DIMETHYLBENZOIC ACID COMPOUNDS
The present invention relates to certain novel dimethylbenzoic 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 COX-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 (PGE ) 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. 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 TxA , a selective
EP4 antagonist may not possess the potential cardiovascular side effects seen with
NSAIDs and COX-2 inhibitors.
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 inhibitors of EP4
and 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 I :
Formula I
wherein A is:
is CH orN;
Y is CH orN;
X is CH orN;
R is CH3, CF3, or F;
R2 is H, F, CI, CH3, CF3, CH2OH, CH2CH2OH, CH2OCH3, OCH3, OCF3,
or CN;
R3 is H or F; or
R2 and R3 together are a OCH 0 group attached to vicinal carbon atoms;
R4 is H, CI, or CH2OH;
or a pharmaceutically acceptable salt thereof.
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 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 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 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 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 a pharmaceutically acceptable salt thereof.
Furthermore, the invention provides a compound of Formula I, or a
pharmaceutically acceptable salt thereof for use in therapy. In addition, the invention
provides a compound of Formula I, or a pharmaceutically acceptable salt thereof for use
in the treatment arthritis. In particular, the invention provides a compound of Formula I,
or a pharmaceutically acceptable salt thereof for use in the treatment of osteoarthritis. In
addition, the invention provides a compound of Formula I, or a pharmaceutically
acceptable salt thereof for use in the treatment of rheumatoid arthritis. The invention also
provides a compound of Formula I, or a pharmaceutically acceptable salt thereof for use
in the treatment of pain associated with arthritis. The invention also provides a
compound of Formula I, 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 a pharmaceutically acceptable
salt thereof, for the manufacture of a medicament for the treatment of arthritis. In
addition, the invention provides the use of a compound of Formula I, 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 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 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 a pharmaceutically acceptable salt thereof, with one or more
pharmaceutically acceptable carriers, diluents, or excipients. This invention also
encompasses novel intermediates and processes for the synthesis of the compound of
Formula I, or a pharmaceutically acceptable salt thereof.
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, cat, 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.
As used herein, the phrase "R2 and R3 together are a OCH20 group attached to
vicinal carbon atoms" refers to the following structures, for example, wherein the
corresponding oxygen atoms are attached to the vicinal carbons on the phenyl:
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 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).
The compounds of Formula I 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 to the new compounds of the
invention, and the treatment methods, uses, and pharmaceutical compositions of the
invention.
Compounds of Formula la and Formula lb are preferred:
Formula la is most preferred.
It is understood by one of ordinary skill in the art, that when the carboxylic acid is
in the meta position to each of the two methyl groups on the phenyl ring as in Formula lb
above, then W must be C and not CH.
It is also preferred that A is:
It is further preferred that A is:
It is especially preferred that A is:
It is preferred thatW is CH.
It is preferred that Y is CH.
It is most preferred that when Wis CH, Y is CH.
It is preferred that R is CH3.
It is preferred that R2 is CH2OH, CH2CH2OH, or
It is further preferred that R2 is CH2OH.
It is also preferred that R2 and R3 together are a OCH20 group attached to vicinal
carbon atoms.
It is further preferred that R is H.
It is preferred that when R2 is CH2OH, CH2CH2OH, or OCH3, R3 is H.
It is further preferred that when when R2 is CH2OH, R3 is H.
It is preferred that R4 is CI.
Preferred compounds are:
3-[[6-(1,3-benzodioxol-5-yl)-3 -methyl-pyridine-2-carbonyl]amino] -2,4-dimethylbenzoic
acid;
3-[[6-[3-(hydroxymethyl)phenyl]-3-methyl-pyridine-2-carbonyl]amino] -2,4-
dimethyl-benzoic acid; and
3-[[3-(3-chlorophenyl)naphthalene- 1-carbonyl]amino]-2,4-dimethyl-benzoic acid;
and the pharmaceutically acceptable salts thereof.
As used herein, "kPag" refers to kilopascals gauge pressure; "Boc" refers to a
tert-butoxycarbonyl protecting group; "DMEM" refers to Dulbecco's Modified Eagle's
Medium; "ACN" refers to acetonitrile; "TFA" refers to trifluoro acetic acid; "DIEA"
refers to N,N-diisopropylethylamine; "DMAP" refers to 4-(N,N-dimethylamino)pyridine;
"DMSO" refers to dimethylsulfoxide; "DMF" refers to N N-dimethylformamide; "EtOH"
refers to ethanol; "THF" refers to tetrahydrofuran; "MeOH" refers to methanol;
"EtOAc" refers to ethyl acetate; "Et20 " refers to diethyl ether; "TBME" refers to tertbutyl
methyl ether; "BOP-C1" refers to bis(2-oxo-3-oxazolidinyl)phosphonic chloride;
"mCPBA" refers to 3-chloroperbenzoic acid; "KHMDS" refers to potassium
bis(trimethylsilyl)amide; "h" refers to hour or hours; "PGE2" refers to prostaglandin E2;
"FBS" refers to Fetal Bovine Serum; "IBMX" refers to (3-isobutyl-l-methylxanthine);
"MES" refers to (2-(N-moholino)ethanesulfonic acid; "HEPES" refers to (2-[4-(2-
hydroxyethyl)piperazin-l-yl]ethanesulfonic acid); "S-Phos" refers to 2-
dicyclohexylphosphino-2',6'-dimethoxybiphenyl; "HTRF" refers to homogeneous timeresolved
fluorescence technology; "HEK" refers to human embryonic kidney; "HBSS"
refers to Hank's Balanced Salt Solution ; "RT" refers to room temperature; "EC 0"
refers to the concentration of an agent that produces 80% of the maximal efficacy
possible for that agent; 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, for example, 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 Berge, S.M., et a ,
"Pharmaceutical Salts," Journal of Pharmaceutical Sciences, 66: 1-19, (1977). One
skilled in the art of synthesis will appreciate that the compounds of the invention are
readily converted to and may be isolated as a pharmaceutically acceptable salt, such as a
hydrochloride salt, using techniques and conditions well known to one of ordinary skill in
the art. In addition, one skilled in the art of synthesis will appreciate that the compounds
of Formula I are readily converted to and may be isolated as the corresponding free base
or free acid from the corresponding pharmaceutically acceptable salt.
The compound 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 compound of Formula I, 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
Methyl 3-amino-2,4-dimethyl-benzoate.
Scheme 1, 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 l-iodo-2,4-dimethyl-3-nitro-benzene
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= 8.2 Hz, 1H), 2.37 (s, 3H), 2.23 (s, 3H).
Scheme 1, 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 MeOH (280 mL). The Parr autoclave is sealed, purged, and
pressurized with CO to 551.6 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. EtOAc (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 methyl 2,4-dimethyl-
3-nitro-benzoate as a red oil that 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).
Scheme 1. step C. To a solution of methyl 2,4-dimethyl-3-nitro-benzoate (37 g,
176.9 mmol) in MeOH (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 silica gel flash chromatography, eluting with
20% EtOAc in hexanes to afford methyl 3-amino-2,4-dimethyl-benzoate as a yellow oil
(20.5 g, 65%). Mass spectrum (m/z): 180.1 (M+H)+.
Scheme 2
Preparation 2
Methyl 4-amino-3,5-dimethyl-benzoate.
Scheme 2. step A. To a solution of 3,5-dimethyl-4-nitro-benzoic acid (10.0 g,
0.0512 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), brought to pH 7-8 with saturated NaHC0 3 solution, and extracted with
EtOAc (2 x 120 mL). The organic layers are combined and dried over anhydrous sodium
sulfate. The solvent is removed under reduced pressure to afford methyl 3,5-dimethyl -4-
nitro-benzoate as a light yellow solid (10.71 g, 98.3%). NMR (400 MHz, DMSO): 
7.83 (s, 2H), 3.88 (s, 3H), 2.30 (s, 6H).
Scheme 2. step B. To a solution of methyl 3,5-dimethyl-4-nitrobenzoate (10.0 g,
0.0478 mol) in MeOH (100 mL), iron powder (15.7 g, 0.2869 mol) and 37% HC1 ( 1.72 g,
0.0478 mol) are added at 0 °C. The reaction mixture is heated at 80 °C for 16 hours. The
mixture is cooled to room temperature, filtered through diatomaceous earth, and washed
with MeOH. The filtrate is concentrated to afford the title compound as brown solid (7.8
g, 99%). Mass spectrum (m/z): 180.2 (M+H)+.
Scheme 3
Preparation 3
6-Chloro-3-methyl-pyridine-2-carboxylic acid.
Scheme 3, step A. To a solution of methyl 3-methylpyridine-2-carboxylate (13.0
g, 0.086 mol) in CH2CI2 (130 mL) is added meta-chloroperoxybenzoic acid (89.05 g,
0.258 mol, 50 % w/w) portionwise at 0 °C. The reaction mixture is stirred for 15 minutes
at 0°C and then gradually warmed to ambient temperature. After 16 hours, saturated
NaHC0 3 solution (100 mL) is added. The mixture is stirred for 30 minutes and is
extracted with CH2CI2. The combined organic layers are washed with 0.5M NaOH
aqueous solution (2 50 mL), dried over sodium sulfate, filtered, and concentrated under
reduced pressure to give methyl 3-methyl-1-oxido-pyridin- 1-ium-2-carboxylate as an offwhite
solid (13.0 g, 89.9%). The residue is used in the next step without further
purification. Mass spectrum (m z): 168.2 (M+H)+.
Scheme 3, step B. POCl3 (30.0 mL) is added slowly to methyl 3-methyl-1-oxidopyridin-
l-ium-2-carboxylate (13.0 g, 0.077 mol) at 0 °C over 30 minutes. The reaction
mixture is stirred for 15 minutes at 0 °C and then gradually warmed to ambient
temperature. After 16 hours, the reaction mixture is cooled to 0 °C and excess POCl3 is
removed under reduced pressure. The crude residue is then quenched by addition of ice
and diluted with water and CH2CI2 (50 mL). The organic layer is washed sequentially
with saturated NaHC0 3 solution, water, and brine; dried over sodium sulfate; filtered; and
concentrated under reduced pressure. The crude product is purified by silica gel flash
chromatography, eluting 5-10% EtOAc in hexanes to afford methyl 6-chloro-3-methylpyridine-
2-carboxylate as a white solid (3.00 g, 20.9%). Mass spectrum (m/z): 186.2
(M+H)+.
