Evaluation of thiazole containing biaryl analogs as diacylglycerol acyltransferase 1 (DGAT1) inhibitors

Article abstract of DOI:10.1016/j.ejmech.2013.05.006

Biphenyl carboxylic acids, exemplified by compound 5, are known potent inhibitors of diacylglycerol acyltransferase, DGAT1, an enzyme involved in the final committed step of triglyceride biosynthesis. We have synthesized and evaluated 2-phenylthiazole, 4-

Full text of DOI:10.1016/j.ejmech.2013.05.006

European Journal of Medicinal Chemistry 65 (2013) 337e347
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Evaluation of thiazole containing biaryl analogs as diacylglycerol
acyltransferase 1 (DGAT1) inhibitors
,
,
Kishorkumar S. Kadam a 1, Ravindra D. Jadhav a 1, Shivaji Kandre a, Tandra Guha a,
M. Mahesh Kumar Reddy b, Manoja K. Brahma b, Nitin J. Deshmukh b, Amol Dixit b,
Lalit Doshi b, Shaila Srinivasan b, Jayendra Devle c, Anagha Damre c, Kumar V.S. Nemmani b,
Amol Gupte a , Rajiv Sharma
,
a
*
a Department of Medicinal Chemistry, Piramal Enterprises Limited, Goregaon (E), Mumbai 400 063, Maharashtra, India
b Department of Pharmacology, Piramal Enterprises Limited, Goregaon (E), Mumbai 400 063, Maharashtra, India
c Drug Metabolism and Pharmacokinetics, Piramal Enterprises Limited, Goregaon (E), Mumbai 400 063, Maharashtra, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Biphenyl carboxylic acids, exemplified by compound 5, are known potent inhibitors of diacylglycerol
acyltransferase, DGAT1, an enzyme involved in the final committed step of triglyceride biosynthesis. We
have synthesized and evaluated 2-phenylthiazole, 4-phenylthiazole, and 5-phenylthiazole analogs as
DGAT1 inhibitors. The 5-phenylthiazole series exhibited potent DGAT1 inhibition when evaluated using
an in vitro enzymatic assay and an in vivo fat tolerance test in mice. Compound 33 (IC50 ¼ 23 nM)
exhibiting promising oral pharmacokinetic parameters (AUCinf ¼ 7058 ng*h/ml, T1/2 ¼ 0.83 h) coupled
with 87 percent reduction of plasma triglycerides in vivo may serve as a lead for developing newer anti-
obesity agents.
Received 11 February 2013
Received in revised form
6 May 2013
Accepted 7 May 2013
Available online 14 May 2013
Keywords:
Diacylglycerol acyltransferase (DGAT)
Obesity
Ó 2013 Elsevier Masson SAS. All rights reserved.
Plasma triglycerides
5-Phenylthiazole
Fat tolerance test
1. Introduction
for the identification of newer chemical scaffolds, devoid of side-
effects, to be developed as anti-obesity agents.
Obesity, the oldest known metabolic disorder, is characterized
by an accumulation of triacylglycerols within the adipocytes. An
enhanced risk of several pathologic conditions such as hyperten-
sion [1], stroke [2], coronary heart disease [3], cancer [4], inflam-
matory disease [5] including arthritis [6], and Type 2 diabetes [7]
have been widely documented amongst the obese. Presently orli-
stat is the only marketed anti-obesity drug that is known to act by
preventing the absorption of fat from human diet [8]. However
adverse effects such as steatorrhea, fecal incontinence, and urgent
bowel movements are commonly observed with the use of orlistat
thereby reducing its appeal within the target population [9]. The
lack of therapeutic alternatives to orlistat presents a pressing need
In humans the biosynthesis of triacylglycerols is catalyzed by
the enzyme Acyl CoA:diacylglycerol acyltransferase (DGAT) [10].
Two isoforms of the DGAT enzyme are presently known namely,
DGAT1 and DGAT2. In mammals, DGAT1 is widely expressed in the
skeletal muscle, intestine, and testis while DGAT2 is expressed
particularly in the liver and adipose tissues. DGAT2 deficiency in
mice knockout models led to death soon after birth apparently
from profound reductions in substrates required for energy meta-
bolism coupled with an impaired permeability barrier function of
the skin Ref. [11]. On the other hand, DGAT1 knockouts were viable,
resistant to weight gain when fed a high-fat diet, and exhibited
increased insulin and leptin sensitivity [12]. Consequently inhibi-
tion of DGAT1 has emerged as a plausible strategy for the treatment
of obesity. Of late, several potent DGAT1 inhibitors have been re-
ported and a few compounds are being clinically evaluated (Fig. 1)
[13e16].
* Corresponding author. 1 Nirlon Complex, Off Western Express Highway, Gor-
egaon (East), Mumbai 400 063, India. Tel.: þ91 22 30818312; fax: þ91 22 30818036.
A series of DGAT1 inhibitors belonging to an amino biphenyl
carboxylic acid scaffold, exemplified by compound 5 [hDGAT1
IC50 ¼ 35 nM, in vitro solubility (pH 7.4) ¼ 0.01 mg/ml] have been
E-mail
addresses:
amol.gupte@piramal.com,
argupte@hotmail.com,
amolrgupte@gmail.com (A. Gupte).
1
Both authors contributed equally to the manuscript.
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.05.006

338
K.S. Kadam et al. / European Journal of Medicinal Chemistry 65 (2013) 337e347
Fig. 1. Clinically studied hDGAT1 inhibitors.
disclosed by Bayer Pharmaceuticals [15,17]. These efforts prompted
us to explore for orally active inhibitors of the DGAT1 enzyme. We
have recently reported a 3-phenylisoxazole scaffold that retains
DGAT1 inhibition and displays a marked improvement in solubility
over compound 5 [18]. In an attempt to identify additional phar-
macophores for DGAT1 inhibition we evaluated the effect of a
thiazole in place of the phenyl ring ‘B’ of compound 5 (Fig. 2). The
thiazole moiety has been positioned either in a 2-phenylthiazole
orientation represented by compound 6, a 5-phenylthiazole orien-
orientation represented by compound 8. Of these three analogs,
compounds 6 (DGAT1 Inhibition [1
M] ¼ 31%) and 8 (DGAT1 In-
hibition [1
M] ¼ 25%) did not exhibit substantial in vitro DGAT1
potency. On the other hand, compound 7 (DGAT1 Inhibition
[1
scaffold exhibits potent DGAT1 inhibition comparable to that of
compound 5. We hence directed our efforts toward the synthesis of
compounds belonging to the 5-phenylthiazole scaffold. Our earlier
efforts have highlighted the advantage of replacing the benzothia-
zole substituent with a phenyl urea as exemplified by structure 9
m
m
m
M] ¼ 81%, IC50 ¼ 10 nM) belonging to the 5-phenylthiazole
tation represented by compound 7, or
a 4-phenylthiazole
Fig. 2. Stepwise development of the 5-phenylthiazole scaffold.

K.S. Kadam et al. / European Journal of Medicinal Chemistry 65 (2013) 337e347
339
(Fig. 2) [18,19]. With such a modification we were able to retain the
DGAT1 activity and improve on the cLogP and solubility. In this
study, we have evaluated the effects of incorporating a phenyl urea
in combination with a 5-phenylthiazole scaffold. Subsequently the
effects of replacing the urea linker with an amide linker have also
been studied. Apart from their effect on the in vitro and in vivo
DGAT1 activity oral absorption pharmacokinetic properties were
also evaluated for selected compounds. Our efforts in this study
were focused toward the development of a novel heteroaryl scaffold
that retains DGAT1 activity and exhibits an acceptable oral phar-
macokinetic profile comparable to that of compound 5.
(22) following
a
treatment with hexamethylenetetramine.
Coupling of compound 22 with ethyl 2-chloro-2-oxoacetate yiel-
ded ethyl 2-((2-(4-nitrophenyl)-2-oxoethyl)amino)-2-oxoacetate
(23) that was subsequently cyclized to ethyl 5-(4-nitrophenyl)
thiazole-2-carboxylate (24) on treatment with Lawesson’s reagent.
Hydrolysis of compound 24 yielded its free acid, 5-(4-nitrophenyl)
thiazole-2-carboxylic acid (25). Compound 25 when coupled with
L-valine methyl ester hydrochloride using isobutyl chloroformate
and N-methyl morpholine yielded methyl 3-methyl-2-(5-(4-
nitrophenyl)thiazole-2-carboxamido)butanoate (26). Reduction of
compound 26 using iron-ammonium chloride in ethanol yielded
the desired amine intermediate, methyl 2-(5-(4-aminophenyl)
thiazole-2-carboxamido)-3-methylbutanoate (27). Coupling of
compound 27 with 2-chloro-6-fluoro benzothiazole and a subse-
quent hydrolysis with 1.0 M lithium hydroxide yielded compound
7. Compounds 28e32 possessing the urea linker were synthesized
by coupling compound 27 with substituted phenyl isocyanates and
subsequent hydrolysis with 1.0 M lithium hydroxide. Similarly
compounds possessing the amide linker (33e36) were synthesized
by coupling compound 27 with substituted acid chlorides followed
by a hydrolysis using 1.0 M lithium hydroxide.
