Synthesis and evaluation of cyclohexane carboxylic acid head group containing isoxazole and thiazole analogs as DGAT1 inhibitors

Article abstract of DOI:10.1016/j.ejmech.2014.03.077

Diacylglycerol acyltransferase 1 (DGAT1) is known to play an important catalytic role in the final step of triglyceride biosynthesis. High fat diet fed DGAT1 knockout mice were resistant to weight gain and exhibited increased insulin and leptin sensitivity thereby indicating a plausible role for DGAT1 inhibitors in the treatment of obesity. 4-Phenylpiperidine-1-carbonyl cyclohexanecarboxylic acid (compound 6, DGAT1 IC₅₀ = 57 nM) has been lately reported as a potent DGAT1 inhibitor. In our search for newer scaffolds possessing potent DGAT1 activity we undertook a systematic diversification of compound 6 to identify a 4-(5-phenylthiazole-2-carboxamido)cyclohexanecarboxylic acid scaffold. Further linker optimization of this scaffold identified compound 9e (DGAT1 IC₅₀ = 14.8 nM) as a potent DGAT1 inhibitor. Coupled with its in vitro potency, compound 9e also exhibited 112 percent plasma triglyceride reduction at a 3 mpk dose in an oral fat tolerance test (FTT) when studied in Swiss mice.

Full text of DOI:10.1016/j.ejmech.2014.03.077

European Journal of Medicinal Chemistry 79 (2014) 203e215
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and evaluation of cyclohexane carboxylic acid head group
containing isoxazole and thiazole analogs as DGAT1 inhibitors
,
Shivaji Kandre a c, Pundlik Rambhau Bhagat c, M. Mahesh Kumar Reddy b, Roda Dalal b,
Amol Dixit b, Nitin J. Deshmukh b, Jessy Anthony b, Julie Bose b, Raghuram Anupindi b,
,
Rajiv Sharma a, Amol Gupte a
*
a Department of Medicinal Chemistry, Piramal Enterprises Limited, 1-Nirlon Complex, Goregaon (E), Mumbai 400063, India
b Department of Pharmacology, Piramal Enterprises Limited, 1-Nirlon Complex, Goregaon (E), Mumbai 400063, India
c Organic Chemistry Division, School of Advanced Sciences, VIT University, Vellore 632014, Tamil Nadu, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Diacylglycerol acyltransferase 1 (DGAT1) is known to play an important catalytic role in the final step of
triglyceride biosynthesis. High fat diet fed DGAT1 knockout mice were resistant to weight gain and
exhibited increased insulin and leptin sensitivity thereby indicating a plausible role for DGAT1 inhibitors
in the treatment of obesity. 4-Phenylpiperidine-1-carbonyl cyclohexanecarboxylic acid (compound 6,
DGAT1 IC50 ¼ 57 nM) has been lately reported as a potent DGAT1 inhibitor. In our search for newer
scaffolds possessing potent DGAT1 activity we undertook a systematic diversification of compound 6 to
identify a 4-(5-phenylthiazole-2-carboxamido)cyclohexanecarboxylic acid scaffold. Further linker opti-
mization of this scaffold identified compound 9e (DGAT1 IC50 ¼ 14.8 nM) as a potent DGAT1 inhibitor.
Coupled with its in vitro potency, compound 9e also exhibited 112 percent plasma triglyceride reduction
at a 3 mpk dose in an oral fat tolerance test (FTT) when studied in Swiss mice.
Received 6 February 2014
Received in revised form
26 March 2014
Accepted 27 March 2014
Available online 28 March 2014
Keywords:
Diacylglycerol acyltransferase (DGAT)
Obesity
Plasma triglycerides
Fat tolerance test (FTT)
Ó 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
LCQ-908 (1), a DGAT1 inhibitor being developed by Novartis is
under evaluation for the treatment of familial chylomicronemia
Diacylglycerol acyltransferase
1
(DGAT1) is abundantly
syndrome (FCS), a condition characterized by elevated triglyceride
levels [5]. Other potent DGAT1 inhibitors include a series of
biphenyl cyclopentane carboxylic acids represented by BAY-744113
(2, IC50 ¼ 73 nM) [6]. Replacing the benzothiazole moiety of 2 with
a phenyl urea resulted in an orally potent carboxylic acid repre-
sented by compound 3 (DGAT1 IC50 ¼ 7 nM) [7]. Other scaffolds
also identified as DGAT1 inhibitors include a bicyclic heterocycle
core (4, DGAT1 IC50 ¼ 15 nM) disclosed by Japan Tobacco/Tularik [8]
and PF-04620110 (5, DGAT1 IC50 ¼ 19 nM) reported by Pfizer [9].
Hoffmann-La Roche has recently reported a 4-phenylpiperidine-1-
carbonyl cyclohexanecarboxylic acid scaffold represented by com-
pound 6, (DGAT1 IC50 ¼ 57 nM) as an active DGAT1 inhibitor [10].
Despite several advances in pre-clinical drug discovery and appli-
cation of stringent selection criteria for advancing clinical candi-
dates, a high attrition rate is observed in case of compounds that
enter clinical development. According to a published study [11] the
success rate for compounds that entered clinical trials in between
1993 and 2004 was 32% for large molecules and a meager 13% for
small molecules. One of the possible scientific approaches for
overcoming such unforeseen developmental hurdles is to invest in
efforts towards the identification of diverse chemical scaffolds for a
expressed in the small intestine, liver, and adipose tissues. It is
known to catalyze the final committed step of triglyceride
biosynthesis [1]. DGAT1 knockout mice phenotypes are viable and
exhibit reduction in the postprandial rise of plasma triglycerides,
resistance to diet induced obesity (DIO), and increased sensitivity
for both insulin and leptin [2, 3]. In contrast, DGAT2 deficient mice
were found to be smaller in size than the wild type and died soon
following their birth [4]. It was observed that the DGAT2 deficient
mice lacked essential fatty acids resulting in skin lipid abnormal-
ities and impaired epidermal barrier function. As a result specific
inhibitors of the DGAT1 enzyme are being explored for metabolic
disorders such as weight gain, dyslipidemia, type 2 diabetes, and
endothelial dysfunction. Of late an increased interest towards this
attractive target has resulted in the identification of several potent
and selective DGAT1 inhibitors (Fig. 1) [5e10].
* Corresponding author.
E-mail addresses: amolrgupte@gmail.com, argupte@hotmail.com (A. Gupte).
http://dx.doi.org/10.1016/j.ejmech.2014.03.077
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

204
S. Kandre et al. / European Journal of Medicinal Chemistry 79 (2014) 203e215
Fig. 1. Selected DGAT1 inhibitors from literature.
validated protein target like DGAT1. The present study highlights
Although this initial modification led to an improvement in DGAT1
potency, it was also observed that this modification led to a mo-
lecular weight increase from 569.5 Da for compound 6 to 584.5 Da
for compound 9. With an aim to reduce the molecular weight of our
resulting molecules to the more acceptable 500 Da range we chose
to replace the trifluoromethyl oxazole amide linker in compound 6
with either amide (compound 9a, Molecular weight ¼ 485.5 Da),
sulfonamide (compound 9b, Molecular weight ¼ 521.5 Da), or urea
(compound 9c, Molecular weight ¼ 500.5 Da) linkers as shown in
Fig. 3. Our efforts involving a systematic diversification of com-
pound 6 were thus focused on developing and optimizing a novel
heteroaryl scaffold that retains DGAT1 inhibitory activity.
our systematic efforts towards the identification of
phenylthiazole-2-carboxamido)cyclohexanecarboxylic
scaffold.
a
4-(5-
acid
In our attempts to develop a novel and selective DGAT1 che-
motype, we undertook the modification of compound 6 (in-house
DGAT1 IC50 ¼ 114 nM). In our hands, compound 6 also demon-
strated 89% triglyceride reduction in an acute fat tolerance test
conducted in mice. Compound 6 appears to be comprised of four
domains (aryl, linker, central core, and acid head) as shown in Fig. 2.
In the present study we have explored replacing the piperidine ring
in the central core with either isoxazole and thiazole structural
motifs thereby resulting in either a 3-phenylisoxazole core (rep-
resented by compound 7), a 4-phenylthiazole core (represented by
compound 8), or a 5-phenylthiazole core (represented by com-
pound 9). Having replaced a six-membered heteroaliphatic piper-
idine ring with five-membered heteroaromatic rings, we
compensated the loss of this singular ring atom by introducing a
nitrogen atom in the acid head portion of the molecule. This
modification resulted in an amide linker preceding the cyclohexyl
acid head. Taken together, these modifications resulted in either a
4-(3-phenylisoxazole-5-carboxamido)cyclohexanecarboxylic acid
scaffold (represented by compound 7, DGAT1 inhibition
2. Chemistry
Compound 11 a key intermediate for the synthesis of compound
6
was prepared following the selective hydrolysis [12] of
commercially available dimethyl trans-1,4-cyclohexane dicarbox-
ylate (10) to trans-1,4-cyclohexanedicarboxylic acid monomethyl
ester (11) as shown in Scheme 1. Compound 6 was prepared
(Scheme 2) following the nitration of commercially available 4-
phenylpiperidine (12) using nitric acid [13] to yield 4-(4-
nitrophenyl)piperidine (13). Compound 13 was coupled with 11
using HATU to give methyl 4-(4-(4-nitrophenyl)piperidine-1-
carbonyl)cyclohexanecarboxylate (14). Reduction of compound 14
using iron-ammonium chloride resulted in the corresponding
amine (15) that on coupling with commercially available 2-phenyl-
5-trifluoromethyloxazole-4-carboxylic acid using HATU followed
by alkaline hydrolysis resulted in the synthesis of compound 6.
[1
hexanecarboxylic acid scaffold (represented by compound 8,
DGAT1 inhibition [1 M] 7%), or 4-(5-phenylthiazole-2-
carboxamido)cyclohexanecarboxylic acid scaffold (represented by
compound 9, DGAT1 inhibition [1
mM]
¼
73%), 4-(4-phenylthiazole-2-carboxamido)cyclo-
m
¼
m
M] ¼ 94%). Amongst these three
heterocyclic scaffolds, the 4-(5-phenylthiazole-2-carboxamido)
cyclohexanecarboxylic acid scaffold thus appeared to exhibit better
DGAT1 potency than the other two heterocyclic scaffolds. We
evaluated the DGAT1 IC50 of compound 9 and found it to be 73 nM.
The
4-(3-phenylisoxazole-5-carboxamido)cyclo-
hexanecarboxylic acid scaffold represented by compound 7 was
synthesized as depicted in Scheme 3. Refluxing 4-Nitro

S. Kandre et al. / European Journal of Medicinal Chemistry 79 (2014) 203e215
205
Fig. 2. Replacement of aliphatic heterocycle by five membered aromatic heterocycles.
Fig. 3. Linker modifications aimed at reducing molecular weight.
cyclized to ethyl 3-(4-nitrophenyl)isoxazole-5-carboxylate (19)
following treatment with ethyl propiolate [14]. Compound 19 was
hydrolyzed to its corresponding acid (20) using sodium hydroxide
that was subsequently coupled with methyl trans-4-
aminocyclohexanecarboxylate to obtain methyl 4-(3-(4-
nitrophenyl)isoxazole-5-carboxamido)cyclohexanecarboxylate
(21). Compound 21 was reduced to its corresponding amino de-
rivative (22). Compound 22 was further coupled with commercially
available 2-phenyl-5-trifluoromethyloxazole-4-carboxylic acid us-
ing HATU as the peptide coupling reagent followed by alkaline
hydrolysis to yield compound 7.
Scheme 1. Synthesis of intermediate 11. Reaction conditions: (a) KOH, methanol reflux,
5 h.
benzaldehyde (16) in methanol with hydroxylamine hydrochloride
yielded the corresponding oxime (17) that was subsequently
treated with N-chlorosuccinimide in DMF to yield N-hydroxy-4-
nitrobenzimidoyl chloride (18). Compound 18 was further