Scheme 3. step C. A solution of aqueous 2N NaOH (5 ml) is added to a stirred
solution of methyl 6-chloro-3-methyl-pyridine-2-carboxylate (0.500 g, 2.702 mmol) in
THF (10 mL) at 0 °C. The mixture is heated at 50°C for 2 hours. The reaction mixture is
acidified with aqueous citric acid solution and extracted with EtOAc (2 x 10 ml). The
combined organic layers are dried over sodium sulfate, filtered, and concentrated under
reduced pressure to give the title compound as an off-white solid (0.45 g, 97%). The
residue is used in next step without further purification. Mass spectrum (m/z): 172.0
(M+H)+.
Scheme 4
Preparation 4
Methyl 3-[(5-bromo-2-methyl-benzoyl)amino] -2,4-dimethyl-benzoate.
Scheme 4. step A. To a solution of 5-bromo-2-methyl-benzoic acid (1.0 g, 4.7
mmol) in THF (5.0 ml), CH2C 12 (5.0 ml) and DMF (50.0 ΐ , 646.6 ΐ ) is added
dropwise oxalyl chloride (2.6 ml, 5.1 mmoles) at 0 °C. The reaction mixture is allowed to
warm gradually to ambient temperature. After 2 hours, the solvent is removed under
reduced pressure. The residue is diluted with CH2C 12 (5 ml), and the mixture is cooled to
0 °C. Methyl 3-amino-2,4-dimethylbenzoate (957.9 mg, 4.70 mmol) is added, followed
by N,N-dimethylpyridin-4-amine (28.4 mg, 0.232 mmoles) and pyridine (1.1 ml, 14.0
mmoles). The cooling bath is removed, and the clear solution is allowed to warm to
ambient temperature. After 2 hours, the solution is concentrated. The residue is diluted
with EtOAc and washed sequentially with IN HC1, saturated solution of sodium
bicarbonate, and brine. The organic layer is dried over anhydrous Na2S0 4, filtered, and
concentrated under reduced pressure. The residue is triturated with 30% EtOAc in
Hexanes and the solids are filtered to provide the title compound (680.0 mg; 38.9%).
Mass spectrum (m/z): 376.0 (M+H)+.
The following compound is prepared essentially by the method above (Scheme 4,
step A), using the appropriate carboxylic acid and amine:
Preparation 6
Methyl 3-[[5-[3-(hydroxymethyl)phenyl]-2-methyl-benzoyl]amino]-2,4-dimethylbenzoate.
Scheme 4. step B. To a solution of methyl 3-[(5-bromo-2-methylbenzoyl)
amino]-2,4-dimethyl-benzoate (0.18 g, 0.478 mmol) in 1,4-dioxane (3.0 ml) and
H20 (0.3 ml) is added (3-(hydroxymethyl)phenyl)boronic acid (87.2 mg, 0.574 mmol)
followed by K2C0 3 (132.2 mg, 0.956 mmol) and PdCl2(dppf>CH 2Cl2 (19.5 mg, 0.024
mmol). The reaction mixture is purged with argon for 5 minutes and then heated at 110
°C. After 2 hours, the reaction is cooled to room temperature, diluted with water, and
extracted with EtOAc. The combined organic layers are dried over sodium sulfate,
filtered, and concentrated. The residue is purified by silica gel flash chromatography
using 40% EtOAc in hexane. The product is triturated with TBME and filtered to afford
the title compound (0.105 g, 54.4%). Mass spectrum (m/z): 404.2 (M+H)+.
The following compounds are prepared essentially by the method above, using the
appropriate carboxylic ester and boronic acid:
MS
Prep. Chemical Name Structure
(m/z)
Methyl 3-[[5-[3-(2-
hydroxyethyl)phenyl] -2- 418.2
7
methyl-benzoyl] amino] -2,4- (M+H)+
dimethyl-benzoate
Methyl 4-[[5-[3-
(hydroxymethyl)phenyl] -2- 404.2
8
methyl-benzoyl] amino] -3,5- (M+H)+
dimethyl-benzoate
0
Methyl 4-[[5-[3-(2-
hydroxyethyl)phenyl] -2- 418.2
9
methyl-benzoyl] amino] -3,5- (M+H)+
dimethyl-benzoate
Methyl 3-[[5-[3-
(hydroxymethyl)phenyl] -2- 458.2
10 (trifluoromethyl)benzoyl] a (M+H)+
mino]-2,4-dimethylbenzoate
Methyl 3-[[5-phenyl-2-
(trifluoromethyl)benzoyl] a 428.2
1 1 mino]-2,4-dimethyl- (M+H)+
benzoate
Methyl 4-[[5-[3-
(hydroxymethyl)phenyl] -2-
456.2
12 (trifluoromethyl)benzoyl] a (M+H)+
mino]-3,5-dimethylbenzoate
Example 1
(Hydroxymethyl)phenyl]-2-methyl-benzoyl]amino]-2,4-dimethyl-benzoic
Scheme 4. Step C. Aqueous IN NaOH (0.5 ml) is added to a stirred solution of
methyl 3-[[5-[3-(hydroxymethyl)phenyl]-2-methyl-benzoyl]amino]-2,4-dimethylbenzoate
(0. 102 g, 0.252 mmol) in THF (2.0 ml ) and MeOH ( 1.0 ml ). After heating at
50 °C for 12 hours, the reaction mixture is acidified to pH 1-2 with aqueous IN HCl
solution. The resulting precipitate is filtered, washed with water, and dried at 40°C in a
vacuum oven for 1 hour to give the title compound as a white solid (95.0 mg, 96.5%).
Mass spectrum (m/z): 390.2 (M+H)+.
The following compounds are prepared essentially by the method described in
Example 1 from the corresponding carboxylic esters described in the preparations:
Preparation 17
Methyl 3-[[5-bromo-2-(trifluoromethyl)benzoyl] amino] -2,4-dimethyl-benzoate.
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-2,4-dimethylbenzoate
(0.44 g, 2.47 mmol) and triethylamine (1.0 ml, 7.06 mmol). After stirringlO minutes, 1-
propanephosphonic acid cyclic anhydride (50% solution in EtOAc, 5.6 ml, 8.83 mmol) is
added via syringe. After 14 hours at ambient temperature, the reaction mixture is diluted
with CH2CI2 and washed with water then brine. The organic layer is dried over
anhydrous Na2S0 4, filtered, and concentrated under reduced pressure. The residue is
purified by silica gel flash chromatography) using 20% EtOAc in hexanes to give the title
compound as white solid (0.6 g, 39 %). Mass spectrum (m/z): 430.0 (M+H)+.
The following compound is prepared essentially by the method described in
preparation 17 above, using the appropriate carboxylic acid and amine:
Example 12
4-[ [5-[3-(Hydroxymethyl)phenyl]-2-(trifluoromethyl)benzoyl]amino] -3,5-dimethylbenzoic
acid.
Scheme 4, Step C. Aqueous 2N NaOH (2.0 ml) is added to a stirred solution of
methyl 4-[ [5-[3-(hydroxymethyl)phenyl]-2-(trifluoromethyl)benzoyl]amino] -3,5 -
dimethyl-benzoate (0.09 g, 0.197 mmol) in THF (8.0 ml ) and MeOH (2.0 ml ). After
stirring at room temperature for 12 hours, the reaction mixture is concentrated and
purified by preparative HPLC using 0.1% TFA in water/acetonitrile (10-90%) to give the
title compound (25.0 mg, 29.7%). Mass spectrum (m/z): 444.2 (M+H)+.
Preparation 19
Methyl 3-[(5-bromo-2-fluoro-benzoyl)amino] -2,4-dimethyl-benzoate.
To a solution of 5-bromo-2-fluoro-benzoic acid (0.5 g, 2.28 mol) in CH2CI2 (5.0
ml) at room temperature are added methyl 3-amino-2,4-dimethylbenzoate (0.38 g, 2.17
mol) and DIEA (1.17 g , 9.13 mol). After stirring 10 minutes, 1-propanephosphonic acid
cyclic anhydride (50% solution in EtOAc, 2.0 ml, 3.42 mol) is added via syringe. After
16 hours at ambient temperature, the reaction mixture is diluted with water and extracted
with CH2CI2. The combined organic layers are dried over sodium sulfate, filtered, and
concentrated under reduced pressure. The residue is purified by silica gel flash
chromatography using 22% EtOAc to give the title compound as off white solid (0.80 g,
93%). Mass spectrum (m/z): 380.0 (M+H)+.
The following compound is prepared essentially by the method described above in
preparation 19, using the appropriate carboxylic acid and amine:
Example 13
3-[[2-Fluoro-5- [3-(hydroxymethyl)phenyl]benzoyl]amino] -2,4-dimethyl-benzoic acid.
Aqueous 4N NaOH (5.0 ml) is added to a stirred solution of methyl 3-[[2-fluoro-
5-[3-(hydroxymethyl)phenyl]benzoyl]amino]-2,4-dimethyl-benzoate (0.30 g, 0.737
mmol) in THF (5.0 ml ) and t-butanol (5.0 ml ). After heating at 60 °C for 16 hours, the
reaction mixture is acidified to pH 1-2 with aqueous IN HCl solution and extracted with
CH2CI2. The combined organic layers are dried over sodium sulfate, filtered, and
concentrated under reduced pressure to give the title compound as off-white solid (0.19 g,
65.5%). Mass spectrum (m/z): 392.1 (M-H)+.
The following compounds are prepared essentially by the method of Example 13
above, using the appropriate carboxylic ester:
Scheme 5
Preparation 23
Methyl 3-methyl-6-phenyl-pyridine-2-carboxylate.
Scheme 5. Step A. To a solution of methyl 6-chloro-3-methyl-pyridine-2-
carboxylate (1.0 g, 5.39 mmol), phenylboronic acid (0.79 g, 6.47 mmol) in 1,4-dioxane
(17 mL) and water (3.0 ml) is added K2CO3 ( 1.64 g, 11.8 mmol). The reaction mixture is
purged with argon for 15 min and PdCl 2(dppf)*CH2Cl2 (131.9 mg, 0.162 mmol) is added.
