DIMETHYL-BENZOIC ACID COMPOUNDS
The present invention relates to novel dimethyl-benzoic acid compounds, to
pharmaceutical compositions comprising the compounds, to methods of using the
compounds to treat physiological disorders, and to intermediates and processes useful in
the synthesis of the compounds.
The present invention is in the field of treatment of inflammatory conditions, such
as arthritis, including osteoarthritis and rheumatoid arthritis, and further including pain
associated with these conditions. Arthritis affects millions of patients in the United States
alone and is a leading cause of disability. Treatments often include NSAIDs
(nonsteroidal anti-inflammatory drugs) or CO -2 inhibitors, which may produce
untoward cardiovascular side effects. As such, patients who have a poor cardiovascular
profile, such as hypertension, may be precluded from using NSAIDs or COX-2 inhibitors.
Thus, there is a need for an alternative treatment of osteoarthritis and rheumatoid arthritis,
preferably without the side effects of the current treatments.
Four prostaglandin E2 (PGE2) receptor subtypes have been identified as the
following: EPl, EP2, EP3 and EP4. It has been disclosed that EP4 is the primary receptor
involved in joint inflammatory pain in rodent models of rheumatoid arthritis and
osteoarthritis (See J. Pharmacol. Exp. Ther., 325 , 425 (2008)). Hence, a selective EP4
antagonist may be useful in treating arthritis, including arthritic pain. In addition, it has
been suggested that since EP4 antagonism does not interfere with biosynthesis of
prostanoids, such as PGI2 and TxA2, a selective EP4 antagonist may not possess the
potential cardiovascular side effects seen with NSAIDs and COX-2 inhibitors. (See for
example Bioorganic & Medicinal Chemistry Letters, 21, 484 (201 1)).
WO 96/02509 discloses certain quinoline derivatives which are selective, nonpeptide
NK3 antagonists useful in treating a variety of disorders including, for example,
pulmonary disorders, CNS disorders, neurogenic inflammation, and inflammatory pain.
In addition, U.S. Patent No. 7,705,035 discloses certain indoline amide derivatives useful
as EP4 ligands, agonists, or antagonists useful in treating various disorders, such as
osteoarthritis, rheumatoid arthritis, and acute and chronic pain.
The present invention provides certain novel compounds that are selective
inhibitors of EP4 relative to EPl, EP2, and EP3. In addition, the present invention
provides certain novel compounds with the potential for reduced cardiovascular or
gastrointestinal side effects in comparison to traditional NSAIDs.
Accordingly, the present invention provides a compound of the Formula II:
Formula II
wherein A is:
R1 is CH3, CF3, or F;
R2 is H, CH3, or F;
R3 is CH3, OCH3, OH, F;
R4 is OH or CH2OH; and
is CH o ;
or a pharmaceutically acceptable salt thereof.
The present invention further provides a compound of the Formula I :
Formula I
or a pharmaceutically acceptable salt thereof.
The invention further provides a hydrated compound of Formula I .
The present invention also provides a method of treating arthritis in a patient,
comprising administering to a patient in need of such treatment an effective amount of a
compound of Formula I or Formula II, or a pharmaceutically acceptable salt thereof. The
present invention also provides a method of treating osteoarthritis in a patient, comprising
administering to a patient in need of such treatment an effective amount of a compound of
Formula I or Formula II, or a pharmaceutically acceptable salt thereof. In addition, the
present invention provides a method of treating rheumatoid arthritis in a patient,
comprising administering to a patient in need of such treatment an effective amount of a
compound of Formula I or Formula II, or a pharmaceutically acceptable salt thereof. The
present invention also provides a method of treating pain associated with arthritis in a
patient, comprising administering to a patient in need of such treatment an effective
amount of a compound of Formula I or Formula II, or a pharmaceutically acceptable salt
thereof. The present invention further provides a method of treating pain associated with
osteoarthritis or rheumatoid arthritis in a patient, comprising administering to a patient in
need of such treatment an effective amount of a compound of Formula I or Formula II, or
a pharmaceutically acceptable salt thereof.
Furthermore, the invention provides a compound of Formula I or Formula II, or a
pharmaceutically acceptable salt thereof for use in therapy, in particular for the treatment
of osteoarthritis. In addition, the invention provides a compound of Formula I or Formula
II, or a pharmaceutically acceptable salt thereof for use in the treatment of rheumatoid
arthritis. The invention also provides a compound of Formula I or Formula II, or a
pharmaceutically acceptable salt thereof for use in the treatment of pain associated with
osteoarthritis or rheumatoid arthritis. Furthermore, the invention provides the use of a
compound of Formula I or Formula II, or a pharmaceutically acceptable salt thereof, for
the manufacture of a medicament for the treatment of osteoarthritis. The invention
provides the use of a compound of Formula I or Formula II, or a pharmaceutically
acceptable salt thereof, for the manufacture of a medicament for the treatment of
rheumatoid arthritis. The present invention also provides the use of a compound of
Formula I or Formula II, or a pharmaceutically acceptable salt thereof, for the
manufacture of a medicament for the treatment of pain associated with osteoarthritis or
rheumatoid arthritis.
The invention further provides a pharmaceutical composition comprising a
compound of Formula I or Formula II, or a pharmaceutically acceptable salt thereof, in
combination with one or more pharmaceutically acceptable carriers, diluents, or
excipients. In a particular embodiment, the composition further comprises one or more
other therapeutic agents. This invention also encompasses novel intermediates and
processes for the synthesis of the compound of Formula I or Formula II, or a
pharmaceutically acceptable salt thereof.
In addition, the invention includes a method of treating inflammatory conditions
such as arthritis, including osteoarthritis and rheumatoid arthritis, in a patient, comprising
administering to a patient in need of such treatment an effective amount of an antagonist
of a proinflammatory prostaglandin, such as an EP4 antagonist, in combination with an
effective amount of a modulator of a lipoxin or resolvin receptor, such as a modulator of
BLT-1, BLT-2, ALX/FPR1, GPR32, CysLTl, CysLT2, or ChemR23.
A further aspect of the invention includes a method of treating inflammatory
disease such as arthritis, including osteoarthritis and rheumatoid arthritis, in a patient,
comprising administering to a patient in need of such treatment an effective amount of an
inhibitor of a proinflammatory prostaglandin synthase, such as an mPGES-1 inhibitor, in
combination with an effective amount of a modulator of a lipoxin or resolvin receptor,
such as a modulator of BLT-1, BLT-2, ALX/FPR1, GPR32, CysLTl, CysLT2, or
ChemR23.
As used herein, the terms "treating" or "to treat" includes restraining, slowing,
stopping, or reversing the progression or severity of an existing symptom or disorder.
As used herein, the term "patient" refers to a mammal, such as a mouse, guinea
pig, rat, dog, or human. It is understood that the preferred patient is a human.
As used herein, the term "effective amount" refers to the amount or dose of the
compound of the invention, or a pharmaceutically acceptable salt thereof which, upon
single or multiple dose administration to the patient, provides the desired effect in the
patient under diagnosis or treatment.
An effective amount can be readily determined by the attending diagnostician, as
one skilled in the art, by the use of known techniques and by observing results obtained
under analogous circumstances. In determining the effective amount for a patient, a
number of factors are considered by the attending diagnostician, including, but not limited
to: the species of mammal; its size, age, and general health; the specific disease or
disorder involved; the degree of or involvement or the severity of the disease or disorder;
the response of the individual patient; the particular compound administered; the mode of
administration; the bioavailability characteristics of the preparation administered; the
dose regimen selected; the use of concomitant medication; and other relevant
circumstances.
The compound of Formula I or Formula II, or pharmaceutically acceptable salt
thereof, are generally effective over a wide dosage range. For example, dosages per day
normally fall within the range of about 0.01 to about 50 mg/kg of body weight. In some
instances dosage levels below the lower limit of the aforesaid range may be more than
adequate, while in other cases still larger doses may be employed with acceptable side
effects, and therefore the above dosage range is not intended to limit the scope of the
invention in any way.
The compounds of the invention are preferably formulated as pharmaceutical
compositions administered by any route which makes the compound bioavailable. Most
preferably, such compositions are for oral administration. Such pharmaceutical
compositions and processes for preparing same are well known in the art. (See, e.g.,
Remington: The Science and Practice of Pharmacy (D.B. Troy, Editor, 21st Edition,
Lippincott, Williams & Wilkins, 2006).
It is understood that Formula II includes Formula Ila and lib:
Formula a Formula lib
The compounds of Formula I and Formula II are particularly useful in the
treatment methods of the invention, but certain groups, substituents, and configurations
are preferred. The following paragraphs describe such preferred groups, substituents, and
configurations. It will be understood that these preferences are applicable both to the
treatment methods and to the new compounds of the invention.
It is preferred that A is:
It is preferred that X is N.
It is further preferred that when R2 is H, that R is OH.
A preferred compound is 4-[[6-(4-hydroxy-l-piperidyl)-3-methyl-pyridine-2-
carbonyl]amino]-3,5-dimethyl-benzoic acid, which is:
or a pharmaceutically acceptable salt thereof.
4-[[6-(4-hydroxy- 1-piperidyl)-3 -methyl-pyridine-2-carbonyl] amino]-3,5-
dimethyl-benzoic acid is especially preferred.
Hydrated 4-[[6-(4-hydroxy-l-piperidyl)-3-methyl-pyridine-2-carbonyl]amino]-
3,5-dimethyl-benzoic acid is further preferred.
Hydrated 4-[[6-(4-hydroxy-l-piperidyl)-3-methyl-pyridine-2-carbonyl]amino]-
3,5-dimethyl-benzoic acid characterized by a substantial peak in the X-ray diffraction
spectrum, at diffraction angle 2-theta, of 9.0°, in combination with two or more peaks at
diffraction angle 2-theta selected from the group consisting of 5.8°, 8.5°, 9.8°, 11.6°,
11.8°, 17.5°, and 24.2°, is also preferred.
As used herein, "DMEM" refers to Dulbecco's Modified Eagle's Medium;
"DMSO" refers to dimethylsulfoxide; "IPA" refers to isopropyl alcohol; "MeOH" refers
to methanol; "EtOH" refers to ethanol; "DMF" refers to dimethylformamide; "THF"
refers to tetrahydrofuran; "EtOAc" referes to ethyl acetate; "FBS" refers to Fetal Bovine
Serum; "PGE " refers to prostaglandin E2; "FBS" refers to Fetal Bovine Serum;
"IBMX" refers to (3-isobutyl-l-methylxanthine); "MES" refers to (2-(Nmorpholino)
ethanesulfonic acid; "HEPES" refers to (2- [4-(2-hydroxy ethyl)piperazin-lyl]
ethanesulfonic acid); "HTRF" refers to homogeneous time-resolved fluorescence
technology; "HEK" refers to human embryonic kidney; and "IC50" refers to the
concentration of an agent that produces 50% of the maximal inhibitory response possible
for that agent.
Pharmaceutically acceptable salts and common methodology for preparing them
are well known in the art. See, e.g., Gould, P.L., "Salt selection for basic drugs,"
International Journal of Pharmaceutics, 33: 201-217 (1986); Bastin, R.J., et al. "Salt
Selection and Optimization Procedures for Pharmaceutical New Chemical Entities,"
Organic Process Research and Development, 4 : 427-435 (2000); and S.M. Berge, et al,
"Pharmaceutical Salts," Journal of Pharmaceutical Sciences, 66: 1-19 (1977). One skilled
in the art of synthesis will appreciate that the compound of Formula I is readily converted
to and may be isolated as pharmaceutically acceptable salts using techniques and
conditions well known to one of ordinary skill in the art.
The compounds of the present invention, or pharmaceutically acceptable salts
thereof, may be prepared by a variety of procedures known in the art, some of which are
illustrated in the schemes, preparations, and examples below. 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 the compounds of Formula I or Formula II,
or pharmaceutically acceptable salt thereof. The products of each step in the schemes
below can be recovered by conventional methods, including extraction, evaporation,
precipitation, chromatography, filtration, trituration, and crystallization. The reagents and
starting materials are readily available to one of ordinary skill in the art. All substituents,
unless otherwise specified, are as previously defined. It is understood that these schemes,
preparations, and examples are not intended to be limiting to the scope of the invention in
any way.
Scheme 1
Preparation 1
Synthesis of 6-chloro-3-methyl-pyridine-2-carboxylic acid.
Scheme 1 step A. A solution of aqueous IN NaOH (10 ml) is added to a stirred
solution of methyl 6-chloro-3-methyl-pyridine-2-carboxylate (1.0 g, 5.39 mmoles) in
THF:MeOH (10ml:2 ml). The mixture is stirred at room temperature for 3 hours. The
organic solvent is removed under reduced pressure and the semi-solid is dissolved in
water and acidified to pH 1-2 with aqueous IN HC1. The resulting precipitate is filtered,
washed with water, and dried at 40°C in a vacuum oven for 12 hours to give the title
compound as a white solid (780 mg, 84%). Mass spectrum (m/z): 172.0 (M+l).
Preparation 2
Synthesis of ethyl 4-[(6-chloro-3-methyl-pyridine-2-carbonyl)amino]-3,5-dimethylbenzoate.
Scheme 1, step B. To a solution of 6-chloro-3-methyl-pyridine-2-carboxylic acid
(0.78 g, 4.55 mmol) in CH2CI2 (15 mL) at room temperature are added ethyl 4-amino-3,5-
dimethyl-benzoate (0.878g, 4.55 mmol) and N,N-diisopropylethylamine (1.98 ml, 11.36
mmol). After stirring the reaction mixture for 10 minutes, 1-propanephosphonic acid
cyclic anhydride (50% solution in ethyl acetate, 3.25 ml, 5.46 mmol) is added via syringe.
After 48 hours, the solvent is removed under reduced pressure and the residue is diluted
with water and extracted with ethyl acetate. The organic layers are combined and dried
over magnesium sulfate, filtered, and concentrated under reduced pressure. The resulting
residue is purified by flash chromatography (silica gel) using a gradient of 0-40% ethyl
acetate in hexanes. After purification, the solid is triturated with 30% ethyl acetate in
hexanes and filtered to give the title compound as a white powder (0.907 g, 57.5%).
Mass spectrum (m/z): 347.2 (M+l).
Preparation 3
Synthesis of tert-butyl-dimethyl-(4-piperidyloxy)silane.
Scheme 1. step C reagent . To a solution of 4-hydroxypiperidine (2.00 g, 9.89
mmoles) in CH2CI2 (30 mL) is added lH-imidazole (2.69 g, 39.55 mmoles) followed by
t-butyldimethylchlorosilane (3.58 g, 23.73 mmoles) and the reaction mixture is stirred at
room temperature. After 12 hours, the reaction mixture is washed with water, saturated
solution of NaHC03, and brine. The organic layers are combined and dried over sodium
sulfate, filtered, and concentrated under reduced pressure. The resulting residue is
purified by flash chromatography (silica gel) over a gradient using 100% ¾ ¾ to 10%
7N ammonia in MeOH/90% CH2C 12 to afford the title compound (3.69 g, 86.3 %). Mass
spectrum (m/z): 216.2 (M+l).
Preparation 4
Synthesis of ethyl 4-[[6-[4-(tert-butyl(dimethyl)silyl)oxy-l-piperidyl]-3-methyl-pyridine-
2-carbonyl]amino]-3,5-dimethyl-benzoate.
Scheme 1 step C. To a solution of ethyl 4-[(6-chloro-3-methyl-pyridine-2-
carbonyl)amino]-3,5-dimethyl-benzoate, (440 mg, 1.27 mmol) in THF (1.6 mL) is added
(iPr)Pd(cinnamyl)Cl (16.43 mg, 0.03 mmol) followed by lithium bis(trimethylsilyl)amide
(3.81 ml). The reaction mixture is purged with nitrogen for 5 minutes and then stirred at
room temperature. After 18 hours, the reaction is diluted with a saturated solution of
aHC0 3 and extracted with ethyl acetate. The organic layers are combined and dried
over magnesium sulfate, filtered, and concentrated under reduced pressure. The resulting
residue is purified by flash chromatography (silica gel) over a gradient using 0 - 30%
ethyl acetate in hexanes to afford the title compound (278 mg, 41%). Mass spectrum
(m/z): 526.2 (M+l).
Preparation 5
Synthesis of ethyl 4-[[6-(4-hydroxy-l-piperidyl)-3-methyl-pyridine-2-carbonyl]amino]-
3,5-dimethyl-benzoate.
Scheme 1, step D. To a solution of ethyl 4-[[6-[4-(tert-butyl(dimethyl)silyl)oxy-lpiperidyl]-
3-methyl-pyridine-2-carbonyl]amino]-3,5-dimethyl-benzoate (342 mg, 0.650
mmol) in THF (4 ml) is added BU4 F 1.0 M in THF (0.975 ml, 0.975 mmol) at 0°C. The
reaction mixture is gradually warmed to ambient temperature. After 12 hours, the
reaction mixture is diluted with ice-water and extracted with ethyl acetate. The organic
layers are combined, dried over sodium sulfate, filtered, and concentrated under reduced
pressure to give a residue. The residue is purified by flash chromatography (silica gel)
over a gradient using 0 - 40% ethyl acetate in hexanes to afford the title compound (223
mg, 83.3%). Mass spectrum (m/z): 412.2 (M+1).