206
S. Kandre et al. / European Journal of Medicinal Chemistry 79 (2014) 203e215
Scheme 2. Synthesis of compound 6. Reaction conditions: (a) HNO3, H2SO4; (b) Compound 11, HATU, TEA, DMF, 16 h; (c) Fe, NH4Cl, THF, EtOH, water, 75 ꢀC, 3 h; (d) 2-phenyl-5-
trifluoromethyloxazole-4-carboxylic acid, HATU, TEA, DMF, 16 h; (e) NaOH, MeOH, 8 h.
Scheme 3. Synthesis of compound 7. Reaction conditions: (a) NH2OH.HCl, MeOH, reflux, 5 h; (b) NCS, DMF, 2 h; (c) Ethyl propiolate, TEA, Toluene, 80 ꢀC, 8 h; (d) NaOH, THF, H2O, 1 h;
(e) Methyl trans-4-aminocyclohexanecarboxylate hydrochloride, N-Methyl morpholin, IBCF, TEA, THF, -20 to 25 ꢀC, 16 h; (f) Fe, NH4Cl, THF, EtOH, water, 75 ꢀC, 3 h; (g) 2-phenyl-5-
trifluoromethyloxazole-4-carboxylic acid, HATU, TEA, DMF, 16 h; (h) NaOH, MeOH, THF, 8 h.
Compound
carboxamido)cyclohexanecarboxylic acid scaffold was synthesized
as shown in Scheme using commercially available 4-
8
representing the 4-(4-phenylthiazole-2-
carboxylate (31). Subsequent hydrolysis of compound 31 yielded
its corresponding acid [15] compound (32) that on coupling with
methyl trans-4-aminocyclohexanecarboxylate yielded methyl 4-(5-
(4-nitrophenyl)thiazole-2-carboxamido)cyclohexanecarboxylate
(33). Reduction of compound 33 with iron-ammonium chloride
yielded the key amine intermediate methyl 4-(5-(4-aminophenyl)
thiazole-2-carboxamido)cyclohexanecarboxylate (34). The amine
4
nitroacetophenone (23). Bromination of compound 23 using
bromine solution yielded 2-bromo-1-(4-nitrophenyl)ethanone (24)
that was cyclized using ethyl thioxoacetate to yield ethyl 4-(4-
nitrophenyl)thiazole-2-carboxylate (25). Subsequent hydrolysis of
25 to its corresponding acid [15] compound (26) followed by
coupling with methyl trans-4-aminocyclohexanecarboxylate yiel-
ded methyl 4-(4-(4-nitrophenyl)thiazole-2-carboxamido)cyclo-
hexanecarboxylate (27). Reduction of 27 to its corresponding amine
(28) was achieved using iron-ammonium chloride. Coupling of
intermediate
(34)
was
coupled
with
2-phenyl-5-
trifluoromethyloxazole-4-carboxylic acid followed by hydrolysis to
yield compound 9. Compound 34 was also diversified using 3,5-
difluorobenzoyl chloride to yield compound 9a, 3,5-
difluorobenzenesulfonyl chloride to yield compound 9b, and
appropriate phenyl isocyanates toyield compounds 9ce9i.Scheme 5
compound
28
with
2-phenyl-5-trifluoromethyloxazole-4-
carboxylic acid followed by hydrolysis resulted in the synthesis of
compound 8.
3. Results and discussion
The synthesis of compounds 9 and 9ae9i possessing the 4-(5-
phenylthiazole-2-carboxamido)cyclohexanecarboxylic acid scaf-
fold (Scheme 4) was initiated with the treatment of 2-bromo-1-(4-
nitrophenyl)ethanone (24) with hexamine and hydrochloric acid
to yield 2-amino-1-(4-nitrophenyl)ethanone hydrochloride (29).
Compound 29 when refluxed with ethyl chloroxoacetate in ethyl
acetate yielded compound 30 that was further cyclized using Law-
esson’s reagent to obtain ethyl 5-(4-nitrophenyl)thiazole-2-
3.1. In vitro pharmacology
The compounds thus synthesized were assayed using an in vitro
enzymatic assay that measured a triolein output from diolein and
radiolabeled oleoyl-CoA [16]. The DGAT1 assay was performed
using 2.5
incubated with 100
m
g of the protein from a post nuclear supernatant pre-
l of the assay buffer [100 mM TriseHCl (pH
m

S. Kandre et al. / European Journal of Medicinal Chemistry 79 (2014) 203e215
207
Scheme 4. Synthesis of compound 8. Reaction condition: (a) Br2, AlCl3, DEE, 1 h; (b) ethyl 2-amino-2-thioxoacetate MeOH, reflux, 3 h; (c) NaOH, THF, H2O, 1 h; (d) Methyl trans-4-
aminocyclohexanecarboxylate hydrochloride, HATU, TEA, DMF, 16 h; (e) Fe, NH4Cl, THF, EtOH, water, 75 ꢀC, 3 h; (f) 2-phenyl-5-trifluoromethyloxazole-4-carboxylic acid, HATU, TEA,
DMF, 16 h; (g) NaOH, MeOH, THF, 8 h.
Scheme 5. Synthesis of compounds 9 and 9ae9i. Reaction condition: (a) Hexamine, Conc. HCl, EtOH, 24 h; (b) Ethyl chloroxoacetate, TEA, EtOAc, reflux, 3 h; (c) Lawesson’s reagent,
dioxane, reflux 1.5 h; (d) 1 N NaOH, THF, 0.5 h; (e) Methyl trans 4-aminocyclohexanecarboxylate hydrochloride, IBCF, N-methyl morpholine, TEA, THF, -20 to 25 ꢀC, 16 h; (f) Fe,
NH4Cl, THF, EtOH, water, 75 ꢀC, 3 h; (g) 2-phenyl-5-trifluoromethyloxazole-4-carboxylic acid, HATU, TEA, DMF, 16 h; (h) 3,5-Difluorobenzoyl chloride; pyridine, DCM, 55 ꢀC, 16 h; (i)
3,5-Difluorobenzenesulfonyl chloride; pyridine, DCM, 55 ꢀC, 16 h; (j) Substituted phenyl isocyanate, THF, 55 ꢀC, 16 h; (k) 1 N NaOH, MeOH, THF, 8 h.
7.5), 250 mM sucrose, and 1.25 mg/ml fatty acid free BSA] con-
taining known concentration of the inhibitor and supplemented
screening of hDGAT1 inhibitors was carried out at 1.0 mM concen-
tration. Subsequent IC50 determinations were undertaken for
compounds exhibiting inhibition greater that 75% in this primary
screening. The IC50 values were determined by evaluating com-
using 2047.5 mM of 1,2-dioleoylglycerol. The hDGAT1 reaction was
initiated following an addition of 16.8 nCi of [14C]-oleoyl CoA and
after 10 min of incubation at 37 ꢀC, the reaction was terminated by
pounds at nine concentrations ranging from 0.1 nM to 1.0
are presented in Table 1.
mM and
adding 300 ml of alkaline ethanol stop solution mix (AESSM) [12.5%
of 100% non-denatured ethanol, 10% deionized water, 2.5% NaOH,
and 75% stop solution (78.4% isopropanol, 19.6% n-heptane, 2%
deionized water)]. The reaction mixture was properly mixed and
Compound 9a (DGAT1 inhibition [1
possessing the amide linker and compound 9c (DGAT1 inhibition
[1
M] ¼ 91%, IC50 ¼ 34 nM) possessing a urea linker exhibited
retention of DGAT1 potency. On the contrary, compound 9b (DGAT1
inhibition [1
M] ¼ 17%) possessing a sulfonamide linker resulted
in greatly diminished DGAT1 potency. Each of these three
m
M] ¼ 86%, IC50 ¼ 76 nM)
m
the 14C triglyceride formed was extracted using 600
m
l of heptane.
l of this extracted heptane was added to the scintillation fluid
and subjected to radioactivity measurement. The primary
250
m
m

208
S. Kandre et al. / European Journal of Medicinal Chemistry 79 (2014) 203e215
compounds possesses 2,4-difluorophenyl as the aryl substituent. In
comparison with compound 6, compound 9a possessing the amide
linker appeared 1.5-fold more active whereas compound 9c pos-
sessing the urea linker appeared 3.3-fold more active, indicating a
preference for the urea linker over the amide linker. Hence we
explored a few additional urea linked analogs (9de9i) belonging to
this
hexanecarboxylic acid scaffold (Table 1). Compound 9i (DGAT1
inhibition [1
substituent appeared well tolerated at the DGAT1 enzyme. The
presence of electron withdrawing halo-substituents on the phenyl
unique
4-(5-phenylthiazole-2-carboxamido)cyclo-
mM] ¼ 89%, IC50 ¼ 55 nM) possessing a phenyl urea
urea as seen in compounds 9e (DGAT1 inhibition [1
IC50 ¼ 15 nM), 9f (DGAT1 inhibition [1 M] ¼ 91%, IC50 ¼ 20 nM), 9g
(DGAT1 inhibition [1
M] ¼ 81%, IC50 ¼ 39 nM), and 9h (DGAT1
inhibition [1
mM] ¼ 93%,
m
m
m
M] ¼ 80%, IC50 ¼ 26 nM) led to an improvement in
Table 1
In vitro evaluation of 4-(5-phenylthiazole-2-carboxamido)cyclohexanecarboxylic
acid analogs.
DGAT1 potency. The presence of an electron donating 2-methoxy
substituent on the phenyl urea as in case of compound 9d
(DGAT1 inhibition [1
mM] ¼ 93%, IC50 ¼ 150 nM) led to a 3-fold loss
in DGAT1 potency. This result indicates that while electronegative
substituents on the phenyl urea led to an increase in DGAT1 po-
tency the presence of electron donating substituent led to a
decrease in the same. Compound 9e was further subjected to an
in vivo acute fat tolerance test and compared alongside compound 6
for its ability to reduce the triglyceride levels following a 10 ml/kg
bolus dose of olive oil.
Compound number
R
In vitro hDGAT1
IC50 (nM)
72.85
inhibition [1
mM]
3.2. Acute fat tolerance test (FTT)
9
93.7%
In an FTT fasted Swiss mice, belonging to the age range of 4e5
weeks and body weight range of 25e30 g, were administered with
either a vehicle (0.5% CMC) or the test compound [3 mg/kg] by oral
gavage. The test compounds (6 and 9e) were formulated as a sus-
9a
9b
86.4%
16.5%
75.59
pension in 0.5% CMC containing Tween 80 (25 mL). An hour later, a
bolus dose of olive oil (10 ml/kg) was given to the animals. Blood
samples were subsequently collected at 1, 2, 3, and 4 h, the plasma
was separated, and triglyceride levels were monitored using a
commercially available kit (Diasys, Germany). Percent reduction in
triglyceride levels were calculated using an area under curve
(AUC0e4h) of the test compounds and comparing it along with an
AUC0e4h of the vehicle group that is considered to be 100 percent
(Fig. 4). In this study, compound 6 exhibited an 89% triglyceride
reduction. Compound 9e, 4-(5-(4-(3-(2-fluorophenyl)ureido)
ND
9c
91.9%
92.7%
34.15
149.9
phenyl)thiazole-2-carboxamido)cyclohexanecarboxylic
acid,
exhibited 112% triglyceride reduction. Thus both the compounds
appear equi-efficacious when evaluated in vivo.
9d
4. Conclusions
In this study, we developed a novel 4-(5-phenylthiazole-2-
carboxamido)cyclohexanecarboxylic acid scaffold exhibiting
improved DGAT1 potency. A preliminary comparison in between
the 3-phenylisoxazole, 4-phenylthiazole, and 5-phenylthiazole
central cores highlighted a preference for the 5-phenylthiazole
scaffold at the DGAT1 enzyme. A subsequent study undertaken to
evaluate the urea, amide, and sulfonamide linkers of this 5-
phenylthiazole scaffold identified the urea and amide linkers to
be well tolerated at the DGAT1 enzyme when analyzed in vitro.
These linker modifications also resulted in bringing down the
molecular weight of this scaffold to the more acceptable 500 Da
range. The in vitro studies identified compound 9e (DGAT1 inhibi-
9e
9f
93.3%
90.9%
61.0%
80.2%
14.8
20.4
38.88
26.2
9g
9h
tion [1
m
M] ¼ 93%, IC50 ¼ 15 nM) to be the most potent amongst the
series of compounds studied. This compound was subsequently
evaluated in vivo for its ability to reduce triglycerides in HFD-fed
Swiss mice when dosed at 3 mg/kg. Compound 9e (Triglyceride
reduction ¼ 112%) appeared equipotent to compound 6 (Triglyc-
eride reduction ¼ 88%) at the same dose. These attributes identify
compound 9e as a potential new lead for developing DGAT1 in-
hibitors for bringing about therapeutic intervention in conditions
involving high triglyceride levels.
9i
89.3%
88.0%
54.85
114
6
e
ND: not determined.