The mixture is purged again with argon for 5 min. After heating at 110 °C for 2 h, the
reaction mixture is cooled to room temperature, diluted with water, and extracted with
EtOAc. The combined organic layers are dried over anhydrous magnesium sulfate,
filtered, and concentrated under reduced pressure. The crude residue is purified by silica
gel flash column chromatography using 0-20% EtOAc in hexanes t to afford the title
compound as a clear oil (985.0 mg, 80.5 %). Mass spectrum ( /z) : 228.0 (M-H)+.
Preparation 24
3-Methyl-6-phenyl-pyridine-2-carboxylic
Scheme 5. Step B. Aqueous IN NaOH (8.7 ml) is added to a stirred solution of
methyl 6-chloro-3-methyl-pyridine-2-carboxylate (985 mg, 4.33 mmol) in THF (5.0 ml)
and MeOH (5.0 ml). The mixture is stirred at ambient temperature for 3 hours,
concentrated under reduced pressure, and acidified with IN HC1 to pH 3. The resulting
precipitate is isolated by filtration, washed with water, and dried in a vacuum oven at 40
°C to give the title compound (685.0 mg, 74.1%). Mass spectrum (m/z): 214.0 (M-H)+.
Preparation 25
Methyl 2,4-dimethyl-3-[(3-methyl-6-phenyl-pyridine-2-carbonyl)amino]benzoate.
Scheme 5, Step C. To a solution of 3-methyl-6-phenyl-pyridine-2-carboxylic acid
(342.0 mg, 1.60 mmol) in CH2C 12 (8 ml) at room temperature are added methyl 3-amino-
2,4-dimethyl-benzoate (287.4 mg, 1.60 mmol) and DIEA (699.3 mg, 4.01 mmol). After
stirring for 10 minutes, 1-propanephosphonic acid cyclic anhydride (50% solution in
EtOAc, 1.22 g, 1.92 mmol) is added via syringe. After 15 hours at 50 °C, the reaction
mixture is triturated with CH2C 12/ TBME and resulting precipitate is isolated by filtration
to give the title compound as a white powder (0.41 g, 68%). Mass spectrum (m/z): 375.2
(M+H)+.
Example 15
2,4-Dimethyl-3-[(3-methyl-6-phenyl-pyridine-2-carbonyl)amino]benzoic acid.
Scheme 5. Step D. To a solution of methyl 2,4-dimethyl-3-[(3-methyl-6-phenylpyridine-
2-carbonyl)amino]benzoate (0.28 g, 0.75 mmol) in THF (3.0 ml), MeOH (3.0
ml), and H20 (1.0 ml) is added LiOH H20 (0.157 g, 3.75 mmol). After heating at 50°C
for 1 hour, the reaction mixture is acidified to pH ~6. The aqueous layer is saturated with
solid NaCl and extracted with EtOAc (4 x 20 mL). The combined organic layers are
dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue
is triturated with diethyl ether to afford the title compound as a white solid (0.231 g,
85.7%). Mass spectrum (m/z): 361.2 (M+H)+.
Alternative synthesis of 2,4-dimethyl-3-[(3-methyl-6-phenyl-pyridine-2-
carbonyl)amino]benzoic acid (Example 15).
Scheme 6
Scheme 6, Step A. To a round bottom flask containing 3-methylpyridine-2-
carbonitrile (16.8 g, 139 mmoles), methylene chloride (70 mL), and methyltrioxorhenium
(VII) (1.42 g, 5.57 mmol), hydrogen peroxide (24 mL, 279 mmoles) is added slowly. The
mixture was stirred overnight and for an additional 24 hours. Stirring is stopped and the
layers are separated. The organic layer is dried over anhydrous magnesium sulfate,
filtered, and concentrated. The residue is triturated with TBME (150 mL), and the solid
was filtered and dried under vacuum to give 3-methyl- 1-oxido-pyridin- l-ium-2-
carbonitrile as a yellow solid (16.2 g, 86.7%). Mass spectrum (m/z): 135.0 (M+H)+.
Scheme 6. Step B. To a round bottom flask is added 3-methyl-l-oxo-pyridine-2-
carbonitrile (16.2 g, 120.77 mmoles), toluene (8 mL), and phosphoryl chloride (16.83
mL; 181.2 mmoles). The mixture is stirred at 90 °C for 90 minutes, cooled to RT, and
added dropwise to aqueous 2M KH2PO4 (483 mL; 966 mmoles). The mixture is stirred
30 minutes, and the layers are separated. The organic layer is dried over MgS04, filtered,
and concentrated to 6-chloro-3-methyl-pyridine-2-carbonitril: Mass spectrum (m/z):
153.0 (M+H)+. Phenylboronic acid (17.94 g, 144.9 mmoles), toluene (130 mL), sodium
carbonate (190.2 g, 362.31 mmoles), bis(triphenylphosphine)palladium(II) chloride (856
mg; 1.21 mmoles) are added to this crude material. The mixture is stirred at 80°C for 1
hour and cooled to RT. The layers are separated, and the organic layer is dried over
MgS04, filtered, and concentrated. The material was purified by silica gel
chromatography (400 g ISCO® cartridge) using methylene chloride in hexanes from 20
to 100% to afford 3-methyl-6-phenyl-pyridine-2-carbonitrile (6.7 g, 28.6%). Mass
spectrum (m/z): 195.1 (M+H)+.
Scheme 6. Step C. To a pale yellow suspension of 3-methyl-6-phenyl-pyridine-2-
carbonitrile (6.10 g, 31.41 mmoles) in ethanol (30 mL) is added aqueous 18N sodium
hydroxide (10.7 mL, 188.43 mmoles) and water (10.98 mL). The yellow suspension is
heated at 100 °C for 18 h and cooled to 22 °C. The mixture is diluted with water (100
mL) and neutralized with aqueous 12M HC1 (18.6 mL, 219.8 mmoles) to give a pH ~ 1.
The solid is filtered, washed with water, and dried under vacuum at 45°C to give 3-
methyl-6-phenyl-pyridine-2-carboxylic acid as a white solid (6.50 g, 94.2%). Mass
spectrum (m/z): 214.1 (M+H)+.
Scheme 6. Step D. To 3-methyl-6-phenyl-pyridine-2-carboxylic acid (5.52 g,
25.89 mmoles) is added thionyl chloride (18.9 mL, 258.9 mmoles) at 22 °C. The yellow
solution is stirred at 22°C for 8 h and concentrated to give 3-methyl-6-phenyl-pyridine-2-
carbonyl chloride as a light brown solid (7.0 g, 25 mmoles). This material is diluted with
THF (70 mL)and treated at 22°C under nitrogen with a solution of methyl 3-amino-2,4-
dimethyl-benzoate (4.54 g, 25.32 mmoles) in THF (35 mL). Pyridine (6.1 mL, 75.97
mmoles) is added slowly, and the reaction mixture is stirred for 45 min. The suspension
is filtered, the solid is rinsed with EtOAc, and the filtrate is concentrated to a brown oil.
The oil is treated with water (50 mL) and placed in an ultrasonic bath for 10 min. The
suspension is filtered, and the solid is washed with water and dried to afford methyl 2,4-
dimethyl-3-[(3-methyl-6-phenyl-pyridine-2-carbonyl)amino]benzoate as a light brown
solid (9.81 g, 95.8%). Mass spectrum (m/z): 375.1 (M+H)+.
Scheme 6. Step E. To a solution of methyl 2,4-dimethyl-3-[(3-methyl-6-phenylpyridine-
2-carbonyl)amino]benzoate (9.00 g, 23.80 mmoles) in THF (90 mL) and MeOH
(36 mL) is added aqueous 1M NaOH solution (71.4 mL, 71.4 mmoles). The mixture is
heated at 50 °C for 4 hours, cooled to 22 °C, and concentrated to remove organic solvents.
The aqueous residue is diluted with water (50 mL), washed with EtOAc (20 mL), and
acidifed with 10% aqueous HC1 solution (13.5 mL, 40.4 mmoles) to a final pH ~ 3. The
solid is filtered, washed with water, recrystallized from acetone/water, and dried in the
vacuum oven at 45 °C for 18 h to afford the title compound as an off-white solid (6.20 g,
72.3%). Mass spectrum (m/z): 361.1 (M+H)+.
Scheme 7
Preparation 26
Methyl 3-[[6-(4-chlorophenyl)-3-methyl-pyridine-2-carbonyl]amino]-2,4-dimethylbenzoate.
Scheme 7, Step A. To a solution of 6-chloro-3-methyl-pyridine-2-carboxylic acid
(0.8 g, 4.6 mmol) in CH2CI2 (4 mL) at room temperature are added methyl 3-amino-2,4-
dimethyl-benzoate (0.84 g, 4.6 mmol) and TEA (945.0 mg, 9.3 mmol). After stirring 10
minutes, 1-propanephosphonic acid cyclic anhydride (50% solution in EtOAc, 2.97 g, 9.3
mmol) is added via syringe. After 2 hours at 35 °C, the reaction mixture is diluted with
saturated NaHC0 3 solution and extracted with CH2C 12. The combined organic layers are
dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue
is purified by silica gel flash chromatography, eluting with 10% EtOAc in hexanes to give
methyl 3-[(6-chloro-3-methyl-pyridine-2-carbonyl)amino]-2,4-dimethyl-benzoate as a
colorless oil (1.1 g, 72%). Mass spectrum (m/z): 333.3 (M+H)+.
Scheme 7. Step B. To a solution of methyl 3-[(6-chloro-3-methyl-pyridine-2-
carbonyl)amino]-2,4-dimethyl-benzoate (230.0 mg, 0.692 mmol) in 1,4-dioxane (5.0 ml)
and H20 (1.0 ml) is added (4-chlorophenyl)boronic acid (120.0 mg, 0.761 mmol)
followed by Na2C0 3 (220.0 mg, 2.08 mmol) and PdCl2(dppf>CH 2Cl2 (60.0 mg, 0.069
mmol). The reaction mixture is purged with argon for 5 minutes and then heated at 100
°C. After 14 hours, the reaction is cooled to RT, diluted with water, and extracted with
EtOAc. The combined organic layers are dried over sodium sulfate, filtered, and
concentrated. The residue is purified by silica gel flash chromatography using 50%
EtOAc in hexane to afford the title compound (0.17 g, 60.7%).