Compound 40, the dimethyl cyano analog, and compound 41,
the diethyl cyano analog, were synthesized (Scheme 5) starting
from methyl 4-(cyanomethyl)benzoate (37). Incorporation of the
dimethyl group was undertaken by reacting compound 37 with
methyl iodide in the presence of potassium tert-butoxide to yield
the dimethyl intermediate, compound 38. Similarly replacing
methyl iodide with ethyl iodide resulted in the synthesis of the
diethyl intermediate, compound 39. Compounds 38 and 39 were
coupled with compound 27 in the presence of trimethyl aluminum
and subsequently deprotected using 1.0 N LiOH to yield the desired
compounds 40 and 41 respectively.
2. Chemistry
Ethyl 2-(4-nitrophenyl)thiazole-4-carboxylate (12), a key inter-
mediate for the synthesis of the 2-phenylthiazole analog, 2-(2-(4-
((6-fluorobenzo[d]thiazol-2-yl)amino)phenyl)thiazole-4-carboxa
mido)-3-methylbutanoic acid (6), was synthesized in two steps
starting from 4-nitrobenzamide (10) as depicted in Scheme 1. Thi-
olation of compound 9 to 4-nitrobenzothioamide (11) followed by
its cyclization in the presence of ethyl bromopyruvate in ethanol
yielded compound 12. Similarly ethyl 4-(4-nitrophenyl)thiazole-2-
carboxylate (15), a key intermediate for the synthesis of the 4-
phenylthiazole analog, 2-(4-(4-((6-fluorobenzo[d]thiazol-2-yl)ami
no)phenyl)thiazole-2-carboxamido)-3-methylbutanoic acid (8),
was synthesized by bromination of 4-nitroacetophenone (13) to 2-
bromo-1-(4-nitrophenyl)ethanone (14) and its subsequent cycli-
zation to compound 15 on refluxing with ethyl 2-amino-2-
thioxoacetate in methanol (Scheme 2).
Synthesis of compound 6 (Scheme 3) involved an hydrolysis
of the key intermediate 12 to its corresponding acid, 2-(4-
nitrophenyl)thiazole-4-carboxylic acid (16). Coupling of 16 with L-
valine methyl ester hydrochloride using isobutyl chloroformate
and N-methyl morpholine yielded methyl 3-methyl-2-(2-(4-nitro
phenyl)thiazole-4-carboxamido)butanoate (18). Reduction of
compound 18 using iron-ammonium chloride yielded the corre-
sponding amine, methyl 2-(2-(4-aminophenyl)thiazole-4-carboxa
mido)-3-methylbutanoate (20). Coupling of 20 with 2-chloro-6-
fluoro benzothiazole under acidic conditions followed by a subse-
quent hydrolysis with 1.0 M lithium hydroxide at room tempera-
ture afforded compound 6, the desired 2-phenylthiazole analog.
Compound 8 was similarly synthesized from its key intermediate
15 (Scheme 3) by following a reaction sequence analogous to that of
compound 6.
3. Results and discussion
3.1. In vitro pharmacology
The synthesized compounds thus synthesized were assayed
using an in vitro enzymatic assay that measured a triolein output
from diolein and radiolabeled oleoyl-CoA [20]. The DGAT1 assay
was performed using 2.5
supernatant preincubated with 100
m
g of the protein from a post nuclear
m
l of the assay buffer [100 mM
Tris-HCl (pH 7.5), 250 mM sucrose, and 1.25 mg/ml fatty acid free
BSA] containing known concentration of the inhibitor and sup-
The synthesis of 2-(5-(4-((6-fluorobenzo[d]thiazol-2-yl)amino)
phenyl)thiazole-2-carboxamido)-3-methylbutanoic acid (7), 5-
phenylthiazole analogs possessing a urea linker (28e32), and the
5-phenylthiazole analogs possessing an amide linker (33e38) has
been depicted in Scheme 4. This synthetic sequence diversifies at a
key amine intermediate, methyl 2-(5-(4-aminophenyl)thiazole-2-
carboxamido)-3-methylbutanoate (27) to yield these different
analogs. Compound 27 was synthesized over six steps starting
from 2-bromo-1-(4-nitrophenyl)ethanone (14). Compound 14 was
converted to 2-amino-1-(4-nitrophenyl)ethanone hydrochloride
plemented 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 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 prop-
erly mixed and the 14C triglyceride formed was extracted using
600
ml of heptane. 250 ml of this extracted heptane was added to the
scintillation fluid and subjected to radioactivity measurement. The
Scheme 1. (a) Lawesson’s reagent, dioxane, 80 ꢀC; (b) ethyl bromopyruvate, EtOH 80 ꢀC.

340
K.S. Kadam et al. / European Journal of Medicinal Chemistry 65 (2013) 337e347
Scheme 2. (a) Bromine, AlCl3, diethyl ether; (b) ethyl 2-amino-2-thioxoacetate, methanol, reflux.
primary screening of hDGAT1 inhibitors was carried out at 1.0
m
M
amide analogs appear to be tolerated at the DGAT1 enzyme. In case of
the 5-phenylthiazole scaffold we have thus observed both the urea
and amide linkers to be well tolerated for DGAT1 inhibition. This
result is contrary to our earlier observations, involving a 3-
phenylisoxazole scaffold, wherein the urea linker appeared to be
well tolerated at the DGAT1 enzyme as compared to the amide linker
[18]. This study has helped identify several DGAT1 inhibitors pos-
sessing potent in vitro activity that belong to the 5-phenylthiazole
scaffold.
Following in vitro evaluation, a few selected 5-phenylthiazole
analogs were assessed for their oral pharmacokinetic parameters
and subsequently evaluated in vivo using an acute fat tolerance test
(FTT). For these evaluations, the most potent phenyl urea analog,
compound 29, and the most potent phenyl amide analog, com-
pound 33, were studied and compared with compound 5.
concentration. Subsequent IC50 determinations were undertaken
for compounds exhibiting inhibition greater that 75% in this pri-
mary screening. The IC50 values were determined by evaluating
compounds at nine concentrations ranging from 0.1 nM to 1.0
and are presented in Table 1.
mM
A
preliminary study involving compounds
6
(Percent
Inhibition ¼ 31%), 7 (Percent Inhibition ¼ 81%, IC50 ¼ 10 nM), and 8
(Percent Inhibition ¼ 25%) identified the 5-phenylthiazole scaffold
represented by compound 7 as an inhibitor of DGAT1. The potent 5-
phenylthiazole scaffold was investigated further by replacing the
amino fluoro benzothiazole subunit of compound 7 with either a
substituted phenyl urea (28e32) or a substituted phenyl amide (33e
38). Following a primary screening at 1.0 mM concentration, these
twelve compounds (28e38) were evaluated for their IC50 values. In
case of the phenyl urea analogs, compounds possessing electron
donating substituents on the phenyl such as 3-trifluoromethyl (28,
IC50 ¼ 22 nM), 3,4-dimethyl (30, IC50 ¼ 20 nM), and 2-phenoxy (31,
IC50 ¼ 23 nM) have been evaluated alongside an electron with-
drawing 2-chloro (29, IC50 ¼ 16 nM) substituent. This study high-
lights an equal preference for both electron donating and electron
withdrawing substituents on the phenyl ring. A 2-phenoxy, 4-chloro
substituted phenyl urea analog (32, IC50 ¼ 58 nM) also exhibits
DGAT1 activity. In case of the phenyl amide analogs a para-tert-butyl
(33, IC50 ¼ 23 nM), a para-isobutyronitrile (40, IC50 ¼ 97 nM), and a
para-2-ethylbutanenitrile (41, IC50 ¼ 101 nM) substituents on the
phenyl ring have been evaluated. The para-tert-butyl substituent
(33) appears to be a potent DGAT1 inhibitor as compared to com-
pounds 40 and 41. Compounds 34 (IC50 ¼ 37 nM) and 35
(IC50 ¼ 82 nM) possessing aliphatic substituents in place of an aro-
matic phenyl substituent and compound 36 (IC50 ¼ 49 nM) pos-
sessing a heteroaromatic ring in place of an aromatic phenyl have
also been evaluated. Each of these substitutions in case of the phenyl
3.2. Pharmacokinetic properties evaluation
Oral pharmacokinetic parameters of the test compounds (5, 29,
33) were assessed following the preparation of a suspension [1 mg/
ml] in 0.5% CMC and Tween 80. Swiss mice were weighed and the
compounds were administered orally (10 mg/kg, n ¼ 4). Blood
samples were withdrawn at 0.08, 0.17, 0.25, 0.5, 0.75, 1.0, 2.0, 6.0,
8.0 and 24 h after dosing. Plasma samples were maintained on ice
before being centrifuged (4 ꢀC for 5 min at 1411 g), and aliquots
were stored at ꢁ80 ꢀC pending the assay. Concentrations of the
compounds were determined using an ESI-LC/MS/MS method
developed at Piramal Enterprises Limited. Pharmacokinetic pa-
rameters were determined by non-compartmental analysis using
WinNonlin Professional (Version 4.1). Cmax and Tmax were taken
directly from the plasma concentration-time profile. The area un-
der the curve from time 0 to the last blood sampling time (AUC0et
was calculated using the linear trapezoidal rule. The area under the
)
Scheme 3. (a) 1.0 N LiOH, THF, rt; (b) NMM, IBCF, L-valine methyl ester hydrochloride, TEA, THF, ꢁ20 ꢀC to rt; (c) Fe, NH4Cl, EtOH, THF, H2O, 80 ꢀC; (d) phenyl isocyanate, THF, rt; (e)
1 N LiOH, THF, rt.

K.S. Kadam et al. / European Journal of Medicinal Chemistry 65 (2013) 337e347
341
Scheme 4. (a) Hexamethylenetetramine, dichloromethane, HCl/ethanol; (b) ethyl 2-chloro-2-oxoacetate, triethylamine, ethyl acetate, reflux; (c) Lawesson’s reagent, dioxane, 80 ꢀC;
(d) aq. NaOH, THF; (e) NMM, IBCF, L-valine methyl ester hydrochloride, TEA, THF, ꢁ20 ꢀC to rt; (f) Fe, NH4Cl, EtOH, H2O; (g) 6-fluoro-2-chlorobenzothiazole, 4.0 M HCl in dioxane,
EtOH, 80 ꢀC, 20 h; (h) substituted phenyl isocyanates, THF, rt, 16 h; (i) substituted acid chloride, pyridine, dichloromethane, rt; (j) 1 N LiOH, THF, rt.
curve extrapolated to infinity (AUCinf) was calculated by using the
plasma concentration at time t divided by slope z, where z is
estimated by linear regression of the terminal log-linear phase of
the plasma concentrationetime curve. Terminal plasma elimina-
tion half life (T1/2) was calculated as 0.693/lz.