S. Kandre et al. / European Journal of Medicinal Chemistry 79 (2014) 203e215
209
5.2. Chemistry
Unless mentioned otherwise all reactions were performed un-
der atmosphere. Unless otherwise specified all reagents were ob-
tained from Aldrich and solvents were obtained from Spectrochem
and used without further purification.
5.2.1. Trans 1,4-cyclohexanecarboxylic acid monomethyl ester (11)
To a solution of trans dimethyl 1,4-cyclohexanedicarboxylate
(9 g, 45 mmol, 1.0 equiv) in methanol (90 ml) was added a solu-
tion of methanolic KOH (3 g, 45 mmol, 1.0 equiv) and the reaction
mixture was refluxed for 5 h. Following reaction completion
methanol was evaporated under reduced pressure. The obtained
residue was dissolved in 50 ml water and extracted using diethyl
ether to remove any unreacted trans dimethyl 1,4-
cyclohexanedicarboxylate. Aqueous layer was acidified to pH 6
using 6.0 N HCl and the precipitated solid was filtered. Solid cake
was washed with 100 ml cold water and dried under high vacuum
to obtain 6.1 g (72%) white solid compound.
1H NMR (DMSO-d6, 300 MHz)
d 12.07 (bs, 1H), 3.58 (s, 3H),
2.25e2.30 (m, 1H), 2.16 (s, 1H), 1.90 (d, J ¼ 7.2 Hz, 4H), 1.25e1.41 (m,
4H); MS (ESI-) m/z 185.2 [M ꢂ H]ꢂ.
5.2.2. 4-(4-Nitrophenyl)piperidine (13)
4-Phenylpiperidine (5 g, 31.0 mmol, 1.0 equiv) was slowly added
to conc. H2SO4 (10.74 ml, 202 mmol, 6.5 equiv) at rt and the reaction
mass was stirred for 10 min. The mixture was then cooled to 0 ꢀC,
fuming nitric acid (1.540 ml, 31.0 mmol, 1.0 equiv) was added to it
over 10 min, and the reaction mixture was stirred at rt for 24 h.
Following reaction completion the reaction mass was slowly
dumped in a mixture of water and ice. The reaction mixture was
basified to pH 8e9 using aqueous 1.0 M NaOH. The resulting
mixture was extracted with EtOAc, the organic layer was washed
with brine, dried over sodium sulfate, and concentrated under
reduced pressure to obtain 4.3 g (67%) yellow colored solid that was
used as such without further purification.
Fig. 4. Effect of compound 6 and compound 9e on plasma triglyceride in an acute fat
tolerance test in Swiss mice.
1H NMR (DMSO-d6, 300 MHz)
d
8.15 (d, J ¼ 8.7 Hz, 2H), 7.51 (d,
J ¼ 8.4 Hz, 2H), 3.02 (d, J ¼ 11.7 Hz, 3H), 2.70e2.78 (m, 1H), 2.57 (t,
J ¼ 11.1 & 12 Hz, 2H), 1.69 (d, J ¼ 11.7 Hz, 2H), 1.48e1.44 (m, 2H); MS
(ESIþ) m/z 207.1 [M þ H]þ.
5. Experimental
5.2.3. Methyl 4-(4-(4-nitrophenyl)piperidine-1-carbonyl)
cyclohexanecarboxylate (14)
5.1. Analytical methods
To a solution of compound 11 (1.8 g, 9.68 mmol, 1.0 equiv) in
DMF (5 ml), HATU (5.50 g, 14.52 mmol, 1.5 equiv), compound 13
(2 g, 9.68 mmol, 1.0 equiv) and triethylamine (2.7 ml, 19.36 mmol,
2.0 equiv) were added sequentially and the reaction mass was
stirred at rt for 2 h. Following reaction completion, water was
added and extracted with EtOAc, the organic layer was washed
with water, brine, dried over sodium sulfate and concentrated
under reduced pressure to obtain pale yellow liquid. The oil was
further purified by column chromatography using 2:8 EtOAc:Pet
ether to get 1.6 g (44%) pale yellow solid compound.
1H NMR and 13C NMR spectras were recorded on a Bruker
spectrometer (300 MHz or 500 MHz) using either CDCl3 or DMSO-
d6 as the solvent. Chemical shifts, d, are reported in ppm relative to
the solvent peak. Multiplicities are indicated by s (singlet),
d (doublet), t (triplet), q (quartet), and m (multiplet). Coupling
constants, J, are reported in Hertz. Mass spectral (MS) data were
obtained on a Bruker Daltonics spectrometer using an electrospray
ionization-quadrapole-time of flight (ESI-QTOF) analyzer. All
melting points have been determined on a manually operated
Veego (VMP-1) melting point apparatus and are reported uncor-
rected. HPLC purities have been determined for the final com-
pounds using a Waters Alliances 2695 system implementing the
following method for chromatographic separation.
1H NMR (CDCl3, 300 MHz)
d
8.20 (d, J ¼ 8.7 Hz, 2H), 7.38 (d,
J ¼ 8.7 Hz, 2H), 4.85 (d, J ¼ 12.9 Hz, 1H), 4.06 (d, J ¼ 13.2 Hz, 1H),
3.69 (s, 3H), 3.18 (t, J ¼ 12.6 Hz, 1H), 2.90 (t, J ¼ 12 & 12.3 Hz, 1H),
2.50e2.68 (m, 2H), 2.32e2.41 (m, 1H), 2.05 (d, J ¼ 12.3 Hz, 2H),
1.84e2.00 (m, 4H), 1.51e1.69 (m, 6H); MS (ESIþ) m/z 375.2
[M þ H]þ.
HPLC method: Elution with 20e80% linear gradient of acetoni-
trile in 6 min followed by 20e80% linear gradient of 0.01 M
NH4OAc þ0.5% TEA, pH 5.0 with AcOH in 1 min that is continued
using an isocratic elution with 80% 0.01 M NH4OAc þ0.5% TEA, pH
5.2.4. Methyl 4-(4-(4-aminophenyl)piperidine-1-carbonyl)
cyclohexanecarboxylate (15)
To a solution of compound 14 (1.6 g, 4.27 mmol, 1.0 equiv) in
ethyl acetate (35 ml) was added 10% Pd-C (160 mg, 10% w/w) and
the mixture was subjected to hydrogenation at 40 psi for 1 h. The
5.0 with AcOH for
3
min using an Ascentis TM Express
mm operated at 1 ml/min, detection at
(50 ꢁ 4.6 mm I.D.), 2.7
288 nm.