The following compounds are prepared essentially by the methods described
above in preparation 26, using the appropriate carboxylic ester and boronic acid:
Prep. Chemical Name Structure MS (m/z)
Methyl 3-[[6-(3-
chlorophenyl)-3 -methyl-
27 409.2
pyridine-2-
(M+H)+
carbonyl]amino]-2,4-
dimethyl-benzoate
methyl 3-[[6-(l, 3-
benzodioxol-5 -yl)-3 -
28 419.9
methyl-pyridine-2-
(M+H)+
carbonyl]amino]-2,4-
dimethyl-benzoate
Methyl 3-[[6-(3-
cyanophenyl) -3-methyl-
29 400.2
pyridine-2-
(M+H)+
carbonyl]amino]-2,4-
dimethyl-benzoate
Methyl 3-[[6-[3-
(hydroxymethyl)phenyl]-3-
30 405.4
methyl-pyridine-2-
carbonyl]amino]-2,4- 1 H (M+H)+
OH
dimethyl-benzoate

Prep. Chemical Name Structure MS (m/z)
Methyl [ - -
benzodio ol- -y - -
419.9
38 methyl-pyridine-2-
(M+H)+
carbonyl]amino]-3,5-
dimethyl-benzoate
Example 16
3-[[6-(4-Chlorophenyl)-3-methyl-pyridine-2-carbonyl]amino]-2,4-dimethyl-benzoic acid.
Scheme 7. Step C. Aqueous IN NaOH (2.0 ml) is added to a stirred solution of
methyl 3-[[6-(4-chlorophenyl)-3-methyl-pyridine-2-carbonyl]amino]-2,4-dimethylbenzoate
(0.17 g, 0.41 mmol) in THF (8.0 ml ) and MeOH ( 2.0 ml ). After heating at 40
°C for 12 hours, the reaction mixture is acidified to pH 1-2 with aqueous IN HC1
solution. The resulting precipitate is filtered, washed with water, and purified by
preparative HPLC using 0.1%TFA in water/acetonitrile to give the title compound as a
white solid (155 mg, 95.9%). Mass spectrum (m/z): 395.1 (M+H)+.
Example 17
3-[[6-(3-Chlorophenyl)-3-methyl-pyridine-2-carbonyl]amino]-2,4-dimethyl-benzoic acid.
Scheme 7. Step C. A solution of aqueous 2N NaOH (2.0 ml) is added to a stirred
solution of methyl 3-[[6-(l ,3-benzodioxol-5-yl)-3-methyl-pyridine-2-carbonyl]amino]-
2,4-dimethyl-benzoate (0.17 g, 0.416 mmol) in THF (8.0 ml) and MeOH (2.0 ml) at 0 °C.
After heating at 60°C for 2 hours, the reaction mixture is acidified to pH 1-2 with aqueous
IN HC1 solution and extracted with CH2C 12. The combined organic layers are dried over
sodium sulfate, filtered, and concentrated under reduced pressure to give the title
compound as a white solid (0.104 g, 64%). Mass spectrum (m/z): 395.2 (M+H)+.
The following compounds are prepared essentially by the method described above
in Example 17, using the appropriate carboxylic ester:
Example 22
3-[[6-[3-(Hydroxymethyl)phenyl]-3-methyl-pyridine-2-carbonyl]amino]-2,4-dimethylbenzoic
acid.
Scheme 7. Step C. A solution of aqueous 2N NaOH (2 ml) is added to a stirred
solution of methyl 3-[[6-[3-(hydroxymethyl)phenyl] -3-methyl-pyridine-2-
carbonyl]amino]-2,4-dimethyl-benzoate (0.13 g, 0.32 mmol) in THF (10 mL) at 0°C.
After heating at 50 °C for 2 hours the reaction mixture is acidified to pH 1-2 with aqueous
IN HCl solution and concentrated. The crude product is purified by preparative HPLC
using 10-90% gradient of 5mM NH4OAC in water/ CH3CN to give the title compound as
a white solid (0.122 g, 97%). Mass spectrum (m/z): 391.12 (M+H)+.
The following compounds are prepared essentially by the method described above
in Example 22, using the appropriate carboxylic ester:
Scheme 8
Alternative synthesis of 3-[[6-[3-(hydroxymethyl)phenyl]-3-methyl-pyridine-2-
carbonyllaminol-2.4-dimethyl-benzoic acid (Example 22).
Scheme 8. Step A. Concentrated sulfuric acid (1.216 L, 21.9 mol) is added over
45 minutes to a solution of 3-methylpyridine-2-carboxylic acid (1000 g, 7.3 mol) in
ethanol (10 L). The reaction is heated at reflux for 19 hours, cooled, and treated with
solid sodium bicarbonate (~4 kg) to attain pH~8. The mixture is diluted with EtOAc (10
L) and filtered through filter cel. The solvent is removed under reduced pressure to give
ethyl 3-methylpyridine-2-carboxylate (939 g, 78%). MS (m/z) 166 [M+H]+.
Scheme 8. Step B. 30% Hydrogen peroxide (1.52 L, 13.7 mol) is added to a
solution of ethyl 3-methylpyridine-2-carboxylate (452 g, 2.74 mol) in HOAc (4.5 L). The
mixture is heated at 60 °C for 4.5 hours, then cooled to room temperature overnight. The
reaction is poured into sodium sulfite/ice water (10 L) and extracted with methylene
chloride (2 x 4 L). The combined organic layers are dried over sodium sulfate and
concentrated under reduced pressure to give ethyl 3-methyl-l-oxido-pyridin-l-ium-2-
carboxylate as a pale yellow oil (502 g, quantitative). MS (m/z) 182 [M+H]+.
Scheme 8. Step C. Phosphorous oxychloride (1.16 L, 12.4 mol) is added dropwise
at 0 °C over ~ 1 hour to a solution of DMF (2.45 L, 24.8 mol) and methylene chloride (4.5
L) The reaction is stirred for 30 minutes at 0 °C, then ethyl 3-methyl- 1-oxido-pyridin-lium-
2-carboxylate (450 g, 2.48 mol) is added. The reaction is allowed to slowly warm
overnight to room temperature, and the reaction mixture is poured into ice water (10 L).
The pH is adjusted with 10% sodium carbonate to pH~8, and the mixture is stirred for 1
hour. The layers are separated, and the aqueous layer is extracted with methylene
chloride (2 x 2 L). The combined organic layers are dried over sodium sulfate, filtered,
and concentrated under reduced pressure to give ethyl 6-chloro-3-methyl-pyridine-2-
carboxylate as a beige semi solid (425 g, 86%). MS (m/z) 200 [M+H]+.
Scheme 8, Step D. Ethyl 6-chloro-3-methyl-pyridine-2-carboxylate (750 g, 4.05
mol) is added in portions over -20 minutes to a solution of KOH (272 g, 4.84 mol) in
isopropanol (14 L). The mixture is stirred for 2 hours, filtered, and washed sequentially
with isopropanol (500 mL) and heptanes (2 L). The solids are dried in vacuum at 50 °C
for 48 hours to give potassium 6-chloro-3-methyl-pyridine-2-carboxylate as a white solid
(767 g, 97%). MS (m/z) 210 [M+H]+.
Scheme 8. Step E. Solid BOP-C1 (662.9 g, 2.604 mol) is added to a mixture of
potassium 6-chloro-3-methyl-pyridine-2-carboxylate (300 g, 1.431 mol) in DMF (4.75 L).
After a mild exotherm of ~5°C, the mixture is stirred for 1 hour. Methyl 3-amino-2,4-
dimethyl-benzoate hydrochloride (277.7 g, 1.288 mol) and diisopropylethylamine (950
mL, 5.438 mol) are added sequentially to the reaction. The mixture is stirred overnight
and poured into water (10 L) and ice (4 Kg). After stirring for 1 hour, the mixture is
filtered, and the solid washed with water and dried in vacuum at 50 °C for 12 hours. The
solids are slurried in heptanes (12 L) for 1 hour, filtered, and dried in vacuum at 50 °C to
give methyl 3-[(6-chloro-3-methyl-pyridine-2-carbonyl)amino]-2,4-dimethyl-benzoate
(430 g, 90%). MS (m/z) 333 [M+H]+.
Scheme 8. Step F. 2 M Na2C0 3 (1.13 L, 2.253 mol) is added to a solution of
methyl 3-[(6-chloro-3-methyl-pyridine-2-carbonyl)amino]-2,4-dimethyl-benzoate (250 g,
0.751 mol) and 3-(hydroxymethyl)phenyl boronic acid (137 g, 0.902 mol) in dioxane (2.5
L). The mixture is warmed to 40 °C and degassed with a stream of nitrogen for 1 hour.
Palladium bistriphenylphosphine dichloride (26.4 g, 0.0376 mol) is added, and the
reaction is degassed with a stream of nitrogen for an additional 20 minutes. The reaction
is heated at reflux for 1.5 hours, cooled to room temperature, filtered through
diatomaceous earth, and concentrated under reduced pressure to remove the organic
solvent. The aqueous mixture is extracted with EtOAc (2 x 2 L). The combined organic
layers are extracted with saturated brine, dried over sodium sulfate, filtered, and
concentrated. This residue is dissolved in toluene (1.5L) and loaded onto silica gel (2 kg).
The column is eluted with a gradient of 0 to 50% EtOAc in heptanes. The product
fractions are concentrated to a beige solid (-300 g). The solid is dissolved in 2-
methyltetrahydrofuran (2.5 L), treated with mercaptopropyl silica gel, heated at 50 °C
with stirring for 2 hours, and cooled overnight to room temperature. The mixture is
filtered, and the silica gel is rinsed with EtOAc (3 L). The solvent is removed under
reduced pressure to give white solid (297 g) that is diluted with isopropanol (1.5 L) and
heated at reflux until a clear solution is obtained. The solution is cooled overnight, and
the precipitate is isolated by filtration and dried in a vacuum oven at 50 °C to give methyl
3-[[6- [3-(hydroxymethyl)phenyl] -3-methyl-pyridine-2-carbonyl]amino] -2,4-dimethylbenzoate
(256 g, 84%). MS (m/z) 405[M+H]+.