Scheme 2
Preparation 6
Synthesis of potassium 6-chloro-3-methyl-pyridine-2-carboxylate.
Scheme 2, step A. Methyl 6-chloro-3-methyl-pyridine-2-carboxylate (50 g, 269.4
mmoles) is added to a solution of potassium hydroxide (18.7 g, 282.9 mmoles) in
isopropyl alcohol (2000 mL). The mixture is stirred at ambient temperature for 1 hour.
Hexanes (500 mL) are added, and the solid is filtered, washed with hexanes, and dried
under reduced pressure at 45°C for 4 hours to give the title compound (52 g, 92 %). Mass
spectrum (m/z): 172.0 (M+1). H NMR (300 MHz, D 0): 7.62 (d, J= 8.0 Hz, 1H), 7.27
(d, J= 8.0 Hz, 1H), 2.23 (s, 3H).
Preparation 7
Synthesis of methyl 4-[(6-chloro-3-methyl-pyridine-2-carbonyl)amino]-3,5-dimethylbenzoate.
Scheme 2. step B. To a suspension of potassium 6-chloropyridine-2-carboxylate
(52 g, 265.8 mmoles) in dimethylformamide (676 mL) is added bis(2-oxo-3-
oxazolidinyl)phosphonic chloride ( 115 g, 451.8 mmoles). The mixture is stirred at
ambient temperature for 30 minutes. Methyl 4-amino-3,5-dimethyl-benzoate (42.9 g,
239.2 mmoles, see preparation 12) and diisopropylethylamine (115.9 mL, 664.5 mmoles)
are added. The reaction is stirred at ambient temperature for 16 hours. The mixture is
then poured into water (2000 mL) and is stirred for 30 minutes. The resulting solid is
filtered and dried under reduced pressure at 45°C. The dry material is triturated with
hexane (1400 mL) over 2 hours. The solid is filtered and dried under vacuum to give the
title compound (69 g, 78 %) as a white solid. Mass spectrum (m/z): 333.05 (M+l). 1H
NMR (300 MHz, DMSO): 10.16 (s, IH), 7.89 (d, J= 8.2 Hz, IH), 7.73 (s, 2H), 7.63 (d,
J= 8.2 Hz, IH), 3.85 (s, 3H), 2.49 (s, 3H), 2.28 (s, 6H).
Example 1
Synthesis of 4-[[6-(4-hydroxy-l-piperidyl)-3-methyl-pyridine-2-carbonyl]
dimethyl-benzoic acid.
Scheme 3, step A. A solution of aqueous IN NaOH (1.08 ml) is added to a stirred
solution of ethyl 4-[[6-(4-hydroxy-l-piperidyl)-3-methyl-pyridine-2-carbonyl]amino]-3,5-
dimethyl-benzoate (223 mg, 0.541 mmol) in THF:MeOH (4ml:2 ml). After heating at
40°C for 12 hours, the organic solvent is removed under reduced pressure and the semi
solid is dissolved in water and acidified to pH 3 with aqueous IN HC1. The resulting
precipitate is filtered, washed with water, and dried at 40°C in a vacuum oven for 12
hours to give the title compound as a white solid (160 mg, 77%). Mass spectrum (m/z):
384.2 (M+l).
Alternative synthesis of 4-[[6-(4-hydroxy-l-piperidyl)-3-methyl-pyridine-2-
carbonyl] amino] -3,5-dimethyl-benzoic acid.
Scheme 3, step B. 4-Hydroxypiperidine (171.2 g, 1660 mmoles) is added to a
solution of methyl 4-[(6-chloro-3-methyl-pyridine-2-carbonyl)amino]-3,5-dimethylbenzoate
(69 g, 207.3 mmoles) in N-methylpyrrolidone (483 mL) and the mixture is
stirred at 150°C for 4 hours. The mixture is cooled to ambient temperature and poured
into water (1000 mL). The mixture is washed with methyl /-butyl ether (300 mL). The
aqueous layer is then acidified to pH 2 with aqueous 36 % HC1. The mixture is extracted
with a solution of 1/1 ethyl acetate/methyl-t-butyl ether (3x250 mL). The organic layer is
evaporated to dryness. Water (500 mL) is added to the residue and the resulting mixture
is stirred for 30 minutes, filtered and the filtered material dried in an oven vacuum at 45°C
overnight. The dried light brown solid is dissolved in acetone (500 mL) and heated to
50°C. Water (1000 mL) is slowly added and the mixture is stirred at 50°C for 2 hours.
The mixture is cooled to ambient temperature, filtered, and the filtered material dried
under reduced pressure at 45°C. The dried material is stirred in ethyl acetate (600 mL) at
50°C for two hours. The mixture is cooled to ambient temperature, filtered, and the
filtered material dried under reduced pressure at 45°C overnight to give the title
compound as a white solid (50.8 g, 64 %). Mass spectrum (m/z): 384.2 (M+l). 1H NMR
(300 MHz, DMSO): 12.85 (s, 1H), 9.87 (s, 1H), 7.71 (s, 2H), 7.49 (d, J= 8.7 Hz, 1H),
7.01 (d, J= 8.7 Hz, 1H), 4.68 (d, J= 4.1 Hz, 1H), 4.1 1-4.04 (m, 2H), 3.70 (m, 1H), 3.15-
3.07 (m, 2H), 2.41 (s, 3H), 2.27 (s, 6H), 1.81-1.77 (m, 2H), 1.44-1.32 (m, 2H).
X-Ray Powder Diffraction
The XRD patterns of crystalline solids are obtained on a Bruker D4 Endeavor Xray
powder diffractometer, equipped with a CuKa source (= 1.54060 A) and a Vantec
detector, operating at 35 kV and 50 mA. The sample is scanned between 4 and 40° in 2,
with a step size of 0.0087° in 2and a scan rate of 0.5 seconds/step, and with 0.6 mm
divergence, 5.28mm 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. It
is well known in the crystallography art that, for any given crystal form, the relative
intensities of the diffraction peaks may vary due to preferred orientation resulting from
factors such as crystal morphology and habit. Where the effects of preferred orientation
are present, peak intensities are altered, but the characteristic peak positions of the
polymorph are unchanged. See, e.g. The U. S. Pharmacopeia 35 - National Formulary 30
Chapter <941> Characterization of crystalline and partially crystalline solids by X-ray
powder diffraction (XRPD) Official December 1, 2012-May 1, 2013. Furthermore, it is
also well known in the crystallography art that for any given crystal form the angular
peak positions may vary slightly. For example, peak positions can shift due to a variation
in the temperature or humidity at which a sample is analyzed, sample displacement, or the
presence or absence of an internal standard. In the present case, a peak position
variability of ± 0.2 in 2will take into account these 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 ° 2), typically the more prominent peaks. The crystal form diffraction patterns,
collected at ambient temperature and relative humidity, were adjusted based on NIST 675
standard peaks at 8.85 and 26.77 degrees 2-theta.
Example A
Preparation of hydrated 4-[[6-(4-hydroxy-l-piperidyl)-3-methyl-pyridine-2-
carbonyl]amino]-3,5-dimethyl-benzoic acid.
4-[[6-(4-Hydroxy-l-piperidyl)-3-methyl-pyridine-2-carbonyl]amino]-3,5-
dimethyl-benzoic acid (240.7 mg) is dissolved in 10 ml of 1:1 acetone:water to give a
clear pale solution. The mixture is concentrated at room temperature and crystalline
hydrated form begins to slowly precipitate from solution over several minutes. The
resulting solids are filtered and air dried to give 197.5mg of the title compound.
A prepared sample of the title compound of Example A is characterized by an Xray
powder diffraction pattern using CuKa radiation as having diffraction peaks (2-theta
values) as described in Table 1 below. Specifically the pattern contains a peak at 9.0° in
combination with two or more of the peaks selected from the group consisting of 5.8°,
8.5°, 9.8°, 11.6°, 11.8°, 17.5°, and 24.2° with a tolerance for the diffraction angles of 0.2
degrees.
Table 1. X-ray powder diffraction peaks of Example A.
Peak Angle (2-Theta °) Intensity (%)
1 5.8 43
2 8.5 16
3 9.0 100
4 9.8 2 1
5 11.6 62
6 11.8 34
7 14. 1 9
8 15.3 15
9 15.4 30
10 17.5 43
11 20.4 11
12 20.8 2 1
13 22.7 13
14 24.2 35
15 24.6 19
16 27.5 9
Scheme 4
Preparation 8
Synthesis of l -iodo-2,4-dimethyl-3-
Scheme 4, step A: To l,3-dimethyl-2-nitro-benzene (68.5 g, 453.2 mmol) is
added sulfuric acid (27.2 mL, 510 mmol), acetic acid (543.8 mL, 9.49 mol), iodine (46 g,
181.3 mmol) and HI0 4 (91.9 g, 403.3 mmol). The reaction is heated to 90°C for 7 days.
The reaction mixture is cooled to ambient temperature and water (500 mL) is added. The
resulting solid is collected by filtration and washed with cold water. The solid is dried
under reduced pressure at 45°C overnight to afford the title compound as a yellow solid
( 119 g, 95 %). 1H NMR (300.16 MHz, CDC13) : 7.80 (d, J= 8.2 Hz, 1H), 6.85 (d, J=
Hz, 1H), 2.37 (s, 3H), 2.23 (s, 3H).
Preparation
Synthesis of methyl 2,4-dimethyl-3-nitro-benzoate.
Scheme 4, Step B: To a 2 L Parr™ autoclave with mechanical stirring is added 1-
iodo-2,4-dimethyl-3-nitro-benzene (70 g, 252.7 mmol), Pd(OAc)2 (2.8 g, 12.6 mmol),
l,4-bis(diphenylphosphino)butane (6.5 g, 15.2 mmol), acetonitrile (462 mL),
triethylamine (88.2 mL), and methanol (280 mL). The Parr™ autoclave is sealed, purged
and pressurized with CO to 55 1.6 kPa (80 psig). The mixture is heated to 100°C for 2
hours. The mixture is cooled to ambient temperature and then vented. The mixture is
then concentrated to dryness under reduced pressure. Ethyl acetate (300 mL) and water
(300 mL) are added. The layers are separated and the aqueous layer discarded. The
organic layer is dried over MgS0 4, filtered, and concentrated to dryness to afford the title
compound as a red oil which crystallizes upon standing (52 g, 98 %). 1H NMR (300.13
MHz, CDC13 ) : 7.89 (d, J= 8.2 Hz, 1H), 7.19 (d, J= 8.2 Hz, 1H), 3.91 (s, 3H), 2.49 (s,
3H), 2.33 (s, 3H).
Preparation 10
Synthesis of methyl 3-amino-2,4-dimethyl-benzoate.
Scheme 4, Step C: To a solution of methyl 2,4-dimethyl-3-nitro-benzoate (37 g,
176.9 mmol) in methanol (370 mL), 10% palladium on carbon 50% wet ( 5.6 g) is added.
The reaction is bubbled with hydrogen and placed under a hydrogen atmosphere for 6
days. The mixture is filtered through diatomaceous earth and the filtrate is evaporated to
dryness. The resulting residue is purified by flash chromatography (silica gel), eluting
with 20% ethyl acetate in hexanes to afford the title compound as a yellow oil (20.5 g,
65%). Mass spectrum (m/z): 180.1 (M+1). 1H NMR (300.16 MHz, DMSO-i¾): 6.89
(s, 2H), 4.78 (s, 2H), 3.76 (s, 3H), 2.23 (s, 3H), 2.12 (s, 3H).
Scheme 5
Preparation 11
Synthesis of methyl 3,5-dimethyl-4-nitro-benzoate.
Scheme 5. step A. To a solution of 3,5-dimethyl-4-nitro-benzoic acid (10.0 g,
0.05 12 mol) in MeOH (150 mL) is added thionyl chloride (10 ml) at 0 °C and the reaction
is heated to 80 °C. After 16 h, the reaction mixture is cooled to room temperature and
solvent is removed under reduced pressure. The residue is diluted with water (50 ml) and
basified with saturated NaHC0 solution to pH 7-8 and extracted with EtOAc (2x120
mL). The organic layers are combined and dried over anhydrous sodium sulfate. The
solvent is removed under reduced pressure to afford the title compound as a light yellow
solid (10.71 g, 98.3%). 1H NMR (400 MHz, DMSO): 7.83 (s, 2H), 3.88 (s, 3H), 2.30
(s, 6H).
Preparation 12
Synthesis of methyl 4-amino-3,5-dimethyl-benzoate.
Scheme 5, step B. To a solution of methyl 3,5-dimethyl-4-nitrobenzoate (10.0 g,
0.0478 mol) in methanol (100 mL), iron powder (15.7 g, 0.2869 mol) and 37% HC1 (1.72
g, 0.0478 mol) is added at 0 °C. The reaction is heated at 80 °C for 16 hours. The
mixture is cooled to room temperature and filtered through Celite™ bed and washed with
methanol followed by evaporation of filtrate to dryness to afford the title compound as a
brown solid (7.8 g, 99%). Mass spectrum (m/z): 180.2 (M+l).
Scheme 6
Preparation 13
Synthesis of methyl 4-[(5-bromo-2-fluoro-benzoyl)amino]-3,5-dimethyl-benzoate.
Scheme 6, step A. To a solution of 5-bromo-2-fluorobenzoic acid (3.50 g, 15.9
mmol) in CH2CI2 (50 niL) at 0 °C are added methyl 4-amino-3,5-dimethylbenzoate (2.86
g, 15.9 mmol, see preparation 12) and N,N-diisopropylethylamine (8.35 ml, 47.9 mmol).
After stirring the reaction mixture for 10 minutes, 1-propanephosphonic acid cyclic
anhydride (50% solution in ethyl acetate, 20.5 ml, 31.9 mmol) is added via syringe and
stirred at ambient temperature. After 36 hours, the solvent is removed under reduced
pressure and the residue is diluted with water and extracted with ethyl acetate. The
organic layers are combined and dried over anhydrous Na2S0 4, filtered, and concentrated
under reduced pressure. The resulting residue is purified by flash chromatography (silica
gel) using 10 % ethyl acetate in hexane to give the title compound as a white solid (4.20
g, 69 %). Mass spectrum (m/z): 380.2 (M+l).
Preparation 14
Synthesis of tert-butyl-dimethyl-(3-piperidylmethoxy)silane.
Scheme 6, step B. To a solution of piperidin-3-ylmethanol (2.10 g, 0.018 mol) in
CH2CI2 (20 mL) is added triethylamine (5.53 g, 0.0547 mol) followed by tbutyldimethylchlorosilane
(4.127 g, 0.0275 mol) and the reaction mixture is stirred at
room temperature. After 24 hours, the reaction mixture is washed with water, saturated
solution of NaHC03, and brine. The combined organic layers are combined and dried
over sodium sulfate, filtered, and concentrated under reduced pressure. The resulting
residue is purified by flash chromatography (silica gel) over a gradient using 0-10%
MeOH in dichloromethane to afford the title compound (2.50 g, 60 %). Mass spectrum
(m/z): 231.2 (M+l).
Preparation 15
Synthesis of methyl 4-[[5-[3-[[tert-butyl(dimethyl)silyl]oxymethyl]-l-piperidyl]-2-fluorobenzoyl]
amino]-3,5-dimethyl-benzoate.
Scheme 6, step B. To a solution of methyl 4-[(5-bromo-2-fluoro-benzoyl)amino]-
3,5-dimethyl-benzoate (0.40 g, 1.05 mmol), tert-butyl-dimethyl-(3-
piperidylmethoxy)silane (0.362 g, 1.57 mmol) and CS2CO (1.04 g, 3.15 mmol) in 1,4-
dioxane (6 ml) is added Pd2(dba)3 (0. 10 g, 0.105 mmol) followed by S-Phos (0.043 g,
0.105 mmol). The reaction mixture is purged with nitrogen for 5 minutes and then heated
at 90 °C. After 16 hours, the reaction is cooled to ambient temperature, filtered through
Celite™, and washed with EtOAc. The combined filtrates are dried over sodium sulfate,
filtered, and concentrated under reduced pressure and purified by flash chromatography
(silica gel) using 10 % EtOAc in hexane to afford the title compound as a pale yellow oil
(0.22 g, 40%). Mass spectrum (m/z): 529.2 (M+l).
Preparation 16
Synthesis of methyl 4-[[2-fluoro-5-[3-(hydroxymethyl)-l-piperidyl]benzoyl]amino]
dimethyl-benzoate.