210
S. Kandre et al. / European Journal of Medicinal Chemistry 79 (2014) 203e215
reaction mixture was filtered through celite and the filtrate was
concentrated to give a yellow residue that was purified by flash
column chromatography (3:7 Ethyl acetate/Chloroform) to obtain
450 mg (30%) off white solid compound.
dried over sodium sulfate, and the solvent was removed under
reduced pressure to obtain a crude solid that was purified by col-
umn using 1:9 Ethyl acetate:Pet ether to obtain 51 g (84%) white
solid compound.
1H NMR (DMSO-d6, 300 MHz)
d
6.87 (d, J ¼ 8.1 Hz, 2H), 6.49 (d,
1H NMR (CDCl3, 300 MHz)
d
8.45 (bs, 1H), 8.26 (d, J ¼ 7.2 Hz, 2H),
J ¼ 8.1 Hz, 2H), 4.84 (s, 2H), 4.51 (d, J ¼ 12.3 Hz, 1H), 4.03 (d,
J ¼ 10.5 Hz, 1H), 3.58 (s, 3H), 3.04 (t, J ¼ 12.3 Hz, 1H), 2.50e2.55 (m,
3H), 2.29 (s, 1H), 1.89 (s, 2H), 1.71 (s, 4H), 1.28e1.42 (m, 6H); MS
(ESIþ) m/z 345.2 (M þ H)þ; HPLC Retention time e 3.23 min, Purity
e 97.45%.
8.04 (d, J ¼ 7.2 Hz, 2H); MS (ESI-) m/z 166.0 [M ꢂ Cl]ꢂ.
5.2.8. Ethyl 3-(4-nitrophenyl)isoxazole-5-carboxylate (19)
To a solution of compound 18 (20.0 g, 10 mmol, 1.0 equiv) and
ethyl propiolate (20.2 ml, 200 mmol, 2.0 equiv) in toluene (250 ml)
was added Et3N (14.6 ml, 105 mmol, 1.05 equiv) over 10 min. The
resulting reaction mixture was heated at 80 ꢀC for 2.5 h. Following
completion the reaction mixture was cooled to rt and then further
diluted with EtOAc (500 ml). The organic layer was washed with
0.1 M HCl, water, and brine. The organic layer was dried over
anhydrous sodium sulfate, the solvent was evaporated, and the
residue was crystallized from chloroform and pet ether to afford the
title compound (23 g, 88%) as off white solid.
5.2.5. 4-(4-(4-(2-Phenyl-5-(trifluoromethyl)oxazole-4-
carboxamido)phenyl)piperidine-1-carbonyl)cyclohexanecarboxylic
acid (6)
To
a
solution of 2-phenyl-5-(trifluoromethyl)oxazole-4-
carboxylic acid 15 (74.7 mg, 0.290 mmol, 1.0 equiv) in DMF
(2 ml), HATU (165 mg, 0.435 mmol, 1.5 equiv), methyl 4-(4-(4-
aminophenyl)piperidine-1-carbonyl)cyclohexanecarboxylate
(100 mg, 0.290 mmol, 1.0 equiv) and triethylamine (0.060 ml,
0.435 mmol, 1.5 equiv) were sequentially added and the reaction
mixture was stirred at rt for 16 h. Following reaction completion,
the mixture was quenched with water and the product was sepa-
rated from the aqueous mixture using EtOAc. The organic layer was
dried over sodium sulfate, filtered, and concentrated under reduced
pressure to obtain an oily residue. The residue was subjected to
column chromatography using 1:9 EtOAc:Chloroform to get a white
solid. This solid was dissolved in THF (0.9 ml) and to it was added
1.0 N NaOH (1.16 ml,1.16 mmol, 4.0 equiv) and the resulting mixture
was stirred at rt for 16 h. Organic solvent was removed and 2 ml
water was added. The reaction mixture was acidified to pH 2 using
1.0 M HCl, precipitated solid was filtered, washed with acetone, and
dried to obtain 93 mg (56%) white solid compound.
1H NMR (CDCl3, 300 MHz)
J ¼ 8.7 Hz, 2H), 7.32 (s, 1H), 4.48 (q, J ¼ 7.2 Hz, 2H), 1.45 (t, J ¼ 7.2 Hz,
3H); MS (ESIþ) m/z 263.1 [M þ H]þ; HPLC Retention time e
5.17 min, Purity e 99.53%.
d
8.35 (d, J ¼ 9.0 Hz, 2H), 8.03 (d,
5.2.9. 3-(4-Nitrophenyl)isoxazole-5-carboxylic acid (20)
To a solution of compound 19 (20 g, 76 mmol, 1.0 equiv) in THF
(200 ml) was added 1.0 M aqueous solution of NaOH (92 ml,
92 mmol, 1.2 equiv) and stirred at rt for 1 h. The reaction mass was
acidified with 1.0 M HCl in water, the precipitated slid was filtered,
washed with water and dried to obtain 10.2 g (57%) white solid
compound.
1H NMR (DMSO-d6, 300 MHz)
d
8.38 (d, J ¼ 7.8 Hz, 2H), 8.26 (d,
J ¼ 7.8 Hz, 2H), 7.97 (s, 1H); MS (ESIþ) m/z 235.1 [M þ H]þ; HPLC
Retention time e 1.59 min, Purity e 99.99%.
1H NMR (DMSO-d6, 300 MHz)
d 12.02 (s, 1H), 10.52 (s, 1H), 8.15
(d, J ¼ 6.9 Hz, 2H), 7.72 (d, J ¼ 8.1 Hz, 2H), 7.64e7.70 (m, 3H), 7.26 (d,
J ¼ 8.1 Hz, 2H), 4.57 (d, J ¼ 15.3 Hz,1H), 4.06 (d, J ¼ 13.8 Hz,1H), 3.10
(t, J ¼ 11.3 & 12.7 Hz, 1H), 2.80 (t, J ¼ 9.6 Hz, 1H), 2.60 (d, J ¼ 12 Hz,
2H), 2.18 (s, 1H), 1.72e1.90 (m, 6H), 1.40 (bs, 6H); 13C NMR (DMSO-
5.2.10. Methyl 4-(3-(4-nitrophenyl)isoxazole-5-carboxamido)
cyclohexanecarboxylate (21)
To a solution of compound 20 (4 g, 17.08 mmol, 1.0 equiv) in THF
(20 ml) was added N-methyl morpholine (1.878 ml, 17.08 mmol,
1.0 equiv) and the reaction mixture was cooled to ꢂ20 ꢀC. To this
was added isobutyl chloroformate (2.243 ml, 17.08 mmol, 1.0 equiv)
and the reaction mixture was stirred for 30 min. To this mixture
were added methyl trans-4-aminocyclohexanecarboxylate hydro-
chloride (3.31 g, 17.08 mmol, 1.0 equiv) and triethylamine (7.14 ml,
51.2 mmol, 3.0 equiv), stirred reaction mixture at rt overnight.
Following reaction completion solvent was removed under reduced
pressure and 20 ml water was added, the product was separated
from the aqueous mixture using EtOAc. The organic layer was
washed with brine, dried over sodium sulfate, filtered, and
concentrated under reduced pressure to get a solid residue. The
residue was subjected to column chromatography using 3:7
EtOAc:Pet ether to get 2 g (32%) an off white solid.
d6, 300 MHz) d 177.04, 173.29, 161.26, 157.01, 142.42, 139.00, 137.55,
136.34, 133.00, 129.89 (2C), 127.62 (2C), 127.43 (2C), 125.25, 121.19
(2C), 120.05, 45.74, 42.51 (2C), 42.06, 41.88, 34.30 (2C), 28.82 (2C),
28.66 (2C); MS (ESIþ) m/z 569.2 (M þ H)þ; HRMS (ESIþ) calcd for
C
30H31F3N3O5 [M þ H]þ 570.2210, found 570.2185, (mean error
4.42 ppm); melting point 142e144 ꢀC; HPLC Retention time e
4.93 min, Purity e 98.42%.
5.2.6. 4-Nitrobenzaldoxime (17)
To a solution of 4-nitronenzaldehyde (50 g, 331 mmol, 1.0 equiv)
in methanol (500 ml) was added hydroxylamine hydrochloride
(34.5 g, 497 mmol, 1.5 equiv) and refluxed for 4 h. Following re-
action completion, the methanol was evaporated under reduced
pressure. The obtained material was dissolved in ethyl acetate,
washed with water, brine, dried over sodium sulfate, and the sol-
vent was removed under reduced pressure. The solid thus obtained
was then recrystallized from ethyl acetate and petroleum ether to
afford the title compound (50 g, 91%) as an off white solid
compound.
1H NMR (DMSO-d6, 300 MHz)
d
8.78 (d, J ¼ 7.8 Hz, 1H), 8.32 (d,
J ¼ 8.1 Hz, 2H), 8.10 (d, J ¼ 8.1 Hz, 2H), 7.88 (s, 1H), 3.70 (s, 1H), 3.54
(s, 3H), 2.29 (s,1H),1.95 (d, J ¼ 15.9 Hz, 4H),1.42e1.96 (m, 4H); Mass
(ESIþ) m/z 374.1 [M þ H]þ.
1H NMR (CDCl3, 300 MHz)
d
8.67 (d, J ¼ 9.0 Hz, 2H), 8.62 (s, 1H),
5.2.11. Methyl 4-(3-(4-aminophenyl)isoxazole-5-carboxamido)
cyclohexanecarboxylate (22)
8.17 (d, J ¼ 9.0 Hz, 2H); MS (ESI-) m/z 165.0 [M ꢂ H]ꢂ.
To a solution of compound 21 (2 g, 5.36 mmol, 1.0 equiv) in
ethanol (20 ml), THF (10.0 ml) and Water (10.0 ml) were added iron
(0.89 g, 16.08 mmol, 3.0 equiv) and ammonium chloride (0.86 g,
16.08 mmol, 3.0 equiv) and reaction mixture was stirred at 75 ꢀC for
3 h. The reaction mixture was filtered through celite and the filtrate
was concentrated to give a yellow residue. To this NaHCO3 solution
was added and, the product was separated from the aqueous
5.2.7. N-hydroxy-4-nitrobenzimidoyl chloride (18)
To a solution of compound 17 (50.0 g, 301 mmol, 1.0 equiv) in
DMF (250 ml) was added N-chlorosuccinimide (52.3 g, 391 mmol,
1.3 equiv) and stirred at rt for 5 h. Following reaction completion
DMF was removed under reduced pressure and the obtained ma-
terial was dissolved in ethyl acetate, washed with water, brine,