The assessed pharmacokinetic parameters of compounds 5, 29,
and 33 following oral administration are presented in Table 2. The
systemic exposure (AUCinf) of compound 33 was approximately 10-
fold higher than that of compound 29 and 1-fold higher than that of
compound 5 following oral administration. The elimination half life
(T1/2) of each of the three compounds was observed to be less than
1 h. However amongst these three compounds, compound 33 (T1/
0.5% CMC containing Tween 80 (25 mL). An hour later, a bolus dose
l
l
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 (AUC0e4 h) of the
test compound and comparing it along with an AUC0e4 of the
h
vehicle group that is considered to be 100 percent. In this study,
compound 5 exhibited a 90% triglyceride reduction. Compound 29,
the 5-phenylthiazole analog possessing a urea linker exhibited 82%
triglyceride reduction. Compound 33 possessing an amide linker
exhibited 87% triglyceride reduction. Thus all three compounds
appear equi-efficacious to each other when evaluated in vivo.
¼ 0.83 h) possessed the longest elimination half life as compared
2
to that of compound 5 (T1/2 ¼ 0.62 h) and compound 29 (T1/
¼ 0.49 h). Thus with respect to oral pharmacokinetic parameters
2
4. Conclusions
the phenyl amide analog 33 appears slightly better than the Bayer
compound 5 and significantly better than the phenyl urea deriva-
tive 29.
In this study, we have developed a 5-phenylthiazole containing
heteroaryl scaffold possessing improved oral pharmacokinetic
properties. A preliminary comparison in between the 2-phenyl
thiazole, 4-phenylthiazole, and 5-phenylthiazole scaffolds high-
lighted a preference for the 5-phenylthiazole scaffold at the DGAT1
enzyme. A subsequent study undertaken to evaluate phenyl urea
and phenyl amide analogs of the 5-phenylthiazole scaffold identi-
fied both these modifications to be well tolerated at the DGAT1
enzyme when analyzed in vitro. Compounds 29 and 33 were
3.3. Acute fat tolerance test (FTT)
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 were formulated as a suspension in
Scheme 5. (a) Methyl iodide (for 38)/ethyl iodide (for 39), potassium tert-butoxide, THF, ꢁ30 ꢀC, 2 h; (b) compound 27, 2.0 M trimethyl aluminum in toluene, 80 ꢀC, 4 h; (c) 1 N
LiOH, THF, rt.

342
K.S. Kadam et al. / European Journal of Medicinal Chemistry 65 (2013) 337e347
Table 1
pharmacokinetic properties the phenyl amide analog, compound
In vitro evaluation of 5-phenylthiazole analogs.
33 (AUCinf ¼ 7058 ng*h/ml, T1/2 ¼ 0.83 h) appeared better than both
compound 29 (AUCinf ¼ 623 ng*h/ml, T1/2 ¼ 0.49 h) and compound
5 (AUCinf ¼ 5344 ng*h/ml, T1/2 ¼ 0.62 h). Thus with the phenyl
amide analog, compound 33, we were able to improve
the pharmacokinetic properties while retaining its in vitro
DGAT1 enzymatic activity and its in vivo ability to reduce plasma
triglyceride levels. These attributes earmark compound 33 as a
potential new lead for developing therapeutically applicable DGAT1
inhibitors.
Compound
number
R
hDGAT1 inhibition
hDGAT1 inhibition
IC50 (nM)
(% inhibition 1
mM)
28
29
81
22
16
5. Experimental
5.1. Chemistry
82
78
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 Thomas Baker
and used without further purification. 1H NMR spectra were
recorded on a Bruker spectrometer (300 MHz) using either CDCl3 or
30
31
20
23
80
81
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 elec-
trospray 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.
32
58
33
34
84
86
23
37
5.2. HPLC method
35
36
40
83
86
88
82
49
97
Elution with 20e80% linear gradient of acetonitrile in 6 min
followed by 20e80% linear gradient of 0.01 M NH4OAc þ 0.5% TEA,
pH 5.0 with AcOH in 1 min that is continued using an isocratic
elution with 80% 0.01 M NH4OAc þ 0.5% TEA, pH 5.0 with AcOH for
3 min using a Ascentis TM Express (50 ꢂ 4.6 mm I.D.), 2.7
mm
operated at 1 ml/min, detection at 288 nm.
5.2.1. 4-Nitrobenzothioamide (11)
41
87
101
To a solution of 4-Nitrobenzamide (4 g, 24.08 mmol,1.0 equiv) in
Dioxane (100 ml) was added Lawesson’s reagent (7.79 g,
19.26 mmol, 0.8 equiv) and the mixture was heated to 80 ꢀC for 2 h.
The reaction mixture was subsequently cooled, added to water and
neutralized with a saturated solution of sodium carbonate. The
product was separated from the aqueous mixture using EtOAc. The
organic layer was dried over sodium sulfate, filtered and concen-
trated under reduced pressure to get a dark brown residue. The
residue was subjected to column chromatography using 2:8
EtOAc:Pet ether to get a dark yellow colored solid that was crys-
tallized in DCM/Pet ether to yield the title compound as a pale
yellow colored solid (3.52 g, 80%).
evaluated for their in vivo ability to reduce triglycerides in fasted
Swiss mice when dosed at 3 mg/kg. Compound 29 (Triglyceride
reduction ¼ 87%) and compound 33 (Triglyceride reduction ¼ 82%)
appear equivalent to compound 5 (Triglyceride reduction ¼ 90%)
at the same dose. However when studied for their oral
Table 2
Plasma triglyceride reduction following an in vivo fat tolerance test [3 mg/kg] and
pharmacokinetic parameters following oral administration [10 mg/kg] to Swiss
mice.
1H NMR (300 MHz, DMSO-d6)
d 10.23 (s, 1H), 9.82 (s, 1H), 8.26
(d, J ¼ 8.4 Hz, 2H), 8.03 (d, J ¼ 8.4 Hz, 2H); MS (ESIþ): m/z 183
[M þ H]þ.
Parameter
Compound 5
Compound 29
Compound 33
Percent TG reduction (FTT)
Cmax (ng/ml)
Cmax (mM)
Tmax (h)
T1/2 (h)
AUC0et (ng*h/ml)
AUCinf (ng*h/ml)
90
2452
5.3
0.25
0.62
5344
5350
82
933
1.9
0.16
0.49
623
672
87
5.2.2. Ethyl 2-(4-nitrophenyl)thiazole-4-carboxylate (12)
5449
11.3
0.17
0.83
7058
7072
To a suspension of compound 11 (3.4 g, 18.66 mmol, 1.0 equiv) in
Ethanol (50 ml) was added Ethyl bromopyruvate (2.348 ml,
18.66 mmol, 1.0 equiv) and the reaction mixture was heated to
80 ꢀC for 4 h. This reaction mass was cooled and TEA (2.60 ml,
18.66 mmol, 1.0 equiv) was added. The solid was filtered, washed

K.S. Kadam et al. / European Journal of Medicinal Chemistry 65 (2013) 337e347
343
with water, and dried to obtain the title compound as a white solid
(4.1 g, 79%).
Reaction mixture was quenched with water and 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 3:7 EtOAc:Pet
ether to get a pale brown colored solid that was crystallized in
CHCl3/Pet ether to yield the product as an off white colored solid
(2.9 g, 66%).
1H NMR (300 MHz, CDCl3)
d
8.35 (d, J ¼ 8.4 Hz, 2H), 8.29 (s, 1H),
8.22 (d, J ¼ 8.4 Hz, 2H), 4.48 (q, J ¼ 6.9 Hz, 2H), 1.46 (t, J ¼ 7.2 Hz,
3H); MS (ESIþ): m/z 279.3 [M þ H]þ.
5.2.3. 2-Bromo-1-(4-nitrophenyl)ethanone (14)
To a solution of 4-Nitroacetophenone (25.0 g, 151 mmol,
1.0 equiv) in ether (250 ml) bromine (7.77 ml, 151 mmol, 1.0 equiv)
was added over 10 min. The reaction mixture was stirred for 30 min
in the presence of catalytic amount of aluminum chloride (1.0 g,
7.5 mmol, 0.05 equiv). Following reaction completion the reaction
mixture was quenched with aqueous sodium bicarbonate. The
ether layer was separated and dried over sodium sulfate and
evaporated under reduced pressure. The off white residue thus
obtained was crystallized in EtOAc:Pet ether to afford the title
compound (25.5 g, 69%).