Scheme 6, step C. To a solution of methyl 4-[[5-[3-[[tertbutyl(
dimethyl)silyl]oxymethyl]-l-piperidyl]-2-fluoro-benzoyl]amino]-3,5-dimethylbenzoate
(0.30 g, 0.54 mmol) in THF (8 ml) is added BU4NF 1.0 M in THF (0.35 g, 1.60
mmol) at 0°C. The reaction mixture is gradually warmed to ambient temperature. After 4
hours, the reaction mixture is diluted with ice-water and extracted with ethyl acetate. The
organic layers are combined, dried over sodium sulfate, filtered, and concentrated under
reduced pressure to give a residue. The residue is purified by flash chromatography
(silica gel) over a gradient using ethyl acetate in hexanes as eluent to afford the title
compound as a white solid (0.10 g, 55%). Mass spectrum (m/z): 414.2 (M+l).
Example 2
Synthesis of 4-[[2-fluoro-5-[3-(hydroxymethyl)-l-piperidyl]benzoyl]amino]-3,5-
dimethyl-benzoic acid.
Scheme 6, step D. A solution of aqueous 2N NaOH (2.00 ml) is added to a stirred
solution of methyl 4-[[2-fluoro-5-[3-(hydroxymethyl)-l-piperidyl]benzoyl]amino]-3,5-
dimethyl-benzoate (0. 18 g, 0.43 mmol) in THF:MeOH (3 ml: 1 ml). After 16 h at 50°C,
the organic solvent is removed under reduced pressure and the residue is diluted with
water, acidified to pH 4 with IN HC1 and extracted with CH2CI2. The organic layers are
combined and dried over anhydrous sodium sulfate. The solvent is removed under
reduced pressure and the resulting precipitate is triturated with diethyl ether and filtered
to afford the title compound as a white solid (0. 12 g, 69%). Mass spectrum (m/z): 401.2
(M+l).
Chiral separation of racemic 4-[[2-fluoro-5-[3-(hydroxymethyl)-l-piperidyl]benzoyl]
amino]-3,5-dimethyl-benzoic acid.
4-[[2-fluoro-5-[3-(hydroxymethyl)-l-piperidyl]benzoyl]amino]-3,5-dimethylbenzoic
acid is separated by chiral chromatography using Chiralpak™ AD-H, 25%
MeOH/C02, 5 ml/min, 225 nm to give:
Example 2A: 4-[[2-fluoro-5-[3-(hydroxymethyl)-l-piperidyl]benzoyl]amino]-3,5-
dimethyl-benzoic acid isomer 1. Peak eluting at 2.52 min. Mass spectrum (m/z): 401.2
(M+l).
Example 2B: 4-[[2-fluoro-5-[3-(hydroxymethyl)-l-piperidyl]benzoyl]amino]-3,5-
dimethyl-benzoic acid isomer 2. Peak eluting at 2.93 min. Mass spectrum (m/z): 401.2
(M+l).
Scheme 7
Preparation 17
Synthesis of methyl 3-[(5-bromo-2-fluoro-benzoyl)amino]-2,4-dimethyl-benzoate.
Scheme 7. step A. To a solution of 5-bromo-2-fluoro-benzoic acid (500 mg, 2.28
mmol) in CH2CI2 (15 mL) at 0 °C are added methyl 3-amino-2,4-dimethylbenzoate (409
mg, 2.28 mmol, see preparation 10) and N,N-diisopropylethylamine (737.6 mg, 5.71
mmol). After stirring the reaction mixture for 10 minutes, 1-propanephosphonic acid
cyclic anhydride (50% solution in ethyl acetate, 1.74 g, 2.74 mmol) is added via syringe
and stirred at 50°C. After 16 hours, the solvent is removed under reduced pressure and
the residue is triturated with MeOH to give the title compound as a white solid (450 mg,
51.8%). Mass spectrum (m/z): 380.2 (M+l).
Preparation 18
Synthesis of methyl 3-[[5-[3-[[tert-butyl(dimethyl)silyl]oxymethyl]-l-piperidyl]-2-fluorobenzoyl]
amino]-2,4-dimethyl-benzoate.
Scheme 7, step B. To a solution of methyl 3-[(5-bromo-2-fluoro-benzoyl)amino]-
2,4-dimethyl-benzoate (0.7 g, 1.84 mmol), tert-butyl-dimethyl-(3-
piperidylmethoxy)silane (2.53 g, 11.03 mmol, see preparation 14) and CS2CO3 (1.81 g,
5.50 mmol) in 1,4-dioxane (15 ml) is added Pd2(dba)3 (0. 168 g, 0.184 mol) followed by
S-Phos (0.075 g, 0.184 mol). The reaction mixture is purged with nitrogen for 5 minutes
and then heated at 120 °C. After 6 hours, the reaction mixture is concentrated under
reduced pressure and residue is carried forward without further purification.
Preparation 19
Synthesis of methyl 3-[[2-fluoro-5-[3-(hydroxymethyl)-l-piperidyl]benzoyl]amino]-2,4-
dimethyl-benzoate.
Scheme 7. step C. To a solution of methyl 3-[[5-[3-[[tertbutyl(
dimethyl)silyl]oxymethyl]- 1-piperidyl]-2-fluoro-benzoyl]amino]-2,4-dimethylbenzoate
(2.9 g, 5.48 mmol) in THF (20 ml) is added BU4NF 1.0 M in THF (5.73 g,
0.0219 mol) at 0°C. The reaction mixture is gradually warmed to ambient temperature.
After 4 hours, the reaction mixture is diluted with ice-water and extracted with ethyl
acetate. The organic layers are combined, dried over sodium sulfate, filtered, and
concentrated under reduced pressure to give a residue. The residue is purified by flash
chromatography (silica gel) over a gradient using 35-45% ethyl acetate in hexane to
afford the title compound as a white solid (0.38 g, 16.8%). Mass spectrum (m/z): 415.2
(M+l).
Example 3
Synthesis of 3-[[2-fluoro-5-[3-(hydroxymethyl)-l-piperidyl]benzoyl]amino]-2,4-
dimethyl-benzoic acid.
Scheme 7. step D . A solution of aOH (0.146 mg, 0.00366 mol) in 1.5 ml of H20
is added to a stirred solution of methyl 3-[[2-fluoro-5-[3-(hydroxymethyl)-lpiperidyl]
benzoyl]amino]-2,4-dimethyl-benzoate (0.38 g, 0.000916 mol) in THF:EtOH (3
ml:2 ml). After 4 hours at ambient temperature, the organic solvent is removed under
reduced pressure and the residue is diluted with water, acidified to pH 4 with IN HCl, and
extracted with ethyl acetate. The organic layer are combined and dried over anhydrous
sodium sulfate. The solvent is removed under reduced pressure to afford the title
compound as a light yellow solid (85 mg g, 36.8%). Mass spectrum (m/z): 401.2 (M+1).
Scheme 8
Preparation 20
Synthesis of methyl 3-[(5-bromo-2-methyl-benzoyl)amino]-2,4-dimethyl-benzoate.
Scheme 8. step A. To a solution of 5-bromo-2-methylbenzoic acid (2.0 g, 0.0093
mol) in CH2CI2 (20 niL) at 0 °C are added methyl 3-amino-2,4-dimethylbenzoate (1.49 g,
0.0084 mol, see preparation 10) and N,N-diisopropylethylamine (4.79 g, 0.0372 mol).
After stirring the reaction mixture for 10 minutes, 1-propanephosphonic acid cyclic
anhydride (50% solution in ethyl acetate, 8.87 g, 0.028 mol) is added via syringe and
stirred at 50°C. After 16 hours, the solvent is removed under reduced pressure and the
residue is diluted with water and extracted twice with ethyl acetate. The organic layers
are combined and dried over anhydrous Na2S0 4, filtered, and concentrated under reduced
pressure. The resulting residue is purified by flash chromatography (silica gel) using 10
% ethyl acetate in hexane to give the title compound as a white solid (2.80 g, 80%). Mass
spectrum (m/z): 376.1 (M+l).
Preparation 21
Synthesis of methyl 3-[[5-[3-[[tert-butyl(dimethyl)silyl]oxymethyl]-l-piperidyl]-2-
methyl-benzoyl]amino]-2,4-dimethyl-benzoate.
Scheme 8, step B. To a solution of methyl 3-[(5-bromo-2-methylbenzoyl)
amino]-2,4-dimethyl-benzoate (0.5 g, 1.32 mmol), tert-butyl-dimethyl-(3-
piperidylmethoxy)silane (0.45 g, 1.99 mmol, see preparation 14) and CS2CO3 (1.29 g,
3.98 mmol) in 1,4-dioxane (15 ml) is added Pd2(dba) (0.12 g, 0.132 mmol) followed by
S-Phos (0.050 g, 0.132 mmol). The reaction mixture is purged with nitrogen for 5
minutes and then heated at 110 °C. After 6 hours, the reaction is cooled to ambient
temperature, filtered through Celite™, and washed with EtOAc. The combined filtrates
are dried over sodium sulfate, filtered, and concentrated under reduced pressure and
purified by flash chromatography (silica gel) using 15 % EtOAc in hexane as eluent to
afford the title compound as a brown semi solid (0.6 g, 85%). Mass spectrum (m/z):
525.2 (M+l).
Preparation 22
Synthesis of methyl 3-[[5-[3-(hydroxymethyl)-l-piperidyl]-2-methyl-benzoyl]amino]-2,4-
dimethyl-benzoate.
Scheme 8. step C. To a solution of methyl 3-[[5-[3-[[tertbutyl(
dimethyl)silyl]oxymethyl]-l-piperidyl]-2-methyl-benzoyl]amino]-2,4-dimethylbenzoate
(0.60 g, 1.14 mmol) in THF (15 ml) is added BU4 F 1.0 M in THF (0.596 g,
2.28 mmol) at 0°C. The reaction mixture is gradually warmed to ambient temperature.
After 1 hour, the reaction mixture is diluted with ice-water and extracted with ethyl
acetate. The organic layers are combined, dried over sodium sulfate, filtered, and
concentrated under reduced pressure to give a residue. The residue is purified by flash
chromatography (silica gel) over a gradient using 4% methanol in dichloromethane as
eluent to afford the title compound as a white solid (0.4 g, 85.5%). Mass spectrum (m/z):
4 11.4 (M+l).
Example 4
Synthesis of 3-[[5-[3-(hydroxymethyl)-l-piperidyl]-2-methyl-benzoyl]amino]-2,4-
dimethyl-benzoic acid.
Scheme 8. step D. A solution of aqueous 4N NaOH (4.00 ml) is added to a stirred
solution of methyl 3-[[5-[3-(hydroxymethyl)-l-piperidyl]-2-methyl-benzoyl]amino]-2,4-
dimethyl-benzoate (0.4 g, 0.975 mmol) in THF:MeOH (10 ml:5 ml). After 4 hours at
ambient temperature, the organic solvent is removed under reduced pressure and the
residue is diluted with water, acidified to pH 4 with IN HC1, and extracted twice with
10% IPA in CH2CI2. The organic layers are combined and dried over anhydrous sodium
sulfate. The solvent is removed under reduced pressure and the resulting precipitate is
triturated with diethyl ether and filtered to afford the title compound as a light brown
solid (0.28 g, 72.5%). Mass spectrum (m/z): 397.2 (M+l).
Chiral separation of racemic 3-[[5-[3-(hydroxymethyl)-l-piperidyl]-2-methylbenzoyl]
amino]-2,4-dimethyl-benzoic acid.
3-[[5-[3-(hydroxymethyl)-l-piperidyl]-2-methyl-benzoyl]amino]-2,4-dimethylbenzoic
acid is separated by chiral chromatography using Chiralcel™ OJ-H, 15%
MeOH/C02, 5 ml/min, 225 nm to give:
Example 4A: 3-[[5-[3-(hydroxymethyl)-l-piperidyl]-2-methyl-benzoyl]amino]-2,4-
dimethyl-benzoic acid isomer 1. Peak eluting at 1.76 min. Mass spectrum (m/z): 397.2
(M+l).
Example 4B: 3-[[5-[3-(hydroxymethyl)-l-piperidyl]-2-methyl-benzoyl]amino]-2,4-
dimethyl-benzoic acid isomer 2. Peak eluting at 2.49 min. Mass spectrum (m/z): 397.2
(M+l).
Scheme 9
Preparation 23
Synthesis of methyl 4-[(5-bromo-2-methyl-benzoyl)amino]-3,5-dimethyl-benzoate.
Scheme 9, step A. To a solution of 5-bromo-2-methylbenzoic acid (2.0 g, 0.008
mol) in CH2CI2 (20 mL) at 0 °C are added methyl 4-amino-3,5-dimethylbenzoate (1.28 g,
0.0072, see preparation 12) and N,N-diisopropylethylamine (4.12 g, 0.032 mol). After
stirring the reaction mixture for 10 minutes, 1-propanephosphonic acid cyclic anhydride
(50% solution in ethyl acetate, 7.63 g, 0.024 mol) is added via syringe and stirred at 50°C.
After 16 hours, the solvent is removed under reduced pressure and the residue is diluted
with water and extracted twice with ethyl acetate. The organic layers are combined and
dried over anhydrous a S04, filtered, and concentrated under reduced pressure. The
resulting residue is purified by flash chromatography (silica gel) using 12 % ethyl acetate
in hexane to give the title compound as a white solid (2.9 g, 97 %). Mass spectrum (m/z):
376.0 (M+l).
Preparation 24
Synthesis of methyl 4-[[5-[3-[[tert-butyl(dimethyl)silyl]oxymethyl]-l-piperidyl]-2-
methyl-benzoyl]amino]-3,5-dimethyl-benzoate.
Scheme 9. step B. To a solution of methyl 4-(5-bromo-2-methylbenzamido)-3,5-
dimethylbenzoate (0.5 g, 0.0013 mol), tert-butyl-dimethyl-(3-piperidylmethoxy)silane
(0.45 g, 0.0019 mol, preparation 14) and Cs2C0 3 (1.29 g, 0.0039 mol) in 1,4-dioxane (15
ml) is added Pd2(dba)3 (0.12 g, 0.00013 mol) followed by S-Phos (0.054 g, 0.00013 mol).
The reaction mixture is purged with nitrogen for 5 minutes and then heated at 110 °C.
After 16 hours, the reaction is cooled to ambient temperature, filtered through Celite™,
and washed with EtOAc. The combined filtrates are dried over sodium sulfate, filtered,
and concentrated under reduced pressure and purified by flash chromatography (silica
gel) using 15 % ethyl acetate in hexane as eluent to afford the title compound as a brown
semi solid (0.4 g, 57.3%). Mass spectrum (m/z): 525.2 (M+l).
Preparation 25
Synthesis of methyl 4-[[5-[3-(hydroxymethyl)-l-piperidyl]-2-methyl-benzoyl]amino]-3,5-
dimethyl-benzoate.
Scheme 9, step C. To a solution of methyl 4-[[5-[3-[[tertbutyl(
dimethyl)silyl]oxymethyl]-l-piperidyl]-2-methyl-benzoyl]amino]-3,5-dimethylbenzoate
(0.40 g, 0.76 mmol) in THF (10 ml) is added BU4 F 1.0 M in THF (0.39 g, 1.52
mmol) at 0°C. The reaction mixture is gradually warmed to ambient temperature. After 1
hour, the reaction mixture is diluted with ice-water and extracted with ethyl acetate. The
organic layers are combined, dried over sodium sulfate, filtered, and concentrated under
reduced pressure to give a residue. The residue is purified by flash chromatography
(silica gel) over a gradient using 4% MeOH in dichloromethane to afford the title
compound as a light brown solid (0.30 g, 96%). Mass spectrum (m/z): 4 11.5 (M+l).
Example 5
Synthesis of 4-[[5-[3-(hydroxymethyl)-l-piperidyl]-2-methyl-benzoyl]amino]-3,5-
dimethyl-benzoic acid.
Scheme 9. step D. A solution of aqueous 4N NaOH (2.00 ml) is added to a stirred
solution of methyl 4-[[5-[3-(hydroxymethyl)-l-piperidyl]-2-methyl-benzoyl]amino]-3,5-
dimethyl-benzoate (0.3 g, 0.73 mmol) in THF:MeOH (10 ml:5 ml). After 16 hours at
ambient temperature, the organic solvent is removed under reduced pressure and the
residue is diluted with water, acidified to pH 4 with IN HCl, and extracted with 10% IPA
in CH2CI2. The organic layers are combined and dried over anhydrous sodium sulfate.
The solvent is removed under reduced pressure and the resulting precipitate is triturated
with diethyl ether and filtered to afford the title compound as a light brown solid (0.27 g,
90%). Mass spectrum (m/z): 397.2 (M+l).
Chiral separation of racemic 4-[[5-[3-(hydroxymethyl)-l-piperidyl]-2-methylbenzoyl]
amino]-3,5-dimethyl-benzoic acid.