S. Kandre et al. / European Journal of Medicinal Chemistry 79 (2014) 203e215
211
mixture using EtOAc, the organic layer was washed with brine,
dried over sodium sulfate, filtered and concentrated under reduced
pressure to get solid residue. The residue was subjected to column
chromatography using 1:9 EtOAc:Chloroform to obtain 1.4 g (76%)
light brown solid compound.
cooled to rt and the precipitated solid was filtered, washed with
cold methanol, and dried to yield 2.9 g (85%) of the desired com-
pound as a white solid.
1H NMR (CDCl3, 500 MHz)
d
8.33 (d, J ¼ 8 Hz, 2H), 8.16 (d,
J ¼ 8 Hz, 2H), 7.96 (s, 1H), 4.55 (q, J ¼ 7 Hz, 2H), 1.50 (t, J ¼ 7 Hz, 3H);
1H NMR (DMSO-d6, 300 MHz)
d
8.76 (d, J ¼ 8.1 Hz, 1H), 7.56 (d,
Mass (ESIþ) m/z 278.6 [M þ H]þ.
J ¼ 8.4 Hz, 2H), 7.39 (s, 1H), 6.63 (d, J ¼ 8.4 Hz, 2H), 5.62 (s, 2H), 3.71
(s, 1H), 3.60 (s, 3H), 2.27 (s, 1H), 1.91 (d, J ¼ 16.8 Hz, 4H), 1.38e1.45
(m, 4H); MS (ESIþ) m/z 344.1 (M þ H)þ; HPLC Retention time e
3.16 min, Purity e 99.22%.
5.2.15. 4-(4-Nitrophenyl)thiazole-2-carboxylic acid (26)
Compound 25 (2.78 g, 10 mmol, 1.0 equiv) was taken in THF
(60 ml) and 1.0 M LiOH (40 ml, 40 mmol, 4.0 equiv) solution in
water was added to it. The reaction mixture was stirred at rt for 4 h.
After removing the solvent the obtained residue was diluted with
water and acidified to pH 2 using 2.0 M HCl. The solid material thus
obtained was filtered, washed with water and dried to obtain 2.4 g
(96%) of the desired compound as a white solid.
5.2.12. 4-(3-(4-(2-Phenyl-5-(trifluoromethyl)oxazole-4-
carboxamido)phenyl)isoxazole-5-carboxamido)
cyclohexanecarboxylic acid (7)
To
a
solution of 2-phenyl-5-(trifluoromethyl)oxazole-4-
carboxylic acid (112 mg, 0.437 mmol, 1.0 equiv) in DMF (2 ml),
HATU (183 mg, 0.481 mmol, 1.1 equiv), compound 22 (150 mg,
0.437 mmol, 1.0 equiv) and triethylamine (0.183 ml, 1.311 mmol,
3.0 equiv) were added sequentially and the reaction mixture was
stirred overnight at rt. Following this reaction the solvent was
removed under reduced pressure to obtain a dark brown residue
that was taken up in water and extracted using ethyl acetate. The
organic layer was separated, washed with brine, dried over anhy-
drous sodium sulfate, and concentrated to obtain a dark brown
residue that was purified using flash column chromatography (3:7
Ethyl acetate/Pet ether) to afford a white solid. This solid was dis-
solved in a mixture of THF (2 ml) and MeOH (2 ml), 1.0 N NaOH
(1.75 ml, 1.75 mmol, 4.0 equiv) was added, and the mixture was
stirred overnight at rt. Following reaction completion, the organic
solvent was removed and 2 ml of water was added. The reaction
mixture was acidified to pH 2.0 using 1.0 M HCl, the precipitated
solid was filtered, washed with acetone, and dried to obtain 64 mg
(56%) of white solid compound.
1H NMR (DMSO-d6, 300 MHz)
d 12.01 (s, 1H), 8.78 (s, 1H), 8.28e
8.32 (m, 4H); Mass (ESIþ) m/z 250.6 [M þ H]þ.
5.2.16. Methyl 4-(4-(4-nitrophenyl)thiazole-2-carboxamido)
cyclohexanecarboxylate (27)
To a solution of compound 26 (800 mg, 3.20 mmol, 1.0 equiv) in
DMF (8 ml), HATU (1337 mg, 3.52 mmol, 1.1 equiv), methyl 4-
aminocyclohexanecarboxylate hydrochloride (619 mg, 3.20 mmol,
1.0 equiv) and triethylamine (0.891 ml, 6.39 mmol, 2.0 equiv) were
added sequentially and the reaction mixture was stirred at rt for
16 h. Following reaction completion, the reaction mixture was
diluted with water and extracted using ethyl acetate. The organic
layer was separated, washed with water, dried over anhydrous
sodium sulfate, and filtered. The solvent was removed under
reduced pressure to obtain a solid that was stirred in methanol at rt
and filtered to obtain the title compound as an off-white solid
(750 mg, 58%).
1H NMR (DMSO-d6, 300 MHz)
1H), 8.36 (bs, 4H), 3.80 (s, 1H), 3.61 (s, 3H), 2.29 (s, 1H), 1.88e2.00
(m, 4H), 1.42e1.60 (m, 4H); Mass (ESIþ) m/z 390.1 [M þ H]þ; HPLC
Retention time e 5.13 min, Purity e 96.28%.
d
8.78 (d, J ¼ 8.4 Hz, 1H), 8.73 (s,
1H NMR (DMSO-d6, 300 MHz)
d
12.10 (s, 1H), 10.81 (s, 1H), 8.87
(d, J ¼ 8.1 Hz, 1H), 8.17 (d, J ¼ 7.2 Hz, 2H), 8.02 (d, J ¼ 8.7 Hz, 2H),
7.92 (d, J ¼ 8.4 Hz, 2H), 7.62e7.70 (m, 3H), 7.56 (s, 1H), 3.72 (s, 1H),
2.16 (s, 1H), 1.91 (d, J ¼ 19.8 Hz, 4H), 1.38e1.44 (m, 4H); 13H NMR
(DMSO-d6, 300 MHz)
d
176.93,162.06,161.00, 158.37, 157.24, 155.58,
5.2.17. Methyl 4-(4-(4-aminophenyl)thiazole-2-carboxamido)
cyclohexanecarboxylate (28)
138.29, 137.10, 133.04, 130.60, 128.70, 129.20 (2C), 127.54 (2C),
127.75 (2C), 123.83, 119.73 (2C), 117.23, 110.53, 48.54, 43.07, 31.38
(2C), 28.43 (2C); MS (ESIþ) m/z 569.2 (M þ H)þ; HRMS (ESIþ) calcd
for C28H24F3N4O6 [M þ H]þ 569.1642, found 569.1626, (mean error
3.03 ppm); melting point >250 ꢀC; HPLC Retention time e
4.89 min, Purity e 93.82%.
Compound 27 (710 mg, 1.82 mmol, 1.0 equiv) was dissolved in a
mixture of ethanol (14 ml), THF (7 ml) and water (7 ml). To this
solution ammonium chloride (294 mg, 5.47 mmol, 3.0 equiv) and
iron powder (306 mg, 5.47 mmol, 3.0 equiv) was added and reac-
tion mixture was stirred at 75 ꢀC for 3 h. Following completion the
reaction mixture was cooled, filtered through celite, and the solvent
was removed under reduced pressure to obtain a dark yellow res-
idue. This residue was taken up in sodium bicarbonate solution and
extracted using ethyl acetate. The organic layer was separated,
dried over anhydrous sodium sulfate, and concentrated to give a
dark brown residue that was purified using flash column chroma-
tography (1:9 ethyl acetate/chloroform) to afford a yellow solid that
was crystallized in DCM:Pet ether to yield the title compound
(533 mg, 81%) as a pale yellow solid.
5.2.13. 2-Bromo-1-(4-nitrophenyl)ethanone (24)
To a solution of 4-nitroacetophenone (50 g, 302 mmol,1.0 equiv)
in diethyl ether (500 ml) was added catalytic amount of aluminum
chloride (2 g, 15 mmol, 0.05 equiv) and the reaction mixture was
stirred for 10 min. To this bromine (15.4 ml, 302 mmol) was added
using an addition funnel over 30 min at rt and the resulting mixture
was stirred for 1 h. Following reaction completion the reaction
mixture was quenched using aqueous sodium bicarbonate. The
ether layer was separated, dried over sodium sulfate, and evapo-
rated under reduced pressure. The solid thus obtained was
recrystallized using ethyl acetate and petroleum ether to afford the
title compound (43 g, 62%).
1H NMR (DMSO-d6, 300 MHz)
d
8.52 (d, J ¼ 8.4 Hz, 1H), 7.96 (s,
1H), 7.75 (d, J ¼ 8.4 Hz, 2H), 6.62 (d, J ¼ 8.4 Hz, 2H), 5.35 (s, 2H), 3.78
(s, 1H), 3.61 (s, 3H), 2.80 (s, 1H), 1.87e1.99 (m, 4H), 1.38e1.60 (m,
4H); Mass (ESIþ) m/z 360.1 [M þ H]þ; HPLC Retention time e
3.80 min, Purity e 99.50%.
1H NMR (CDCl3, 300 MHz)
d
8.36 (d, J ¼ 8.1 Hz, 2H), 8.18 (d,
J ¼ 8.1 Hz, 2H), 4.48 (s, 2H); Mass (ESIþ) m/z 245.0 [M þ H]þ.
5.2.18. 4-(4-(4-(2-Phenyl-5-(trifluoromethyl)oxazole-4-
carboxamido)phenyl)thiazole-2-carboxamido)
cyclohexanecarboxylic acid (8)
5.2.14. Ethyl 4-(4-nitrophenyl)thiazole-2-carboxylate (25)
To a solution of compound 24 (3 g, 12.2 mmol, 1.0 equiv) in
methanol (60 ml) was added ethyl 2-amino-2-thioxoacetate
(1.64 g, 12.2 mmol, 1.0 equiv) and reaction mixture was refluxed
for 2 h. Following reaction completion, the reaction mixture was
To
a
solution of 2-phenyl-5-(trifluoromethyl)oxazole-4-
carboxylic acid (107 mg, 0.417 mmol, 1.0 equiv) in DMF (5 ml),
HATU (175 mg, 0.459 mmol, 1.1 equiv), compound 28 (150 mg,

212
S. Kandre et al. / European Journal of Medicinal Chemistry 79 (2014) 203e215
0.417 mmol, 1.0 equiv) and triethylamine (0.116 ml, 0.835 mmol,
2.0 equiv) were added sequentially and reaction mixture was stir-
red overnight at rt. The reaction was then quenched with water and
extracted using ethyl acetate. The organic layer was separated,
dried over sodium sulfate, and evaporated under reduced pressure
to obtain a crude compound that was purified using flash column
chromatography (3:7 Ethyl acetate/Petroleum ether) to afford a
white solid. This solid was taken in THF (5 ml) and added 1.0 M
aqueous solution of NaOH (1.67 ml, 1.67 mmol, 4.0 equiv) and re-
action mixture was stirred at rt overnight. The solvent was removed
and the obtained residue was diluted with water and acidified to
pH 2 using 2.0 M HCl. The solid material thus obtained was filtered,
washed with water and acetone to obtain 87 mg (36%) of the title
compound as an off white solid.
subsequently cooled, added to water, and neutralized with a satu-
rated solution of sodium carbonate. The product was separated
from the aqueous mixture using EtOAc. The organic layer was dried
over sodium sulfate, filtered, and concentrated under reduced
pressure to get a dark brown residue. The residue was subjected to
column chromatography using 2:8 EtOAc:Pet ether to yield a dark
brown colored solid that was stirred in methanol and filtered to
obtain the title compound as a light brown solid (3.4 g, 69%).
1H NMR (DMSO-d6, 300 MHz)
d
8.34 (d, J ¼ 8.4 Hz, 2H), 8.30 (s,
1H), 7.82 (d, J ¼ 8.4 Hz, 2H), 4.54 (q, J ¼ 7.2 Hz, 2H),1.49 (t, J ¼ 7.2 Hz,
3H); Mass (ESIþ) m/z 279.0 [M þ H]þ; HPLC Retention time e
7.02 min, Purity e 96.90%.
5.2.22. 5-(4-Nitrophenyl)thiazole-2-carboxylic acid (32)
1H NMR (DMSO-d6, 300 MHz)
d
12.12 (s, 1H), 10.69 (s, 1H), 8.66
To a solution of compound 31 (3 g, 10.78 mmol) in THF (250 ml)
was added 1.0 M aqueous NaOH (21.5 ml, 21.56 mmol, 2.0 equiv)
and the reaction mixture was stirred for 30 min. The reaction
mixture was acidified to pH 2 using 1.0 M HCl. The resulting
mixture was extracted with EtOAc, the organic layer was washed
with brine, dried over sodium sulfate, and concentrated under
reduced pressure to obtain a brown colored solid. Recrystallization
with EtOAc/Pet ether yielded the desired product as a light brown
solid (2.2 g, 82%).
(d, J ¼ 8.1 Hz, 1H), 8.36 (s, 1H), 8.17 (d, J ¼ 6.6 Hz, 2H), 8.13 (d,
J ¼ 8.1 Hz, 2H), 7.97 (d, J ¼ 7.8 Hz, 2H), 7.66e7.68 (m, 3H), 3.77 (s,
1H), 2.18 (s, 1H), 1.88e1.99 (m, 4H), 1.35e1.56 (m, 4H); 13H NMR
(DMSO-d6, 300 MHz)
d 176.83 (2C), 164.18, 161.28, 158.67, 157.16,
155.28, 138.51, 137.40, 133.04, 130.22, 129.91 (2C), 127.64 (2C),
127.25 (2C), 125.23, 121.03 (2C), 120.83, 119.13, 48.44, 42.17, 31.48
(2C), 28.23 (2C); Mass (ESIþ) m/z 585.1 [M þ H]þ; HRMS (ESIþ)
calcd for C28H24F3N4O5S [M þ H]þ 585.1414, found 585.1377, (mean
error 5.91 ppm); melting point: 270e272 ꢀC; HPLC Retention time
e 4.64 min, Purity e 98.51%.
1H NMR (DMSO-d6, 300 MHz)
d 14.14 (bs, 1H), 8.68 (s, 1H), 8.32
(d, J ¼ 8.7 Hz, 2H)), 8.08 (d, J ¼ 8.7 Hz, 2H); Mass (ESIþ) m/z 251.0
[M þ H]þ.
5.2.19. 2-amino-1-(4-nitrophenyl)ethanone hydrochloride (29)
To a stirred solution of compound 24 (40 g, 164 mmol, 1.0 equiv)
in DCM (600 ml) was added hexamine (27.6 g, 196 mmol, 1.2 equiv)
and reaction mixture was stirred at rt for 8 h. Following completion
the reaction mass was filtered, washed with 200 ml DCM, and dried
on a rotavap to give an off-white solid that was dissolved in ethanol
(600 ml) and conc. HCl (35 ml, 410 mmol, 2.5 equiv). The reaction
mixture was stirred at rt for 16 h. The off white solid thus obtained
was filtered, washed with water, and dried to give an off white solid
that was crystallized in water to yield 23 g (65%) of the title com-
pound as a light brown solid.
5.2.23. Methyl 4-(5-(4-nitrophenyl)thiazole-2-carboxamido)
cyclohexanecarboxylate (33)
To a solution of 5-(4-nitrophenyl)thiazole-2-carboxylic acid 32
(1.6 g, 6.39 mmol, 1.0 equiv) in THF (10 ml) was added N-methyl
morpholine (0.703 ml, 6.39 mmol, 1.0 equiv) at rt and the reaction
mixture was cooled to ꢂ20 ꢀC. To this isobutyl chloroformate
(0.840 ml, 6.39 mmol, 1.0 equiv) was added and the reaction
mixture was stirred for 20 min at ꢂ20 ꢀC. To this reaction mass was
added a solution of methyl trans 4-aminocyclohexanecarboxylate
hydrochloride (1.238 g, 6.39 mmol, 1.0 equiv) in THF (20 ml) after
neutralizing with triethylamine (1.33 ml, 9.58 mmol,1.5 equiv). This
mixture was stirred at ꢂ20 to ꢂ30 ꢀC for 5 min. Following this the
reaction mixture was gradually warmed up to rt over a period of
1 h. Following reaction completion, the organic solvent was
removed under reduced pressure and the crude material was
subjected to column chromatography using 1:9 EtOAc:CHCl3 to get
the desired compound as an off white solid (1.65 g, 66%).
1H NMR (DMSO-d6, 300 MHz)
d
8.53 (bs, 2H), 8.40 (d, J ¼ 9 Hz,
2H), 8.27 (d, J ¼ 9 Hz, 2H), 4.68 (s, 2H); Mass (ESIþ) m/z 181.1
[M þ H]þ.
5.2.20. Ethyl 2-((2-(4-nitrophenyl)-2-oxoethyl)amino)-2-
oxoacetate (30)
To a solution of 2-amino-1-(4-nitrophenyl)ethanone hydro-
chloride 29 (20 g, 92 mmol, 1.0 equiv) in ethyl acetate (400 ml) was
added triethylamine (15.5 ml, 111 mmol, 1.2 equiv) and reaction
mixture was cooled to 5 ꢀC. To this mixture ethyloxalylchloride
(12.4 ml, 111 mmol, 1.2 equiv) was added dropwise and the reaction
mixture was refluxed for 3 h. The reaction mass was cooled,
quenched with water, and the resulting organic layer was sepa-
rated, dried over sodium sulfate, and evaporated under reduced
pressure to obtain a dark brown oil. The oil was further purified by
column chromatography using 3:7 EtOAc:Pet ether to get a yellow
solid. The solid was further crystallized in EtOAc/Pet ether to yield
the desired compound as a yellow colored solid (8.9 g, 34%).
1H NMR (DMSO-d6, 300 MHz)
d
8.84 (d, J ¼ 8.7 Hz, 1H), 8.65 (s,
1H), 8.31 (d, J ¼ 8.7 Hz, 2H), 8.08 (d, J ¼ 8.7 Hz, 2H), 3.76 (m, 1H),
3.60 (s, 3H), 2.55 (s, 1H), 1.95 (d, J ¼ 10.5 Hz, 2H), 1.86 (d, J ¼ 10.5 Hz,
2H), 1.40e1.53 (m, 4H); Mass (ESIþ) m/z 390.1 [M þ H]þ.
5.2.24. Methyl 4-(5-(4-aminophenyl)thiazole-2-carboxamido)
cyclohexanecarboxylate (34)
To a solution of compound 33 (1.6 g, 4.11 mmol, 1.0 equiv) in
EtOH (25 ml), THF (12.5 ml) and water (12.5 ml) were added iron
(0.918 g, 16.43 mmol, 4.0 equiv) and ammonium chloride (0.879 g,
16.43 mmol, 4.0 equiv). The resulting reaction mixture was stirred
at 75 ꢀC for 3 h. Following this the resulting mixture was cooled,
filtered through celite, and solvent was removed under pressure to
get a dark brown residue. The residue was taken in aqueous sodium
bicarbonate solution and extracted using ethyl acetate. The organic
layer was dried using sodium sulfate and concentrated to obtain a
brown residue that was purified by column chromatography using
2:8 EtOAc:CHCl3 to afford the title compound as a yellow solid
(1.06 g, 72%).
1H NMR (DMSO-d6, 300 MHz)
d
9.19 (t, J ¼ 5.4 Hz, 1H), 8.35 (d,
J ¼ 8.7 Hz, 2H), 8.20 (d, J ¼ 8.7 Hz, 2H), 4.75 (d, J ¼ 5.4 Hz, 2H), 4.26
(q, J ¼ 7.2 Hz, 2H), 1.27 (t, J ¼ 7.2 Hz, 3H); Mass (ESIþ) m/z 303.1
[M þ Na]þ; HPLC Retention time e 2.83 min, Purity e 91.74%.
5.2.21. Ethyl 5-(4-nitrophenyl)thiazole-2-carboxylate (31)
To a solution of ethyl 2-((2-(4-nitrophenyl)-2-oxoethyl)amino)-
2-oxoacetate 28 (5 g, 17.84 mmol, 1.0 equiv) in 1,4-Dioxane (100 ml)
was added Lawesson’s Reagent (7.9 g, 19.64 mmol, 1.1 equiv) and
the reaction mixture was refluxed for 2 h. The reaction mixture was
1H NMR (CDCl3, 300 MHz)
7.06 (d, J ¼ 8.4 Hz, 1H), 6.72 (d, J ¼ 8.4 Hz, 2H), 3.94e3.96 (m, 1H),
d
7.83 (s, 1H), 7.41 (d, J ¼ 8.4 Hz, 2H),

