmid constitutively expressing the Gal4
DNA binding domain (DBD) and the RARa ligand binding domain (LBD), Gal4 (DBD)
RARy (LBD), or the Gal4 (DBD) RAR (LBD) hybrid receptor using a SV40 promoter.
Cells are transfected in poly-d- lysine coated T175 cm flasks in DMEM media with 5%
charcoal-stripped Fetal Bovine Serum (FBS). After an overnight incubation, transfected
cells are trypsinized, plated in opaque 96 well dishes in DMEM media containing 5%
charcoal-stripped FBS, incubated for 4h, and then exposed to 0.17 nM to 10 mM of test
compound in half log dilutions. To determine the antagonist activity of the test
compound, ECso concentrations of agonist for each receptor is also added to the media
(15 nM all-trans retinoic acid, ATRA, for RARa and RARy, 10 nM of ATRA for RAR ).
After 24 hours of incubation with compounds, cells are lysed and luciferase activity is
determined. Data are fitted to a four parameter- fit logistics to determine IC50 values. The
maximum % inhibition is determined versus the cellular response to 0.25% DMSO in the
absence of ATRA. All of the compounds of the Examples disclosed herein demonstrate
activity in the Gal4 reporter assay substantially as described herein with a measured ¾ , of
less than 250 nM. The results of four of the compounds are shown in Table 10.
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Table 10
Gal4 Reporter Assay
The results of the Gal4 reporter assay support that the Examples disclosed herein
are RARy antagonists.
RARy SRC-2 Coactivator Recruitment Assay (Agonist Mode)
The RARy SRC-2 Coactivator recruitment assay utilizes the ligand binding
domain (LBD) of RARy with its binding partner RXRa to determine the ability of a
compound to enhance the recruitment of the co-activator SRC-2 to the receptor complex.
Enhanced recruitment of SRC2 is known to be reflective of an agonist confirmation of the
RARy receptor. The RARy LBD and SRC2 peptides are covalently linked to
AlphaScreen® beads such that enhanced protein-protein interactions can be assessed by
energy transfer. Coactivator recruitment assays are performed using AlphaScreen®
technology (Perkin Elmer USA) using a 6X-Histidine tagged human RARy LBD and
GST tagged hSRC-2 protein. Unlabelled RXRa LBD is added as a silent heterodimer
partner. Nickel chelated donor beads are used to bind RARy LBD and anti-GST acceptor
beads are used to bind SRC-2. Serially diluted test compound is added in concentrations
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ranging from 10 mM to 500 pM to 20 nM human RARy receptor, 25 nM RXRa LBD, and
5 nM SRC-2 protein in a buffer containing 25 mM HEPES (pH 7.5), 100 mM NaCl, 0.1%
Bovine Serum Albumin (fraction V), and 2 mM DTT containing 16.67 mg/ml of nickel
chelated donor beads and 16.67 mg/ml of anti GST acceptor beads in a final volume of 15
mΐ per well in a white 384 shallow well proxiplate. The RAR agonist, TTNPB, is used as
a standard on each plate and is added in concentrations ranging from 100 nM to 5 pM.
After incubating for 12 hours at room temperature, read the plate on a Perkin Elmer
Envision using standard AlphaScreen® parameters for excitation and fluorescence. Use
the data to calculate an EC50 for compounds after fitting the dose-response curves to a
4-parameter logistic fit. Calculate the percent stimulation using the fitted top of the
TTNPB standard curve as a comparator. All of Examples disclosed herein demonstrate
less than 50 % maximum stimulation in this assay. The results of four of the compounds
are shown in Table 10.
Table 11
Percent Stimulation in the RARy SRC-2 Coactivator Recruitment Assay (Agonist Mode)
The results of this assay show that the Examples disclosed herein do not exhibit
significant RARy agonistic activity.