4-[[5-[3-(hydroxymethyl)-l-piperidyl]-2-methyl-benzoyl]amino]-3,5-dimethylbenzoic
acid is separated by chiral chromatography using Chiralpak AD-H, 30%
EtOH/C02, 5 ml/min, 225 nm to give:
Example 5A: 4-[[5-[3-(hydroxymethyl)-l-piperidyl]-2-methyl-benzoyl]amino]-3,5-
dimethyl-benzoic acid isomer 1. Peak eluting at 4.22 min. Mass spectrum (m/z): 397.2
(M+l).
Example 5B: 4-[[5-[3-(hydroxymethyl)-l-piperidyl]-2-methyl-benzoyl]amino]-3,5-
dimethyl-benzoic acid isomer 2. Peak eluting at 5.30 min. Mass spectrum (m/z): 397.2
(M+l).
Scheme 10
Preparation 26
Synthesis of methyl 3-[[5-[3-[[tert-butyl(dimethyl)silyl]oxymethyl]pyrrolidin-l-yl]-2-
methyl-benzoyl]amino]-2,4-dimethyl-benzoate.
Scheme 10, step A. To a solution of methyl 3-[(5-bromo-2-methylbenzoyl)
amino]-2,4-dimethyl-benzoate (0.5 g, 1.32 mmol), tert-butyl-dimethyl-
(pyrrolidin-3-ylmethoxy)silane (0.4 g, 1.99 mmol) and CS2CO (1.2 g, 3.99 mmol) in 1,4-
dioxane (15 ml) is added Pd2(dba)3 (0.12 g, 0.132 mmol) followed by S-Phos (0.054 g,
0.132 mmol). The reaction mixture is purged with nitrogen for 5 minutes and then heated
at 100 °C. After 16 hours, the reaction is cooled to ambient temperature, filtered through
Celite™, and washed with EtOAc. The combined filtrates are dried over sodium sulfate,
filtered, and concentrated under reduced pressure and purified by flash chromatography
(silica gel) using 20 % EtOAc in hexane to afford the title compound as a brown semi
solid (0.36 g, 53%). Mass spectrum (m/z): 5 11.2 (M+l).
Preparation 27
Synthesis of methyl 3-[[5-[3-(hydroxymethyl)pyrrolidin-l-yl]-2-methyl-benzoyl]amino]-
2,4-dimethyl-benzoate.
Scheme 10. step B. To a solution of methyl 3-[[5-[3-[[tertbutyl(
dimethyl)silyl]oxymethyl]pyrrolidin-l-yl]-2-methyl-benzoyl]amino]-2,4-dimethylbenzoate
(0.35 g, 0.686 mmol) in THF (50 ml) is added BU4 F 1.0 M in THF (0.537 g,
2.05 mmol) at 0°C. The reaction mixture is gradually warmed to ambient temperature.
After 2 hours, the reaction mixture is diluted with ice-water and extracted twice with
ethyl acetate. The organic layers are combined, dried over sodium sulfate, filtered, and
concentrated under reduced pressure to give a residue. The residue is purified by flash
chromatography (silica gel) over a gradient using 50% ethyl acetate in hexane to afford
the title compound as a brown solid (150 mg, 51%).
Example 6
Synthesis of 3-[[5-[3-(hydroxymethyl)pyrrolidin-l-yl]-2-methyl-benzoyl]amino]-2,4-
dimethyl-benzoic acid.
Scheme 10, step C. A solution of aqueous 5N NaOH (5.00 ml) is added to a
stirred solution of methyl 3-[[5-[3-(hydroxymethyl)-l-piperidyl]-2-methylbenzoyl]
amino]-2,4-dimethyl-benzoate (0.4 g, 0.975 mmol) in MeOH (5 ml). After 5
hours at ambient temperature, the organic solvent is removed under reduced pressure and
the residue is diluted with water, acidified to pH 4 with IN HCl, and extracted twice with
10% IPA in CH2CI2. The organic layers are combined and dried over anhydrous sodium
sulfate. The solvent is removed under reduced pressure and the resulting precipitate is
triturated with diethyl ether and filtered to afford the title compound as a brown solid (70
mg, 56%). Mass spectrum (m/z): 381.2 (M-l).
Scheme 11
Preparation 28
Synthesis of methyl 3-[(5-bromo-2-methyl-benzoyl)amino]-2,4-dimethyl-benzoate.
Scheme 11, step A. To a solution of 5-bromo-2-methyl-benzoic acid (2.0 g,
0.0093 mol) in CH2CI2 (20 ml) at room temperature are added methyl 3-amino-3,5-
dimethylbenzoate (1.49 g, 0. 0083 mol, see preparation 12) and N,Ndiisopropylethylamine
(4.79 g, 0.037 mol). After stirring the reaction mixture for 10
minutes, 1-propanephosphonic acid cyclic anhydride (50% solution in ethyl acetate, 8.87
g, 0.027 mol) is added via syringe and heated at 50°C. After 16 hours, the reaction
mixture is diluted with ¾ (¾ washed with water and brine. The organic layers are
combined and dried over anhydrous Na2S0 4, filtered, and concentrated under reduced
pressure. The residue is purified by flash chromatography (silica gel) using 10% EtOAc
in hexanes to give the title compound as a white powder (2.8 g, 80 %).
Preparation 29
Synthesis of methyl 3-[[5-[3 -[tert-butyl(dimethyl)silyl] oxy- 1-piperidyl] -2-methylbenzoyl]
amino]-2,4-dimethyl-benzoate.
Scheme 11, step B. To a solution of methyl 3-[(5-bromo-2-methylbenzoyl)
amino]-2,4-dimethyl-benzoate (0.5 g, 1.32 mmol), tert-butyl-dimethyl-(3-
piperidyloxy)silane (0.425 g, 1.98 mmol) and CS2CO3 (1.28 g, 3.96 mmol) in 1,4-dioxane
(15 ml) is added Pd2(dba)3 (0.12 g, 0.132 mmol) followed by S-Phos (0.054 g, 0.132
mmol). The reaction mixture is purged with nitrogen for 5 minutes and then heated at
100 °C. After 16 hours, the reaction is cooled to ambient temperature, filtered through
Celite™, and washed with EtOAc. The combined filtrates are dried over sodium sulfate,
filtered, and concentrated under reduced pressure and purified by flash chromatography
(silica gel) using 10% EtOAc in hexane to afford the title compound as a brown semi
solid (0.4 g, 60%).
Preparation 30
Synthesis of methyl 3-[[5-(3-hydroxy-l-piperidyl)-2-methyl-benzoyl]amino]-2,4-
dimethyl-benzoate.
Scheme 11. step C. To a solution of methyl 3-[[5-[3-[tertbutyl(
dimethyl)silyl]oxy-l-piperidyl]-2-methyl-benzoyl]amino]-2,4-dimethyl-benzoate
(0.4 g, 0.78 mmol) in THF (20 ml) is added BU4 F 1.0 M in THF (0.409 g, 1.56 mmol)
at 0°C. The reaction mixture is gradually warmed to ambient temperature. After 1 hour,
the reaction mixture is diluted with ice-water and extracted twice with ethyl acetate. The
organic layers are combined, dried over sodium sulfate, filtered, and concentrated under
reduced pressure to give a residue. The residue is purified by flash chromatography
(silica gel) over a gradient using 3% methanol in dichloromethane to afford the title
compound as a white solid (0.3 g, 97%).
Example 7
Synthesis of 3-[[5-(3-hydroxy-l-piperidyl)-2-methyl-benzoyl]amino]-2,4-dimethylbenzoic
acid.
Scheme 11, step D. A solution of aqueous 4N NaOH (2.00 ml) is added to a
stirred solution of methyl 3-[[5-(3-hydroxy-l-piperidyl)-2-methyl-benzoyl]amino]-2,4-
dimethyl-benzoate (0.3 g, 0.75mmol) in THF:MeOH (5 ml:2 ml). After 3 hours at
ambient temperature, the organic solvent is removed under reduced pressure and the
residue is diluted with water, acidified to pH 4 with IN HCl, and extracted with 10% IPA
in CH2CI2. The organic layers are combined and dried over anhydrous sodium sulfate.
The solvent is removed under reduced pressure and the resulting precipitate is triturated
with diethyl ether and filtered to afford the title compound as an off-white solid (0.274 g,
94%). Mass spectrum (m/z): 383.2 (M+l).
Scheme 12. step A. To a solution of 5-bromo-2-methyl-benzoic acid (1.35 g, 6.15
mmol) in THF (15 ml), CH2C 12 (15 ml) and DMF (14.27 ΐ , 184.57 1 is added
dropwise oxalyl chloride (587.12 ΐ , 6.77 mmoles) at 0 °C and reaction mixture is slowly
allowed to warm to ambient temperature. After 2 hours, the solvent is removed under
reduced pressure. To the residue CH2CI2 (30 ml) is added and reaction mixture is cooled
to 0 °C, then ethyl 4-amino-3,5-dimethyl-benzoate (1.19 g, 6.15 mmol) is added followed
by 4-pyridinamine, N,N-dimethyl- (37.58 mg, 307.61 ) and pyridine (1.49 ml,
18.46 mmoles). The cooling bath is removed and the clear solution is allowed to warm to
ambient temperature. After 5 hours, the solvent is removed and the reaction mixture is
diluted with ethyl acetate and washed with IN HC1, saturated solution of sodium
bicarbonate, and brine. The organic layers are combined and dried over anhydrous
a2S04, filtered, and concentrated under reduced pressure. The resulting residue is
triturated with hexanes and the resulting solids were collected by filtration to give the title
compound as a white solid (1.80 g; 75.2%). Mass spectrum (m/z): 390.2 (M+l).
Preparation 32
Synthesis of ethyl 4-[[5-(l,4-dioxa-8-azaspiro[4.5]decan-8-yl)-2-methyl-benzoyl]amino]-
3,5-dimethyl-benzoate.
Scheme 12, step B. To a solution of ethyl 4-[(5-bromo-2-methyl-benzoyl)amino]-
3,5-dimethyl-benzoate (0.5 g, 1.28 mmol), l,4-dioxa-8-azaspiro(4.5)decane (0.22 g, 1.54
mmol) and Cs2C0 3 (1.25 g, 3.84 mmol) in THF (10 ml) is added Pd(OAc)2 (0.04 g, 0.19
mmol) followed by racemic-2,2'-bis(diphenylphosphino)-l,l'-binaphthyl ( 119.7 mg, 0.19
mmol). The reaction mixture is purged with nitrogen for 5 minutes and then heated at 90
°C. After 16 hours, the reaction is cooled to ambient temperature and diluted with
ammonium chloride and extracted twice with EtOAc. The combined organic layers are
dried over sodium sulfate, filtered, and concentrated under reduced pressure. The
resulting residue is purified by flash chromatography (silica gel) using 0-100 % ethyl
acetate in hexane to afford the title compound as a brown solid (0.21 g, 36.2%). Mass
spectrum (m/z): 453.2 (M+l).
Preparation 33
Synthesis of ethyl 3,5-dimethyl-4-[[2-methyl-5-(4-oxo-lpiperidyl)
benzoyl]amino]benzoate.
Scheme 12, step C. To a solution of ethyl 4-[[5-(l,4-dioxa-8-azaspiro[4.5]decan-
8-yl)-2-methyl-benzoyl]amino]-3,5-dimethyl-benzoate (205 mg, 452.99 ) in
acetone (5 ml) is added a mixture of 5M HC1 ( 1 mL) in ]¾0 ( 1 mL) and heated to 60 °C.
After 12 hours, the mixture is cooled to ambient temperature, diluted with 2M NaOH to
pH 6 and extracted with ethyl acetate. The combined organic layers are dried over
sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue
is purified by flash chromatography (silica gel) using 0-40 % ethyl acetate in hexane to
afford the title compound as an off-white foam (0. 11 g, 61%). Mass spectrum (m/z):
409.2 (M+l).
Preparation 34
Synthesis of 3,5-dimethyl-4-[[2-methyl-5-(4-oxo-l-piperidyl)benzoyl]amino]benzoic
acid.
Scheme 12. step D. A solution of aqueous 2N NaOH (2.00 ml) is added to a
stirred solution of ethyl 3,5-dimethyl-4-[[2-methyl-5-(4-oxo-lpiperidyl)
benzoyl]amino]benzoate (0.11 g, 269.28 ΐ) in THF:MeOH (2.4 ml: 1.2 ml).
After 16 hours at ambient temperature, the organic solvent is removed under reduced
pressure and the residue is diluted with water, acidified to pH 7 with IN HC1, and
extracted twice with ethyl acetate. The organic layers are combined with aqueous layer
and concentrated under reduced pressure. The resulting solids are triturated with 1:1
mixture of acetonitrile and ethanol to give white slurry that is filtered through Celite™
bed. The filtrate is concentrated under reduced pressure and the precipitate is triturated
with acetone and filtered to afford the title compound as an off-white solid (99 mg, 99%).
Mass spectrum (m/z): 381.2 (M+l).
Example 8
Synthesis of 4-[[5-(4-hydroxy-l-piperidyl)-2-methyl-benzoyl]amino]-3,5-dimethylbenzoic
acid.
Scheme 12, step E. To a solution of 3,5-dimethyl-4-[[2-methyl-5-(4-oxo-lpiperidyl)
benzoyl]amino]benzoic acid (69.5 mg, 182.68 ) in methanol (1.83 ml) is
added sodium tetrahydroborate (13.82 mg, 365.36 ) and the resulting mixture is
stirred at ambient temperature. After 1 hour, the mixture is quenched with saturated
solution of NH4C 1 (0.2 ml) and extracted with CHC13 (3 ml):IPA ( 1 ml). The combined
organic layers are dried over sodium sulfate, filtered, and concentrated under reduced
pressure. The resulting solid is triturated with H20 (5ml), filtered, and washed with H20
(5ml). The residue is purified by reverse phase chromatography (CI 8) using 20 %
acetonitrile in ¾0 with 0.1% formic acid to afford the title compound as a white powder
(0.01 g, 15.7%). Mass spectrum (m/z): 383.2 (M+l).
Scheme 13
Preparation 35
Synthesis of methyl 3-[(5-bromo-2-methyl-benzoyl)amino]-2,4-dimethyl-benzoate.
Scheme 13, step A. To a solution of 5-bromo-2-methyl-benzoic acid (2.45 g,
11.16 mmol) in THF (10 ml), CH2C 12 (10 ml) and DMF (20.00 ΐ , 258.65 ΐ ) is
added dropwise oxalyl chloride (1.16 ml, 13.39 mmoles) at 0 °C and the reaction mixture
is slowly allowed to warm to ambient temperature. After 2 hours, the solvent is removed
under reduced pressure. To the residue is added CH2CI2 (40 ml) and the reaction mixture
is cooled to 0 °C, then ethyl 4-amino-3,5-dimethyl-benzoate (2 g, 11.16 mmol) is added
followed by N,N-dimethylpyridin-4-amine (13.63 mg, 0.1 1 mmoles) and pyridine (2.71
ml, 33.48 mmoles). The cooling bath is removed and the clear solution is allowed to
warm to ambient temperature. After 2 hours, the solvent is removed and the reaction
mixture is diluted with ethyl acetate and washed with IN HC1, a saturated solution of
sodium bicarbonate, and brine. The organic layers are combined, dried over anhydrous
Na2S0 4, filtered, and concentrated under reduced pressure to give the title compound as a
light yellow solid (4.0 g; 95.5%). Mass spectrum (m/z): 376.0 (M+l).
Preparation 36
Synthesis of methyl 3-[[5-(l,4-dioxa-8-azaspiro[4.5]decan-8-yl)-2-methylbenzoyl]
amino]-2,4-dimethyl-benzoate.
Scheme 13, step B. To a solution of methyl 3-[(5-bromo-2-methylbenzoyl)
amino]-2,4-dimethyl-benzoate (0.5 g, 1.33 mmol), l,4-dioxa-8-
azaspiro(4.5)decane (228.34 mg, 1.59 mmol) and CS2CO (1.3 g, 3.99 mmol) in 1,4-
dioxane (10 ml) is added Pd(OAc)2 (29.84 mg, 132.89 ΐ) followed by racemic-2,2'-
bis(diphenylphosphino)-l,l'-binaphthyl (82.75 mg, 132.89 ΐ) . The reaction mixture
is purged with nitrogen for 10 minutes and then heated to 90°C. After 3 hours, very small
amount of the product is formed. To the reaction mixture is added Pd(OAc)2 (29.84 mg,
132.89 ΐ) followed by racemic-2,2'-bis(diphenylphosphino)-l,l'-binaphthyl (82.75
mg, 132.89 ΐ) and heated to 100°C. After 12 hours, the reaction is cooled to ambient
temperature and diluted with water and extracted with EtOAc. The combined organic
layers are washed with brine, dried over sodium sulfate, filtered, and concentrated under
reduced pressure. The resulting residue is purified by flash chromatography (silica gel)
using 0-100 % ethyl acetate in hexane to afford the title compound as a brown solid (0.26
g, 44.62%). Mass spectrum (m/z): 439.2 (M+l).