Scheme 8, Step G. Potassium hydroxide (86 g, 1.537 mol, 3 equiv) is added to a
solution of methyl 3-[[6-[3-(hydroxymethyl)phenyl]-3-methyl-pyridine-2-
carbonyl]amino]-2,4-dimethyl-benzoate (207 g, 0.512 mol) in MeOH (2 L). The reaction
is heated at reflux for 16 hours, cooled to room temperature, and concentrated to dryness
under reduced pressure. The residue is partitioned between water (2 L) and TBME. The
aqueous layer is adjusted to pH~2 with 10% HCl, and the precipitate is filtered, washed
with water ( 1 L) and heptanes ( 1 L) and dried in vacuum at 50 °C (197 g, 98%). The
solids are combined with material from another run (226 g total) and refluxed in ethanol
(2 L) for 2 hours. After cooling to room temperature, the solids are filtered and dried in a
vacuum at 50 °C for 16 hours to give the title compound (21 1 g). MS (m/z) 391[M+H]+.
Example 25
4-[[6-(l,3-Benzodioxol-5-yl)-3-methyl-pyridine-2-carbonyl]amino]-3,5-dimethyl-benzoic
acid.
Scheme 4. Step C. To a solution of aqueous LiOH (50 mg, 1.18 mmol) in water
(1.0 ml) is added methyl 4-[[6-(l,3-benzodioxol-5-yl)-3-methyl-pyridine-2-
carbonyl]amino]-3,5-dimethyl-benzoate (240.0 mg, 0.573 mmol) in THF (2.0 ml) and
MeOH (2.0 ml). After 3 hours at ambient temperature, the reaction mixture is acidified to
pH 1-2 with aqueous IN HCl solution and extracted with EtOAc. The combined organic
layers are dried over sodium sulfate, filtered, and concentrated under reduced pressure.
The residue is crystallized from diethyl ether to give the title compound (0.2 g, 86.4%).
MS (m/z): 405.2 (M+l) +.
Scheme 9
Preparation 39
Ethyl 4-amino-3 ,5-dimethyl-pyridine-2-carboxylate.
Scheme 9. Step A. To a solution of 3,5-dimethylpyridine (20.0 g, 186.65 mmol)
in acetic acid (8 ml) at room temperature is added hydrogen peroxide (60.0 ml, 1.96 mol).
After heating at 70°C for 24 hours, the reaction mixture is diluted with water and
extracted with 5%MeOH:CH2Cl2 . The organic layers are combined and dried over
anhydrous Na2S0 4, filtered, and concentrated under reduced pressure. The residue is
purified by silica gel chromatography using 10%MeOH in CH C 12 to give 3,5-dimethyl-
1-oxido-pyridin-l-ium as off white powder (12.2 g, 53.1%). Mass spectrum (m/z): 124.1
(M+l) +.
Scheme 9, Step B. To nitric acid (20.0 ml, 463.08 mmol) is slowly added sulfuric
acid (60 ml, 1.13 mol) at 0°C, followed by 3,5-dimethyl-l-oxido-pyridin-l-ium (8.0 g,
64.96 mmol). The reaction mixture is heated to 70°C for 6 hours, diluted with water, and
extracted with 5%MeOH:CH Cl2 . The organic layers are combined and dried over
anhydrous Na2S0 4, filtered, and concentrated under reduced pressure. The residue is
purified by silica gel chromatography using 10%MeOH in CH2Cl2 to give 3,5-dimethyl-
4-nitro-l-oxido-pyridin-l-ium (3.8 g, 34.8 %). Mass spectrum (m/z): 169.09 (M+l) +.
Scheme 9. Step C. To a solution of 3,5-dimethyl-4-nitro-l-oxido-pyridin-l-ium
(3.5 g, 20.69 mmol) and zinc cyanide (4.86 g, 41.38 mmol) in acetonitrile (30 ml) is
added dropwise under argon ,-dimethylcarbamoyl chloride (2.86 ml, 31.04 mmol).
After heating at 100°C for 16 hours, the reaction mixture is diluted with ice-water and
extracted with EtOAc. The organic layers are combined and dried over anhydrous
Na2S0 4, filtered, and concentrated under reduced pressure. The residue is purified by
silica gel chromatography using 20%EtOAc/ in Hexanes to give two compounds: 3,5-
dimethyl-4-nitro-pyridine-2-carbonitrile (0.47 g, 12.8 %). 1H NMR (300.16 MHz,
CDC13): 8.58 (s, 1H), 2.52 (s, 3H), 2.39 (s, 3H) and 4-chloro-3,5-dimethyl-pyridine-2-
carbonitrile (1.05 g, 30.5%). Mass spectrum (m/z): 167.07 (M+l) +.
Scheme 9. Step D. To a solution of 3,5-dimethyl-4-nitro-pyridine-2-carbonitrile
(0.45 g, 2.54 mmol) in ethanol (2 ml) is added dropwise concentrated sulfuric acid (2 ml,
37.52 mmol) at 0 °C. After heating at 90°C for 18 hours, the reaction mixture is poured
onto ice and extracted with EtOAc. The organic layers are combined and dried over
anhydrous Na2S0 4, filtered, and concentrated under reduced pressure. The residue is
purified by silica gel flash chromatography using 40% EtOAc in Hexanes to give ethyl
3,5-dimethyl-4-nitro-pyridine-2-carboxylate (0.35 g, 61.5%). Mass spectrum (m/z): 225.1
(M+l) +.
Scheme 9. Step E. To a solution of ethyl 3,5-dimethyl-4-nitro-pyridine-2-
carboxylate (0.30 g, 1.34 mmol), in ethanol (5 ml) is added 10% Pd on activated carbon
(0.06 g), and the mixture is purged with argon for 10 min, followed by the addition of
hydrogen gas. After 5 hours, the reaction mixture is filtered through diatomaceous earth
and washed with ethanol. The filtrate is concentrated under reduced pressure to give
ethyl 4-amino-3,5-dimethyl-pyridine-2-carboxylate (0.24 g, 92.3%). Mass spectrum
(m/z): 195.17 (M+l) +.
Scheme 9, Step F. To a solution of 4-chloro-3,5-dimethyl-pyridine-2-carbonitrile
(0.6 g, 3.60 mmol) and phenylmethylamine (0.58 g, 5.40 mmol) in 1,4-dioxane (6 ml)
purged with argon is added cesium carbonate (3.52 g, 10.8 mmol), S-Phos (147.5 mg,
0.36 mmol), and Tris(dibenzylideneacetone)dipalladium(0) (329.7 mg, 0.60 mmol).
After heating at 100°C for 4 hours, the reaction mixture is filtered through filter eel and
washed with EtOAc. The filtrate is concentrated under reduced pressure, and the residue
is purified by silica gel chromatography using 40% EtOAc in Hexanes to give 4-
(benzylamino)-3, 5-dimethyl -pyridine-2-carbonitrile (0.61 g, 71.4%). Mass spectrum
(m/z): 238.06 (M+l) +.
Scheme 9, Step G. To a solution of 4-(benzylamino)-3,5-dimethyl-pyridine-2-
carbonitrile (0.6 g, 2.53 mmol) in ethanol (5.0 ml) at 0°C is added dropwise concentrated
sulfuric acid (5.0 ml), and the reaction mixture is warmed to room temperature. After
heating at 100°C for 32 hours, the reaction mixture is diluted with saturated solution of
NaHC0 3 and extracted with EtOAc. The combined organic layers are dried over
anhydrous Na2S0 4, filtered, and concentrated under reduced pressure. The residue is
purified by silica gel flash chromatography using 10%MeOH in CH2C 12 to give the title
compound (0.4 g, 81.4%). Mass spectrum (m/z): 195.1 (M+l) +.
Example 26
4-[[6-[3-(Hydroxymethyl)phenyl] -3-methyl-pyridine-2-carbonyl]amino] -3,5 -dimethylpyridine-
2-carboxylic acid.
Scheme 10, Step A. To a solution of 6-chloro-3-methyl-pyridine-2-carboxylic
acid (220 mg, 1.28 mmol) in THF (15 mL) under argon at 0°C are added isobutyl
chloroformate (192.6 mg, 1.41 mmol) and N-methylmorpholine (259.4 mg, 2.56 mmol).
The reaction mixture is stirred at 0°C for 40 min, and a solution of ethyl 4-amino-3,5-
dimethyl-pyridine-2-carboxylate (249 mg, 1.28 mmol) in THF (1.5 ml) is added. After
18 hours at 50°C, the reaction mixture is poured onto a column packed with neutral
alumina and purified by flash chromatography eluting with 40% EtOAc in Hexanes to
give ethyl 4-[(6-chloro-3 -methyl-pyridine-2-carbonyl)amino] -3,5-dimethyl -pyridine-2-
carboxylate (0.03 g, 7.6%). Mass spectrum (m/z): 348.0 (M+l) +.
Scheme 10, Step B. To a solution of ethyl 4-[(6-chloro-3-methyl-pyridine-2-
carbonyl)amino]-3,5-dimethyl-pyridine-2-carboxylate (30 mg, 0.09 mmol) in 1,4-dioxane
(1.0 mL) and water (0.5 ml) is added (3-(hydroxymethyl)phenyl)boronic acid (14.4 mg,
0.09 mmol) followed by Cs2C0 3 (56.2 mg, 0.172 mmol) and PdCl2(dppf>CH 2Cl2 (14.1
mg, 0.017 mmol). The reaction mixture is purged with argon for 5 minutes and then
heated at 100°C. After 2 hours, the reaction mixture is cooled to room temperature,
filtered through diatomaceous earth, and washed with EtOAc. The filtrate is
concentrated, and the residue is purified by silica gel flash chromatography using 50%
EtOAc in hexane to afford ethyl 4-[[6-[3-(hydroxymethyl)phenyl]-3-methyl-pyridine-2-
carbonyl]amino]-3,5-dimethyl-pyridine-2-carboxylate as a colorless oil (0.02 g, 60.8%).
Mass spectrum (m/z): 420.3 (M+l) +.
Scheme 10, Step C. To a solution of aqueous LiOH (18.01 mg, 0.429 mmol) in
water ( 1 ml) is added ethyl 4-[[6-[3-(hydroxymethyl)phenyl]-3-methyl-pyridine-2-
carbonyl]amino]-3,5-dimethyl-pyridine-2-carboxylate (36 mg, 0.09 mmol) in THF ( 1 ml)
and MeOH ( 1 ml). After 3 hours at ambient temperature, the reaction mixture is acidified
to pH 1-2 with aqueous IN HC1 solution and extracted with EtOAc. The combined
organic layers are dried over sodium sulfate, filtered, and concentrated under reduced
pressure. The residue is triturated with pentane and filtered to give the title compound
(0.02 g, 56.8%). Mass spectrum (m/z): 392.2 (M+l) +.