1H NMR (300 MHz, DMSO-d6)
d 8.53 (s, 1H), 8.51e8.48 (d,
J ¼ 8.4 Hz 1H), 8.37 (bs, 4H), 4.39 (t, J ¼ 7.5 Hz, 1H), 3.69 (s, 3H),
2.30e2.23 (m, 1H), 0.96 (t, J ¼ 6.6 Hz, 6H); MS (ESIþ): m/z 364
[M þ H]þ.
5.2.8. Methyl 3-methyl-2-(4-(4-nitrophenyl)thiazole-2-carboxamido)
butanoate (19)
To a solution of 4-(4-nitrophenyl)thiazole-2-carboxylic acid
(4.5 g, 18 mmol, 1.0 equiv) in THF (75 ml) was added N-methyl
morpholine (1.8 g, 18 mmol,1.0 equiv) and cooled the reaction mass
to ꢁ20 ꢀC. To this was added isopropyl chloroformate (2.4 g,
18 mmol,1.0 equiv) and the reaction mixture was stirred for 20 min.
1H NMR (300 MHz, CDCl3)
d
8.36 (d, J ¼ 8.1 Hz, 2H), 8.18 (d,
J ¼ 8.1 Hz, 2H), 4.48 (s, 2H); MS (ESIþ) m/z 245.0 [M þ H]þ.
5.2.4. Ethyl 4-(4-nitrophenyl)thiazole-2-carboxylate (15)
In another flask containing a solution of D-valine hydrochloride
To a stirred solution of 2-bromo-1-(4-nitrophenyl)ethanone
(9.2 g, 37.7 mmol, 1.0 equiv) in methanol (200 ml) was added ethyl
2-amino-2-thioxoacetate (5.0 g, 37.7 mmol, 1.0 equiv) and reaction
mixture was refluxed for 2 h. Following reaction completion the
reaction mass was cooled to rt and the precipitated solid was
filtered, washed with cold methanol, and dried to yield (7.1 g, 67%)
of the title compound as a white solid.
(7.2 g, 43 mmol, 2.4 equiv) in THF (20 ml) was neutralized using
triethylamine (4.3 g, 43 mmol, 2.4 equiv) by stirring the mixture for
5 min. This neutralized valine ester solution was added into the
reaction mass and stirred at rt for 3 h. Following reaction
completion solvent was removed under reduced pressure. The
desired compound was isolated by flash column chromatography
using 20:80 EtOAc:Pet ether (4.4 g, 67%).
1H NMR (300 MHz, CDCl3):
d
8.33 (d, J ¼ 8.0 Hz, 2H), 8.16 (d,
1H NMR (300 MHz, DMSO-d6)
d
8.34 (d, J ¼ 8.1 Hz, 2H), 8.13 (d,
J ¼ 8.0 Hz, 2H), 7.96 (s, 1H), 4.55 (q, J ¼ 7.0 Hz, 2H), 1.50 (t, J ¼ 7.0 Hz,
J ¼ 8.1 Hz, 2H), 7.94 (s, 1H), 7.79 (d, J ¼ 6.9 Hz, 1H), 4.76 (t, J ¼ 4.8 Hz,
1H), 3.83 (s, 3H), 2.36 (m, 1H), 1.07 (s, 6H); MS (ESIþ) m/z 364
[M þ H]þ.
3H); MS (ESIþ) m/z 276.6 [M þ H]þ.
5.2.5. 2-(4-Nitrophenyl)thiazole-4-carboxylic acid (16)
To a solution of ethyl 2-(4-nitrophenyl)thiazole-4-carboxylate
(4 g, 14.37 mmol, 1.0 equiv) in THF (80 ml) was added 1.0 M aq.
Lithium hydroxide (57.5 ml, 57.5 mmol, 4.0 equiv) and the reaction
mixture was stirred at rt for 2 h. The reaction mixture was acidified
to pH ¼ 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 pale
yellow colored solid. Recrystallization with EtOAc/Pet ether yielded
the desired product as an off white solid (3.12 g, 87%).
5.2.9. Methyl 2-(4-(4-aminophenyl)thiazolidine-2-carboxamido)-
3-methylbutanoate (20)
To a solution of Methyl 3-methyl-2-(2-(4-nitrophenyl)thiazole-
4-carboxamido)butanoate (1.8 g, 4.95 mmol, 1.0 equiv) in EtOH
(36 ml), THF (16 ml), and water (16 ml) were added Iron (0.830 g,
14.86 mmol, 3.0 equiv) and Ammonium chloride (0.795 g,
14.86 mmol, 3.0 equiv) and the mixture was refluxed at 80 ꢀC for
4 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 water and extracted
using ethyl acetate. The organic layer was dried using sodium sul-
fate and concentrated to obtain a dark brown residue which was
purified by column chromatography using 4:6 EtOAc:Pet ether to
afford the title compound as a pale yellow solid (1.34 g, 81%).
1H NMR (300 MHz, DMSO-d6)
d 13.30 (bs, 1H), 8.66 (s, 1H), 8.37
(d, J ¼ 8.4 Hz, 2H), 8.26 (d, J ¼ 8.4 Hz, 2H); MS (ESIþ): m/z 251
[M þ H]þ.
5.2.6. 4-(4-Nitrophenyl)thiazole-2-carboxylic acid (17)
To a solution of ethyl 4-(4-nitrophenyl) thiazole-2-carboxylate
(7.0 g, 25.0 mmol, 1.0 equiv) in THF (70 ml) was added 1.0 N
lithium hydroxide (1.05 g, 25.0 mmol, 1.0 equiv) solution and the
reaction mixture was stirred at rt for 4 h. After reaction completion
THF was removed under reduced pressure and dil. HCl was added
to the mixture. The precipitated solid was filtered, washed with
water, and dried to yield (6.2 g, 98%) of the title compound as an off
white solid.
1H NMR (300 MHz, DMSO-d6)
d
8.20e8.17 (d, J ¼ 8.4 Hz,1H), 8.10
(s, 1H), 7.73 (d, J ¼ 8.4 Hz, 2H), 6.65 (d, J ¼ 8.4 Hz, 2H), 5.78 (bs, 2H),
4.39 (t, J ¼ 7.8 Hz, 1H), 3.68 (s, 3H), 2.28e2.21 (m, 1H), 0.94 (t,
J ¼ 3.9 Hz, 6H); MS (ESIþ): m/z 334.4 [M þ H]þ.
5.2.10. Methyl 2-(4-(4-aminophenyl)thiazole-2-carboxamido)-3-
methylbutanoate (21)
To a solution of compound 19 (4.27 g, 11.78 mmol) in ethanol
(40 ml) and water (20 ml) were added iron (1.97 g, 35.34 mmol) and
ammonium chloride (1.89 g, 35.36 mmol) and the reaction mixture
was stirred at 80e85 ꢀC for 3 h. Following reaction completion, the
mass was cooled to rt and filtered through Celite. Organic solvent
was removed and a saturated sodium bicarbonate solution was
added to obtain a pH of 7.0 and the compound was extracted
using ethyl acetate. The solvent was evaporated to yield a solid
which was further purified by column chromatography using 5:95
EtOAc:chloroform to yield (2.7 g, 68%) the title compound as an off
white solid.
1H NMR (300 MHz, DMSO-d6)
MS (ESIþ) m/z 251.6 [M þ H]þ.
d 8.78 (s, 1H), 8.32e8.28 (m, 4H);
5.2.7. Methyl 3-methyl-2-(2-(4-nitrophenyl)thiazole-4-carboxamido)
butanoate (18)
To a solution of 2-(4-nitrophenyl)thiazole-4-carboxylic acid
(3.0 g, 11.99 mmol, 1.0 equiv) in DMF (60 ml) was sequentially
added HATU (6.84 g, 17.98 mmol, 1.5 equiv), methyl 2-amino-3-
methylbutanoate hydrochloride (2.412 g, 14.39 mmol, 1.2 equiv)
and TEA (2.507 ml, 17.98 mmol, 1.5 equiv) and stirred for 24 h.

344
K.S. Kadam et al. / European Journal of Medicinal Chemistry 65 (2013) 337e347
1H NMR (300 MHz, DMSO-d6)
d
8.70 (d, J ¼ 7.8 Hz, 1H), 8.03 (s,
5.2.13. 2-Amino-1-(4-nitrophenyl)ethanone hydrochloride (22)
1H), 7.76 (d, J ¼ 7.5 Hz, 2H), 6.63 (d, J ¼ 7.8 Hz, 2H), 5.38 (s, 2H), 4.36
(t, J ¼ 7.2 Hz, 1H), 3.96 (s, 3H), 2.30e2.28 (m, 1H), 0.96 (d, J ¼ 5.7 Hz,
6H); MS (ESIþ) m/z 334 [M þ H]þ.