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Monosodium Iodoacetate (MIA) Model of Pain
The injection of monoiodoacetic acid (MIA) into the knee joint of rats produces
an acute inflammatory insult, which then develops into chronic degeneration of the joint
tissues in the injected joint. The pain resulting from the joint injury can be measured via
differential weight bearing of the hind legs using an incapacitance tester. The MIA model
has been well-described in the literature and has been used to demonstrate efficacy versus
pain for a variety of mechanisms and compounds. Efficacy is routinely measured by the
ability of a compound to partially normalize weight distribution. The maximal efficacy
that can be achieved in the standard MIA is dependent on the mechanism being studied;
however, for many mechanisms the maximal efficacy achieved results in a 25% - 50%
reduction in the weight bearing differential.
Use Male Lewis rats approximately 150-170 g and between 6-8 weeks of age. Let
the animals acclimate to the environment for at least 72 h. Record body weight as needed
for dosing schedules and for calibration of Incapacitance Testers. Animals are assigned
to treatment groups using the Block Randomized Allocation Tool (BRAT).
MIA sodium salt (from Sigma). Store MIA salt at -80 °C. Prepare the MIA, 0.3
mg in 50 mΐ , in sterile 0.9% saline. Load the syringes with the prepared MIA solution the
day the rats are to be injected.
Anesthetize the animals with Isoflurane. Flex the knee joint to locate the joint
space between the tibia and the femur. Clean the injection site with 70% ethanol and
slowly inject the MIA or saline into the joint space. Inject the right knee with MIA (50
mΐ) and inject the left knee (contralateral control) with sterile saline (50 mΐ) .
Incapacitance Tester Readings - Incapacitance Testers (Columbus Instruments
International, Columbus, OH) for weight bearing measurements. Place rats in a
plexiglass chamber so that each hind paw rests on a separate force plate (pressure sensor).
Allow the rats to acclimate to the chamber for at least 5 minutes. A total of three one
second readings are taken to reflect the amount of pressure exerted on both the left and
right hind paw while the rat is positioned in the chamber. The force exerted by each hind
paw is measured in grams and calculated as the left hind paw weight distribution-right
hind paw weight distribution. Thus, the final paw weight distribution for each animal is
an average of the three one second readings.
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Studies for RARy antagonists: Dose rats with RARy antagonist once on day 9
post MIA injection and measure each rat for pain 2 h post dosing. Allow 10-15 min
between dosing for each rat to allow 10-15 minutes for pain measurements. Most
compounds are initially screened for reduction of pain in a single dose study at 1 or 3
mg/kg of compound before advancing to dose response studies. Generate dose response
curves and ED 0 values for the RARy antagonist, by performing either separate dose
response studies and combining the results where the 2 studies are: 1) vehicle, RARy
antagonist at 0.1, 0.3 and 1.0 mg/kg, and 2) vehicle, RARy antagonist at 1, 3, and 10
mg/kg; or as a dose response study where all animals are tested in the same study at 0.1,
0.3, 1.0, 3.0, and 10 mg/kg (Example 1 only). The dose volume for either type of study is
5 ml/kg.
In a dose response study in the standard MIA model, the compound of Example 1
significantly inhibits pain when compared to vehicle at a dose of 0.1 mg/kg.
Exemplified compounds of the present invention can be readily formulated into
pharmaceutical compositions in accordance with accepted practice such as found in
Remington's Pharmaceutical Sciences, Gennaro, Ed., Mack Publishing Co. Easton Pa.
1990. Oral administration is typically the preferred route of administration for
osterarthritis therapy. Preferred pharmaceutical compositions can be formulated as a
tablet or capsule for oral administration. The tablet or capsule can include a compound of
the present invention in an effective amount.
The pharmaceutical composition is administered to a patient in amounts effective
to treat arthritis, more particularly osteoarthritis and still more preferable for pain
associated with osteorarthritis. An appropriate amount or dose effective to treat a patient
can be determined by a health care provider and may be dependent upon the age, health,
and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment,
and the nature of the effect desired. Typical dosage levels can be optimized using
standard clinical techniques and will be dependent on the mode of administration and the
condition of the patient.