S. Kandre et al. / European Journal of Medicinal Chemistry 79 (2014) 203e215
213
3.91 (s, 2H), 3.70 (s, 3H), 2.27e2.35 (m, 1H), 2.18 (d, J ¼ 9.6 Hz, 2H),
2.10 (d, J ¼ 15.6 Hz, 2H), 1.57e1.68 (m, 2H), 1.27e1.40 (m, 2H); Mass
(ESIþ) m/z 360.1 [M þ H]þ; HPLC Retention time e 3.75 min, Purity
e 99.83%.
Mass (ESIþ) m/z 486.1 [M þ H]þ; HRMS (ESIþ) calcd for
24H22F2N3O4S [M þ H]þ 486.1294, found 486.1277, (mean error
C
3.25 ppm); melting point >250 ꢀC; HPLC Retention time e
3.90 min, Purity e 98.24%.
5.2.25. 4-(5-(4-(2-Phenyl-5-(trifluoromethyl)oxazole-4-
carboxamido)phenyl)thiazole-2-carboxamido)
cyclohexanecarboxylic acid (9)
5.2.27. 4-(5-(4-(3,5-difluorophenylsulfonamido)phenyl)thiazole-2-
carboxamido)cyclohexanecarboxylic acid (9b)
To a solution of compound 34 (90 mg, 0.250 mmol, 1.0 equiv) in
DCM (2 ml) were added pyridine (0.06 ml, 0.750 mmol, 3.0 equiv)
and 3,5-difluorobenzenesulfonyl chloride (80 mg, 0.376 mmol,
1.5 equiv) and the resulting mixture was stirred at 55 ꢀC for 16 h.
Following completion the reaction mass was diluted with DCM,
washed with water, followed by brine and dried over anhydrous
sodium sulfate. The solvent was removed under reduced pressure
and the crude solid so obtained was purified by flash column
chromatography (3:7 EtOAc/Petroleum ether) to provide the
methyl ester as a white solid. This solid was taken in THF (5 ml) and
to it 1.0 M aqueous solution of LiOH (1 ml, 1.0 mmol, 4.0 equiv) was
added and the reaction mixture was stirred overnight at rt. The
solvent was removed and the resulting residue was diluted with
water and acidified to pH 2 using 2.0 M HCl. The solid material thus
obtained was filtered, washed with water and acetone to obtain
70 mg (54%) as off white solid.
To
a solution of 2-phenyl-5-(trifluoromethyl) oxazole-4-
carboxylic acid (64.4 mg, 0.250 mmol, 1.0 equiv) in DMF (3 ml),
HATU (105 mg, 0.275 mmol, 1.1 equiv), methyl 4-(5-(4-
aminophenyl)thiazole-2-carboxamido)cyclohexanecarboxylate 34
(90 mg, 0.250 mmol, 1.0 equiv) and triethylamine (0.070 ml,
0.501 mmol, 2.0 equiv) were added sequentially and the reaction
mixture was stirred at rt for 16 h. Following completion the reac-
tion mixture was quenched with water and extracted using ethyl
acetate. The organic layer was separated, dried over sodium sulfate,
and evaporated under reduced pressure to obtain a crude com-
pound that was purified using flash column chromatography (3:7
Ethyl acetate/Pet ether) to afford a white solid. This solid was taken
in THF (5 ml), 1.0 M aqueous solution of NaOH (1.0 ml, 1.0 mmol,
4.0 equiv) was added, and the resulting mixture was stirred over-
night at rt. The solvent was removed and the obtained residue was
diluted with water and acidified to pH 2 using 2.0 M HCl. The solid
material thus obtained was filtered, washed with water and
acetone to obtain the title compound (52 mg, 35%) as an off white
solid.
1H NMR (DMSO-d6, 300 MHz)
d 12.08 (s, 1H), 10.78 (s, 1H), 8.66
(d, J ¼ 8.4 Hz, 1H), 8.29 (s, 1H), 7.69 (d, J ¼ 8.4 Hz, 2H), 7.62e7.65 (m,
1H), 7.49e7.51 (m, 2H), 7.21 (d, J ¼ 8.4 Hz, 2H), 3.73 (s, 1H), 2.12 (s,
1H), 1.94 (d, J ¼ 11.4 Hz, 2H), 1.80 (d, J ¼ 17.1 Hz, 2H), 1.36e1.50 (m,
1H NMR (DMSO-d6, 300 MHz)
d 12.09 (bs, 1H), 10.77 (s, 1H), 8.68
4H); 13C NMR (DMSO-d6, 300 MHz)
d 176.82, 164.20, 162.82, 160.90,
(d, J ¼ 8.7 Hz, 1H), 8.38 (s, 1H), 8.17 (d, J ¼ 6.3 Hz, 2H), 7.96 (d,
J ¼ 8.7 Hz, 2H), 7.82 (d, J ¼ 8.4 Hz, 2H), 7.62e7.70 (m, 3H), 3.75 (s,
1H), 2.13 (bs, 1H), 1.95 (d, J ¼ 12 Hz, 2H), 1.85 (d, J ¼ 12.6 Hz, 2H),
158.60, 143.32, 142.93, 139.88, 138.12, 128.38 (2C), 126.97, 121.12
(2C), 111.18, 110.81, 109.61, 48.38, 42.08, 31.36 (2C), 28.16 (2C); Mass
(ESIþ) m/z 522.1 [M þ H]þ; HRMS (ESIþ) calcd for C23H22F2N3O5S2
[M þ H]þ 522.0963, found, 522.0932 (mean error 6.00 ppm);
melting point >250 ꢀC; HPLC Retention time e 3.58 min, Purity e
98.98%.
1.38e1.55 (m, 4H); 13H NMR (DMSO-d6, 300 MHz)
d 175.59, 162.63,
161.29, 158.69, 157.30, 143.79, 139.82 (2C), 139.09, 137.26, 133.06,
129.92 (2C), 127.76 (2C), 127.65 (2C), 126.79, 125.22, 121.47 (2C),
117.22, 51.85, 48.28, 41.92, 31.26, 29.47, 28.10; Mass (ESIþ) m/z
585.2 [M þ H]þ; HRMS (ESIþ) calcd for C28H24F3N4O5S [M þ H]þ
585.1414, found 585.1380, (mean error 4.78 ppm); melting point
>280 ꢀC; HPLC Retention time e 5.21 min, Purity e 99.18%.
5.2.28. General procedure for the synthesis of urea derivatives of
phenylthiazole series
To a solution of compound 34 (90 mg, 0.250 mmol, 1.0 equiv) in
THF (5 ml) was added appropriate phenyl isocyanate (1.1 equiv) and
reaction mixture was stirred at 55 ꢀC for 16 h. Following reaction
completion the precipitated solid was filtered and washed with
diethyl ether to obtain a white solid as ester compound. To a so-
lution of ester compound (1.0 equiv) in THF (3 ml) and methanol
(3 ml) was added 1 N aqueous NaOH (4 equiv) solution and stirred
at rt for 16 h. The solvent was removed and the obtained residue
was diluted with water and acidified to pH 2.0 using 2.0 M HCl. The
material thus obtained was filtered and washed with acetone to
obtain the desired product.
5.2.26. 4-(5-(4-(3,5-Difluorobenzamido)phenyl)thiazole-2-
carboxamido)cyclohexanecarboxylic acid (9a)
To a solution of compound 34 (90 mg, 0.250 mmol, 1.0 equiv) in
DCM (2 ml) were added pyridine (0.06 ml, 0.750 mmol, 3.0 equiv)
and 3,5-difluorobenzoyl chloride (0.045 ml, 376 mmol, 1.5 equiv)
and the resulting mass was stirred at 55 ꢀC for 16 h. Following
completion the reaction mass was diluted with DCM, washed with
water followed by brine, and dried over anhydrous sodium sulfate.
The solvent was removed under reduced pressure and the crude
solid so obtained was purified by flash column chromatography
(3:7 EtOAc/Pet ether) to provide the methyl ester as a white colored
solid. This solid was taken in THF (5 ml) and added to 1.0 M aqueous
NaOH solution (1.0 ml, 1.0 mmol, 4.0 equiv) and the reaction
mixture was stirred at rt overnight. The solvent was removed, the
residue was diluted with water, and acidified to pH 2 using 2.0 M
HCl. The resulting solid was filtered, washed with water and
acetone to obtain (66.6 mg, 55%) of the title compound as an off
white solid.
5.2.28.1. 4-(5-(4-(3-(3,5-Difluorophenyl)ureido)phenyl)thiazole-2-
carboxamido)cyclohexanecarboxylic
acid
(9c). Prepared
as
described above in the general procedure using 3,5-difluorophenyl
isocyanate (43 mg, 0.275 mmol, 1.1 equiv) as the substituted iso-
cyanate to afford the title compounds (81.5 mg, 65%) as off white
solid.
1H NMR (DMSO-d6, 300 MHz)
d 12.09 (s, 1H), 9.16 (s, 1H), 9.11 (s,
1H NMR (DMSO-d6, 300 MHz)
d
12.09 (s, 1H), 10.54 (s, 1H), 8.67
1H), 8.65 (d, J ¼ 8.7 Hz, 1H), 8.30 (s, 1H), 7.71 (d, J ¼ 8.4 Hz, 2H), 7.57
(d, J ¼ 8.4 Hz, 2H), 7.23 (s, 1H), 7.19 (s, 1H), 6.78e6.84 (m, 1H), 3.74
(s, 1H), 2.13 (s, 1H), 1.95 (m, J ¼ 12.6 Hz, 2H), 1.83 (d, 2H), 1.37e1.51
(d, J ¼ 8.4 Hz, 1H), 8.36 (s, 1H), 7.89 (d, J ¼ 8.7 Hz, 2H), 7.80 (d,
J ¼ 8.7 Hz, 2H), 7.69e7.72 (m, 2H), 7.52e7.58 (m, 1H), 3.75 (s, 1H),
2.13 (s, 1H), 1.95 (d, J ¼ 12.3 Hz, 2H), 1.84 (d, 2H), 1.37e1.52 (m, 4H);
(m, 4H); 13C NMR (DMSO-d6, 300 MHz)
d 176.86, 164.76, 162.07,
13C NMR (DMSO-d6, 300 MHz)
d
176.87, 164.38, 164.23, 163.44,
166.55, 158.71, 152.50, 144.17, 142.71, 140.64, 139.14, 127.93 (2C),
124.55, 119.28 (2C), 101.73, 101.34, 97.40, 48.36, 42.10, 31.39 (2C),
28.18 (2C); Mass (ESIþ) m/z 501.1 [M þ H]þ; HRMS (ESIþ) calcd for
162.53, 161.10, 160.93, 158.67, 143.88, 139.92, 139.62, 127.72 (2C),
126.35, 121.25 (2C), 111.83, 111.61, 48.40, 42.10, 31.39 (2C), 28.18
(2C).
C
24H23F2N4O4S [M þ H]þ 501.1403, found 501.1380, (mean error