Scheme 11
Preparation 40
Methyl 5-amino-4,6-dimethyl-pyridine-2-carboxylate.
Scheme 11. Step A. To a solution of 3-amino-2,4-dimethylpyridine ( 1 g, 8.19
mmol) in methylene chloride (40.0 ml) is added a solution of bromine (0.55 ml, 40.64
mmol) in methylene chloride (10.0 ml) at 0 °C over 5 min. After 12 hours at ambient
temperature, the reaction mixture is diluted with water and extracted with EtOAc. The
combined organic layers are dried over sodium sulfate, filtered, and concentrated under
reduced pressure. The residue is purified by silica gel chromatography using 10-100%
EtOAc in hexanes to afford 6-bromo-2,4-dimethyl-pyridin-3-amine (992 mg, 60.3 %).
Mass spectrum (m/z): 201.0 (M+l) +.
Scheme 11. Step B. To a 100 ml Parr autoclave with mechanical stirring is added
6-bromo-2,4-dimethyl-pyridin-3-amine (0.99 g, 4.92 mmol), Pd(OAc)2 ( 112 mg, 0.50
mmol), ), PdCl2(dppf>CH 2Cl2 (331 mg, 0.6 mmol), CH3CN (30.0 ml), triethylamine
(1.75 ml), and MeOH (20.0 ml). The Parr autoclave is sealed, purged, and pressurized
with CO to 689.5 kPa (100 psig). The mixture is heated at 85 °C for 3 hours. The
mixture is cooled to ambient temperature and vented. The mixture is filtered, rinsed with
MeOH, and concentrated to dryness under reduced pressure. The residue is purified by
silica gel flash chromatography using a gradient of 0-5% 2M N MeOH in methylene
chloride as eluent. The product fractions are combined and concentrated. The residue is
triturated with EtOAc and filtered. The filtrate is concentrated to give the title compound
(537.0 mg, 60.4 %). Mass spectrum (m/z): 181.0 (M+l) +.
Scheme 1
Example 27
5-[[6-[3-(Hydroxymethyl)phenyl]-3-methyl-pyridine-2-carbonyl]amino]-4,6-dimethylpyridine-
2-carboxylic acid.
Scheme 12, Step A. To a solution of methyl 6-chloro-3-methyl-pyridine-2-
carboxylate (1.0 g, 5.39 mmol) in 1,4-dioxane (17.9 ml) and H20 (2.9 ml) is added (3-
(hydroxymethyl)phenyl)boronic acid (0.982 g, 6.47 mmol) followed by K C0 3 (1.86 g,
13.47 mmol) and PdCl2(dppf)*CH Cl2 (131.9 mg, 0.13 mmol). The reaction mixture is
purged with argon for 5 minutes and then heated at 110°C. After 2 hours, the reaction is
cooled to room temperature, diluted with water and extracted with EtOAc. The combined
organic layers are dried over magnesium sulfate, filtered, and concentrated. The residue
is purified by silica gel flash chromatography using 70% EtOAc in hexane to afford
methyl 6-[3-(hydroxymethyl)phenyl]-3-metliyl-pyridine-2-carboxylate (0.713 g, 51.4%).
Mass spectrum (m/z): 258.0 (M+l) +.
Scheme 12. Step B. To a solution of methyl 6-[3-(hydroxymethyl)phenyl]-3-
methyl-pyridine-2-carboxylate (713.0 mg, 2.77 mmoles) in CH2CI2 (13.9 ml) is added
lH-imidazole (285.8 mg, 4.16 mmoles) followed by t-butyldimethylchlorosilane (516.7
mg, 3.33 mmoles). The reaction mixture is stirred at room temperature for 2 hours and
then washed sequentially with water, saturated NaHC03 solution, and brine. The
combined organic layers are dried over magnesium sulfate, filtered, and concentrated to
afford methyl 6-[3-[[tert-butyl(dimethyl)silyl]oxymethyl]phenyl]-3-methyl-pyridine-2-
carboxylate (920.0 mg, 89.4 %). Mass spectrum (m/z): 372.2 (M+l) +.
Scheme 12. Step C. A solution of aqueous IN LiOH (207.8 mg, 4.95 mmol) is
added to a stirred solution of methyl 6-[3-[[tert-butyl(dimethyl)silyl]oxymethyl]phenyl]-
3-methyl-pyridine-2-carboxylate (920.0 mg, 2.48 mmol) in THF (5.0 ml) and MeOH (5.0
mL). After stirring at ambient temperature for 2 hours, the mixture is diluted with water
and extracted with 10% MeOH/methylene chloride. The combined organic layers are
dried over magnesium sulfate, filtered and concentrated. The residue is purified by silica
gel flash chromatography using 0-100% EtOAc in hexanes to give 6-[3-[[tert-
Butyl(dimethyl)silyl]oxymethyl]phenyl]-3-methyl-pyridine-2-carboxylic acid (589.0 mg,
66.5%). Mass spectrum (m/z): 358.2 (M+l) +.
Scheme 12, Step D. To a solution of 6-[3-[[tertbutyl(
dimethyl)silyl]oxymethyl]phenyl]-3-methyl-pyridine-2-carboxylic acid (300.0 mg,
0.839 mmol) in THF (8.4 ml) at 0 °C is added isobutyl chloroformate (128.6 mg, 0.923
mmol) and N-methylmorpholine (101.8 mg, 1.01 mmol). The reaction mixture is stirred
at 0 °C for 20 minutes, and a solution of methyl 5-amino-4,6-dimethyl-pyridine-2-
carboxylate (151.2 mg, 0.839 mmol) in THF (2.0 ml) is added. The reaction mixture is
warmed up to ambient temperature. After 12 hours, the reaction mixture is diluted with
water and extracted with EtOAc. The combined organic layers are dried over sodium
sulfate, filtered, and concentrated. The residue is purified by silica gel flash
chromatography using 0-40% EtOAc in hexanes to give methyl 5-[[6-[3-[[tertbutyl(
dimethyl)silyl]oxymethyl]phenyl]-3-methyl-pyridine-2-carbonyl]amino]-4,6-
dimethyl-pyridine-2-carboxylate (192 mg, 44%). Mass spectrum (m/z): 520.2 (M+l) +.
Scheme 12. Step E. To a solution of methyl 5-[[6-[3-[[tertbutyl(
dimethyl)silyl]oxymethyl]phenyl]-3-methyl-pyridine-2-carbonyl]amino]-4,6-
dimethyl-pyridine-2-carboxylate (190 mg, 0.366 mmol) in THF (2.0 ml) is added Bu4NF
1.0 M in THF (0.548 ml, 0.548 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 with EtOAc. The combined organic layers are dried over sodium sulfate,
filtered, and concentrated. The residue is purified by silica gel flash chromatography
using 0-40% EtOAc in hexane to afford methyl 5-[[6-[3-(hydroxymethyl)phenyl]-3-
methyl-pyridine-2-carbonyl]amino] -4,6-dimethyl-pyridine-2-carboxylate (133.0 mg,
89.8%). Mass spectrum (m/z): 406.2 (M+l) +.
Scheme 12. Step F. A solution of aqueous IN NaOH (0.64 ml) is added to a
stirred solution of methyl 5-[[6-[3-(hydroxymethyl)phenyl]-3-methyl-pyridine-2-
carbonyl]amino]-4,6-dimethyl-pyridine-2-carboxylate (130.0 mg, 0.32 mmol) in THF
(2.0 ml ) and MeOH ( 2.0 ml ). After 2 hours at ambient temperature, the reaction
mixture is acidified to pH 1-2 with aqueous IN HCl solution. The resulting precipitate is
filtered, washed with water, and dried at 40°C in vacuum oven for 1 hour to give the title
compound as a white solid (88.0 mg, 70.0%). Mass spectrum (m/z): 392.2 (M+l) +.
Preparation 4 1
Methyl 4-[[6-chloro-3-(1rifluoromethyl)pyridine-2-carbonyl]amino]-3,5-dimethylbenzoate.
To a solution of 6-chloro-3-(trifluoromethyl)pyridine-2-carboxylic acid (0.40 g,
1.77 mmol) in C 2 (8.0 ml) at room temperature are added methyl 4-amino-3,5-
dimethylbenzoate (0.318 mg, 1.77 mmol) and diisopropylethylamine (0.573 g , 4.43
mmol). After stirring the mixture for 10 minutes, 1-propanephosphonic acid cyclic
anhydride (50% solution in EtOAc, 1.35 g, 2.13 mmol) is added via syringe. After 36
hours at ambient temperature, the reaction mixture is heated at 50°C for 7 days. The
reaction mixture is concentrated and triturated with MeOH to give the title compound as
white solid (456.0 mg, 66.5%). Mass spectrum (m/z): 387.2 (M+l).
The following compound is prepared essentially by the method described above in
preparation 41, using the appropriate carboxylic acid and amine:
Scheme 13
Preparation 43
Methyl 4-[[6-(3-methoxyphenyl)-3-methyl-pyridine-2-carbonyl]amino] -3,5 -dimethylbenzoate.
Scheme 13, Step A. A mixture of methyl 4-[(6-chloro-3 -methyl-pyridine-2-
carbonyl)amino]-3,5-dimethyl-benzoate (200 mg, 1.18 mmol), (3-methoxyphenyl)boronic
acid (198 mg, 1.3 mmol), potassium carbonate (170 mg, 1.23 mmol), and ),
PdCl2(dppf>CH 2Cl2 (20 mg) in dioxane (4 mL) and water ( 1 mL) is heated to 110 °C in a
microwave reactor for 120 minutes. The mixture is filtered through a hydrophobic frit
and washed with methylene chloride (2 x 10 mL). The filtrate is concentrated and
purified by silica gel chromatography using 15% ethyl acetate in hexane to provide
methyl 4-[[6-(3-methoxyphenyl)-3-methyl-pyridine-2-carbonyl]amino]-3,5-dimethylbenzoate
as product (194 mg, 41% yield). Mass spectrum (m/z): 405.2 (M+H)+.
The following compounds are prepared essentially by the method described above
in preparation 43, using the appropriate carboxylic ester and boronic acid:
carbonyl]amino]benzoate
Example 28
4-[[6-(3-Methoxyphenyl)-3-methyl-pyridine-2-carbonyl]amino] -3,5-dimethyl-benzoic
acid.