Preparation 37
Synthesis of methyl 2,4-dimethyl-3-[[2-methyl-5-(4-
piperidyl)benzoyl]amino]benzoate.
Scheme 13. step C. To a solution of methyl 3-[[5-(l,4-dioxa-8-
azaspiro[4.5]decan-8-yl)-2-methyl-benzoyl]amino]-2,4-dimethyl-benzoate (250 mg,
570.09 moles) in THF (2 ml) is added 4M HC1 (1ml) and stirred at ambient temperature.
The mixture is concentrated under reduced pressure and re-dissolved in acetone (5 ml)
followed by the addition of 5N HC1 ( 1 ml). After 22 hours at 60°C, the mixture is cooled
to ambient temperature and diluted with 2M NaOH to pH 6 and extracted twice with ethyl
acetate. The combined organic layers are dried over sodium sulfate, filtered, and
concentrated under reduced pressure. The resulting residue is purified by flash
chromatography (silica gel) using 0-50 % ethyl acetate in hexane to afford the title
compound as an off-white foam (0.12 g, 53.36%). Mass spectrum (m/z): 395.2 (M+1).
Preparation 38
Synthesis of 2,4-dimethyl-3-[[2-methyl-5-(4-oxo-l-piperidyl)benzoyl]amino]benzoic
acid.
Scheme 13. step D. A solution of aqueous 2N NaOH ( 1.00 ml) is added to a
stirred solution of methyl 2,4-dimethyl-3-[[2-methyl-5-(4-oxo-lpiperidyl)
benzoyl]amino]benzoate (0.12 g, 304.20 ΐ) in THF:MeOH (2 ml:l ml).
After 16 hours at ambient temperature, the organic solvent is removed under reduced
pressure and the residue is diluted with water, acidified to pH 6 with IN HC1, and
extracted with ethyl acetate. The organic layers are combined with aqueous layer and
concentrated under reduced pressure. The resulting solids are dissolved in
acetonitrile/H20 and lyophilized to afford the title compound as an off-white solid (58
mg, 50.12%). Mass spectrum (m/z): 381.2 (M+l).
Example 9
Synthesis of 3-[[5-(4-hydroxy-l-piperidyl)-2-methyl-benzoyl]amino]-2,4-dimethylbenzoic
acid.
Scheme 13. step E. To a solution of 2,4-dimethyl-3-[[2-methyl-5-(4-oxo-lpiperidyl)
benzoyl]amino]benzoic acid (51 mg, 134.05 ) in methanol (1.34 ml) is
added sodium tetrahydroborate (10. 14 mg, 268. 11 ) and the mixture is stirred at
ambient temperature. After 1 hour, the mixture is quenched and adjusted to pH7 with
saturated solution of NH4C 1 and extracted twice with CHCI3 (3 ml):IPA(l ml) . The
combined organic layers are dried over sodium sulfate, filtered, and concentrated under
reduced pressure. The solid is re-dissolved in 3:1 ¾ :and diluted with HC1 to
pH2 and extracted with 3:1 ¾ :. The combined organic layers are dried over
sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue
is taken up in MeOH and filtered through cotton plug. The filtrate is concentrated under
reduced pressure followed by dissolution in H20 and lyophilized for 12 hours to afford
the title compound as an off-white powder (0.04 g, 81.9%). Mass spectrum (m z): 383.2
(M+l).
Preparation 39
Synthesis of 6-chloro-3-methyl-pyridine-2-carboxylic acid.
Scheme 14. step A. A solution of aqueous IN NaOH (10.00 ml) is added to a
stirred solution of methyl 6-chloro-3-methyl-pyridine-2-carboxylate (1.0 g, 5.39 mmoles)
in THF:MeOH (10 ml:2 ml). The mixture is stirred at room temperature for 3 hours. The
organic solvent is removed under reduced pressure and the semi-solid is dissolved in
water and acidified to pH 1-2 with aqueous IN HCl. The resulting precipitate is filtered,
washed with water, and dried at 40°C in vacuum oven for 12 hours to give the title
compound as a white solid (780 mg, 84%). Mass spectrum (m/z): 172.0 (M+l).
Preparation 40
Synthesis of methyl 3-[(6-chloro-3-methyl-pyridine-2-carbonyl)amino]-2,4-dimethylbenzoate.
Scheme 14. step B. To a solution of 6-chloro-3-methyl-pyridine-2-carboxylic acid
(0.35 g, 0.20 mmol) in CH2CI2 (6 ml) at room temperature are added methyl 3-amino-3,5-
dimethylbenzoate (0.36 g, 0.20 mmol, see preparation 12), and ,Ndiisopropylethylamine
(0.77 g, 0.60 mmol). After stirring the reaction mixture for 10
minutes, 1-propanephosphonic acid cyclic anhydride (50% solution in ethyl acetate, 2.54
g, 0.80 mmol) is added via syringe. After 16 hours at ambient temperature, the reaction
mixture is diluted with ¾ (¾ washed with water and brine. The organic layers are
combined and dried over anhydrous Na2S0 4, filtered, and concentrated under reduced
pressure to give the title compound as a white powder (0.52 g, 76 %). Mass spectrum
(m/z): 333.2 (M+l).
Preparation 41
Synthesis of tert-butyl-dimethyl-(4-piperidyloxy)silane.
Scheme 14, step C. To a solution of 4-hydroxypiperidine (2.00 g, 9.89 mmoles)
in CH2CI2 (30 mL) is added lH-imidazole (2.69 g, 39.55 mmoles) followed by tbutyldimethylchlorosilane
(3.58 g, 23.73 mmoles) and the reaction mixture is stirred at
room temperature. After 12 hours, the reaction mixture is washed with water, saturated
solution of NaHC03, and brine. The organic layers are combined and dried over sodium
sulfate, filtered, and concentrated under reduced pressure. The resulting residue is
purified by flash chromatography (silica gel) over a gradient using 100% CH2CI2 to 10%
7N ammonia in MeOH/90% CH2C 12 to afford the title compound (3.69 g, 86.3 %). Mass
spectrum (m/z): 216.2 (M+l).
Preparation 42
Synthesis of methyl 3-[[6-(4-hydroxy-l-piperidyl)-3-methyl-pyridine-2-carbonyl]amino]-
2,4-dimethyl-benzoate.
Scheme 14. step C. To a solution of methyl 3-(6-chloro-3-methylpicolinamido)-
2,4-dimethylbenzoate (0.50 g, 0.0015 mol), tert-butyl-dimethyl-(4-piperidyloxy)silane
(0.90 g, 0.0045 mol) and Cs2C0 3 (2.00 g, 0.006 mol) in toluene (6 ml) is added Pd2(dba)3
(0.14 g, 0.00015 mol) followed by BINAP (0.086 g, 0.00015 mol). The reaction mixture
is purged with nitrogen for 5 minutes and then heated at 120 °C. After 4 hours, the
reaction is cooled to ambient temperature, filtered through Celite™, and washed with
EtOAc. The combined filtrate is dried over sodium sulfate, filtered, and concentrated
under reduced pressure to afford the title compound as a white semi-solid (0.220 g, 29
%). Mass spectrum (m/z): 398.2 (M+l).
Example 10
Synthesis of 3-[[6-(4-hydroxy-l-piperidyl)-3-methyl-pyridine-2-carbonyl]amino]-2,4-
dimethyl-benzoic acid.
Scheme 14. step D. A solution of aqueous 2N NaOH (2.00 ml) is added to a
stirred solution of methyl 3-[[6-(4-hydroxy-l-piperidyl)-3-methyl-pyridine-2-
carbonyl]amino]-2,4-dimethyl-benzoate (0.21 g, 0.52 mmol) in THF:MeOH (3 ml:l ml).
After 16 h at ambient temperature, the organic solvent is removed under reduced pressure
and the residue is diluted with water, acidified to pH 6 with aqueous IN HC1, and
extracted with ethyl acetate (2 x 20 ml). The organic layers are combined and dried over
anhydrous sodium sulfate. The solvent is removed under reduced pressure and the
resulting precipitate is triturated with pentane and filtered to afford the title compound as
a white solid (0.082 g, 39%). Mass spectmm (m/z): 384.2 (M+1).
Scheme 15
Preparation 43
Synthesis of methyl 4-[(6-chloro-3-methyl-pyridine-2-carbonyl)amino]-3,5-dimethylbenzoate.
Scheme 15. step A. To a solution of 6-chloro-3-methyl-pyridine-2-carboxylic
acid (0.70 g, 4.093 mmol, see preparation 1) in CH2C 12 (6 mL) at 0 °C are added methyl
4-amino-3,5-dimethylbenzoate (0.74 g, 4.093 mmol, see preparation 12), and
triethylamine (0.83 g, 8.187 mmol). After stirring the reaction mixture for 10 minutes, 1-
propanephosphonic acid cyclic anhydride (50% solution in ethyl acetate, 2.60 g, 8.187
mmol) is added via syringe and stirred at ambient temperature. After 12 hours, the
solvent is removed under reduced pressure and the residue is diluted with water and
extracted with ethyl acetate. The organic layers are combined and dried over anhydrous
Na2S0 4, filtered, and concentrated under reduced pressure. The resulting residue is
purified by flash chromatography (silica gel) using a gradient of 0-40% ethyl acetate in
hexanes to give the title compound as an off-white solid (1.10 g, 8 1 %). Mass spectrum
(m/z): 333.2 (M+l).
Preparation 44
Synthesis of tert-butyl-dimethyl-(3-piperidylmethoxy)silane.
Scheme 15, step B. To a solution of piperidin-3-ylmethanol (2.10 g, 0.018 mol) in
CH2C 12 (20 mL) is added triethylamine (5.53 g, 0.0547 mol) followed by
t-butyldimethylchlorosilane (4.127 g, 0.0275 mol) and the reaction mixture is stirred at
room temperature. After 24 hours, the reaction mixture is washed with water, saturated
solution of aHC0 , and brine. The combined organic layers are dried over sodium
sulfate, filtered, and concentrated under reduced pressure. The resulting residue is
purified by flash chromatography (silica gel) over a gradient using 0-10% MeOH in
dichloromethane to afford the title compound (2.50 g, 60 %). Mass spectrum (m/z):
231.2 (M+l).
Preparation 45
Synthesis of methyl 4-[[6-[3-[[tert-butyl(dimethyl)silyl]oxymethyl]-l-piperidyl]-3-
methyl-pyridine-2-carbonyl]amino]-3,5-dimethyl-benzoate.
Scheme 15, step B. To a solution of methyl 4-(6-chloro-3-methylpicolinamido)-
2,4-dimethylbenzoate (0.50 g, 0.0015 mol), tert-butyl-dimethyl-(3-
piperidylmethoxy)silane (0.42 g, 0.0018 mol) and Cs2C0 3 (1.96 g, 0.0060 mol) in 1,4-
dioxane (20 ml) is added Pd2(dba)3 (0. 14 g, 0.00015 mol) followed by S-Phos (0.062 g,
0.00015 mol). The reaction mixture is purged with nitrogen for 5 minutes and then
heated at 80 °C. After 24 hours, the reaction is cooled to ambient temperature, filtered
through Celite™, and washed with EtOAc. The combined filtrates are dried over sodium
sulfate, filtered, and concentrated under reduced pressure. The resulting residue is
purified by flash chromatography (silica gel) to afford the title compound as a white semi
solid (0.230 g, 30 %). Mass spectrum (m/z): 526.5 (M+l).
Preparation 46
Synthesis of methyl 4-[[6-[3-(hydroxymethyl)-l-piperidyl]-3-methyl-pyridine-2-
carbonyl]amino]-3,5-dimethyl-benzoate.
Scheme 15. step C. To a solution of methyl 4-[[6-[3-[[tertbutyl(
dimethyl)silyl]oxymethyl]-l-piperidyl]-3-methyl-pyridine-2-carbonyl]amino]-3,5-
dimethyl-benzoate (0.22 g, 0.873 mmol) in THF (3 ml) is added BU4 F 1.0 M in THF
(2.00 ml) at 0°C. The reaction mixture is gradually warmed to ambient temperature.
After 8 hours, the reaction mixture is diluted with ice-water and extracted twice with
ethyl acetate. The organic layers are combined, dried over sodium sulfate, filtered, and
concentrated under reduced pressure to give a residue. The residue is purified by flash
chromatography (silica gel) over a gradient using 0 - 40% ethyl acetate in hexanes to
afford the title compound as a white solid (0.06 g, 30%). Mass spectrum (m/z): 412.2
(M+l).
Example 11
Synthesis of 4-[[6-[3-(hydroxymethyl)-l-piperidyl]-3-methyl-pyridine-2-
carbonyl]amino]-3,5-dimethyl-benzoic acid.
Scheme 15. step D. A solution of aqueous 2N NaOH (2.00 ml) is added to a
stirred solution of methyl 4-(6-(3-(hydroxymethyl)piperidin-l-yl)-3-methylpicolinamido)-
3,5-dimethylbenzoate (0.06 g, 0.146 mmol) in THF:MeOH (4 ml:l ml). After 12 hours at
ambient temperature, the organic solvent is removed under reduced pressure and the
residue is diluted with water, acidified to pH 3 with aqueous citric acid solution, and
extracted with ethyl acetate (2 x 10 ml). The organic layers are combined and dried over
anhydrous sodium sulfate. The solvent is removed under reduced pressure and the
resulting precipitate is triturated with pentane and filtered to afford the title compound as
an off-white solid (0.03 g, 48%). Mass spectrum (m/z): 398.2 (M+l).
Scheme 16
Preparation 47
Synthesis of methyl 3-[[6-[3-[[tert-butyl(dimethyl)silyl]oxymethyl]-l-piperidyl]-3-
methyl-pyridine-2-carbonyl] amino]-2,4-dimethyl-benzoate.
i—
Scheme 16, step A. To a solution of methyl 3-[(6-chloro-3-methyl-pyridine-2-
carbonyl)amino]-2,4-dimethyl-benzoate (0.5 g, 0.0015 mol, see preparation 40), tertbutyl-
dimethyl-(3-piperidylmethoxy)silane (0.41 g, 0.0018 mol, see preparation 14) and
Cs2C0 3 (1.95 g, 0.006 mol) in 1,4-dioxane (20 ml) is added Pd2(dba)3 (0.137 g, 0.00015
mol) followed by S-Phos (0.06 g, 0.00015 mol). The reaction mixture is purged with
nitrogen for 5 minutes and then heated at 80 °C. After 24 hours, the reaction is cooled to
ambient temperature, filtered through Celite™, and washed with EtOAc. The combined
filtrates are dried over sodium sulfate, filtered, and concentrated under reduced pressure
and purified by flash chromatography (silica gel) using 20 % EtOAc in hexane to afford
the title compound as a brown semi-solid (0.2 g, 38%). Mass spectrum (m/z): 525.2
(M+l).
Preparation 48
Synthesis of methyl 3-[[6-[3 -(hydroxymethyl)- 1-piperidyl]-3 -methyl-pyridine-2-
carbonyl]amino]-2,4-dimethyl-benzoate.
Scheme 16. step B. To a solution of methyl 3-[[6-[3-[[tertbutyl(
dimethyl)silyl]oxymethyl]-l-piperidyl]-3-methyl-pyridine-2-carbonyl]amino]-2,4-
dimethyl-benzoate (0.20 g, 0.38 mmol) in THF (4 ml) is added BU4 F 1.0 M in THF (1.5
ml) at 0°C. The reaction mixture is gradually warmed to ambient temperature. After 8
hours, the reaction mixture is diluted with ice-water and extracted with ethyl acetate. The
organic layers are combined, dried over sodium sulfate, filtered, and concentrated under
reduced pressure. The resulting residue is purified by flash chromatography (neutral
alumina) over a gradient using 50% ethyl acetate in hexane to afford the title compound
as a white solid (0.18 g, 115%). Mass spectrum (m/z): 412.2 (M+l).
Example 12
Synthesis of 3-[[6-[3-(hydroxymethyl)-l-piperidyl]-3-methyl-pyridine-2-
carbonyl] amino]-2,4-dimethyl-benzoic acid.
Scheme 16, step C. A solution of aqueous 2N NaOH (3.00 ml) is added to a
stirred solution of methyl 3-[[6-[3-(hydroxymethyl)-l-piperidyl]-3-methyl-pyridine-2-
carbonyl]amino]-2,4-dimethyl-benzoate (0.18 g, 0.437 mmol) in THF (8 ml). After 12
hours at ambient temperature, the organic solvent is removed under reduced pressure and
the residue is diluted with water, acidified to pH 4 with citric acid, and extracted with
ethyl acetate. The organic layers are combined and dried over anhydrous sodium sulfate.
The solvent is removed under reduced pressure and the resulting residue is purified by
preparative HPLC to afford the title compound as off white solid (57 mg, 14%). Mass
spectrum (m/z): 398.2 (M+l).