To a solution of 2-bromo-1-(4-nitrophenyl)ethanone (25.0 g,
102 mmol, 1.0 equiv) was in DCM (250 ml) was added hexameth-
ylenetetramine (20.1 g, 143 mmol, 1.4 equiv) and the reaction
mixture was stirred for 1 h. Following completion the reaction was
filtered to yield 30.0 g of an off-white solid that was dissolved in
ethanol (162 ml) and conc. HCl (40 ml). The mixture was stirred for
3 h and then left standing for two days. The off white solid thus
obtained was filtered, washed with water, and dried to yield the
title compound (11.8 g, 72%).
5.2.11. 2-(2-(4-((6-Fluorobenzo[d]thiazol-2-yl)amino)phenyl)thiazole-
4-carboxamido)-3-methylbutanoic acid (6)
Compound 20 (300 mg, 0.900 mmol, 1.0 equiv) and 2-chloro-6-
fluorobenzo[d]thiazole (203 mg, 1.080 mmol, 1.2 equiv) were taken
in EtOH (10 ml) and heated at 55e60 ꢀC to obtain a clear solution.
To this solution was added 4.0 M aq. HCl in 1,4-Dioxane HCl
(0.027 ml, 0.900 mmol, 1.0 equiv) and the resulting mixture was
refluxed at 80 ꢀC for 20 h. Following this the EtOH was removed
completely under pressure and the residue was purified by column
chromatography using 3:8 EtOAc:Pet ether to obtain an pale brown
solid. The solid was crystallized in EtOAc/Pet ether to yield the
corresponding methyl ester. To a solution of this methyl ester
(1 equiv) in THF was added 1.0 M solution of LiOH in water (5 equiv)
and stirred for 16 h 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 material thus obtained was extracted using ethyl acetate,
washed with water, and dried over anhydrous sodium sulfate. The
solvent was removed under reduced pressure to obtain a solid that
was crystallized using ethyl acetate to yield the title compound as
off-white solid (171 mg, 40%).
1H NMR (300 MHz, DMSO-d6)
d
8.57 (bs, 3H), 8.38 (d, J ¼ 9.0 Hz,
2H), 8.25 (d, J ¼ 9.0 Hz, 2H), 4.68 (s, 2H); MS (ESIþ) m/z 181
[M þ H]þ.
5.2.14. Ethyl 2-((2-(4-nitrophenyl)-2-oxoethyl)amino)-2-oxoacetate
(23)
To a solution of 2-amino-1-(4-nitrophenyl)ethanone hydrochlo-
ride (11.5 g, 53.1 mmol, 1.0 equiv) in EtOAc (115 ml), was added
triethylamine (8.88 ml, 63.7 mmol, 1.2 equiv). To this mixture eth-
ylchlorooxoacetate (7.11 ml, 63.7 mmol, 1.2 equiv) dissolved in ethyl
acetate(35 ml)wasaddeddropwise andrefluxed for 2 h. Thereaction
mass was cooled, quenched with water, and the resulting organic
layer was separated, dried over sodium sulfate and evaporated under
reduced pressure to get 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 wasfurthercrystallizedin EtOAc/Petethertoyieldthe
desired compound as a yellow colored solid (8.9 g, 59%).
1H NMR (300 MHz, DMSO-d6)
d 11.03 (s, 1H), 8.21e8.18 (m, 2H),
8.04 (d, J ¼ 8.4 Hz, 2H), 7.99 (d, J ¼ 8.4 Hz, 2H), 7.78e7.52 (m, 1H),
7.66e7.61 (m,1H), 7.22e7.16 (m,1H), 4.18 (m,1H), 2.23 (m,1H), 0.93
1H NMR (300 MHz, DMSO-d6)
d
9.21 (m, 1H), 8.35 (d, J ¼ 9.0 Hz,
(d, J ¼ 6.3 Hz, 6H); 13C NMR (75 MHz, DMSO-d6):
d
173.23, 167.74,
2H), 8.24 (d, J ¼ 9.0 Hz, 2H), 4.78 (d, J ¼ 6.0 Hz, 2H), 4.29 (q,
161.48, 160.30, 156.96, 150.83, 149.01, 143.20, 131.90, 127.88 (2C),
126.35,123.51,120.72,118.31 (2C),114.14,108.63; 58.63, 31.27,19.97,
18.63; HRMS (ESIþ) calcd for C22H19FN4O3S2 [M þ H]þ 471.0955,
found 471.0930 (error 5.27 ppm); mp 240e242 ꢀC; HPLC Retention
time e 3.95 min, Purity e 97.98%.
J ¼ 6.0 Hz, 2H),1.29 (t, J ¼ 6.0 Hz, 3H); MS (ESIꢁ) m/z 278.8 [M ꢁ H]ꢁ.
5.2.15. Ethyl 5-(4-nitrophenyl)thiazole-2-carboxylate (24)
To a solution of ethyl 2-(2-(4-nitrophenyl)-2-oxoethylamino)-2-
oxoacetate (5 g, 17.84 mmol, 1.0 equiv) in 1,4-Dioxane (100 ml) was
added Lawesson’s reagent (7.22 g, 17.84 mmol, 1.0 equiv) and the
mixture was refluxed for 2 h. The reaction mixture was subse-
quently cooled, added to water and neutralized with a saturated
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 chromatographed
using 0.5:9.5 EtOAc:CHCl3 to get a dark yellow colored solid that
was crystallized in CHCl3/Pet ether to yield the product as a pale
yellow colored solid (3.65 g, 73%).
5.2.12. 2-(4-(4-((6-Fluorobenzo[d]thiazol-2-yl)amino)phenyl)
thiazole-2-carboxamido)-3-methylbutanoic acid (8)
To a solution of compound 21 (300 mg, 0.900 mmol,1.0 equiv) in
n-butanol (5 ml) was added 2-chloro-6-fluorobenzo[d]thiazole
(202 mg, 1.1 mmol, 1.1 equiv) and the reaction mixture was stirred
at 70 ꢀC followed by the addition of 4.0 M HCl (131 mg). The
resulting reaction mixture was stirred at 90 ꢀC for 16 h. Following
reaction completion the solvent was removed under reduced
pressure and the compound was purified by flash column chro-
matography using 10:90 EtOAc:Pet ether to yield methyl 2-(4-(4-
((6-fluorobenzo[d]thiazol-2-yl)amino)phenyl)thiazole-2-
carboxamido)-3-methylbutanoate (130 mg, 29%) as a solid com-
pound. To this butanoate ester (105 mg, 0.216 mmol) in THF (2 ml)
was added 1.0 N lithium hydroxide solution (45 mg, 1.1 mmol,
1.1 equiv) and the reaction mixture was stirred at rt for 4 h.
Following reaction completion THF was removed under reduced
pressure and dil. HCl was added to acidify the reaction mixture. The
solid that precipitated as a result was filtered, washed with water,
and dried to yield (70 mg, 68%) of the title compound as a white
solid compound.
1H NMR (300 MHz, CDCl3)
d
8.33 (d, J ¼ 9.0 Hz, 2H), 8.30 (s, 1H),
7.83 (d, J ¼ 9.0 Hz, 2H), 4.54 (q, J ¼ 6.0 Hz, 2H), 1.49 (t, J ¼ 6.0 Hz,
3H); MS (ESIþ): m/z 279 [M þ H]þ.
5.2.16. 5-(4-Nitrophenyl)thiazole-2-carboxylic acid (25)
To a solution of ethyl 5-(4-nitrophenyl)thiazole-2-carboxylate
(3.6 g, 12.94 mmol, 1.0 equiv) in THF (90 ml) was added 1.0 M aq.
NaOH (52 ml) and the reaction mixture was stirred at rt for 15e
20 min. The reaction mixture was acidified to pH 2.0 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 pale yellow
colored solid. Recrystallization with EtOAc/Pet ether yielded the
desired product as an off white solid (2.48 g, 76%).
1H NMR (300 MHz, DMSO-d6)
d
10.71 (s, 1H), 8.41 (d, J ¼ 8.1 Hz,
1H), 8.29 (s, 1H), 8.05 (d, J ¼ 7.8 Hz, 2H), 7.88 (d, J ¼ 8.1 Hz, 2H), 7.74
(d, J ¼ 7.5 Hz, 1H), 7.60 (m, 1H), 7.17 (t, J ¼ 6.9 Hz, 1H), 4.24 (m,
1H), 2.27e2.25 (m, 1H), 0.94 (d, J ¼ 6.0 Hz, 6H); 13C NMR
1H NMR (300 MHz, DMSO-d6)
d 14.21 (bs,1H), 8.70 (s,1H), 8.31 (d,
(300 MHz, DMSO-d6):
d
172.86, 163.38, 161.65, 159.19, 157.52,
J ¼ 9.0 Hz, 2H), 8.10 (d, J ¼ 9.0 Hz, 2H); MS (ESIþ): m/z 251 [M þ H]þ.
155.75, 149.18, 141.29, 131.76, 127.69, 127.56 (2C), 120.53, 118.62,
118.26 (2C), 114.01, 108.57, 58.82, 30.84, 19.87, 18.77; HRMS (ESIþ)
calcd for C22H19FN4O3S2 [M þ H]þ 471.0955, found 471.0935 (mean
error ¼ 4.28); mp 226e228 ꢀC; HPLC Retention time e 3.891 min,
Purity e 98.9%.