214
S. Kandre et al. / European Journal of Medicinal Chemistry 79 (2014) 203e215
4.62 ppm); melting point >250 ꢀC; HPLC Retention time e
3.98 min, Purity e 99.23%.
1H NMR (DMSO-d6, 300 MHz)
d 12.10 (s,1H), 9.27 (s,1H), 8.64 (d,
J ¼ 8.4 Hz, 1H), 8.58 (s, 1H), 8.29 (s, 1H), 8.03e8.11 (m, 1H), 7.71 (d,
J ¼ 8.1 Hz, 2H), 7.56 (d, J ¼ 8.4 Hz, 2H), 7.29e7.35 (m, 1H), 7.00e7.06
(m,1H), 3.74 (s, 1H), 2.13 (s, 1H), 1.95 (d, J ¼ 11.1 Hz, 2H),1.83 (d, 2H),
5.2.28.2. 4-(5-(4-(3-(2-Methoxyphenyl)ureido)phenyl)thiazole-2-
1.37e1.51 (m, 4H); 13C NMR (DMSO-d6, 300 MHz)
d 176.88, 162.00,
carboxamido)cyclohexanecarboxylic
acid
(9d). Prepared
as
158.72, 152.59, 144.21, 140.86, 139.07, 127.99 (2C), 124.33, 122.70,
122.61, 118.88 (2C), 111.68, 111.37, 104.61, 103.98, 48.37, 42.12, 31.40
(2C), 28.18 (2C); Mass (ESIþ) m/z 501.1 [M þ H]þ; HRMS (ESIþ)
calcd for C24H23F2N4O4S [M þ H]þ 501.1403, found 501.1380, (mean
error 4.54 ppm); melting point ꢃ 250 ꢀC; HPLC Retention time e
3.71 min, Purity e 99.16%.
described above in the general procedure using 2-methoxyphenyl
isocyanate (41 mg, 0.275 mmol, 1.1 equiv) as the substituted iso-
cyanate to afford the title compounds (74.8 mg, 60%) as off white
solid.
1H NMR (DMSO-d6, 300 MHz)
d 12.09 (s, 1H), 9.56 (s, 1H), 8.64
(d, J ¼ 8.4 Hz, 1H), 8.29 (s, 2H), 8.13 (d, J ¼ 7.5 Hz, 1H), 7.69 (d,
J ¼ 8.7 Hz, 2H), 7.56 (d, J ¼ 8.7 Hz, 2H), 7.03 (d, J ¼ 8.1 Hz, 1H), 6.88e
6.99 (m, 2H), 3.89 (s, 3H), 3.74 (s,1H), 2.13 (s,1H),1.94 (d, J ¼ 11.1 Hz,
2H), 1.85 (d, J ¼ 10.8 Hz, 2H), 1.37e1.51 (m, 4H); 13C NMR (DMSO-d6,
5.2.28.6. 4-(5-(4-(3-(2,4,5-Trifluorophenyl)ureido)phenyl)thiazole-2-
carboxamido)cyclohexanecarboxylic
acid
(9h). Prepared
as
300 MHz)
d 176.84, 161.90, 158.74, 152.62, 148.16, 144.32, 141.29,
described above in the general procedure using 2,4,5-
trifluorophenyl isocyanate (48 mg, 0.275 mmol, 1.1 equiv) as the
substituted isocyanate to afford the title compounds (90.7 mg, 70%)
as off white solid.
138.96, 128.86, 127.99 (2C), 124.00, 122.49, 121.01, 118.82, 118.65,
(2C), 111.21, 56.24, 48.37, 42.10, 31.40 (2C), 28.18 (2C); Mass (ESIþ)
m/z 495.1 [M þ H]þ; HRMS (ESIþ) calcd for C25H27N4O5S [M þ H]þ
495.1697, found 495.1649, (mean error 9.53 ppm); melting point
>250 ꢀC; HPLC Retention time e 3.71 min, Purity e 98.64%.
1H NMR (DMSO-d6, 300 MHz)
d 12.09 (s,1H), 9.32 (s,1H), 8.78 (s,
1H), 8.65 (d, J ¼ 8.4 Hz, 1H), 8.30 (s, 1H), 8.14e8.24 (m, 1H), 7.72 (d,
J ¼ 8.4 Hz, 2H), 7.60e7.66 (m, 1H), 7.56 (d, J ¼ 8.4 Hz, 2H), 3.74 (s,
1H), 2.13 (s, 1H), 1.95 (d, J ¼ 12.3 Hz, 2H), 1.83 (d, 2H), 1.37e1.55 (m,
5.2.28.3. 4-(5-(4-(3-(2-Fluorophenyl)ureido)phenyl)thiazole-2-
4H); 13C NMR (DMSO-d6, 300 MHz)
d 176.85, 162.09, 158.70, 152.29,
carboxamido)cyclohexanecarboxylic
acid
(9e). Prepared
as
144.12, 140.48, 139.15, 128.01 (2C), 124.61, 124.60, 118.99 (2C),
109.09, 108.81, 106.34, 105.98, 105.71, 48.36, 42.10, 31.39 (2C), 28.18
(2C); Mass (ESIþ) m/z 519.1 [M þ H]þ; HRMS (ESIþ) calcd for
described above in the general procedure using 2-fluorophenyl
isocyanate (38 mg, 0.275 mmol, 1.1 equiv) as the substituted iso-
cyanate to afford the title compounds (75.7 mg, 63%) as off white
solid.
C
24H22F3N4O4S [M þ H]þ 519.1308, found 519.1283, (mean error
4.80 ppm); melting point ꢃ 250 ꢀC; HPLC Retention time e
4.08 min, Purity e 98.29%.
1H NMR (DMSO-d6, 300 MHz)
d 12.09 (s, 1H), 9.31 (s, 1H), 8.64
(d, J ¼ 9.0 Hz, 2H), 8.30 (s, 1H), 8.15 (t, J ¼ 7.8 & 8.4 Hz, 1H), 8.71 (d,
J ¼ 8.4 Hz, 2H), 7.55 (d, J ¼ 8.7 Hz, 2H), 7.22e7.28 (m, 1H), 7.16 (t,
J ¼ 7.5 & 7.8 Hz 1H), 7.02e7.05 (m, 1H), 3.75 (s, 1H), 2.13 (s, 1H), 1.94
(m, J ¼ 11.4 Hz, 2H), 1.83 (d, 2H), 1.37e1.51 (m, 4H); 13C NMR
5.2.28.7. 4-(5-(4-(3-Phenylureido)phenyl)thiazole-2-carboxamido)
cyclohexanecarboxylic acid (9i). Prepared as described above in the
general procedure using phenyl isocyanate (33 mg, 0.275 mmol,
1.1 equiv) as the substituted isocyanate to afford the title com-
pounds (74 mg, 64%) as off white solid.
(DMSO-d6, 300 MHz) d 176.85,162.01,158.72,152.45,144.21,140.86,
139.08, 128.01 (2C), 127.83, 127.69, 124.98, 124.33, 123.23, 121.15,
118.86 (2C), 115.60, 48.36, 42.10, 31.39 (2C), 28.18 (2C); Mass (ESIþ)
m/z 483.1 [M þ H]þ; HRMS (ESIþ) calcd for C24H24F1N4O4S
[M þ H]þ 483.1497, found 483.1469, (mean error 6.72 ppm);
melting point >250 ꢀC; HPLC Retention time e 3.63 min, Purity e
98.33%.
1H NMR (DMSO-d6, 300 MHz)
d 12.10 (s, 1H), 8.93 (s, 1H), 8.74 (s,
1H), 8.65 (d, J ¼ 8.7 Hz, 1H), 8.29 (s, 1H), 7.70 (d, J ¼ 8.4 Hz, 2H), 7.57
(d, J ¼ 8.7 Hz, 2H), 7.46 (d, J ¼ 8.1 Hz, 2H), 7.29 (t, J ¼ 7.5 Hz & 8.1 Hz,
2H), 6.99 (t, J ¼ 6.9 Hz & 7.2 Hz, 1H), 3.75 (s, 1H), 2.13 (s, 1H), 1.94 (d,
J ¼ 11.4 Hz, 2H), 1.83 (d, 2H), 1.37e1.51 (m, 4H); 13C NMR (DMSO-d6,
300 MHz)
d 176.84, 161.92, 158.73, 152.78, 144.31, 141.19, 139.91,
5.2.28.4. 4-(5-(4-(3-(2-Chlorophenyl)ureido)phenyl)thiazole-2-
carboxamido)cyclohexanecarboxylic acid (9f). Prepared as described
above in the general procedure using 2-chlorophenyl isocyanate
(42 mg, 0.275 mmol, 1.1 equiv) as the substituted isocyanate to
afford the title compounds (78.5 mg, 63%) as off white solid.
138.99, 126.26 (2C), 127.93 (2C), 124.06, 122.49, 118.91 (2C), 118.79
(2C), 48.36, 42.10, 31.40 (2C), 28.18 (2C); Mass (ESIþ) m/z 465.1
[M þ H]þ; HRMS (ESIþ) calcd for C24H25N4O4S [M þ H]þ 465.1591,
found 465.1570, (mean error 2.87 ppm); melting point >280 ꢀC;
HPLC Retention time e 3.38 min, Purity e 99.98%.
1H NMR (DMSO-d6, 300 MHz)
d 12.09 (s, 1H), 9.65 (s, 1H), 8.65
(d, J ¼ 8.4 Hz, 1H), 8.37 (s, 1H), 8.30 (s, 1H), 8.17 (d, J ¼ 8.1 Hz, 1H),
7.72 (d, J ¼ 8.4 Hz, 2H), 7.58 (d, J ¼ 8.7 Hz, 2H), 7.48 (d, J ¼ 7.8 Hz,
1H), 7.32 (t, J ¼ 7.5 Hz & 7.8 Hz, 1H), 7.05 (t, J ¼ 7.8 Hz, 1H), 3.75 (s,
1H), 2.13 (s, 1H), 1.94 (d, J ¼ 10.8 Hz, 2H), 1.85 (d, J ¼ 11.1 Hz, 2H),
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.03.077.
1.33e1.55 (m, 4H); 13C NMR (DMSO-d6, 300 MHz)
d 176.84, 162.02,
158.72, 152.41, 144.21, 140.87, 139.10, 136.20, 129.70, 128.02 (3C),
124.39, 123.99, 122.55, 121.88, 118.95 (2C), 48.36, 42.10, 31.39 (2C),
28.18 (2C); Mass (ESIþ) m/z 499.1 [M þ H]þ; HRMS (ESIþ) calcd for
References
C
24H24Cl1N4O4S [M þ H]þ 499.1201, found 499.1199, (mean error
[1] S. Cases, S.J. Smith, Y.W. Zheng, H.M. Myers, S.R. Lear, E. Sande, S. Novak,
C. Collins, C.B. Welch, A.J. Lusis, S.K. Erickson, R.V. Farese Jr., Identification of a
gene encoding an acyl CoA: diacylglycerol acyltransferase, a key enzyme in
triacylglycerol synthesis, Proceedings of the National Academy of Sciences of
the United States of America 95 (1998) 13018e13023.
1.10 ppm); melting Point >250 ꢀC; HPLC Retention time e 3.98 min,
Purity e 99.89%.
[2] S.J. Smith, S. Cases, D.R. Jensen, H.C. Chen, E. Sande, B. Tow, D.A. Sanan,
J. Raber, R.H. Eckel, R.V. Farese Jr., Obesity resistance and multiple mechanism
of triglyceride synthesis in mice lacking DGAT, Nature Genetics 25 (2000) 87e
90.
[3] H.C. Chen, S.J. Smith, Z. Ladha, D.R. Jensen, L.D. Ferreira, L.K. Pulawa,
J.G. McGuire, R.E. Pitas, R.H. Eckel, R.V. Farese Jr., Increased insulin and leptin
sensitivity in mice lacking acyl CoA: diacylglycerol acyltransferase 1, Journal of
Clinical Investigation 109 (2002) 1049e1055.
5.2.28.5. 4-(5-(4-(3-(2,4-Difluorophenyl)ureido)phenyl)thiazole-2-
carboxamido)cyclohexanecarboxylic
acid
(9g). Prepared
as
described above in the general procedure using 2,4-difluorophenyl
isocyanate (43 mg, 0.275 mmol, 1.1 equiv) as the substituted iso-
cyanate to afford the title compounds (61.8 mg, 49%) as off white
solid.