Scheme 17
Preparation 49
Synthesis of ethyl 4-[(6-chloro-3-methyl-pyridine-2-carbonyl)amino]-3,5-dimethylbenzoate.
Scheme 17, step A. To a solution of 6-chloro-3-methyl-pyridine-2-carboxylic
acid (465 mg, 2.71 mmol) in THF (5 ml), CH2C 12 (5 ml) and DMF (0.01 ml, 129.33
) is added dropwise oxalyl chloride (0.3 ml, 3.46 mmoles) at 0 °C and reaction
mixture is slowly allowed to warm to ambient temperature. After 2 hours, the solvent is
removed under reduced pressure and the residue. To the residue CH2CI2 (20 ml) is added
and reaction mixture is cooled to 0 °C, then ethyl 4-amino-3,5-dimethyl-benzoate (520
mg, 2.69 mmol) is added followed by N,N-dimethylpyridin-4-amine (15 mg, 122.78
) and Pyridine (0.66 ml, 8. 16 mmoles). The cooling bath is removed and the clear
yellow solution is allowed to warm to ambient temperature. After 1 hour, the solvent is
removed and the reaction mixture is diluted with ethyl acetate and washed with 0.5N HCl,
saturated solution of sodium bicarbonate, and brine. The organic layers are combined and
dried over anhydrous Na2S0 4, filtered, and concentrated under reduced pressure. The
residue is purified by flash chromatography (silica gel) using 0-50% ethyl acetate in
hexanes to give the title compound as a solid (790 mg, 84%). Mass spectrum (m/z):
347.2 (M+l).
Preparation 50
Synthesis of ethyl 4-[[6-(4-hydroxy-4-methyl-l-piperidyl)-3-methyl-pyridine-2-
carbonyl]amino]-3,5-dimethyl-benzoate.
Scheme 17. step B. To a solution of ethyl 4-[(6-chloro-3-methyl-pyridine-2-
carbonyl)amino]-3,5-dimethyl-benzoate (275 mg, 792.93 ), 4-methylpiperidin-4-ol
(120 mg, 1.04 mmol) and CS2CO (0.8 g, 2.46 mmol) in 1,4-dioxane (6 ml) is added
Pd(OAc)2 (27 mg, 120.26 ) followed by racemic-2,2'-bis(diphenylphosphino)-
, -binaphthyl (54 mg, 86.72 ^). The reaction mixture is purged with nitrogen for
5 minutes and then heated to reflux. After 2 hours, the reaction is cooled to ambient
temperature and diluted with ammonium chloride and extracted twice with EtOAc. The
combined organic layers are dried over sodium sulfate, filtered, and concentrated under
reduced pressure. The resulting residue is purified by flash chromatography (silica gel)
using 0-70 % ethyl acetate in hexane to afford the title compound as a yellow oil (0.27 g,
80%). Mass spectrum (m/z): 426.2 (M+l).
Example 13
Synthesis of 4-[[6-(4-hydroxy-4-methyl-l-piperidyl)-3-methyl-pyridine-2-
carbonyl] amino] -3,5-dimethyl-benzoic acid.
Scheme 17, step C. A solution of aqueous 2N NaOH (5.00 ml) is added to a
stirred solution of ethyl 4-[[6-(4-hydroxy-4-methyl-l-piperidyl)-3-methyl-pyridine-2-
carbonyl]amino]-3,5-dimethyl-benzoate (0.27 g, 634.51 mmol) in THF:MeOH (10 ml:5
ml). After 1 hour at 50 °C, the organic solvent is removed under reduced pressure and the
residue is diluted with water, acidified to pH 6 with 5N HC1, and extracted with ethyl
acetate. The organic layers are combined with aqueous layer and concentrated under
reduced pressure. The solids are triturated with acetone/acetonitrile and filtered. The
filtrate is dried over sodium sulfate, filtered, and concentrated under reduced pressure to
afford the title compound as an off-white solid (0. 11 g, 43.6%). Mass spectrum (m/z):
398.2 (M+l).
Preparation 51
Synthesis of methyl 3-[(6-chloro-3-methyl-pyridine-2-carbonyl)amino]-2,4-dimethylbenzoate.
Scheme 18, step A. To a solution of 6-chloro-3-methyl-pyridine-2-carboxylic
acid (465 mg, 2.71 mmol) in THF (5 ml), CH2C 12 (5 ml) and DMF (0.01 ml, 129.33
) is added dropwise oxalyl chloride (0.3 ml, 3.46 mmoles) at 0 °C and the reaction
mixture is slowly allowed to warm to ambient temperature. After 2 hours, the solvent is
removed under reduced pressure. To the residue CH2CI2 (20 ml) is added and reaction
mixture is cooled to 0 °C, then methyl 3-amino-3,5-dimethylbenzoate (490 mg, 2.73
mmol) is added followed by N,N-dimethylpyridin-4-amine (15 mg, 122.78 ) and
pyridine (0.66 ml, 8.16 mmoles). The cooling bath is removed and the clear solution is
allowed to warm to ambient temperature. After 1.5 hours, the solvent is removed and the
reaction mixture is diluted with ethyl acetate and washed with 0.5N HC1, saturated
solution of sodium bicarbonate, and brine. The organic layers are combined and dried
over anhydrous Na2S0 4, filtered, and concentrated under reduced pressure to give the title
compound as a light yellow solid (664 mg, 73%). Mass spectrum (m/z): 333.2 (M+l).
Preparation 52
Synthesis of methyl 3-[[6-(4-hydroxy-4-methyl-l-piperidyl)-3-methyl-pyridine-2-
carbonyl] amino] -2,4-dimethyl-benzoate.
Scheme 18. step B. To a solution of methyl 3-[(6-chloro-3-methyl-pyridine-2-
carbonyl)amino]-2,4-dimethyl-benzoate (315 mg, 946.55 ^), 4-methylpiperidin-4-ol
(130 mg, 1.13 mmol) and CS2CO (0.9 g, 2.76 mmol) in 1,4-dioxane (7 ml) is added
Pd(OAc)2 (25 mg, 89.93 ) followed by racemic-2,2'-bis(diphenylphosphino)-l,l'-
binaphthyl (56 mg, 89.93 ^). The reaction mixture is purged with nitrogen for 5
minutes and then heated to reflux. After 2 hours, the reaction is cooled to ambient
temperature and diluted with ammonium chloride and extracted with EtOAc. The
combined organic layers are dried over sodium sulfate, filtered, and concentrated under
reduced pressure. The resulting residue is purified by flash chromatography (silica gel)
using 0-100 % ethyl acetate in hexane to afford the title compound as a brown solid (0.27
g, 69%). Mass spectrum (m/z): 412.2 (M+l).
Example 14
Synthesis of 3-[[6-(4-hydroxy-4-methyl-l-piperidyl)-3-methyl-pyridine-2-
carbonyl] amino] -2,4-dimethyl-benzoic acid.
Scheme 18, step C. A solution of aqueous 2N NaOH (5.00 ml) is added to a
stirred solution of methyl 3-[[6-(4-hydroxy-4-methyl-l-piperidyl)-3-methyl-pyridine-2-
carbonyl]amino]-2,4-dimethyl-benzoate (0.27 g, 656.13 mmol) in THF:MeOH (10 ml:5
ml). After 1 hour 50 °C, the organic solvent is removed under reduced pressure and the
residue is diluted with water, acidified to pH 6 with 5N HC1, and extracted with ethyl
acetate. The organic layers are combined and concentrated under reduced pressure. The
solids are triturated with acetone/acetonitrile and filtered. The filtrate is dried over
sodium sulfate, filtered and concentrated under reduced pressure to afford the title
compound as an off-white solid (75 mg, 29%). Mass spectrum (m/z): 398.2 (M+l).
Preparation 53
Synthesis of methyl 4-[[6-[3-[[tert-butyl(dimethyl)silyl]oxymethyl]pyrrolidin-l-yl]-3-
methyl-pyridine-2-carbonyl]amino]-3,5-dimethyl-benzoate.
Scheme 19. step A. To a solution of methyl 4-(6-chloro-3-methylpicolinamido)-
2,4-dimethylbenzoate (0.4 g, 1.2 mmol, see preparation 2), tert-butyl-dimethyl-
(pyrrolidin-3-ylmethoxy)silane (0.775 g, 3.6 mmol) and CS2CO (1.17 g, 3.6 mmol) in
1,4-dioxane (5 ml) is added Pd2(dba)3 (0.1 g, 0.12 mmol) followed by S-Phos (0.05 g,
0.12 mmol). The reaction mixture is purged with nitrogen for 5 minutes and then heated
at 90 °C. After 24 hours, the reaction is cooled to ambient temperature, filtered through
Celite™, and washed with EtOAc. The combined filtrates are dried over sodium sulfate,
filtered, and concentrated under reduced pressure. The resulting residue is purified by
flash chromatography (silica gel) using 10% ethyl acetate in hexanes to afford the title
compound as a white semi-solid (1.2 g, crude). Mass spectrum (m/z): 512.2 (M+l).
Preparation 54
Synthesis of methyl 4-[[6-[3-(hydroxymethyl)pyrrolidin-l-yl]-3-methyl-pyridine-2-
carbony 1] amino]-3,5-dimeth 1-benzoate.
Scheme 19, step B. To a solution of methyl 4-[[6-[3-[[tertbutyl(
dimethyl)silyl]oxymethyl]pyrrolidin-l-yl]-3-methyl-pyridine-2-carbonyl]amino]-
3,5-dimethyl-benzoate (1.1 g, 2.15 mmol) in THF (7 ml) is added BU4 F 1.0 M in THF
(5.00 ml) at 0°C. The reaction mixture is gradually warmed to ambient temperature.
After 6 hours, the reaction mixture is diluted with ice-water and extracted with ethyl
acetate. The organic layers are combined, dried over sodium sulfate, filtered, and
concentrated under reduced pressure. The resulting residue is purified by flash
chromatography (silica gel) over a gradient using 0 - 50% ethyl acetate in hexanes to
afford the title compound as an off-white solid (0.36 g, 42%). Mass spectrum (m/z):
398.2 (M+l).
Example 15
Synthesis of 4-[[6-[3-(hydroxymethyl)pyrrolidin-l-yl]-3-methyl-pyridine-2-
carbonyl] amino]-3,5-dimethyl-benzoic acid.
Scheme 19, step C. A solution of NaOH (0.16 g) in H20 ( 1 ml) is added to a
stirred solution of methyl 4-[[6-[3-(hydroxymethyl)pyrrolidin-l-yl]-3-methyl-pyridine-2-
carbonyl]amino]-3,5-dimethyl-benzoate (0.35 g, 0.88 mmol) in THF:MeOH (5 ml:l ml).
After 8 hours at ambient temperature, the organic solvent is removed under reduced
pressure and the residue is diluted with water, acidified to pH 3 with aqueous citric acid
solution, and extracted with ethyl acetate (2 10 ml). The organic layers are combined
and dried over anhydrous sodium sulfate. The solvent is removed under reduced pressure
and the resulting precipitate is triturated with diethyl ether and filtered to afford the title
compound as an off-white solid (73 mg, 33.7%). Mass spectrum (m/z): 384.2 (M+l).
Scheme 20
Preparation 55
Synthesis of methyl 4-[(6-chloro-3-methyl-pyridine-2-carbonyl)amino]-3,5-dimethylbenzoate.
Scheme 20, step A. To a solution of 6-chloro-3-methyl-pyridine-2-carboxylic
acid (6.00 g, 0.03508 mol) in CH2C 12 (50 ml) at 0 °C are added methyl 4-amino-3,5-
dimethylbenzoate (5.65 g, 0.03157 mol), and N,N-diisopropylethylamine (15.1 ml,
0.0877 mol). After stirring the reaction mixture for 10 minutes, 1-propanephosphonic
acid cyclic anhydride (50% solution in ethyl acetate, 22.32 g, 0.07016 mol) is added via
syringe and stirred at 60 °C. After 3 hours, the solvent is removed under reduced pressure
and the residue is diluted with water and extracted with ethyl acetate. The organic layers
are combined and dried over anhydrous Na2S0 4, filtered, and concentrated under reduced
pressure. The resulting residue is purified by flash chromatography (silica gel) using a
gradient of 0-40% ethyl acetate in hexanes to give the title compound as an off-white
solid (8.70 g, 74%). Mass spectrum (m/z): 333.2 (M+l).
Preparation 56
Synthesis of methyl 4-[[6-(4-methoxy-l-piperidyl)-3-methyl-pyridine-2-carbonyl]amino]-
3,5-dimethyl-benzoate.
Scheme 20. step B. To a solution of methyl 4-(6-chloro-3-methylpicolinamido)-
2,4-dimethylbenzoate (0.30 g, 0.90 mmol), 4-methoxypiperidine (0.178 g, 1.8 mmol) and
Cs2C0 3 (0.88 g, 2.7 mmol) in 1,4-dioxane (5 ml) is added Pd2(dba)3 (0.082 g, 0.09 mmol)
followed by S-Phos (0.037 g, 0.09 mmol). The reaction mixture is purged with nitrogen
for 5 minutes and then heated at 130 °C. After 16 hours, the reaction is cooled to ambient
temperature, filtered through Celite™, and washed with EtOAc. The combined filtrates
are dried over sodium sulfate, filtered, and concentrated under reduced pressure. The
resulting residue is purified by flash chromatography (silica gel) using 20% ethyl acetate
in hexane to afford the title compound as a light yellow solid (0. 14 g, 34 %).
Example 16
Synthesis of 4-[[6-(4-methoxy-l-piperidyl)-3-methyl-pyridine-2-carbonyl]amino]-3,5-
dimethyl-benzoic acid.
Scheme 20. step C. Lithium hydroxide (LiOH H20 , 0.026 g, 0.62 mmol) is added
to a stirred solution of methyl 4-[[6-(4-methoxy-l-piperidyl)-3-methyl-pyridine-2-
carbonyl]amino]-3,5-dimethyl-benzoate (0.129 g, 0.313 mmol) in THF:H 20:MeOH (0.7
ml:0.3 ml:0.7 ml). After 2 hours at 50 °C, the organic solvent is removed under reduced
pressure and the residue is diluted with water, acidified to pH 6 with aqueous IN HC1,
and extracted with ethyl acetate (3 x 20 ml). The organic layers are combined and dried
over anhydrous sodium sulfate. The solvent is removed under reduced pressure and the
resulting precipitate is triturated with diethyl ether and filtered to afford the title
compound as an off-white solid (0. 1lg, 88%). Mass spectrum (m/z): 398.2 (M+l).
Scheme 21
Preparation 57
Synthesis of methyl 4-[[6-(4,4-difluoro-l-piperidyl)-3-methyl-pyridine-2-
carbonyl]amino]-3,5-dimethyl-benzoate.
Scheme 1 step A. To a solution of methyl 4-(6-chloro-3-methylpicolinamido)-
2,4-dimethylbenzoate (0.30 g, 0.90 mmol, see preparation 43), 4,4-difluoropiperidine
(0.283 g, 1.8 mmol) and CS2CO (1.17 g, 3.6 mmol) in 1,4-dioxane (5 ml) is added
PdCi2(dpp CH2C 2 (0.082 g, 0.0903 mmol) followed by S-Phos (0.037 g, 0.0903 mmol).
The reaction mixture is purged with nitrogen for 5 minutes and then heated at 130 °C.
After 16 hours, the reaction is cooled to ambient temperature, filtered through Celite™,
and washed with EtOAc. The combined filtrates are dried over sodium sulfate, filtered,
and concentrated under reduced pressure to afford the title compound as a light yellow
solid (0.240 g, 63 %). Mass spectrum (m/z): 418.2 (M+l).
Example 17
Synthesis of 4-[[6-(4,4-difluoro-l-piperidyl)-3-methyl-pyridine-2-carbonyl]amino]-3,5-
dimethyl-benzoic acid.
Scheme 21. step B. Lithium hydroxide (LiOH H20 , 0.048 g, 0.00115 mol) is
added to a stirred solution of methyl 4-[[6-(4,4-difluoro-l-piperidyl)-3-methyl-pyridine-2-
carbonyl]amino]-3,5-dimethyl-benzoate (0.24 g, 0.575 mmol) in THF:H 20:MeOH (1.5
ml:0.7 ml: 1.5 ml). After 3 hours at 50 °C, the organic solvent is removed under reduced
pressure and the residue is diluted with water, acidified to pH 6 with aqueous IN HC1,
and extracted with ethyl acetate (3 x 20 ml). The organic layers are combined and dried
over anhydrous sodium sulfate. The solvent is removed under reduced pressure and the
resulting precipitate is triturated with diethyl ether and filtered to afford the title
compound as a white solid (0.166 g, 71%). Mass spectrum (m/z): 404.2 (M+l).
Scheme 22
Preparation 58
Synthesis of methyl 4-[(2-chloroquinoline-4-carbonyl)amino]-3,5-dimethyl-benzoate.