Scheme 13, Step B. A slurry of methyl 4-[[6-(3-methoxyphenyl)-3-methylpyridine-
2-carbonyl]amino]-3,5-dimethyl-benzoate (194 mg, 0.479 mmol) in MeOH (3
mL), THF (2 mL), and IN NaOH (1.2 mL) is stirred at RT over 3 nights. The reaction
mixture is treated with IN HC1 ( 1.2 mL) and diluted with water. The solid is collected by
vacuum filtration and dried under reduced pressure to provide the title compound (172
mg, 92% yield). Mass spectrum (m/z): 391.2 (M+H)+.
The following compounds are prepared essentially by the method described above
in Example 28, using the appropriate carboxylic ester:
Scheme 14
Example 36
3,5-Dimethyl-4-[(2-phenylquinoline-4-carbonyl)amino]benzoic acid.
Scheme 14. Step A. To a solution of 2-phenylquinoline-4-carboxylic acid (193.5
g, 0.776 mmol) in CH2CI2 (6.0 ml) at 0°C are added ethyl 4-amino-3,5-dimethyl-benzoate
(150.0 g, 0.776 mmol), and N,N-diisopropylethylamine (0.338 ml, 1.94 mmol). The
reaction mixture is stirred for 10 minutes and 1-propanephosphonic acid cyclic anhydride
(50% solution in ethyl acetate, 0.555 ml, 0.932 mmol) is added via syringe. The mixture
is stirred at ambient temperature for 48 hours, diluted with water, and extracted with
EtOAc. The combined organic layers are dried over magnesium sulfate, filtered, and
concentrated under reduced pressure. The residue is triturated with CH2CI2 and filtered to
give ethyl 3,5-dimethyl-4-[(2-phenylquinoline-4-carbonyl)amino]benzoate as a white
solid (93.5 mg, 28.4 %). Mass spectrum (m/z): 425.2 (M+H)+.
Scheme 14. Step B. A solution of aqueous IN NaOH (1.0 ml) is added to a stirred
solution of ethyl 3,5-dimethyl-4-[(2-phenylquinoline-4-carbonyl)amino]benzoate (93.5
mg, 0.220 mmol) in THF (3.0 ml ). After heating at 50°C for 8 hours, the reaction
mixture is acidified to pH 1-2 with aqueous IN HCl solution. The resulting precipitate is
filtered, washed with water, and dried at 40°C in vacuum oven for 1 hour to give the title
compound as a white solid (70.0 mg, 80.2%). Mass spectrum (m/z): 397.2 (M+H)+.
Preparation 1
Ethyl 4-[(2-chloroquinoline-4-carbonyl)amino] -3,5-dimethyl-benzoate.
Scheme 15. Step A. To a solution of 2-chloroquinoline-4-carboxylic acid (1.07 g,
5.17 mmol) in CH2CI2 (15 ml) at 0°C are added ethyl 4-amino-3,5-dimethyl-benzoate
(1.00 g, 5.17 mmol), and N,N-diisopropylethylamine (2.26 ml, 12.94 mmol). After
stirring the reaction mixture for 10 minutes, 1-propanephosphonic acid cyclic anhydride
(50% solution in ethyl acetate, 3.70 ml, 6.21 mmol) is added via syringe. The reaction
mixture is stirred at ambient temperature for 24 hours, diluted with water, and extracted
with EtOAc. The combined organic layers are dried over magnesium sulfate, filtered, and
concentrated under reduced pressure. The resulting residue is triturated with MeOH and
filtered. The filtrate is purified by high pH reverse phase chromatography (0-100%
lOmM ammonium bicarbonate in water with 5% MeOH/CH3CN) to give the title
compound as white solid (0.49 g, 25 %). Mass spectrum (m/z): 383.1 (M+H)+.
The following compounds are prepared essentially by the method described above
in preparation 51, using the appropriate carboxylic acid and amine:
Preparation 54
Ethyl 4-[[2-[3-(hydroxymethyl)phenyl] quinoline-4-carbonyl] amino] -3,5 -dimethylbenzoate.
Scheme 15. Step B. To a solution of ethyl 4-[(2-chloroquinoline-4-
carbonyl)amino]-3,5-dimethyl-benzoate (0.49 g, 1.27 mmol) in 1,4-dioxane (17.0 ml) and
H20 (2.5 ml) is added (3-(hydroxymethyl)phenyl)boronic acid (231.9 mg, 1.53 mmol)
followed by K2C0 3 (439.2 mg, 3.18 mmol) and PdCl2(dppf>CH 2Cl2 (103.9 mg, 0.0127
mmol). The reaction mixture is purged with argon for 5 minutes and then heated at
110°C. After 3 hours, the mixture is cooled, diluted with water, and extracted with ethyl
acetate. The combined organic layers are dried over sodium sulfate, filtered, and
concentrated. The residue is purified by silica gel flash chromatography using 40% ethyl
acetate in hexane to afford the title compound (0.42 g, 72.3%). Mass spectrum (m/z):
455.2 (M+H)+.
The following compounds are prepared essentially by the method described above
in preparation 54, using the appropriate carboxylic ester and boronic acid:

a) H NMR (400 MHz, DMSO): 10.23 (s, 1H), 8.37-8.36 (m, 1H), 8.32-8.30 (m,
1H), 8.12 (d, J= 1.9 Hz, 1H), 8.11-8.08 (m, 1H), 7.83-7.83 (m, 1H), 7.76-7.74 (m,
3H), 7.62-7.59 (m, 2H), 7.49 (t, J= 7.7 Hz, 1H), 7.38-7.36 (m, 1H), 5.29 (t, J= 5.7
Hz, 1H), 4.61-4.60 (m, 2H), 4.30 (q, J= 7.1 Hz, 2H), 2.39 (s, 6H), 1.31 (t, J= 7.1
Hz, 3H).
Example 37
4-[[2- [3-(Hydroxymethyl)phenyl] quinoline-4-carbonyl] amino] -3,5-dimethyl-benzoic
acid.
Scheme 15. Step C. A solution of aqueous IN NaOH (1.9 ml) is added to a stirred
solution of ethyl 4-[[2-[3-(hydroxymethyl)phenyl]quinoline-4-carbonyl]amino]-3,5-
dimethyl-benzoate (0.42 g, 0.920 mmol) in THF (10.0 ml ) and MeOH ( 3.0 ml ). After
heating at 40 °C for 12 hours, the reaction mixture is acidified to pH 1-2 with aqueous IN
HCl solution. The resulting precipitate is filtered, washed with water, and dried at 40°C
in vacuum oven for 1 hour to give the title compound as white solid (333.0 mg, 84.9%).
Mass spectrum (m/z): 427.2 (M+H)+.
The following compounds are prepared essentially by the method described above
in Example 37, using the appropriate carboxylic ester:

Preparation 6 1
Methyl 3-[(3-bromonaphthalene- 1-carbonyl)amino] -2,4-dimethyl-benzoate.
To a solution of 3-bromonaphthalene- 1-carboxylic acid (0.42 g, 1.67 mmol) and
methyl 3-amino-2,4-dimethylbenzoate (0.30 g, 1.67 mmol) in CH3CN (10.0 ml) at 0 °C is
added phosphorus trichloride (0.292 ml, 3.35 mmol). After heating at 100°C in a
microwave reactor for 1.5 hours, the reaction mixture is diluted with water and extracted
with EtOAc. The combined organic layers are dried over magnesium sulfate, filtered, and
concentrated under reduced pressure to give the title compound as off white foam (0.60 g,
86.7%). Mass spectrum (m/z): 412.0 (M+H)+.
Scheme 16
Alternate Preparation of 3-[[3-(3-ChlorophenvDnaphthalene-1-carbonyl]amino] -2,4-
dimethyl-benzoic acid (Example 43).
Scheme 16. Step A. Charge a reactor with 10 L of water and 585 g (6.970 mol) of
sodium bicarbonate. Heat the solution to 50 °C and sparge with nitrogen for 15 min.
Add 500 g (1.991 mol) of 3-bromo-1 -naphthalene carboxylic acid and heat the mixture to
75 °C. Add 4.47 g (19.9 mmol) of palladium acetate. Add 358 g (2.290 mol) of 3-
chlorophenylboronic acid by portions of 50 g over lh. Stir for 2 h at 75 °C. Cool to 25°C
and add 2 L of water, 0.5 L of 25% aqueous sodium hydroxide and 1.5 L of THF. Wash
the aqueous solution with 2 x 2 L of heptanes. Add 10 L of THF and 2.5 L of ethyl
acetate to the washed aqueous solution. Add 890 g of 37% HCl to bring the pH to approx
2. Separate the phases and extract the aqueous phase with a mixture of 2 L of THF and
0.5 L of ethyl acetate. Wash the combined organic solutions with 1 L of brine. Treat the
solution with 100 g of activated carbon at 20°C for 1 h and filter the mixture over
diatomaceous earth. Add 400 g of metal scavenging silica and heat the suspension to
50°C, then stir at 50°C for 4 h. Cool to 20°C and remove the silica by filtration. Repeat
the operation with another charge of 400 g part of scavenger. Cool to 20°C, remove the
scavenger by filtration and evaporate the solution to dryness under reduced pressure.
Dissolve the crude material in 8 L of THF and add 12 L of heptanes. Filter the solution
over a pad of 900g of silica and elute with 4 x 900mL of a mixture of THF and heptanes
2-3. Evaporate the filtrate under reduced pressure to give a suspension. Collect the solids
by filtration and dry at 50°C under 2-10 mBar for 4 h to give 502 g (89%) of 3-(3-
chlorophenyl)naphthalene- 1-carboxylic acid. H NMR (400 MHz, DMSO) 7.48 - 7.5 1
(m, 1 H), 7.56 (t, J = 8.1 Hz, 1 H), 7.61 - 7.69 (m, 2 H), 7.81- 7.83 (m, 1H), 7.92 (d, J =
2.2 Hz, 1 H), 8.10 - 8.13 (m, 1 H), 8.42 (d, J =2.2 Hz, 1 H), 8.54 (d, J =2.2 Hz, 1 H),
8.85 (d, J = 8.4 Hz, 1 H), 13.35 (br s, 1 H).