5.2.17. Methyl 3-methyl-2-(5-(4-nitrophenyl)thiazole-2-carboxamido)
butanoate (26)
To a solution of 5-(4-nitrophenyl)thiazole-2-carboxylic acid
(2.3 g, 9.19 mmol, 1.0 equiv) in THF (72 ml) was added N-methyl

K.S. Kadam et al. / European Journal of Medicinal Chemistry 65 (2013) 337e347
345
morpholine (1.01 ml, 9.19 mmol, 1.0 equiv) and the reaction
mixture was stirred for 10 min at rt. The resulting mixture was
cooled to ꢁ20 ꢀC and to this isobutyl chloroformate (1.19 ml,
9.19 mmol, 1.0 equiv) was added and stirred for 15e20 min at ꢁ20
found 471.0939 (error 3.02 ppm); mp 244e246 ꢀC; HPLC Retention
time e 3.81 min, Purity e 99.72%.
5.2.20. General procedure for the synthesis of urea analogs
to ꢁ30 ꢀC. To this reaction mass was added a solution of
L-valine
To a solution of Compound 27 (0.36 mmol, 1.0 equiv) in THF
(3 ml) was added the appropriately substituted phenyl isocyanate
(1.1 equiv) and the mixture was stirred for 16 h at rt. The reaction
mixture was then concentrated and purified using flash column
chromatography (2:8 EtOAc/Pet ether) to afford the desired methyl
ester. To a solution of the methyl ester (1 equiv) in THF was added
1.0 M solution of LiOH in water (5 equiv) and stirred for 16 h at rt.
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 extracted with ethyl acetate, washed with water,
and dried over anhydrous sodium sulfate. The solvent was removed
under reduced pressure to obtain a solid that was crystallized in
ethyl acetate to yield the desired acid.
methyl ester hydrochloride (2.15 g, 12.87 mmol, 1.4 equiv) in THF
(20 ml) after neutralizing with triethylamine (1.8 ml, 12.87 mmol,
1.4 equiv). This mixture was stirred at ꢁ20 to ꢁ30 ꢀC for 5 min.
Following this the reaction mixture was gradually warmed up to rt
over a period of 1 h. The organic solvent was removed under
reduced pressure and the crude material was subjected to column
chromatography using 15:85 EtOAc:CHCl3 to get a pale yellow
colored solid. The title compound (2.25 g, 67%) was obtained as an
off white solid on crystallization with EtOAc/Pet ether.
1H NMR (300 MHz, DMSO-d6)
d
8.95 (d, J ¼ 9.0 Hz, 1H), 8.70 (s,
1H), 8.34 (d, J ¼ 9.0 Hz, 2H), 8.09 (d, J ¼ 9.0 Hz, 2H), 4.33 (m, 1H),
3.68 (s, 3H), 2.27 (m,1H), 0.95 (t, J ¼ 6.0 Hz, 6H); MS (ESIþ): m/z 364
[M þ H]þ.
5.2.20.1. 3-Methyl-2-(5-(4-(3-(3-(trifluoromethyl)phenyl)ureido)
phenyl)thiazole-2-carboxamido)butanoic acid (28). Prepared as
described above in the general procedure using 3-trifluorophenyl
isocyanate (74.12 mg, 0.39 mmol, 1.1 equiv) as the substituted iso-
cyanate to afford the title compound (94 mg, 52%) as an off white
solid.
5.2.18. Methyl 2-(5-(4-aminophenyl)thiazole-2-carboxamido)-3-
methylbutanoate (27)
To a solution of methyl 3-methyl-2-(5-(4-nitrophenyl)thiazole-
2-carboxamido)butanoate (2.15 g, 5.92 mmol, 1.0 equiv) in EtOH
(21.5 ml), THF (8.6 ml), and water (8.6 ml) were added ammonium
chloride (1.04 g, 17.75 mmol, 3.0 equiv) and iron (777 mg,
13.90 mmol, 2.35 equiv) and the mixture was refluxed at 80 ꢀ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 water and extracted
using ethyl acetate. The organic layer was dried using sodium sul-
fate and concentrated to obtain a dark brown residue which was
purified by column chromatography using 2.5:7.5 EtOAc:CHCl3 to
afford the title compound as a sticky yellow solid (1.82 g, 91%).
1H NMR (300 MHz, DMSO-d6)
d 12.97 (bs, 1H), 9.18 (s, 1H), 9.13
(s, 1H), 8.37 (m, 1H), 8.03 (bs, 1H), 7.71 (d, J ¼ 9.0 Hz, 2H), 7.61 (d,
J ¼ 9.0 Hz, 2H), 7.55e7.50 (m, 2H), 7.33 (d, J ¼ 6.0 Hz, 1H), 4.31, (m,
1H), 2.28 (m, 1H), 0.95 (d, J ¼ 6.0 Hz, 6H); 13C NMR (75 MHz, DMSO-
d6):
d 172.91, 161.71, 159.49, 152.82, 144.85, 141.05, 140.89, 139.37,
130.38, 128.03 (3C), 124.19, 122.43, 119.20 (3C), 118.71, 114.72, 58.33,
30.35, 19.63, 18.74; HRMS (ESIþ) calcd for C23H21F3N4O4S [M þ H]þ
507.1308, found 507.1286 (error 4.30 ppm); mp 220e222 ꢀC; HPLC
Retention time e 3.78 min, Purity e 99.14%.
1HNMR (300 MHz, DMSO-d6)
d
8.59 (d, J ¼ 9.0 Hz, 1H), 8.15 (s,
1H), 7.42 (d, J ¼ 9.0 Hz, 2H), 6.63 (d, J ¼ 9.0 Hz, 2H), 5.76 (bs, 2H),
4.31 (m, 1H), 3.67 (s, 3H), 2.27 (m, 1H), 0.91 (d, J ¼ 6.0 Hz, 6H); MS
(ESIþ): m/z 334 [M þ H]þ.
5.2.20.2. 2-(5-(4-(3-(2-Chlorophenyl)ureido)phenyl)thiazole-2-
carboxamido)-3-methylbutanoic acid (29). Prepared as described
above in the general procedure using 2-chlorophenyl isocyanate
(60.84 mg, 0.396 mmol, 1.1 equiv) as the substituted isocyanate to
afford the title compound (108 mg, 63%) as an off white solid.
5.2.19. 2-(5-(4-((6-Fluorobenzo[d]thiazol-2-yl)amino)phenyl)thia
zole-2-carboxamido)-3-methylbutanoic acid (7)
1H NMR (300 MHz, DMSO-d6)
d 9.67 (s, 1H), 8.38e8.34 (m, 3H),
Compound 27 (150 mg, 0.45 mmol, 1.0 equiv) and 2-chloro-6-
fluorobenzo[d]thiazole (101 mg, 0.54 mmol, 1.2 equiv) were taken
in EtOH (3 ml) and heated at 55e60 ꢀC to obtain a clear solution. To
this solution was added 4.0 M aq. HCl in 1,4-Dioxane (0.11 ml,
0.45 mmol, 1.0 equiv) and the resulting mixture was refluxed at
80 ꢀC for 20 h. Following this the EtOH was removed completely
under pressure and the residue was purified by column chroma-
tography using 2:8 EtOAc:Pet ether to obtain an yellow solid. The
solid was crystallized in EtOAc/Pet ether to yield the corresponding
methyl ester. To a solution of this methyl ester (1 equiv) in THF was
added 1.0 M solution of LiOH in water (5 equiv) and stirred for 16 h
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
material thus obtained was extracted using ethyl acetate, washed
with water, and dried over anhydrous sodium sulfate. The solvent
was removed under reduced pressure to obtain a solid that was
crystallized using ethyl acetate to yield the title compound as a pale
yellow solid (87 mg, 41%).
8.18 (d, J ¼ 9.0 Hz, 1H), 7.74 (d, J ¼ 9.0 Hz, 2H), 7.59 (d, J ¼ 9.0 Hz,
2H), 7.49 (d, J ¼ 9.0 Hz, 1H), 7.32 (m, 1H), 7.06 (m, 1H), 4.33, (m, 1H),
3.68 (s, 3H), 2.26 (m, 1H), 0.95 (d, J ¼ 6.0 Hz, 6H); 13C NMR (75 MHz,
DMSO-d6):
d 172.83, 160.71, 159.48, 152.43, 144.83, 141.04,
139.37, 136.20, 129.70, 128.12 (3C), 124.20, 124.00, 122.60, 121.93,
118.95 (2C), 58.31, 30.34, 19.64, 18.73; HRMS (ESIþ) calcd
for C22H21ClN4O4S [M þ H]þ 473.1045, found 473.1028 (error
3.59 ppm); mp 206e208 ꢀC; HPLC Retention time e 3.49 min,
Purity e 99.65%.
5.2.20.3. 2-(5-(4-(3-(3,4-Dimethylphenyl)ureido)phenyl)thiazole-2-
carboxamido)-3-methylbutanoic acid (30). Prepared as described
above in the general procedure using 3,4-Dimethylphenyl isocya-
nate (58.21 mg, 0.396 mmol,1.1 equiv) as the substituted isocyanate
to afford the title compound (95 mg, 56%) as an off white solid.