S. Kandre et al. / European Journal of Medicinal Chemistry 79 (2014) 203e215
215
[4] S.J. Stone, H.M. Myers, S.M. Watkins, B.E. Brown, K.R. Feingold, P.M. Elias,
R.V. Farese Jr., Lipopenia and skin barrier abnormalities in DGAT2-deficient
mice, Journal of Biological Chemistry 279 (2004) 11767e11776.
carboxylic acid derivatives of 2-phenyl-5-trifluoromethyloxazole-4-
carboxamide as potent diacylglycerol acyltransferase-1 inhibitors for the
potential treatment of obesity and diabetes, Journal of Medicinal Chemistry 54
(2011) 2433e2446.
[5] R.J. DeVita, S. Pinto, Current status of the research and development of diac-
ylglycerol O-acyltransferase
Chemistry 56 (2013) 9820e9825.
1
(DGAT1) Inhibitors, Journal of Medicinal
[11] J.A. DiMasi, L. Feldman, A. Seckler, A. Wilson, Trends in risks associated with
new drug development: success rates for investigational drugs, Clinical
Pharmacology & Therapeutics 87 (2010) 272e277.
[12] C.A. Blum, X. Zheng, S.D. Lombaert, Design, synthesis, and biological evalua-
tion of substituted 2-cyclohexyl-4-phenyl-1H-imidazoles: potent and selec-
tive neuropeptide Y Y5-receptor antagonists, Journal of Medicinal Chemistry
47 (2004) 2318e2325.
[13] G.A. Rogers, S.M. Parsons, D.C. Anderson, L.M. Nilsson, B.A. Bahr,
W.D. Kornreich, R. Kaufman, R.S. Jacobs, B. Kirtman, Synthesis, in vitro
acetylcholine-storage-blocking activities, and biological properties of de-
rivatives and analogs of trans-2-(4-phenylpiperidino)cyclohexanol (vesami-
col), Journal of Medicinal Chemistry 32 (1989) 1217e1230.
[14] R.D. Jadhav, K.S. Kadam, S. Kandre, T. Guha, M.M.K. Reddy, M.K. Brahma,
N.J. Deshmukh, A. Dixit, L. Doshi, N. Potdar, A.A. Enose, R.A. Vishwakarma,
H. Sivaramakrishnan, S. Srinivasan, K.V.S. Nemmani, A. Gupte,
A.K. Gangopadhyay, R. Sharma, Synthesis and biological evaluation of iso-
xazole, oxazole, and oxadiazole containing heteroaryl analogs of biaryl ureas
as DGAT1 inhibitors, European Journal of Medicinal Chemistry 54 (2012)
324e342.
[6] R. Smith, A.M. Campbell, P. Coish, M. Dai, S. Jenkins, D. Lowe, S. O’Connor,
N. Su, G. Wang, M. Zhang, L. Zhu, Preparation and Use of Aryl Alkyl Acid
Derivatives for the Treatment of Obesity US 2004/0224997, 2004.
[7] G. Zhao, A.J. Souers, M. Voorbach, H.D. Falls, B. Droz, S. Brodjian, Y.Y. Lau,
R.R. Iyengar, J. Gao, A.S. Judd, S.H. Wagaw, M.M. Ravn, K.M. Engstrom,
J.K. Lynch, M.M. Mulhern, J. Freeman, B.D. Dayton, X. Wang, N. Grihalde, D. Fry,
D.W.A. Beno, K.C. Marsh, Z. Su, G.J. Diaz, C.A. Collins, H. Sham, R.M. Reilly,
M.E. Brune, P.R. Kym, Validation of diacyl glycerolacyltransferase I as a novel
target for the treatment of obesity and dyslipidemia using a potent and se-
lective small molecule inhibitor, Journal of Medicinal Chemistry 51 (2008)
380e383.
[8] A.M. Birch, S. Birtles, L.K. Buckett, P.D. Kemmitt, G.J. Smith, T.J.D. Smith,
A.V. Turnbull, S.J.Y. Wang, Discovery of a potent, selective, and orally effica-
cious
pyrimidinooxazinyl
bicyclooctaneacetic
acid
diacylglycerol
acyltransferase-1 inhibitor, Journal of Medicinal Chemistry 52 (2009) 1558e
1568.
[9] R.L. Dow, M. Andrews, G.E. Aspnes, G. Balan, E.M. Gibbs, A. Guzman-Perez,
K. Karki, J.L. LaPerle, J.C. Li, J. Litchfield, M.J. Munchhof, C. Perreault, L. Patel,
Design and synthesis of potent, orally-active DGAT-1 inhibitors containing a
dioxino[2,3-d]pyrimidine core, Bioorganic & Medicinal Chemistry Letters 21
(2011) 6122e6125.
[15] K.S. Kadam, R.D. Jadhav, S. Kandre, T. Guha, M.M.K. Reddy, M.K. Brahma,
N.J. Deshmukh, A. Dixit, L. Doshi, S. Srinivasan, J. Devle, A. Damre,
K.V.S. Nemmani, A. Gupte, R. Sharma, Evaluation of thiazole containing biaryl
analogs as diacylglycerol acyltransferase
1 (DGAT1) inhibitors, European
[10] Y. Qian, S.J. Wertheimer, M. Ahmed, A.W. Cheung, F. Firooznia, M.M. Hamilton,
S. Hayden, S. Li, N. Marcopulos, L. McDermott, J. Tan, W. Yun, L. Guo,
A. Pamidimukkala, Y. Chen, K.S. Huang, G.B. Ramsey, T. Whittard, K. Conde-
Knape, R. Taub, C.M. Rondinone, J. Tilley, D. Bolin, Discovery of orally active
Journal of Medicinal Chemistry 65 (2013) 337e347.
[16] R.R. Ramharack, M.A. Spahr, Diacylglycerol Acyltransferase (DGAT) Assay,
US20020127627, 2002.