Scheme 16. Step B. Charge a 20 L reactor with 485 g (1.715 mol) of 3-(3-
chlorophenyl)naphthalene-l -carboxylic acid, 136 g of pyridine (1.715 mol) and 4.85 L of
toluene. Heat the mixture to 75 °C and add 306 g (2.573 mol) of thionyl chloride over
about 15 min. Stir the reaction mixture for 3 h at 75°C and add 102 g (0.858 mol) of
thionyl chloride to consume remaining starting acid. After stirring the reaction mixture
for 1 h, decant the upper layer and discard the lower waste layer at 75°C. Add 102 g
(0.858 mol) of thionyl chloride and 34g (0.429 mol) of pyridine. Stir the reaction mixture
for 1 h at 75°C. Hold the reaction mixture overnight at 20-25°C. Wash the organic layer
with 2 0.5 L of water. Add a solution of 370 g (1.715 mol) of methyl 3-amino-2,4-
dimethyl-benzoate in 2.425 L of water to the reactor in one portion. Add a solution of
859 g (8.577 mol) of potassium bicarbonate in 4.850 L of water over 40 min. Stir the
reaction mixture for 2 h at 20-25°C. Add 4.850 L of THF, and stir until all the solids
dissolve. Decant and discard the lower aqueous phase. Wash the upper organic phase
with a solution of 2.57 g of Na2S-9H20 in 0.970 L of water and discard the aqueous
phase. Wash the organic layer three times with 0.970 L of brine, then with 0.970 L of IN
hydrochloric acid, and finally with 0.970 L of brine. Dry the organic phase over sodium
sulfate. Concentrate the product solution (THF-Toluene) under reduced pressure to
remove the THF. Seed the batch with crystalline product and allow the temperature to
decrease from 45 °C to 37 °C over approx 1 h. As crystallization progresses, add 1.94 L
of toluene and lower the temperature to 20°C. Collect the product by filtration after 2 h
of stirring. Wash the product cake with 1.94 L of TBME and dry at 80°C under 2-10 Torr
to constant weight. Dissolve the solid in 0.970 L of acetone at 55°C and add 1.940 L of
TBME. Cool the solution to 20°C over 1 h to crystallize the product. Collect the white
crystalline solid by filtration after 2 h of stirring at 20°C and dry at 80°C under 2-10 Torr
until constant weight. Purify the material by chromatography over a short pad of silica
(1.5kg, 3 parts) eluted with dichloromethane to give 442 g (58%) of methyl 3-[[3-(3-
chlorophenyl)naphthalene-l-carbonyl]amino]-2,4-dimethyl-benzoate as a white solid. H
NMR (400 MHz, DMSO) 2.41 (s, 3 H), 2.53 (s, 3 H), 3.85 (s, 3 H), 7.30 (d, J = 8.0 Hz,
1 H), 7.51 (d, J = 8.0 Hz, 1 H), 7.59 (d, J = 8.0 Hz, 1 H), 7.63 - 7.65 (m, 2 H), 7.69 (d, J
= 8.0 Hz, 1 H), 7.90 (d, J = 8.0 Hz, 1 H), 8.02 (s, 1 H), 8.10 - 8.14 (m, 1H), 8.20 (s, 1 H),
8.37 - 8.40 (m, 1 H), 8.46 (s, 1 H), 10.20 (br s, 1 H).
Scheme 16. Step C. Charge a 20 L reactor with 178 g (3.176 mol) of potassium
hydroxide and 1.59 L of water. Stir the mixture until complete dissolution. Add 4.70 L
of isopropanol and heat the solution 65 °C. Add a solution of 470 g (1.059 mol) of
methyl 3-[[3-(3-chlorophenyl)naphthalene-l-carbonyl]amino]-2,4-dimethyl-benzoate in
1.88 L of THF to the reactor at 65 °C in about 1 h. After the end of the addition, stir the
reaction mixture for 2 h at 65 °C. Add 23.5 g of active carbon and stir the mixture at 65
°C for 1 h. Cool the mixture to 20°C and filter over diatomaceous earth. Rinse the cake
with 0.94 L of isopropanol followed by 2x0.94 L of water. Concentrate the solution
under reduced pressure to remove the organic solvents. Transfer the aqueous solution to
a 10 L reactor and dilute with 3.76 L of water. Acidify to pH 3.0 by adding 0.95 L of 3 N
HC1. Add 0.94 L of isopropanol and stir the suspension overnight at 20 °C. Collect the
product by filtration and wash with 2.82 L of water. Dry the crude product at 50 °C over
the weekend. Transfer the dried solid to a 20 L reactor, add 4.70 L of acetone and heat
the suspension to reflux for 2 h. Cool the suspension to 20 °C over 3 h and hold at this
temperature overnight. Cool the suspension to 8 °C and stir for 1 h. Collect the product
by filtration and wash the cake with 1.41 L of acetone. Dry at 50 °C under 2-10 Tonovernight
to give the title compound as a white solid (378.0 g, 83% yield). Mass
spectrum (m/z): 430.3 (M+H)+.
In Vitro binding to human EP1. EP2. EP3 and EP4
hEPl and hEP4 membranes are prepared from recombinant HEK293 cells stably
expressing human EP1 (Genbank accession number AY275470) or EP4 (Genbank
accession number AY429109) receptors. hEP2 and hEP3 membranes are prepared from
HEK293 cells transiently transfected with EP2 (Genbank accession number AY275471)
or EP3 (isoform VI: Genbank accession number AY429108) 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 [ H]-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. Non-specific binding is calculated by comparing
binding in the presence and absence of 2 of PGE . The membranes are harvested by
filtration (TomTek harvester), washed 4 times with cold buffer (10 mM MES pH 6.0 with
KOH, 10 mM MgCl2), 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 PGE . 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 ( = /(1 +
[L] i¾) where [L] is the ligand concentration). Results are expressed as the geometric
mean ± standard deviation; n = number of independent determinations. The standard
deviation is calculated by the delta method, being SDio gKi x geometric mean x ln(10).
The compounds of Examples 1-43 herein are tested essentially as described above
and exhibit a K value for hEP4 of lower than 1 .
Table 1: In vitro binding of Example 1 to human EP1, EP2, EP3 and EP4
The data in Table 1 demonstrate the compounds of Example 1 and Example 22
bind to hEP4 more strongly than to hEPl, 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% C0 2. Cells are
harvested using 2.5% Trypsin-EDTA, suspended in freeze media (FBS with 6% DMSO)
at 107cells/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 PGE -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 EC 0 of PGE (0.188 nM
PGE 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 (See Murase, A., et al., Life Sciences, 82:226-232 (2008)). To
measure the cAMP levels, cAMP-d2 conjugate and anti cAMP-cryptate 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.
Results are expressed as the geometric mean ± standard deviation; n = number of
independent determinations. The standard deviation is calculated by the delta method,
being SDiogIC 5o x geometric mean x ln(10).
Following the procedures essentially as described above, the compound of
Example 1 has an IC50 of 2.31+2.02 nM (n=2) demonstrating that the compound of
Example 1 is an 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 EC 0) at ratio of
DMSO/buffer at 1/100. The cells are treated with compounds in the presence of PGE
(EC oconcentration) 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 1.5 1 nM measured at rat EP4. This demonstrates that the
compound of Example 1 is an 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 PGE 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% C0 2 humidified atmosphere, for 30 minutes, after
which 1 of a solution of 1 mg/mL of lipopolysaccharide (LPS) (Sigma 0111 :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 PGE . The plates are
incubated for 20-24 hours at 37 °C in a 5% C0 humidified atmosphere. The plates are
centrifuged at 1800 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 ΐ 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 A450-A650 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. The standard deviation is
calculated by the delta method, being SDiogicso x geometric mean x ln(10).
Following the procedures essentially as described above, the compound of
Example 1 has an IC50 of 0.0595±0.0378 (n=3). This demonstrates that the
compound of Example 1 is an EP4 antagonist in the human blood TNFa induction assay.
WE CLAIM:
1. A compound of the formula:
wherein A is:
is CH orN;
Y is CH orN;
X is CH orN;
R is CH3, CF3, or F;
R2 is H, F, CI, CH3, CF3, CH2OH, CH2CH2OH, CH2OCH3, OCH3, OCF3,
or CN;
R3 is H or F; or
R2 and R3 together are a OCH20 group attached to vicinal carbon atoms;
R4 is H, CI, or CH2OH;
or a pharmaceutically acceptable salt thereof.
2. The compound or salt according to claim 1 of the formula:
3. The compound or salt according to either claim 1 or claim 2 of the
formula:
4. The compound or salt according to claims 1 to 3 wherein A is:
5. The compound or salt according to claims 1 to 4 wherein R is CH3.
6. The compound or salt according to claims 1 to 5 wherein R is H.
7. The compound or salt according to claims 1 to 6 wherein R2 is CH2OH,
CH2CH2OH, or OCH3.
8 . The compound or salt according to claims 1 to 7 wherein wherein R is
CH2OH.
9. The compound or salt according to claims 1 to 5 wherein R2 and R3
together are a OCH 0 group attached to vicinal carbon atoms.
10. The compound or salt according to claims 1 to 4 wherein R4 is CI.
11. The compounds or salts thereof according to claim 1 which are:
3-[[6-(l,3-benzodioxol-5-yl)-3-methyl-pyridine-2-carbonyl]amino]-2,4-
dimethyl-benzoic acid;
3-[[6-[3-(hydroxymethyl)phenyl]-3-methyl-pyridine-2-carbonyl]amino]-
2,4-dimethyl-benzoic acid; and
3-[[3-(3-chlorophenyl)naphthalene- 1-carbonyl]amino]-2,4-dimethylbenzoic
acid.
12. 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 11.
13. 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 11.
14. 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 tol 1.
15. A compound or pharmaceutically acceptable salt thereof according to
claims 1 to 11 for use in therapy.
16. A compound or pharmaceutically acceptable salt thereof according to
claims 1 to 11 for use in the treatment of osteoarthritis.
17. A compound or pharmaceutically acceptable salt thereof according to
claims 1 to 11 for use in the treatment of rheumatoid arthritis.
18. A compound or pharmaceutically acceptable salt thereof according to
claims 1 to 11 for use in the treatment of pain associated with osteoarthritis
or rheumatoid arthritis.
19. A pharmaceutical composition, comprising a compound or a
pharmaceutically acceptable salt thereof according to claims 1 to 11 with
one or more pharmaceutically acceptable carriers, diluents, or excipients.