1H NMR (300 MHz, DMSO-d6)
d 12.98 (bs, 1H), 8.91 (s, 1H), 8.56
(s, 1H), 8.36 (m, 2H), 8.34 (s, 1H), 7.71 (d, J ¼ 9.0 Hz, 2H), 7.57 (d,
J ¼ 9.0 Hz, 2H), 7.24 (bs, 1H), 7.19e7.16 (m, 1H), 7.04 (d, J ¼ 9.0 Hz,
1H), 4.31, (m, 1H), 2.27 (m, 1H), 2.23 (s, 3H), 2.20 (s, 3H), 0.95 (d,
1H NMR (300 MHz, DMSO-d6)
d 12.99 (bs, 1H), 10.76 (s, 1H), 8.36
(s, 1H), 8.34 (d, 1H), 7.88 (d, J ¼ 9.0 Hz, 2H), 7.81e7.76 (m, 3H), 7.65
J ¼ 6.0 Hz, 6H); 13C NMR (75 MHz, DMSO-d6):
d 172.80, 160.50,
(dd, J ¼ 6.0 Hz, 1H), 7.20 (m, 1H), 4.32, (m, 1H), 2.27 (m, 1H), 0.97 (d,
159.54, 152.78, 144.99, 141.50, 139.22, 137.56, 136.82, 130.11 (2C),
128.01 (2C), 123.71, 120.11, 118.80 (2C), 116.34, 58.25, 30.28, 20.10,
19.62, 19.14, 18.74; HRMS (ESIþ) calcd for C24H26N4O4S [M þ H]þ
467.1748, found 467.1724 (error 4.91 ppm); mp 188e190 ꢀC; HPLC
Retention time e 3.58 min, Purity e 99.63%.
J ¼ 6.0 Hz, 6H); 13C NMR (75 MHz, DMSO-d6):
d 172.79, 161.49,
160.66, 159.56, 157.60, 149.07, 144.91, 141.85, 139.38, 131.76, 128.24
(2C), 124.08, 120.69, 118.54 (2C), 114.12, 108.63, 58.25, 30.31, 19.62,
18.74; HRMS (ESIþ) calcd for C22H19FN4O3S2 [M þ H]þ 471.0955,

346
K.S. Kadam et al. / European Journal of Medicinal Chemistry 65 (2013) 337e347
5.2.20.4. 3-Methyl-2-(5-(4-(3-(2-phenoxyphenyl)ureido)phenyl)
thiazole-2-carboxamido)butanoic acid (31). Prepared as described
above in the general procedure using 2-phenoxyphenyl isocyanate
(83.55 mg, 0.396 mmol, 1.1 equiv) as the substituted isocyanate to
afford the title compound (83 mg, 44%) as an off white solid.
58.24, 35.18, 31.39 (3C), 30.29, 19.63, 18.76; HRMS (ESIþ) calcd for
C
26H29N3O4S [M þ H]þ 480.1952, found 480.1928 (error 3.95 ppm);
mp 226e228 ꢀC; HPLC Retention time e 4.28 min, Purity e 99.95%.
5.2.21.2. 2-(5-(4-(3-Cyclohexylpropanamido)phenyl)thiazole-2-
carboxamido)-3-methylbutanoic acid (34). The methyl ester was
synthesized by general procedure III using 3-cyclohexylpropanoyl
chloride (118 mg, 0.67 mmol, 1.5 equiv) as the substituted acid
chloride at the first step and subsequently deprotected by following
general procedure II at the second step to afford the title compound
(78 mg, 38%) as a white solid.
1H NMR (300 MHz, DMSO-d6)
d 12.98 (bs, 1H), 9.53 (s, 1H), 8.54
(s, 1H), 8.37 (m, 2H), 8.30e8.27 (m, 1H), 7.72 (d, J ¼ 9.0 Hz, 2H), 7.56
(d, J ¼ 9.0 Hz, 2H), 7.43 (dd, J ¼ 9.0 Hz, 2H), 7.19e7.13 (m, 2H), 7.06
(d, J ¼ 9.0 Hz, 2H), 7.00 (m, 1H), 6.86 (m, 1H), 4.31 (m, 1H), 2.27 (m,
1H), 0.94 (d, J ¼ 6.0 Hz, 6H); 13C NMR (75 MHz, DMSO-d6):
d 172.78,
160.57, 159.54, 157.17, 152.59, 145.52, 144.91, 141.21, 139.30, 131.54,
130.52 (2C), 128.10 (2C), 124.45, 124.04, 123.97, 122.91, 120.19,
118.81 (3C), 118.75 (2C), 58.24, 30.26, 19.61, 18.74; HRMS (ESIþ)
1H NMR (300 MHz, DMSO-d6)
d 13.01 (bs, 1H), 10.08 (s, 1H),
8.36e8.33 (m, 2H), 7.74e7.68 (m, 4H), 4.30 (d, J ¼ 6 Hz, 1H), 2.34 (t,
J ¼ 7.5 Hz, 2H), 2.29e2.20 (m, 1H), 1.73e1.54 (m, 4H), 1.50 (q,
J ¼ 6.9 Hz, 2H), 1.25e1.09 (m, 4H), 1.94 (d, J ¼ 6.9 Hz, 6H), 0.94e0.91
calcd for
C
28H26N4O5S [M
þ
H]þ 531.1697, found 531.1665
(error 6.02 ppm); mp 172e174 ꢀC; HPLC Retention time e 4.28 min,
Purity e 99.59%.
(m, 3H); 13C NMR (300 MHz, DMSO-d6):
d 172.77, 172.25, 160.82,
159.51, 144.75, 140.83, 139.53, 172.88 (2C), 124.98, 119.81 (2C),
58.24, 37.24, 34.50, 33.05 (2C), 32.94, 30.25, 26.56, 26.22 (2C), 19.61,
18.74; HRMS (ESIþ) calcd for C24H32N3O4S [M þ H]þ 458.2108,
found 458.2092 (error ¼ 4.57); mp 198e200 ꢀC; HPLC Retention
time e 4.39 min, Purity e 96.4%.
5.2.20.5. 2-(5-(4-(3-(4-Chloro-2-phenoxyphenyl)ureido)phenyl)thia-
zole-2-carboxamido)-3-methylbutanoic acid (32). Prepared as des
cribed above in the general procedure using 2-phenoxy, 4-
chlorophenyl isocyanate (97.25 mg, 0.396 mmol, 1.1 equiv) as the
substituted isocyanate to afford the title compound (85 mg, 40%) as
an off white solid.
5.2.21.3. 2-(5-(4-(4,4-Difluorocyclohexanecarboxamido)phenyl)thia-
zole-2-carboxamido)-3-methylbutanoic acid (35). The methyl ester
was synthesized by general procedure III using 4,4-difluoro
cyclohexanecarbonyl chloride (123 mg, 0.67 mmol, 1.5 equiv) as
the substituted acid chloride at the first step and subsequently
deprotected by following general procedure II at the second step to
afford the title compound (116 mg, 55%) as a white solid.
1H NMR (300 MHz, DMSO-d6)
d 12.99 (bs, 1H), 9.61 (s, 1H), 8.73
(s, 1H), 8.39 (d, J ¼ 9.0 Hz,1H), 8.37e8.34 (m, 2H), 7.73 (d, J ¼ 9.0 Hz,
2H), 7.56 (d, J ¼ 9.0 Hz, 2H), 7.47e7.42 (m, 2H), 7.2 (dd, J ¼ 9.0 Hz,
1H), 7.10 (d, J ¼ 9.0 Hz, 2H), 7.03e6.99 (m, 1H), 6.86 (d, J ¼ 9.0 Hz,
1H), 4.31 (m, 1H), 2.27 (m, 1H), 0.94 (d, J ¼ 6.0 Hz, 6H); 13C NMR
(75 MHz, DMSO-d6): d 172.80,160.72, 159.49,156.74,152.44,144.79,
144.41, 140.85, 139.40, 132.74, 130.64 (2C), 128.12 (2C), 128.06,
124.49, 124.27, 122.19, 119.84, 119.10 (3C), 118.90 (2C), 58.30, 30.32,
19.64, 18.73; HRMS (ESIþ) calcd for C28H25ClN4O5S [M þ H]þ
565.1307, found 565.1303 (error 0.70 ppm); mp 180e182 ꢀC; HPLC
Retention time e 4.89 min, Purity e 99.77%.
1H NMR (300 MHz, DMSO-d6)
d 12.99 (bs, 1H), 10.19 (s, 1H), 8.36
(s, 2H), 7.72 (s, 4H), 4.29 (d, J ¼ 6.3 Hz,1H), 2.27e2.25 (m, 1H), 2.20e
2.00 (m, 2H), 1.95e1.91 (m, 3H), 1.80e1.67 (m, 3H), 0.95 (d, J ¼ 6 Hz,
6H); 13C NMR (300 MHz, DMSO-d6):
d 173.61, 172.85, 160.97, 159.47,
144.68, 140.71, 139.60, 127.87 (2C), 125.21, 120.04 (2C), 58.32,
42.48, 32.68, 30.35 (2C), 26.03, 25.95 (2C), 19.64, 18.73; HRMS
(ESIþ) calcd for C22H26F2N3O4S [M þ H]þ 466.1607, found 466.1596
(error ¼ 1.40); MS (ESIþ): m/z 466 [M þ H]þ; mp 254e256 ꢀC; HPLC
Retention time e 3.15 min, Purity e 99.7%.