Scheme 22, step A. To a solution of 2-chloroquinoline-4-carboxylic acid (5.00 g,
0.0024 mol) in CH2C 12 (50 mL) at 0°C are added methyl 4-amino-3,5-dimethylbenzoate
(3.88 g, 0. 02167 mol, see preparation 12) and N,N-diisopropylethylamine (12.5 ml,
0.07225 mol). After stirring the reaction mixture for 10 minutes, 1-propanephosphonic
acid cyclic anhydride (50% solution in ethyl acetate, 31.0 ml, 0.048 mol) is added via
syringe and heated at 40°C. After 5 hours, the reaction mixture is diluted with water and
extracted with dichloromethane. The organic layers are combined and dried over
magnesium sulfate, filtered, and concentrated under reduced pressure. The resulting
residue is purified by flash chromatography (silica gel) using a gradient of 0-40% ethyl
acetate in hexanes. After purification the solid is triturated with 20 % diethyl ether in
pentane and dried to give the title compound as a white solid (8.50 g, 96 %). Mass
spectrum (m/z): 369.1 (M+l).
Preparation 59
Synthesis of methyl 4-[[2-[4-[tert-butyl(dimethyl)silyl]oxy-l-piperidyl]quinoline-4-
carbonyl]amino]-3,5-dimethyl-benzoate.
Scheme 22, step B. To a solution of methyl 4-(2-chloroquinoline-4-
carboxamido)-3,5-dimethylbenzoate, (0.50 g, 0.00135 mol), tert-butyl-dimethyl-(4-
piperidyloxy)silane (0.32 g, 0.0015 mol, see preparation 3) and CS2CO (1.77 g, 0.0054
mol) in 1,4-dioxane (5 ml) is added Pd2(dba)3 (0.124 g, 0.000135 mol) followed by SPhos
(0.060 g, 0.000135 mol). The reaction mixture is purged with nitrogen for 5 minutes
and then stirred at 80 °C. After 4 hours, the reaction is cooled to ambient temperature,
filtered through Celite™, and washed with EtOAc. The organic layers are combined and
dried over sodium sulfate, filtered, and concentrated under reduced pressure to afford the
title compound (0.21 g, 28 %). Mass spectrum (m/z): 548.2 (M+l).
Preparation 60
Synthesis of methyl 4-[[2-(4-hydroxy-l-piperidyl)quinoline-4-carbonyl]amino]-3,5-
dimethyl-benzoate.
Scheme 22, step C. To a solution of methyl 4-(2-(4-((tertbutyldimethylsilyl)
oxy)piperidin-l-yl)quinoline-4-carboxamido)-3,5-dimethylbenzoate
(0.21 g, 0.00038 mol) in THF (4 ml) is added BU4 F 1.0 M in THF (4.0 ml) at 0°C. The
reaction mixture is gradually warmed to ambient temperature. After 14 hours, the
reaction mixture is diluted with ice-water and extracted with ethyl acetate. The organic
layers are combined, dried over sodium sulfate, filtered, and concentrated under reduced
pressure to give a residue. The residue is purified by flash chromatography (silica gel)
over a gradient using 0 - 40% ethyl acetate in hexanes to afford the title compound as a
light yellow solid (0.13 g, 78%). Mass spectrum (m/z): 434.2 (M+l).
Example 18
Synthesis of 4-[[2-(4-hydroxy-l-piperidyl)quinoline-4-carbonyl]amino]-3,5-dimethylbenzoic
acid.
Scheme 22, step D . A solution of aqueous 2N NaOH (2.00 ml) is added to a
stirred solution of methyl 4-(2-(4-hydroxypiperidin-l-yl)quinoline-4-carboxamido)-3,5-
dimethylbenzoate (0.13 g, 0.0003 mol) in THF:MeOH (5 ml:2 ml). After 5 h at 50°C, the
organic solvent is removed under reduced pressure and the residue is diluted with water
acidified to pH 6 with aqueous IN HCl and extracted with ethyl acetate (2 x 20 ml). The
organic layers are combined and dried over anhydrous sodium sulfate. The solvent is
removed under reduced pressure and the resulting precipitate is triturated with pentane
and diethyl ether and filtered to afford the title compound as a light yellow solid (0.05 g,
40 %). Mass spectrum (m/z): 420.2 (M+l).
Scheme 23
Preparation 61
Synthesis of methyl 3-[(2-chloroquinoline-4-carbonyl)amino]-2,4-dimethyl-benzoate.
Scheme 23. step A. To a solution of 2-chloroquinoline-4-carboxylic acid (0.40 g,
0.0019 mol) in CH2C 12 (8 mL) at 0°C are added methyl 3-amino-2,4-dimethylbenzoate
(0.3 1 g, 0. 0017 mol, see preparation 10) and triethylamine (0.80 ml, 0.0058 mol). After
stirring the reaction mixture for 10 minutes, 1-propanephosphonic acid cyclic anhydride
(50% solution in ethyl acetate, 2.45 ml, 0.0038 mol) is added via syringe and stirred at
room temperature. After 16 hours, the reaction is diluted with water and extracted twice
with dichloromethane. The organic layers are combined and dried over magnesium
sulfate, filtered, and concentrated under reduced pressure. The resulting residue is
triturated with 20 % diethyl ether in pentane and filtered to give the title compound as an
off-white solid (0.55 g, 77 %). Mass spectrum (m/z): 369. 1 (M+l).
Preparation 62
Synthesis of methyl 3-[[2-[4-[tert-butyl(dimethyl)silyl]oxy-l-piperidyl]quinoline-4-
carbonyl]amino]-2,4-dimethyl-benzoate.
Scheme 23. step B. To a solution of methyl 3-(2-chloroquinoline-4-
carboxamido)-2,4-dimethylbenzoate (0.54 g, 0.00146 mol), tert-butyl-dimethyl-(4-
piperidyloxy)silane (0.63 g, 0.00293 mol, see preparation 3) and CS2CO (1.43 g, 0.0044
mol) in 1,4-dioxane (8 ml) is added Pd2(dba)3 (0.134 g, 0.000146 mol) followed by SPhos
(0.060 g, 0.000146 mol). The reaction mixture is purged with nitrogen for 5
minutes and then stirred at 80 °C. After 16 hours, the reaction is cooled to ambient
temperature, filtered through Celite™, and washed with EtOAc. The organic layers are
combined and dried over sodium sulfate, filtered, and concentrated under reduced
pressure to afford the title compound as a black semi-solid (1.3 1 g, 60%, crude). Mass
spectrum (m/z): 548.2 (M+l).
Preparation 63
Synthesis of methyl 3-[[2-(4-hydroxy-l-piperidyl)quinoline-4-carbonyl]amino]-2,4-
dimethyl-benzoate.
Scheme 23, step C. To a solution of methyl 3-(2-(4-((tertbutyldimethylsilyl)
oxy)piperidin-l-yl)quinoline-4-carboxamido)-2,4-dimethylbenzoate
(1.30 g, 0.00087 mol) in THF (2.0 ml) is added Bu4NF 1.0 M in THF (10.0 ml) at 0°C.
The reaction mixture is gradually warmed to ambient temperature. After 14 hours, the
reaction mixture is diluted with ice-water and extracted with ethyl acetate. The organic
layers are combined, dried over sodium sulfate, filtered, and concentrated under reduced
pressure to give a residue. The residue is purified by flash chromatography (silica gel)
with ethyl acetate in hexanes to afford the title compound as a yellow solid (0. 10 g, 27%)
Mass spectrum (m/z): 434.3 (M+l).
Example 19
Synthesis of 3-[[2-(4-hydroxy-l-piperidyl)quinoline-4-carbonyl]amino]-2,4-dimethylbenzoic
acid.
Scheme 23, step D. A solution of aqueous 2N NaOH (2.00 ml) is added to a
stirred solution of methyl 3-(2-(4-hydroxypiperidin-l-yl)quinoline-4-carboxamido)-2,4-
dimethylbenzoate (0.10 g, 0.00023 mol) in THF:MeOH (4 ml:2 ml). After 5 h at 50°C,
the organic solvent is removed under reduced pressure and the residue is diluted with
water, acidified to pH 6 with aqueous IN HC1, and extracted with ethyl acetate (2 20
ml). The organic layers are combined and dried over anhydrous sodium sulfate. The
solvent is removed under reduced pressure and the resulting precipitate is triturated with
pentane and diethyl ether and filtered to afford the title compound as a light yellow solid
(0.03 g, 31%). Mass spectrum (m/z): 420.2 (M+l).
Scheme 24
Preparation 64
Synthesis of methyl 3-[(3-bromonaphthalene-l-carbonyl)amino]-2,4-dimethyl-benzoate.
Scheme 24, step A. To a solution of 3-bromo-1 -naphthoic acid (0.40 g, 1.59
mmol) in CH2CI2 (8 mL) at 0°C are added methyl 3-amino-2,4-dimethylbenzoate (0.26 g,
1.43 mmol, see preparation 10) and triethylamine (0.78 ml, 5.38 mmol). After stirring the
reaction mixture for 10 minutes, 1-propanephosphonic acid cyclic anhydride (50%
solution in ethyl acetate, 2.03 ml, 3.18 mmol) is added via syringe and stirred at room
temperature. After 16 hours, the reaction is diluted with water and extracted with ethyl
acetate. The organic layers are combined and dried over magnesium sulfate, filtered, and
concentrated under reduced pressure. The resulting residue is purified by flash
chromatography (silica gel) with 5 % EtOAc in hexane as eluent to afford the product as a
white solid (0.52 g, 79 %). Mass spectrum (m/z): 412.3 (M+l).
Preparation 65
Synthesis of methyl 3-[[3-[4-[tert-butyl(dimethyl)silyl]oxy-l-piperidyl]naphthalene-lcarbonyl]
amino]-2,4-dimethyl-benzoate.
Scheme 24, step B. To a solution of methyl 3-(3-bromo-l-naphthamido)-2,4-
dimethylbenzoate (0.48 g, 0.0011 mol), tert-butyl-dimethyl-(4-piperidyloxy)silane (0.75
g, 0.0034 mol, see preparation 3) and Cs2C0 3 (1.00 g, 0.0033 mol) in 1,4-dioxane (8 ml)
is added Pd2(dba)3 (0.10 g, 0.0001 1 mol) followed by S-Phos (0.045 g, 0.0001 1 mol).
The reaction mixture is purged with nitrogen for 5 minutes and then stirred at 80 °C.
After 16 hours, the reaction is cooled to ambient temperature, filtered through Celite™,
and washed with EtOAc. The organic layers are combined and dried over sodium sulfate,
filtered, and concentrated under reduced pressure to afford the title compound as a black
semi-solid (0.52 g, crude). Mass spectrum (m/z): 547.5 (M+l).
Preparation 66
Synthesis of methyl 3-[[3-(4-hydroxy-l-piperidyl)naphthalene-l-carbonyl]amino]-2,4-
dimethyl-benzoate.
Scheme 24, step C. To a solution of methyl 3-[[3-[4-[tertbutyl(
dimethyl)silyl]oxy-l-piperidyl]naphthalene-l-carbonyl]amino]-2,4-dimethylbenzoate
(0.30 g, 0.54 mmol) in THF (8.0 ml) is added BU4 F 1.0 M in THF (0.35 g,
1.60 mmol) at 0°C. The reaction mixture is gradually warmed to ambient temperature.
After 14 hours, the reaction mixture is diluted with ice-water and extracted with ethyl
acetate. The organic layers are combined, dried over sodium sulfate, filtered, and
concentrated under reduced pressure to give a residue. The residue is purified by flash
chromatography (silica gel) with ethyl acetate in hexanes to afford the title compound as a
white solid (0.10 g, 55%). Mass spectrum (m/z): 433.0 (M+l).
Example 20
Synthesis of 3-[[3-(4-hydroxy-l-piperidyl)naphthalene-l-carbonyl]amino]-2,4-dimethylbenzoic
acid.
Scheme 24, step D. A solution of aqueous 2N NaOH (3.00 ml) is added to a
stirred solution of methyl 3-(3-(4-hydroxypiperidin-l-yl)-l-naphthamido)-2,4-
dimethylbenzoate (0.10 g, 0.23 mmol) in THF:MeOH (3 ml:l ml). After 12 hours at
room temperature, the organic solvent is removed under reduced pressure and the residue
is diluted with water, acidified to pH 4 with aqueous IN HC1 and extracted with
dichloromethane (2 x 20 ml). The organic layers are combined and dried over anhydrous
sodium sulfate. The solvent is removed under reduced pressure and the resulting
precipitate is triturated with diethyl ether and filtered to afford the title compound as a
white solid (0.028 g, 29%). Mass spectrum (m/z): 419.2 (M+l).
Scheme 25
Preparation 67
Synthesis of methyl 3-[[5-bromo-2-(trifluoromethyl)benzoyl]amino]-2,4-dimethylbenzoate.
Scheme 25. step A. To a solution of 5-bromo-2-(trifluoromethyl)benzoic acid (1.0
g, 3.53 mmol) in CH2CI2 (6 ml) at room temperature are added methyl 3-amino-3,5-
dimethylbenzoate (0.44 g, 2.47 mmol, see preparation 12) and trimethylamine (1.0 ml,
7.06 mmol). After stirring the reaction mixture for 10 minutes, 1-propanephosphonic
acid cyclic anhydride (50% solution in ethyl acetate, 5.6 ml, 8.83 mmol) is added via
syringe. After 14 hours at ambient temperature, the reaction mixture is diluted with
CH2CI2, washed with water and brine. The organic layers are combined and dried over
anhydrous Na2S0 4, filtered, and concentrated under reduced pressure. The resulting
residue is purified by flash chromatography (silica gel) using 20% ethyl acetate in
hexanes to give the title compound as a white solid (0.6 g, 39 %).
Preparation 68
Synthesis of methyl 3-[[5-[4-[tert-butyl(dimethyl)silyl]oxy-l-piperidyl]-2-
(trifluoromethyl)benzoyl]amino]-2,4-dimethyl-benzoate.
Scheme 25. step B. To a solution of methyl 3-[[5-bromo-2-
(trifluoromethyl)benzoyl]amino]-2,4-dimethyl-benzoate (0.20 g, 0.46 mmol), tert-butyldimethyl-(
3-piperidylmethoxy)silane (0.11 g, 0.50 mmol, see preparation 3) and CS2CO3
(0.45 g, 1.39 mmol) in 1,4-dioxane (15 ml) is added Pd2(dba)3 (43 mg, 0.046 mmol)
followed by S-Phos (19.3 mg, 0.046m mol). The reaction mixture is purged with nitrogen
for 5 minutes and then heated at 110 °C. After 12 hours, the reaction is cooled to ambient
temperature, filtered through Celite™, and washed with EtOAc. The combined filtrates
are dried over sodium sulfate, filtered, and concentrated under reduced pressure. The
resulting residue is purified by flash chromatography (silica gel) using 30 % ethyl acetate
in hexane to afford the title compound as a brown semi-solid (180 mg, 68%). Mass
spectrum (m/z): 565.2 (M+l).
Preparation 69
Synthesis of methyl 3-[[5-(4-hydroxy-l-piperidyl)-2-(trifluoromethyl)benzoyl]amino]-
2,4-dimethyl-benzoate.
Scheme 25, step C. To a solution of methyl 3-[[5-[4-[tertbutyl(
dimethyl)silyl]oxy-l-piperidyl]-2(trifluoromethyl)benzoyl]amino]-2,4-dimethylbenzoate
(180 mg, 0.32 mmol) in THF (10.0 ml) is added BU4 F 1.0 M in THF (2.0 ml)
at 0°C. The reaction mixture is gradually warmed to ambient temperature. After 8 hours,
the reaction mixture is diluted with ice-water and extracted with ethyl acetate. The
organic layers are combined, dried over sodium sulfate, filtered, and concentrated under
reduced pressure to give a residue. The residue is purified by flash chromatography
(neutral alumina) over a gradient using 100% ethyl acetate in hexane to afford the title
compound as a white solid (0.11 g, 76%). Mass spectrum (m/z): 451.2 (M+l).
Example 21
Synthesis of 3-[[5-(4-hydroxy-l-piperidyl)-2-(trifluoromethyl)benzoyl]amino]-2,4-
dimethyl-benzoic acid.
Scheme 25, step D. A solution of aqueous 2N NaOH (2 ml) is added to a stirred
solution of methyl 3-[[6-[3-(hydroxymethyl)-l-piperidyl]-3-methyl-pyridine-2-
carbonyl]amino]-2,4-dimethyl-benzoate (0.1 1 g, 0.24 mmol) in THF (8 ml). After 12
hours at ambient temperature, the organic solvent is removed under reduced pressure and
the residue is diluted with water, acidified to pH 4 with IN HCl, and extracted twice with
dichloromethane. The organic layers are combined and dried over anhydrous sodium
sulfate. The solvent is removed under reduced pressure and the resulting precipitate is
triturated with pentane and diethyl ether and filtered to afford the title compound as an
off-white solid (95 mg, 89%). Mass spectrum (m/z): 437.2 (M+l).