A compound of the present invention can be employed in combination with one or
more therapeutic agents, such as, analgesics and/or NSAIDS (nonsteroidal anti
inflammatory drug) or COX-2 inhibitors for example, such as aspirin, acetaminophen,
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celecoxib, diclofenac, ibuprofen, indomethacin, and naproxen, or other anti inflammatory
agents.
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What is claimed is:
1. A compound having a formula below:
is CH o ;
Rl is selected from: -S0 2CH3, -S0 2N(CH3)2, -C(0)N(R3) 2, -C(0)R4, and
-NHS0 2CH3;
R2 is selected from: -C3-4 alkyl, -OCH(CH3)2, and -SCH(CH3)2;
each R3 is independently selected from: H and -CH3;
R4 is selected from: 4-morpholinyl, l-piperidinyl, 4-thiomorpholinyl,
-NH(CH2)3OH, and 4-methyl-l-piperazinyl; and
provided that when one of A or X is N, the other one of A or X is CH;
or a pharmaceutically acceptable salt thereof.
2. A compound according to claim 1 wherein A is CH.
3. A compound according to claim 1 or 2 wherein X is CH.
4. A compound according to any one of claims 1 to 3 wherein Rl is
-C(0)N(R3) 2 or -C(0)R4, or a pharmaceutically acceptable salt thereof.
5. A compound according to any on of claims 1 to 4 wherein R2 is
selected from: -C3_4 alkyl and -SCH(CH3)2; or a pharmaceutically acceptable salt
thereof.
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6. A compound according to to any one of claims 1 to 5 wherein R2 is
selected from: isopropyl, -butyl, and -SCH(CH )2, or a pharmaceutically acceptable
salt thereof.
7. A compound according to any one of claims 1 to 6 wherein R2 is
isopropyl or -butyl, or a pharmaceutically acceptable salt thereof.
8. A compound according to any one of claims 1 to 7 wherein each R3 is
-CH3, or a pharmaceutically acceptable salt thereof.
9. A compound according to any one of claims 1 to 7 wherein each R3 is
H, or a pharmaceutically acceptable salt thereof.
10. A compound according to any one of claims 1 to 9 wherein R4 is
selected from: 4-morpholinyl, 1-piperidinyl, 4-thiomorpholinyl, and
4-methyl- 1-piperazinyl, or a pharmaceutically acceptable salt thereof.
11. A compound according to any one of claims 1 to 10 wherein R4 is 4-
morpholinyl or 4-methyl- 1-piperazinyl, or a pharmaceutically acceptable salt thereof.
12. A compound according to any one of claims 1 to 11 wherein R4 is
4-methyl- 1-piperazinyl, or a pharmaceutically acceptable salt thereof.
13. A compound according to claim 1 wherein:
A is CH;
X is CH;
Rl is -C(0)N(R3) 2, or -C(0)R4;
R2 is selected from: -C3-4 alkyl, -OCH(CH3)2, and -SCH(CH3)2;
each R3 is independently H or CH ; and
R4 is selected from: 4-morpholinyl, 1-piperidinyl, 4-thiomorpholinyl,
-NH(CH2)3OH and 4-methyl- 1-piperazinyl;
or a pharmaceutically acceptable salt thereof.
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14. A compound according to claim 13 wherein,
A is CH;
X is CH;
Rl is -C(0)N(R3) 2, or -C(0)R4;
R2 is selected from: -C3-4 alkyl;
each R3 is independently H or -CH3; and
R4 is selected from: 4-morpholinyl, l-piperidinyl, 4-thiomorpholinyl,
-NH(CH2)3OH and 4-methyl- 1-piperazinyl;
or a pharmaceutically acceptable salt thereof.
15. A compound according to claim 1 wherein:
A is CH;
is N;
Rl is -C(0)N(R3) 2;
R2 is -C3 _4 alkyl; and
R3 is H or -CH3; or
a pharmaceutically acceptable salt thereof.
16. A compound according to claim 1 wherein:
A is N;
X is CH;
Rl is -C(0)R3 or -C(0)R4;
R2 is -C3 _4 alkyl;
R3 is H or CH3;
R4 is 4-morpholinyl, or
a pharmaceutically acceptable salt thereof.