5.2.21. General procedure for the synthesis of thiazole analogs
possessing an amide linker
To a solution of compound 27 (150 mg, 0.45 mmol, 1 equiv) in
DCM (3 ml) was added pyridine (0.11 ml, 1.35 mmol, 3.0 equiv) and
stirred for 5 min. To this reaction mixture the appropriate acid
chloride (1.5 equiv) was added and stirred for 1 h. The reaction
mixture was then concentrated and purified using flash column
chromatography using 2:8 EtOAc:CHCl3 to yield the corresponding
methyl ester. To a solution of the methyl ester (1 equiv) in THF was
added 1.0 M solution of LiOH in water (5 equiv) and stirred for 16 h
at rt. 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 extracted with ethyl acetate, washed
with water, and dried over anhydrous sodium sulfate. The solvent
was removed under reduced pressure to obtain a solid that was
crystallized in ethyl acetate to yield the desired acid.
5.2.21.4. 2-(5-(4-(5-Butylpicolinamido)phenyl)thiazole-2-carboxami
do)-3-methylbutanoic acid (36). The methyl ester was synthesized by
general procedure III using 5-butylpicolinoyl chloride (133 mg,
0.67 mmol, 1.5 equiv) as the substituted acid chloride at the first step
and subsequently deprotected by following general procedure II at
the second step to afford the title compound (64 mg, 30%) as a white
solid.
1H NMR (300 MHz, DMSO-d6)
d 12.96 (bs, 1H), 10.81 (s, 1H), 8.59
(s, 1H), 8.40e8.35 (m, 2H), 8.07e7.98 (m, 3H), 7.91 (d, J ¼ 7.8 Hz,
1H), 7.79 (d, J ¼ 7.2 Hz, 2H), 4.30 (m, 1H), 2.73e2.62 (m, 2H), 1.35e
1.26 (m, 1H), 1.61 (m, 2H), 1.33e1.32 (m, 2H), 0.94 (s, 9H); 13C NMR
(300 MHz, DMSO-d6):
d 172.81, 163.26, 161.11, 159.49, 148.84,
148.01, 144.65, 142.04, 139.97, 139.83, 138.05, 127.80 (2C), 125.87,
122.71, 121.12 (2C), 58.30, 33.04, 32.21, 30.35, 22.14, 19.64, 18.73,
14.15; HRMS (ESIþ) calcd for C25H29N4O4S [M þ H]þ 481.1904,
found 481.1877 (error ¼ 4.56); MS (ESIþ): m/z 481.2 [M þ H]þ; mp
138e140 ꢀC; HPLC Retention time e 4.75 min, Purity e 88.1%.
5.2.21.1. 2-(5-(4-(4-(tert-Butyl)benzamido)phenyl)thiazole-2-carbo
xamido)-3-methylbutanoic acid (33). The methyl ester was syn-
thesized by general procedure III using 4-tert-butylbenzoyl chloride
(0.125 ml, 0.68 mmol, 1.5 equiv) as the substituted acid chloride at
the first step and subsequently deprotected by following general
procedure II at the second step to afford the title compound
(126 mg, 59%) as a white solid.
5.2.22. Methyl 4-(2-cyanopropan-2-yl)benzoate (38)
To a solution of potassium tert-butoxide (8.0 g, 0.08 mmol,
2.5 equiv) in THF (25 ml) at ꢁ30 ꢀC was added a solution of methyl
iodide (5.35 ml, 0.09 mmol, 3.0 equiv) and methyl 4-(cyanomethyl)
benzoate (5.0 g, 0.03 mmol,1.0 equiv) in THF (25 ml) under nitrogen
atmosphere over 20 min. The cooling bath was removed and the
reaction mixture was allowed to warm to rt and stirred for 2 h.
Following this the reaction was quenched with water (10 ml) and
1H NMR (300 MHz, DMSO-d6)
d 13.02 (bs, 1H), 10.38 (s, 1H),
8.40e8.36 (m, 2H), 7.94 (d, J ¼ 9.0 Hz, 2H), 7.91 (d, J ¼ 9.0 Hz, 2H),
7.79 (d, J ¼ 9.0 Hz, 2H), 7.57 (d, J ¼ 9.0 Hz, 2H), 4.32 (m, 1H), 2.26 (m,
1H), 1.33 (s, 9H), 0.95 (d, J ¼ 6.0 Hz, 6H); 13C NMR (75 MHz, DMSO-
d6):
d 172.78, 166.13, 161.00, 159.53, 155.12, 144.73, 140.81, 139.73,
134.90, 132.46, 128.07 (2C), 127.79 (2C), 125.68 (2C), 121.02 (2C),

K.S. Kadam et al. / European Journal of Medicinal Chemistry 65 (2013) 337e347
347
ethyl acetate was added and the organic and aqueous layers were
1H NMR (300 MHz, DMSO-d6)
d
13.03 (bs, 1H), 10.52 (s, 1H),
separated. The organic layer was washed with water, brine, and
dried over sodium sulfate. The organic solvent was removed under
vacuum to obtain a violet residue that was purified by column
chromatography using 20:80 EtOAc:Pet ether to obtain an off white
solid that was crystallized in CHCl3:Pet ether to afford the title
compound (4.0 g, 69%) as a white solid.
8.40e8.37 (m, 2H), 8.02 (d, J ¼ 9.0 Hz, 2H), 7.92 (d, J ¼ 9.0 Hz, 2H),
7.80 (d, J ¼ 9.0 Hz, 2H), 7.61 (d, J ¼ 9.0 Hz, 2H), 4.29 (m, 1H), 2.25
(m, 1H), 2.12e1.99 (m, 4H), 0.96 (d, J ¼ 6.0 Hz, 6H), 0.81 (t,
J ¼ 6.0 Hz, 6H); 13C NMR (75 MHz, DMSO-d6):
d 172.77, 165.71,
161.13, 159.47, 144.64, 142.19, 140.63, 139.78, 134.61, 128.86 (2C),
127.81 (2C), 126.64 (2C), 125.78, 122.38, 121.12 (2C), 58.34, 50.05,
33.06 (2C), 30.78, 19.65, 18.73, 10.03 (2C); HRMS (ESIþ) calcd
for C28H30N4O4S [M þ H]þ 519.2061, found 519.2042 (error
3.27 ppm); mp 242e244 ꢀC; HPLC Retention time e 3.91 min,
Purity e 98.27%.
1HNMR (300 MHz, CDCl3):
d
8.08 (d, J ¼ 9.0 Hz, 2H), 7.57 (d,
J ¼ 9.0 Hz, 2H), 3.97 (s, 3H), 1.76 (s, 6H); MS (ESIþ): m/z 204.1
[M þ H]þ.
5.2.23. Methyl 4-(3-cyanopentan-3-yl)benzoate (39)
Synthesized compound 39 using the same procedure as
described for compound 38 by replacing ethyl iodide (4.11 ml,
0.05 mmol, 3.0 equiv) in place of methyl iodide to afford the title
compound (2.5 g, 63%) as a white solid.
Appendix A. Supplementary data
Supplementary data related to this article can be found at
http://dx.doi.org/10.1016/j.ejmech.2013.05.006.
1H NMR (300 MHz, CDCl3):
d
8.07 (d, J ¼ 9.0 Hz, 2H), 7.50 (d,
J ¼ 9.0 Hz, 2H), 3.94 (s, 3H), 2.0 (m, 2H), 2.00e1.89 (m, 2H), 0.91 (t,
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To
a solution of methyl 2-(5-(4-aminophenyl)thiazole-2-
carboxamido)-3-methylbutanoate (150 mg, 0.45 mmol, 1.0 equiv)
and methyl 4-(2-cyanopropan-2-yl)benzoate (100 mg, 0.495 mmol,
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the reaction mixture was sealed and heated to 80 ꢀC for 4 h.
Following this the reaction mass was cooled to rt and neutralized
with saturated aqueous solution of ammonium chloride. The re-
action mixture was extracted with CH2Cl2 and the organic layer was
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saturated sodium bicarbonate and brine solution. The organic layer
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under reduced pressure to obtain dark brown residue which was
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pale brown colored solid. The solid was crystallized from CHCl3:Pet
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ester (1 equiv) in THF was added 1.0 M solution of LiOH in water
(4 equiv) and stirred for 16 h at rt. Following removal of the solvent
obtained residue was diluted with water and acidified to pH 2 using
2.0 M HCl. The material thus obtained was extracted with EtOAc,
washed with water, and dried over anhydrous sodium sulfate. The
solvent was removed under reduced pressure to obtain a solid that
was crystallized in EtOAc to yield the desired acid as white solid
(25 mg, 12%).
1H NMR (300 MHz, DMSO-d6)
d
13.00 (bs, 1H), 10.49 (s, 1H),
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d6):
d 172.78, 165.63, 161.07, 159.53, 145.49, 144.66, 140.60, 139.80,
134.66, 128.85 (2C), 127.82 (2C), 125.79 (3C), 124.78, 121.12 (2C),
58.28, 37.31, 30.29 (2C), 28.63, 19.63, 18.76; HRMS (ESIþ) calcd for
C
26H26N4O4S [M þ H]þ 491.1748, found 491.1730 (error 3.43 ppm);
mp 256e258 ꢀC; HPLC Retention time e 3.46 min, Purity e 92.97%.
5.2.25. 2-(5-(4-(4-(3-Cyanopentan-3-yl)benzamido)phenyl)thiazole-
2-carboxamido)-3-methylbutanoic acid (41)
Synthesized following a similar procedure as described for
compound 40 by replacing methyl 4-(3-cyanopentan-3-yl)benzo-
ate (114 mg, 0.495 mmol, 1.1 equiv) in place of methyl 4-(2-
cyanopropan-2-yl)benzoate to afford the title compound (58 mg,
25%) as an off-white solid.
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