Scheme 26
Preparation 70
Synthesis of methyl 4-[[5-bromo-2-(trifluoromethyl)benzoyl]amino]-3,5-dimethylbenzoate.
Scheme 26, step A. To a solution of 5-bromo-2-(trifluoromethyl)benzoic acid
(1.00 g, 0. 00371 mol) in ¾ ¾ (15 mL) at room temperature are added methyl 4-
amino-3,5-dimethylbenzoate (0.46 g, 0.00260 mol, see preparation 12) and triethylamine
(1.10 ml, 0.00743 mol). After stirring the reaction mixture for 10 minutes, 1-
propanephosphonic acid cyclic anhydride (50% solution in ethyl acetate, 6.00 ml,
0.00929 mol) is added via syringe and stirred at room temperature. After 14 hours, the
reaction mixture is diluted with water and extracted with dichloromethane. The organic
layers are combined and dried over magnesium sulfate, filtered, and concentrated under
reduced pressure. The resulting residue is purified by flash chromatography (silica gel)
with 10-15 % EtOAc to give the title compound as an off-white solid (0.45 g, 27.67 %).
Mass spectrum (m/z): 430.0 (M+l).
Preparation 71
Synthesis of methyl 4-[[5-[4-[tert-butyl(dimethyl)silyl]oxy-l-piperidyl]-2-
(trifluoromethyl)benzoyl]amino]-3,5-dimethyl-benzoate.
Scheme 26, step B. To a solution of methyl 4-[[5-bromo-2-
(trifluoromethyl)benzoyl]amino]-3,5-dimethyl-benzoate (0.10 g, 232.44 ΐ), tert-butyldimethyl-(
4-piperidyloxy)silane (55.08 mg, 255.68 ΐ , see preparation 3) and CS2CO
(227.2 mg, 697.32 ΐ) in 1,4-dioxane (2.5 ml) is added Pd2(dba)3 (21.28 mg, 23.24
ΐ) followed by 2-dicyclohexylphosphino-2',6'-dimethoxybiphenyl (S-Phos) (9.54 mg,
23.24 ΐ) . The reaction mixture is purged with nitrogen for 5 minutes and then heated
at 120 °C. After 8 hours, the reaction is cooled to ambient temperature, filtered through
Celite™, and washed with EtOAc. The combined filtrates are dried over sodium sulfate,
filtered, and concentrated under reduced pressure and purified by flash chromatography
(neutral alumina) using of 15% - 85% ethyl acetate in hexanes as eluent to afford the title
compound as a pale yellow oil (0. 10 g, 80 %). Mass spectrum (m/z): 565.4 (M+l).
Preparation 72
Synthesis of methyl 4-[[5-(4-hydroxy- 1-piperidyl)-2-(trifluoromethyl)benzoyl]amino]-
3,5-dimethyl-benzoate.
Scheme 26, step C. To a solution of methyl 4-[[5-[4-[tertbutyl(
dimethyl)silyl]oxy-l-piperidyl]-2-(trifluoromethyl)benzoyl]amino]-3,5-dimethylbenzoate
(0.10 g, 177.08 ΐ ) in THF (4 ml) is added BU4 F 1.0 M in THF (1.0 ml)
at 0°C. The reaction mixture is gradually warmed to ambient temperature. After 6 hours,
the reaction mixture is diluted with ice-water and extracted with ethyl acetate. The
organic layers are combined, dried over sodium sulfate, filtered, and concentrated under
reduced pressure to give a residue. The residue is purified by flash chromatography
(silica gel) over a gradient using 40 - 60% ethyl acetate in hexanes to afford the title
compound as a light yellow solid (0.06 g, 81.5%). Mass spectrum (m/z): 451.2 (M+l).
Example 22
Synthesis of 4-[[5-(4-hydroxy-l-piperidyl)-2-(trifluoromethyl)benzoyl]amino]-3,5-
dimethyl-benzoic acid.
Scheme 26, step D . A solution of aqueous 2N NaOH (1.5.00 ml) is added to a
stirred solution of methyl 4-[[5-[4-[tert-butyl(dimethyl)silyl]oxy-l-piperidyl]-2-
(trifluoromethyl)benzoyl]amino]-3,5-dimethyl-benzoate (60.0 mg, 133.20 ) in
THF:MeOH (5 ml:0.5 ml). After 12 h at ambient temperature, the organic solvent is
removed under reduced pressure and the residue is diluted with water, acidified to pH4
with aqueous citric acid, and extracted with ethyl acetate (2 x 20 ml). The organic layers
are combined and dried over anhydrous sodium sulfate. The solvent is removed under
reduced pressure and the resulting precipitate is triturated with pentane and diethyl ether
and filtered to afford the title compound as an off-white solid (0.04 g, 60 %). Mass
spectrum (m/z): 437.2 (M+l).
In Vitro binding to human EP1, EP2, EP3 and EP4
hEP l and hEP4 membranes are prepared from recombinant HEK293 cells stably
expressing human EP1 (Genbank accession number AY275470) or EP4 (Genbank
accession number AY429 109) receptors. hEP2 and hEP3 membranes are prepared from
HEK293 cells transiently transfected with EP2 (Genbank accession number AY27547 1)
or EP3 (isoform VI: Genbank accession number AY429 108) receptor plasmids. Frozen
cell pellets are homogenized in homogenization buffer using a Teflon/glass homogenizer.
Membrane protein is aliquoted and quick frozen on dry ice prior to storage at -80°C.
Homogenization buffer contained 10 mM Tris-HCl, pH 7.4, 250 mM sucrose, 1mM
EDTA, 0.3 mM indomethacin and plus Complete™, with EDTA, obtained from Roche
Molecular Biochemicals (Catalog Number 1 697 498).
Kd values for [3HJ-PGE2 binding to each receptor are determined by saturation
binding studies or homologous competition. Compounds are tested in a 96-well format
using a three-fold dilution series to generate a 10-point curve. Diluted compound is
incubated with 20 EP2, 1 ug/well EP3 or 10 to 20 EP4
membrane for 90 minutes at 25 C in the presence of 0.3 to 0.5 nM [ H]-PGE2
(PerkinElmer, 118 to 180 Ci/mmol). The binding reaction is performed in 200 MES
buffer (10 mM MES pH 6.0 with KOH, 10 mM MgCl2 and 1mM EDTA) using 0.5 mL
polystyrene 96-well deep-well plates. Nonspecific binding is calculated by comparing
binding in the presence and absence of 2 of PGE2. The membranes are harvested by
filtration (TomTek harvester), washed 4 times with cold buffer (10 mM MES pH 6.0 with
KOH, 10 mM MgCy, dried in a 60°C oven, and the radioactivity is quantified as counts
per minute (CPM) using a TopCount detector. Percent specific binding is calculated as
the percent of the binding in the absence of any inhibitor, corrected for binding in the
presence of 2 uM of PGE2. Data are analyzed using a 4-parameter nonlinear logistic
equation (ABase Equation 205) as shown: y = (A+((B-A)/(l+((C/x) D)))) where, y = %
specific inhibition, A = bottom of the curve; B = top of the curve; C = relative IC50 =
concentration causing 50% inhibition based on the range of the data from top to bottom;
D = Hill, Slope = slope of the curve. K conversion from IC50 Values K = IC o (l +
[L]/A ) where [L] is the ligand concentration). The compounds of Examples 1-22 herein
are tested essentially as described above and exhibit a K value for hEP4 of lower than
about 2 .
Table 2 : In vitro binding of Example 1 to human EP1, EP2, EP3 and EP4
Following the procedures essentially as described above, the data in table 2
demonstrate that the compound of Example 1 binds to hEP4 at low nanomolar
concentrations. The data in table 2 also demonstrate the compound of Example 1 binds to
hEP4 more strongly than to hEP 1, hEP2, and hEP3 indicating selectivity for the hEP4
receptor.
In Vitro human EP4 functional antagonist activity
Assays are conducted in recombinant HEK293 cells stably expressing human EP4
receptor. The cell lines are maintained by culturing in DMEM with high glucose and
pyridoxine hydrochloride (Invitrogen) supplemented with 10% fetal bovine serum (FBS),
1mM sodium pyruvate, lOmM HEPES, 500 g/ml geneticin and 2 mM L-glutamine.
Confluent cultures are grown at 37°C in an atmosphere containing 5% CO2. Cells are
harvested using 2.5% Trypsin-EDTA, suspended in freeze media (FBS with 6% DMSO)
at 10 cells/mL and aliquots are stored in liquid nitrogen. Just before assay, cells are
thawed in DMEM, centrifuged, and resuspended in cAMP buffer.
The inhibition of PGE2-stimulated cAMP production by EP4 antagonists is
measured using HTRF; (Cisbio catalog # 62AM4PEB). An aliquot equivalent to 4000
cells is incubated with 50cAMP assay buffer containing ECso of PGE2 (0.188 nM
PGE2 from Sigma, catalog # P5640-10mg) and antagonists at room temperature for 20
minutes. cAMP assay buffer contains 500 mL HBSS (Hank's Balanced Salt Solution),
0.1 % BSA, 20 mM HEPES and 200 IBMX (Sigma 15879). CJ-042794 (4-{(lS)-l-
[({5-chloro-2-[(4-fluorophenyl)oxy]phenyl} carbonyl)amino]ethyl}benzoic acid) serves
as a positive control. To measure the cAMP levels, cAMP-d2 conjugate and anti cAMPcryptate
conjugate in lysis buffer are incubated with the treated cells at room temperature
for 1 hour. The HTRF signal is detected using an EnVision ® plate reader (Perkin-Elmer)
to calculate the ratio of fluorescence at 665 nm to 620 nm. The raw data are converted to
cAMP amount (pmole/well) using a cAMP standard curve generated for each experiment.
Data are analyzed using a 4-parameter nonlinear logistic equation (ABase Equation 205)
as shown: y = (A+((B-A)/(l+((C/x) D)))) where, y = % specific inhibition, A = Bottom
of the curve, B = Top of the curve, C = Relative IC50 = concentration causing 50%
inhibition based on the range of the data from top to bottom, D = Hill, Slope = slope of
the curve.
Following the procedures essentially as described above, the compound of
Example 1 has an IC50 of 5.6 + 2.8 nM (n=8) measured at human EP4. This demonstrates
that the compound of Example 1 is a potent antagonist of human EP4 in vitro.
In Vitro rat EP4 functional antagonist activity
Rat EP4 cDNA (Genebank Accession# NM_03276) is cloned into pcDNA 3.1
vector and subsequently transfected in HEK293 cells for receptor expression. Rat EP4
stable clone is scaled up and then frozen down as cell bank for future compounds
screening. To test EP4 antagonist compounds in rEP4 cells, thaw the frozen cells and
then resuspend cells in cAMP assay buffer. The cAMP buffer is made by HBSS without
Phenol Red (Hyclone, SH30268) supplemented with 20 mM HEPES (Hyclone,
SH30237), 0.1% BSA (Gibco, 15260) and 125 IBMX (Sigma, 15879). The cells are
plated into 96-well half area flat-bottom polystyrene black plates (Costar 3694).
Compounds are serial diluted with DMSO to give 10-point concentration response curves.
Then diluted compounds are added into cAMP assay buffer which contains PGE2
(Cayman 14010, in a concentration predetermined to produce an ECso) at ratio of
DMSO/buffer at 1/100. The cells are treated with compounds in the presence of PGE2
(ECso concentration) for 30 minutes at room temperature. The cAMP levels generated
from the cells are quantified by a cAMP HTRF assay kit (Cisbio 62AM4PEC). The
plates are read on an EnVision plate reader using HTRF optimized protocol
(PerkinElmer). ICso's are calculated using Graphpad Prism (v. 4) nonlinear regression,
sigmoidal dose response curve fitting.
Following the procedures essentially as described above, the compound of
Example 1 has an IC50 of 12 nM measured at rat EP4. This demonstrates that the
compound of Example 1 is a potent antagonist of rat EP4 in vitro.
In Vitro antagonist activity in human whole blood
The inhibitory effects of PGE2 on LPS-induced TNFa production from
macrophages/monocytes are believed to be mediated by EP4 receptors (See Murase, A.,
et al, Life Sciences, 82:226-232 (2008)). The ability of the compound of Example 1 to
reverse the inhibitory effect of PGE2 on LPS-induced TNFa production in human whole
blood is an indicia of functional activity.
Blood is collected from normal volunteer donors into sodium heparin vacutainer
tubes. Donors have not taken NSAIDs or celecoxib within 48 hours or glucocorticoids
within two weeks of the donation. All tubes/donor are pooled into 50 mL Falcon conical
centrifuge tubes and 98 is distributed into 96-well tissue culture plates (Falcon
3072). Compounds are diluted into DMSO to 100 X final and 1 in triplicate is
added to the blood to give 7 point concentration response curves. The blood is pretreated
with the compounds at 37 °C, in a 5% CO2 humidified atmosphere, for 30 minutes, after
which 1 of a solution of 1 mg/mL of lipopolysaccharide (LPS) (Sigma 0 111:B4)
in 0.2 mg/mL bovine serum albumin (BSA)/PBS +/- 1mM PGE2 (Cayman 14010) is
added to give a final LPS concentration of 10 g/mL +/- 10 nM PGE2. The plates are
incubated for 20-24 hours at 37 °C in a 5% CO2 humidified atmosphere. The plates are
centrifuged at 1800 x g, 10 minutes at 22°C, in an Eppendorf 5810R centrifuge. Plasma
is removed from the cell layer and is transferred to v-bottom polypropylene plates.
TNFa levels in 2 plasma are quantified by a commercially available enzyme
immunoassay (R&D Systems DY210), using Immulon 4 HBX plates (Thermo 3855) and
3,3',5,5' tetramethylbiphenyl-4,4'-diamine substrate (KPL 50-76-03). The plates are read
at A 4 0-A 0 on a plate reader (Molecular Devices Versamax) using SOFTmaxPRO (v.
4.3.1) software. IC50S are calculated using Graphpad Prism (v. 4) nonlinear regression,
sigmoidal dose response curve fitting. Results are expressed as the geometric mean ±
standard deviation; n = number of independent determinations.
Following the procedures essentially as described above, the compound of
Example 1has an IC50 of 123 + 8 1 nM (n=8) measured at human EP4. This
demonstrates that the compound of Example 1 is a potent EP4 antagonist in the human
blood TNFa induction assay.
WE CLAIM:
1. A compound of the formula:
wherein A is:
R1 is C¾, CF3, or F;
R2 is H, CH3, or F;
R3 is CH3, OCH3, OH, F;
R4 is OH or CH2OH; and
is CH or ;
or a pharmaceutically acceptable salt thereof.
A compound or salt according to claim 1wherein A
A compound or salt according to either claim 1 or claim 2 wherein R1 is
CH3.
4. A compound or salt according to any of claims 1to 3 wherein X is N.
5. A compound or salt according to any of claims 1to 4 wherein R2 is H and
R3 is OH.
The compound according to claim 1which is:
or a pharmaceutically acceptable salt thereof.
7. The compound according to claim 1which is:
8. Hydrated compound according to claim 7.
The hydrated compound according to claim 8 characterized by a
substantial peak in the X-ray diffraction spectrum, at diffraction angle 2-
theta, of 9.0°, in combination with two or more peaks at diffraction angle
2-theta selected from the group consisting of 5.8°, 8.5°, 9.8°, 11.6°, 11.8°,
17.5°, and 24.2°.
A method of treating osteoarthritis in a patient, comprising administering
to a patient in need of such treatment an effective amount of a compound,
or pharmaceutically acceptable salt thereof, according to claims 1 to 9.
A method of treating rheumatoid arthritis in a patient, comprising
administering to a patient in need of such treatment an effective amount of
a compound or pharmaceutically acceptable salt thereof, according to
claims 1 to 9.
A method of treating pain associated with osteoarthritis or rheumatoid
arthritis in a patient, comprising administering to a patient in need of such
treatment an effective amount of a compound or a pharmaceutically
acceptable salt thereof, according to claims 1 to 9.
A compound or pharmaceutically acceptable salt thereof according to
claims 1 to 9 for use in therapy.
A compound or pharmaceutically acceptable salt thereof according to
claims 1 to 9 for use in the treatment of osteoarthritis.
A compound or pharmaceutically acceptable salt thereof according to
claims 1 to 9 for use in the treatment of rheumatoid arthritis.
16. A compound or pharmaceutically acceptable salt thereof according to
claims 1 to 9 for use in the treatment of pain associated with osteoarthritis
or rheumatoid arthritis.
17. A pharmaceutical composition, comprising a compound or a
pharmaceutically acceptable salt thereof according to claims 1 to 9 in
combination with one or more pharmaceutically acceptable carriers,
diluents, or excipients.