17. A compound according to either claim 15 or 16 wherein R2 is tertbutyl,
or a pharmaceutically acceptable salt thereof.
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18. A compound which is 4-[5-(3,5-Di-tert-butylphenyl)-l-[4-(4-
methylpiperazine-l-carbonyl)phenyl]pyrazol-3-yl]benzoic acid, or a pharmaceutically
acceptable salt thereof.
19. A pharmaceutical composition comprising a compound according to
any one of claims 1 to 18, or a pharmaceutically acceptable salt thereof, and at least one
pharmaceutically acceptable carrier, excipient, or diluent.
20. A pharmaceutical composition comprising a compound as claimed
byclaim 18 and additionally comprising one or more therapeutic agents.
21. A method of treating osteoarthritic pain in a patent in need of
treatment comprising administering to said patient an effective amount of a
pharmaceutical composition according to claim 19 or 20.
22. A compound or a pharmaceutically acceptable salt thereof according
to any one of claims 1 to 18 for use in therapy.
23. A compound, or a pharmaceutically acceptable salt thereof, according
to any one of claims 1 to 18, for use in the treatment of osteoarthritis.
24. Use of a compound, or a pharmaceutically acceptable salt thereof,
according to any one of claims 1 to 18, for use in the manufacture of a medicament.
25. A compound according to formula II
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R is selected from C1-4 alkyl, C1-4 haloalkyl, C3-6 cycloalkyl, C1-4 alkyl-C3-6
cycloalkyl, phenyl, and C1-5 alkylphenyl;
A is CH o ;
is CH orN
Rl is selected from: -S0 2CH3, -S0 2N(CH3)2, -C(0)N(R3) 2, -C(0)R4, and
-NHS0 2CH3;
R2 is selected from: -C3-4 alkyl, -OCH(CH3)2, and -SCH(CH3)2;
each R3 is independently selected from: H and -CH3;
R4 is selected from: 4-morpholinyl, 1-piperidinyl, 4-thiomorpholinyl,
-NH(CH2)3OH, and 4-methyl-l-piperazinyl; and
provided that when one of A or X is N, the other one of A or X is CH;
or a pharmaceutically acceptable salt thereof.
26. A process of preparing a compound of formula I or a pharmaceutically
acceptable salt thereof,
wherein:
A is CH orN;
X is CH orN;
Rl is selected from: -S0 2CH3, -S0 2N(CH3)2, -C(0)N(R3) 2, -C(0)R4, and
-NHS0 2CH3;
R2 is selected from: -C3 4 alkyl, -OCH(CH3)2, and -SCH(CH3)2;
each R3 is independently selected from: H and -CH3;
R4 is selected from: 4-morpholinyl, 1-piperidinyl, 4-thiomorpholinyl,
-NH(CH2)3OH, and 4-methyl-l-piperazinyl; and
provided that when one of A or X is N, the other one of A or X is CH;
said method comprising de-esterifying a compound of formula II;
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wherein Rl to R4 is as above; and
R is selected from C1-4 alkyl, C1-4 haloalkyl, C 3-6 cycloalkyl, C1-4 alkyl-C3-6
cycloalkyl, phenyl, and C1-5 alkylphenyl to provide a compound of formula I, or a
pharmaceutically acceptable salt thereof
27. A compound which is 4-[5-(3,5-Di-tert-butylphenyl)-l-[4-(4-
methylpiperazine-l-carbonyl)phenyl]pyrazol-3-yl]benzoic acid in crystalline form
characterized by an X-ray powder diffraction pattern obtained from a CuKa source
(l=1.54056 A) which comprises peaks at:
a) 5.4, 7.5, 14.6, and 19.9 +/-0.2 in 2Q; or
b) 5.4, 7.5, 14.6, 16.0, 19.4, and 19.9 +/- 0.2 in 2Q; or
c) 5.4, 7.5, 14.6, 15.7, 16.0, 19.4, 19.9 and 22.1 +/-0.2 in 2Q.